id stringlengths 3 9 | source stringclasses 1 value | version stringclasses 1 value | text stringlengths 1.54k 298k | added stringdate 1993-11-25 05:05:38 2024-09-20 15:30:25 | created stringdate 1-01-01 00:00:00 2024-07-31 00:00:00 | metadata dict |
|---|---|---|---|---|---|---|
211988999 | pes2o/s2orc | v3-fos-license | Applications of deep learning in stock market prediction: recent progress
Stock market prediction has been a classical yet challenging problem, with the attention from both economists and computer scientists. With the purpose of building an effective prediction model, both linear and machine learning tools have been explored for the past couple of decades. Lately, deep learning models have been introduced as new frontiers for this topic and the rapid development is too fast to catch up. Hence, our motivation for this survey is to give a latest review of recent works on deep learning models for stock market prediction. We not only category the different data sources, various neural network structures, and common used evaluation metrics, but also the implementation and reproducibility. Our goal is to help the interested researchers to synchronize with the latest progress and also help them to easily reproduce the previous studies as baselines. Base on the summary, we also highlight some future research directions in this topic.
Introduction
Stock market prediction is a classical problem in the intersection of finance and computer science. For this problem, the famous efficient market hypoth-esis (EMH) gives a pessimistic view and implies that financial market is efficient (Fama, 1965), which maintains that technical analysis or fundamental analysis (or any analysis) would not yield any consistent over-average profit to investors. However, many researchers disagree with EMH (Malkiel, 2003).
Some studies are trying to measure the different efficiency levels for mature and emerging markets, while other studies are trying to build effective prediction models for stock markets, which is also the scope of this survey.
The effort starts with the stories of fundamental analysis and technical analysis. Fundamental analysis evaluates the stock price based on its intrinsic value, i.e., fair value, while technical analysis only relies on the basis of charts and trends. The technical indicators from experience can be further used as handcrafted input features for machine learning and deep learning models. Afterwards, linear models are introduced as the solutions for stock market prediction, which include autoregressive integrated moving average (ARIMA) (Hyndman & Athanasopoulos, 2018) and generalized autoregressive conditional heteroskedasticity (GARCH) (Bollerslev, 1986). With the development of machine learning models, they are also applied for stock market prediction, e.g., Logistic regression and support vector machine (Alpaydin, 2014).
Our focus in this survey would be the latest emerging deep learning, which is represents by various structures of deep neural networks (Goodfellow et al., 2016). Powered by the collection of big data from the Web, the parallel processing ability of graphics processing units (GPUs), and the new convolutional neural network family, deep learning has achieved a tremendous success in the past few years, for many different applications including image classification (Rawat & Wang, 2017;Jiang & Zhang, 2020), object detection (Zhao et al., 2019), time series prediction (Brownlee, 2018;Jiang & Zhang, 2018), etc. With a strong ability of dealing with big data and learning the nonlinear relationship between input features and prediction target, deep learning models have shown a better performance than both linear and machine learning models on the tasks that include stock market prediction.
In the past few years, both the basic tools for deep learning and the new prediction models are undergoing a rapid development. With the continuous improved programming packages, it becomes easier to implement and test a novel deep learning model. Also, the collection of online news or twitter data provides new sources of predicting stock market. More recently, graph neural networks using various knowledge graph data appear as new ideas. The study for stock market prediction is not limited to the academia. Attracted by the potential profit by stock trading powered by the latest deep learning models, asset management companies and investment banks are also increasing their research grant for artificial intelligence which is represented by deep learning models nowadays.
Since there are many new developments in this area, this situation makes it difficult for a novice to catch up with the latest progress. To alleviate this problem, we summarize the latest progress of deep learning techniques for stock market prediction, especially those which only appear in the past three years.
We also present the trend of each step in the prediction workflow in these three years, which would help the new-comers to keep on the right track, without wasting time on obsolete technologies.
We focus on the application of stock market, however, machine learning and deep learning methods have been applied in many financial problems. It would be beyond the scope of this survey to cover all these problems. However, the findings presented in this survey would also be insightful for other time series prediction problems in the finance area, e.g., exchange rate or cryptocurrency price prediction.
We also pay a special attention to the implementation and reproducibility of previous studies, which is often neglected in similar surveys. The list of open data and code from published papers would not only help the readers to check the validity of their findings, but also implement these models as baselines and make a fair comparison on the same datasets. Based on our summary of the surveyed papers, we try to point out some future research directions in this survey, which would help the readers to choose their next movement.
Our main contribution in this survey are summarized as follows: 1. We summarize the latest progress of applying deep learning techniques to stock market prediction, especially those which only appear in the past three years.
2. We give a general workflow for stock market prediction, based on which the previous studies can be easily classified and summarized. And the future studies can refer to the previous work in each step of the workflow.
3. We pay a special attention to implementation and reproducibility, which is often neglected in similar surveys.
4. We point out several future directions, some of which are on-going and help the readers to catch up with the research frontiers.
The rest of this survey is organize as follows: Section 2 presents related work; Section 3 gives an overview of the papers we cover; Section 4 describes the major findings in each step of the prediction workflow; Section 5 gives the discussion about implementation and reproducibility; Section 6 points up some possible future research directions; We conclude this survey in Section 7.
Related Work
Stock market prediction has been a research topic for a long time, and there are some review papers accompanied with the development and flourishment of deep learning methods prior to our work. While their focus could also be applications of deep learning methods, stock market prediction could only be one example of many financial problems in these previous surveys. In this section, we list some of them in a chronological order and discuss our motivation and unique perspectives.
Back to 2009, Atsalakis & Valavanis (2009) surveys more than 100 related published articles that focus on neural and neuro-fuzzy techniques derived and applied to forecast stock markets, with the discussion of classifications of input data, forecasting methodology, performance evaluation and performance measures used. Li & Ma (2010) gives a survey on the application of artificial neural networks in forecasting financial market prices, including the forecast of stock prices, option pricing, exchange rates, banking and financial crisis. Nikfarjam et al. (2010) surveys some primary studies which implement text mining techniques to extract qualitative information about companies and use this information to predict the future behavior of stock prices based on how good or bad are the news about these companies. Aguilar-Rivera et al. (2015) presents a review of the application of evolutionary computation methods to solving financial problems, including the techniques of genetic algorithms, genetic programming, multi-objective evolutionary algorithms, learning classifier systems, co-evolutionary approaches, and estimation of distribution algorithms. Cavalcante et al. (2016) gives an overview of the most important primary studies published from 2009 to 2015, which cover techniques for preprocessing and clustering of financial data, for forecasting future market movements, for mining financial text information, among others. Tkáč & Verner (2016) provides a systematic overview of neural network applications in business between 1994 and 2015 and reveals that most of the research has aimed at financial distress and bankruptcy problems, stock price forecasting, and decision support, with special attention to classification tasks. Besides conventional multilayer feedforward network with gradient descent backpropagation, various hybrid networks have been developed in order to improve the performance of standard models.
More recently, Xing et al. (2018) reviews the application of cutting-edge NLP techniques for financial forecasting, which would be concerned when text including the financial news or twitters is used as input for stock market prediction. Rundo et al. (2019) covers a wider topic both in the machine learning techniques, which include deep learning, but also the field of quantitative finance from HFT trading systems to financial portfolio allocation and optimization systems. Nti et al. (2019) focuses on the fundamental and technical analysis, and find that support vector machine and artificial neural network are the most used machine learning techniques for stock market prediction. Based on its review of stock analysis, Shah et al. (2019) points out some challenges and research opportunities, including issues of live testing, algorithmic trading, self-defeating, long-term predictions, and sentiment analysis on company filings. Different from other related works that cover more papers from the computer science community, Reschenhofer et al. (2019) reviews articles covered by the Social Sciences Citation Index in the category Business, Finance and gives more insight on economic significance. It also points out some problems in the existing literature, including unsuitable benchmarks, short evaluation periods, and nonoperational trading strategies.
Some latest reviews are trying to cover a wider range, e.g., Shah et al. (2019) covers machine learning techniques applied to the prediction of financial market prices, and covers more financial instruments. However, our motivation is to catch up with the research trend of applying deep learning techniques, which have been proved to outperform traditional machine learning techniques, e.g., support vector machine in most of the publications, with only a few exceptions, e.g., Ballings et al. (2015) finds that Random Forest is the top algorithm followed by Support Vector Machines, Kernel Factory, AdaBoost, Neural Networks, K-Nearest Neighbors and Logistic Regression, and Ersan et al. (2019) finds that K-Nearest Neighbor and Artificial Neural Network both outperform Support Vector Machines, but there is no obvious pros and cons between the performances of them. With the accumulation of historical prices and diverse input data types, e.g., financial news and twitter, we think the advantages of deep learning techniques would continue and it is necessary to keep updated with this trend for the future research.
Compared with , whose focus is deep learning for financial time series forecasting and a much longer time period (from 2005 to 2019 exactly), we focus on the recent progress in the past three years (2017)(2018)(2019) and a narrower scope of stock price and market index prediction. For readers who are also interested in other financial instruments, e.g., commodity price, bond price, cryptocurrency price, etc., we would refer them to this work. We also care more about the implementation workflow and result reproducibility of previous studies, e.g., dataset and code availability, which is a problem that has drawn the attention from the AI researchers (Gundersen & Kjensmo, 2018). We would also pay more attention to the uniqueness of stock market prediction (or financial time series forecasting) from general time series prediction problems, e.g., the evaluation of profitability besides prediction accuracy.
Overview
In this section, we give an overview of the papers we are going to review in this study. All the works are searched and collected from Google Scholar, with searching keywords such as deep learning, stock prediction, stock forecasting, etc. Most of the covered papers (115 out of 124) are published in the past three years (2017-2019). In total, we cover 56 journal papers, 58 conference papers and 10 preprint papers. These preprint papers are all from arXiv.org, which is a famous website for e-print archive and we cover these papers to keep updated with the latest progress. The top source journals & conferences sorted by the number of papers we cover in this study are shown in Table 1 and Table 2, respectively. In this study, the major focus would be the prediction of the close prices of individual stocks and market indexes. Some financial instrument whose price is bounded to the market index is also covered, e.g., some exchange-traded fund (ETF) or equity index futures that track the underlying market index. For intraday prediction, we would also cover mid-price prediction for limit order books. Other financial instruments are not mentioned in this study, e.g., bond price and cryptocurrency price. More specifically, if the target to predict the specific value of the prices, we classify it as a regression problem, and if the target is to predict the price movement direction, e.g., going up or down, we classify it as a classification problem. Most studies are considering the daily prediction (105 of 124) and only a few of them are considering the intraday prediction (18 of 124), e.g., 5-minute or hourly prediction. Only one of the 124 papers is considering both the daily and intraday situations (Liu & Wang, 2019).
Based on the target output and frequency, the prediction problems can be classified into four types: daily classification (52 of 124), daily regression (54 of 124), intraday classification (8 of 124) and intraday regression (11 of 124). A detailed paper count of different prediction problem types is shown in Figure 1.
The reason behind this could be partially justified by the difficulty of collecting the corresponding data. The daily historical prices and news titles are easier to collect and process for research, while the intraday data is very limited in the academia. We would further discuss the data availability in Section 5. Surveyed markets as well as the most famous stock market index in these markets are shown in the Table 3. The paper count of different surveyed markets is shown in Figure 2 1 . Most of the studies would focus on one market, while some of them would evaluate their models on multiple markets 2 . Both mature markets (e.g., US) and emerging markets (e.g., China) are gaining a lot of attention from the research community in the past three years.
Prediction Workflow
Given different combinations of data sources, previous studies explored the use of deep learning models to predict stock market price/movement. In this section, we summarize the previous studies in a general workflow with four steps that most of the studies follow: Raw Data, Data Processing, Prediction Model and Model Evaluation. In this section, we would discuss each step separately and reveal a general approach that the future work can easily reproduce.
Raw Data
The first step of predicting is to collect proper data as the basis. It could be the intrinsic historical prices with the assumption that history repeats itself, or the extrinsic data sources that affect the stock market. In the efficient-market hypothesis, asset prices already reflect all available information. However, in practice many researchers do not agree with this conclusion, thus many different extrinsic sources of data are used for stock market prediction, e.g., Weng et al. (2017) compares the usage of market data, technical indicators, Wikipedia traffic, Google news counts, and generated features, and Liu et al. (2019b) covers market data, fundamental data, knowledge graph, and news.
Data Types
In this part, we categories the raw data that are commonly used for stock market prediction into seven types: • Market data: market data includes all trading activities that happen in a stock market, e.g., open/high/low/close prices, trading volume, etc. It is used as both input features (e.g., the historical prices in a look-back window) and prediction target (e.g., the close price of the next day).
• Text data: text data refer to the text contributed by individuals, e.g., social media, news, web searches, etc. As a type of alternative data, these data are hard to collect and process, but may provide useful information that is not included in market data. Sentiment analysis can be applied on these text data and produce a sentiment factor (e.g., positive, neural, or negative) that can be further used for prediction.
• Macroeconomics data: macroeconomics data reflects the economic circumstances of a particular country, region or sector, e.g., Consumer Price Index (CPI), Gross domestic product (GDP), etc. These indicators are related with the stock market in the sense that they indicate how healthy the overall stock market is and can provide confirmation as to the quality of a stock market advance or decline.
• Knowledge graph data: there are some kind of relationship between different companies and different markets, e.g., the movement of stocks in the same sector may be affected by the same news. Powered by the recently developed graph neural networks, the knowledge graph data from open sources such as FreeBase (Bollacker et al., 2008) and Wikidata 3 can now be used to improve the prediction performance.
• Image data: inspired by the success of convolutional neural networks in 2D image processing, e.g., classification and object detection, candlestick charts are used as input images for stock prediction. While satellite and CCTV images or videos are used to monitor the situation of companies and may be helpful for stock price prediction, they are never used in the surveyed papers because of the prohibitive cost of collection and the potential privacy leakage risk.
• Fundamental data: the most common type of fundamental data is the accounting data, which is reported quarterly, e.g., assets, liabilities, etc.
It is less used in studies with deep learning models because the low frequency of reporting and also the inaccuracies of the reporting date, e.g., the fundamental data published is indexed by the last date included in the report and precedes the date of the release, which brings in a risk of using future information.
• Analytics data: analytics data refers to the data that can be extracted from reports (e.g., recommendation for selling or buying a stock) that are provided by investment banks and research firms, who make an indepth analysis of companies' business models, activities, competitions, etc.
These reports provide valuation information, while they may be costly and shared among different consumers, who all want to use this information to make a profit.
Different types or raw data are accompanied with different levels of difficulty for obtaining and processing, and the usage of different data types is shown in Figure 3. For deep learning models, a huge amount of input data is necessary for the training of a complex neural network model. In this case, market data is the best choice and used for the most as it provides the largest amount of data sample, while the other data types usually have a smaller size. Text data is used for the second most, with the popularity of social media and online news website and the easier use of web crawlers to get the text data. An extreme case is the analytics data, which is never used in the surveyed studies, because of both the data sparsity and the high cost to access.
There is also a trend in Figure 3 that more diverse data types are used in 2018 and 2019, compared to the studies in 2017. It indicates the fact that it is harder to get a better prediction result based on only the market data. It also reflects the development of new tools so that new types of data can be used for prediction, e.g., graph neural networks for knowledge graph data. Figure 3: The usage of different raw data types.
Data Length
To evaluate the performance of different models, historical data is necessary for evaluation. However, there is a tradeoff of choosing the data length. A short time period of data is not sufficient to show the effective and has a higher risk of overfitting, while a long time period takes the risk of traversing different market styles and present out-of-dated results. Besides, the data availability and cost are factors that needs to be taken into consideration when choosing the data length.
The distribution of time periods of data used in the surveyed papers is shown in Figure 4. It is more expensive to get intraday data with a good quality and most of the previous studies involving intraday prediction would use a time period less than one year. For a single prediction, lag is used to denote the time length of the input data to be used by the model, e.g., in the daily prediction, a lag of 30 days means the data in the past 30 days are used to build the input features. For technical indicators, lag is often set as an input parameters and vary a lot in previous studies from 2 to 252 time periods. Correspondingly, horizon is used to denote the time length of the future to be predicted by the model. Most of the studies focus on a short-term prediction horizon, e.g., one day or five minutes, with only a few exceptions for a longer horizon such as five days or ten days.
Missing Data Imputation
The problem of missing data is not as severe as in other domains, e.g., sensor data, because the market data is more reliable and well supported and maintained by the trading markets. However, to align multiple types of data with different sampling frequencies, e.g., market data and fundamental data, the data with a lower sampling frequency should be inserted in a forward way by propagating the last valid observation forward to next valid, to avoid data leakage of the future information.
Denoising
With many irrational behaviors in the stock trading process, the market data is filled with noise, which may misrepresent the trend of price change and misguide the prediction. As a signal processing technique, wavelet transform has been used to eliminate noise in stock price time series (Bao et al., 2017;Liang et al., 2019). Another approach to eliminate noisy data in Sun et al. (2017) is the use of the kNN-classifiers, based on two training sets with different labels in a data preparation layer.
Feature Extraction
For machine learning models, feature engineering is the process of extracting input features from raw data based on domain knowledge. Combined with raw data, these handcrafted features are used as input for the prediction models and can substantially boost machine learning model performance.
For market data, technical analysis is a feature extraction approach that builds various indicators for forecasting the direction of prices based on his-torical prices and volumes, e.g., moving average, or moving average convergence/divergence (MACD). These technical indicators can be further used to design simple trading strategies. Technical indicators are also used to build image inputs, e.g., 15 different technical indicators with a 15-days periods are used to construct a 15 × 15 sized 2D images in Sezer & Ozbayoglu (2018).
While the feature extraction techniques represented by technical analysis for market data have been used and validated for many years, the tools for extracting features from text data have made a greater progress in the past few years, owing to the various deep learning models developed for natural language processing. Before the popularity of machine learning models, the bag-of-words (BoW) model (Harris, 1954) is used as a representation of text that describes the occurrence of words within a document. In recent years, machine learning and deep learning models show an improved performance for word embedding.
Given the sequence of words, the word2vec model (Mikolov et al., 2013), which are shallow and two-layer neural networks, can be used to embed each of these words in a vector of real numbers and has been used in Liu For knowledge graph data used more recently, the TransE model (Bordes et al., 2013) is a computationally efficient predictive model that satisfactorily represents a one-to-one type of relationship and has been used in Liu et al. (2019c).
Based on the input raw data and extracted features, we show the distribution of different combinations of input features in Figure 5 and the detailed article lists in Table 4. From Figure 5, historical prices and technical indicators are the most commonly used input features and followed by text and macroeconomics data. This could be explained by the easier accessing and processing of market data than other data types. Feature selection is another way of dimensionality reduction, by choosing only a subset of input features. Chi-square method (Zheng et al., 2004) and maximum relevance and minimum redundancy (MRMR) (Peng et al., 2005) are two common used feature selection techniques. and maximum relevance and minimum redundancy is used in Kumar et al. (2019).
Feature Normalization & Standardization
Given different input features with varying scales, feature normalization and standardization are used to guarantee that some machine learning models can work and also help to improve the model's training speed and performance. or between -1 and 1 (Ticknor, 2013;Zhang et al., 2017b;Sezer & Ozbayoglu, 2018). Feature standardization means subtracting a measure of location and dividing by a measure of scale, e.g., the z-score method that subtracts the mean and divides by the standard deviation (Tsantekidis et al., 2017a,b;Zhang et al., 2019b;Li et al., 2019a).
Data Split
For evaluation of different prediction models, in-sample/out-of-sample split or train/validation/test split of data samples is commonly used in machine learning and deep learning fields. The model is trained with the training or in-sample data set, the hyper-parameters is fine-tuned on the validation data set optional, and the final performance is evaluated on the test or out-of-sample data set. k-fold cross validation is further used to split the dataset into k consecutive folds, and k-1 folds is used as the training set, while the last fold is then used as a test set.
As a special case, train-validation-test split with a rolling (or sliding, moving, walk-forward) window is also often used for time series tasks including stock prediction (
Data Augmentation
Data augmentation techniques have been widely used for image classification and object detection tasks and proved to effectively enhance the classification and detection performance. However, it is less used for time series tasks including stock prediction, even though the size of stock price time series is not comparable to the size of public image datasets, which usually have millions of sample and even more in recent years.
There are still a few works that explore the usage of data augmentation. Zhang et al. (2017a) firstly clusters different stocks based on their retracement probability density function and combines all the day-wise information of the same stock cluster as enlarged training data. In the ModAugNet framework proposed in Baek & Kim (2018), the authors choose 10 companies' stock that are highly correlated to the stock index and augment the data samples by using the combinations of 10 companies taken 5 at a time in an overfitting prevention LSTM module, before feeding the data samples to a prediction LSTM module for stock market index prediction.
Prediction Model
Most of the prediction models belong to a supervised learning approach, when training set is used for the training and test set is used for evaluation.
Only a few of the studies use semi-supervised learning when the labels are not available in the feature extraction step. We further classify the various prediction models into three types: standard models and their variants, hybrid models, and other models. For standard models, three families of deep learning models, namely, feedforward neural network, convolutional neural network and recurrent neural network, are used a lot. And we category the use of generative adversarial network, transfer learning, and reinforcement learning into other models. These models only appear in recent years and are still in an early stage of being applied for stock market prediction.
In this part, we are focusing on the usage of different types of deep learning models, instead of diving into the details of each model. For a more detailed introduction to deep learning models, we refer the readers to Goodfellow et al. (2016). The abbreviations of machine learning and deep learning methods are shown in Table 5. (Niaki & Hoseinzade, 2013;de Oliveira et al., 2013;Zhong & Enke, 2017). In this survey, we use ANN to refer to the neural networks which only have one or zero hidden layers, and DNN to refer to those which have two or more hidden layers. The list of standard models or their variants is shown in Table 6. We organize the standard models into three major types: While standard models perform well at early stages of research, their variants are further developed to improve the prediction performance. One approach is to use stacked models, where neural network sub-models are embedded in a larger stacking ensemble model for training and prediction. Another approach is to introduce the attention mechanism (Treisman & Gelade, 1980) into recurrent neural network models, in which attention is a generalized pooling method with bias alignment over inputs.
There are also some types that we list separately: • Sequence to sequence (seq2seq) model is based on the encoder-decoder architecture to generate a sequence output for a sequence input, in which both the encoder and the decoder use recurrent neural networks. Seq2seq model has been used in Liu & Wang (2019) for stock prediction.
While our focus in this survey is not the linear models or the traditional machine learning models, they are often used as baselines for comparison with deep learning models.
Some often used linear prediction models are as follows: • Linear regression (LR). Linear regression is a classical linear model that tries to fit the relationship between the predicted target and the input variables with a linear model, in which the parameters can be learned in the least squares approach.
• Autoregressive integrated moving average (ARIMA). ARIMA is a generalization of the autoregressive moving average (ARMA) model, which describes a weakly stationary stochastic process with two parts, namely, the autoregression (AR) and the moving average (MA). Compared with ARMA, ARIMA is capable of dealing with non-stationary time series, by introducing an initial differencing step, which is referred as the integrated part in the model. Similarly, some often used machine learning models are as follows: • Logistics regression (Logit). Logistics regression can be seen as a generalized linear model, in which a logistic function is used to model the probabilities of a binary target of being 0 or 1. It is suitable for the classification of price movements, e.g., going up or down.
• Support vector machine/regression (SVM/SVR). Support vector machine is a classical and powerful tool for classification with a good theoretical performance guarantee and has been widely adopted before the popularity of deep learning models. SVM tries to learn a hyperplane to distinguish the training samples that maximize distance of the decision boundary from training samples. Combine with the kernel trick, which maps the input training samples into high-dimensional feature spaces, SVM can efficiently perform non-linear classification tasks. Support vector regression is the regression version of SVM.
• k-nearest neighbor (kNN). kNN is a non-parametric model for both classification and regression, in which the output is the class most common or the average of the values among k nearest neighbors. A useful technique is to assign weights to the neighbors when combing their contributions.
Given the predicted movement direction or prices, a long-short strategy can be further designed to perform trading based on the prediction model, e.g., if the predicted direction is going up, long it, otherwise short it. A simple baseline is the Buy&Hold Strategy, which buys the asset at the beginning and hold it to the end of the testing period, without any further buying or selling operations (Niaki & Hoseinzade, 2013;Sezer & Ozbayoglu, 2018).
Technical indicators are also often used for designing baseline trading strategies, e.g., momentum strategy, which is introduced in Jegadeesh & Titman We further categories the hybrids into two classes, namely, the hybrid models between deep learning models and traditional models, and the hybrid models between different deep learning models.
The list of hybrid models between deep learning models and traditional models is shown in Table 7 We list other types of models in Table 9. We category five types of models in this part, which have not been fully explored for stock market prediction but already show some promising results.
• Generative adversarial network (GAN). GAN is introduced by Goodfellow et al. (2014), in which a discriminative net D learns to distinguish whether a given data instance is real or not, and a generative net G learns to confuse D by generating high quality fake data. This game between G and D would lead to a Nash equilibrium. Since the introduction of GAN, it has been applied in multiple image-related tasks, especially for image generation and enhancement, and generates a large family of variants.
Inspired the success of GANs, Zhou et al. (2018) proposes a generic GAN framework employing LSTM and CNN for adversarial training to predict high-frequency stock market.
• Graph neural network (GNN). GNN is designed to utilize graph-structured data, thus capable of utilizing the network structure to incorporate the interconnectivity of the market and make better predictions, compared to relying solely on the historical stock prices of each individual company or on hand-crafted features (Matsunaga et al., 2019). Chen et al. (2018c) first constructs a graph including 3,024 listed companies based on investment facts from real market, then learns a distributed representation for each company via node embedding methods applied on the graph, and applies three-layer graph convolutional networks to predict. uses LSTM for the individual stock prediction task and GRU for the index movement prediction task where an additional graph pooling layer is needed.
• Capsule Network. Different from the method of CNNs and RNNs, the capsule network increases the weights of similar information through its dynamic routing, which is proposed by Sabour et al. (2017) and displaces the pooling operation used in conventional convolution neural network. Liu et al. (2019a) is the first to introduce the capsule network for the problem of stock movements prediction based on social media and show that the capsule network is effective for this task.
• Reinforcement learning. Unlike supervised learning, reinforcement learning trains an agent to choose the optimal action given a current state, with the goal to maximize cumulative rewards in the training process. Reinforcement learning can be applied for stock prediction with the advantage of using information from not only the next time step but from all subsequent time steps . Reinforcement learning is also used for building algorithmic trading systems (Deng et al., 2016). shown in Figure 9. With the further exploration of deep learning models for stock prediction, their ratio as baselines keeps increasing in the past three years.
Model Evaluation
In this part, we category the evaluation metrics for the prediction models mentioned in the last part into four types: • Classification metrics. Classification metrics are used to measure the model's performance on movement prediction, which is modeled as a classification problem. Common used metrics include accuracy (which is the correct number of prediction for directional change), precision, recall, sensitivity, specificity, F1 score, macro-average F-score, Matthews correlation coefficient (which is a discrete case for Pearson correlation coefficient), average AUC score (area under Receiver Operating Characteristic curves (Fawcett, 2006)), Theil's U coefficient, hit ratio, average relative variance, etc. Confusion matrices and boxplots for daily accuracy are also used for classification performance analysis (Guang et al., 2019;Zhong & Enke, 2017;Zhang et al., 2019b). • Profit Analysis. Profit analysis evaluates whether the predicted-based trading strategy can bring a profit or not. It is usually evaluated from two aspects, the return and the risk. The return is the change in value on the stock portfolio and the risk can be evaluated by maximum drawdown (Zhou et al., 2019a), which is the largest peak-to-trough decline in the value of a portfolio and represents the max possible loss, or the annualized volatility (Karathanasopoulos & Osman, 2019). Sharpe Ratio is a comprehensive metric with both the return and risk into consideration, which is the average return earned in excess of the risk-free per unit of volatility (Sharpe, 1994). More detailed analysis about the transactions is given in Sezer & Ozbayoglu (2018. • Significance Analysis. In order to determine if there is significant differ-ence in terms of predictions when comparing the deep learning models to the baselines, Kruskal-Wallis (Kruskal & Wallis, 1952) and Diebold-Mariano (Diebold & Mariano, 2002) tests can be used to test the statistical significance, which decides a statistically better model. They are not used often for stock prediction, with only a few studies in 2019 (Kumar et al., 2019;Zhang et al., 2019b).
The detailed list of studies using each metrics (as well as the metrics' abbreviations) is shown in Table 10.
Implementation
In this section, we pay a special attention to the implementation details of the papers we survey, which is less discussed before in previous surveys.
We firstly investigate the programming language used for the implementa- which provides a bunch of packages and frameworks for model implementation purpose, e.g., Keras 4 , TensorFlow 5 , PyTorch 6 , Theano 7 , scikit-learn 8 . Other choices include R, Matlab, Java, etc. We show the specific paper list using different programming languages and frameworks in Table 11. From Deep learning models require a larger amount of computation for training, and GPU has been used to accelerate the convolutional operations involved.
With the need of processing multiple types of input data, especially the text data, the need for GPU would keep increasing in this research area. We give a list of different types of GPU used in the surveyed papers in Table 12. Cloud computing is another solution when GPU is not available locally. There are many commercial choices of cloud computing services, e.g., Amazon Web Services 9 , Google Cloud 10 , and Microsoft Azure 11 . However, they are not widely adopted in current study of stock market prediction and no previous study covered in this survey mentions the usage of cloud service explicitly.
Result Reproducibility
While deep learning techniques have been proved to be effective in many These studies also question the reproducibility and replicability in the previous papers which use ML methods.
While it is beyond the scope of this study to check the result of each paper, we instead investigate the data and code availability of the surveyed papers, which are two important aspects for the result reproducibility. Some of the source journals would require or recommend the data and code submitted as supplementary files for peer review, e.g., PLOS ONE. In other cases, the authors would share their data and code proactively, for the consideration that following works can easily use them as baselines, which gains a higher impact for their publications.
Data Availability
There are many free data sources on the Internet for the research purpose of stock market prediction. For historical price and volume, the first choice should be the widely used Yahoo! Finance 12 , which provides free access to data including stock quotes, up-to-date news, international market data, etc., and has been mentioned at least in 25 out of 124 papers. Other similar options include Even though most of the data sources are available on the Internet, it would be more convenient for replicability if the authors could release the exact dataset they use. In Table 13, we list those with the data description and link, for those data which is hosted in software host websites such as Github 33 , cloud services, researcher's own website, and data competition websites such as Kaggle.
For the mid-price prediction of limit order book data, there is a benchmark dataset provided by Ntakaris et al. (2017) and has been used in the following studies (Tran et al., 2018).
Code Availability
Github has been the mainstream platform of hosting source code in the computer science field. However, only a small number of studies would release their code for now, in the area of stock market prediction. In Table 14, we list the articles with public code repositories. A short description of each method is mentioned, and the details can be found in Section 4 and the original documents.
Future Directions
Based on our review of recent works, we give some future directions in this section, which aims to bring new insight to interested researchers.
New Models
Different structures of neural networks are not fully studied for stock prediction, especially those who only appear in recent years. There are two steps where deep learning models involve in stock prediction, namely, Data Processing and Prediction Model in Section 4. While we already covered some latest effort of applying new models in this survey, e.g., the attention mechanism and generative adversarial networks, there are still a huge space to explore for new et al., 2018) are widely used in natural language process-ing, but is less discussed for financial news analysis.
Multiple Data Sources
Observed from our discussion in Section 4, it is not wise to design a stock prediction solution based on a single data source, e.g., market data, as it has been heavily used in previous studies and it would be very challenging to outperform existing solutions. A better idea is to collect and use multiple data sources, especially those which are less explored in the literature (Zhou et al., 2019b).
Cross-market Analysis
Most of the existing studies focus on only one stock market, in the sense that stock markets differ from each other because of the trading rules, while different markets may share some common phenomenon that can be leveraged
Algorithmic Trading
The prediction is not the end of the journey. Good prediction is one factor to make money in the stock market, but not the whole story. Some of the studies have evaluated the profit and risk of the trading strategies based on the prediction result, as we discussed in Section 4.4. However, these strategies are simple and intuitive, which may be impractical limited by the trading rules.
The transaction cost is often omitted or simplified, which makes the conclusion less persuasive. Another problem is the adaption for different market styles, as the training of deep learning models is time-consuming. These studies are not sufficient for building a practical algorithmic trading system. One possible direction is deep reinforcement learning, which has recent successes in a variety of applications and is also been used in a few studies for stock prediction and trading (Xiong et al., 2018;Lee et al., 2019). It has advantages of simulating more possible cases and making a faster and better trading choice than human traders.
Conclusion
Inspired by the rapid development and increasing usage of deep learning models for stock market prediction, we give a review of recent progress by surveying more than 100 related published articles in the past three years. We cover each step from raw data collection and data processing to prediction model | 2020-03-05T02:41:36.269Z | 2020-02-29T00:00:00.000 | {
"year": 2020,
"sha1": "00c7a21bd4d7c2c67ae54efbd2f6336cd5dc17e6",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/2003.01859",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "00c7a21bd4d7c2c67ae54efbd2f6336cd5dc17e6",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Computer Science",
"Economics"
]
} |
17061107 | pes2o/s2orc | v3-fos-license | An Unusual Type of Localized Hypertrophic Cardiomyopathy With Wolf Parkinson White Syndrome Presenting With Pulmonary Edema
Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease that is the most common genetic cardiac disorder. The disease is characterized by excessive thickening of the left ventricular myocardium. The anterior portion of the interventricular ventricular septum is often involved. Asymmetric hypertrophy of apical site, left ventricular free wall, and right ventricle are less common in hypertrophic cardiomyopathy that occur in 1% cases. We report a case of a patient with an unusual type of hypertrophic cardiomyopathy and Wolf Parkinson White (WPW) presenting with pulmonary edema.
Introduction
Hypertrophic cardiomyopathy (HCM) which occurs at a prevalence of one case per 500 population (0.2%), is characterized by an asymetrical wall thickening of the heart that can lead to severe cardiac problems such as progressive heart failure, embolic stroke, and sudden cardiac death [1]. The extent and localization of the wall thickening is highly variable. The interventricular septum is often involved. Asymmetric hypertrophy without involvement of interventricular septum is less common which occurs in 1% cases [2]. We report a case of a patient with an unusual type of hypertropic cardiomyopathy and Wolf Parkinson White (WPW) presenting with pulmonary edema.
Case Report
A 32-year-old woman was admitted to the emergency room with suddenly occured dispnea and orthopnea. On admission she had a blood pressure of 110/70 mmHg, a regular pulse of 134 beaps per minute, and a breath rate of 34 per minute with 68% of partial oxygen saturation. There was a grade 2 midsystolic murmur in the mesocardiac region and bilateral pulmonary ralles throughout the lung fields.
The electrocardiogram (ECG) revealed sinus tachycardia (Fig. 1A). Chest X-ray showed bilateral extended hilar and paranchimal infiltration and lines of Kerley B. The patient was dramatically rapidly improved with diuretic therapy and partial oxygen saturation increased up to 95%. Transthoracic echocardiography (TTE) showed normal left ventricle ejection fraction with severe hypertrophy of left ventricular posterior, lateral free wall, and mid ventricular septum ( Fig. 2 A, B). The maximal wall thickness of posterior free wall and mid ventricular septum were 25 mm and 19 mm respectively. There was a significant peak intraventricular gradient of 91 mmHg at rest (Fig. 2C). The color doppler imaging demonstrated flow acceleration due to mid ventricular obstruction (Fig. 2D). The coronary angiography performed because of elevated troponin I levels, that was demostrated normal coronary arteries. Left ventriculography showed totally midventricular obtruction at systolic phase (Fig. 3). The cardiac MRI was also performed to confirm the diagnosis. The cardiac MRI images were clearly showed the asymetric thickening of the left ventricular posterior, lateral free wall, and mid ventricular septum (Fig. 4). The 24 hour Holter ECG monitoring performed and any significant arrhythmias was detected. She was discharged with the prescription of metoprolol and acetyl salisilic acid. After one on the first admission to the hospital (Fig. 1B). The electrophysiologic study performed and radiofrequency ablation of accessory pathway was applied. Two weeks after hospital discharge, she was admitted to our hospital with ventricular tachycardia which was converted to sinus rhythm after electrical cardioversion. According to the European Society of Cardiology guidelines, ICD implanted because of the risk of sudden death.
Discussion
Hypertrophic cardiomyopathy (HCM) is one of the most common genetically transmitted cardiovascular disease, and is a leading cause of sudden death amoung adults [1]. The characteristic morphologic feature is asymmetric septal hypertrophy; but asymmetric thickness of apical segments or left ventricular free wall are rare types of HCM [2]. The diversity of the morphologic expression of HCM were due to racial, genetic factors, and differences in phenotypic expression [2]. Andreini et al; described a very atypical form of HCM. In this case the abnormal myocardial thickness was localized to the anterior and lateral free wall of the left ventricle without any involvement of interventricular septum [3]. Duncan et al; described second case of unusual form of HCM with massive midventricular obstruction and an akinetic apical chamber [4]. Lewis et al; described a subgroup of 17 patient with hypertrophic cardiomyopathy, and an un-usual and distinctive patern of left ventricular hypertrophy characterized on echocardiography by marked thickening of the posterior free wall and virtually normal or modest increased ventricular septal thickness [5].
Although echocardiography is the tool choice for the diagnosis of HCM, maximal wall thickness and determination of the left ventricular muscle mass can also be determined using cardiac MRI. Myocardial scar tissue can be detected using delayed-enhancement cardiac MRI [6].
Wolf Parkinson White (WPW) syndrome is the most common congenital cardiac abnormality results from an anomalous atrioventricular conduction pathway that can produce ventricular preexcitation and paroxysmal reentrant tachyarrhythmias. The occurence of WPW syndrome is very rare in patients with HCM. It is estimated that 5-10% of patient with HCM are reported to have ventricular preexcitation. The relationship between HCM and WPW syndrome has not clear yet, but there are some clinical evidence to suggest that development of ventricular preexcitation in individuals with HCM may reflect a distinct genetic etiology [7]. A less common type of HCM, known as metabolic HCM that is typically associated with ventricular preexcitation (WPW), is not related to defects in the cardiac sarcomere, but due to glycogen accumulation in cardiac myocytes [8]. There are a few reports about the co-occurrence of HCM and WPW syndrome. Firstly, Shibata et al; described two patients with familial HCM associated with WPW syndrome who showed progression to left ventricle dilatation [9]. Bobkowski at al; reported a case of HCM with asymmetric hypertrophy of ventricular septum and left ventricular posterior wall that was associated with WPW syndrome [10].
To our knowledge this is the first report; the asymetric hypertrophy was limited to the posterior and lateral free wall, and mid ventricular septum in patient with HCM and WPW with acute pulmonary edema. In our case midventricular obstruction and suspected paroxysmal tachyarryhtmia can be responsible from the development of acute pulmonary edema. Since no linkage between the atypical form of HCM with WPW syndrome has been showed in case reports published to date, we cannot conclude whether the preva- lance of WPW syndrome is higher in patient with atypical morphologic feature of HCM or an incidental co-occurence of atypically localized HCM and WPW syndrome. | 2018-04-03T06:22:31.281Z | 2012-05-20T00:00:00.000 | {
"year": 2012,
"sha1": "997fe11672f49f7613e85f8a24218a3e0355c54b",
"oa_license": "CCBY",
"oa_url": "https://cardiologyres.org/index.php/Cardiologyres/article/download/138/186",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "997fe11672f49f7613e85f8a24218a3e0355c54b",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
234282212 | pes2o/s2orc | v3-fos-license | The F19W mutation reduces the binding affinity of the transmembrane Aβ11–40 trimer to the membrane bilayer
Alzheimer's disease is linked to the aggregation of the amyloid-β protein (Aβ) of 40 or 42 amino acids. Lipid membranes are known to modulate the rate and mechanisms of the Aβ aggregation. Point mutations in Aβ can alter these rates and mechanisms. In particular, experiments show that F19 mutations influence the aggregation rate, but maintain the fibril structures. Here, we used molecular dynamics simulations to examine the effect of the F19W mutation in the 3Aβ11–40 trimer immersed in DPPC lipid bilayers submerged in aqueous solution. Substituting Phe by its closest (non-polar) aromatic amino acid Trp has a dramatic reduction in binding affinity to the phospholipid membrane (measured with respect to the solvated protein) compared to the wild type: the binding free energy of the protein–DPPC lipid bilayer increases by 40–50 kcal mol−1 over the wild-type. This is accompanied by conformational changes and loss of salt bridges, as well as a more complex free energy surface, all indicative of a more flexible and less stable mutated trimer. These results suggest that the impact of mutations can be assessed, at least partially, by evaluating the interaction of the mutated peptides with the lipid membranes.
Introduction
According to the World Alzheimer Report 2016, there were 46.8 million patients with Alzheimer's disease (AD) in 2015, and the number of global cases was predicted to reach 131.5 million by 2050. 1 AD is a neurodegenerative disease, which is pathologically characterized by amyloid plaques resulting from the aggregation of extracellular amyloid-b (Ab) peptide, and by neurobrillary tangles made by the accumulation of intracellular tau protein in the hippocampus and cerebral cortex. 2-5 AD progressively affects normal brain functions such as memory, judgement, and cognition, and results in the failure of crucial cellular processes. 6 Amyloid plaques consist of the extracellular accumulation of the Ab 40 and Ab 42 peptides derived from the transmembrane amyloid precursor protein (APP), which is located in the lipid-rich microdomains (lipid ras) of endosome and the plasma membrane, 7,8 aer cleavage by band g-secretases. 9 The Ab 42 peptide is known to be more insoluble and with a higher probability of polymerization than the Ab 40 peptide. The latter is considered the primary constituent in cerebral amyloid angiopathy and is generally more abundant in plaques. 10 The accumulation of soluble Ab oligomers can cause neurovirulence and impair the synaptic transition. 11,12 Experimental and computational investigations in the Ab peptide grew over the past years due to Ab's dual nature: high intrinsic disorder and high aggregation propensity. 5,[13][14][15][16][17] Indeed, the diversity and exibility of Ab bring many challenges in its structural characterization by experiments, especially because the interaction between Ab and the phospholipids in the cell membrane plays a crucial role in the aggregation mechanisms. Using a single electron method to study the interaction between the Ab 40 peptide and anionic lipid membranes, Ding et al. reported that trimers and tetramers may be the smallest Ab 40 oligomers in the lipid bilayers, and could lead to the initial neurotoxicity. 18 Later, Jana et al. demonstrated that membrane-bound tetramer and trimer Ab 40 oligomeric species are associated with toxicity in cultured neurons. 19 Several U-shape bril models of Ab 40 that form in-register parallel b-sheets have been experimentally reported. [20][21][22][23] In general, residues 1-10 in these models are disordered. Even though the conformations of Ab 40 depend on the peptide sequence and lengths, 24 the contribution of the 1-10 residues is negligible. Additional experimental evidence suggests that the truncated Ab peptide can capture the oligomerization/ brillation behavior just as well as the full-length Ab 40 peptide. 25 A scheme of the Ab peptide is shown in Fig. 1, where the two hydrophobic patches (red) L17-A21 (central hydrophobic core, CHC) and A30-V40 (C-terminus) are separated by a hydrophilic (blue) loop region (E22-G29). The Nterminus (E11-K16) is also very hydrophilic, and plays an important role in metal ion interactions together with the residues H13 and H14. 5 In order to complement experimental endeavors, Molecular Dynamics (MD) simulations have been employed successfully, such as in the search of potential inhibitors 26,27 against the aggregation of wild-type Ab oligomers. [14][15][16][17] In particular, numerical studies employing replica exchange MD (REMD) have provided insight into the truncated Ab peptide and its corresponding trimer (3Ab ) in its wild type and mutant forms [28][29][30][31] in solution. In addition to its soluble conformations, the insertion of the Ab oligomers in membranes has been investigated via MD. [32][33][34][35] For instance, for the transmembrane 4Ab 17-42 tetramer a helical structure 35 has been described.
Mutations in the Ab peptide modify its toxicity, assembly, and rate of bril formation. Specically, the mutations in the CHC and loop regions, including F19W, 36 F20W, 36 L17A/F19A, 37 Flemish A21G, 38 Dutch E22Q, 39 Italian E22K, 40 Arctic E22G, 41,42 E22D, 43 and Iowa D23N 44 could affect the conformational changes in Ab oligomers. Another example shown that the combination of mutation A2V in N-terminal and histidine tautomerism can affect the Ab monomer structures and its aggregation process. 45 Thus, numerous mutation studies have been carried out both experimentally and employing MD simulations. These studies and their main results are summarized in Table S1. † In particular, the inuence of local physical interactions on the brillation kinetics and the structure and dynamics of Ab 40 has been characterized by experimental studies that include uorescence, transmission electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. 46 The hydrophobic contact between F19-L34 was modied by a series of mutations on the residues F19 and L34, including F19G, F19P, F19E, F19K, F19Y, F19W, L34E, and L34K. These mutants were studied to understand the effect of local interactions, including electrostatic interactions (F19E and F19K mutations); hydrophobic interactions (F19Y and F19W mutations); conformational exibility (F19G and F19P mutations); and the salt-bridge interactions (L34E and L34K mutations). These local interactions were found to impact the brillation kinetics, the intermolecular hydrogen bonds and the dynamics of the Ab 40 , without changing the general bril structure. The results also demonstrated that the non-local F19-L34 contact plays an important role in early-oligomers of Ab 40 . The F19W mutation showed slower brillation kinetics than the wild-type. While both F19Y and F19W mutations replace the Phe ring by another aromatic ring, the tryptophan mimicked Phe better. A subsequent study investigated how the F19K mutation altering the F19-L34 contact affects the bril structure and the toxicity of the Ab 40 . 47 This mutation was found to alter the local structure of the bril and to abolish cytotoxicity. In addition, computational studies have characterized the A21G mutation in Ab 40 and Ab 42 , 48 and also in transmembrane 3Ab 11-40 . 33 It has been shown that the "susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane". 49 These experiments included relatively complex physiological scenarios that included Ca 2+ inux and cellular damage and opened new lines of questioning, for instance, how mutations affect the binding to the membrane and which minimal models can capture changes in binding. In this work we set out to nd a simple model that can show mutations affecting the binding affinity of the aggregates. For this, we noticed that the impact of F19 mutations on the oligomers' structure of Ab 40 in membrane lipid bilayers has not been characterized. Also, if one wants to characterize a sort of "threshold" for binding differences in the mutated oligomer, it is better to choose a "subtle mutation" that is close to the original amino acid and does not change its polar/non-polar nature. For the case of Phe, the closest non-polar one is Trp, as it lacks the -OH group of Tyr and therefore mimicks Phe better. We notice that MD simulations have been used to study the conformations of the 3Ab 11-40 trimer in both solution 28 and dipalmitoylphosphatidylcholine (DPPC) lipid environment. 34 In addition, the F19W mutant of the 11-40 truncated Ab trimer (F19W 3Ab in aqueous solution was recently characterized via MD simulations. 31 In the present study, the (F19W 3Ab trimer with an initial conformation obtained from the Ab bril was inserted into a DPPC lipid bilayer, solvated and then simulated using REMD techniques. The metastable structures of the transmembrane F19W 3Ab were deduced using a combination of free energy surface and clustering methods. Our results provide detailed structural conformations of the transmembrane F19W 3Ab and how they differ from the wildtype transmembrane 3Ab obtained in previous studies. 34 The binding free energy of the mutated oligomer clearly shows that the even the subtle F19W mutation greatly destabilizes the 3Ab 11-40 trimer with respect to its wild type counterpart.
Temperature-REMD simulations
The conformation of the transmembrane 3Ab inserted in the DPPC membrane bilayers 50 was taken from a previous study 34 in which the crystal structure of the 3Ab 11-40 was obtained from a bril-like structure. 20 PYMOL tools 51 were then used to create the mutated F19W version for the 3Ab . Finally, the F19W 3Ab was inserted in the DPPC lipid bilayer. The mutant trimer was then represented using the united atom GROMOS 53a6 force eld. 52 While there are other IDP-specic force elds 53,54 that may be used to simulate intrinsically disordered proteins (IDP) in solution, we used the united atom GROMOS 53a6 force eld because it is known to be quite good for amyloid beta transmembrane proteins. 55,56 In addition, to save computational time and compare with the 3Ab 11-40 wild-type results, 34 we used united atom GROMOS 53a6 force eld.
The system was solvated using the simple point charge (SPC) water model. 57 The solvated system was neutralized with three Na + ions. The initial conformation of the transmembrane F19W 3Ab is presented in Fig. 2, in which Na + ions are represented by three black balls and the mutant points were highlighted. The entire solvated transmembrane F19W 3Ab system consists of 16 987 atoms, including the F19W Ab oligomer, 3293 water molecules, 125 DPPC molecules and three Na + atoms.
The initial structures for F19W 3Ab were based on the previously deduced bril-like structure. This choice is justied because it would take more than several microseconds per replica to simulating the aggregation of the Ab from monomers. In this work, the transmembrane F19W 3Ab was simulated using the T-REMD method with 32 replicas with temperatures varying from 321 to 423 K in the isothermal-isobaric (NPT) ensemble. The GROMACS version 5.1.3 was used with a periodic boundary condition (PBC) box with dimensions of 6.028 Â 6.052 Â 7.134 nm 3 and a time step of 2 fs using a leap-frog algorithm. 58 The electrostatic interactions were calculated using the particle mesh Ewald method with a 0.9 nm cut-off. 59 The van der Waals interactions also had a 0.9 nm cut-off. The nonbonded pair lists were updated every 10 fs. The velocityrescaling thermostat 60 was used to control temperature, and the Parrinello-Rahman barostat 61 was used to control pressure. All bonds were constrained by the LINear Constraint Solver (LINCS) 62 with an order of 4. The individual temperatures were generated using a Web server. 63 Exchanges between neighboring replicas were attempted every 1 ps, leading to mean acceptance ratios ranging from 18 to 25% (Fig. S2 †). It was conrmed that all replicas have an efficient exchange rate over the whole temperature range, as is illustrated by the two examples shown in Fig. S3 † which plots the temperature indices for the rst replica (which has the lowest initial temperature) and last replica (which has the highest initial temperature), respectively. Each replica ran for 400 ns, resulting in a total of 12 800 ns of MD simulations. Data was recorded every 10 ps. The results were analyzed for the last 150 ns of REMD simulations. The rst 250 ns of the simulations were removed to avoid any starting bias. During the simulation, the membrane DPPC lipid bilayer was stable ( Fig. S4 †), in agreement with the nonmutated transmembrane 3Ab 11-40 . 34 Secondary structure analysis The secondary structure parameters of the transmembrane F19W 3Ab , including coil, beta, turn, and helix contents, were predicted using DSSP tool. 64 Free energy surface (FES) The free energy surface of the mutant trimer was constructed using the "gmx sham" tools 65 of GROMACS with root-meansquare deviation (RMSD) and radius of gyration (R g ) serving as reaction coordinates.
Free energy perturbation (FEP) method
The binding free energy between the mutant F19W 3Ab and DPPC bilayer was predicted using the FEP method 34,66 as described in the ESI. †
Collision cross section (CCS)
The Ion Mobility Projection Approximation Calculation Tool (IMPACT) with the trajectory method was employed to compute the mutant trimer CCS. 67
Solvent-accessible surface area (SASA)
We calculated the total solvent-accessible surface area (SASA) for the transmembrane F19W 3Ab with the double cubic lattice method 68 as implemented in GROMACS.
Computational analysis tools
The clustering method was carried out with a C a RMSD cut-off of 0.3 nm. 65,69 A non-bonded contact between heavy atoms of different residues was counted when their distance was smaller than 0.45 nm. A polar contact between two charged groups was counted when the distance between two specic atoms was equal or less than 0.46 nm. The intermolecular contact between heavy atoms of the F19W 3Ab with the phosphate groups of the DPPC lipid bilayer was calculated by evaluating the minimum distance of the corresponding atoms with a cut-off of 0.45 nm. The contacts between side-chains (SCs) of the neighboring chains inside the trimer peptide were also counted if the distances between the SC were smaller than 0.45 nm. The lipid order parameters were computed using the formula S CD ¼ 1 2 3 cos 2 q À 1 where C is carbon, D is deuterium, and q is the angle between the molecular axis provided by the C iÀ1 À C i+1 vector and the bilayer normal and the results were averaged over the membrane during the simulation interval.
Results and discussion
Sampling convergence of the REMD simulations of the F19W 3Ab REMD convergence at 324 K, above the phase transition 315 K temperature of the membrane DPPC lipid bilayer, was assessed by eight metrics calculated over time intervals of 250-350 ns and 250-400 ns. These included the percentage of secondary structures (beta, coil, turn and alpha contents), the radius of gyrations (R g ), the RMSD, the total solvent-accessible surface area, and the salt bridge D23-N27 of chain A of F19W 3Ab . Fig. 3 shows that the system had reached equilibrium at 324 K aer 250 ns, with all metrics remaining consistent over the two time windows. Overall, the b content value varies in the range of 24-53% with the mean value of 44.36 AE 3.74% (Fig. 3a), while the random coil content spans the range of 27-59% with the average value of 41.7 AE 3.8% (Fig. 3b). The mean value of turn and helix contents are 1.18 AE 1.18% (Fig. 3c) and $0% (Fig. 3d), respectively. The mean R g value of F19W 3Ab 11-40 is 1.47 AE 0.02 nm (Fig. 3e), which is larger than that in the wild type 3Ab (1.42 AE 0.02 nm). 34 The majority (71%) of the F19W 3Ab 11-40 population has a R g higher than 1.45 nm (Fig. 3e), while the wild type 3Ab 11-40 has a R g smaller than 1.45 nm. 34 The mean RMSD value of the F19W 3Ab 11-40 is 0.53 AE 0.05 nm (Fig. 3f), larger than that of the wild 3Ab (0.47 AE 0.07 nm). 34 The distribution of total solvent accessible surface area of the F19W 3Ab is rather broad, with the average value of 70.43 AE 3 nm 2 (Fig. 3g) signicantly higher than that of wild 3Ab 11-40 (64.73 AE 3.07 nm 2 ). The distribution of the salt-bridge D23-N27 of chain A of the F19W 3Ab is also broad, with the mean value of 1.04 AE 0.14 nm (Fig. 3h). The F19W 3b does not have a well-dened population with D23-N27 polar contacts, as was found in the wild 3Ab 11-40 (see below). As D23-N27 polar contacts play a crucial role in stabilizing the structures of the Ab peptides and their fragments, 34,70 the difference indicates that the F19W 3Ab 11-40 forms more extended structures that are less stable than those associated with wild 3Ab . Similar behavior of salt-bridge D23-N27 is also found in chain B and chain C (see Fig. S5 in the ESI †).
Distribution of secondary structures of the transmembrane F19W 3Ab
per residue
The averages of the random coil, beta, turn and a-helix structures are presented in Fig. 4. During our simulations, on average, the a-helix was rarely observed, comprising only $0.03% over the REMD simulations, which decreased in comparison with the wild type transmembrane 3Ab 11-40 $ 0.2% over the simulations. This data conrms that the a-helix is an intermediate step in the Ab aggregation process. 24,71,72 The turn population also decreased to $1.18% (it was $3% in the wild type transmembrane). The random coil conformation decreased from 57% in the wild type to 41.7% in the mutant type. The error bars of coil conformation for residues 22-29 is large due to the different behavior of the three chains (see Fig. S6 †). In contrast, the b-content was dominant and increased from 40% in the wild type to 44.36% in the mutant type. This result is in good agreement with the solvated F19W 3Ab . 31 Again, the error bars of the b-content is large for residues 20, 30-32 due to the fact that the population of bcontent is quite different for each of the three chains (see Fig. S6 †).
We also investigated the secondary structure along the sequence of each chain of the transmembrane F19W 3Ab obtained in the last 150 ns of REMD simulations at 324 K (Fig. S6 †). All chains were divided into ve main sequences, in which sequences 14-20 and 30-36 exhibit mostly b-structures, while sequences 11-13, 21-29, and 37-40 exhibit mostly random coil structures. Overall, the two b-structure domains are separated by the three random coil regions. Turns are observed in the region dominant by coils, at residues 12-13, 24-32 and 36-37. The majority of turns were found at residues 24-26. The negligible amount of helical population was mostly found around residues 25-27 of chain B and 29-31 of chain C. In comparison with the wild-type truncated trimer, 34 there are some slight shis in the b-domains and random coils. In the wild type, b sheets were found in sequences 14-19 and 31-37, while random coils were observed at sequences 11-13, 20-30 and 38-40.
Interactions of the F19W Ab 11-40 chains with the other chains and with the lipid bilayer
To quantify the interactions of the peptides within the mutant truncated trimer, we constructed the backbone-backbone (BB-BB) and side-chain-side-chain (SC-SC) inter contact maps (Fig. 5) over the equilibrated snapshots. Looking at the interpeptide SC-SC contact maps between neighboring chains of the transmembrane F19W 3Ab 11-40 , we found diverse interactions between chain A-chain B (Fig. 5a) and chain B-chain C (Fig. 5b). In both Fig. 5a and b, these contacts can be divided into various regions: the interactions between CHC-CHC, Cterminal-C-terminal, C-terminal-CHC, loop-loop, N-terminal-CHC, N-terminal-C-terminal, C-terminal-loop, loop-CHC regions. The details of the contact probabilities between these chain pairs are given in Table S2. † By dening an 80% threshold, we found that the dominant interactions between both pairs chain A-chain B and chain Bchain C involves CHC-CHC, C-terminal-C-terminal and a part of N-terminal-N-terminal regions. The most populated residuepair contacts are shown in Table S2. † Other interaction regions have probability in the range of 10-80%. Importantly, the interpeptide SC-SC contact maps reveal many interactions between side-chains of residues L17 and W19 and side-chains of residues I32, L34 and V36 in both chain pairs: L34 B -W19 C (64.78%), V36 B -L17 C (61.65%), L34 A -L17 B (46.44%), etc. (Table S2 †). These C-terminal-CHC interactions in early oligomers have been recently reported by experiments. 21,23,46,73,74 The long-range contacts between CHC-CHC occur between V18-F20, A21-W19, W19-L17 in both chain pairs with populations between 35.83% to 79.65%. In addition, the Cterminal-C-terminal contacts show many long-range interactions between residues I32, L34, V36, V39 with residues A30, I32, L34, V39 and M35 in both chain pairs with populations varying from 10.71% to 50.71%. The strongest interactions are I32 A -A30 B (50.71%), L34 A -I32 B (26.90%) and V36 A -L34 B (21.12%), which have been studied by both experiments 21,23,73 and computations. 5,14 For C-terminal-N-terminal contacts, the interaction between V36-Q15 indicates that even polar side chains can be tolerated to a certain degree in the hydrophobic region. Finally, the N-terminal-N-terminal contacts cannot be ignored, with the strongest interactions occurring in Q15-H13 and V12-H14 between different chains. The details of the interactions for different residue pairs are shown in Table S1. † The C-terminal-loop, loop-loop and N-terminal-CHC contacts show interactions with much lower population (<30%) ( Table S2 †).
Unlike the diverse contacts between chain A-chain B and chain B-chain C, the chain A-chain C contact map (Fig. 5c) is rather sparse, indicating the lack of strong interactions. The hydrophobic interactions with highest probability appeared in the C-terminal-CHC regions between L34 and L17 (31.64%). The contacts in C-terminal-loop and C-terminal-N-terminal give interactions with probabilities around 20%. The contact probabilities of N-terminal-N-terminal, N-terminal-CHC and loop-C-terminal vary from 3.08% to 11.17%. Fig. 5d-f shows the inter-peptide BB-BB contacts between adjacent chains of the transmembrane F19W 3Ab . The BB-BB contacts between chain A-chain B and chain B-chain C occur in the N-terminal-N-terminal, CHC-CHC, loop-loop and C-terminal-C-terminal regions. Details of contact probabilities between these chain pairs are given in Table S3. † Fig. 5d shows that the dominant BB-BB interactions are located in residues 15-19 of chain A with residues 14-20 of chain B and residues 30-36 of both chains, with probabilities in the range of 80% to 100% (Table S3 †). Fig. 5e shows the dominant (probabilities of 80-100%) inter-peptide BB-BB interactions between chain Bchain C involve residues 16-21, 33-36 of both chains, and Q15 B -H16 C and V36 B -G37 C (Table S3 †). Weaker, long-range interactions appear in the C-terminal-loop and loop-loop contacts, with probability ranging from 10% to 80%. The weakest interactions (probability lower than 10%) were all found in the loop regions. In Fig. 5f, the contact map is rather sparse, indicating that the contacts between chain A-chain C are negligible.
Overall, both SC-SC and BB-BB contact maps of chain pairs show diverse and strong interactions between chain A-chain B and chain B-chain C, while interactions between chain A-chain C are negligible. This implies that chain B stays in the middle between chain A and chain C during the simulation, contacting both A and C chains, which are thus separated from each other. In addition, the highest probabilities in both inter-peptide SC-SC and BB-BB contact maps appear in the parallel interactions between CHC-CHC, C-terminal-C-terminal and a small part of N-terminal-N-terminal of chain A-chain B and chain B-chain C which mainly correspond to the b sheets (sequences 14-20 and [30][31][32][33][34][35][36]. This also support the fact that the trimer forms parallel b sheets, in good agreement with previous solid state nuclear magnetic resonance (ss-NMR), electron microscopy (EM) and electron paramagnetic resonance (EP) experimental studies on the structures of Ab 1-40 brils. 23,74 The weakest interactions occur in the random coils (residues 11-13, 21-29, and 37-40) and helical contents regions (residues 25-27 of chain B and 29-31 of chain C).
The D23-N27, D23-K28 polar contacts have been shown in some previous studies 21,22,34,70,75 to considerably contribute to stabilizing a loop that facilitates Ab folding in solution. The distributions of the intra-molecular polar contacts of the transmembrane mutant F19W 3Ab peptide are shown in Fig. 6, where upper and lower panels show results for D23-N27 and D23-K28, respectively. While chain A and B do not form D23-N27 polar contacts (Fig. 6a), chain C does with very low population (2.02%). The D23-K28 polar contact cannot be observed in chains A and C (Fig. 6b), and it rarely occurs in chain B (population 0.21%). Polar contacts are rarely observed in the chains of the F19W 3Ab 11-40 trimer because both K28 and N27 form contacts with the phosphate atoms of the DPPC lipid bilayers (see Fig. 7 below). This is in agreement with computational studies about the effect of lipid bilayers on the conformational changing of the Ab 40 monomer. 76,77 Unlike the mutant, the wild-type truncated trimer has these polar contacts in all three chains, with very high population in chain C for D23-K28 contacts and in chains A and B 34 for D23-N27 polar contacts. This suggests that the transmembrane wild-type trimer is more stable than the mutant, and that the F19W mutation would destabilize the folded trimer. In addition, the breakdown of essential salt-bridges can also lead to the lag of brillation. Sciarretta et al. studied the brillation rate of the Ab 1-40 and Ab 1-40 Lactam (D23/K28). 78 They proved that although Ab 1-40 -Lactam (D23/K28) forms brils similar to those formed by Ab , the brillogenesis rate increased to 1000-fold by suppressing the lag period. They highlighted that in Ab 1-40 Lactam (D23/ K28), the Lactam linkage resulted a bend-like structure in the peptide.
To understand how the protein interacts with the membrane lipid bilayers, we calculated the probabilities of intermolecular contacts between phosphate atoms of DPPC lipid bilayers and heavy atoms of the truncated trimer F19W (Fig. 7 and S8 †), and the contact map between phosphate atoms of DPPC bilayers and each residue (Fig. S7 †). Fig. 7 shows that in all three chain residues E11, K16 and K28 have contacts populations higher than 100%. This means that aer the F19W mutation, residues E11, K16 and K28 in all chains contact the membrane in all the conformations. This is in agreement with previous studies showing that K16 and K28 form the most regular contacts with lipid phosphate head groups. 76,77 In comparison to the wild-type 3Ab 11-40 , 34 there are some remarkable changes in the membrane contacts of several regions of the mutant. In the F19W 3Ab 11-40 trimer, residues 22-23 in the random coil in the loop region of the chain (Fig. S6 †) do contact the lipid bilayers, which does not happen in the wildtype 3Ab trimer. In addition, there are fewer contacts in residues 17-21 and 35-36 in the mutant, while residues E11 and K28 increase their contact with the lipid bilayers, thus decreasing the D23-K28 polar contacts. Similarly, the increase of contacts between the N27 and D23 residues and the membrane results in the disappearance of the D23-N27 saltbridge. In short, F19W mutation leads to more proteinmembrane contacts and precludes the formation of crucial saltbridges, which may decrease the aggregation rate.
Free energy surface and representative structures of the transmembrane F19W 3Ab 11-40 trimer To characterize the conformations of the transmembrane F19W 3Ab 11-40 trimer, we constructed the free energy surface (FES) as a function of RMSD and radius gyration R g and then used clustering methods 65 to identify the metastable states. The FES is shown in Fig. 8, with the RMSD values in the range of 0.37-0.66 nm, and R g values between 1.39-1.56 nm. The FES reveals twelve minima, denoted as S1-S12 with the representative structures shown in Fig. 9. The twelve states have populations varying from 17.29% to 0.51%, with their total population accounting for 67% of the system's uctuation. All the twelve states form U-shaped conformations with three parallel bstrands in CHC and with the C-terminal regions separated by random coils in the loop region. Strong hydrophobic interpeptide contacts in these b-strands regions create a b-core of the trimer aligning parallelly to the lipid bilayer. In contrast, the random coil regions located at the end of the b-core strongly interact with phosphate head groups of DPPC lipid bilayers. This result captures the experimental structures of Ab [25][26][27][28][29][30][31][32][33][34][35] in membranes, in which the hydrophobic b-sheets are inserted into membranes while hydrophilic regions interact with the membrane surface. 79 The result is also consistent with computational studies of the Ab 10-40 peptide. 80 Based on all the conformations of each state, properties of the twelve states were computed and are presented in Table 1, which includes populations, the RMSD, the R g , the secondary structures, the collision cross sections and solvent-accessible surface areas. Dominant secondary structures are b-strands and random coils. On average, the populations are 45% bstrand, 39.58% random coil, 0.42% turn and 0% helix.
The rst four states, S1-S4, account for 47.29% of the ensemble. The detailed topological characterization of those states are shown in Table S4, † including the positions for bstrands and coils, the orientation of the two b-strands and the inter-peptide contacts that stabilized the two b-domains. S1 with a population of 17.29% is characterized by parallel bstrands spanning residues 15-20, 28-36 in chain A, residues 15-19, 28-36 in chain B and residues 15-19, 28-35 in chain C. The two b-strands in each chain form two antiparallel b-sheets, resulting in two antiparallel, three-stranded b-sheets for the trimer. Random coils are present at residues 11-14, 37-40 in all three chains, and loop regions at residues 20-27 in chain A and 21-27 in chains B and C. Propensities for b-strands, coils, turns and helices are 47%, 39%, 1% and 0%, respectively. The state is stabilized by the inter-peptide contacts CHC-CHC, C-ter-C-ter, CHC-C-ter and N-ter-N-ter between chains A-B and chains B-C. The collision cross section is 1416Å 2 , while the solventaccessible surface area is 72.46 nm 2 . Each state in S2, S3, and S4 has also two three-stranded b-sheets spanning different residues in the chains, separated by 3 coils domains, and packed either perpendicularly (state S2), or in antiparallel (state S3 and S4) fashion (Table S4 †). S1 and S2 have similar b populations, and these values are higher than those obtained in S3, S4 (Table 1). In addition, S1 and S3 have similar values of CCS and SASA, which are higher than those found in S2 and S4 (Table 1). Only S1 and S4 have turn contents with very low populations.
Among these twelve states, S2 and S9 have the highest b population (49%). States S11 and S3 rank highest in random coil (46%, 45%). Turn populations are very low in all cases. State S11 is most exposed to water, with a solvent accessible surface area of 73.96 nm 2 , while the states with least access to water are S6 and S7, with SASA 65.34 nm 2 and 65.37 nm 2 . Each state among S1, S3, S4, S5, S8, S11 and S12 has two antiparallel b- Fig. 8 The FES of the transmembrane F19W 3Ab 11-40 as a function of RMSD and radius gyration R g . Twelve minima are noted from S1 to S12 with those representative structures shown in Fig. 9. For clarity, several minima very closed to each other are shown in only one line with the names of the minima are noted.
sheets facing to each other, while the two b-sheets are located in two perpendicular planes for states S2, S6, S7, S9 and S10. The two b-sheets form the most in-register antiparallel in states S5, S8, S11, and they are the most perpendicular in states S2, S7, S10.
Average values over for the twelve states of the transmembrane F19W 3Ab trimer are 45% b sheet, 39.58% coil, 0.42% turn, 0% helix, 1389Å 2 for CCS, and 69.33 nm 2 for SASA. In comparison, those numbers in the truncated wild-type trimer are 44%, 54%, 2%, 0%, 1340Å 2 and 64.18 nm 2 for b, coil, turn, helix contents, CCS and SASA, respectively. The only signicant difference between the two is the decrease of the coil content, and increases for both the CCS and the SASA values for the mutant trimer with respect to the wild type.
Finally, the free energy values for these twelve states S1-S12 are À13.70, À12.56, À11.40, À13.13, À12.56, À12.56, À11.98, À13.7, À10.27, À13.7, À12.56 and À11.41 kJ mol À1 , respectively. The global minimum, À13.70 kJ mol À1 is comparable to the wild type minimum in DPPC lipid bilayers (about $0.2 kJ mol À1 higher 34 ) and it is about $0.9 kJ mol À1 lower than those found in solvated F19W 3Ab . 31 The membrane truncated F19W trimer is more exible than the wild-type trimer, Fig. 9 Representative structures of the twelve minima shown in Fig. 8. The population of each state is calculated using FES and clustering methods and is given in parentheses. Here, the residues b-contents are represented in yellow, those of mutant F19W are shown in blue, and coil conformations are shown as grey and purple colors, respectively. due to the higher number of minima with smaller free energy barriers. This is consistent with the disappearance of essential polar contacts. Three states S1, S8 and S10 have the same lowest free energy value, À13.7 kJ mol À1 , however, the populations are 17.29%, 1.66% and 1.29%, correspondingly. It indicates that the global representative structure of the system is S1. The total population of three states found in the global minimum is 20.24% of the ensemble. Meanwhile, four states were found in the global minimum of the wild-type truncated trimer (3Ab , with populations of 29%, 21%, 13% and 9%, 34 resulting the total population 72% of the ensemble. From that point of view, the latter has a higher exibility than the wild-type 3Ab .
Binding free energy of the F19W 3Ab trimer to the DPPC lipid bilayer To quantify the interactions between the mutant trimer and the lipid membrane, we calculated the binding free energy of the truncated mutant trimer and the DPPC using the doubleannihilation binding free energy method. In this method, the peptide is annihilated by both the solvated and transmembrane systems. As the structure S1 has the highest population in the total ensemble, it was adopted as the initial structure for FEP computations. 66 The binding free energy (DG bind ) was estimated by the difference in the annihilation energy between the transmembrane protein and that of the corresponding solvated protein. DG bind consists of two terms, the Coulomb interaction energy DG Cou and the van der Waals interaction energy DG vdW : For F19W 3Ab is signicantly higher than that obtained for the wild type trimer using the same method, indicating that the mutant has less binding affinity to the membrane than the wild type. The signicant difference in DG bind arises from the difference of collective Coulomb and van der Waals binding energies of the two proteins with DPPC bilayer. In comparison with the trimer wild type, both DG Cou and DG vdW increase for the mutant type. In particular, the increase in DG Cou is consistent with the increasing interactions between the phosphate groups of DPPC and the charged residues E11, E22 and D23 discussed previously ( Fig. 7 and S8 †).
Comparison with other studies
The collision cross section (CCS) is an important parameter for describing proteins in both experimental and computational methods. In experiments, CCS can be estimated by ion mobility mass spectrometry (IM-MS), 81 Our CCS values are smaller than that obtained from Ngo's paper due to the different point mutations between the two studies (F19W in our case, and F20W in Ngo's paper). Also, the starting structures in Ngo's paper are both U-shaped conformations (parallel b-sheets) and b-hairpin (antiparallel b-sheets), while the initial structure in our study is U-shaped. CD experiments with different sample preparations have shown that the b content of the Ab trimer in solution is around 50% (ref. 24) or 40.8%. 87 Our simulations gives 44.36%, in between the two CD-derived values. Also, during our simulations, a helices were rarely observed ($0.03% over the simulations). In a previous study based on REMD simulations with the AMBER96 force eld to study trimer Ab 10-35 , Jang et al. reported the propensity of b-strands was $50% with negligible a helices. 71 More recently, using the four-bead coarse-grained discrete molecular dynamics simulations to study the Ab oligomers, 88 Urbanc et al. found that the b-strand populations of 17% and 19%, and turn populations of 44% and 43% for the wild-type Ab 1-40 and Ab 1-42 trimers, respectively. Besides, based on REMD simulations to study the stability of the F20W Ab 11-40 trimer transmembrane with antiparallel and parallel b-sheet organizations, Ngo et al. reported that the b-contents is in the range of 44% to 60%, with a mean value of 49.71%, the coilcontents population of 37% to 58%, with an average value of 48%, and turn 2%. 86 Our results, consistent with the rst and third REMD studies and experimental results, do not support this: in particular, the b-strand propensities of residues 14-20 and 30-36 have an average value of 85%, while the largest bstrand content never exceeds 30% in the ref. 88. In addition, our computational studies found many non-local contacts between W19 and L34, and an increase in b population consistent with Huster and Hoffmann's experimental studies 46,47 that were focused on the structure of the mature brils. The increase of b population also consistent with Ngo's results in F20W 3Ab transmembrane studies. 86 Our investigation provides a better understanding the structure of oligomers aer the F19W mutation, specically related to the conformational changes of the transmembrane F19W 3Ab in DPPC lipid bilayers. In particular, the mutated residue inserted itself into the bril core, in agreement with previous Thioavin and tryptophan uorescence and transmission electron microscopy experimental studies. 36 Additionally, we found that F19W mutation destabilized the structure of 3Ab in the membrane. It is consistent with Ngo's previous studies shown that F19P mutation destabilized the structure of 3Ab in the DPPC lipid bilayer. 86 We also found U-shaped conformations with two threestranded b-sheets in the CHC and C-terminal regions that oriented in (i) the antiparallel form captured in many ss-NMR studies for Ab oligomers varying between 4 and 33 chains 5these forms may act as nucleation sites for antiparallel b-sheets brils as observed in Ab 1-40 , 23 and Ab [16][17][18][19][20][21][22] ; 15 (ii) perpendicular orientation, consistent with the observations in coarsegrained [15][16][17] and all-atom simulations 14,89 of amyloid-peptides. In addition, the two b regions are completely inserted in the membrane, stabilized by the hydrophobic inter-peptide contacts, and separated by three random coils that interact with the phosphate head groups on the surface of the membrane. These ndings are consistent with experimental studies of Ab [25][26][27][28][29][30][31][32][33][34][35] in membranes 79 and with computational studies of Ab 10-40 in membranes. 80 Finally, because the amyloid landscape is highly heterogeneous and sensitive to the experimental conditions, we cannot neglect that the b-hairpin conformations may also exist. 86
Conclusions
In AD, the Ab peptide is involved in neuronal toxicity via interactions with the cell membrane. Lipid membranes are known to modulate the rate and mechanisms of Ab selfassembly by having the lipid molecules interact specically with the growing brils, and thus accelerate the bril growth rate. 90 In addition, the vulnerability of cells to the effects of oligomeric aggregates is directly associated to the oligomer binding affinity to the cell membrane. 49 Given these ndings, we presented a minimal oligomer model, the 3Ab 11-40 trimer, with a "subtle mutation" F19W, where the Trp residue preserves the aromatic, non-polar character of the Phe residue, in order to investigate whether such minimal mutation can alter the binding affinity and the conformations of the mutated trimer compared to the wild type trimer. In order to achieve this, we ran extensive all-atom REMD simulations of the mutated F19W 3Ab trimer both in solution and transmembrane DPPC lipid bilayers and computed the all-atom free energy landscape in terms of two order parameters (radius of gyration and RMSD). We then computed the F19W trimer binding free energies to the lipid bilayer.
We found that the mutation brought about some nonnegligible conformational changes with respect to the wild type. In particular, the average populations of alpha and turn motifs slightly decreased, but they were almost negligible in the wild type to start with. The random coil population decreased by 15.3%, from 57% in the wild type to 41.7% in the mutant. In contrast, the dominant b content increased by 4.4%, from 40% in wild type to 44.4% in the mutant type. Both the radius of gyration and the RMSD slightly increased, while the SASA increased from 64.73 nm 2 in 3Ab 11-40 to 70.43 nm 2 in the transmembrane F19W 3Ab . The latter suggests that the F19W 3Ab 11-40 trimer may aggregate more slowly than the wild type, which would be consistent with experiments 46 showing that the F19W mutation slows down the brillation kinetics. The essential salt-bridges of 3Ab 11-40 disappeared in F19W 3Ab , indicating that the F19W mutation could destabilize the truncated trimer within the membrane. The interactions between the phosphate atoms of the DPPC lipid bilayers and the heavy atoms of the F19W 3Ab 11-40 trimer differ along the sequences and the residues of the trimer, and the total amount of contacts between the protein and membrane increases.
More important differences were found in the free energy surface in terms of the R g and RMSD order parameters. This surface displayed twelve minima that account for 67% of the ensemble; by comparison, the wild-type free energy surface only displayed ve that accounted for 100% of total conformations. 34 The free energy values of the twelve states vary from À13.70 to À11.41 kJ mol À1 , and the global minimum free energy is about $0.2 kJ mol À1 higher than those found in the minima of wildtype 3Ab . Although the free energy difference is small, the mutant is more exible, due to the population of global minimum in F19W 3Ab is much lower (20.24%) than that in wild-type truncated trimer (72%). 34 The representative states are consistent with many other simulations' results and may act as nucleation sites for the brillation process. Finally, rather dramatic differences were found in the F19W 3Ab binding free energy to the DPPC bilayer, computed using the FEP method. Our results indicate that this binding free energy is $40-50 kcal mol À1 higher than that in the wild-type 3Ab trimer.
Altogether, our studies provide insight into the effect of mutation F19W on transmembrane 3Ab . The disappearance of crucial salt-bridges, the increase of the interactions between the peptides and the membrane as well as the greater structural diversity with higher free energy values indicate that the mutant is more exible than the wild type, while the binding free energy indicates that F19W 3Ab is considerably less stable in the lipid environment than its wild-type counterpart. These results suggest that the impact of mutations can be assessed, at least partially, by evaluating the interactions of both the wild-type and the mutated oligomers with the lipid membranes.
Conflicts of interest
There are no conicts to declare. | 2021-05-11T00:04:16.415Z | 2021-01-11T00:00:00.000 | {
"year": 2021,
"sha1": "3e84ef8d787ae779abc4ed8b7218b28c2ee36667",
"oa_license": "CCBYNC",
"oa_url": "https://pubs.rsc.org/en/content/articlepdf/2021/ra/d0ra08837d",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "df01d73d7ed23520943452e0cfd3fefe28907735",
"s2fieldsofstudy": [
"Medicine",
"Biology",
"Chemistry"
],
"extfieldsofstudy": [
"Medicine",
"Chemistry"
]
} |
220907235 | pes2o/s2orc | v3-fos-license | Elevated depression and anxiety symptoms among pregnant individuals during the COVID-19 pandemic
Background Anxiety and depression symptoms in pregnancy typically affect between 10 and 25% of pregnant individuals. Elevated symptoms of depression and anxiety are associated with increased risk of preterm birth, postpartum depression, and behavioural difficulties in children. The current COVID-19 pandemic is a unique stressor with potentially wide-ranging consequences for pregnancy and beyond. Methods We assessed symptoms of anxiety and depression among pregnant individuals during the current COVID-19 pandemic and determined factors that were associated with psychological distress. 1987 pregnant participants in Canada were surveyed in April 2020. The assessment included questions about COVID-19-related stress and standardized measures of depression, anxiety, pregnancy-related anxiety, and social support. Results We found substantially elevated anxiety and depression symptoms compared to similar pre-pandemic pregnancy cohorts, with 37% reporting clinically relevant symptoms of depression and 57% reporting clinically relevant symptoms of anxiety. Higher symptoms of depression and anxiety were associated with more concern about threats of COVID-19 to the life of the mother and baby, as well as concerns about not getting the necessary prenatal care, relationship strain, and social isolation due to the COVID-19 pandemic. Higher levels of perceived social support and support effectiveness, as well as more physical activity, were associated with lower psychological symptoms. Conclusion This study shows concerningly elevated symptoms of anxiety and depression among pregnant individuals during the COVID-19 pandemic, that may have long-term impacts on their children. Potential protective factors include increased social support and exercise, as these were associated with lower symptoms and thus may help mitigate long-term negative outcomes.
Introduction
Since it was first recognized in December 2019, the 2019 novel coronavirus has spread rapidly throughout the world. The health consequences of this virus are distressing: death, strained health care systems, and economic uncertainty. The psychological and social consequences may be equally devastating. People have been physically isolated from family, friends, and community, and schools and daycares around the world have been closed. There is a growing urgency to understand the impact of the COVID-19 pandemic on mental health to best prevent the emergence of serious mental illness as a secondary consequence (Cullen et al., 2020;Geraldo da Silva et al., 2020).
Although limited, previous work shows that infectious disease outbreaks increase symptoms of depression and anxiety. A study of 129 individuals quarantined during the 2003 severe acute respiratory (SARS) outbreak in Toronto, Canada found that 29% of individuals had symptoms of post-traumatic stress disorder and 31% had symptoms of depression approximately one month following their quarantine; longer periods of quarantine were associated with more severe symptoms (Hawryluck et al., 2004). In the early phase of the COVID-19 outbreak, 53.8% of respondents in China's Wuhan region reported moderate or severe psychological impact, with 17% and 29% reporting moderate to severe depression and anxiety symptoms, respectively (Wang et al., 2020). A survey by the Kaiser Family Foundation in late March 2020 found that 53% of women and 37% of men said that stress related to coronavirus had a negative impact on their mental health (Hamel and Salganicoff, 2020).
Pregnancy is a particularly vulnerable time when psychological distress can have negative consequences for both mother and baby. Since women tend to report higher symptoms of anxiety and depression during disease outbreaks than men (Al-Rabiaah et al., 2020;Hamel and Salganicoff, 2020;Wang et al., 2020), women who are pregnant during the COVID-19 pandemic may be especially affected. Sustained, elevated prenatal anxiety and depression symptoms increase the risk of postpartum depression, as well as prenatal infection and illness rates (Bayrampour et al., 2016;Coussons-Read, 2013). Prenatal anxiety and depression symptoms may also cause changes in physical activity, nutrition, and sleep, which in turn affect maternal mood and fetal development (Coussons-Read, 2013). Prenatal anxiety and depression also increases the risk of miscarriage, preterm birth, lower birthweight, and lower Apgar scores at birth (Accortt et al., 2015;Grigoriadis et al., 2018;Qu et al., 2017;Rondo et al., 2003;Stein et al., 2014). Children of mothers who experienced high stress during pregnancy are more likely to have cognitive and behavioural problems, and are at higher risk for later mental health problems themselves (Glover, 2014;MacKinnon et al., 2018;Stein et al., 2014;Van den Bergh, Dahnke, and Mennes, 2018;Van den Bergh et al., 2017). Prenatal anxiety and depression are also associated with changes to brain structure and function in infants and children (Adamson et al., 2018;Lebel et al., 2016;Qiu et al., 2013;Sandman et al., 2015). These long-lasting psychological and neurological effects highlight the importance of mitigating prenatal distress now, to support both pregnant individuals and their babies.
It is also important to look for potential resilience factors that may help protect against high prenatal stress. Social support can buffer the effects of prenatal stress, and has been shown to mitigate the impacts of prenatal anxiety and depression symptoms on maternal and infant stress response systems (Thomas, et al., 2018). Physical activity is also associated with reduced depressive and anxiety symptoms in pregnant individuals (Demissie et al., 2011), and thus may provide another resilience factor.
Given the potential negative psychological sequelae of psychological, health and financial uncertainty coupled with social isolation, there is an urgent need to determine the prevalence of psychological distress among pregnant individuals during this pandemic and identify protective factors so that targeted interventions can be quickly implemented. The aims of the current study were to determine the prevalence of anxiety and depression symptoms in pregnant people during the COVID-19 pandemic and identify potential resilience factors associated with lower symptoms. The social distancing universally recommended by governments around the world may be especially problematic during pregnancy because social support has a wellrecognized role in buffering the negative effects of stress (Reid and Taylor, 2015).
Participants
The current study reports data collected from an ongoing study: Pregnancy during the COVID-19 Pandemic. This study recruited pregnant individuals across Canada via social media to complete an online survey. Study advertisements and the study website were shared via Twitter, Facebook, and Instagram. Ads were distributed to groups for expecting mothers, young parents, and midwifery and obstetric groups, and participants were encouraged to share the study with their friends and family. Inclusion criteria were: living in Canada, able to read and write English, and having a confirmed pregnancy <35 weeks gestation.
The data reported here were collected between April 5-20, 2020. This study was approved by the Conjoint Health Research Ethics Board (CHREB) at the University of Calgary, REB20-0500.
Demographics
Participants provided comprehensive demographic information including their birth month and year, postal code, education level, household income range, their baby's due date, and number of other children.
COVID-19
Participants completed a questionnaire about COVID-19 infections and isolations, as well as COVID-19-related life changes such job loss. This questionnaire was developed specifically for this study, based on previous work assessing stress during natural disasters (King and Laplante, 2015). Participants were asked specifically about concerns due to COVID-19 with the following statements/questions: "During the COVID-19 pandemic, I have felt more alone than usual", "How much do you think your life is in danger during the COVID-19 pandemic?", "How much are you worried that exposure to the COVID-19 virus will harm your unborn baby?", and "Are you concerned that you or your baby are not receiving the care that you need?". Participants answered on a scale of 0 (not at all) to 100 (very much so). Participants were also asked "How has the COVID-19 pandemic affected your relationship with your partner?" on a scale of 0-100, with 0 (it has strained our relationship), 50 (not much has changed) and 100 (it has brought us closer together).
Anxiety and depression symptoms
Maternal depressive symptoms were assessed using the Edinburgh Depression Scale (EPDS) (Cox et al., 1987;Kozinszky and Dudas, 2015), a self-report questionnaire with possible scores ranging from 0 to 30. Scores ≥13 are used to identify women with clinically concerning depression symptoms and have been shown to have maximal consistency with a diagnosis of major depressive disorder (Cox et al., 1987). For a cut-off of 13 on the EPDS, sensitivity ranges from 38 to 43% (depending on trimester) and specificity is 98-99% (Bergink et al., 2011). The PROMIS Anxiety Adult 7-item short form was used to assess general anxiety symptoms; T-scores 60-69.9 are considered moderately elevated anxiety symptoms and scores at or above 70 are considered severely elevated (Cella et al., 2010); possible scores range from 36.3-82.7. Pregnancy-related anxiety symptoms were assessed with a 10-item questionnaire about feelings surrounding the health of the baby and circumstances of the birth (Rini et al., 1999); possible scores on this questionnaire range from 10 to 40 . On all measures, higher scores indicate worse symptoms. There is no cut score for the pregnancy-related anxiety scale, but previous treatment studies used a median split to define groups with higher versus lower pregnancy anxiety symptoms (Urizar et al., 2019). In our sample the median was 19, which we used to divide the sample into groups with higher and lower pregnancy-related anxiety symptoms.
Social support
Participants completed the social support effectiveness questionnaire (SSEQ) (Rini et al., 2006), which evaluates the type and selfperceived effectiveness of the support they receive from their partner or another support person, and the interpersonal support evaluation list (ISEL) (Cohen and Hoberman, 1983), which measures broader perceived social support from friends, family, and others. Shephard Leisure-Time Exercise Questionnaire (Godin, 2011), which is a validated self-report measure of exercise frequency in which participants report the number of times per week they engaged in mild, moderate, and strenuous exercise of more than 15 min. A total score was calculated, per standard procedure, by multiplying episodes of mild exercise by 3, moderate by 5, and strenuous exercise by 9. Individuals with scores below 14 are considered sedentary, 14-23 are moderately active, and 24 or more are considered active.
Data analysis
Survey data were manually checked for accuracy and consistency before analysis. From an original 2225 respondents, we identified and removed 238 invalid records because either participants had not provided consent or they provided invalid due dates (i.e., their gestation fell outside the range of 1-35 weeks).
All analyses were conducted using SPSS 26.0. Descriptive statistics were computed for demographics and main study variables. An analysis of covariance (ANCOVA) was used to compare nulliparous to primiparous and multiparous pregnant individuals on measures of mental health symptoms (EPDS, PROMIS anxiety, pregnancy-related anxiety). Mental health symptoms were included as continuous variables. Age and gestation were included as covariates. The significance was set at p<0.017 using Bonferroni correction for 3 multiple comparisons.
Bivariate correlations were used to determine relationships between mental health symptoms measures and social support measures. Multivariate binomial logistic regression was used to identify how COVID-19 related stressors (loss of employment, social isolation, relationship strain) and worries (concern about threat to own life, harm to baby, and not receiving the care needed) were associated with clinically elevated mental health symptoms (EPDS, PROMIS anxiety, pregnancyrelated anxiety). Clinically elevated mental health symptoms were defined using cutoffs from previous literature: ≥13 on the EPDS (Cox et al., 1987), and T-scores ≥ 60 for the PROMIS anxiety scale (Cella et al., 2010). The loss of employment variable was binomial (yes/ no); all other variables were measured from 0 to 100. The significance threshold was set at p<0.0028 using Bonferroni correction for 18 multiple comparisons. The multivariate model was used to determine unique associations between COVID-19 factors and anxiety and depression symptoms. Parity was included as a covariate in the pregnancy-related anxiety model because of its significant association with pregnancy-related anxiety symptoms. No covariates were included in the other models. Univariate models were conducted as supplementary analysis, also with Bonferroni correction at p<0.00028.
A logistic regression was used to identify resilience factors (physical activity, perceived partner support, perceived general social support) that were associated with lower odds of clinically elevated symptoms of anxiety and depression. Partner support was operationalized as the Total Support score from the SSEQ and general social support was operationalized as the Total Support from ISEL; both were continuous variables. The total score from the Godin was our measure of physical activity. The significance was set at p<0.0056 using Bonferroni correction for 9 multiple comparisons.
Participants
A total of 1987 individuals provided data for at least one measure on the survey between April 5-20, 2020 and were included in the current analysis; specific numbers of individuals providing data for each measure are listed in Table 1. Not all participants provided data for each question, so numbers included in each analysis vary between 1581 and 1987. Missing data were handled with listwise deletion for each separate analysis; n is provided in the tables for each analysis.
51% of participants had other children (37% had one child, 11% had two children, and 3.5% had three or more other children While Alberta residents are over-represented in the data compared to Alberta's population within Canada (11.6% of Canadian residents live in Alberta), there were no significant differences in weeks gestation or maternal age between Alberta respondents and the rest of the sample, Alberta respondents were equally likely to be born in Canada, and had a similar breakdown by ethnicity (p>0.05). Alberta residents had higher incomes (p<0.001; median=$125,000-149,000/year vs $100,000-125,000/year), different education profiles (p = 0.002; higher proportion of high school diplomas and bachelor degrees), and were more likely to be married than respondents from elsewhere (p<0.001; 83% vs 74%), which is consistent with population demographics in Alberta and Canada (Statistics Canada, 2013.
COVID-19 stressors
One participant had a confirmed case of COVID-19; 25 others reported suspected but unconfirmed cases. At the time of this initial survey, most provinces in Canada were only testing serious cases (i.e., in hospital) or healthcare workers. None of these individuals were hospitalized. 11 individuals reported other people with COVID-19 infections within their household (6 partners; 1 child, 1 housemate; 3 unspecified).
At the time of the survey, 18.3% of participants reported job loss due to COVID-19 (16.1% laid off, 2.2% indicated their employment was terminated).
Participants rated their social isolation as 64 +/-26, their worries that their own life was in danger due to COVID-19 as 46 +/-24, and worries that the virus would cause harm to their unborn baby as 52 +/-25, all on a scale of 0-100 (not at all to very much so). Average score on relationship strain (where scores <50 indicate more strain and scores >50 indicate the pandemic brought them closer with their partner) were 56 +/-21.
Most participants (89%) reported changes in prenatal care due to the pandemic, including canceled appointments (36%), or not being allowed to bring a support person (90%). Average scores on the question of whether participants believed that the quality of their prenatal care had decreased were 36 +/-28, on a scale of 0-100. 35% of respondents made changes to their birth plan because of the pandemic, including the location (11%), support people (25%), and childcare arrangements (11%). 74% had trouble accessing other healthcare during their pregnancy, most commonly reporting they could not access massage therapy services (58%), followed by chiropractic (26%); 9% reported that they were unable to access psychological counselling services.
Anxiety and depression symptoms
Mean scores are shown in Table 1. 37.0% of participants had clinically elevated symptoms of depression (EPDS scores ≥13). 46.3% of participants had moderately elevated anxiety symptoms (T-scores 60-69), and 10.3% severely elevated anxiety symptoms (T-scores>70). 56.6% total had clinically elevated anxiety symptoms. As expected, measures of anxiety and depression symptoms were moderately to strongly associated with each other, and negatively associated with perceived social support (Table 2).
COVID-19 worries and stressors in association with anxiety and depression symptoms
We used binomial logistic regression to determine which COVID-19 related worries (threat to own life, harm to baby, not getting needed care) and stressors (loss of employment, changes to relationship with partner, feelings of isolation) were associated with and clinically elevated anxiety and depression symptoms. The odds for clinically elevated depression symptoms were increased by COVID-19-related worries and by partner relationship strain, but not by loss of employment. Odds for clinically elevated depression symptoms increased by 1% for each unit increase in perceived threat to own life, harm to baby and not getting care needed, 5% for each unit increase in feelings of isolation, and 2% for each unit increase in relationship strain (all measures on 0-100 scale). Loss of employment did not increase the odds of clinically elevated depression symptoms. Similar findings were observed for general anxiety and pregnancy-related anxiety symptoms (Table 4). For both depression and general anxiety symptoms, the largest effects were for social isolation. Results of univariate binomial logistic regression models showed significant associations between most COVID-19 factors and depression, anxiety, and pregnancy-related anxiety symptoms (Supplementary Table 1). Only loss of employment (for all 3 symptoms) and relationship strain (for pregnancy-related anxiety symptoms) were not significant in the univariate analysis.
Resilience factors
The mean physical activity score on the Godin was 33, indicating that the sample could be considered 'active', using the classifications established by the Godin. Average scores on the SSEQ Total Support (partner social support) were 55.8 +/-14.9. Average scores on the ISEL Total Support (general social support) were 34.1 +/-6.3, which are consistent with previous reports in pregnant women, M = 35.4-38.7 Table 1 Sample characteristics. Mean, standard deviation, and range are provided for key demographic characteristics and depression and anxiety symptoms in the sample. The number of datapoints available for each comparison is also given. . Significant results (p<0.017) are indicated in bold.
C. Lebel, et al. Journal of Affective Disorders 277 (2020) (Chou et al., 2008;Christian et al., 2009;Messer et al., 2013). The odds of clinically elevated depression and anxiety symptoms were lower if participants and had better perceived social support (independent effects for partner and general support) ( Table 5). The odds of clinically elevated anxiety symptoms (both general anxiety and pregnancy-related anxiety) were lower if participants reported more physical activity.
Discussion
Pregnant participants reported high levels of depression, general anxiety, and pregnancy-specific anxiety symptoms. Higher symptoms were associated with more concern about threats of COVID-19 to the life of the mother and baby, as well as concerns about not getting the necessary prenatal care, relationship strain, and social isolation due to the COVID-19 pandemic. These findings suggest that the COVID-19 pandemic presents serious psychological challenges for pregnant individuals, with the potential for both short term (e.g., preterm birth, postpartum depression) and long-lasting impacts on the developing fetus. These findings highlight the urgent need to reduce psychological distress during pregnancy. Increased perceived social support and increased physical activity were associated with reduced symptoms, and thus may be possible targets for intervention. Elevated symptoms (above cut-off scores) of depression (37%), anxiety (59%) were higher than expected based on previous pre-COVID-19 cohort studies assessing symptoms in pregnant women with similar demographic profiles. Prenatal depression is estimated to affect 9-11% of individuals at any given time, with 18% of individuals experiencing a depressive episode at some point during pregnancy (Gavin et al., 2005;Woody et al., 2017). The England-based Avon Longitudinal Study of Parents and Children (ALSPAC) found that 17% of 2390 pregnant women reported clinically elevated depressive symptoms (≥13 on the EPDS) in the first wave of the study (1990)(1991)(1992), while 25% of 180 women in the second generation (2012-2016) reported clinically elevated depressive symptoms (Pearson et al., 2018). In the Canadian Alberta Pregnancy Outcomes and Nutrition (APrON) study (Kaplan et al., 2014), a study with similar demographic profiles to those seen here, 11% of women had clinically elevated depression symptoms on the EPDS (Leung et al., 2017). Normative data for the United States indicates prevalence of clinically elevated depression symptoms in 8% of adults (Brody et al., 2018). These comparisons suggest that symptoms of depression have increased substantially during the COVID-19 pandemic (Fig. 1). In a survey of Chinese residents early in the COVID-19 outbreak (Jan 31-Feb 2, 2020), 17% of respondents reported moderate or severe depression, and 29% reporting moderate to severe anxiety (C. Wang et al., 2020). The rates of elevated depressive and anxiety symptoms in our pregnancy cohort are even higher, suggesting that the psychological impact of the outbreak may be of particular concern for pregnant individuals.
Pregnancy-related anxiety symptoms were similarly elevated in our cohort (mean=19.1) compared to recent studies with similar demographics, which reported mean scores of 7.3 (Tomfohr-Madsen et al., 2019) and 7.5 (Thomas et al., 2017). General anxiety was elevated compared to a meta-analysis of pregnancy which reports 18-25% prevalence (Dennis et al., 2017) and the general US population prevalence of 16% (Cella et al., 2019); see Fig. 1. High levels of prenatal distress, particularly pregnancy anxiety, are concerning due to unique associations with elevated risk of preterm birth (Bussieres et al., 2015). The elevated anxiety and depression symptoms appear to be, at least in part, a consequence of the COVID-19 pandemic given that COVID-19-related worries were associated with higher symptoms. The odds of depression increased by 1-5% for each unit (on a 0-100 scale) increase in COVID-19 worries, results that were mirrored in the anxiety outcomes. Importantly, participants' worries that they were not getting adequate prenatal care due to COVID-19 were associated with higher symptoms in all categories, with the largest effect for pregnancy-related anxiety symptoms. This suggests that maintaining high quality prenatal care is a priority for pregnant individuals, and changes to care may lead to increased anxiety symptoms.
Consistent with the broader literature, better social support was associated with lower symptoms of depression and anxiety. The finding that higher perceived support and support effectiveness are associated with decreased depression and anxiety symptoms is consistent with the notion that social support buffers the effects of stress on anxiety and depression symptoms (Cohen, 2004) and previous research showing decreased prenatal and postnatal anxiety and depression among women with higher levels of social support (Akiki et al., 2016;Friedman et al., 2020). Social support is an important determinant of physical and psychological well-being, especially during pregnancy when individuals take on new responsibilities and roles (Dunkel Schetter, 2011). Supportive social relationships directly affect mental health by encouraging positive health behaviors, increasing positive feelings, and enhancing emotion regulation (Cohen and Wills, 1985) and indirectly by reducing the physiological stress response (Giesbrecht et al., 2013). Social support also reduces the effects of prenatal maternal stress on infant stress responses, suggesting that positive social relationships buffer the biological cascade of stress from mother to infant (Thomas et al., 2018).
Previous studies in multiple populations (Cotman and Berchtold, 2002;Erickson et al., 2011;Vankim and Nelson, 2013), including pregnant individuals (Demissie et al., 2011), indicate that physical activity is associated with reduced depression and anxiety symptoms. Our results were highly consistent, although the effect did not reach significance for depression after Bonferroni correction, but was at trend-level (p = 0.01). These associations have implications for pandemic control measures that limit opportunities for physical activity (e.g., closure of parks, beaches, and gyms) and suggests that encouraging physical activity among pregnant individuals may help reduce feelings of anxiety and depression.
The high anxiety and depression symptoms reported by participants are concerning for both maternal and child health. Children whose mothers experienced high prenatal stress are at higher risk of cognitive and behavioural problems, as well as mental illness in their own lives (Brouwers et al., 2001;DiPietro et al., 2006;Glover, 2014;Huizink et al., 2004;Kinsella and Monk, 2009;Mennes et al., 2006;O'Connor et al., 2003;Stein et al., 2014;Van den Bergh et al., 2005;Weinstock, 2008). Prenatal anxiety and depression symptoms are also associated with changes to brain structure and function in children (Adamson et al., 2018;Lebel et al., 2016;Sandman et al., 2015). Such changes in offspring development are known to occur via multiple mechanisms, including epigenetic, hormonal (e.g., cortisol), behavioral (e.g., lifestyle factors), and social (e.g., lack of adequate support) factors (Beijers et al., 2014), all of which are modifiable and therefore represent potential intervention/prevention targets.
Given the potentially serious consequences of untreated anxiety and depression symptoms in pregnancy on physical and psychological outcomes, interventions are urgently needed to reduce symptoms and build resilience. Psychological interventions for preventing and treating depression and anxiety in pregnancy are effective, with cognitive behavior therapy (CBT) emerging as a front-line treatment and interpersonal therapy (IPT) potentially offering additional benefits to reduce depression and increase social support (Field, 2017;Manber et al., 2019;O'Connor et al., 2019). Preliminary evidence also provides support for Fig. 1. To understand depression and anxiety symptoms in context, we compared results to published meta-analyses and normative scores on our measures of depression and anxiety. The prevalence of clinically elevated anxiety (blue) and depression (red) symptoms in the current study was substantially higher compared to meta-analyses (green boxes indicate full range of estimates) and the US population norms (green circles). References 1: (Dennis et al., 2017); 2: (Cella et al., 2019) 3: (Gavin et al., 2005) 4: (Brody et al., 2018). e-health interventions; however, trials to date are relatively small and scaling for widespread dissemination is urgently needed (Felder et al., 2020;Heller et al., 2020;Loughnan et al., 2019). Evaluation of e-health treatments should be a priority, given that in-person psychological treatments are currently not available or severely limited. Treatment of pregnancy anxiety is also effective with brief midwife or obstetric lead interventions; however, the ability to deliver these via telehealth has not been tested (Stoll et al., 2018). There is also suggestion that online programs improve partner social support and satisfaction, but to our knowledge these have not been tested in pregnancy (Doss et al., 2020). Psychological treatments may require specific investments from government to ensure wide access but could have large future returns.
One of the factors that may be closely associated with pregnancyspecific anxiety symptoms is parity, as first-time mothers tend to report greater pregnancy-related anxiety than parous women (Huizink et al., 2016). Indeed, in our sample, nulliparous individuals had significantly higher pregnancy-related anxiety symptoms than primiparous and multiparous individuals. Parity was not significantly related to EPDS or PROMIS Anxiety scores.
Our sample was slightly older and more likely to be married or cohabitating than the Canadian averages for pregnancy (Chalmers et al., 2008;Provencher et al., 2018). While this suggests that our data may not be entirely representative of Canadian pregnant individuals, samples with higher education, older age, and where more individuals are partnered tend to have fewer prenatal anxiety and depression symptoms. Thus, the elevated anxiety and depression symptoms seen here would be highly unexpected under normal circumstances. Given the low sociodemographic risks in our sample and the fact that Canada has had a relatively contained outbreak and universal health care, the results may be worse in populations with higher sociodemographic risks (e.g., low education, low income) or living in countries with larger outbreaks and/or worse containment measures. Symptoms of anxiety and depression can vary across pregnancy (Bayrampour et al., 2016;Bennett et al., 2004;Gavin et al., 2005), and the rapid changes in government policies and outbreak risks during the current pandemic could add further confounding. Therefore, longitudinal studies with multiple assessment points will be necessary to better understand the nature of anxiety and depression symptoms in pregnant women during the current pandemic. Future studies should consider other factors that may additionally contribute to anxiety and depression symptoms such as history of mental health problems.
Conclusions
Pregnant individuals are experiencing substantially elevated anxiety and depression symptoms during the COVID-19 pandemic that are significantly related to COVID-19 specific worries about threats to their own lives, their baby's health, not getting enough prenatal care, and social isolation. These levels far exceed those normally expected during pregnancy and those experienced by other groups of people during the current pandemic. Social support and physical activity appear to be protective resilience factors. Given the known effects of stress on pregnancy, infant, and child outcomes, there is an urgent need to support pregnant individuals during this critical time to mitigate longterm negative outcomes.
Declaration of Competing Interest
The authors report no conflicts of interest.
Funding
This research was supported by the Alberta Children's Hospital Research Institute and the Owerko Centre; the funder had no role in the research. | 2020-08-02T13:05:06.386Z | 2020-08-01T00:00:00.000 | {
"year": 2020,
"sha1": "950dd33c9529e900a8687c41a277c8fbc987e077",
"oa_license": null,
"oa_url": "https://doi.org/10.1016/j.jad.2020.07.126",
"oa_status": "BRONZE",
"pdf_src": "PubMedCentral",
"pdf_hash": "349b21fc48fb377497bb83b05c06dfd36e3a222d",
"s2fieldsofstudy": [
"Psychology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
6429409 | pes2o/s2orc | v3-fos-license | Glucagon regulates hepatic lipid metabolism via cAMP and Insig-2 signaling: implication for the pathogenesis of hypertriglyceridemia and hepatic steatosis
Insulin induced gene-2 (Insig-2) is an ER-resident protein that inhibits the activation of sterol regulatory element-binding proteins (SREBPs). However, cellular factors that regulate Insig-2 expression have not yet been identified. Here we reported that cyclic AMP-responsive element-binding protein H (CREBH) positively regulates mRNA and protein expression of a liver specific isoform of Insig-2, Insig-2a, which in turn hinders SREBP-1c activation and inhibits hepatic de novo lipogenesis. CREBH binds to the evolutionally conserved CRE-BP binding elements located in the enhancer region of Insig-2a and upregulates its mRNA and protein expression. Metabolic hormone glucagon and nutritional fasting activated CREBH, which upregulated expression of Insig-2a in hepatocytes and inhibited SREBP-1c activation. In contrast, genetic depletion of CREBH decreased Insig-2a expression, leading to the activation of SREBP-1c and its downstream lipogenic target enzymes. Compromising CREBH-Insig-2 signaling by siRNA interference against Insig-2 also disrupted the inhibitory effect of this signaling pathway on hepatic de novo triglyceride synthesis. These actions resulted in the accumulation of lipid droplets in hepatocytes and systemic hyperlipidemia. Our study identified CREBH as the first cellular protein that regulates Insig-2a expression. Glucagon activated the CREBH-Insig-2a signaling pathway to inhibit hepatic de novo lipogenesis and prevent the onset of hepatic steatosis and hypertriglyceridemia.
tissues and organs 5,7 . Although these two isoforms differ in the enhancer regions of their mRNA structures, with Insig-2a mRNA containing a non-coding exon-1 and an approximate intron that are missing in the mRNA of Insig-2b, both isoforms are eventually spliced to give the same mRNA that encodes identical proteins, Insig-2. Metabolic hormone, insulin, and metabolites, namely sterols, have been shown to regulate Insig-2a cellular abundance. However, little is known about the cellular protein(s) involved in conveying signaling from metabolic hormone to Insig-2 expression thus far.
Although the mechanism of how hepatic SREBP-1c is induced by insulin in the fed state is well-established, little is known about how hepatic SREBP-1c is suppressed during the fasting state. Glucagon, a metabolic hormone released from pancreatic α cells, is the principal regulatory hormone that counters the actions of insulin. Exogenous glucagon reduces liver TG content and prevents the development of fatty liver in dairy cows 8,9 , whereas reduced glucagon action is associated with the development of fatty liver 10,11 . Emerging evidence has further demonstrated that, in the fasted state, glucagon action is essential for multiple pathways regulating lipid homeostasis [12][13][14] . Its inhibitory effect on hepatic de novo lipogenesis was proposed to be mediated by the cAMP/protein kinase A pathways. However, the mechanism of action between glucagon/cAMP signaling and SREBP activation is not well defined. cAMP-responsive element-binding protein H (CREBH) is a recently identified transcription factor that is structurally related to the SREBPs 15 . Similar to Insig-2a, CREBH is selectively and highly expressed in the liver 16 . CREBH is synthesized as an ER-resident precursor protein and activated by S1P and S2P proteases in the Golgi apparatus in a mode similar to SREBP activation 15,17 . Activation of CREBH is induced by nutritional factors, such as fasting and free fatty acids, and suppressed by refeeding [18][19][20] . Genetic depletion of CREBH in mice induced fasting hypoglycemia and hypertriglyceridemia compared to wild type littermates 20,21 . Heterozygous nonsynonymous or insertional mutations in human CREBH (CREB3L3) caused severe hypertriglyceridemia in these individuals 21 . These evidences suggest that CREBH may play a negative regulatory role in hepatic de novo lipid synthesis. However, the intrinsic association between CREBH and SREBPs, particularly the link between these two lipid metabolic transcription factors and their impacts in regulating hepatic lipid synthesis, haven't been reported. In this study, we identify CREBH as the first cellular molecule that regulates Insig-2a at the transcriptional level. We further demonstrated that the CREBH-Insig-2a pathway inhibits hepatic de novo lipogenesis during the nutritional fasting state and is regulated by glucagon. Specifically, CREBH suppressed hepatic de novo lipid synthesis by upregulating the mRNA and protein expressions of Insig-2a. Chromatin Immunoprecipitation (ChIP) assay demonstrated the functional association between CREBH and the CRE-BP binding elements within the enhancer region of the Insig-2a gene. Nutritional signaling, fasting, and metabolic hormone glucagon activated CREBH, which subsequently induced Insig-2a expression to inhibit SREBP-1c activation. In contrast, compromising CREBH activity through genetic depletion decreased hepatic abundance of Insig-2a. Depletion of Insig-2 by siRNA interference disrupted the inhibitory effect of CREBH-Insig-2a on hepatic lipid synthesis. These actions led to the activation of SREBP-1c and its downstream lipogenic target enzymes as well as the subsequent de novo lipid synthesis upon fasting, resulting in hepatic steatosis and systemic hyperlipidemia.
Results
Insig-2a mRNA and protein expression are regulated by CREBH in response to fasting and refeeding. Previously, mRNA expression of Insig-2a was reported to be induced by fasting and suppressed by refeeding by Drs. Goldstein and Brown's group 22 . Subsequently, Danno et al. showed that CREBH is elevated during nutrition depletion 18 . To investigate whether there is intrinsic association between these two ER-resident and liver-expressed proteins, we subjected two groups of mice (n = 6/group) to a fasting and refeeding protocol, as described in the Methods. After determining the mRNA levels of CREBH and Insig-2a in the livers of the mice by quantitative real-time PCR, we found that both CREBH and Insig-2a mRNA levels concomitantly rose at the fasting state and fell upon refeeding ( Supplementary Fig. S1A). In contrast, mRNA level of Insig-2b, the other isoform of Insig-2, which is expressed ubiquitously, was not affected by the nutrient switch ( Supplementary Fig. S1A). Consistent with a previous report 22 , Insig-1 mRNA was significantly reduced or induced by fasting and refeeding, respectively ( Supplementary Fig. S1A). Immunoblot analysis further showed that activation of CREBH induced by fasting, shown through the increased expression of the N-terminus of CREBH (CREBH-N), was accompanied by marked upregulation of Insig-2 protein mass, compared to the refed state (Fig. 1A). Reciprocally, protein level of Insig-1 was significantly lower during the fasting state but rose upon refeeding (Fig. 1A). In vitro, mimicking the fasted state by depleting serum from the culture medium (glucose 1 g/ml) of a human hepatoma cell line, HepG2 cells, for 18 hours activated CREBH, indicated by the increased abundance of active CREBH, N-terminal CREBH (CREBH-N) (Fig. 1B). Fasting further stimulated expression of Insig-2a mRNA (Fig. 1C). Refeeding the fasted HepG2 cells with complete medium (FBS 5%; glucose 4.5 g/ml) suppressed CREBH activation and inhibited Insig-2a mRNA expression (Fig. 1B,C). In contrast, refeeding enhanced Insig-1 mRNA expression in HepG2 cells (Fig. 1C). These results suggested the potential regulatory effect of CREBH on Insig-2a.
To further explore the intrinsic physiological association between CREBH and Insig-2a, Insig mRNAs were measured in the liver tissues from CrebH knockout mice (CREBH-KO) and their wild type littermates (WT) under chow-diet conditions. We reasoned that if the mRNA abundance of Insig-2a is regulated by CREBH, depletion of CREBH should be able to prevent the responsive changes of Insig-2a mRNA upon stimulation with fasting and refeeding. Indeed, we found that compared to the WT controls, Insig-2a mRNAs in the livers of the KO mice were expressed at a significantly lower level, which failed to respond to the stimulation of fasting and refeeding (Fig. 1D). In contrast, Insig-1 mRNAs in the KO mice were significantly increased compared to that of the WT controls. This may suggest a compensatory feedback response on this Insig sister protein. mRNAs of Insig-2b remained unchanged in both WT and KO mice (Fig. 1D). Modification of Insig-1 and Insig-2a mRNA transcripts by CREBH depletion were further reflected on the protein expression of hepatic Insig-1 and -2. Immunoblot analysis showed that Insig-1 protein expression was upregulated at both fasted and refed states in the KO mice, whereas Insig-2 protein mass was significantly lower compared to the WT at the fasted state, where CREBH was Scientific RepoRts | 6:32246 | DOI: 10.1038/srep32246 activated ( Fig. 1E and Supplementary Fig. S1B). Expression of Insig-1 mRNA has been shown to be upregulated by insulin 5 , we thus measured the plasma insulin contents and found that plasma insulin levels were significantly elevated in the fasted-KO mice compared to the fasted-WT mice ( Supplementary Fig. S1C). Refeeding stimulated insulin secretion in both WT and KO mice but a higher insulin level was noted in the refed-KO compared to refed-WT mice ( Supplementary Fig. S1C). The regulatory effect of CREBH occurred specifically in the liver as we failed to detect statistically significant changes in Insig-2b mRNAs in the white adipose tissues of both WT and KO mice, while Insig-2a mRNA was undetectable in this tissue ( Supplementary Fig. S1D). This observation is in line with the physiological distribution of both CREBH and Insig-2a, which are selectively and highly expressed in the liver 5,16 . Together, these results indicated that activation of CREBH specifically enhances mRNA and protein expression of Insig-2a but not Insig-2b or Insig-1 in response to nutritional starvation.
Impairment of CREBH and Insig-2a signaling augments hepatic de novo lipogenesis in the fasting state in CREBH-KO mice.
To determine the regulatory role of the CREBH-Insig-2 axis on hepatic lipid metabolism, we first investigated whether CREBH regulates SREBPs on the transcriptional level. Overexpression of a control vector or a cDNA encompassing CREBH wild type (CREBH WT) in McA-RH7777 (McA) cells, a rat hepatoma cell line, was unable to stimulate mRNA transcription of SREBP-1c and SREBP-2 ( Supplementary Fig. S2A), indicating that CREBH is unlikely to regulate SREBPs at the transcriptional level. Since Insigs are the key proteins that sequester the SCAP-SREBP complex in the ER and inhibit SREBP activation 5,22 , we thus investigated the consequence of the defective CREBH and Insig-2a activities on hepatic lipid metabolism by determining the activation of SREBPs and their target enzymes involved in lipid synthesis during the fasting and refeeding states. In the fasting state, although the precursor of SREBP-1c was comparable between WT and KO mice, the abundance of the active SREBP-1c (SREBP-1c-n) was significantly elevated in the fasted-KO mice compared to the fasted-WT littermates ( Fig. 2A), suggesting a post-translational activation of SREBP-1c in the CREBH-KO mice during fasting. More importantly, activation of SREBP-1c in the fasted-KO mice stimulated de novo lipid synthesis signaling in hepatocytes, indicated by the significantly higher mRNAs of the lipogenic enzymes that are involved in free fatty acid synthesis, including fasn, acc and scd-1, in the livers of the fasted-KO mice relative to those in the fasted-WT mice (Fig. 2B). Refeeding stimulated activation of SREBP-1c and SREBP-2 in both groups of mice ( Fig. 2A), which in turn augmented hepatic de novo lipogenesis signaling by significantly enhancing the mRNA transcription of the lipogenic enzymes, fasn, acc and scd-1, as well as hmgcs, hmgcr and ldl-receptor Supplementary Fig. S2B). The biological consequence of hyperactivation of SREBP-1c was further determined by measuring hepatic de novo lipogenesis using 3 H 2 O as tracer by the procedure described in ref. 23. Figure 2C shows that depletion of CREBH significantly increased incorporation of 3 H 2 O into fatty acids in the livers of the fasted-KO mice, suggesting an increased rate of hepatic de novo lipid synthesis (Fig. 3C). Previously, we have showed that a chemical compound, lipoic acid (LA), activates CREBH in vitro and in vivo 24 . To further determine the specific role of the CREBH-Insig-2a axis in regulating hepatic lipid metabolism, we used siRNA against Insig-2 to silence the endogenous Insig-2 in McA cells and activated CREBH by LA. We reasoned that, if CREBH-Insig-2 signaling is critical in regulating hepatic de novo fatty acid synthesis, silencing Insig-2 would disrupt the inhibitory effect of CREBH-Insig-2 signaling on glucose-induced lipogenesis. Indeed, after delivering a control siRNA or a siRNA specifically against Insig-2 into McA cells for 36 hours followed by treating the cells with glucose (6 mM) and LA (200 nM) for 36 hours, we found that mRNAs of fasn and acc were significantly higher in the Insig-2 siRNA-transfected cells compared to the control-siRNA transfected cells (Fig. 2D). Determination of the lipids secreted by McA cells revealed that TG content was significantly higher in the Insig-2 siRNA-transfected cells compared to the controls while CHOL content had no significant change (Fig. 2E). Taken together, these results strongly demonstrated that the CREBH-Insig-2a signaling pathway inhibits hepatic de novo lipid synthesis at the interface of the fasting and refeeding.
Defective CREBH and Insig-2a signaling induces hepatic steatosis and hyperlipidemia in the fasting state. To further assess the outcome of the hyperactivation of hepatic lipogenic pathways, we analyzed hepatic lipid contents with oil red O staining. Figure 3A shows the marked accumulation of lipid droplets in the livers of the fasted-KO mice compared to the fasted-WT mice (Fig. 3A, a, b compared with e, f). Refeeding resulted in accumulation of lipid droplets in the livers of both refed-WT and refed-KO mice (Fig. 3A, c, d and g, h). Quantification of the hepatic lipid contents was done by extracting and determining lipid contents in the mouse liver tissues using the protocol described in the Methods. Consistent with the oil red O staining results, fasting resulted in significant accumulation of TG in the KO liver compared to the WT (Fig. 3B). Refeeding raised hepatic TG in the WT mice but failed to further elevate TG in the KO mice (Fig. 3B). Depletion of CREBH did not significantly alter CHOL contents in the livers of either WT or KO mice at the fasting state (Fig. 3B). In contrast, refeeding significantly raised CHOL contents in livers of WT but not the KO mice (Fig. 3B). Upon further examining the plasma lipid contents, we found that plasma TG was significantly elevated in the fasted-KO, refed-WT and refed-KO mice compared to fasted-WT (Fig. 3C). Depletion of CREBH did not significantly alter plasma CHOL level in the fasted-KO mice compared to the fasted-WT (Fig. 3C). Refeeding markedly elevated plasma CHOL levels in WT but not in the KO mice (Fig. 3C) which was consistent with the liver CHOL contents (Fig. 3B). At a glance, this result is at odds with the hyperactivation of HMG-CoA reductase and HMG-CoA synthase (Supplementary Fig. S2B). However, it has been reported that binding of Insig-1 to the sterol-sensing domain of HMG-CoA reductase accelerates the degradation of this rate-limiting enzyme in cholesterol biosynthesis 25 . Because mRNA and protein levels of Insig-1 is significantly induced in the KO mice ( Fig. 1D and Supplementary Fig. S1), whether or not the decrease of plasma CHOL in the KO mice is caused by the aberrant elevated level of Insig-1 requires further investigation. To investigate whether Insig-2 directly regulates hepatic VLDL-apoB synthesis, McA cells were transfected with a mock empty vector or a cDNA encompassing Insig-2 for 48 hours. Upon examining the secreted VLDL-TG and VLDL-apoB100 (apoB), a key structural apolipoprotein in the VLDL particles, in the culture media, we found that VLDL-TG was significantly lower in the Insig-2 transfected-cell media compared to the mock transfected cell media, whereas VLDL-apoB was comparable between these two treatments ( Fig. 3D and Supplementary Fig. S3). This result suggests that reduced VLDL-TG secretion in the Insig-2 transfected-cells was more likely a consequence of lowered lipid substrates availablity caused by the reduction of hepatic de novo lipid synthesis rather than the suppression of apoB expression. Taken together, these data demonstrated that the CREBH-Insig-2a signaling pathway is critical for maintaining hepatic lipid homeostasis. Compromising the action of CREBH-Insig-2a signaling induces hepatic steatosis and systemic hyperlipidemia in the fasting state.
Insig-2 is a target gene of CREBH which functionally interacts with the CREB-biding elements within the promoter of Insig-2. To further delineate the regulatory mechanism of CREBH on Insig-2a, we (Fig. 4A). Protein mass of Insig-2 but not Insig-1 was also markedly induced in the presence of Flag-CREBH-WT compared to the mock transfection (Fig. 4B). These results indicated that CREBH positively regulates Insig-2a expression on the transcriptional and translational levels.
To further investigate the potential functional association between CREBH and the Insig-2 gene promoter, we analyzed the promoters and transcriptional enhancer regions of Insig-2 in three different species -human, rat and mouse -and identified two potential CRE-BP (− 62) and CREB (+ 99) binding elements that are located proximally to the non-coding exon-1 of human Insig-2a ( Supplementary Fig. S4A). More importantly, these two binding motifs and the gene sequences flanking these two binding sites are evolutionally conserved among human, rat and mouse, which may indicate their biological significances in regulating cellular activities (Supplementary Fig. S4A).
To determine whether CREBH is physically associated with these binding sites, a chromatin immunoprecipitation (ChIP) assay was performed using specific anti-CREBH antibody to immunoprecipitate fragments of genomic DNA from the liver homogenates from fasted and refed mice. The result given in Fig. 4C shows the PCR amplification of a specific DNA fragment that encompassed the binding sites (Fig. 4C, upper panel). Activation of CREBH by fasting induced stronger interaction of CREBH with Insig-2 promoter in the liver of the fasted mice than in the refed mice (Fig. 4C, upper panel). The amplification of this fragment was undetectable in the negative control samples utilizing normal rabbit control IgG for immunoprecipitation (Fig. 4C, upper panel), although this fragment was present in the whole cell lysates (Fig. 4C, lower panel). A negative control primer pair was unable to amplify the DNA fragment immunoprecipitated by anti-CREBH antibody or control IgG (Fig. 4C, middle panel). The interaction of CREBH with the CRE-BP binding motifs within the Insig-2a promoter was further demonstrated in another rodent species, rat, by a ChIP assay as well (Supplementary Fig. S4B). Taken together, these data demonstrated that CREBH functionally associates with the CRE-BP binding elements in the enhancer region of the Insig-2 gene and positively regulates Insig-2 mRNA transcription.
Glucagon inhibits hepatic lipid synthesis via the mediation of CREBH and Insig-2 signaling.
Glucagon is a major metabolic hormone secreted during the fasted state that counters insulin signaling and promotes gluconeogenesis and glycogenolysis to maintain normal glycemia in the fasted state 5,12 . This action is mediated through the activation of a Gs protein, which leads to the stimulation of adenylate cyclase activity, cAMP production, and CREB activation 5,12 . To investigate whether glucagon alone is sufficient to activate CREBH and induce Insig-2a expression, we treated HepG2 cells with glucagon at a concentration of 2 ng/ml for 24 hours. Incubation with glucagon induced activation of CREBH in HepG2 cells, as evidenced by the elevated levels of the active form of CREBH (CREBH-N) (Fig. 5A, upper panel). The activation of CREBH in the glucagon-treated cells was accompanied by increased mRNA and protein expression of Insig-2a (Fig. 5A lower panel and B) but not Insig-1 (Fig. 5A, middle panel), suggesting that glucagon may be involved in the activation of CREBH and Insig-2a during the fasted sate. To further determine the specific role of CREBH in mediating the activation of Insig-2a by glucagon in vivo, we treated the WT and KO mice with glucagon (30 μ g/kg) for a 4 hour time course (Fig. 5C). Glucagon treatment also induced marked reduction of plasma VLDL-TG, indicating the inhibitory effect of glucagon on VLDL secretion (Supplementary Fig. S5A). mRNA expression of apoC4, a direct target gene of CREBH 26 , was also determined in this experiment to serve as a positive indicator of CREBH activation. As shown in Supplementary Fig. 5B, mRNA of apoC4 but not apoC2 or apoA5 was raised about 6 fold upon glucagon treatment compared to the untreated mice ( Supplementary Fig. S5B). Expression of Insig-1 mRNA was not significantly affected by glucagon treatment (Fig. 5C). These data strongly support that Insig-2 is a target gene of CREBH and glucagon activates the CREBH-Insig-2 axis to regulate hepatic lipid metabolism.
Discussion
The present study unveils a novel signaling pathway in hepatic lipid metabolism at the nutrient transition state between fasting and refeeding, which is regulated by glucagon (Fig. 6). This novel pathway involves CREBH and Insig-2a, two proteins that are located proximally to each other on the ER membrane and that are expressed tissue-specifically, mainly in hepatocytes. We demonstrated that activation of CREBH during the fasting state or by glucagon suppresses hepatic de novo lipid synthesis by enhancing the mRNA and protein expression of Insig-2a to hinder the activation of SREBP-1c. This regulatory signaling exerts its greatest effect on hepatic de novo triglyceride synthesis at the fasting state where glucagon is one of the major metabolic hormones. Genetic depletion of CREBH in mice reduces Insig-2a expression, leading to the activation of SREBP-1c and the subsequent activation of its lipogenic target enzymes. Blunting CREBH-Insig-2 signaling by siRNA interference against Insig-2 disrupted the inhibitory effect of this signaling pathway on hepatic de novo lipid synthesis, leading to aberrant accumulation of lipid droplets in hepatocytes and systemic hypertriglyceridemia during the nutritional fasting state (Fig. 6).
Hepatic lipogenesis, in which SREBPs and their target lipogenic enzymes are essential components, is regulated by nutritional status 27 . Insig-1, Insig-2 and SCAP are proteins that are associated with and regulate activation of SREBPs 28,29 . The anti-lipogenic actions of Insig-1 and Insig-2 have been reported in studies where overexpression of recombinant Insig-1 or -2 cDNA in Zucker diabetic fatty (ZDF) (fa/fa) rats substantially attenuated hepatic steatosis and hyperlipidemia 30,31 . Human studies further revealed that Insig-2 promoter polymorphism is an obesity-predisposing genotype that is present in 10% of obese individuals 32 . While these studies suggest that perturbation of Insig-2 protein abundance contributes to the development of obesity, hepatic steatosis and hyperlipidemia, little is known about the underlying mechanism and the cellular regulator(s) of Insig-2. In this study, we identified the first cellular protein, the transcription factor CREBH, as a positive regulator of Insig-2. Furthermore, glucagon activates the CREBH-Insig-2a signaling pathway in the liver. CREBH exerts it regulatory effect on Insig-2 through functionally associated with the CRE-BP binding sites at the enhancing region of Insig-2 and positively regulated the transcription and translation of Insig-2(a). Increased abundance of Insig-2 inhibits activation of SREBP-1c in the liver. Of note, we further noticed that although depletion of CREBH reduces Insig-2a, the expressions of Insig-1 mRNA and protein are significantly above that of the WT controls at both fasted and refed states. We further noticed that the elevated Insig-1 in the KO mice may be associated with the increased insulin secretion in KO mice as plasma insulin contents were significantly higher in these mice. Increased Insig-1 in this context could be a metabolic response in an attempt to compensate for the deficiency of Insig-2 in the KO mice and to hinder the aberrant activation of SREBP-1c. However, despite the compensatory increase of Insig-1, de novo lipid synthesis rate in hepatocytes of KO mice was still significantly higher compared to the WT mice. This finding may indicate that Insig-2a plays a more significant role in regulating lipid metabolism. Moreover, we further noticed that, although the protein abundance of nuclear SREBP-1c were comparable between fasted-KO and refed-KO mice, mRNA expression of the lipogenic genes, fasn and acc, in the refed-KO mice were significantly higher in the refed-KO than that in the fasted-KO mice. The mechanism for this observation is currently unknown. One possibility is the participation of other transcription factor(s) that directly induce mRNA transcription of fasn and acc. For instance, the transcription factor, liver X receptor (LXR), is able to directly interact with the fasn gene promoter and induce its transcription 33 . It is also possible that insulin induced by refeeding modulates the phosphorylation status of nuclear SREBP-1c and enhances its association with the promoter of its target gene to increase transcriptional efficiency [34][35][36] . More research is definitely warranted to delineate the mechanism for this phenotype.
Insulin has been shown to enhance the turnover rate of Insig-2 mRNA, which causes depletion of Insig-2, and the subsequent activation of lipogenic signaling. Glucagon stimulates cAMP formation and cAMP response element binding protein activation and inhibits lipogenic gene expression via the mediation of PPARγ 37 . In this study, for the first time, we demonstrated that glucagon upregulates Insig-2 mRNA and protein expression through the mediation of a new member of the CREB family, CREBH, which in turn suppressed hepatic lipid synthesis. Depletion of CREBH abolished the response of Insig-2 to glucagon treatment. Silencing the expression of Insig-2 by siRNA interference disrupted the inhibitory effect of CREBH-Insig-2a signaling on hepatic de novo lipid synthesis. Taken together, these findings may have clinical implications for therapeutic strategies to use glucagon to activate the cAMP signaling molecule CREBH and increase hepatic Insig-2a abundance in the treatment of hepatic steatosis and hyperlipidemia.
Animal protocols. All animal experiments were approved by the University of Nebraska-Lincoln Institutional Animal Care and Use Committee and were carried out under the institutional guidelines for ethical animal use. Mice used in this study were 10-14 weeks old. WT (C57BL/6J) mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). CREBH knockout mice with exons 4-7 of the CrebH gene deleted were previously described 16 . Animals were housed on alternating 12-hour light and dark cycles with free access to food and water and placed on a chow diet (Dyets Inc., USA). After a week of acclimatization, mice were subjected to fasting for 12 hour or fasting for 12 hour followed by refeeding for 6 hour, n = 3-16/ per group. For glucagon treatment, mice were fasted for 8 hours followed by IP injection with glucagon (30 μ g/kg body weight in 10% gelatine) or 10% gelatine alone. This treatment involved a total of 6 injections over a 4-hour period. Mice were then administrated a final dose (7 th dose) of glucagon via portal vein two minutes before being euthanized. Plasmas and liver tissues were collected and livers were homogenized in solubilization buffer as previously described 38 . Immunoblot analyses. Immunoblotting was performed as previously described 39 . The following antibodies were used in this study: anti-CREBH, anti-Insig-1 and anti-Insig-2 (Santa Cruz, USA); anti-SREBP-1 and anti-SREBP-2 (Novus, USA); anti-Flag (Cell signaling, USA). All antibodies were used at a final concentration of 0.1-1 μ g/ml. After incubation with the appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (1:5000 dilution; GE Healthcare UK), proteins were visualized by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham Biosciences, Pittsburgh, PA, USA).
Plasmids and transfection.
Plasmid pFlag-CREBH WT and pFlag-CREBH-DN were kindly provided by Dr. Randal J. Kaufman (Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan, USA) and were previously described 15 . pCMV-Insig-2-Myc encoding human Insig-2 was kindly provided by Dr. Jin Ye (University of Texas Southwestern Medical Center, Dallas, Texas, USA) and was previously described 40 . For cell transfection, 1.5 μ g of plasmid DNAs were transfected into McA cells as previously described 38 . For siRNA transfection, 100 nmol of siRNA against rat Insig-2 or control siRNA were transfected into McA cells as previous described 38 . 36 hours after the siRNA transfection, cells were treated with glucose (6 mM) and lipoic acid (LA) (200 μ M) for additional 36 hours as described in ref. 24. Cells were collected for total RNA extraction and cell media were used for lipid extraction.
Chromatin Immunoprecipitation assay. 4 × 10 7 cells were subjected to ChIP assay using Simple ChIP TM Enzymatic Chromatin IP Kit (Cell Signaling, USA) and anti-CREBH polyclonal antibodies or normal-rabbit IgG (Santa Cruz, USA). The assay was performed according to the manufacturer's instructions with minor modification to the PCR repeating cycles (36 cycles). The PCR primer sets used for analysis of the mouse Insig-2 Scientific RepoRts | 6:32246 | DOI: 10.1038/srep32246 promoter were ACACCGGAAGTCCTTTTGCC (forward) and AGCTCCTCTTCCCAAAAGCC (reverse), which flank the CRE-BP binding elements in the mouse Insig-2 promoter. Measurement of hepatic de novo lipogenesis. The rate of hepatic de novo lipogenesis was determined by measuring the amount of newly synthesized fatty acids present in the liver 1 hour after intraperitoneal injection of 2 mCi/mouse of 3 H 2 O (PerkinElmer) as described 23 . 3 H-labeled fatty acids were isolated by saponification of liver samples in KOH. After extraction of nonsaponifiable lipids, and acidification with H 2 SO 4 , the 3 H-labeled fatty acids were extracted and separated by thin layer chromatography (TLC). The plate was stained with iodine; the fatty acid "spot" was scraped off the plate, and the isolated fatty acid was added to scintillation fluid and counted in a liquid scintillation counter. De novo lipogenesis was shown as CPM of 3 H 2 O incorporated into fatty acid /h/g liver protein.
Lipid extraction and TG and CHOL mass measurement from cell, tissue and plasma. Liver lipid extraction and analysis were performed as previously described 39 . Briefly, approximately 300 mg of liver tissue was added to 20 volumes of 2:1 chloroform:methanol mixture and incubated for 24 h at room temperature. Following the incubation period, 0.2 volumes of 0.9% NaCl were added to the solvent mixture. The samples were thoroughly vortexed then centrifuged at 2000 rpm for 3 min. The upper aqueous phase was removed and the solvent layer was allowed to evaporate. The dried lipid was resuspended in 1 ml of 100% ethanol. Cell Lipids were extracted using a hexane:isopropanol (3:2) solvent mixture as described in ref. 41. TG and CHOL concentrations were determined using an Enzymatic/GPO endpoint method (Pointe Scientific, Canton, MI) as per the manufacturer's instructions. Lipid data are expressed in milligrams of lipid per gram of liver tissue.
Plasma insulin measurement. Plasmas were collected from mice after 12 hour fasting or 12 hour fasting followed by 6 hour refeeding. Plasma insulin contents were determined using an ultrasensitive mouse insulin ELISA kit (Crystal Chem, IL, USA) as per the manufacturer's instructions.
RNA isolation and qRT-PCR. Total RNA was isolated from tissues and cells using TRIzol (Life Technologies, Grand Island, NY). First strand cDNA was synthesized with oligo (dT) and random primers using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Life Technologies). Q-RT-PCR reactions were carried out using SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies). Relative quantities of mRNA were calculated from threshold cycle (C T ) values with the comparative C T method, using 18S rRNA as an internal reference. Primer sequences are provided in the supplementary materials.
Statistical analyses. Data obtained by densitometry or fluorography were evaluated using one-way ANOVA (GraphPad Prism 5, La Jolla, CA, USA). Post-test analysis was performed to determine the significance between groups, using unpaired two-way Student t-tests. All results are presented as means ± SEM. Asterisks (* or **) indicate statistically significant differences of P < 0.05 or P < 0.01, respectively, compared to controls. | 2016-12-22T08:44:57.161Z | 2016-09-01T00:00:00.000 | {
"year": 2016,
"sha1": "acdf1371718570886cc4e9abc637fb540845bbba",
"oa_license": "CCBY",
"oa_url": "https://www.nature.com/articles/srep32246.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "96246e5e119c3a10d6226d290e40bb40c37a654e",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
266997525 | pes2o/s2orc | v3-fos-license | Upper partial sternal split for pediatric cardiac surgery
Objectives We introduced the use of an upper partial sternal split for pediatric cardiac surgical procedures in our unit in 2016. We report the outcomes of our experience in 51 patients using this approach. Methods From February 2016 to September 2022, 51 patients underwent congenital cardiac surgical procedures using an upper partial sternal split including vascular ring repair (n = 20), subaortic membrane (n = 12), ventricular septal defect closure with aortic valve resuspension (n = 9), aortic arch repair (n = 4), pulmonary artery band (n = 2), pulmonary artery sling (n = 1), supravalvular aortic stenosis (n = 1), aortic valve replacement (n = 1), and pulmonary artery plasty (n = 1). The surgical approach involved a midline skin incision, based on the manubrium, followed by an upper manubriotomy. No special surgical instrumentation was required. Median patient age was 2.9 years (IQR 1.3, 6.0); median body weight was 15 kg (IQR 9.8, 20). Results There was no mortality and no patient required intraoperative conversion to full sternotomy. One patient required re-exploration for bleeding when the incision was converted to a full sternotomy. There were no wound complications in any patient. Twenty-one patients (41%) were extubated on the table and of the remaining 30 patients, 23 patients (76%) were extubated within 24 h of surgery. Eleven patients did not require intensive care unit (ICU) admission. Median ICU and hospital stay was 1 day (IQR 1, 1.25) and 5 days (IQR 4, 8) ,respectively. Conclusion An upper partial sternal split approach is straightforward and can be performed safely with a preferable cosmetic result in selected pediatric cardiac operations. Supplementary Information The online version contains supplementary material available at 10.1007/s11748-023-01996-7.
Introduction
Minimally invasive cardiac surgery (MICS) for adult cardiac surgery has become increasingly popular due to advances in surgical instrumentation, wound retractors, cannulas and cannulation techniques, and myocardial protection.This allows surgeons to perform procedures using videoassisted thoracoscopy or through small incisions under direct vision [1].Reported benefits include shorter hospital stay, less postoperative pain, as well as an improved cosmetic appearance [2].MICS procedures for pediatric patients have been increasingly adopted since the 2000s, especially for managing simple congenital heart defects, such as atrial septal defects (ASD) and ventricular septal defects (VSD) using partial lower sternal splits or thoracotomies [3].However, existing techniques are not well suited for managing aortic arch anomalies and aortic valve lesions, due to the limited exposure of the aortic arch and left ventricular outflow tract and potentially suboptimal surgical outcomes.The upper partial sternal split approach for the management of congenital heart defects was initially reported by Miyaji et al. who described the outcomes of subaortic membrane resection procedures using video-assisted thoracoscopic surgery (VATS) [4].
Our interest in developing this approach was triggered by an increase in the number of requests from parents and patients for smaller skin incisions.Consequently, one surgeon in the unit began using the upper partial sternal split approach at Queensland Children's Hospital since 2016.
The aim of our study was to describe our technique for an upper partial sternal split in congenital heart surgery and report the outcomes of our initial experience with this approach.
Methods
All patients under 18 years old who underwent congenital heart surgery using an upper partial sternal split at the Queensland Children's Hospital from February 2016 to September 2022 were included in this study.Median patient age was 2.9 years (IQR 1.3,6.0)and median body weight was 15 kg (IQR 9.8,20).Operations without the use of cardiopulmonary bypass (CPB) included repair of vascular ring with reimplantation of aberrant subclavian artery and repair of double aortic arch.Those involving the use of CPB included resection of subaortic membrane, repair of distal hypoplastic aortic arch/coarctation, and closure of a subarterial VSD with aortic valve resuspension.The study was deemed a low and negligible risk project and the need for informed consent was waived by our institutional ethics committee.
Upper partial sternal split
Through a manubrium based, limited, midline skin incision (approximately 3-5 cm), an upper manubriotomy is performed with an oscillating sternal saw.We did not need an additional transverse sternal cut at the inferior end of the partial sternotomy.Fixation sutures taken through the lower edges of the divided manubrium are used to stabilize the sternal retractor.A second single blade sternal retractor is placed to widen the surgical field vertically.After a subtotal thymectomy, the innominate vein is dissected and retracted with a silastic tape.We do not use any special surgical instrumentation for this approach.Video 1 demonstrates a subaortic membrane resection through an upper partial sternal split.
Repair of a vascular ring
The commonest types of vascular rings we have operated include right aortic arch with a retro-esophageal aberrant left subclavian artery (SCA), Kommerell diverticulum, and a double aortic arch [5].A pre-operative bronchoscopy is performed on table after induction of anesthesia if the pre-operative CT suggests compression of the trachea and bronchus by the vascular ring.Insertion of a nasogastric tube helps to identify the esophagus and an arterial line is placed in the left upper limb to confirm the patency of the reimplanted aberrant SCA after the procedure.Through an upper partial sternum split, the ascending aorta, proximal descending aorta, and all the neck vessels [including the left and right common carotid arteries (CCA), right subclavian artery and aberrant left SCA] are dissected.The ligamentum arteriosum is divided.aorta.A curved vascular clamp is applied just below the origin of the left SCA, and the distal end is temporary occluded using loosely applied titanium liga clips.The artery is divided, leaving enough length for closure of the aortic origin to avoid stenosis of the descending aorta.The SCA is dissected along its retro-esophageal course.It is then brought anterior to the trachea and esophagus on the left side.A side-biting clamp is placed across the left CCA.Near-infrared spectroscopy (NIRS) monitoring is used to confirm unimpaired brain perfusion.The left SCA is anastomosed to the left CCA in an end-to-side fashion with a running polypropylene suture.Patency of the anastomosis is assessed from pulse waves in the arterial tracing of left upper limb arterial monitoring line.Relief of tracheal compression is assessed by flexible bronchoscopy on table.An aortopexy to the under surface of the manubrium is performed if required.
Cardiopulmonary bypass
After systemic heparinization, CPB is instituted by cannulating the ascending aorta and right atrium with a DLP onepiece arterial cannula and a DLP metal tip single venous cannula (Medtronic, Minnesota, USA), respectively.
Subaortic membrane resection
The patient is maintained at normothermia during CPB.The left heart is vented through the main pulmonary artery.The aorta is cross clamped and cardioplegia is delivered antegrade into the ascending aorta.An oblique aortotomy is made into the non-coronary sinus.Stay sutures are placed above each aortic commissure to bring the aortic valve and the subaortic area into view.A flat malleable retractor is used to retract the valve leaflets.The fibromuscular shelf is resected en bloc and a myectomy is performed below the left half of the right coronary cusp.Video 1 demonstrates subaortic membrane resection through a partial sternal split.
Closure of subarterial ventricular septal defect with aortic valve resuspension
Using a similar approach as for subaortic membrane resection, the VSD is exposed through an oblique aortotomy and closed directly with three pledgeted polypropylene sutures in such a way that the right coronary cusp is resuspended at the same time (Yacoub technique) [6].
Repair of distal hypoplastic aortic arch/coarctation on beating heart
After initiation of CPB, the patient is cooled to 32 °C.The ascending aorta, innominate artery, left CCA, left SCA, descending thoracic aorta, and the ductus arteriosus are dissected.The ductus is ligated and divided.Titanium ligaclips are used to temporarily occlude the left CCA and left SCA.The distal aortic arch beyond the left SCA is ligated with a silk suture and divided.A cross clamp is placed across the descending thoracic aorta.At 32 °C, selective antegrade cerebral perfusion is commenced by maneuvering the arterial cannula into the innominate artery and snaring it.The juxta ductal segment is resected.A cardioplegia cannula is placed in the aortic root.Another cross clamp is placed beyond the cardioplegia cannula, and the heart is perfused with blood.An incision is made on the underside of the proximal aortic arch.The descending thoracic aorta is anastomosed to this opening in an end-toside manner with continuous 6-0 polypropylene sutures.The arch is de-aired through the cardioplegia cannula, and the clamps are released.The set up for this operation is depicted in Fig. 1.
Drain insertion and postoperative pain management
Bilateral intercostal nerve blocks are administered parasternally using injections of levobupivacaine prior to skin incision.A non-luminal channeled drain (Blake® drain, Ethicon, Inc, Somerville, NJ) is introduced from the left side of the incision into the mediastinum and/or pleural cavities (if opened) in every patient.Following the development of a pericardial effusion in a few patients, we modified our drain insertion technique in 2019 to ensure that a loop of the drain drains the retro-cardiac space (Fig. 2).The sternum is approximated with delayed absorbable sutures.After skin closure, On-Q PainBuster soaker catheters (I-Flow Corporation, Lake Forest, CA) are placed bilaterally into the subcutaneous tissues parallel to the sternotomy incision.The elastomeric pump is filled with levobupivacaine and connected to the PainBuster catheters for continuous infusion.Dexmedetomidine, patient-controlled analgesia with morphine or fentanyl, and oral paracetamol are administered postoperatively by a dedicated Acute Pain Service Team.The PainBuster systems are removed 2 days after surgery.The postoperative wound appearance is depicted in Fig. 3.
Twenty-one patients (41%) were extubated on the table and of the remaining 30 patients, 23 patients (76%) were extubated within 24 h of surgery.For these 30 patients who were intubated on transfer to the intensive care unit (ICU), the median ventilation time was 8.5 h (IQR 3, 16).Eleven patients did not require ICU admission.The median ICU and hospital stay was 1 day (IQR 1, 1.25) and 5 days (IQR 4, 8), respectively.
There were no wound complications in any patient and no patient needed intraoperative conversion to a full sternotomy.One patient who underwent aortic valve replacement required re-exploration for bleeding during when the incision was converted to a full sternotomy.Three patients (8%) needed drainage of pericardial effusion out of which two were accomplished using needle pericardiocentesis while one patient needed surgical drainage.After revising the technique of pericardial drain placement in February 2019, no patient has required drainage of a pericardial effusion.
Discussion
MICS techniques in adult cardiac surgery cannot be easily implemented in children as peripheral vessels are often too small to be cannulated.A lower partial sternal split for simple congenital heart defects in children, with or without transmediastinal cannulation, is a well-established approach.
A previous report has demonstrated a return to school and engagement in high-intensity activities such as gymnastics earlier than after a conventional median sternotomy approach [7].In addition, MICS in children can potentially reduce not only the physical trauma related to surgery, but also the emotional stress caused by a large skin scar and potential breast deformities, especially in pubertal girls [8].
A transverse sternal split and thoracotomy approach has been previously reported for pediatric cardiac cases [9,10].However, this method requires peripheral cannulation and concerns remain about distal perfusion of the cervical or femoral vessels.We introduced the upper partial sternal split approach in selected pediatric cardiac operations at QCH in 2016, and gradually extended its application to more complex lesions, including repair of hypoplastic aortic arch.One additional benefit of our approach is that it does not require special instruments or peripheral cannulation, in contrast to other minimally invasive approaches described in children [5,8,9].It is important to note that regarding the exposure of a target treatment zone of the procedures, the surgical view obtained from upper partial sternal split is same as that of conventional full sternotomy.Lower structures of the heart, especially the inferior vena cava, are not easily accessible through the upper partial sternal split approach but these parts are of no particular interest for the patients in whom an upper partial split is utilized.Nonetheless, the upper partial sternal split can easily be converted to a full sternotomy, should hemodynamics intraoperatively deteriorate or if the surgical exposure is suboptimal.A manubriotomy alone also results in improved sternal stability as compared to a full median sternotomy with potentially reduced postoperative patient discomfort.Moreover, upper partial sternal split has an obvious merit from cosmetic perspective with a far shorter incision than median sternotomy.Upper partial sternal split also offers a clear view for both the operator and assistant, in sharp contrast to thoracotomy.This inevitably correlates with the success and safety of the procedure.MICS through a right axillary thoracotomy in pediatric patients can make it difficult for assistants to visualize the detailed cardiac anatomy, which may impair their ability to assist, even during intraoperative complications.In contrast, MICS with UPSS allows the surgeon and assistant to share the same view and respond immediately to intraoperative issues.In addition, MICS with UPSS is well-visualized by assistants during surgery, facilitating instruction of pediatric cardiac fellows during case sharing and making it easier to learn than MICS through a right thoracotomy approach.Although it may initially be challenging, UPSS is a method that anyone can learn, as it is possible to quickly switch to full sternotomy and respond to intraoperative issues.
The only disadvantage is that being a minimally invasive procedure, the upper partial sternal split is definitely technically more challenging, where surgeons need to operate in a limited space with already tight working area in pediatric patients.However, this becomes less challenging and reproducible with experience.
Surgeries for subaortic membrane resection, doubly committed subarterial type VSD, and vascular ring are feasible indications for UPSS if the case does not require re-sternotomy.In the case of aortic arch, UPSS could be considered if the shape and size of aortic arch are suitable for end-to-side anastomosis.In the case of aortic valve stenosis, this method
Vascular ring
Approaches for vascular ring repair include median sternotomy, thoracotomy, and video-assisted thoracoscopic surgery (VATS).Thoracotomy is the preferred approach in many centers [11,12].A good surgical view of the intrathoracic course of the aberrant SCA is achieved through a thoracotomy.However, this method often requires children to be kept on single-lung ventilation during the procedure.In contrast, using a midline approach allows the patient to remain fully ventilated which is beneficial for patients with severe respiratory symptoms [13].Herrin et al. reported their experience of 115 vascular ring repairs using VATS in Boston Children's Hospital with excellent long-term results.The advantages of VATS included shorter operation time and hospital stay compared to thoracotomy [5].Patients who were operated on using VATS were older and heavier (median age and weight: 2.7 years and 14.2 kg) than those operated on through a thoracotomy (median age and weight: 0.9 years and 8.9 kg).In our experience, the upper partial sternal split can be used even in small children.In our series, the youngest patient was 47 days and the lowest weight was 3.1 kg.
With the VATS approach, it can be difficult to reimplant an aberrant SCA to the CCA.No patients underwent reimplantation of SCA via the VATS approach in Herrin's study and this would seem to corroborate our observation.As stated by Backer et al., division of the ligamentum alone is not adequate for the repair of complex vascular rings.Reimplantation avoids potential left upper limb ischemia and subclavian steal syndrome later in life [14].The advantage of our approach is that the resection of Kommerell diverticulum and translocation of left SCA to left CCA can be performed with an excellent surgical view.
Backer et al. also recommended complete resection of the Kommerell diverticulum in cases where it is more than 1.5 times the size of the distal SCA, to avoid compression of the esophagus and trachea as well as reduce the chances of aneurysmal rupture or dissection later in life [12].Furthermore, Luciano et al. found cystic medial necrosis from resected Kommerell diverticulum walls, in at least 50% of specimens, even in very young children.This further reinforces the need to resect the diverticulum during the initial operation [13,15,16].
This technique also provides with the option of performing aortopexy if there is vascular compression of the airway as guided by intraoperative bronchoscopy.
Aortic arch repair with beating heart
To avoid potential tracheal or left pulmonary artery compression, we routinely dissect and mobilize the descending aorta to the level of highest intercostal branches [17].The view of the surgical field is not different, when compared to a conventional median sternotomy, in terms of recognizing structures around the descending aorta, such as the recurrent laryngeal nerve and left phrenic nerve.We keep the heart beating during aortic cross clamping by perfusing the coronary arteries through an aortic root cannula.We prefer to advance the arterial cannula into the innominate artery for selective antegrade cerebral perfusion and have not found it necessary to suture a Gore-Tex shunt to the innominate artery for arterial cannulation.
Cosmetic benefits of upper partial sternal split
A review by Konstantinov et al. on minimally invasive procedures in children emphasized the importance of cosmetically superior incisions, especially in the pediatric population, provided that it does not affect patient safety [18].Observed cosmetic benefits included a smaller incision length, reduced overall wound healing time, and a smaller scar.A study on mini-sternotomy procedures by Vieites et al. also reported improved cosmetic outcomes as well as patient satisfaction, indicating that the perceived benefits certainly manifest in the patients undergoing these operations [19].
The key to ensuring a clear view during surgery is to use chest retractors to spread the wound open in a crisscross pattern, as shown in Fig. 3b.This allows for a wider view without damaging the wound.This makes it possible to apply the UPSS method to the aortic arch repair with end-to-side anastomosis.
Conclusion
An upper partial sternal split approach is straightforward and can be performed safely with good surgical outcomes and a preferable cosmetic result in selected pediatric cardiac operations.
Fig. 1
Fig. 1 Set-up for arch repair without cardioplegic arrest through an upper partial sternal split Fig. 2 Chest X-ray showing loop of non-luminal channeled drain ensuring drainage of retro-cardiac space (black arrow)
Fig. 3
Fig. 3 Wound appearance after upper partial sternal split.a Incision for upper partial sternotomy.b Operative view of subaortic membrane resection.c After skin closure with drain exiting through upper part of wound.d 1-month post-surgery
Table 2
Procedures performed through an upper partial sternal split CPB cardiopulmonary bypass, ICU intensive care unit, IQR interquartile range, VSD ventricular septal defect can be used if the disease is unlikely to require conversion to Ross procedure, Konno procedure, or the root replacement. | 2024-01-17T06:16:53.159Z | 2024-01-16T00:00:00.000 | {
"year": 2024,
"sha1": "31a82f40edb80d9f2c75dd1b29f1add6880387aa",
"oa_license": "CCBY",
"oa_url": "https://link.springer.com/content/pdf/10.1007/s11748-023-01996-7.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "61162dbb89aa9d9ee1dd27e1551e351cef6a88d4",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
216649336 | pes2o/s2orc | v3-fos-license | Reinventing Ethics: Inventing Right and Wrong
I offer new arguments for an unorthodox reading of J. L. Mackie’s Ethics: Inventing Right and Wrong, one on which Mackie does not think all substantive moral claims are false, but allows that a proper subset of them are true. Further, those that are true should be understood in terms of a “hybrid theory”. The proposed reading is one on which Mackie is a conceptual pruner, arguing that we should prune away error-ridden moral claims but hold onto those already free of error. This reading is very different from the standard ones found in the literature. I build on recent work by Moberger and argue that this reading is better corroborated by close attention to the way in which Mackie argues at length that terms like “good” and “ought” are systematically context-sensitive, as well as by considerable additional textual evidence. This reading, however, faces an important challenge—to explain in what sense, if any, morality retains its “normativity” on the proposed reading. I argue that this challenge can be met, at least given some of Mackie’s further assumptions about the nature of rationality.
Michael Ridge
Chapter one of J. L. Mackie's Ethics: Inventing Right and Wrong is part of the "canon" of modern metaethics, appearing on any Introduction to Metaethics syllabus worth its salt. The orthodox reading has Mackie offering a powerful battery of arguments for the "moral error theory", according to which all substantive moral claims are untrue. Ordinary moral judgments, Mackie is taken to argue, commit us to an ontologically "queer" kind of objective value for which a plausible epistemology is problematic. Moreover, Mackie argues, such an objectivist interpretation of our moral practice does not fit well with the kind of widespread moral disagreement within and between communities. A better explanation is that our judgments involve a kind of projective fallacy, whereby in Humean fashion we "gild and stain" the world with our sentiments, taking what are really our subjective reactions to be objective features in the world. These arguments influenced generations of future self-styled error theorists, who typically take Mackie's work as their jumping off point. There is an elephant in the room, though. The rest of Mackie's book does not sit comfortably with the orthodox reading of chapter one. After supposedly having argued that all of morality is bunk, Mackie goes on to defend a number of substantive moral claims. On the standard reading of chapter one, this flagrantly contradicts chapter one. As one commentator put it, it would I do not mean to suggest, of course, that Mackie is the first philosopher to defend (or be taken to defend, anyway) an error theory of this sort. Moral nihilism has a rich and storied history in philosophy, one which I cannot even usefully summarize here. The point remains, though, that contemporary discussions of the error theory take Mackie's work as canonical. For a useful discussion of the broader history, see Olson ( ).
be as if someone argued that "astrology is all the rankest, most hopeless nonsense, only to go on, in Part II, to argue that you can never trust Librans" (Lenman , ). Somewhat scandalously, discussions of Mackie often ignore this apparently glaring contradiction. The few commentators who have discussed the issue have tried to resolve the contradiction in one of two main ways: either by reading Mackie as a "conservationist", who implicitly encourages us to live with the contradiction (cf. Olson , chap. ) or by reading him as a "fictionalist", who encourages us to continue to make moral claims but only as a sort of make-believe. Neither reading is very plausible (Section ).
In my view, a much more credible interpretation has recently been put forward. In particular Victor Moberger has offered an interpretation of Mackie as encouraging us to engage in what I shall here refer to as "conceptual pruning" (Moberger ). This is a form of conceptual reform, but not the standard form associated with so-called "reforming definitions" (cf. Brandt ; Köhler and Ridge ). Rather than offering entirely new definitions for moral terms and recommending them, Moberger reads Mackie as holding that our moral terms already have senses which are entirely error-free. To this extent, Mackie is somewhat surprisingly, not an error theorist-at least not in the standard contemporary sense of "error theory". The problem with our common-sense moral practice, on this reading, is simply that we do not typically use those terms in their error-free sense. Instead, our default is to use moral language in a problematically objective sense. If we simply prune away the error-ridden uses of moral language, then we can get on with moralizing by using moral terms in their error-free, subjective senses. As it happens, the error-free meanings of moral terms are such that they are best understood not only as adverting to subjective values, but in terms of what nowadays is called a "hybrid theory" (cf. Ridge , chap. ; Fletcher and Ridge ). A hybrid theory in the relevant sense is one on which the relevant judgments are partly constituted by non-cognitive attitudes (other hybrid views build the non-cognitive attitude only into the implicatures of speechact, but these views are less similar to Mackie's).
Although this reading is very promising, Moberger's case for it is, in my view, incomplete in two important respects. First, the positive case for this "conceptual pruning" interpretation can be substantially amplified by connecting that reading to Mackie's more extensive semantic treatment of evaluative and deontic terms ("good", "reason", "ought", etc.) as systematically context-sensitive, as well as by noting the textual evidence in sources beyond Ethics: Inventing Right and Wrong and Mackie's own characterization of the works which heavily influenced his metaethical views. Here the influence of Hume on Mackie's thinking cannot be underestimated.
Second, Moberger's reading raises a pressing question: If the only moral truth worth taking seriously is a purely subjective or culturally relative one, then what sorts of reasons do we have for "reinventing" morality? The proposed reinvention is meant to be one undertaken for good reasons-that would be rational, as Mackie sometimes puts it. Are the reasons to embrace Mackie's proposed reform moral, prudential, both or some other kind altogether? Finally, in what sense, if any, are the reasons we have for reforming morality "normative" for Mackie? This last question might seem anachronistic, given that the turn from morality to the more broadly "normative" came into full swing well after Mackie's death. However, Mackie still would have wanted some way of distinguishing the reasons we have for re-inventing morality one way rather than another as being less arbitrary than reasons fixed by some arbitrary principle or convention-"good", the reasons given by some "holy text", the wishes of some tyrant or the reasons laid down by a thieves' code. The suggestion is that it would be most charitable if the kinds of reasons we attribute to him are normative in some recognizable sense; this appeal to the principle of charity does not presuppose that Mackie himself traded in the language of normativity.
Fortunately, both these lacunae in Moberger's argument for the "conceptual pruning" interpretation can be filled, and in this essay I do just that. Here is the plan. First, I briefly explain why rival interpretations (conservationism and fictionalism) are implausible (Section ). I then lay out Moberger's interpretation and his arguments for it (Section ). I then bolster those arguments with additional positive arguments and textual evidence, showing that the conceptual pruning interpretation can be justified much more directly via Mackie's broader semantic theory (Section ). Finally, I turn to the challenge of explaining what sorts of reasons we have for re-inventing morality on this interpretation, and the sense in which those reasons are normative; this leads to a discussion of Mackie's implicit view of practical rationality (Section ). Here again, Hume's influence on Mackie is evident.
Rival Interpretations
In this section, I consider fictionalist and conservationist readings of Mackie in turn. Because the debate over these interpretations is well-worn, I will be brief.
Fictionalism is a revisionary doctrine-a doctrine about how we ought to use moral discourse once we have been convinced that our existing discourse is thoroughly error-ridden. Revisionary fictionalism itself comes in at least two varieties: contentfictionalism and attitude/force fictionalism. According to content fictionalism, moral judgments should be ordinary beliefs with contents about what is true in a given fiction-"the morality fiction", or some such. According to attitude/force fictionalism, moral judgments are not ordinary beliefs, but are some other propositional attitude, perhaps of the sort we characteristically take when engaging with proper fiction (novels, films, etc.) in various ways-something akin to pretence or make-believe. My critique of fictionalist readings of Mackie will not depend much on which of these two versions is attributed to Mackie.
What is the textual evidence in favour of a fictionalist reading? Richard Joyce offers the following evidence: On the very last page of his book Ethics: Inventing Right and Wrong, John Mackie ( ) suggests that moral discourse-which he has argued is deeply error-laden-can continue with the status of a "useful fiction". (Joyce , xx) This is not convincing. First, Mackie's remark is presented in a somewhat offhand way, at the very end of the book, with no elucidation. Second, and much more importantly, what Mackie actually says in this passage is not that we "can continue" with morality as a useful fiction, as Joyce glosses the passage, but rather that "the objectification of moral values and obligations is not only a natural but also a useful fiction" (Mackie , ). Mackie here says nothing about continuing with the relevant fiction, but instead uses the present tense, and is pretty clearly describing our current practice, which he clearly took not to be one which embodies fictionalism-if it did, then ordinary practice would be guilty of no error! When Mackie attributes a "useful fiction" to ordinary practice here, he is not attributing any of the fancy machinery of fictionalism, but is instead using the phrase in one of its vernacular senses, as when one attributes a false belief to someone but allows that the belief, while false, is useful. That Mackie here is discussing our existing practice, rather than any intended reforming fictionalism, is even more clear in that he immediately considers the worry that it might be dangerous "to expose it as a fiction".
It may seem churlish to focus on this single piece of textual evidence at length. I have done so in part because it is one of the few pieces of text used to warrant this reading of Mackie, and this is not surprising. There are very few other passages that could be used to support such a reading. In fact, the word 'fiction' does not appear anywhere else in Ethics: Inventing Right and Wrong. Moreover, when Mackie in another work (The Miracle of Theism) characterizes religion as a fiction, he clearly means that it is a systematically false system of beliefs (Mackie , ), suggesting that he tends to use this idiom as colourful way of expressing an error theory rather than some fancy form of fictionalism. Indeed, in that work Mackie makes fairly dismissive comments about a form of fictionalism in the religious realm, expressing the worry that would plausibly carry over to the moral case, suggesting that "one could not consistently make a big thing of praising and glorifying a god that one at the same time recognized to exist only in one's own mind, or even jointly in the minds of many believers like a figure in a widely current myth or legend" (Mackie , ). One suspects that Mackie would also think it hard to "make a big thing" of moral values one at the same time knows are merely part of a shared myth.
Moreover, there are strong reasons to reject a fictionalist reading. First, it is anachronistic; fictionalist treatments of various forms of discourse was not really "in vogue" when Mackie was writing. There is a risk of reading currently fashionable doctrines back into Mackie's s context. Second, Mackie never explicitly says that he favours a fictionalist approach, nor does he argue for one. Given that the fictionalist proposal is a bold and ambitious one, effectively calling for widespread sociocultural engineering, one would have expected Mackie to have been explicit about this and argued for it more explicitly and carefully. It is not as if moral fictionalism is not open to prima facie powerful objections, objections Mackie was surely sharp enough to anticipate, yet he does not even canvass those objections much less try to refute them. To that extent, the fictionalist interpretation is uncharitable, attributing a clearly inadequately defended view to Mackie. The fact that he only mentions a "useful fiction" in an offhand way on the very last page of the book, and nowhere else, corroborates just how uncharitable this reading is (cf. Kalf ). Third, the fictionalist interpretation does not fit well with the way in which Mackie characterizes the project of (re-)inventing morality. In particular, Mackie repeatedly suggests that we can better refashion our moral practices if we do so without any recourse to the idea of objective values, e.g.: "My hope is that concrete moral issues can be argued out without appeal to any mythical objective values or requirements or obligations or transcendental necessities" (Mackie , ). Mackie makes similar remarks in Hume's Moral Theory: What, as Hume saw, holds for the duty of allegiance holds also for morality as a whole. We are more likely to get its benefits without its disadvantages if we see through its claim to absolute or objective authority.
(Mackie , ) If Mackie were a fictionalist, then he would instead explain how we can refashion our moral practices around a self-consciously fictional conception of objective values-in effect, making up new "stories" about these mythical objective values. Whereas what Mackie actually suggests, repeatedly, is that we can better reinvent our moral practices by doing without any appeal to objective values, which presumably includes any in a fictional mode.
What, then, about a conservationist reading of Mackie? What evidence is there that Mackie intended for us to continue using a mode of thought and discourse riddled with error? One argument for this reading, offered by Caroline West, is that conservationism is the best explanation of Mackie's thesis that first-order and second-order questions are completely independent: Mackie himself seemed to take this "business as usual" view . . . First-order and second-order moral questions, he says, "are not merely distinct, but completely independent". (West , -) The idea seems to be that Mackie's second-order error theory does not entail that we ought, all things considered, quit making moral judgments. Since he does, himself, happily go on to advance numerous moral judgments in the second half of the book, the simplest explanation of this presumably is that he thinks we have good reasons to go on making moral judgments even while knowing at some level they are all untrue.
Of course, to do this while accepting the error theory we would have to learn to live with a contradiction. Perhaps this is possible, though. Jonas Olson, who also defends a conservationist reading of Mackie, argues that we can manage to do this through a kind of compartmentalization, effectively believing the error theory "in the seminar room" but making positive moral judgments in other settings (Olson , -). Moreover, whether this sort of compartmentalization is in fact possible, Mackie seems himself, in the discussion of religious belief, to allow that it is (cf. Mackie , -). Even if it is psychologically possible (on Mackie's view) to believe flagrantly contradictory things in this way, it must be admitted that it is epistemologically unhealthy and weird. This alone should create at least a presumptive case against this reading. More to the point, conservationism fits very poorly with Mackie's characterizing the project of Ethics: Inventing Right and Wrong being, in part, one of "conceptual reform". Mackie first made this observation in his earlier book, Problems From Locke, suggesting that we need conceptual reform both in the case of personal identity (his topic there) and ethics: A fairly plausible suggestion is that we should adopt the revised Lockean account, openly admitting that it is not a correct analysis of our present concept, but proposing it as a conceptual reform and as a factual analysis, an account of all that is true and relevant in this area. [Footnote :] A similar conceptual reform, rather than mere analysis of our present concepts is, I believe, needed in ethics. I hope to discuss this topic in another book. (Mackie , ) It is pretty clear that this other book was to be Ethics: Inventing Right and Wrong. Moreover, Mackie's main aim in the second half of Ethics: Inventing Right and Wrong was to (re)-invent morality A point I owe to an anonymous referee footnoted by Victor Moberger in his discussion (see Moberger , n ). Mackie's view here is quite different from the one defended by Bart Streumer, also an error theorist; see Streumer ( ).
(cf. Moberger
, -). For Mackie, it is important that we "remake" morality: [M]orality is not to be discovered but to be made; we cannot brush this aside by adding 'but it has been made already, long ago'. It may well need to be in part remade.
(Mackie , ) Remaking morality does not sound much like "business as usual". The conservationist might reply that Mackie's aim is to remake morality in terms of its first-order content, and doing that is compatible with "business as usual" in the sense of continuing to use moral language in a way that encodes a false presupposition of objective values. Indeed, in the sentences just following the quotation above, Mackie argues that while the duty of fidelity is worth preserving, the virtue of patriotism may have "outlived its usefulness". A much more plausible interpretation is that Mackie is inviting us to reject the error-ridden moral concepts which presuppose objective values and to deploy some other moral concepts. It is with those error-free concepts that we should reinvent morality, developing new first-order moral views which are informed by our rejection of objective value. Just what these error-free moral concepts are, though, is a very good question, a question to which I turn in the next section.
Conceptual Pruning and Hybrid Theory: Moberger's Reading
Given that Mackie is in the business of conceptual reform, what sort of reform does he advocate? On some ways of understanding concepts, they are abstract entities, not capable of change, so talk of conceptual reform is perhaps best not taken literally. It is therefore often associated instead with changing the meanings of terms, so that they come to express new concepts. The most common approach to conceptual reform (or "conceptual engineering", as it is sometimes called) takes the form of offering a "reforming definition". Here one takes some vexed philosophical term, like "free will", and argues that the ordinary concept it expresses is defective in some way. One then offers a new "reforming" definition for the corresponding term, arguing that the new definition allows the discourse to do the job it was in some sense "meant to do" but without the problems associated with its original meaning. A standard worry about such approaches is that they simply change the subject. Interestingly, this is not the only project worthy of the name "conceptual reform". Another approach might instead be called "conceptual pruning". Here one takes some vexed ordinary language term, and argues that for at least a wide (perhaps the predominant) range of meanings, the term is defective in some way. One then argues that the term is, however, ambiguous in some way, and that in some of its ordinary senses, it is not defective. One then argues that we should "prune away" the defective uses, and shift over to using the term more consistently in its error-free way. One advantage of this approach is that the worry that one has simply "changed the subject" does not arise; no new definitions are being proposed "from the armchair".
It might be useful to consider a more down to earth example. Consider the term "luck". It is sometimes used to refer to a purported "projectable" property, such that some people are lucky and if they are lucky then they not only have a track record of doing better than average or than you would expect given their abilities, you can also base predictions about their likelihood to do well, win bets, etc., in the future on their luckiness. On this view, luckiness is a robust property whose instantiation increases the odds of success for the lucky person. In this sense of "luck" we should be error theorists about luck. In another, not completely unrelated, sense of "luck", though, someone is lucky with respect to some domain if and only if they did better Cf. Cappelen ( ), Scharp ( ). The phrase "conceptual engineering" stems from Blackburn ( ).
than one should have rationally predicted ex ante, taking into account their abilities, etc. In this sense, someone who gets dealt great hands in poker over and over is lucky even if that in no way grounds any predictions about what cards they will be dealt in the future. Here we should prune away the error-ridden (first) sense of "lucky" but retain the second, naturalistically acceptable sense of the word. Although he does not use the label "conceptual pruning", this is basically the interpretation of Mackie offered by Victor Moberger in a recent paper (Moberger ). In this section, I summarize the main lines of his interpretation and his arguments for it.
Moberger argues that Mackie is a "semantic pluralist" about moral discourse. On his reading, Mackie holds that there are "two different strands of moral discourse", about which two different stories should be told. The first strand presupposes objective values and is therefore error-ridden. This is, for Mackie, by far the most pervasive form of moral discourse. The second strand, though, does not presuppose objective values and is error-free. On this interpretation, it is misleading to call Mackie an error theorist in the modern idiom, since that is usually defined in unqualified, universal terms-as holding that all substantive [putting to one side tautologies] moral claims are untrue. On Moberger's reading, Mackie is an error theorist only in a slightly weaker sense; he holds that most forms of ordinary moral discourse are error-ridden. Moberger usefully compares Mackie's views to the kind of pluralism defended by Gill ( ), according to which ordinary moral discourse is not as semantically uniform as much metaethical theorizing seems to suppose.
How does Moberger argue for this somewhat surprising interpretation? He begins with some textual evidence, pointing out that Mackie very consistently qualifies his claims about ordinary moral discourse by saying only, e.g., that "many" or " Thanks to Guy Fletcher for suggesting this nice example. in the main" or that "ordinarily" moral judgments presuppose objectivity, citing these passages [emphasis added]: [It] can plausibly be maintained at least that many moral judgments contain a categorically imperative element . . . (Mackie , ) [M]ost people in making moral judgments implicitly claim, among other things, to be pointing to something objectively prescriptive.
(Mackie , ) [ As Moberger points out, it would be very odd for Mackie so consistently to qualify his attribution of error to folk moral discourse if he did not think that at least some of our moral judgments do not presuppose objective value. A second piece of evidence Moberger offers for Mackie's semantic pluralism is the way in which he formulates his ontological claim. Mackie never says there are no moral values, only that the there are no objective values (and so ipso facto no objective moral values). Moreover, he sometimes formulates his positive view as "moral subjectivism", and entitles the first chapter of his book "The Subjectivity of Values", strongly suggesting that he thinks there are subjective moral values.
A third reason Moberger offers for reading Mackie as a semantic pluralist is that it allows us to read his conceptual reform programme in the second half of the book as a form of what I am calling "conceptual pruning". Given that at least some of our actual moral discourse is amenable to a subjectivist reading, we can in principle just abandon the objectivist strands of moral discourse and switch over to more consistently speaking in a subjectivist idiom. This is an advantage of reading Mackie as a semantic pluralist insofar as it can explain why the second half of Mackie's book does not contradict the first half better than rival interpretations.
Fourthly, Moberger argues that reading Mackie as a semantic pluralist helps vindicate his otherwise confusing claims in chapter one about how second-order and first-order claims are entirely independent of one another. On the standard reading of Mackie as an error-theorist, this is an odd claim, since it seems like his second-order view logically contradicts our first-order moral claims, since it asserts that they are all untrue. Whereas on Moberger's reading, independence makes sense; so long as we stick to purely subjective construals of our first-order claims, Mackie's error theory is logically compatible with whatever firstorder view one likes.
Actually, this line of argument is complicated by a remark Mackie makes in a paper only published posthumously, in a volume edited by Joan and Penelope Mackie. In "Bootstraps Enterprises", he clarifies his claims about neutrality as follows: Can what I say here be reconciled with what I say in my book (p. ) about the complete independence of first and second order views? Yes, because the first-order views referred to there were construed more widely than the views internal to a bootstraps enterprise . . . The first order moral views with which any second order view is compatible are identified simply as approval of and support for certain things and condemnation of others.
(Mackie b, ) Taken at face value, this passage suggests that Mackie had in mind an expressivist reading of moral discourse when allowing for the compatibility of his second-order view with any first-order view one likes. It is only on an expressivist view that moral views are literally identical with one's attitudes of approval/support/condemnation, as Mackie suggests here. By contrast, on the hybrid subjectivist view Mackie also thinks applies to ordinary language moral judgments are only partly con-stituted by such attitudes. They are also partly constituted by representational beliefs about the requirements of contextually specified moral institutions. I do not think this spoils the overarching case for Moberger's reading. At most, it shows that he cannot appeal to Mackie's remarks about neutrality as evidence for his reading. But there is ample evidence independently of that. My own view is that the evidence for the subjectivist reading is on the whole much stronger than the slender evidence for an expressivist reading supplied by this one sentence in a reply to Dworkin that Mackie didn't himself properly polish/work up for publication and which therefore may not represent his considered view. Further, Mackie's remark here is also compatible with my reading and Moberger's, in that Mackie might have allowed that 'morally good' (etc.) admits of both a subjectivist and an expressivist reading in ordinary language already, albeit these uses are very rare compared to the error-infected ones. He could then be read as pruning away the error-theoretic ones but keeping both of these. In fact, this could help with the puzzle about "normativity" I discuss in Section below. However, I am not sure whether to attribute this expressivist view to Mackie to help resolve that problem given how limited the evidence is for that reading. Readers who are more tempted by that reading will find it even easier to resolve the problem discussed in Section .
Given that, on the whole, there seems to be a reasonable case for reading Mackie as a semantic pluralist and a conceptual pruner, an obvious question is what sense should be attached to our moral claims in their subjectivist idiom? On Moberger's reading, Mackie takes subjective moral judgments to be about the requirements (etc.) of some contextually specified moral institution, where typically the relevant institution is one the speaker occupies. Institutions are, for Mackie, social practices in which participants conform to certain patterns of behaviour and deploy socially enforced sanctions on those who deviate. Mackie offers many examples of non-moral institutions, such as chess, and ar-gues that morality is an institution which functions to help us counteract our limited sympathy (Mackie , ). Mackie distinguishes between speaking about an institution "from the outside", in the idiom of a sociologist commenting on its requirements, on the one hand, and speaking "within the institution", on the other. When we speak within a given institution in Mackie's sense, our claims are not purely descriptive (as in the external mode), but instead are infused with evaluative/prescriptive force. Mackie illustrates this distinction by arguing that Searle's famous attempt to derive an "ought" from an "is" fails because Searle conflates these two modes of thought/discourse with respect to the institution of promising (Mackie , -). Moberger reads Mackie as taking what in modern terminology would be a "hybrid" theory of moral claims made within the institution. Hybrid theories of moral discourse, in the relevant sense, hold that the relevant moral judgments are constituted not merely by representational beliefs (here, about one's moral institution, say) but also by associated non-cognitive attitudesattitudes of endorsement of the behaviour required by the institution, in favour of sanctioning those who deviate from those norms, etc. Moberger offers considerable and in my view convincing textual evidence for this reading. Mackie was in this sense "ahead of his time", since hybrid theories were not much discussed when he was writing, but have become something of a cottage industry more recently.
Finally, Moberger notes a further subtlety. Mackie does not think ordinary discourse sharply distinguishes the subjective and the objective elements. Rather, he takes typical moral claims to be composites of the subjective (and hybrid theoretical) and the objective. Typical moral claims are made from within a moral institution, both describe and endorse that institution's demands For some discussion of the variety of modern hybrid theories, see Fletcher and Ridge ( ). See also Ridge ( , chap. ). and claim (or perhaps presuppose) that those demands also have objective validity. Having distinguished adverting to objective requirements and speaking within a moral institution, Mackie adds that these elements "do not normally occur in isolation, and views which single out any one of them as the meaning of moral terms are implausible" (Mackie , ). So far, so good. Moberger's interpretation is a huge step forward, and sheds great light on what is going on in the transition from chapter of Mackie's book to the second half of the book. However, there is more work to be done. First, although Moberger makes a strong case for his reading, the positive case can be made much stronger and more direct, as I explain in the following section. Second, the conceptual pruner/hybrid theory reading of Mackie leads naturally into the question of what sorts of reasons Mackie is offering for refashioning morality in one way rather than another. I turn to these remaining tasks in the next two sections.
Strengthening the Positive Case: Mackie's Contextualist Semantics
Moberger's case for reading Mackie as a semantic pluralist rests primarily on ( ) the ways in which Mackie consistently qualifies his attribution of error to ordinary moral thought/discourse and his formulation of the main ontological claim and ( ) the explanatory power such a reading provides when it comes to understanding Mackie's broader project. These arguments are well-taken, but they are also somewhat indirect. As it happens, a much more direct and stronger case can be made for reading Mackie as a semantic pluralist if we also attend to his positive semantic views about evaluative and deontic language more generally. Moreover, attending to these aspects of Mackie's view reveal that it is a slight oversimplification to speak of "two strands" of moral judgment. Mackie spends two chapters (chaps. and ) laying out a se-mantics for "good", "ought", and "reason". In all three cases, the meaning of the term should be understood in terms of some contextually specified requirement(s). To say something is good is to say it is "such as to satisfy requirements (etc.) of the kind in question" (Mackie , -). To say one ought or must do something is (roughly; I gloss over Mackie's distinction between "ought" and "must") to say that there is some contextually specified requirement that one do it. To say there is a reason to do something is (roughly) to say that doing it would tend to lead to the satisfaction of some contextually specified requirement. Requirements can be provided by desires or interests, but also by institutions (Mackie , -). Mackie thinks that in moral contexts speakers typically have in mind the elusive idea of requirements that are "just there", or "part of the fabric of the universe", but our interest is in uses that avoid those errors.
Mackie's semantics predicts that claims about what is good, what one ought to do, and what there is reason to do are relative to contextually specified requirements, and these requirements can be provided by institutions. Mackie also very clearly thinks of morality as a kind of institution (Mackie , ). These commitments together entail that a speaker can felicitously use moral language to make claims about what a contextually specified moral institution requires. Indeed, this is no surprise, since we already saw that Mackie thinks most ordinary moral claims do advert to such institutions. However, he also thinks most ordinary claims introduce a presupposition that those requirements also track what is objectively good.
Even if most uses of moral language carry this presupposition of objective value, the semantic theory predicts that it should be at least possible for a speaker to use, e.g., "morally ought" to advert to the requirements of a moral institution without introducing any presupposition of objective value. Mackie is quite explicit that nothing compels us to add a presupposition of objective validity to our claims about institutional requirements, presumably including moral institutions (indeed, Mackie in the following passage is discussing our reasons to alleviate the suffering of others, and so has moral reasons in view): [C]ertainly nothing compels us to reinterpret the requirements of an institution, however well established, however thoroughly enshrined in our ordinary ways of thinking and speaking, as objective, intrinsic, requirements of the nature of things.
(Mackie , -) That this possibility is already present, if merely latent, in our discourse, would already be enough for the "conceptual pruning" approach to work, since Mackie could be urging us to drop the problematic uses of moral language and start using moral language in ways already available to us, without any need for a "reforming definition". So, semantic pluralism can be argued for much more directly than the more indirect argument found in Moberger. Note, moreover, that the kind of systematic context-sensitivity for moral terms found in Mackie's theory is not well understood in terms of there being simply "two strands" of moral discourse, one objective and one subjective, as Moberger suggests. At least, this oversimplifies dramatically. Claims about what is morally good, e.g., can be relative to the moral institutions of one's society, some other society, a merely possible society, one's own personal standards, the institutions implicit in some sacred text, and so on. Any of these moral claims can, in turn, be combined with a presupposition that the relevant institutions track objective value or not. We can in principle make claims about what requirements there are "in the fabric of the universe", bringing in objective value without any reference to institutions. Finally, Mackie also draws a distinction Mackie between morality in the broad sense, which is a fully general theory of conduct, and morality in the narrow sense, which functions more specifically to help us overcome our limited sympathies.
Of course, the conceptual pruning interpretation would be bolstered if it could be shown that Mackie thought the possibility of purely subjective uses of moral language were not merely latent in the semantics for moral vocabulary, but that we actually already use moral discourse in this way sometimes. Here the textual evidence mobilized by Moberger is very much to the point. The fact that Mackie so consistently hedges his formulation of the error theory with locutions like "many moral judgments", what is "ordinarily" claimed, what "most people" mean to claim, and so on, strongly suggests that Mackie thinks that people do sometimes use moral language in a purely subjective way. Indeed, Mackie seems to explicitly endorse this in his discussion of "justice", which he says in one sense requires only fidelity to the relevant standards, and no queer value entities: [T]here is an objective distinction which applies in many such fields, and yet would itself be regarded as a peculiarly moral one: the distinction between justice and injustice. In one important sense of the word, it is a paradigm case of an injustice if a court declares someone to be guilty of an offence of which it knows him to be innocent. More generally, a finding is unjust if it is at variance with what the relevant law and the facts together require . . . justice or injustice of decisions relative to standards can be a thoroughly objective matter.
(Mackie , ; emphasis added) When Mackie says this is an objective matter, he does not mean it involves objective value. Rather, he means that the justice or injustice of a decision can be derived in an objectively valid way from the relevant descriptive facts. Here Mackie clearly countenances error-free uses of "justice". In fact, there is even more textual evidence than Moberger provides for his proposed reading, some of it arising in the chapters in which Mackie offers his broader semantic theory. For example, in his discussion of the semantics of "good", Mackie allows that we sometimes use "good" to engage in what he calls "egocentric commendation", where this "should, perhaps, be called not purely descriptive, since an essential element in it is the speaker's implicit endorsing of the requirements . . . which the thing commended is being said to be such as to satisfy. But it is partly descriptive, in that it claims both that the thing has the intrinsic characteristics, whatever they are, and (hence) that it bears this relation to those requirements" (Mackie , ). The most straightforward reading of this is that "good" is sometimes used to make descriptive claims about what satisfies certain requirements and at the same time express approval of those requirements and commending the object of evaluation.
We are also told in Hume's Moral Theory that a "mixed account" could provide a more plausible conceptual reform than either pure descriptivism or pure expressivism (Mackie , ), and Mackie in that passage footnotes his own Ethics: Inventing Right and Wrong, suggesting this is his own view. Further, Mackie elsewhere endorses such a hybrid view of legal terms and talk of responsibility: [L]egal terms and terms like 'responsibility' contain a prescriptive element, which must be distinguished from their descriptive element, and also if we say that in certain circumstances it is correct for a judge to give such-and-such a decision, we are ourselves making or quoting a further, higher-level, prescription: we are prescribing when the judge is to prescribe.
(Mackie , ) Even more clearly, Mackie explicitly endorses a hybrid theory of aesthetic value judgments, and in the same breath indicates that this approach has been "worked out" in the case of ethics: Perhaps the pure subjectivist theory, and the pure objectivist one, have both gone wrong in the same way. Each of them has assumed that an aesthetic judgment is made in one move. But again, we can usefully borrow an account of value judgments which has been worked out and discussed particularly with regards to ethics, the core of which is that a value judgment involves at least two moves . . . in making an evaluative judgment we are at once saying or hinting that the object judged has certain natural characteristics, and commending it, or perhaps condemning it, on that account. . . (Mackie , ) In addition to all of this direct textual evidence from other sources, the proposed reading of Mackie fits well with the figures Mackie says most profoundly influenced his views in ethics: From the modern period, Stevenson, Ayer, and Westermarck, and more historically the "British moralists", but most especially David Hume, whose moral philosophy Mackie wrote an entire book about (Mackie , ). Stevenson can plausibly be read as a kind of hybrid theorist, holding that moral claims state that the speaker approves of something and then includes a "do so as well!" element, which expresses the speaker's approval (cf. Stevenson ). Ayer famously emphasizes the use of moral language to express as well as report one's attitudes.
Furthermore, there are many other passages in which Mackie's claims about our moral concepts being infected with error are hedged to include only "typical" or "most" uses. For example, in addition to the passages mentioned by Moberger, we find the remark that "typical moral statements do not seem to mean what any dispositionally descriptive account . . . says that they mean" (Mackie , ), where this is because they typically involve a presupposition of objective value. Again, we are told that the error theory goes against assumptions "built into some of the ways in which language is used" (Mackie , ). Mackie also contrasts European ethical concepts with the "ethical concepts of Plato and Aristotle" (Mackie , ), further corroborating his commitment to semantic pluralism.
One piece of text which might seem to count against Moberger's reading is Mackie's seemingly unqualified claim that in moral contexts "good" "is used as if it were the name of a supposed non-natural quality" (Mackie , ). Even here, though, Mackie qualifies this claim, actually saying only that it "would not be so far wrong" to say that "good" has this meaning in moral contexts. The inclusion of this caveat makes this passage compatible with Moberger's reading, since the claim could be partly wrong because some moral contexts are not such that "good" is used in this objective sense. Similarly, the suggestion that objectivism "has a basis . . . in the meanings of moral terms" (Mackie , ) is compatible with some strands of moral discourse not involving this commitment.
Another reading of this passage consistent with the spirit of Moberger's One worry about this reading of Mackie is how it squares with his advocacy of "remaking" morality. One tempting reading of Mackie's talk of "remaking" morality is as calling for replacing our existing moral concepts with new ones, where these new ones have a different content-a content suitably informed by our rejection of objective value. Indeed, it seems clear that Mackie's remaking project essentially involves changing the content of morality in a deliberate way, and it might seem obscure how the content could change if we retain the same concepts. Moberger himself has very little to say about how this remaking should go on his account.
This objection can be found in Wouter Kalf's critique of Moberger. Kalf offers the following objection: [Moberger's] interpretation fails to mention that after latching on to morality in the narrow sense, we should change the content of reading would be that the commitment to objective value is built into the meanings of moral terms in the form of a conventional implicature a la Grice. Conventional implicatures are part of conventional meaning and cannot be cancelled (unlike their conversational cousins), but they are also not part of the literal content. On this reading, the conceptual pruning would simply be to drop the conventional implicature but keep the strict, literal semantic content. This would be like no longer using 'but', but only using 'and' as one's conjunctive connective, e.g., I prefer Moberger's reading simply because the conventional implicature reading would predict that all uses of moral language implicate objective value, whereas Mackie very carefully and repeatedly says only that "typical", "most", etc. uses do. It is, though, worth noting that if someone thought we should dismiss those passages for some reason and put more weight on the one or two passages in which Mackie makes claims about what is built into conventional meanings, then a reading very much like Moberger's, in spirit, would remain available in the form of a conventional implicature approach. Generalized conversational implicature is yet another view one could explore here, though that would predict that only contexts in which the implicature of objective value is explicitly cancelled are exceptions to Mackie's generalization. I do not think there is adequate textual evidence for that reading, but it is more promising than the conventional implicature approach (cf. Fletcher and Ridge ). Both of these readings would have the very surprising upshot that even moral claims which do encode an assumption of objective value need not be untrue, since implicatures are distinct from literal, truth-conditional content. morality in light of changes in the circumstances of justice, and that we should change morality's content by replacing our concept of the good 'with some other concept of the good' ( ). Moberger's interpretation is therefore . . . incomplete in this crucial respect because it fails to do justice to Mackie's remark that after the discovery that we should be error theorists, we should also change the content of our new schmoral discourse. (Kalf , ) One strand of Kalf's objection here is textual, citing Mackie's apparent reference to the need to replace our concept of good with some other concept. However, Kalf takes this passage out of context. In the quoted passage (on p. ), Mackie is clearly talking about the utilitarian's concept of "the good" rather than our own concept, and he is floating an option he clearly doesn't endorse, namely keeping the consequentialist conception of right action but tweaking the associated value theory. This clearly provides no support for reading Mackie as supporting a reforming definition for our own moral concepts.
Still, the core of Kalf's worry might be independent of this appeal to the text. Clearly, Mackie thinks we need to change the foundational content of morality. It might seem obscure how he could do that without providing us with new concepts. Same concepts, same truth-conditions, one might worry. More formally: ( ) Mackie's proposal is that we change the truth-conditional content of our most basic moral judgments.
( ) The only way to change the truth-conditional content of our most basic moral judgments is by replacing our existing moral concepts with new ones [same concepts, same basic truth-conditions, since concepts fix truth-conditions].
( ) Therefore, Mackie's proposal requires that we replace our existing moral concepts with new ones.
The problem with this argument should be apparent once it is stated, so long as we remember the content of the moral judg-ments which survive Mackie's "pruning". Those moral judgments will characteristically be about the moral institutions of the speaker's community. Institutions, though, are human inventions in a broad sense-patterns of behaviour, sanctions. As such, they can themselves be reinvented. We can thus change the truth-conditional content of our most basic moral judgment by changing the relevant institutions. For example, Mackie thinks patriotism as a virtue may be past its "sell by" date. Taking his advice, we can change our moral institutions so that they no longer encourage patriotism and make "patriotism is a virtue" (which we may take as a basic moral truth-one not derived from within our institutions from some more basic norm) go from true to false. We can, in this way, change the truth-conditional content of our most basic moral judgments by changing our institutions and without replacing our existing moral concepts with new ones. By way of analogy, we can change the content of 'legally permissible now in Scotland' by changing the law, rather than by changing our concept of the law. Premise ( ) in the argument above is thus false.
Although Kalf's objection can be dispatched easily enough, it suggests another challenge for Moberger's interpretation, and this challenge is not so easily met. Moreover, by seeing how to meet this challenge we can fill an important lacuna in the proposed interpretation. I turn to this challenge and how to meet it in the final section.
Why Remake Morality? (And in What Sense of 'Morality'?)
Mackie's proposal that we remake morality is not presented as an arbitrary suggestion, something we can take or leave as we like. Rather, he presents it as something we have strong reason to do. But what sorts of reasons is Mackie offering? Talk of reasons is, for Mackie, always relative to contextually specified requirements. Requirements, in turn, can be derived from institutions, desires, the law, conventions and other sources. What, then, is the source of the requirements which ground our reasons to remake morality and why do they matter? A plausible constraint on Mackie's argument for remaking morality is that the reasons he offers are ones which in some sense are "normative". Of course, 'normative' is a term of art, open to multiple interpretations, and the turn from morality to the normative in metaethics came decades after Mackie was writing. There is, therefore, a risk of anachronism in formulating the challenge in this way. Indeed, T. M. Scanlon specifically reference's Ethics: Inventing Right and Wrong when remarking on the shift in metaethics as a field from focusing on morality in particular to the normative: Contemporary metaethics differs in two important ways from the metaethics of the s and s and even the later s, when John Mackie wrote Ethics: Inventing Right and Wrong. In that earlier period, discussion in metaethics focused almost entirely on morality . . . Today . . . a significant part of the debate concerns practical reasoning and normativity more generally . . . (Scanlon , ) However, this concern about anachronism notwithstanding, it also seems clear that Mackie thought the reasons he offered were not chosen at random. For example, suppose Mackie had offered reasons derived from the requirements of the "Vory v Zakone", or "thieves' code", loosely translated from the Russian-a code of conduct governing Russian organized crime. This would rightly strike us as bizarre and philosophically uninteresting. Given Mackie's semantics for 'reason', though, there will trivially be true claims about what reasons there are relative to the Vory v Zakone. Clearly, Mackie thinks the reasons he offers us to remake morality have more of a "grip" on us in some intuitive sense-that they matter-than the reasons grounded by the requirements of the Vory v Zakone. A plausible constraint on Mackie's project, then, is to explain why the reasons he offers are privileged in some way that, e.g., the reasons grounded by the Vory v Zakone are not.
It is important to see that Mackie's project taken on its own terms requires some specification of the kinds of reasons on offer here; this puts the worry about anachronism in the right light. Crucially, on Mackie's contextualist semantics, any use of 'reason', including his own use of 'reason' when claiming we have reason to reinvent morality, is indexed to some contextually specified requirement or set of requirements. So we need some account of which requirements we are meant to have in mind when Mackie tells us we have good reason to remake morality. My suggestion is that whatever requirements we do read Mackie as implicitly having in mind, it would be more plausible, and hence more charitable if those requirements were plausibly seen as normative. That interpretative argument crucially does not require that Mackie himself was implicitly thinking in terms of a normative/non-normative distinction. Rather, it requires only that the interpreter be guided in their reading by the idea that the requirement(s) Mackie did have in mind are ones we would classify as normative, as that would track with their not seeming, e.g., as arbitrary as the Vory v Zakone requirements. Lest we read Mackie as having in mind some totally arbitrary set of requirements when he tells us we have reason to reform morality, which seems highly implausible, this constraint seems like a good way to ensure that our interpretation is charitable.
Before trying to see what kind of reasons Mackie might be offering for remaking morality, we should be clear about in what sense of "morality", he thinks it needs to be remade. Recall that Mackie distinguishes morality in the broad sense from morality in the narrow sense. A morality in the broad sense "would be a general, all-inclusive theory of conduct", while morality in the narrow sense "is a system...of constraints on conduct . . . whose central task is to protect the interests of persons other than the agent and which presents themselves to an agent as checks on his natural inclinations" (Mackie , ). Interestingly, morality in the broad sense tends to be person-relative, and the morality to which a person subscribes in this sense "would be whatever body of principles he allowed ultimately to guide or determine his choices of action" (Mackie , ). By contrast, morality in the narrow sense is an essentially social phenomenonmorality in the narrow sense is clearly an institution, constituted by human practices. This is because the whole point of morality in the narrow sense is to help people coordinate with one another peacefully; in this sense a purely private morality would be "worthless": If a morality is to perform the sort of function described in Chapter , it must be adopted socially by a group of people in their dealings with one another . . . Privately imagined rules or principles of action are worthless . . . What counts is rules that are actually recognized by the members of some social circle, large or small, and that thus sets up expectations and claims.
(Mackie , -) The context makes it clear that the "sort of function" Mackie invokes here is the one associated with morality in the narrow sense, and that he therefore thinks morality in the narrow sense must be a social reality. When Mackie discusses the need to "remake" morality, it is pretty clear that he primarily has in mind morality in the narrow sense. His arguments for remaking morality in one way rather than another tend to appeal to considerations relating the proposed reforms to the function of morality in the narrow sense-helping us get along with one another. Furthermore, he typically characterizes remaking morality as a shared projectone we undertake together. This would not make much sense if his aim were morality in the broad sense, which need not be shared. Indeed, Mackie explicitly takes a sort of "to each his own" approach to morality in the broad sense, allowing that here there need not be agreement: [I]t should be expected that different individuals and different groups should have different ideals and values. Each person's special values will help to determine his morality in the broad sense; his actions will be guided not simply by what he wants but also, to some extent, by the endeavour to realize in some degree whatever he sees as good.
We should therefore read Mackie's proposal to remake morality as concerning morality in the narrow sense. This does not mean his proposal is entirely independent of morality in the broad sense. If most people's morality in the broad sense prescribed acting in ways counter to the sort of morality in the narrow sense Mackie proposes, then that proposal would be unrealistic and unstable. Mackie therefore appeals to fairly widespread values (e.g., avoiding pain, autonomy) that will inform just about everyone's morality in the broad sense in arguing for his proposed reform to morality in the narrow sense. What sorts of reasons, then, does Mackie offer for remaking morality [in the narrow sense] in one way rather than another? Typically, he offers prudential reasons. Here, for example, he It is striking how much this passage seems to anticipate Rawls's views on the extent of "reasonable pluralism" when it comes to what he calls "comprehensive views", though these include more than morality in the broad sense (e.g., theological and metaphysical views; cf. Rawls , Lecture ). The fact that Mackie seems happy for us to continue making such judgments also undermines Kalf's reading, on which it is only morality in the broad sense which is committed to objective values, and which we should jettison. Mackie actually thinks that both forms of moral thinking (broad and narrow) are amenable to the presupposition of objective value, and that both have purely subjectivist versions that are worth preserving; cf. Kalf ( , § ). A further interesting question is what Mackie takes to fix the meaning of moral claims in the broad sense. He seems most likely to have in mind a subjectivist hybrid theory, where the content of those judgments is about one's own norms (or, perhaps, about the content of those norms) but where making the judgment requires actually having the norms and attitudes in question. That would fit well with Mackie's characterization of a person's morality being fixed by the principles which guide his conduct and also with his more general theory of the meanings of evaluative and prescriptive terms. The other reading worth exploring would be an expressivist one, since it is not obvious that Mackie's objections to expressivism earlier in the book would apply as forcefully to morality in the broad sense. On balance, though, a subjectivist hybrid theory seems most likely what Mackie implicitly had intended for discourse about morality in the broad sense. appeals to each person's "own interest" to explain why we should welcome the existence of morality in the narrow sense and to explain why each of us should try to modify it to better "suit" ourselves where it is not in our interest: Everyone should, in his own interest, welcome the fact that there is, and hope there will continue to be some system of morality, and why, even if the existing system does not suit him, his aim should be to modify it, at least locally, rather than to destroy it. (Mackie , ) Mackie allows that prudence and morality in the narrow sense can in principle come apart, but is cautiously optimistic that it will not do so too often, given that it will tend to be in our interests to cultivate a conscience; those without one tend to be shunned by others, and most amoralists are not good at hiding their shamelessness. Having a conscience, in turn, itself partly constitutes one's well-being, for Mackie: If someone . . . has at least fairly strong moral tendencies, the prudential course, for him, will almost certainly coincide with what he sees as the moral one, simply because he will have to live with his conscience.
(Mackie , -) Mackie's proposal for how we should remake morality also is heavily informed by the need for realism-where realism here means ensuring that morality dramatically depart from what individual's perceived self-interest (this is part of why he rejects utilitarianism; see Mackie , ). Still, Mackie allows that for some people, acting morally will sometimes and perhaps often run counter to their interests. He simply suggests that this will be sufficiently rare as to not undermine the tenability of his proposed reforms.
Having some sense of the kinds of reasons Mackie offers for remaking morality, we can now return to the "Vory v Zakone" challenge. What is so special about prudence? Why is showing that we have prudential reasons to remake morality as Mackie recommends any more interesting that we have Vory v Zakone reasons to remake it in a very different way? One option Mackie explicitly rejects is the idea that prudence is a necessary requirement of rationality: Even the rationality of prudence-in the sense of equal concern for the interests and welfare at all future times of this same person, oneself-is not quite as self-evident as is commonly supposed. Personal identity is not absolute, as it is believed to be: as I argued in Chapter , our concept of personal identity through time itself functions as a sort of institution, aided by a contingent present desire for our own future welfare. (Mackie , ) Interestingly, Mackie here characterizes our concept of personal identity as functioning "as a sort of institution" and refers the reader back to Chapter . There he compares the concept of personal identity to "an institution like promising" and suggests that this concept introduces a "requirement for attention to the future well-being of what will be the same human being as the agent in question" (Mackie , ). Mackie is here no doubt drawing on his broadly Lockean view of personal identity, one developed in more detail elsewhere (e.g., Mackie , chap. ). There he defends a "conceptual reform" of our ordinary concept of personal identity which he argues is infected with error, having its identity over time absolutely ("like a Lockean atom"; Mackie , ), rather than relative to psychological or physical continuity of the subject. On his proposed reforming definition, our concept of personal identity should be understood as a natural kind term and given an externalist semantics. The nominal essence of personal identity is, on this view, "whatever underlies and makes possible the unity of consciousness". The real essence underlying this is, for Mackie, a species of bodily continuity, and he thinks this is at least part of our ordinary concept, but that our ordinary concept also includes dualist strands which he thinks we must jettison.
Interestingly, in this earlier work (Problems from Locke), Mackie also suggested that prudence has no special claim to rationality: [T]hat there is no factual basis for the employment of our present absolute concept . . . has an important bearing on moral philosophy, especially in bringing it about that there is no exclusive rationality in having an equal concern for all of one's future selves. (Mackie , ) One can also here as well see the profound influence Hume has had on Mackie, since these passages also strongly echo Hume's infamous remark that "'Tis not contrary to reason to prefer the destruction of the whole world to the scratching of my finger" (Hume -, . . . ). Reason for both Mackie and Hume is always relative to one's present desires.
Putting these pieces of the puzzle together, we can see that Mackie takes the rationality of prudence to depend on the contingent but systematic and widespread fact that each of us, at each moment in time, tends to care deeply about the welfare of future time-slices of the human being which constitutes oneself at that moment in time. In taking part in the pseudo-institution of personal identity, by deploying the concept of prudence, we take up an "internal" perspective on that pseudo-institution. Just as someone who takes an "internal" perspective on the institution of promising thereby endorses the requirement to keep their promises, and so tends to be appropriately motivated, someone who takes an "internal" perspective on personal identity thereby endorses the requirement to care equally about the welfare of future time-slices of the human being which underlies their current unity of consciousness. In both cases, there is no categorical requirement of rationality to care about either (promises or one's future welfare); the rational requirement follows only from an agent's contingent present desire.
This, in turn, gives us a clue as to how Mackie might answer the "Vory v Zakone" challenge. Implicit in the passages in which Mackie discusses the merely conditional rationality of prudence is the view that at least a sufficient condition for an agent's being prima facie rationally required to do something is that doing it would promote the satisfaction of one or more of the agent's present desires. That would explain why the rationality of caring about one's future self depends on one's contingently caring about the welfare of the human being which currently constitutes oneself. This hypothesis is corroborated by passages like the following: The rationality of morality (in the narrow sense) consists in the fact . . . that men need moral rules and principles and dispositions if they are to live together and flourish in communities . . . The rationality of prudence consists in the fact that a man is more likely to flourish if he has, at any one time, some concern for the welfare of later phases of this same human being . . . Both these contrast with the more basic rationality of the hypothetical imperative, rationality in the sense in which it is rational to do whatever will satisfy one's own present desires.
(Mackie , -) The rationality of doing "whatever will satisfy one's present desires" is taken to be "more basic" than the rationality of morality and prudence. This passage, and connection with those quoted above, strongly suggest the following understanding of how Mackie could meet the "Vory v Zakone" challenge: ( ) Practical rationality is [at least in part] a matter of doing what will best promote the satisfaction of one's present desires.
( ) Most people (for evolutionary reasons Mackie also canvasses), throughout most of their lives, care a great deal about the well-being of their future selves (they take an "internal perspective" on the pseudo-institution of prudence).
( ) For most people, at most times, prudence is, therefore a rational requirement.
( ) Therefore, establishing that the proposed reforms to morality in the narrow sense (one's socially constructed moral institutions) are such that they would be prudentially good for most people is enough to establish a prima facie rational requirement to support those reforms insofar as individuals can effectively do so.
( ) Rationality is normative; establishing that the proposed reforms are (generally) rationally required is enough to meet the Vory v Zakone challenge.
The fifth stage of this response to the challenge is the only one I have not yet discussed. Unfortunately, Mackie never much discusses practical rationality and "the normative". It is, however, commonplace for those working on the normative to characterize genuinely normative reasons as ones that an agent cannot ignore, on pain of practical irrationality. Theorists often disagree about which, if either, is more basic-normative reasons or rationality. The idea that the two are intimately linked in some way is, though, widespread, and has been endorsed both by moral realists, Kantian rationalists and expressivists. To that extent, insofar as Mackie can demonstrate that his proposed reforms are rationally required, that is enough to show that they matter, in some intuitive sense-that they are "normative". Moreover, even if this link to the normative can be reasonably contested, rationality is plausibly an essential feature of human nature. To that extent, showing how his proposed reforms are rational is already enough to meet the Vory v Zakone challenge. The Russian thieves' code (and other such arbitrary conventions) are no essential part of human nature in the way that rationality plausibly is. To the extent that we take the study of human nature to be part of philosophy's core mission (as Mackie's historical influences, like David Hume, certainly did), it will be philosophically interesting to show that the proposed reforms are rational in a way that showing, e.g., that they would be supported by the norms of the Vory v Zakone would not.
Of course, the present aim theory of practical rationality, or more modestly (perhaps all that Mackie needs) the thesis that satisfying an agent's present desire(s) is sufficient in itself to ground a prima facie rational requirement, is itself highly con-See, e.g., Scanlon ( ), Korsgaard ( ), Smith ( ), and Ridge ( ). For further useful discussion, see Dreier ( ).
troversial. But it is also a view with some attractions and interesting philosophical justifications. My main aim here, though, is not to defend Mackie's overall view, but to offer an interpretation that adequately balances charity and fidelity to the text. The present aim conception of rationality has enough plausibility that attributing that view to Mackie does not seem terribly uncharitable. It also has considerable textual support. Moreover, this is a broadly Humean conception of practical rationality. Hume thought reason was a real faculty of the mind, but a fairly anaemic one which simply functioned to keep our beliefs and desires consistent. For Hume, reason famously is and ought to be the slave of the passions, and Mackie here takes a similar view. This is no coincidence. Mackie makes it clear both in the footnotes to Ethics: Inventing Right and Wrong and in Hume's Moral Theory just how indebted he is to Hume's approach here. I suspect that if Mackie had discussed practical rationality in more detail that he would have also found traces of the errorridden notion of "objective value", and urged us to excise that from our discourse and "prune" our talk of rationality as well, but that is admittedly a speculative hypothesis.
Conclusion
Mackie's Ethics: Inventing Right and Wrong has long been misunderstood. Mackie is not an error theorist in the modern sense; he does not think all moral discourse is riddled with error. Rather, he thinks much and perhaps most ordinary moral discourse is riddled with error, but that important pockets of ordinary moral discourse are entirely in good working order. This explains how he can go on, in the second half of the book, to make numerous first-order moral claims, why he emphasizes the neutrality of his second-order position from first-order ethics, etc. As it happens, See Schroeder ( ) for a recent defense of a view in this neighborhood. Derek Parfit ( ) famously discusses the present aim theory.
the pocket of ordinary moral discourse which is error free is subjectivist in its content and amenable to a "hybrid theory" in that the relevant judgments are partly constituted by endorsing some contextually specified set of moral institutions. Mackie thus should be understood as both a hybrid theorist and as a "conceptual pruner", urging us to do away with those strands of moral thought and discourse which are indefensible. Moberger made a reasonable initial case for this reading of Mackie, but left out what I consider some of the strongest and most direct evidence in its favour, relying instead too heavily on indirect clues arising from caveats Mackie included in stating the error theory. Most notably, the semantic pluralism needed for this reading can be justified much more directly by attending to Mackie's semantic theory for terms like "good", "ought", "must", and "reason". That discussion also further corroborates reading Mackie as a hybrid theorist, given his discussion of using "good" for "egocentric commendation", as do passages from Hume's Moral Theory. Moreover, there is considerable additional textual evidence for the view, some of it in other sources, and it fits well with Mackie's own account of the figures who most influenced his thinking (Stevenson, Ayer, and especially Hume).
All of that said, the resulting view does face a challenge: to explain in what sense the reasons we have for "remaking" morality (in the narrow sense) are somehow "special"-why they have a grip on us or "are normative". I have argued that there is an implicit answer to this in Mackie's work, albeit a controversial one. The reasons Mackie offers for reforming morality are prudential, but prudence is not itself essentially rational. Rather, it is grounded in the rationality of promoting the satisfaction of one's present desires and the contingent but deeply ingrained and widespread desire most people have for their own future welfare. Why rationality itself counts "as normative", is of course a further question, but here at least Mackie is in good company, since theorists of many different stripes take the normativity of rationality as a fundamental posit. Moreover, even if rationality is not in any suitable sense normative, it is plausibly an essential feature of human nature, and so grounding his proposed reforms in rationality should make the project philosophically interesting to that extent. | 2020-04-23T09:09:39.424Z | 2020-04-20T00:00:00.000 | {
"year": 2020,
"sha1": "6f143b8887f46df660509509b4c860146719543a",
"oa_license": "CCBYNC",
"oa_url": "https://jhaponline.org/jhap/article/download/4000/3515",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "358cb45c5484a2254a35caffa538770afb169cda",
"s2fieldsofstudy": [
"Philosophy"
],
"extfieldsofstudy": [
"Philosophy"
]
} |
140473061 | pes2o/s2orc | v3-fos-license | STUDIES ON MILD STEEL PARTICULATES REINFORCED DURALUMIN COMPOSITE FABRICATED THROUGH POWDER METALLURGY ROUTE
In the present study Duralumin(DA) based Mild Steel particles (MSp-6% wt. and 12% wt.) reinforced composite
are fabricated by cold compacting the milled powders. The mechanically mixed powders are compacted in a die cavity by
applying a uniaxial steady load in the range 22-38 T. The hardness of green compacts produced are increased by
sintering. The SEM images showed an even dispersion of MSp in the duralumin alloy and elemental composition is
confirmed by the EDAX spectrum. The sintered specimens were further subjected to aging treatment. Compacts were
soaked at 5500C for a span of 2 h and then are quenched in water at ambient temperature. The temperatures selected for
aging treatment are 100, 150, and 2000C and hardness was checked for every 60 minutes of time. The peak hardness
obtained because of aging heat treatment is found to be much better than the corresponding sintered compacts. Compacts peak aged at 1000C exhibited better tensile strength than the rest of the other specimens
INTRODUCTION
Aluminium and its alloys have been extensively preferred for many applications because of their excellent mechanical properties. They are the first choice of researchers and engineers because of their low density, good heat and electric conductivity, resistance to corrosion and high endurance [1]. Addition of compatible reinforcements even in small quantities would further enhance the stiffness, hardness, fatigue resistance and tribological properties of aluminum alloys in specific [2]. In the present day situation aluminum matrix composites are being used in automobile, aerospace, marine and defense industries because of superior properties [1,3] exhibited by these composites. Even with the various available methods like stir casting, pressure infiltration etc. to produce metal matrix composite Powder Metallurgy (PM) has been the most popular technique as it is simple and flexible in terms of designing the constituents [4]. The PM method greatly avoids the formation of reinforcement clusters on the matrix and reaction between the reinforcement and matrix. PM route is advantageous as higher quantity of reinforcement can be dispersed in the matrix and also a better control of microstructure phases is achievable [5]. Also in specific by aluminum PM it is possible to produce high performance, net or nearly net to shapes of components, hence bringing down or avoiding the investment and running costs attached with complex machining operations [6]. In the recent year's researchers have used ceramic particles for producing the composites. But the major drawback with ceramics reinforced MMC's are their relatively lower toughness and toughness. Also, the relatively higher costs would make them economically unsuitable for various applications. In contrast, steels are competitively low cost, which has made them attractive in a wide range of applications [7].
In the light of above, the present investigation concentrates on to develop the composite by dispersing the mild steel particles in different proportions in duralumin matrix by employing the cold compacting approach. The composite developed is expected to provide superior mechanical properties suitable for high strength, heat and wear resistant applications.
Powders Chosen for Compaction
Matrix material chosen for the present investigation is duralumin powder borrowed from Varsha bullion & elemental analab, Mumbai. The average particle size of the duralumin powder is 50 microns and are irregular in shape. The apparent density is found to be 1.2448 g/cc. The elemental composition of this material has been shown in Table 1. As seen from this table, the major alloying element in duralumin is copper with 3.9% wt. This matrix exhibits a superior blend of strength and damage resistance at higher and cryogenic temperatures [8]. Duralumin is extensively used for aircraft components like fittings, couplings, transmission shafts and for gears. The morphology of powder particles is shown in the SEM micrograph (figure 1). Micrographs were captured by SEM (EVO MA18 with Oxford EDS(X-act)) having magnification 1X and maximum 100000X.
Water atomized MS powder used as a reinforcement contains irregular particle shapes with dark grey color exhibiting high hardness with particle size in the range 40-45 microns. It is borrowed from Parshwamani metals, Mumbai. Table 2 gives the chemical components of mild steel powder.
Details of Compaction Tools
The tooling used for cold compacting include simple die and punches and are as shown in figure 2. The length to diameter ratio of the die is maintained as 4 in the design. The die has been fabricated from En24 steel and the punches from En8 material. In order to induce adequate strength, the die and punches were subjected to heat treatment after machining and TIG welding operations.
Mixing of Powders
The duralumin and MS p powders are mechanically mixed in a ball milling equipment [2]. The equipment preloaded with steel balls is charged with two types of powders such that Ball to Charge ratio(BCR) is 5:1. This ratio is essential for obtaining the homogeneous mixture of powders [9]. The powders are milled for 60 min and at a constant In order to apply the load on powders to compact them, UTM of 60 T capacity is used. The steadily increasing load is axially applied through the punch as shown in figure 4. The compacts of duralumin and composite (6% and 12% 38 T. In order to avoid the seizure of compact in the die and to prevent the direct surface contact between the punch and die the surfaces of the tools are applied with a layer of zinc was removed undamaged from the die cavity by a simple ejection system that is as shown in figure 4. Some amount of minute surface cracks was observed for green composite with MS p . The
Sintering Process
The green compacts will not have sufficient strength to withstand the external load, hence in the present study they were subjected to sintering operation in a PID controlled muffle furnace. The compacts are held at 580 0 C for 5 h in furnace and were let to cool down in the furnace for 18 h. The surface oxidation of compacts is prevented by loading them into furnace after the inside temperature was stabilized. The process of sintering of mainly brings about the bonding between powder particles at the contact areas and thus enhances overall strength of compact [8]. At the atomic level, sintering process provides increased bonding among atoms and diffusion of atoms and weldzonesare formed while compaction will enhance the bonding [10].
Density and Hardness of Compacts
Density of DA and reinforced compacts was determined using a digital electronic weighing machine(0.0001 g) by adopting Archimedes' principle according to ASTM B311-08 [8,10,11]. The theoretical values of densitiesare arrived by employing rule of mixtures. The specimens of DA, DA+ 6% MS p and DA+12%MS p are further cut into smaller cylinders in the wire EDM machine. The process of parting is shown in figure 6 and the specimens for hardness test are as shown in figure 7. Scratches and surface defects on the sample were eliminated by thoroughly polishing the test specimens with emery papers (400, 600, 800, and 1000 grit). The hardness measurement was done at normal room temperature (30 0 C) and the hardness was measured at four spots on the surface of each sample to know the average hardness. In order to measure the hardness of specimens micro Vickers hardness tester was used [2,10,11] Table 4.
Aging Heat Treatment Process
The sintered compacts were made to undergo to aging treatment. The heat treatment was carried out in a PID controlled muffle furnace. The furnace temperature was initially made stable to the required temperature and then specimens were loaded to avoid the surface oxidation. Compacts are initially subjected to soaking heat treatment at 550ᴼC for span of 2 h, followed by quenching in water at ambient temperature. Hardness was measured before starting aging Table 14.
Tension Test
The tensile test specimens were fabricated from the cylindrical peak hardened compacts as per E8/E8M-13a standard [12]. The tensile test was carried out in a Tensometer which was computer integrated to generate the on line plot of load-vs-extension. The results of tension test are tabulated in Table 15.
Density of Compacted Specimens
As the quantity of MS p is increased, an increase in the density of the composites is noticed. This behavior could be because of relatively high packing factor of MS p , increased concentration within the duralumin resulting in increase of the density of the composites. This behavior is furthermore increased by an effective fusion of MS p while sintering to form [13]. Figure 9 depicts the variation of density of composite compacts with increasing amount of MS p . This behavior is increased by the proper fusion of MS p while sintering with the matrix to form a coherent phase. Relative density is calculated by using the relation, Relative density=sintered density/theoretical density [12] and its variation is depicted in figure 10. A decrease in the level of porosity is observed as the amount of MS p is increased ( figure 11) and is due to strong interfacial bonding between the particles during sintering at elevated temperature. Lower porosity levels basically provide composite materials with improved mechanical properties. As a result of lower porosity in the compacts, they definitely exhibit superior strengths, high load carrying ability, and good corrosion resistance. The variation of porosity of composites in the present investigation matches with the observations noticed by investigators to develop brake pads [14].
Microstructure Study on Sintered Compacts
As seen from the SEM images shown in figure 12, there is fairly homogeneous dispersion of mild steel particles in the entire duralumin matrix with no surface porosity or cracks. Also, it appears to be a fair bondage among DA and MS p that could certainly result in the improved load transfer from the matrix to reinforcements in the composites. As observed from the micrographs, there is no clustering of MS p in DA, which is very common in the liquid state processing techniques. Figure 13 depicts the EDAX spectrum taken on sintered composite compact which confirms the elemental composition.
Hardness in Sintered and Aged Condition
As can be seen in figure 14, as the amount of reinforcement is increased in matrix the hardness of composite will also increase. The enhancement of hardness could be because of higher quantity of MS p and also higher amount of intermetallic phases appearing with steel particle addition during heat treatment. Earlier studies have noticed that iron enhances the strength in compression and composite hardness. The strengthening mechanism isbecause of refinement of grains of DA matrix, the homogeneous dispersion reinforced particles, as well as the appearance of Al 13 Fe 4 intermetallic compound [15].
Both DA and DA-6% and 12% wt. aged samples are subjected to hardness measurement (VHN). Variation of hardness as a function of temperature of aging and duration for the above composites are depicted in figures 15-20.
Hardness is found to increase gradually with time for both the DA and its composites. It reaches peak hardness value followed by over-aging that results in decrease of hardness [16]. The hardness increases with increase in amount of mild steel particulates. The increase in hardness is because of hard reinforcement particles that positively contributes to the hardness of composites [17]. Since matrix is strong age hardenable alloy, it is expected that mild steel reinforced DA alloy composites may be very sensitive to age hardening regardless of temperature of aging. Among all the aging temperatures, aging at 100°C for both base alloy and its composites show higher peak hardness values although the time intervals for achieving peak aging are considerably higher. Artificial aging at lower temperature (100°C) pays to the enhanced hardness due to escalation in the quantity of intermediate zones throughout precipitation, higher amount of fine intermetallics and reduced interparticle distances. The hardness reduces after peak aged state, could be because of increased size of intermetallic precipitates that appear while aging process and existence of incoherency between matrix and intermetallics termed as over-aging [18]. Higher temperatures (150 and 200°C) accelerate the aging rate because of increased diffusion rate in the matrix. Higher the temperature of aging, lower will be the duration required to achieve peak hardness [16].
Comparison of variation of peak hardness with quantity of mild steel particles for sintered condition and for different aging temperatures is shown in figure 21.
Tensile Test
The specimens are observed to have undergone primarily the ductile fracture, with cup and cone shape formed on the fractured halves of the samples. Tension test is performed on DA and peak aged samples. Figure 22 shows the comparison of variation of UTS with quantity of MS p for sintered condition and for different aging temperatures of compacts. Uniform distribution of the reinforcement particles is an important requirement for various high performance engineering applications. A homogeneous distribution of individual and hybrid reinforcements is essential to improve the composites mechanical properties achieved by strong interfacial bonding, which contributes for effective load transfer and distribution from the matrix to the reinforcements exhibiting enhanced elastic modulus and strength. The improvement in tensile strength by the presence of hard secondary phases on soft matrix leads to alloy strengthening, which also helps to decrease grain size of the matrix resulting further improvement of mechanical properties [ 19,20]. Higher particle concentration and lower aging temperature result in a higher density of dislocations and particle interaction dislocations.
When load is applied, the presence of the hard particles and intermetallics contribute to dislocation pileup, increasing back Fabricated Through Powder Metallurgy Route www.tjprc.org SCOPUS Indexed Journal editor@tjprc.org stress, and work hardening the matrix due to restricted plastic flow in the ductile matrix. The synergetic effect of dislocation interaction with the reinforcement, intermetallic and grain boundary provides a positive contribution to alloy strengthening [21,22].
CONCLUSIONS
In the present investigation the compacts of duralumin and the duralumin based mild steel particles reinforced composites were successfully fabricated by cold compacting the powders and the sintered composite specimens showed a homogeneous dispersion of the MS p in DA matrix. An increase in the densities of the composites was noticed as the amount of mild steel particulates is increased and this may be due to a relatively higher packing factor of MS p . The porosity level is observed to have decreased with increased amount of MS p and this behavior may be because of strong bond at the interface among particles through elevated temperature sintering. Because of accelerated aging rate at higher temperatures (150 and 200 0 C as compared to 100 0 C) the time required to attain peak hardness was observed to be less comparatively. Specimen peak aged at 100 0 C displayed a good improvement in UTS than the counterparts peak aged at 150 and 200 0 C. | 2019-05-01T13:04:45.216Z | 2019-01-01T00:00:00.000 | {
"year": 2019,
"sha1": "0fe68030e2e515a2454cd6bbc27fd9bf735a7872",
"oa_license": null,
"oa_url": "https://doi.org/10.24247/ijmperdapr201988",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "5771f347779bba05d950031dd86fd476ff316315",
"s2fieldsofstudy": [
"Materials Science",
"Engineering"
],
"extfieldsofstudy": [
"Materials Science"
]
} |
90186683 | pes2o/s2orc | v3-fos-license | Evaluating Metagenome Assembly on a Simple Defined Community with Many Strain Variants
We evaluate the performance of three metagenome assemblers, IDBA, MetaSPAdes, and MEGAHIT, on short-read sequencing of a defined “mock” community containing 64 genomes (Shakya et al. (2013)). We update the reference metagenome for this mock community and detect several additional genomes in the read data set. We show that strain confusion results in significant loss in assembly of reference genomes that are otherwise completely present in the read data set. In agreement with previous studies, we find that MEGAHIT performs best computationally; we also show that MEGAHIT tends to recover larger portions of the strain variants than the other assemblers.
Introduction
using the script trim-low-abund.py script with -C 5 from khmer v2 [27, 140 28]. 141 Assemblers 142 We assembled the quality-filtered reads using three different assemblers: [17], we used --pre correction to perform pre-correction before assembly 145 and -r for the pe files. IDBA could not ingest orphan sequences so singleton 146 reads were omitted from this assembly. where -l is for maximum read length, -m is for max memory in bytes to 153 be used in constructing the graph, and --cpu-only uses only the CPU 154 and no GPUs. We also used --presets meta-large for large and complex 155 metagenomes, and --12 and -r to specify the interleaved-paired-end and 156 single-end files respectively. MEGAHIT allows the specification of a memory limit and we used -M 1e+10 for 10 GB. 158 All three assemblies were executed on the same XSEDE Jetstream in-159 stance (S1.Xxlarge) at Indiana University, running Ubuntu 16.04 (install Unless otherwise mentioned, we eliminated all contigs less than 500 bp 167 from each assembly prior to further analysis. 168 Mapping 169 We aligned all quality-filtered reads to the reference metagenome with bwa 170 aln (v0.7.7.r441) [23]. We aligned paired-end and orphaned reads separately. 171 We then used samtools (v0.1.19) [29] to convert SAM files to BAM files for 172 both paired-end and orphaned reads. To count the unaligned reads, we 173 included only those records with the "4" flag in the SAM files [29]. 174 Assembly analysis using NUCmer 175 We used the NUCmer tool from MUMmer3.23 [30] to align assemblies to the 176 reference genome with options -coords -p. Then we parsed the generated 177 ".coords" file using a custom script analyze assembly.py, and calculated 178 several analysis metrics across all three assemblies at a 99% alignment iden-179 tity.
180
Reference-based analysis of the assemblies 181 We conducted reference-based analysis of the assemblies under two condi- The script summarize-coords2.py was used to calculate aligned cov-187 erage from the loose alignment conditions: each base in the reference was 188 marked as "covered" if it was included in at least one alignment. The script 189 analyze ng50.py was used to calculate NGA 50 for each individual reference genome.
191
Analysis of chimeric misassemblies 192 We analyzed each assembly for chimeric misassemblies by counting the num-193 ber of contigs that contained matches to two distinct reference genomes. In 194 order to remove secondary alignments from consideration, we included only 195 the longest non-overlapping NUCmer alignments for each contig at a mini-196 mum alignment identity of 99%. We then used the script analyze chimeric2.py 197 to find individual contigs that matched more than one distinct reference 198 genome. As a negative control on our analysis, we verified that this ap-199 proach yielded no positive results when applied to the alignments of the 200 reference metagenome against itself.
201
Analysis of unmapped reads 202 We conducted assembly and analysis of unmapped reads with MEGAHIT, 203 NUCmer, and sourmash as above. The new GenBank genomes are listed in 204 the Zenodo archive at the file accession-list-unmapped.txt and for con-205 venience are available for download at the archival URL https://osf.io/34ef8/.
207
The raw data is high quality. present at some minimal coverage. 245 We excluded the remaining 13 genomes (see Table 3) from any fur-246 ther reference-based analysis because interpreting recovery and misassembly 247 statistics for these genomes would be confounding; also see the discussion of 248 strain variants, below.
249
MEGAHIT is the fastest and lowest-memory assembler eval-250 uated 251 We ran three commonly used metagenome assemblers on the QC data set: The assemblies contain most of the raw data 261 We assessed read inclusion in assemblies by mapping the QC reads to 262 the length-filtered assemblies and counting the remaining unmapped reads.
263
Depending on the assembly, between 2.7 million and 3.9 million reads (2.5-264 3.5%) did not map to the assemblies ( Table 5). All of the assemblies included 265 the large majority of high-abundance 51-mers (more than 96.8% in all cases).
266
Much of the reference is covered by the assemblies. A '*' after the name indicates the presence of at least one other genome with > 2% Jaccard similarity at k=31 in the community. Where NGA50 cannot be calculated due to poor coverage, a marker is placed at 1kb.
Individual genome statistics vary widely in the assemblies. 283 We computed the NGA50 for each individual genome and assembly in order 284 to compare assembler performance on genome recovery (see left panel of Fig-285 ure 2). The NGA50 statistics for individual genomes vary widely, but there 286 are consistent assembler-specific trends: IDBA yields the lowest NGA50 for 287 28 of the 51 genomes, while MetaSPAdes yields the highest NGA50 for 32 288 of the 51 genomes. 289 We also evaluated aligned coverage per genome for each of the three 290 assemblies (right panel, Figure 2). We found that 13 of the 51 genomes were 291 missing 5% or more of bases in at least one assembly, despite all 51 genomes 292 having 99% or higher read-and 51-mer coverage.
293
There are 12 genomes with k=31 Jaccard similarity greater than 2% 294 to other genomes in the community, and these (denoted by '*' after the 295 name) typically had lower NGA50 and aligned coverage numbers than other 296 genomes. In particular, these constituted 12 of the 13 genomes missing 5% 297 or more of their content, and the lowest eight NGA50 numbers.
298
Longer contigs are less likely to be chimeric. and assembled these reads in isolation using MEGAHIT, yielding 6.5 Mbp 311 of assembly in 1711 contigs > 500bp in length. We then did a k-mer in-312 clusion analysis of this assembly against all of the GenBank genomes at 313 k=31, and estimated the fraction of the k-mers that belonged to different 314 species (Table 9). We find that 51.1% of the k-mer content of these contigs 315 positively match to a genome present in GenBank but not in the reference 316 metagenome.
317
To verify these assignments, we aligned the MEGAHIT assembly of un-318 mapped reads to the GenBank genomes in Table 9 with NUCmer using 319 "loose" alignment criteria. We found that 1.78 Mbp of the contigs aligned 320 at 99% identity or better to these GenBank genomes. We also confirmed 321 that, as expected, there are no matches in this assembly to the full updated 322 reference metagenome. 323 We note that all but the two P. ruminis matches and the E. coli isolate 324 YS are strain variants of species that are part of the defined community 325 but are not completely present in the reads (see Table 2). For Proteiniclas-326 ticum ruminis, there is no closely related species in the mock community 327 design, and very little of the MEGAHIT assembly aligns to known P. ru-328 minis genomes at 99%. However, there are many alignments to P. ruminis Assembly recovers basic content sensitively and accurately.
336
All three assemblers performed well in assembling contigs from the con-337 tent that was fully present in reads and k-mers. After length filtering, 338 all three assemblies contained more than 95% of the reference (Table 6); 339 even with removal of secondary alignments, more than 87% was recovered 340 by each assembler (Table 7). About half the constituent genomes had an 341 NGA50 of 50kb or higher (Figure 2), which, while low for current Illumina 342 single-genome sequencing, is sufficient to recover operon-level relationships 343 for many genes.
344
The presence of multiple closely related genomes confounds 345 assembly.
346
In agreement with CAMI, we also find that the presence of closely related that one or more assembler will fail to recover 5% or more -of the 13/51 354 genomes for which less than 95% is recovered, 12 of them have close genomes 355 in the community. Interestingly, very little similarity is needed -all genomes 356 with Jaccard similarity of 2% or higher at k=31 exhibit these problems.
357
The Shewanella baltica OS185 genome is a good example: there are two 358 strain variants, OS185 and OS223, present in the defined community. Both 359 are present at more than 99% in the reads, and more than 98% in 51-mers, 360 but only 75% of S. baltica OS185 and 50% of S. baltica OS223 are recovered 361 by assemblers. This is a clear case of "strain confusion" where the assemblers 362 simply fail to output contigs for a substantial portion of the two genomes.
363
Another interest of this study was to examine cross-species chimeric as-364 sembly, in which a single contig is formed from multiple genomes. In Table 8, 365 we show that there is relatively little cross-species chimerism. Surprisingly, 366 what little is present is length-dependent: longer contigs are less likely to 367 be chimeric. This might well be due to the same "strain confusion" effect 368 as above, where contigs that share paths in the assembly graphs are broken 369 in twain.
370
MEGAHIT performs best by several metrics.
MetaSPAdes and IDBA in memory and time (Table 4). The MEGAHIT 373 assembly also included more of the reads than either IDBA or MetaSPAdes, 374 and omitted only 0.4% more of the unique 51-mers from the reads than 375 IDBA. MEGAHIT covered more of the reference genome with both loose 376 and strict alignments (Table 6 and Table 7 Figure 2 suggest that much of 396 this differential assembly content is due to the impact of strains.
397
The missing reference may be present in strain variants of the 398 intended species.
399
Several individual genomes are missing in measurable portion from the QC 400 reads (Table 2), and many QC reads (4.4% of 108m) did not map to the full 401 reference metagenome. These appear to be related issues: upon analysis of 402 the unmapped reads against GenBank, we find that many of the contigs as-403 sembled from the unmapped reads can be assigned to strain variants of the 404 species in the mock community (Table 9) An additional concern is that metagenome assemblies are often performed after pooling data sets to increase coverage (e.g. [4,32]); this pooled 445 data is more likely to contain multiple strains, which would then in turn 446 adversely affect assembly of strains. This may not be resolvable within the 447 current paradigm of assembly, which focuses on outputting linear assem-448 blies that cannot properly represent strain variation. | 2019-04-02T13:08:26.130Z | 2017-07-02T00:00:00.000 | {
"year": 2017,
"sha1": "de0b6aff005f2f3e8dee54e347096811fdab196c",
"oa_license": "CCBY",
"oa_url": "https://www.biorxiv.org/content/biorxiv/early/2017/07/03/155358.full.pdf",
"oa_status": "GREEN",
"pdf_src": "BioRxiv",
"pdf_hash": "e7197423edab6ffffaadd4c43e4b8e6e06f0c94f",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology"
]
} |
2277536 | pes2o/s2orc | v3-fos-license | A centrosomal scaffold shows some self-control
The scaffolding protein AKAP350A is known to localize to the centrosome and the Golgi, but the molecular details of its function at the centrosome remain elusive. Using structure–function analyses, protein interaction assays, and super-resolution microscopy, Kolobova et al. now identify AKAP350A's specific location and protein partners at the centrosome. The authors further define an autoregulatory mechanism that likely controls AKAP350A's ability to nucleate microtubule growth.
The scaffolding protein AKAP350A is known to localize to the centrosome and the Golgi, but the molecular details of its function at the centrosome remain elusive. Using structurefunction analyses, protein interaction assays, and super-resolution microscopy, Kolobova et al. now identify AKAP350A's specific location and protein partners at the centrosome. The authors further define an autoregulatory mechanism that likely controls AKAP350A's ability to nucleate microtubule growth.
Protein kinase A-anchoring proteins (AKAPs) 2 constitute a large family of scaffolding proteins that bind protein kinase A and mediate cAMP signaling. One member of this family, AKAP350 (AKAP9/AKAP450/CG-NAP/hyperion), is thought to regulate microtubule (MT) nucleation from the surface of two organelles-the Golgi and the centrosome (1,2). AKAP350 is related to pericentrin in that these are the only mammalian proteins to contain the pericentrin-AKAP450 centrosome-targeting (PACT) domain (3), and pericentrin has garnered significant attention for its role in MT nucleation at the centrosome. However, AKAP350's role remains largely unexplored, which could limit our overall understanding of MT nucleation at centrosomes. A new study by Kolobova et al. (4) provides significant insight into the molecular architecture of the longest AKAP350 isoform, AKAP350A, and its binding partners at the centrosome. It also identifies an intriguing autoinhibition mechanism that may regulate multiple functions of AKAP350A.
The centrosome is a non-membrane-bound organelle composed of a core pair of centrioles surrounded by pericentriolar material (PCM), a complex protein assembly responsible for nucleating and anchoring MTs. Pioneering work by several labs in 2012 used structured illumination microscopy (SIM) to investigate the spatial distribution of many centrosome proteins (5). These studies showed that pericentrin is an extended molecule with its C terminus (containing the PACT domain) near the centriole wall and its N terminus extending outward. Surprisingly, when Kolobova et al. (4) performed SIM on centrosomal AKAP350A, they found that it does not assemble into an extended molecule like pericentrin. Rather, AKAP350A localizes to the intercentriolar linker (Fig. 1A), thus establishing a distinct localization for AKAP350A from pericentrin, likely driven by unique PACT domain-mediated protein interactions. In support of such a mechanism, the authors show that AKAP350A interacts with Cep68, a known intercentriolar linker component required for centriole cohesion. This result raises the possibility that AKAP350A may regulate centriole linkage (Fig. 1B, Q1), perhaps complementing the recentlyidentified role for pericentrin (6).
To investigate AKAP350A's function further, Kolobova et al. (4) expressed the 4000-amino acid AKAP350A as three more technically manageable fragments: an N-terminal F1 fragment, a central F2 fragment, and a C-terminal F3 fragment. Overexpression of F3 led to an unexpected result: ectopic assembly of multiple MT-nucleating centers (MTNCs) that contained known PCM proteins such as Cdk5RAP2. Although these MTNCs are not normally present in cells, this observation led the authors to wonder if AKAP350A might play a significant and unappreciated MT nucleation function at centrosomes. However, when Kolobova et al. (4) tested the role of full-length AKAP350A at centrosomes, they observed that AKAP350A depletion resulted in a reduction of Cep68 and Cep170 but did not impact levels of the PCM proteins Cdk5RAP2 and ␥-tubulin. Thus, although AKAP350A can initiate MTNCs, its role in MT nucleation at the centrosome remains unclear (Fig. 1B, Q2). Combinatorial loss-of-function experiments may help elucidate AKAP350's PCM recruitment role at the centrosome, which may be masked by the presence of pericentrin.
The authors next mapped a "promoting" domain within F3 to amino acids 2762-3458 that is required to induce MTNC formation and interacts directly with Cdk5RAP2 based on yeast two-hybrid analysis. Together, these data suggest that AKAP350A's C terminus not only targets the full-length protein to the centriole linker but also promotes centrosome MT nucleation by directly recruiting Cdk5RAP2 upstream of ␥-tubulin. This centrosome-based MT nucleation pathway differs from the Golgibased pathway, which relies on AKAP350's N terminus for both Golgi targeting and the recruitment of GCP2/3, a component of the ␥-tubulin ring complex (2, 7). Of note, a second Golgitargeting domain within AKAP350A has been identified just upstream of the PACT domain (8). It would seem, therefore, that AKAP350A can utilize organelle-specific targeting domains to promote spatially distinct MT nucleation pathways.
Based on the differences in behavior between the truncated and full-length AKAP350 sequences, the authors suspected that full-length AKAP350 contains a sequence that prevents unchecked MTNC formation. Indeed, mapping efforts identi- fied an "inhibitory" domain (amino acids 1881-2183) that suppresses MTNC formation. Thus, AKAP350A cannot only promote PCM recruitment, it can also suppress this recruitment via an autoinhibition mechanism. Consistent with this idea, yeast two-hybrid analyses, biochemical experiments, and proteomics analysis performed by Kolobova et al. (4) showed that the interaction landscape for AKAP350A changes in the presence of the inhibitory domain. Specifically, the authors show that the presence of the inhibitory domain reduced binding of Cep170 to AKAP350A but conversely increased AKAP350A's binding to RII␣, a subunit of protein kinase A. This fascinating result suggests that the interaction of AKAP350A with RII␣ in vivo might engage the autoinhibition mechanism, thereby preventing MT nucleation (Fig. 1B). Relevant to this finding is previous work showing that the Cdk1-dependent phosphorylation of RII␣ mediates its dissociation from AKAP350 during mitotic onset and that this regulation is critical for spindle formation (9). Considering the data in aggregate, we suggest a two-step regulatory mechanism for AKAP350. During interphase, the AKAP350-RII␣ interaction triggers cAMP signaling, prevents MTNC formation, and possibly plays a role in attenuating MT nucleation at the centrosome by enhancing the function of the inhibitory domain. As cells approach mitosis, the Cdk1-dependent phosphorylation of RII␣ serves to reduce its interaction with AKAP350 (9), thereby relieving AKAP350 autoinhibition. The promoting domain would then be free to upregulate binding to Cep170, Cep68, and Cdk5RAP2. While many questions remain unanswered, including the precise function of these newly-identified interactions, a clearer model is beginning to take shape. The discovery of these regulatory domains, coupled with the autoinhibitory mechanism within AKAP350, provides an exciting framework around which all future studies of AKAP350 and pericentrin should be centered. A, the distinct localizations of AKAP350 and pericentrin (both of which contain PACT domains (black dots) at the centrosome. Specifically, AKAP350 (blue) localizes to the intercentriole linkage (orange), while pericentrin (pink) localizes to the proximal end of centrioles (olive). Cdk5RAP2 (green) is anchored to the centrosome by pericentrin; it remains unknown if AKAP350 also positions Cdk5RAP2 at the centrosome. B, a rough sequence map captures AKAP350A's autoregulatory mechanism as identified by Kolobova et al. (4). The gray-shaded area, which was the focus of the authors' work, contains the newly discovered inhibitory domain (Inh, red), which regulates the ability of the newly discovered promoting domain (green) to interact with Cep68, Cep170, and Cdk2RAP5. The inhibitory domain may be sensitive to cell state based on overlap with a previously identified site that interacts with RII␣ (see text for details (9)). The precise role of AKAP350 at the intercentriole linker (Q1) and the centrosome (Q2) remains unclear. | 2018-04-03T03:19:12.842Z | 2017-12-15T00:00:00.000 | {
"year": 2017,
"sha1": "0922d7afff3f450e36d39585c1ad89432cc80d4a",
"oa_license": "CCBY",
"oa_url": "http://www.jbc.org/content/292/50/20410.full.pdf",
"oa_status": "HYBRID",
"pdf_src": "Highwire",
"pdf_hash": "881240a0f91be08fdc1cba1c49558ef4066bebb8",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Chemistry"
]
} |
28413873 | pes2o/s2orc | v3-fos-license | Redetermination of 1,4-dimethoxybenzene
The structure of the centrosymmetric title compound, C8H10O2, originally determined by Goodwin et al. [Acta Cryst.(1950), 3, 279–284], has been redetermined to modern standards of precision to aid in its use as a model compound for 13C chemical-shift tensor measurements in single-crystal NMR studies. In the crystal structure, a C—H⋯O interaction helps to establish the packing.
The structure of the centrosymmetric title compound, C 8 H 10 O 2 , originally determined by Goodwin et al. [Acta Cryst. (1950), 3, 279-284], has been redetermined to modern standards of precision to aid in its use as a model compound for 13 C chemical-shift tensor measurements in single-crystal NMR studies. In the crystal structure, a C-HÁ Á ÁO interaction helps to establish the packing.
Data collection: COLLECT (Hooft, 1998); cell refinement: DENZO-SMN (Otwinowski & Minor, 1997); data reduction: DENZO-SMN; program(s) used to solve structure: SIR97 (Altomare et al., 1999); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: WinGX (Farrugia, 1999) and ORTEP-3 (Farrugia, 1997); software used to prepare material for publication: publCIF (Westrip, 2009 (Carter et al., 1988). In 1950 Goodwin et al. obtained the first X-ray diffraction structure for 1,4-dimethoxybenzene. This structure (R-factor = 0.12) is shown in Fig. 1 and reported an unusual H-C-C angle of 75.7°, which prompted the acquisition of a second structure (Carter et al., 1988). More typical H-C-C angles were observed with this new refinement and this structure (R-factor = 0.067) was used to assign tensor orientations in the single-crystal NMR analysis. Inadvertently, the second structure was never submitted to the Cambridge Crystallographic database. Here, the acquisition of a third structure is reported to correct this oversight. The new structure (R-factor = 0.038) is shown in Fig. 2. The unit-cell and space group of the previous studies are confirmed.
Acquisition of this third, more accurate, structure is beneficial to NMR studies because the 13 C chemical shift tensor data of 1,4-dimethoxybenzene continue to serve as a standard to evaluate new chemical-shift tensor measurement methods as well as to assess electronic structure methods for computing magnetic properties of molecules.
Refinement
The H atoms were located in difference maps and their positions and U iso values were freely refined. Fig. 1. The structure of (I) according to Goodwin et al. (1950).
Special details
Experimental. The program DENZO-SMN (Otwinowski & Minor, 1997) uses a scaling algorithm (Fox & Holmes, 1966) which effectively corrects for absorption effects. High redundancy data were used in the scaling program hence the 'multi-scan' code word was Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. | 2017-06-18T07:06:02.642Z | 2009-01-08T00:00:00.000 | {
"year": 2009,
"sha1": "960be504a76f4541c0bf5555e4921e307f0b72b1",
"oa_license": "CCBY",
"oa_url": "http://journals.iucr.org/e/issues/2009/02/00/hb2878/hb2878.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "3f50f20cdccb34566a06ab0cacfd06250a23e39a",
"s2fieldsofstudy": [
"Chemistry"
],
"extfieldsofstudy": [
"Chemistry",
"Medicine"
]
} |
260746598 | pes2o/s2orc | v3-fos-license | Non-Electroneutrality Generated by Bacteriorhodopsin-Incorporated Membranes Enhances the Conductivity of a Gelatin Memory Device
We have previously demonstrated the potential of gelatin films as a memory device, offering a novel approach for writing, reading, and erasing through the manipulation of gelatin structure and bound water content. Here, we discovered that incorporating a bacteriorhodopsin (BR)–lipid membrane into the gelatin devices can further increase the electron conductivity of the polypeptide-bound water network and the ON/OFF ratio of the device by two folds. Our photocurrent measurements show that the BR incorporated in the membrane sandwiched in a gelatin device can generate a net proton flow from the counter side to the deposited side of the membrane. This leads to the establishment of non-electroneutrality on the gelatin films adjacent to the BR-incorporated membrane. Our Raman spectroscopy results show that BR proton pumping in the ON state gelatin device increases the bound water presence and promotes polypeptide unwinding compared to devices without BR. These findings suggest that the non-electroneutrality induced by BR proton pumping can increase the extent of polypeptide unwinding within the gelatin matrix, consequently trapping more bound water within the gelatin-bound water network. The resulting rise in hydrogen bonds could expand electron transfer routes, thereby enhancing the electron conductivity of the memory device in the ON state.
Introduction
Finding suitable materials to bridge electronic devices and biological systems is a crucial challenge in various biomedical applications [1][2][3][4]. Electronic devices rely on electron conduction, while biological systems operate in an aqueous environment that traditionally hinders efficient long-distance electron transfer. In our previous work, we demonstrated that gelatin hydrogels can function as an electron-conductive resistor by removing unbound free water while retaining bound water [5]. The formation of a proteinbound water network can facilitate long-distance electron transfer in gelatin films.
We used gelatin as the device material since it contains numerous sites capable of forming hydrogen bonds with water molecules [6]. Since previous studies have reported that hydrogen bonds can transfer electrons [7,8], it is possible that the bound-water-mediated hydrogen bonds can transfer electrons across different polypeptide chains. In a typical wet gel below the gelation temperature, bound water primarily resides within gelatin triple helices, limiting its ability to connect different helices. The dynamic free bulk water between different helices could also hinder the electron transfer and therefore causes low electron conductivity. The property to switch the electron conductivity based on the controllable structural change and bound water connection allows gelatin to have the ability to become a resistive memory.
Since the extent of polypeptide unwinding can significantly increase the electron conductivity and therefore the ON/OFF ratio of a gelatin resistive memory, it is important Gels 2023, 9, 635 2 of 12 to investigate what factors can promote unwinding. Previous studies have demonstrated that the introduction of electrostatic repulsion can influence the winding and unwinding of biopolymers [9][10][11][12]. In this study, we incorporated bacteriorhodopsin (BR) into the sandwiched membrane in a gelatin memory device to induce non-electroneutrality under illumination, thereby promoting polypeptide unwinding. BR is a membrane protein that can pump protons under light illumination [13,14] and has been used for various photoelectrical applications [15][16][17][18][19]. When incorporated into a membrane, BR can transport protons across the membrane, generating spatial non-electroneutrality [20][21][22].
In this study, we discovered that the non-electroneutrality caused by BR proton pumping could promote the unwinding of the triple-helical structure and enhance the formation of protein-bound water, thereby facilitating electron transfer and creating a more conductive memory device. To demonstrate the relationship between BR proton pumping and conductivity, we conducted voltage sweep current-voltage (I-V) measurements on the gelatin devices. Our results clearly show that the conductivity of the devices in the ON state is positively correlated with the BR density ratio and that the presence of active proton pumping induced by light is essential for the observed conductivity enhancement. We also employed Raman spectroscopy to investigate the amount of bound water and the extent of polypeptide unwinding in the gelatin devices both with and without BR membranes. Our results suggested that the non-electroneutrality caused by BR pumping leads to an increase in bound water and unwound polypeptides, thereby significantly enhancing the conductivity of the gelatin devices.
Addition of BR Membranes to Gelatin Devices Significantly Enhances Electron Conductivity
We prepared BR-incorporated gelatin devices with varying BR density ratios of 100%, 50%, and 0% (definitions in Materials and Methods). Figure 1a provides an overview of the device structure and the proposed mechanism for the writing process. Initially, the gelatin hydrogel possesses a triple-helical structure surrounded by free water molecules, acting as an insulator for electron transport. During the writing process, the temperature increases above the gelling temperature, causing the gelatin triple helices to unwind into single strands. As water content decreases, the gelatin single strands can compact together along with bound water molecules trapped within the matrix. Simultaneously, BRs actively pump protons across the membrane under illumination, resulting in an increase in protons on one side of the gelatin and a decrease on the other side. We propose that the alteration of the proton gradient by BRs further disrupts the gelatin secondary structure, allowing more amino acids in the gelatin polypeptides capable of forming hydrogen bonds to interact with bound water. The increased hydrogen bonds facilitated by bound water among the gelatin single strands provide additional routes for electron transfer within the protein-bound water network. Consequently, the BR-incorporated gelatin memory device could exhibit enhanced conductivity in the ON state.
To fabricate the BR-incorporated device, we deposited a BR membrane on an ITO plate with a gelatin film, which was then sandwiched with another ITO plate with a gelatin film, as illustrated in Figure 1b. The gelatin film on each of the ITO plates had a thickness of 152 µm, and the dried gelatin film after the water loss had a thickness of about 100 µm. The protruding ITO plates on both sides were clamped with alligator clips for subsequent electrical measurements. Due to possible preferential orientation during deposition, the gelatin surfaces on the deposition side and the counter side may experience different environments. More information can be found in the next subsection.
Resistive memory devices store and access information by encoding "0" and "1" based on the electrical bistability of the active layer. Achieving a high current ratio of the ON state to the OFF state is often desirable [23,24]. Figure 1b,c show the electrical memory characteristics of the BR-incorporated gelatin memory device. The voltage sweep measurement, ranging from −0.09 to +0.09 V at a sweep rate of 0.36 V/s, was used to record the current. Figure 1b shows that the typical BR-incorporated gelatin hydrogel Gels 2023, 9, 635 3 of 12 behaves as an ideal capacitor in the OFF state, similar to bare gelatin gel. Following the illumination writing process, the current of the 100% BR ratio sample increases to 4.0 × 10 −3 A at 0.09 V, and it acts as a resistor in the ON state. The ON/OFF current ratio reaches 1.6 × 10 5 , which is two orders of magnitude higher than those in previous studies using bio-related materials for constructing memory devices [25,26]. Table 1 displays the conductivity of BR-incorporated gelatin hydrogel with different BR density ratios in the ON state. Increasing the density ratio of BR leads to enhanced conductivity. The 100% BR ratio sample exhibits a conductivity of 6.9 × 10 −5 S/cm (n = 3), which is in the range of the conductivity of a semiconductor or electrically conductive polymers [27]. One-way analysis of variance (ANOVA) with a p-value of 0.012 demonstrates statistically significant differences in electron conductivity among the 100%, 50%, and 0% BR ratio samples. Notably, incorporating BR into a gelatin device increases the ON/OFF current ratio by approximately 2-fold, which is likely due to the additional protons and proton vacancies introduced during the writing process.
Resistive memory devices store and access information by encoding "0" and "1" based on the electrical bistability of the active layer. Achieving a high current ratio of the ON state to the OFF state is often desirable [23,24]. Figure 1b,c show the electrical memory characteristics of the BR-incorporated gelatin memory device. The voltage sweep measurement, ranging from −0.09 to +0.09 V at a sweep rate of 0.36 V/s, was used to record the current. Figure 1b shows that the typical BR-incorporated gelatin hydrogel behaves as an ideal capacitor in the OFF state, similar to bare gelatin gel. Following the illumination writing process, the current of the 100% BR ratio sample increases to 4.0 × 10 −3 A at 0.09 V, and it acts as a resistor in the ON state. The ON/OFF current ratio reaches 1.6 × 10 5 , which is two orders of magnitude higher than those in previous studies using bio-related materials for constructing memory devices [25,26]. Table 1 displays the conductivity of BRincorporated gelatin hydrogel with different BR density ratios in the ON state. Increasing the density ratio of BR leads to enhanced conductivity. The 100% BR ratio sample exhibits a conductivity of 6.9 × 10 −5 S/cm (n = 3), which is in the range of the conductivity of a semiconductor or electrically conductive polymers [27]. One-way analysis of variance (ANOVA) with a p-value of 0.012 demonstrates statistically significant differences in electron conductivity among the 100%, 50%, and 0% BR ratio samples. Notably, incorporating BR into a gelatin device increases the ON/OFF current ratio by approximately 2-fold, which is likely due to the additional protons and proton vacancies introduced during the writing process. This type of memory also demands repeatable switching between the ON and OFF states, and it often relies on voltage or current for the reprogrammability [24,28]. To assess the stability and reprogrammability of the BR-incorporated gelatin device, we conducted tests for retention time and WRER (write-read-erase-reread) cycles, as depicted in Figures 2a and 2b, respectively. Figure 2a demonstrates that at 0.09 V, the increased current of the 100% BR-incorporated gelatin device can be maintained in the ON state for up to 5 days. Furthermore, Figure 2b reveals that the device is capable of enduring five WRER cycles, signifying its potential application in non-volatile memories. This type of memory also demands repeatable switching between the ON and OFF states, and it often relies on voltage or current for the reprogrammability [24,28]. To assess the stability and reprogrammability of the BR-incorporated gelatin device, we conducted tests for retention time and WRER (write-read-erase-reread) cycles, as depicted in Figure 2a and Figure 2b, respectively. Figure 2a demonstrates that at 0.09 V, the increased current of the 100% BR-incorporated gelatin device can be maintained in the ON state for up to 5 days. Furthermore, Figure 2b reveals that the device is capable of enduring five WRER cycles, signifying its potential application in non-volatile memories.
Net Proton Flow toward the Deposited Side of the BR-Incorporated Membrane Revealed by Photocurrent Measurements
We hypothesized that the enhanced performance of the gelatin device can be attributed to the non-electroneutrality in the gelatin films caused by BR proton pumping. During the writing process, if the BRs in the sandwiched membrane can provide a net proton flow toward either the deposition side or the counter side of the gelatin films, the imbalanced proton concentrations can give rise to non-electroneutral states in both gelatin films.
To determine the direction of the proton flow during the writing process, photocurrent measurements were conducted in the OFF state gelatin devices which still have abundant free bulk water for BRs to properly function. The presence of a photocurrent would indicate the existence of a net proton flow, with the direction of the photocurrent indicating the direction of proton movement [29,30]. Figure 3 illustrates the photocurrent results obtained from BR-incorporated devices with two different density ratios of BR (100% and 50%) as well as a device containing only a DOPC membrane without BR (0%). The device geometries are depicted in Figure 3a. During the illumination period of 3 to 8 s for the BRincorporated gelatin devices, photocurrents were observed flowing from the deposited
Net Proton Flow toward the Deposited Side of the BR-Incorporated Membrane Revealed by Photocurrent Measurements
We hypothesized that the enhanced performance of the gelatin device can be attributed to the non-electroneutrality in the gelatin films caused by BR proton pumping. During the writing process, if the BRs in the sandwiched membrane can provide a net proton flow toward either the deposition side or the counter side of the gelatin films, the imbalanced proton concentrations can give rise to non-electroneutral states in both gelatin films.
To determine the direction of the proton flow during the writing process, photocurrent measurements were conducted in the OFF state gelatin devices which still have abundant free bulk water for BRs to properly function. The presence of a photocurrent would indicate the existence of a net proton flow, with the direction of the photocurrent indicating the direction of proton movement [29,30]. Figure 3 illustrates the photocurrent results obtained from BR-incorporated devices with two different density ratios of BR (100% and 50%) as well as a device containing only a DOPC membrane without BR (0%). The device geometries are depicted in Figure 3a. During the illumination period of 3 to 8 s for the BR-incorporated gelatin devices, photocurrents were observed flowing from the deposited side electrode to the counter side in the external circuit (Figure 3b). The magnitude of the photocurrent increased with the BR density ratio in the membrane. These findings suggest that the BR-incorporated membrane, positioned in the middle of the device, facilitates a net proton flow from the counter side to the deposited side of the gelatin film.
It is important to note that the physical state of the BR-incorporated membrane within the gelatin device has not been elucidated. Whether the prepared liposomes rupture to form supported lipid bilayers or crumple on the gelatin film remains unclear. Additionally, the specific orientation of BR within the membrane is also unclear. Nevertheless, what we can affirm is the presence of an overall net proton flow from the counter side to the deposition side of the gelatin device, leading to non-electroneutrality on both sides of the gelatin films.
The Proton Pumping Enhances the Conductivity of the Gelatin Device in the ON State
To further investigate how the incorporation of BRs increases electron conductivity, we employed two different heating methods for the writing processes: hotplate heating and illumination heating. During hotplate heating, BRs remain inactive, since they require light absorption to pump protons. In contrast, during illumination heating, BRs contribute additional protons and proton vacancies to the writing process. The voltage sweep curve in Figure 4a demonstrates that the conductivity values for all devices were nearly identical Conversely, in the bare gelatin device and the 0% BR device, small negative currents were observed. The negative currents are likely attributed to the delocalization of electrons of the ITO plates, which is induced by the ultraviolet band in the light [31]. Since the system was illuminated from the deposited side, the ITO at the deposited side had a higher likelihood of electron delocalization and pressure compared to the ITO plate at the counter side. As a result, electron movement occurred from the deposited side electrode to the counter side electrode in the external circuit, which is equivalent to a current from the counter side electrode to the deposited side electrode in the external circuit.
It is important to note that the physical state of the BR-incorporated membrane within the gelatin device has not been elucidated. Whether the prepared liposomes rupture to form supported lipid bilayers or crumple on the gelatin film remains unclear. Additionally, the specific orientation of BR within the membrane is also unclear. Nevertheless, what we can affirm is the presence of an overall net proton flow from the counter side to the deposition side of the gelatin device, leading to non-electroneutrality on both sides of the gelatin films.
The Proton Pumping Enhances the Conductivity of the Gelatin Device in the ON State
To further investigate how the incorporation of BRs increases electron conductivity, we employed two different heating methods for the writing processes: hotplate heating and illumination heating. During hotplate heating, BRs remain inactive, since they require light absorption to pump protons. In contrast, during illumination heating, BRs contribute additional protons and proton vacancies to the writing process. The voltage sweep curve in Figure 4a demonstrates that the conductivity values for all devices were nearly identical when hotplate heating was used, indicating that inactive BRs did not significantly contribute to the conductivity. On the other hand, when the illumination writing process was applied, the conductivities increased with the BR density ratio in the membrane (Figure 4b). These findings support the notion that the increase in conductivity in the ON state is primarily Gels 2023, 9, 635 6 of 12 attributed to the proton pumping activity of BRs rather than the mere presence of the BR material. Gels 2023, 9, 635 6 of 12 when hotplate heating was used, indicating that inactive BRs did not significantly contribute to the conductivity. On the other hand, when the illumination writing process was applied, the conductivities increased with the BR density ratio in the membrane ( Figure 4b). These findings support the notion that the increase in conductivity in the ON state is primarily attributed to the proton pumping activity of BRs rather than the mere presence of the BR material.
The Increased Conductivity Is Not Due to the pH Change
As BR proton pumping can potentially alter the pH across the membrane [29,30], we investigated whether the increased conductivity was a result of pH change. We conducted experiments using gelatin films prepared at pH = 4.5 and pH = 7.6 to simulate varying pH values on either side of the BR membrane. We chose these two pH conditions since one was more acidic and the other was more basic than our typical working condition at pH = 5.8. Figure 5a,b display the electrical characteristics of these gelatin films in the OFF and ON states. The conductivities of the gelatin films at different pH values were found to be similar and are summarized in Table 2. It is apparent that the conductivity values of the gelatin films at pH = 4.5, 7.6, and the combination of pH 4.5 and 7.6 were comparable to that of the gelatin films at pH = 5.8. None of these conditions can elevate the conductivity to the level achieved by BR-incorporated gelatin films. These findings strongly suggest that the improved conductivity is not attributable to variations in the acidic or basic conditions.
The Increased Conductivity Is Not Due to the pH Change
As BR proton pumping can potentially alter the pH across the membrane [29,30], we investigated whether the increased conductivity was a result of pH change. We conducted experiments using gelatin films prepared at pH = 4.5 and pH = 7.6 to simulate varying pH values on either side of the BR membrane. We chose these two pH conditions since one was more acidic and the other was more basic than our typical working condition at pH = 5.8. Figure 5a,b display the electrical characteristics of these gelatin films in the OFF and ON states. The conductivities of the gelatin films at different pH values were found to be similar and are summarized in Table 2. It is apparent that the conductivity values of the gelatin films at pH = 4.5, 7.6, and the combination of pH 4.5 and 7.6 were comparable to that of the gelatin films at pH = 5.8. None of these conditions can elevate the conductivity to the level achieved by BR-incorporated gelatin films. These findings strongly suggest that the improved conductivity is not attributable to variations in the acidic or basic conditions. 3.2 ± 0.5 4.5/7. 6 3.7 ± 0.7 7.6/7. 6 3.3 ± 0.6
Raman Spectroscopy Analysis Shows Increased Amounts of Bound Water and a Mor dered Gelatin Structure in the ON State Gelatin Devices
To investigate whether the presence of BR proton pumping activity could e the bound water content in the ON state gelatin devices, the Raman spectra of devices at pH = 4.5, 5.8, and 7.6 without BR membranes were compared with that w membranes. The peaks observed at 3320 cm -1 and 3450 cm -1 correspond to the OH ing vibrations of tightly bound water [32][33][34], which can be utilized to quantify the of bound water. The peaks in the range of 2800-3000 cm -1 originate from the CH str of gelatin. To ensure comparability, all spectra were scaled to achieve the same in of the CH stretching, thereby allowing for the normalization of bound water OHing intensities by the gelatin content. Figure 6 presents the Raman spectra of the gelatin films in the BR-incorporate tin devices, either the deposited side or the counter side, and they exhibited higher water contents compared to the devices without BR membranes at various pH There were no significant differences in the bound water contents among the gel vices with different pH values. The higher bound water content suggests that the polypeptides were in a more unwound state, thereby providing more amino acids of forming hydrogen bonds with water molecules. The presence of additional wa diated hydrogen bonds within the protein-bound water network could facilitate e transfer, resulting in enhanced conductivity. These findings align with the increas ductivity observed through BR proton pumping, as indicated in Table 1.
Raman Spectroscopy Analysis Shows Increased Amounts of Bound Water and a More Disordered Gelatin Structure in the ON State Gelatin Devices
To investigate whether the presence of BR proton pumping activity could enhance the bound water content in the ON state gelatin devices, the Raman spectra of gelatin devices at pH = 4.5, 5.8, and 7.6 without BR membranes were compared with that with BR membranes. The peaks observed at 3320 cm −1 and 3450 cm −1 correspond to the OH stretching vibrations of tightly bound water [32][33][34], which can be utilized to quantify the amount of bound water. The peaks in the range of 2800-3000 cm −1 originate from the CH stretching of gelatin. To ensure comparability, all spectra were scaled to achieve the same intensity of the CH stretching, thereby allowing for the normalization of bound water OH-stretching intensities by the gelatin content. Figure 6 presents the Raman spectra of the gelatin films in the BR-incorporated gelatin devices, either the deposited side or the counter side, and they exhibited higher bound water contents compared to the devices without BR membranes at various pH values. There were no significant differences in the bound water contents among the gelatin devices with different pH values. The higher bound water content suggests that the gelatin polypeptides were in a more unwound state, thereby providing more amino acids capable of forming hydrogen bonds with water molecules. The presence of additional water-mediated hydrogen bonds within the protein-bound water network could facilitate electron transfer, resulting in enhanced conductivity. These findings align with the increased conductivity observed through BR proton pumping, as indicated in Table 1.
Raman spectra covering the protein amide region were also examined to study how the protein secondary structure is influenced by the BR proton pumping. Previous studies have indicated that 1530-1650 cm −1 range in the Amide I spectrum region corresponds to the ordered secondary structure. A decrease in the peak intensity suggests the increased disorder of a protein structure [35][36][37]. In addition, the intensity reduction at 1268 cm −1 in the Amide III spectrum region indicates the loss of the triple-helix structure [38,39]. Figure 7a depicts the Raman spectra of a representative gelatin device without BR membranes. Comparing the ON state with the OFF state, lower intensities at 1530-1650 cm −1 and 1268 cm −1 suggest a loss of the triple-helix structure [38,39]. Figure 7b,c present the Raman spectra of the gelatin films at the deposited and counter sides of a 100% BR ratio memory device. In both cases, the spectra in the ON state exhibit significantly reduced intensities at 1530-1650 cm −1 and 1268 cm −1 compared to the OFF state spectra, indicating the loss of secondary structure. Notably, the decreases in the Raman intensities in the ON state BR-incorporated devices are more pronounced than those in the ON state gelatin device with no BR membranes, suggesting a more complete loss of secondary structure in the presence of BR membranes. Furthermore, an increase in intensity within the 1350-1460 cm −1 range, corresponding to CH, CH2, and CH3 vibrations, was observed in the ON state BR-incorporated gelatin device compared to the OFF state, implying greater flexibility in Raman spectra covering the protein amide region were also examined to study how the protein secondary structure is influenced by the BR proton pumping. Previous studies have indicated that 1530-1650 cm -1 range in the Amide I spectrum region corresponds to the ordered secondary structure. A decrease in the peak intensity suggests the increased disorder of a protein structure [35][36][37]. In addition, the intensity reduction at 1268 cm -1 in the Amide III spectrum region indicates the loss of the triple-helix structure [38,39]. Figure 7a depicts the Raman spectra of a representative gelatin device without BR membranes. Comparing the ON state with the OFF state, lower intensities at 1530-1650 cm -1 and 1268 cm -1 suggest a loss of the triple-helix structure [38,39]. Figure 7b,c present the Raman spectra of the gelatin films at the deposited and counter sides of a 100% BR ratio memory device. In both cases, the spectra in the ON state exhibit significantly reduced intensities at 1530-1650 cm -1 and 1268 cm -1 compared to the OFF state spectra, indicating the loss of secondary structure. Notably, the decreases in the Raman intensities in the ON state BR-incorporated devices are more pronounced than those in the ON state gelatin device with no BR membranes, suggesting a more complete loss of secondary structure in the presence of BR membranes. Furthermore, an increase in intensity within the 1350-1460 cm -1 range, corresponding to CH, CH2, and CH3 vibrations, was observed in the ON state BR-incorporated gelatin device compared to the OFF state, implying greater flexibility in the gelatin polypeptide backbones. This increased flexibility supports the notion of more extensive unwinding of the gelatin polypeptides in the ON state BRincorporated devices. Raman spectra covering the protein amide region were also examined to study how the protein secondary structure is influenced by the BR proton pumping. Previous studies have indicated that 1530-1650 cm -1 range in the Amide I spectrum region corresponds to the ordered secondary structure. A decrease in the peak intensity suggests the increased disorder of a protein structure [35][36][37]. In addition, the intensity reduction at 1268 cm -1 in the Amide III spectrum region indicates the loss of the triple-helix structure [38,39]. Figure 7a depicts the Raman spectra of a representative gelatin device without BR membranes. Comparing the ON state with the OFF state, lower intensities at 1530-1650 cm -1 and 1268 cm -1 suggest a loss of the triple-helix structure [38,39]. Figure 7b,c present the Raman spectra of the gelatin films at the deposited and counter sides of a 100% BR ratio memory device. In both cases, the spectra in the ON state exhibit significantly reduced intensities at 1530-1650 cm -1 and 1268 cm -1 compared to the OFF state spectra, indicating the loss of secondary structure. Notably, the decreases in the Raman intensities in the ON state BR-incorporated devices are more pronounced than those in the ON state gelatin device with no BR membranes, suggesting a more complete loss of secondary structure in the presence of BR membranes. Furthermore, an increase in intensity within the 1350-1460 cm -1 range, corresponding to CH, CH2, and CH3 vibrations, was observed in the ON state BR-incorporated gelatin device compared to the OFF state, implying greater flexibility in the gelatin polypeptide backbones. This increased flexibility supports the notion of more extensive unwinding of the gelatin polypeptides in the ON state BRincorporated devices.
Proposed Mechanism of How Non-Electroneutrality Influences the Gelatin-Bound Water Network in the ON State
Based on the electrical performance measurements and the Raman spectra obtained under various conditions, we propose a mechanism (Figure 8) in which the non-electroneutrality induced by BR proton pumping enhances the conductivity of a gelatin device in the ON state. During the illumination writing process, proton pumping across the membrane leads to non-electroneutrality on both sides of the gelatin films. This non-electroneutrality could induce electrostatic repulsion between individual gelatin polypeptide strands, promoting the unwinding of the triple-helical structure. This structural change, facilitated by nonelectroneutrality, likely prevents the self-association of gelatin single strands as the triple helices unwind. With more unwound strands, more bound water molecules can trap and stay in the gelatin matrix during the writing process. This process allows more routes for electron transport in the gelatin-bound water network, and therefore, the conductivity increases. troneutrality could induce electrostatic repulsion between individual gelatin polypeptide strands, promoting the unwinding of the triple-helical structure. This structural change, facilitated by non-electroneutrality, likely prevents the self-association of gelatin single strands as the triple helices unwind. With more unwound strands, more bound water molecules can trap and stay in the gelatin matrix during the writing process. This process allows more routes for electron transport in the gelatin-bound water network, and therefore, the conductivity increases. Figure 8. Illustration of the effect of non-electroneutrality on gelatin structure after the writing process. During the writing process, non-electroneutrality induces a more thorough unwinding of the triple-helical structure into single polypeptide strands. This unwinding exposes a greater number of hydrogen-bonding sites on the polypeptides, facilitating the formation of hydrogen bonds with bound water molecules. As a result, the gelatin-bound water network exhibits a higher bound water content, enabling multiple pathways for electron transport, which in turn leads to an increase in device conductivity.
Conclusions
Our study revealed that the incorporation of the proton-pumping protein bacteriorhodopsin (BR) through an illumination writing process has a significant impact on the gelatin-bound water network, leading to enhanced conductivity in the gelatin memory device. We demonstrated that the conductivity in the ON state can be further increased by augmenting the BR density ratio in the membrane incorporated in a gelatin device. To establish the underlying mechanism, we employed different writing methods and created varying pH environments for the gelatin, enabling us to attribute the conductivity enhancement to the non-electroneutrality induced by BR proton pumping. Raman spectroscopy analyses supported our findings by indicating that BR proton pumping promotes the unwinding of the gelatin's triple-helical structure. The more unwound gelatin polypeptide chains could be capable of forming additional hydrogen bonds with water molecules. This phenomenon allows for the trapping of a greater number of bound water mol- Figure 8. Illustration of the effect of non-electroneutrality on gelatin structure after the writing process. During the writing process, non-electroneutrality induces a more thorough unwinding of the triple-helical structure into single polypeptide strands. This unwinding exposes a greater number of hydrogen-bonding sites on the polypeptides, facilitating the formation of hydrogen bonds with bound water molecules. As a result, the gelatin-bound water network exhibits a higher bound water content, enabling multiple pathways for electron transport, which in turn leads to an increase in device conductivity.
Conclusions
Our study revealed that the incorporation of the proton-pumping protein bacteriorhodopsin (BR) through an illumination writing process has a significant impact on the gelatin-bound water network, leading to enhanced conductivity in the gelatin memory device. We demonstrated that the conductivity in the ON state can be further increased by augmenting the BR density ratio in the membrane incorporated in a gelatin device. To establish the underlying mechanism, we employed different writing methods and created varying pH environments for the gelatin, enabling us to attribute the conductivity enhancement to the non-electroneutrality induced by BR proton pumping. Raman spectroscopy analyses supported our findings by indicating that BR proton pumping promotes the unwinding of the gelatin's triple-helical structure. The more unwound gelatin polypeptide chains could be capable of forming additional hydrogen bonds with water molecules. This phenomenon allows for the trapping of a greater number of bound water molecules within the gelatin-bound water network, thereby enhancing the efficiency of electron transfer. Our findings provide valuable insights into the influence of non-electroneutrality on gelatin structure and its impact on the conductivity and performance of the memory devices. This research opens up promising opportunities for enhancing the functionality and conductivity of gelatin-based devices by using the non-electroneutrality effects induced by proton-pumping proteins such as BR.
BR-Incorporated Liposome Preparation
The HmBRI/D94N bacteriorhodopsin mutant was obtained from Professor Chii-Shen Yang's group at National Taiwan University. To express HmBRI, the mutant plasmid was transformed into E. coli and allowed to undergo overexpression [40]. Subsequently, HmBRI was purified using octyl glucoside as a detergent. To incorporate HmBRI into liposomes, purified HmBRI was mixed with a 50 mg/mL solution of DOPC (1,2-dioleoyl-sn-glycero-3phosphocholine) liposomes at a weight ratio of 1:100. The mixture was then dialyzed for 24 h at 4 • C to remove the octyl glucoside detergent, allowing HmBRI to insert into the Gels 2023, 9, 635 10 of 12 DOPC liposomes. To obtain liposomes with various BR density ratios, a mixture of the prepared BR-incorporated DOPC liposomes and pure DOPC liposomes at a concentration of 50 mg/mL was prepared, using different volume ratios as required.
Preparation of Gelatin Solution
To prepare a 10 wt% gelatin solution, 1 g of Type B gelatin powder (Sigma-Aldrich, St. Louis, MO, USA) was added to 10 g of 100 mM NaCl solution (pH = 5.8). The mixture was gently stirred for 2 h at 60 • C to ensure complete dissolution and achieve a homogeneous gelatin solution.
Fabrication of BR-Incorporated Gelatin Memory Devices
First, 200 µL of 60 • C gelatin solution was placed on an ITO plate (7 Ω, 2 × 2.5 cm 2 in size Ruilong-glass Co., Ltd., Bengbu, China) and immediately spincoated at 2000 rpm for 20 s at 25 • C. The gelatin thin films were then dried at 4 • C for 30 min. Subsequently, liposomes were added onto the gelatin films for 1 h, and then, the samples were washed with 100 mL of 100 mM NaCl solution (pH 5.8). To fabricate the BR-incorporated gelatin memory device, the gelatin-coated ITO plate with the deposited BR membrane was sandwiched with another gelatin-coated ITO plate.
Electrical Performance Measurements
Electrical performance measurements were conducted using a Keithley 2636B sourcemeter from TEKTRONIX, Inc. The voltage sweep current-voltage (I-V) characteristics were recorded under a sweep rate of 0.36 V/s without light illumination. For the measurement of current-time characteristics, the gelatin-based devices were illuminated using a Xenon lamp system (Prosper OptoElectronic Co., Ltd., Yilan, Taiwan) of AM1.5. The stability performance of the gelatin-based memory device was tested at a reading voltage of +0.09 V. Prior to the reading measurements, the devices were illuminated for 1 h during the writing process and then cooled to ambient temperature. The erasing process was accomplished by immersing the devices in a 100 mM NaCl solution (pH = 5.8) for 1 h. All measurements were performed under environmental conditions with a relative humidity of 40%.
Raman Spectroscopy to Examine the Bound Water Content and Gelatin Secondary Structure
Raman measurements were carried out using the inVia™ confocal Raman microscope (Renishaw, Wotton-under-Edge, UK) equipped with a He-Ne laser for 633 nm excitation. The average laser power was set at 14.1 mW. Renishaw WiRE 5.0 software was used for data retrieval and analysis. All Raman spectra, including the gelatin and water OHstretch regions, were normalized to have the same intensity for the CH-stretch band at 2950 cm −1 , representing the gelatin content in the samples. Additionally, the spectra containing the amide band were normalized to obtain the same intensity for the CH 2 wag band at 1450 cm −1 . All spectra were subjected to smoothing and baseline subtraction to eliminate the background signal. | 2023-08-10T15:06:45.831Z | 2023-08-01T00:00:00.000 | {
"year": 2023,
"sha1": "c456533f8a5696a71d6a2c71023b612c7b444afb",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2310-2861/9/8/635/pdf?version=1691463475",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "1f14f88019b71c729c3bcb5ce5125bf6b872636c",
"s2fieldsofstudy": [
"Materials Science",
"Chemistry"
],
"extfieldsofstudy": [
"Medicine"
]
} |
245567309 | pes2o/s2orc | v3-fos-license | Minimizing misclassification bias with a model to identify acetabular fractures using health administrative data
Abstract Acetabular fractures (AFs) are relatively uncommon thereby limiting their study. Analyses using population-based health administrative data can return erroneous results if case identification is inaccurate (‘misclassification bias’). This study measured the impact of an AF prediction model based exclusively on administrative data upon misclassification bias. We applied text analytical methods to all radiology reports over 11 years at a large, tertiary care teaching hospital to identify all AFs. Using clinically-based variable selection techniques, a logistic regression model was created. We identified 728 AFs in 438,098 hospitalizations (15.1 cases/10,000 admissions). The International Classification of Disease, 10th revision (ICD-10) code for AF (S32.4) missed almost half of cases and misclassified more than a quarter (sensitivity 51.2%, positive predictive value 73.0%). The AF model was very accurate (optimism adjusted R2 0.618, c-statistic 0.988, calibration slope 1.06). When model-based expected probabilities were used to determine AF status using bootstrap imputation methods, misclassification bias for AF prevalence and its association with other variables was much lower than with International Classification of Disease, 10th revision S32.4 (median [range] relative difference 1.0% [0%–9.0%] vs 18.0% [5.4%–75.0%]). Lone administrative database diagnostic codes are inadequate to create AF cohorts. The probability of AF can be accurately determined using health administrative data. This probability can be used in bootstrap imputation methods to importantly reduce misclassification bias.
Introduction
Although pelvic and acetabular fractures (AFs) account for only 1.5% of adult fractures, they are complex to treat. [1] AF incidence ranges between 3 and 9.5 per 100,000 and typically follows a bi-modal age distribution. [2][3][4] Mechanisms of injury typically varies by age with high-energy trauma primarily responsible for AF in the young and while low-energy injuries, such as falling from a standing height, commonly cause AF in the elderly. [2] AF incidence has quadrupled over the past 4 decades but seems to have recently stabilized. [5] Epidemiological studies focusing on AF are relatively uncommon (Table 1). These studies involve multiple countries including Finland, [2] Scotland, [3] France, [6] and the United States. [4,7] These data suggest that the preferred AF management in the young clearly consists of open reduction and internal fixation. In the elderly population, however, best management remains unclear due to the anatomical and medical implications involved in their treatment. AFs in the elderly have been of particular interest to trauma and arthroplasty surgeons alike since such elderly patients, compared to age matched patients with hip fracture, have longer hospital stays and higher risk-adjusted mortality. [8] To date, outcome studies of the elderly AFs are limited to retrospective cohorts of limited size. [9][10][11] The lack of epidemiological studies focusing on management and outcomes of elderly AF patients hampers its study and advancement. This issue could be solved with population-based health administrative databases. Several population-based studies have created AF cohorts using diagnostic codes within hospitalization abstracts (Table 1). To our knowledge, however, the accuracy of diagnostic codes for AF has not been measured and no algorithms to identify patients with AF within health administrative data have been published or assessed. Measuring and optimizing the accuracy of AF identification using administrative data is essential to accurately study AF using population-based health administrative data with bias from case misclassification. In this study, we derived and internally validated a model using health administrative data which returns the probability that an AF was identified during a hospitalization. We then compared misclassification bias with AF identification using this model to that using single diagnostic codes for AF.
Study setting
The study took place at The Ottawa Hospital, a 1000-bed teaching hospital with 2 campuses that is the tertiary referral institution and trauma center in our region of approximately 1.3 million people. Annually, The Ottawa Hospital has more than 175,000 emergency department visits, 40,000 non-psychiatric admissions, and 50,000 surgical cases. The study was approved by the Ottawa Health Science Network Research Ethics Board (File: 20210026-01H).
Case identification
Our goal was to detect all AFs diagnosed at The Ottawa Hospital by reviewing the text reports of all computerized tomographic (CT) studies of the pelvis. To create this text search algorithm, we first collated a sample of AFs identified in our hospital's trauma registry. The trauma registry includes all patients who: presented to the hospital as a trauma code; were admitted under the trauma service; or had an injury severity score of 12 or higher upon presentation to the hospital. Detailed diagnostic, radiological, procedural, and outcome information is collected prospectively on each patient. For a separate analysis, we had retrieved the CT pelvis reports of all patients with radiographically confirmed AF in the trauma registry from January 2008 to December 2013 and January 2016 to December 2018. Using clinical experience and text analysis of this sample, we derived a text search algorithm that identified all CT pelvis reports indicating AF in this sample.
We then applied this text search algorithm to the reports of all CT pelvis studies at our hospital between January 1, 2008 and December 31, 2018. This time frame was chosen because of data availability. The reports of all CTs that were AF screen-positive were then manually reviewed (by AA) to identify patients with true AF. These people constituted all AF cases radiographically diagnosed at The Ottawa Hospital during the study period.
Creation of the AF model
We assumed that diagnostic and procedural codes would be important for a model that returned the probability of AF based exclusively on administrative data. However, the number of distinct codes present in the discharge abstract of a large group of patients can be extensive. To identify diagnostic and procedural codes that might identify AF, we retrieved for the AF cases (identified in the previous step) all International Classification of Diseases, 10 th revision (ICD-10) diagnostic codes and all Canadian Classification of Intervention procedural codes registered in their hospital discharge abstracts. Diagnostic and procedural codes were grouped by their first 3 and 5 alphanumerics, respectively, with the exception of the ICD-10 code specific to AF (S32.4). Code groups that were present in at least 5% of cases were independently ranked by 2 study members (AA, GG) regarding their clinical sensibility and likelihood to distinguish between patients with and without AF.
We then identified all non-psychiatric adult hospitalizations (defined as age exceeding 14) between January 1, 2008 and December 31, 2018 using our hospital's discharge abstract database. This dataset was linked to all AF cases to determine patients who were diagnosed with AF during their admission. We determined for each person the values of covariates from the hospital discharge abstract that we felt might be important to identify AF status including: age and sex; encounter urgency and ambulance status; hospitalization service; status of the diagnostic and procedural codes identified in the previous step; hospital length of stay; and death status.
Binomial logistic regression was then used to create a model that returned the probability of AF during each admission. To help prevent over-fitting, we limited the number of variables offered to the model to ensure no fewer than 20 degrees of freedom per AF case. To account for possible non-linear associations between continuous variables (i.e., age and hospital length of stay) and AF status, we used fractional polynomials with 2 terms identified with a transformation identification macro from Sauerbrei et al, [12] thereby consuming a total of 4 degrees of freedom for each continuous variable. We did not use univariate inferential testing to screen for variable inclusion; instead, we ranked all variables by potential model relevance based on clinical experience and offered to the model all variables whose cumulative sum (degrees of freedom) was no less than one twentieth the total number of AF cases in our cohort. These variables were then all entered in the model with no subsequent variable selection based on their statistical association with AF status. This approach was used to minimize biased parameter estimates from variable selection using inferential testing ("testimation bias"). [13] We did not use other binary modelling methods, such as classification trees, random forests, neural networks, or support vector machines, because they do not necessarily improve model calibration. [14] Model performance was internally validated using optimism corrected c-statistics (for discrimination) and calibration in the large (for calibration) using methods described by Steyer- Table 1 Description of published acetabular fracture cohort studies.
Study
Country Case identification method Sampling frame Time period N Rinne [2] Finland ICD-10 S32.4 (1 * or 2 * ) All hospitals 1997-2014 5022 Laird [3] Scotland Trauma registry query Single hospital 1998-2003 351 Melhem [6] France Not reported All hospitals 2006-2016 32,614 Ferguson [7] USA Case registry all surgeries by single surgeon Single hospital 1980-2007 1309 Best [4] USA ICD-9-CM 808.0 or 808. [13,15] using 200 bootstrap samples. We used Youden method to identify the expected AF probability having the greatest ability to discriminate between patients by AF status. [16] We also measured the sensitivity of our model by using it to determine the expected AF probability of all AF patients from the trauma registry who were not used to derive our AF text search algorithm for pelvic CTs (i.e., all AF in the trauma registry admitted from January 1, 2014 to December 31, 2015). We then quantified misclassification bias. First, we used the reference standard AF status to calculate true values of 9 statistics (prevalence of AF in study cohort; the association of AF with 2 continuous variables [age, hospital length of stay]; and the association of AF with 6 categorical variables [sex, admission urgency and ambulance status, packed red blood cell transfusions during the hospitalization, death status, and hospital procedure status]). With the exception of the latter (which was deemed present if patients had any Canadian Classification of Interventions code starting with '1' [indicating therapeutic intervention]), none of these statistics relied on administrative database codes and are very accurately measured. [17] We repeated measurement of these 9 statistics again after determining AF status using the ICD-10 code of S32.4 and the AF model. The latter approach used bootstrap imputation methods. [18][19][20] Bootstrap imputation started by creating 1000 random bootstrap samples (with replacement) of the study cohort, each with a sample size identical to the original cohort. For each patient within each bootstrap sample, a uniformly distributed number between 0 and 1 was randomly selected; AF was imputed to be present if the random number was below the expected probability of AF for that patient (as determined from the AF model). Within each bootstrap sample, we measured all 9 statistics with the median value of all 1000 bootstrap samples used as the final point estimate and the 2.5 th and 97.5 th percentiles as the confidence intervals. We quantified misclassification bias as the unsigned relative difference in each of the 9 statistics compared to that achieved using true AF status. All analyses were conducted using SAS 9.4 (Cary, NC, USA).
Results
We initially identified 207 AF patients from our hospital's trauma registry. One hundred seventy-six of these patients (85.0%) had their acetabular imaging conducted at our hospital with CT reports available for analysis. All reports had at least 1 of the 4 text combinations that we found were important to identify AFs. This screen was then applied to the radiological reports of all pelvic CT studies at our hospital during the study period (n = 296,588) and identified 1804 screen-positive reports conducted on 1558 patients. Manual review of these reports identified 1117 AFs in 908 patients. Of the latter, 728 unique patients were hospitalized when they were diagnosed with AF; the other cases had been transferred directly from our emergency department to another institution following their assessment at our center or were diagnosed based on ambulatory imaging.
During the 11-year study period, there were 438,098 nonpsychiatric adult admissions to the hospital resulting in an AF incidence of 15.1 per 10,000 hospitalizations per year. AF admissions were distinct from other hospitalizations ( Table 2). AF patients were slightly older and were much more likely to be male, arrive to the hospital by ambulance, or be admitted urgently. More than two-thirds of AFs were treated by the orthopedic or trauma service compared to only 10.1% of non-AF admissions. The diagnostic code for AF (i.e., ICD-10 "S32.4") was present in only 51.2% of AFs and only 73.0% of patients with this code had an AF (i.e., S32.4 sensitivity and positive predictive value of 51.2% and 73.0%, respectively). A diagnostic code starting with "S32" was present in 33.9% of fractures but less than 1% of non-cases. Other relevant diagnostic codes most commonly present in AF related to co-injuries or injury mechanisms commonly found in AF. Similarly, the most common procedural codes identified in AF patients dealt with fixation of the pelvis or other loco-regional bones as well as local imaging studies. Hospital length of stay for AFs was much longer than the average but death risk was not distinctive.
Most of the selected covariates were significantly associated with AF status (Table 3). After adjustment for all model covariates, AF likelihood increased slightly with age but did not vary by sex. AF was notably more likely when patients were admitted urgently and under the orthopedics or trauma team. .56]) were the procedural codes having the strongest association with AF. The optimism-corrected overall model fit (Nagelkirke R 2 : 0.618), discrimination (c-statistic: 0.988), and calibration (calibration slope: 1.06) was excellent. In patients without and with AF, the median (interquartile range; 5 th -95 th percentile) expected AF probability distribution was 0.01% (0.001%-0.03%; 0.00007%-0.008%) and 45.9% (6.2%-85.6%; 0.07%-98.5%), respectively. In 60 patients with AF from our trauma registry who were not used to generate our text search algorithm, the median (interquartile range; 5 th -95 th percentile) expected probability of AF was similar at 33.5% (7.1-71.3, 1.5%-99.4%).
Despite a very strong model, however, considerable misclassification occurred when we categorized the model-based expected AF probability (Table 4). We found that the most discriminating model-based expected AF probability was 0.09%. Using this cutpoint captured all but 44 of the 728 AF cases in the cohort (sensitivity 94.0%). However, only 3.7% of people with this expected AF probability or more actually had an AF (i.e., positive predictive value of 3.7%).
When AF status was determined using the AF model and bootstrap imputation, misclassification bias was always smallerwith 1 exceptionwhen compared to that using the ICD-10 code S32.4 (Fig. 1). True AF incidence was 16.6 per 10,000 admissions; using the AF model and bootstrap imputation returned an identical value (Fig. 1A). In contrast, ICD-10 code of S32.4 significantly underestimated incidence by 29.5%. With the exception of "any procedure" (Fig. 1F), misclassification bias of association measures using the AF model and bootstrap imputation (median [range] unsigned relative difference 1.0% [0%-9.0%]) was always smaller than that when ICD-10 code S32.4 was used for case identification (median [range] unsigned relative difference 18.0% [5.4%-75.0%]). In most cases, estimates were biased towards the null; however, the association of AF using S32.4 with sex was significantly greater than true values (Fig. 1C).
Discussion
Population-based health administrative databases are very attractive for studying uncommon conditions like AF. However, accurately identifying rare conditions in administrative data is always a challenge. In this study, we used our hospital's data warehouse to identify every AF case diagnosed at our hospital over 11 consecutive years. Using health administrative data, we found that the diagnostic code for AF had a sensitivity and positive predictive value of only 51% and 73%, respectively. Using this code to determine AF status underestimated AF prevalence by almost 30% and returned biased associations with other covariates. Using data found exclusively within health administrative databases, we created a very accurate model that returned the probability of AF for hospitalizations. When these expected probabilities were used to determine AF status using bootstrap imputation methods, misclassification bias was greatly reduced compared to that from using the ICD code for AF. Our study makes several important points. First, our results highlight the potential misclassification resulting from using a single diagnostic code to identify AF. Although S32.4 (the ICD-10 code for AF) was very strongly associated with AF (with an aOR of 1783 [95% confidence interval 1289-2465]), the creation of an AF cohort using this code alone would miss almost half of cases and a quarter of this cohort would not actually have AF. In addition, we found that associations measured using this code for AF case identification frequently returned values that were importantly distinct from true values (Fig. 1). These results indicate the caution one must use when interpreting results from studies using non-validated codes for case identification. Second, our model was well constructed using methods that addressed all of the key aspects highlighted in the PROBAST criteria [21] for predictive model assessment. These include factors involving study participants (appropriate data sources and inclusion criteria), predictors (defined and available predictors), outcomes (determined appropriately and standardized, independent of predictors or model), and analysis (reasonable number of participants, appropriate handling of continuous variables, inclusion of all enrolled participants in the analysis, model predictors selected without univariate screening, and model performance measured adjusting for optimism). The AF model demonstrated exemplary optimism-adjusted performance explaining more than 66% of the observed variation in the cohort. It also had almost perfect discrimination (c-statistic 0.988) and was very well calibrated. Despite having such an accurate model, there was extensive misclassification when we categorized the model's expected AF probability for case identification (Table 3). This seemingly paradoxical resulta very accurate case-probability model returning misclassified disease status when a probability cutpoint is usedhas been illustrated in other studies. [18,20] These results highlight the need to use analytical methods, such as bootstrap imputation, that account for uncertainty of case ascertainment when using health administrative data. When case probability estimates from our AF model (Table 3) were applied using bootstrap imputation methods, prevalence estimates and measures of association with (Fig. 1). These results highlight the power of applying an accurate case-identification model using statistical methods that account for determination uncertainty. Third, it is commonly believed that misclassification will bias estimates towards the null. Our results indicate that this is not always the case (Fig. 1C). Several issues should be kept in mind when assessing our results. First, our model has not been externally validated. This step will be important before it is applied to identify AF at a population-level. Second, our model can only be applied to health jurisdictions using other coding methods if 'cross-walks' are used to transform the codes used in our model to those native to the study center. Obviously, model accuracy should be reassessed if such steps are taken to confirm the validity of using this model. Third, it is likely that our AF case identification method will have missed some cases treated in our hospital during the study period. In our cohort of AF from the hospital's trauma registry, we found that 15% of patients did not have any imaging done at the hospital because imaging had been done at the referring hospital. When patients have no imaging done at our hospital, they will be missed by our case identification methods. However, the bias introduced into our model by this misclassification is unlikely to be extensive because of the overwhelming number of people in our cohort without AF.
In summary, we found that health administrative database diagnostic codes for AF are inadequate by themselves to create AF cohorts. We derived and internally validated a model that exclusively uses information available within health administrative database to return an accurate probability that AF is present during a particular hospitalization. When AF probability estimates were used to determine AF status using bootstrap imputation methods, misclassification bias was greatly reduced compared to that from using the ICD code for AF. If this model is validated in other centers, it could be used along with statistical methods accounting for its probabilistic nature to study AF at a population level. Table 4 Operating characteristics of categorized expected acetabular fracture probability. We used Youden method to identify the most discriminative threshold for expected AF probability from the AF model (Table 3). This returned a sensitivity of 94.0% but positive predictive value of only 3.7%. AF = acetabular fracture. This table presents parameter estimates, P value, and adjusted odds ratio (with 95%) of all variables in the acetabular fracture model. Adjusted odds ratios exceeding 1 are associated with an increased likelihood of acetabular fracture. Odds ratios for length of stay are not presented because its association with AF is non-linear. AF = acetabular fracture, CI = confidence interval, CT = computerized tomography, MRI = magnetic resonance imaging, US = ultrasound. † Excludes acetabular fractures (S32.4).
Adamczyk et al. Medicine (2021) 100:52 www.md-journal.com Figure 1. Misclassification bias when determining acetabular fracture status using the AF model or diagnostic code. This figure presents values for 9 statistics when AF status was determined with reference standard methods ("True"), with the AF model ( Table 2) using bootstrap imputation ("BI"), or with the ICD-10 code for AF ("S32.4"). These statistics include AF incidence and the association of AF with continuous variables (AGE, LENGTH OF STAY) or binary variables (remaining variables). Associations are presented with 95% confidence intervals and were measured using linear regression for continuous variables (presented as the parameter estimate "Estimate") or logistic regression for binary variable (presented as the odds ratios "OR"). AF = acetabular fracture, ICD = International Classification of Disease. | 2021-12-31T06:16:10.305Z | 2021-12-30T00:00:00.000 | {
"year": 2021,
"sha1": "239d1d919386a6a26e5c6597b49301465ef76ee7",
"oa_license": "CCBYNC",
"oa_url": "https://doi.org/10.1097/md.0000000000028223",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "6e4845fcefad93c549b6075952e210552e903f7a",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
254366534 | pes2o/s2orc | v3-fos-license | Determination of superexchange interactions for the CuO$_2$ chains in LiCu$_2$O$_2$
Starting from \textit{ab-initio} calculations, we derive a five-band Hubbard model to describe the CuO$_2$ chains of LiCu$_2$O$_2$. This model is further simplified to a low-energy effective Heisenberg model with nearest-neighbor (NN) $J_1$, and next-nearest-neighbor (NNN) $J_2$ interactions, combining perturbation theory, exact diagonalization calculations and Density Matrix Renormalization Group results. For realistic parameters we find the corresponding values of these interactions. The obtained effective model is consistent with a spiral-magnetic ground state as experimentally observed. Using symmetry arguments, the spiral state is a sufficient condition for the ferroelectricity observed in the system.
I. INTRODUCTION
Ferroelectric and magnetic materials have led to some of the most important technological advances to date. Ferroelectricity and magnetism combine in rather unusual materials called multiferroic, which offer the possibility to control the polarization by magnetic means and are expected to have technological applications [1][2][3][4][5][6][7][8] . In noncollinear magnets, one expects, on general symmetry arguments, a contribution to the electric polarization P ∼ e × S 1 × S 2 where e is a unit vector connecting sites 1 and 2 and S i , i = 1, 2 are the spins on each site 9,10 . Therefore, the existence of spiral magnetic order in chains is naturally expected to lead to ferroelectricity. Recently, the relevance of noncollinear antiferromagnets for low-field spin caloritronics and magnonics has been stressed 11 . In addition, the presence of a magnetic field is expected to lead to a considerable effect in the electric polarization of the compound 1,12 .
This is the case of the quantum quasi-one-dimensional compound LiCu 2 O 2 . There is experimental evidence of the emergence of ferroelectricity when the spiralmagnetic state of the spin 1/2 chains sets in 6 . Therefore, the system belongs to type-II multiferroics. The magnetic structures of the chains have been investigated by several experimental techniques [13][14][15][16][17][18][19][20][21] and ab-initio calculations 15 . In spite of some uncertainty in the parameters, the spiral order is believed to result from the competition of nearest-neighbor (NN) J 1 and next-nearestneighbor (NNN) J 2 interactions in a spin-1/2 Heisenberg chain. Classically, a spiral magnetic order takes place when J 2 > 0 (antiferromagnetic) and |J 1 | < 4J 2 22,23 . The quantum cases have been studied by numerical [22][23][24] and field-theoretical 23,[25][26][27] techniques and the spiral or-der is confirmed.
Nevertheless, a justification of the microscopic model is lacking. The ab-initio calculations 15,28 fail to reproduce the experimentally observed charge gap 29 . In addition, angle-resolved photoemission (ARPES) and optical measurements in single crystals of LiCu 2 O 2 29 show features that cannot be reproduced by the existing calculations of the electronic structure and point to the need to resort to strongly correlated models.
In this work we study the electronic structure of the CuO 2 chains and derive an effective Heisenberg model for them, taking into account the strongly correlated nature of the system, using a combination of different techniques. Using hopping matrix elements obtained with maximally localized Wannier functions derived from abinitio calculations, on-site Coulomb repulsions typical of the superconducting cuprates, and atomic values for the exchange energy of O 2p orbitals, we derive a five-band Hamiltonian for the CuO 2 chains, with one orbital per Cu atom and two orbitals per O atom. Using perturbation theory in the hopping matrix elements, we derive the effective Heisenberg model.
While this calculation sheds light on the different processes involved, due to the covalent nature of the compound, the perturbative results for the exchange interactions J 1 and J 2 are not accurate. Therefore, the values of these interactions are obtained by fitting the energies of the Heisenberg model to those of a multiband Hubbard model for a CuO chain with eight unit cells, for each wave vector and total spin projection.
The paper is organized as follows. In Section II we use first-principle calculations to determine the relevant orbitals and physical parameters of the CuO 2 chains and obtain the effective five-band Hubbard model, which in-cludes local Coulomb interactions. In Section III, by taking into account the experimental filling of one hole per unit cell, we perform a low-energy reduction of this model to one in which the degrees of freedom are the spins 1/2 at the Cu sites, giving rise to a Heisenberg model with NN and NNN exchange interactions. These interactions are calculated by perturbation theory in the hopping matrix elements. As this turns out to be inaccurate for realistic values of the parameters, in Section IV we obtain the Heisenberg parameters by fitting the lowest energy levels of a multiband Hubbard model, which we calculate numerically using exact diagonalization. For the determination of the value of the difference between on-site energies of holes at O and Cu sites ∆ we solve the fiveband Hubbard model using the Density Matrix Renormalization Group (DMRG) 30 . We present a summary and conclusions in Section V.
II. DERIVATION OF THE FIVE-BAND HUBBARD MODEL
The atomic structure of LiCu 2 O 2 , neglecting the spin spiral symmetry, is orthorhombic with space group P nma, where the short side is in the CuO 2 chain direction (a = 5.72Å, b = 2.86Å, c = 12.40Å). In the unit cell, the atoms are arranged in six planes perpendicular to the c-axis, where three are a mirror image of the others, with an offset in a and b-crystal directions of 1.52Åand 1.44Å, respectively 28 .
As is well known, there are two types of Cu atoms 17,21 ; one type is a non-magnetic monovalent cation (Cu + ), while the other type is a magnetic divalent cation (Cu 2+ ). The Cu + are located on planes perpendicular to the cdirection separating the Cu 2+ that form the CuO 2 chains in the b-direction. In addition, these CuO 2 chains are separated in the a-direction by chains of Li + ions, as can be seen in Fig 1a. As a consequence of this distribution, a natural cleavage plane exists between the CuO 2 chains, implying a relatively weak bonding between them. In the chain, the Cu 2+ atoms have four O nearest neighbors, forming a Cu-O-Cu angle of 94 degrees. This square of O atoms around the Cu 2+ ions is where all the dominant magnetic interactions occur. Since we focus on the magnetic chains, the Cu + lose relevance in our model and, henceforth, we will refer to the Cu 2+ ions as the Cu atoms.
In order to have an insight of the magnetic interactions in the chain, we perform ab-initio calculations within density-functional theory (DFT) by means of the Quantum Espresso code 31 . We use PAW pseudopotential with an energy cut of 80 eV for the Bloch wave functions along with GGA as the chosen exchange-correlation potential and the mesh in reciprocal space is 16x32x7. As expected, the relevant orbitals for the electronic structure of the CuO 2 chains are the 3d orbitals of Cu that point towards their NN O atoms and the two 2p orbitals of O that point towards their NN Cu atoms. We choose the direction of the chain in terms of unit vectors asx +ŷ, so that these relevant orbitals have symmetries d xy and p x , p y . See Fig. 1b. Density of state analysis (not shown) of a collinear magnetic configuration shows a strong hybridization between Cu-d xy and O-p x , p y orbitals. The magnetic moments of the atoms in the chain are 0.53µ B and 0.19µ B for Cu and O atoms, respectively. Right: density of states projected on each atom type. Note the difference between the Cu 2+ that belong to the chain, and the Cu + that belong to the plane.
The Hamiltonian takes the form
where H 0 contains the difference ∆ between on-site ener-gies of holes at O and Cu sites and the interaction terms: where the sum over i (j) runs over all Cu (O) sites, iσ creates a hole on the d xy orbital of Cu at site i with spin σ. The Coulomb interaction between two holes in the Cu (O) ions is U Cu (U O ) and J O is the Hund's coupling between oxygen orbitals, as well as the hopping between singlet pairs [last term of Eq. (2)].
The O part of the interaction has been calculated in terms of two parameters U O = F 0 + 4F 2 and J O = 3F 2 in a similar way as for d electrons 32 . We determined the value of F 2 = 0.279 eV from a fit of the atomic energy levels of neutral O 33 . We have taken U Cu = 10 eV and U O = 3 eV from typical values in the cuprates 34 but also study the dependence of the results with U O , since it affects the results in a sensitive way.
The hopping matrix elements have the following form where s = ±1 and R is the Cu-O distance in the chains. The phase of the p jα orbitals displaced from the Cu in thex or −ŷ directions (below the Cu-Cu line, see Fig. 1b) has been changed by a factor -1 so that the Cu-O hopping has the same sign in all directions. For the hopping matrix elements we follow a two step procedure. First, we perform a spin-unpolarized DFT calculation where the Bloch states are obtained. The band structure is shown in Fig. 1c, where the O and Cu atoms of the chain share a peak at the Fermi level, suggesting a strong hybridization between them.
In order to have an atomic-like description, our second step is to use the method proposed by Marzari and Vanderbilt 35 to find the Wannier functions in real space. This method consists in projecting the Bloch states into a set of Wannier functions that are maximally localized in the target atoms.
The wannierization process needs to define an energy window where the projections take place. In Fig. 1c, the energy window is enclosed within the dotted lines. As it is evident from the figure, it is not necessary to perform a disentanglement procedure because the bands have no overlap with other bands outside the defined region. From an analysis of the projected density of states (shown in Fig. 1c), we found that the main contribution to the bands in that energy window comes from p orbitals of O and d orbitals from Cu. Furthermore, the obtained Wannier Hamiltonian does not show appreciable contribution to the Cu-O hopping from the Cu that lies outside the spin chain. This fact supports our model that only takes into account the Cu-O hopping in the chains. The obtained Wannier basis is well localized in the atoms of the spin chain and the wave functions respect the p and d symmetry for each atom case.
Changing the basis to that used in Eqs. (2) and (3) we obtain t = 1.1eV, t O = 0.36eV.
III. EFFECTIVE HEISENBERG MODEL AND PERTURBATION THEORY
Since the occupancy of the CuO 2 chains is 1 per unit cell, in the limit in which t, t O ∆ the only relevant degrees of freedom are the spins 1/2 at each Cu site. The hopping terms lead to effective Heisenberg interactions between two spins. Collecting the most relevant terms, the low-energy effective Hamiltonian takes the form where i, j indicates NN and i, j NNN. The contribution to order N in H hop to the effective matrix element between two states |g 1 and |g 2 , which are part of the degenerate ground state of H 0 with energy E g , is given by J 2 (the former being two times larger) roughly of order Fig. 3).
In the first case (a), the holes of two NN Cu ions jump to different 2p orbitals of the same O atom, paying a lower energy cost if the spins form a triplet, compared to the case in which the spins form a singlet, resulting in an effective ferromagnetic interaction proportional to the Hund exchange J O . There are eight contributions of this type depending on the relative order between processes 1 and 2, between 3 and 4, and the two O atoms involved (above or below the Cu-Cu line).
In case (b), after three hopping processes, the holes meet at the same Cu or O orbital and then return to the original Cu positions, or interchange them. For the parallelogram shown in Fig. 3, there are 20 of such processes which differ in the sequence of intermediate states.
There is, in addition, a factor 2 because the parallelogram can be below or above the Cu-Cu line and an additional factor 2 from the contributions of the parallelogram obtained from that shown in Fig. 3 after reflection through a vertical plane between two Cu ions. For J 2 , the parallelogram changes to an isosceles trapezoid, with the base of two unit cells, and the latter reflection does not change the figure, then the corresponding factor 2 is lacking and the number of different contributions is exactly half of those for J 1 .
Note that the usual antiferromagnetic exchange found in the cuprates is forbidden for a Cu-O-Cu angle of 90 degrees.
Adding carefully all contributions we obtain: The terms proportional to J 2 account for the antiferromagnetic contributions and the last term in Eq. (6) is the ferromagnetic contribution to J 1 .
To check that all antiferromagnetic contributions have been added correctly, we have calculated exactly the energies of a Cu 2 O 2 molecule containing the relevant orbitals of the parallelogram of Fig. 3, and obtained the correct singlet-triplet splitting for t, t O → 0 37 .
IV. NUMERICAL RESULTS
Due to the covalency of the system, the perturbative results obtained in the previous section for the exchange parameters J i with i = 1, 2 are not accurate for realistic values of the parameters of the five-band Hubbard Hamiltonian. To derive the J i in this case, we follow an approach which has been proved successful for example in mapping the three-band model for the cuprates into a spin-fermion model 36 : we assume that the effective model H Heis given by Eq. (4) retains this form and obtain the J i fitting the low lying energy levels of the multiband Hubbard Hamiltonian. We have checked that adding an exchange interaction to third nearest neighbors practically does not improve the fitting, which supports the above mentioned assumption.
Due to the rapid increase of the Hilbert space with the size of the system, we have done the fitting using a three-band model in a CuO chain with eight unit cells, eliminating one row of the original CuO 2 chain. Smaller systems lead to either frustration (6 unit cells) or underestimation (4 unit cells) of the NNN interaction. While DMRG can be used to obtain the ground-state of the 5-band model with 8 unit cells, our code does not allow to obtain excited states with definite wave vector k. According to perturbation theory, the results for J i should by multiplied by two to reach to the correct answer.
We have calculated exactly the energies of the threeband model for the parameters obtained as described in section II for each wave vector and the lowest total spin projections. The sum of the squares of the differences with the corresponding energies of H Heis was minimized with respect to both J i using the Nelder-Mead procedure, starting from the perturbative regime and varying parameters of the multiband model in an adiabatic way to avoid reaching local non-physical minima. Periodic boundary conditions were used for H Heis and antiperiodic ones for the three-band model to compensate for the fermionic sign in a state of eight particles when the last particle is moved to the first place.
A. Perturbative regime
In order to check the regime of validity of the perturbative calculations, we have compared energies in this regime for different choices of parameters.
Ferromagnetic contribution to J1
From Eqs. (6), setting t O = 0 the only contribution to the perturbative calculation corresponds to the case (a) of section III, which leads to a ferromagnetic NN exchange J 1 < 0, and zero NNN exchange J 2 = 0.
In Fig. 4 we show energies for both models for parameters of the multiband model in the perturbative regime and t O = 0. Here the parameters of H Heis are taken from Eqs. (6) and not by minimization. From these results we can see that perturbative calculations are consistent with the numerical result, with a slight difference between energies, being the energies of the effective model around 12% higher.
Antiferromagnetic contributions to Ji
We have also analyzed the antiferromagnetic contribution to both exchange interactions, J 1 and J 2 by considering a finite hopping between the oxygen atoms, t O and J O = 0, so that the ferromagnetic contributions to J 1 vanish. The energies for both models are shown in Fig. 5 for ∆ = 10eV where we have used the optimized parameters J 1 and J 2 . The Heisenberg model reproduces accurately the energies of the multiband model.
To check the perturbative results for the antiferromagnetic contributions to the NN and NNN exchange parameters, we have compared the perturbative results J i with the corresponding values J i obtained from the minimization procedure using the Cu 8 O 8 ring, for different choices of ∆. The results for the exchange parameters are shown in Table I. The ratios 2J i /J i indicate how much the minimization results differ from the perturbative results of Eqs. (6). We remind the reader that since the Cu 8 O 8 ring has half the O atoms of the CuO 2 chain, one expects that the resulting exchange iterations are half the correct ones. For J 1 , in the case ∆ = 10eV, J 1 is barely higher (only 0.06%) from the perturbative result. When ∆ increases, the minimization result tends to grow around 30% higher. On the other hand, the minimiza- tion results for J 2 tend to improve when ∆ is increased, but for the case ∆ = 15eV there is still a factor 2 between both, being the minimization result smaller than the perturbative one. Nevertheless, both minimization results for NN and NNN couplings remained of the same order of magnitude of the corresponding perturbative calculation. We believe that the discrepancy is likely due to the effect of perturbative processes of higher order (probably not contained in the Cu 2 O 2 molecule mentioned at the end of Section III 37 ), which are still important for large values of ∆. One fact that contributes to this effect is the rapid increase of the number of different perturbative processes of the same order involved, as the order increases. In particular, for the calculation of the antiferromagnetic contribution to J 1 , the results of 80 different processes have been added.
B. Non-perturbative regime
In this subsection, we report the resulting values of J i obtained from our fitting procedure using realistic parameters of the multiband Hamiltonian. To this end we use the experimental determination of the charge-transfer (CTG) gap 29 of 1.95eV to fit the values of ∆ in Eq. (2) for a set of realistic values for U O . These values were obtained by extrapolating the finite-size CTGs for L = 4 and L = 8 cells using the five-orbital model, solved with the DMRG. In Table II The value of the pitch angle observed in neutron experiments is near 63 degrees 6,13 . This points to a ferromagnetic J 1 and of larger magnitude than our results.
We find that for smaller values of U O , the ferromagnetic term in Eq. (2) is favored and this leads to negative values of J 1 . This, in turn, leads to a pitch angle θ < 90 degrees since there is a larger ferromagnetic alignment between neighboring spins that competes with the antiferromagnetic NNN interaction. For larger values of U O , the ferromagnetic term in Eq. (2) is weakened, leading to a larger predominance of the antiferromagnetic interaction between NN spins.
A comparison of the energies in both models is shown in Fig. 6 and Fig. 7, where we find an excellent correspondence between the realistic multiband Hamiltonian and the effective spin model.
V. SUMMARY AND DISCUSSION
In summary, we have derived from first principles an effective model to describe the CuO 2 chains in the ferroelectric material LiCu 2 O 2 . We began with an ab-initio calculation to determine the relevant orbitals and physical parameters from which we obtain a five-band Hubbard model containing local Coulomb and Hund inter- actions. Using perturbation theory in the hopping parameters we obtained the effective low-energy spin model for one hole per Cu site, leading to a one-dimensional spin-1/2 Heisenberg model with NN (J 1 ) and NNN (J 2 ) exchange interactions. These calculations shed light on the underlying physical mechanisms behind these interactions.
However, for realistic values of the parameters the system is not in the perturbative regime. Therefore, we obtained corrected values of the effective spin interactions J 1 and J 2 by fitting the lowest energy levels of a multiband Hubbard model, calculated using exact diagonalization. We rely on experimentally obtained values of the charge-transfer gap to obtain the difference between onsite energies of Cu and O, for which we solve the five-band Hubbard model using the Density Matrix Renormalization Group. The magnitude of J 2 is, in general, larger than J 1 and is always antiferromagnetic, while J 1 changes from ferro-to antiferromagnetic as the local Coulomb repulsion on the oxygen atoms increases. The large value of J 2 leads to a spiral spin order, which is a sufficient condition for the emergence of ferroelectricity in this material, on the basis of symmetry arguments.
The pitch angle observed in neutron experiments, that has a value of around 63 degrees, is smaller than our results. This would require larger values for ∆ (smaller values of the local Coulomb interaction) which would lead to a more negative J 1 and a smaller positive J 2 . Since a very large screening of the Coulomb interaction is unlikely, we believe that the discrepancy might be due to an underestimation of the difference between on-site energies of holes at O and Cu sites ∆ as a consequence of finite-size effects.
Nevertheless, to our knowledge, this is the first manybody calculation of the effective exchange interactions of the material.
ACKNOWLEDGMENTS
N. Aucar Boidi acknowledges support from ICTP through the STEP program, the ESI through the longterm program "Tensor Networks: Mathematical Structures and Novel Algorithms" and the "Autumn School on Correlated Electrons: Dynamical Mean-Field Theory of Correlated Electrons" in which part of this work has been done. AAA is supported by PICT 2017-2726 and AAA and KH by PICT 2018-01546 of the ANPCyT, and by PIP 2015-0538 of CONICET, Argentina. | 2022-12-08T06:42:05.596Z | 2022-12-06T00:00:00.000 | {
"year": 2022,
"sha1": "141483a1a54254365e201c0097e1da49aac3bf06",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Arxiv",
"pdf_hash": "141483a1a54254365e201c0097e1da49aac3bf06",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
10114514 | pes2o/s2orc | v3-fos-license | Classification of normal/abnormal heart sound recordings based on multi-domain features and back propagation neural network
This paper aims to classify a single PCG recording as normal or abnormal for computer-aided diagnosis. The proposed framework for this challenge has four steps: preprocessing, feature extraction, training and validation. In the preprocessing step, a recording is segmented into four states, i.e., the first heart sound, systolic interval, the second heart sound, and diastolic interval by the Springer Segmentation algorithm. In the feature extraction step, the authors extract 324 features from multi-domains to perform classification. A back propagation neural network is used as predication model. The optimal threshold for distinguishing normal and abnormal is determined by the statistics of model output for both normal and abnormal. The performance of the proposed predictor tested by the six training sets is sensitivity 0.812 and specificity 0.860 (overall accuracy is 0.836). However, the performance reduces to sensitivity 0.807 and specificity 0.829 (overall accuracy is 0.818) for the hidden test set.
Introduction
Heart sounds provide important initial clues in cardiovascular disease evaluation. It is necessary to develop automatic algorithms for computer-aid diagnosis. The previous studies used frequency spectrum [1], wavelet coefficients [2] to classify heart sound recordings based on self-organization map, grow and learn network, linear vector quantization. However, the recordings in these studies were limited by the recording number, length, signal frequency range, or the environments of data collection. To address the problem, the physioNet/Cinc Challenge 2016 provides a large collection of heart sound recordings for this purpose [3]. The authors extract multi-domain features to perform classification.
Descriptions of the heart sound databases
The opened training set consists of six databases (A through F) containing a total of 3153 PCG recordings, collected at either a clinical or nonclinical environment, from both healthy subjects and pathological patients. Each recording has been manually labeled as normal (-1) and abnormal (1). The training and test sets have each been divided so that they are two sets of mutually exclusive population. The details of the databases and challenge can be found in [4].
Preprocessing
A heart sound signal is filtered with pass band [20 120] Hz to remain the main part of heart sounds. Then it is inversely filtered to avoid group delay. Springer's segmentation algorithm [5] is applied to the filtered signal for segmenting. The heart sound signal sequence is labeled by four states (S1, systole, S2 and diastole) for each beat. The feature extraction is therefore carried on based on the state labels.
Extracted features
In this study, 324 features are extracted from each recording from multi-domains. They are listed in detail in the follows. 9. m_IntDia: mean of diastolic intervals 10. sd_IntDia: SD of diastolic intervals 11. m_Ratio_SysRR: mean of the ratio of systolic interval to RR of each heart beat 12. sd_Ratio_SysRR: SD of the ratio of systolic interval to RR of each heart beat 13. m_Ratio_DiaRR: mean of ratio of diastolic interval to RR of each heart beat 14. sd_Ratio_DiaRR: SD of ratio of diastolic interval to RR of each heart beat 15. m_Ratio_SysDia: mean of the ratio of systolic to diastolic interval of each heart beat 16. sd_Ratio_SysDia: SD of the ratio of systolic to diastolic interval of each heart beat 17. m_Amp_SysS1: mean of the ratio of the mean absolute amplitude during systole to that during the S1 period in each heart beat 18. sd_Amp_SysS1: SD of the ratio of the mean absolute amplitude during systole to that during the S1 period in each heart beat 19. m_Amp_DiaS2: mean of the ratio of the mean absolute amplitude during diastole to that during the S2 period in each heart beat 20. sd_Amp_DiaS2: SD of the ratio of the mean absolute amplitude during diastole to that during the S2 period in each heart beat The authors add another two features: 21. m_Ratio_IntS1S2: mean value of the ratio of S1 interval to S2 interval 22. sd_Ratio_IntS1S2: SD of the ratio of the mean.
10 Features in energy domain 1. Ratio_energy_HSTotal: Ratio of the sum of heart sounds' energy to the total energy of a recording 2. Ratio_magnitude_HSTotal: Ratio of sum of heart sounds' absolute magnitude to the sum of absolute magnitude of a recording 3. Ratio_energy_HSRemain: Ratio of the sum of heart sounds' energy to the energy of the remain parts 4. Ratio_magnitude_HSRemain: Ratio of the sum of heart sounds' absolute magnitude to the sum of the absolute remain parts 5. m_Ratio_energy_SysCycle: mean value of the ratio of energy in systolic interval to cardiac cycle energy of each heart beat 6. sd_energy_SysCycle: SD of the ratio of the mean 7. m_Ratio_energy_DiaCycle: mean value of the ratio of energy in diastolic interval to cardiac cycle energy of each heart beat 8. sd_energy_DiaCycle: SD of the ratio the mean 9. m_Ratio_HSCycle: mean value of the Heart sounds energy to cardiac cycle energy of each heart beat 10. sd_energy_HSCycle: SD of the ratio of the mean 82 Features in frequency spectrum 1-12. m_Fre_Spec_S1_f: mean of frequency spectral values of S1 at frequency f. The frequency f is considered from 10 Hz to 120 Hz with interval of 10 Hz 13-24. m_Fre_Spec_S2_f: mean of spectral values of S2 at frequency f. The frequency f is considered from 10 Hz to 120 Hz with interval of 10 Hz. 25-53. m_Fre_Spec_Sys_f: mean of spectral values of systole signal at f of each heart beat where the frequency is from 10 Hz to 290 Hz with interval of 10 Hz 54-82. m_Fre_Spec_Dia_f: mean of spectral values of diastole of each heart beat from 10 Hz to 290 Hz with interval of 10 Hz 2 Features in heart rate sequence 1. m_HR: Mean of heart cycle period. The method to calculate m_HR is different from that to calculate m_RR. The heart rate is based on analysis of the autocorrelation function and the positions of the peaks therein. 2. sd_HR: SD of the mean 57 Features in frequency spectrum of heart rate sequence 1-19. spec_HR_seq_f: Spectral values of heart rate sequence from 0.05 Hz to 1 Hz with interval 0.05 Hz. The heart rate sequence is non-uniformly sampled in time domain due to heart rate variability. So, nonlinear interpolation by 'cubic' method is used in this study.
where E(.) is an expectation operator.
Features in cyclostationarity
1. m_cyclostationarity_1: mean value of the degree of cyclostationarity. The definition of "degree of cyclostationarity" can be found in [6]. This feature indicates the degree of signal repetition. It will be infinite if the events occurred in heart beating were exact periodic.
However, it will be a small number if the events are random alike. Let's assume γ(α) is the cycle frequency spectral density of a heart sound signal at cycle frequency α. This feature is defined as where β is the maximum cycle frequency considered and η is the basic cycle frequency indicated by the first peak location of γ(α). A heat sound signal is equally divided into subsequence. The feature can be estimated for each subsequence, then the mean and standard deviation can be obtained. 2. sd_cyclostationarity_1: SD of the mean 3. m_cyclostationarity_2: The definition of this indicator is the sharpness of the peak of cycle frequency spectral density. It is )) (3) The operators, max(.) and median(.) are the maximum magnitude and median of the cycle frequency spectral density. It is obviously that the sharper of the peak is, the greater the feature is. Similarly, the feature can be calculated for each sub-sequence of the heat sound signal and then get the mean and SD. 4. sd_cyclostationarity_2: SD of the mean 82 Features in power spectral density 1-12. m_Pow_Spec_S1_f: mean of frequency spectral values of S1 at frequency f of each heartbeat. The frequency f is considered from 10 Hz to 120 Hz with interval of 10 Hz 13-24. m_Pow_Spec_S2_f: mean of spectral values of S2 at frequency f of each heartbeat. The frequency f is considered from 10 Hz to 120 Hz with interval of 10 Hz. 25-53. m_Pow_Spec_Sys_f: mean of spectral values of systole signal at f of each heart beat where the frequency is from 10 Hz to 290 Hz with interval of 10 Hz 54-82. m_Pow_Spec_Dia_f: mean of spectral values of diastole of each heart beat from 10 Hz to 290 Hz with interval of 10 Hz 57 Features in power spectral density of heart rate sequence [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]
Prediction model
A back propagation artificial neural network is applied to approximate the link between the features and blood pressure, whose structure with two hidden layers is shown in Fig. 1. A back propagation neural network is a feedforward network with its weights adjusted through the method of back propagation learning algorithm and it can achieve arbitrary nonlinear mapping from input to output, generally having a good performance. Each net node in the network is a neuron whose function is to calculate the inner product of the input vector and weight vector by a nonlinear transfer function to get a scalar result. The "logsig" function is applied to the hidden layer 1. The "purelin" function is applied to the hidden layer 2 and the output layer. The number of the neurons for the hidden layer 1 and hidden layer 2 are empirically chosen to be 10 and 5, respectively. The training algorithm applied is the "trainlm".
Threshold to classify normal and abnormal
The prediction model outputs a real number. According the link trained by the input-output pairs, the output of the prediction model will approach -1 if a recording is normal and approach 1 if a recording is abnormal. To determine an optimal threshold for classifying, it is necessary to investigate the statistic of outputs for both normal and abnormal. The all inputs are selected to train the model, histogram analysis is performed for the outputs of both normal and abnormal recordings, see the arrow in Fig. 2. It can be found that the cross point of the two curves is at about -0.4. However, the position of the cross point changes if the percent number of input varies. To investigate this change, firstly, 50% of the inputs were randomly selected to feed into the model, and then 60% of inputs were to put into the model, and repeat the operation with increment 10% each time. The position of the cross point with respect to the percent inputs is listed in Table 1. It is found that the position of the cross point varied little with the percent number. So, the threshold is finally set to the average, -0.52. Those recordings whose model output greater than the threshold are classified as abnormal. To the contrary, those recordings whose output less or equal to the threshold are classified as normal.
Performance of classification
The training set is exclusively divided into two parts. The first part is used to train the model and the other part is used to test the model. The percent to train is from 30% to 90% of the training set, and the performances were listed in table 2. It is found that the prediction performance of the model is lightly increasing with the percent to train.
Conclusions
This paper tries to classify heart sound signal recordings into normal or abnormal based on multidomain features. 324 features were extracted to train a back propagation neural network for the prediction. The best performance of the model based on training set is: sensitivity 0.812, specificity 0.860, overall score 0.836. However, the performance tested by the hidden set is: sensitivity 0.807, specificity 0.829, overall score 0.818. The authors believe that the performance would be improved if a prediction model based on deep learning is used. | 2017-03-27T21:14:56.807Z | 2016-09-14T00:00:00.000 | {
"year": 2018,
"sha1": "44f08fd7e287e3b1aebec3f929d560876b9cb862",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Arxiv",
"pdf_hash": "44f08fd7e287e3b1aebec3f929d560876b9cb862",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Engineering",
"Computer Science",
"Mathematics"
]
} |
258652677 | pes2o/s2orc | v3-fos-license | In Vivo Antiviral Activity of Baloxavir against PA/I38T-Substituted Influenza A Viruses at Clinically Relevant Doses
Although the prevalence of polymerase acidic (PA)/I38T strains of influenza virus with reduced susceptibility to baloxavir acid is low, there is a possibility of emergence under selective pressure. Furthermore, the virus may be transmitted between humans. We investigated the in vivo efficacy of baloxavir acid and oseltamivir phosphate against influenza A subtypes H1N1, H1N1pdm09, and H3N2, with PA/I38T substitution, at doses simulating human plasma concentrations. A pharmacokinetic/pharmacodynamic analysis was performed to strengthen the validity of the findings and the applicability in a clinical setting. Although the antiviral effect of baloxavir acid was attenuated in mice infected with PA/I38T-substituted viral strains compared with the wild type (WT), baloxavir acid significantly reduced virus titers at higher—but clinically relevant—doses. The virus titer reduction with baloxavir acid (30 mg/kg subcutaneous single dose) was comparable to that of oseltamivir phosphate (5 mg/kg orally twice daily) against H1N1 and H1N1pdm09 PA/I38T strains in mice, as well as the H3N2 PA/I38T strain in hamsters. Baloxavir acid demonstrated an antiviral effect against PA/I38T-substituted strains, at day 6, with no further viral rebound. In conclusion, baloxavir acid demonstrated dose-dependent antiviral effects comparable to that of oseltamivir phosphate, even though the degree of lung virus titer reduction was diminished in animal models infected with PA/I38T-substituted strains.
Introduction
Influenza is one of the most problematic acute respiratory infections worldwide, and along with its associated complications, it results in significant morbidity and mortality [1,2]. Antiviral drugs, including neuraminidase inhibitors (NAIs), adamantane derivatives, and a cap-dependent endonuclease (CEN) inhibitor, have been developed over the years for the treatment and prevention of influenza [2,3]. As most circulating influenza A viruses are resistant to the adamantane derivatives, rimantadine and amantadine, this group of drugs is no longer recommended [3]. NAIs, such as oseltamivir, zanamivir, and peramivir, are widely prescribed [2,3], as they are known to reduce the duration of influenza symptoms in otherwise healthy patients [4]. However, different mutant strains show reduced susceptibility to different NAIs. For example, the H1N1pdm09 NA/H275Y (N1 numbering; H274Y, N2 numbering) variant has decreased susceptibility to oseltamivir and peramivir but not to zanamivir [5]. Reduction in virus susceptibility to available antiviral drugs raises clinical concerns and the need to develop novel therapeutic strategies, such as CEN inhibitors.
Baloxavir marboxil is currently the only CEN inhibitor available for clinical use [6]. It is a prodrug that is hydrolyzed to its active form, baloxavir acid, following oral administration, and it acts by inhibiting the CEN residing in the polymerase acidic (PA) subunit of the influenza virus polymerase. In animal models and clinical trials, compared with placebo, a single dose of
Compounds
Baloxavir acid was synthesized at Shionogi & Co., Ltd. (Osaka, Japan). Baloxavir suspension was prepared by suspending baloxavir acid in 0.5% (w/v) methylcellulose using an agate mortar and pestle. Oseltamivir phosphate was purchased from Sequoia Research Products Ltd. (Pangbourne, UK) and dissolved in 0.5% (w/v) methylcellulose. For the PK/PD studies, baloxavir acid was dissolved in 10% (w/v) Tween 80 and 0.5% (w/v) vinylpyrrolidone-vinyl acetate copolymer in sodium carbonate-sodium hydrogen carbonate buffer under heating, and the pH was adjusted to~9. For dosing, each solution or suspension was diluted with the same respective vehicle.
Animals
All animal studies were conducted under applicable laws and guidelines and with the approval of the Shionogi Animal Care and Use Committee. The study is reported in accordance with ARRIVE guidelines.
Sample Size
A total of 1441 mice and 84 hamsters were used in the study. The number of animals to be used was decided based on the results of preliminary tests while considering the reduction in the number of animals as much as possible. There were no excluded experimental units or data points.
Inclusion and Exclusion Criteria
Animals with any abnormalities in initial body weight or general condition were not included. There were no other inclusion/exclusion criteria. As a result, of the 1541 mice and 84 hamsters prepared for the experiments, 1441 mice (≈94%) and all the hamsters were used.
Animal Housing
All mice and hamsters were maintained in a temperature and relative humiditycontrolled environment (20-26 • C and 30-70%, respectively), with a normal 12 h light and 12 h dark cycle. There were two hamsters housed per sterile cage, with chip paper bedding, paper towel, and cardboard tubes for environmental enrichment. Between 5 and 10 mice were housed per cage, with chip paper bedding and wooden bite stick for environmental enrichment. Standard chow diet (CE-2, CLEA Japan) and water were available ad libitum.
Cells and Viruses
Madin-Darby canine kidney (MDCK) cells were obtained from the European Collection of Authenticated Cell Cultures (Wiltshire, UK) and maintained using the method described previously [9].
Plaque Reduction Assay
The susceptibility of H1N1pdm09.WT and H1N1pdm09.PA/I38T was determined using the plaque reduction assay described previously [14].
Anesthesia
Mice and hamsters were anesthetized upon virus inoculation by the intramuscular administration of an anesthetic solution containing medetomidine hydrochloride, midazolam, and butorphanol tartrate. For experiments analyzing plasma concentrations of baloxavir acid in non-infected animals, mice were anesthetized with isoflurane, and hamsters were unanesthetized upon blood sampling.
Drug Administration
When administered orally, baloxavir marboxil results in a shorter baloxavir acid plasma half-life in animal models than that observed in humans [11,27]. To maintain the comparable plasma concentrations, we therefore administered baloxavir acid suspension subcutaneously. We used clinically relevant doses of baloxavir acid in mice to mimic human exposure. For doses ≤ 10 mg/kg, one injection was administered subcutaneously to the "back of the neck". In the case of 20 or 30 mg/kg doses, baloxavir acid was administered subcutaneously to two or three sites on the "back", respectively. Oseltamivir phosphate was administered at a corresponding therapeutic dose of 5 mg/kg, orally, twice daily. The vehicle was an aqueous solution of 0.5% [w/v] methylcellulose that could be administered either subcutaneously or orally.
Antiviral (H1N1) Studies in Mice 2.7.1. Examination of Virus Titers
Mice were euthanized by cervical dislocation under anesthesia, and virus titers in lung homogenates were determined 6 days post-infection (24 h after treatment). Lung homogenates were prepared by adding 2 mL of DPBS to approximately 200 mg of lungs. To measure lung virus titers, serial dilutions of lung homogenates were inoculated onto confluent MDCK cells, as described previously [9]. The presence of cytopathic effects was determined microscopically, and the virus titers were calculated as log 10 median tissue culture infectious doses (TCID 50 )/mL. When no cytopathic effect was observed in the lowest dilution (10-fold), the titer of undetected virus was defined as 1.5 log 10 TCID 50 /mL, except for the experiment assessing the time course of virus titers (Supplementary Figure S1). In this study, when no cytopathic effect was observed in the lowest dilution (5-fold), the titer of undetected virus was defined as 1.35 log 10 TCID 50 /mL.
Effect on Body Weight in Mice
Anesthetized mice were intranasally inoculated with 10,000 TCID 50 of rgH1N1.WT, rgH1N1.PA/I38T, or rgH1N1.NA/H275Y. The initial body weights of all the mice were recorded on the day of infection. Mice were assigned to each group one day prior to or on the first day of treatment. Assignment was based upon the uniformity of mean body weight and the mean proportion of body weight to initial body weight among groups. From each group, 10-20 infected mice were treated with baloxavir acid (10 or 30 mg/kg subcutaneous single dose and vehicle, 10 mL/kg orally, twice daily, for 5 days), oseltamivir phosphate (5 mg/kg orally, twice daily, for 5 days and vehicle 10 mL/kg subcutaneous single dose), or vehicle (10 mL/kg subcutaneous single dose and orally, twice daily, for 5 days). For 10 days and 14 days after infection, mice were examined daily for body weight and survival. Mice were regarded as dead if their body weights were lower than 70% of the initial body weight according to the humane endpoint. These mice were euthanized. In the analysis of the evaluation of the effect on body weight change, mice who died or reached the endpoint were extrapolated as 70% of the body weight on the day of infection.
Antiviral (H3N2) Studies in Hamsters
Anesthetized hamsters were intranasally inoculated with 300 TCID 50 of rgH3N2.WT or rgH3N2.PA/I38T virus suspension. At 1 day post-infection, infected hamsters were treated with baloxavir acid (10 or 30 mg/kg subcutaneous single dose; n = 8/group), oseltamivir phosphate (5 mg/kg orally twice daily; n = 8/group), or vehicle (5 mL/kg subcutaneous single dose; n = 8/group). Hamsters were euthanized by exsanguination, and the lung tissues were removed. The lung homogenates were prepared by adding 6 mL of DPBS to approximately 600 mg of lungs. Virus titers in lung homogenates were determined 2 days post-infection (24 h after treatment).
2.9. PK/PD Analysis 2.9.1. PK Studies in Non-Infected Mice To determine whether blood exposure of baloxavir acid could be increased linearly by injecting the drug into multiple sites, baloxavir acid (prepared using baloxavir acid in suspension: 1 mg/mL for mice; 5 mg/mL for hamsters) was injected subcutaneously at different sites in mice (n = 3/group) and hamsters (n = 3/group).
For mice, ≈10 mg/kg (0.2 mg/mouse) of the suspension was administered to one (≈10 mg/kg), two (≈20 mg/kg), or three (≈30 mg/kg) sites. Blood (≈0.025-0.4 mL/time point) was taken from the inferior vena cava, heart, or tail vein at 2, 4, 8, 24, 48, 120, and 216 h (two and three sites) or 2, 4, 8, 24, 48, 120, 168, and 216 h (one site) after dosing. The maximum plasma concentration (C max ) and area under the curve, from 0 to the time of the last quantifiable concentration (AUC last ), were calculated by WinNonlin (Certara USA Inc., Princeton, NJ, USA) based on a non-compartment model with uniform weighting. In hamsters, 10 mg/2 mL/kg of the suspension was administered to one (10 mg/kg) or three (total of 30 mg/kg) sites. Blood (≈0.1 mL/time point) was col-Viruses 2023, 15, 1154 6 of 18 lected from the jugular vein at 1,4,8,24,48, and 72 h after dosing. In both mice and hamsters, the plasma concentration was determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that administering 1, 2, and 3 subcutaneous injections (≈10, 20, and 30 m/kg) to separate sites on the back of the mice resulted in a linear increase in baloxavir acid at 24 h (C 24 ) levels (26.1 ± 1.3, 47.4 ± 4.9, and 70.8 ± 13.9 ng/mL, respectively, n = 3; Supplementary Figure S2). Similarly, administering one and three subcutaneous injections (≈10 and 30 mg/kg) to separate sites on the back of the hamsters resulted in a linear increase in C 24 levels (17.3 ± 3.3 and 45.7 ± 19.7 ng/mL, respectively, n = 3; Supplementary Figure S2). To achieve optimal plasma concentrations with a dose of 30 mg/kg, we therefore administered three subcutaneous injections of 10 mg/kg to separate sites in this study.
To compare the PK parameters of baloxavir acid in humans, mice, and hamsters, the human oral PK data were derived from a study on population PK parameters and exposure-response analyses in adults and adolescents [26]. All mice (n = 3) and hamsters (n = 3) received one (10 mg/kg) subcutaneous suspension of baloxavir acid (1 mg/mL for mice; 2 mg/mL for hamsters). Plasma concentrations of baloxavir acid were determined by LC-MS/MS, using blood (mice: ≈0.025-0.4 mL/time point; hamster: ≈0.1 mL/time point) taken from the inferior vena cava or heart of anesthetized mice (at 2, 4, 8, 24, 48, 120, 168, and 216 h after dosing) or from the jugular vein of unanesthetized hamsters (at 1, 4, 8, 24, 48, and 72 h after dosing). Assuming dose linearity, plasma concentrations of baloxavir acid for a 30 mg/kg suspension in mice and hamsters were estimated from the measured plasma concentration data for the 10 mg/kg suspension (Supplementary Figure S3).
To obtain the PK data, mice infected with rgH1N1.PA/I38T were treated with baloxavir acid (0.25, 1, 4, 16, or 64 mg/kg, subcutaneous single dose) and blood was sequentially taken from the tail vein at 0.25, 0.5, 1, 2, 4, 6, 8, or 24 h after dosing (n = 3/group). Plasma concentrations of baloxavir acid were determined by LC-MS/MS and averaged by dose and nominal time. The mean plasma concentrations, at each sampling time and dose level, were used for the PK analysis. The C max , plasma concentration at the end of the dosing interval after the first dosing (C τ ), and area under the curve, from time 0 to 24 h after dosing (AUC 0-24 ), were calculated by WinNonlin based on a non-compartment model with uniform weighting. The same PK parameters for the doses that were not administered to infected mice for PK evaluation (0.125, 0.5, 2, 8, and 32 mg/kg) were mathematically scaled by extrapolation or interpolation, based on the PK parameters in infected mice dosed at 0.25, 1, 4, 16, or 64 mg/kg, using the method described previously [11]. Each parameter's value at the 0.125 mg/kg dose was calculated as 1/2 × the parameter's value at the 0.25 mg/kg dose; the parameters for the remaining values (0.5, 2, 8, and 32 mg/kg) were calculated as 2/3 × the value at the nearest lower dose + 1/3 × the value at the nearest higher dose, as described previously 11 (Supplementary Table S2).
The linear model (y = E 0 -βx) was used to investigate the relationship between antiviral activity and PK parameters, following subcutaneous baloxavir acid dosing, in the rgH1N1.PA/I38T infection model. This model was applied to virus titer data, derived from individual mice in the PD studies, and to the PK parameters that were calculated from the observed mean plasma concentrations at each dose and time point (for doses 0.25, 1, 4, 16, or 64 mg/kg), as well as the PK parameters for 0.125, 0.5, 2, 8, and 32 mg/kg doses that were mathematically estimated by extrapolation or interpolation based on the PK parameters at 0.25, 1, 4, 16, or 64 mg/kg. E 0 is the baseline effect, and β is a regression coefficient. In this case, AUC 0-24 , C max , C 24 , and C τ were used as the logarithmic values. Model fitness was evaluated by the coefficient of determination, R 2 , adjusted for degrees of freedom (Supplementary Table S2).
Statistical Analyses
Virus titers and body weights on each day, after treatment with baloxavir acid, were compared with the respective control groups (oseltamivir phosphate and vehicle) and analyzed using the Dunnett's multiple comparison method. Statistical analyses were performed using SAS Studio version 9.4 for Windows (SAS Institute, Cary, NC, USA). The two sided-adjusted p-values below 0.05 were considered statistically significant.
In the statistical analysis, it was assumed that the data showed a normal distribution. The normal distribution of the data was confirmed in the histogram, and we judged that the data met the assumption.
In Vitro Drug Susceptibility of Influenza a Viruses to Baloxavir Acid and Oseltamivir Acid
The in vitro drug susceptibility of H1N1pdm09 WT and PA/I38T strains to baloxavir acid was tested using a plaque reduction assay. The drug sensitivity of H1N1pdm09.PA/I38T to baloxavir acid (measured as the half-maximal effective concentration [EC 50 ]) was reduced by 25.74-fold (Table 1). Data from a previous study [14] showed that the presence of PA/I38T mutation in H1N1 (rgA/WSN/33) and H3N2 (rgA/Victoria/3/75) strains decreased the drug sensitivity to baloxavir acid by 27.24-fold and 56.59-fold, respectively. The drug sensitivity of rgH1N1.PA/I38T and rgH3N2.PA/I38T to oseltamivir acid (measured as the half-maximal inhibitory concentration [IC 50 ] was comparable to respective WT strains (1.00-fold and 1.21-fold, respectively). The presence of NA/H275Y mutation in the H1N1 (rgA/WSN/33) strain decreased the drug sensitivity to oseltamivir acid by 226.12-fold, but it did not affect the drug sensitivity to baloxavir acid (0.77-fold change). Data for the drug sensitivity of the H1N1pdm09 (A/Osaka/129/2009) to oseltamivir acid were not available (Table 1). The EC 50 of baloxavir acid against H1N1pdm09 WT and PA/I38T strains was determined by a plaque reduction assay. The remaining data were taken from a previous study [14]. Fold change (FC) was calculated as relative EC 50 or IC 50 of each tested virus to that of the WT virus. Bold type, FC > 10; EC 50 , half-maximal effective concentration; IC 50 , half-maximal inhibitory concentration; N/A, not applicable; ND, no data; SD, standard deviation; WT, wild type.
Virus Growth Kinetics in a Non-Lethal Mouse Model
In a previous study [11], we established a non-lethal mouse model, using TCID 50 of 100 with the natural strains, to evaluate the rapid reduction in virus burden following baloxavir marboxil treatment. Treatment was initiated 5 days post-infection followed by lung virus titer quantification at 6 days post-infection (24 h after treatment) [11]. However, Figure S4). We therefore established a similar model using 300 TCID 50 with the H1N1 (rgA/WSN/33) and H1N1pdm09 (A/Osaka/129/2009) strains. The viral growth kinetics for each virus tested (H1N1 and H1N1pdm09), from 3 to 6 days post-infection, were similar across the different strains, with minimal impact on body weight ( Supplementary Figures S5 and S6).
PK Studies in Non-Infected Mice
To increase the validity of results in humans, we used baloxavir acid and oseltamivir phosphate at doses that achieved plasma concentrations similar to those recorded in humans. We compared human oral PK data derived from a previous study (on population PK parameters and exposure-response analyses in adults and adolescents) [26] to plasma concentrations of baloxavir acid in the animal models. The C 24 in mice following a 10 mg/kg single dose was 26.1 ng/mL, and that of a simulated dose of 30 mg/kg was 78.3 ng/mL. These concentrations were within the 10th-90th percentile of the human C 24 (Supplementary Figure S3). For oseltamivir phosphate, we used a corresponding therapeutic dose of 5 mg/kg, twice daily in mice, which has been shown to achieve the same AUC as an oral dose of 75 mg, twice daily, in humans in a previous study [28]. Baloxavir acid showed a dose-dependent decrease in lung virus titers in both strains ( Figure 1). In mice infected with the rgH1N1.WT, the virus titers in baloxavir acid-treated groups were significantly lower than vehicle-treated groups at all doses (p < 0.05). A single dose of baloxavir acid at 0.1 mg/kg showed a similar antiviral effect to that of oseltamivir phosphate (5 mg/kg, twice daily). The antiviral activity of baloxavir acid was significantly greater than oseltamivir phosphate at doses of 0.3 mg/kg and above (p < 0.0001). In rgH1N1.PA/I38T-infected mice, baloxavir acid demonstrated a significant decrease in virus titers, with doses of 10 mg/kg or more, compared with vehicle (p < 0.05) and a similar antiviral effect to oseltamivir phosphate at the 30 mg/kg dose, given once daily (p = 0.2925). To confirm dose dependency in this model, we used a high dose of 120 mg/kg/day (30 mg/kg, four times daily for 1 day, over a clinically equivalent dose). Notably, in PA/I38T-infected mice, this dose (120 mg/kg/day) reduced virus titers by 1.88 log 10 TCID 50 /mL compared with vehicle ( Figure 1). There was an overall shift in the amount of baloxavir acid needed to achieve the same level of inhibition for the PA/I38T strain as the WT strain. However, the doses of 10 and 30 mg/kg (single dose) still resulted in concentrations that were within the human C 24 range (Supplementary Figure S3). These doses were, therefore, used in further experiments examining the antiviral effect of baloxavir acid. (Figures 2 and 3A).
To confirm dose dependency in this model, we used a high dose of 120 mg/kg/day (30 mg/kg, four times daily for 1 day, over a clinically equivalent dose). Notably, in PA/I38Tinfected mice, this dose (120 mg/kg/day) reduced virus titers by 1.88 log10 TCID50/mL compared with vehicle ( Figure 1). There was an overall shift in the amount of baloxavir acid needed to achieve the same level of inhibition for the PA/I38T strain as the WT strain. However, the doses of 10 and 30 mg/kg (single dose) still resulted in concentrations that were within the human C24 range (Supplementary Figure S3). These doses were, therefore, used in further experiments examining the antiviral effect of baloxavir acid. . The green dotted line shows the LLOQ. Each bar represents the mean ± standard deviation of 10 mice. * p < 0.05 and ** p < 0.0001 vs. vehicle, † p < 0.01 and † † p < 0.0001 vs. oseltamivir phosphate (Dunnett's test) bid, twice daily; BXA, baloxavir acid; LLOQ, lower limit of quantification; OSP, oseltamivir phosphate; qd, once daily; qid, four times daily; TCID50, median tissue culture infectious dose; WT, wild type. Baloxavir acid resulted in a significantly large drop in virus titers to below the lower limit of quantification (LLOQ; 1.5 log10 TCID50/mL) in mice infected with the rgH1N1.WT and rgH1N1.NA/H275Y, as well as almost to the LLOQ in mice infected with the H1N1pdm09.WT. The antiviral effect of baloxavir acid was diminished in mice infected with the PA/I38T strains, but the virus titers remained significantly lower than vehicle at all doses except with the 10 mg/kg dose against the H1N1pdm09.PA/I38T.
Effect of Baloxavir Acid and Oseltamivir Phosphate on Lung Virus Titers in Mice
Oseltamivir phosphate demonstrated numerically different virus titer reduction across the different strains, but the virus titers were statistically lower compared with vehicle against all strains. The degree of virus titer reduction in oseltamivir phosphate was numerically smaller in the rgH1N1.NA/H275Y (0.38 log10TCID50/mL) than in the WT strain (1.15 log10TCID50/mL), suggesting reduced susceptibility to oseltamivir phosphate by NA/H275Y substitution.
Importantly, the reduction in lung virus titers with baloxavir acid (30 mg/kg, single dose) and oseltamivir phosphate (5 mg/kg, twice daily) was statistically similar for rgH1N1.PA/I38T and H1N1pdm09.PA/I38T (p = 0.4233 and p = 0.3078, respectively; Figures 2 and 3A). Similar results were noted in the dose-dependency experiment for rgH1N1.PA/I38T (p = 0.2925; Figure 1). In the case of the NA/H275Y mutant variant (rgH1N1.NA/H275Y), baloxavir acid showed a significantly higher antiviral activity compared with oseltamivir phosphate (p < 0.0001; Figure 2). . The green dotted line shows the LLOQ. Each bar represents the mean ± standard deviation of 19-20 mice. * p < 0.01 and ** p < 0.0001 vs. vehicle, † p < 0.05 and † † p < 0.0001 vs. oseltamivir phosphate (Dunnett's test) bid, twice daily; BXA, baloxavir acid; LLOQ, lower limit of quantification; OSP, oseltamivir phosphate; qd, once daily; TCID 50 , median tissue culture infectious dose; WT, wild type. Baloxavir acid resulted in a significantly large drop in virus titers to below the lower limit of quantification (LLOQ; 1.5 log 10 TCID 50 /mL) in mice infected with the rgH1N1.WT and rgH1N1.NA/H275Y, as well as almost to the LLOQ in mice infected with the H1N1pdm09.WT. The antiviral effect of baloxavir acid was diminished in mice infected with the PA/I38T strains, but the virus titers remained significantly lower than vehicle at all doses except with the 10 mg/kg dose against the H1N1pdm09.PA/I38T.
Oseltamivir phosphate demonstrated numerically different virus titer reduction across the different strains, but the virus titers were statistically lower compared with vehicle against all strains. The degree of virus titer reduction in oseltamivir phosphate was numerically smaller in the rgH1N1.NA/H275Y (0.38 log 10 TCID 50 /mL) than in the WT strain (1.15 log 10 TCID 50 /mL), suggesting reduced susceptibility to oseltamivir phosphate by NA/H275Y substitution.
Virus Titer Kinetics after Dosing
To determine whether mice infected with 300 TCID 50 H1N1pdm09.PA/I38T and rgH1N1.PA/I38T remain virus free post-baloxavir acid treatment, virus titers were evaluated up to 8 and 14 days post-infection, respectively ( Figure 3B and Supplementary Figure S1).
We did not observe any increase in virus titers during this time window ( Figure 3B and Supplementary Figure S1). Moreover, the overall virus titers in H1N1pdm09.PA/I38Tinfected mice continued to decrease over time; they were significantly lower with baloxavir than the vehicle-treated mice, on days 6 and 7 post-infection, at the dose of 30 mg/kg, and they were reduced to near the LLOQ on day 8 (1.87 log 10 TCID 50 /mL).
To monitor the emergence of further substitutions in addition to PA/I38T, during or after treatment with baloxavir acid, we conducted a Sanger sequence analysis of PA genes. We did not find any genotype changes, by observation, up to 14 days post-infection (9 days after baloxavir administration) in mice infected with the rgH1N1.PA/I38T and 8 days post-infection (3 days after baloxavir administration) in mice infected with the H1N1pdm09.PA/I38T.
Antiviral Effect of Baloxavir Acid in H3N2-Infected Hamsters
As mice are not susceptible to seasonal H3N2 viruses, we examined the effect of baloxavir acid on rgH3N2.WT and rgH3N2.PA/I38T using hamster models, in which H3N2 more efficiently proliferates [17].
In rgH3N2.WT-infected hamsters, baloxavir acid showed a significantly higher reduction in virus titers (at both 10 and 30 mg/kg single doses) than vehicle and oseltamivir phosphate (both p < 0.0001; Figure 4). Although the PA/I38T mutation significantly lowered the drug susceptibility towards baloxavir acid, a single dose of 30 mg/kg resulted in a statistically significant reduction in virus titers compared with the vehicle (p < 0.0001) and oseltamivir phosphate (p < 0.05; Figure 4).
PK/PD Analysis in Infected Mice
We performed a PK/PD analysis in a non-lethal mouse model infected with the rgH1N1.PA/I38T and treated it with baloxavir acid suspension.
PK parameters for the 0.25, 1, 4, 16, or 64 mg/kg doses of baloxavir acid in mice infected with rgH1N1.PA/I38T were calculated from the observed mean plasma concentration. Those for the 0.125, 0.5, 2, 8, and 32 mg/kg doses were mathematically scaled, assuming a linear relationship between the dose and PK parameters. These were correlated with the PD data on antiviral activity of baloxavir acid, determined from the virus titer data, using the linear model. Model fitness was evaluated by the coefficient of determination R 2 adjusted for degrees of freedom. The adjusted R 2 values were 0.633, 0.606, 0.627, and 0.672 for AUC0-24, Cmax, C24, and Cτ, respectively. The Cτ was the parameter that demonstrated the highest R 2 , and it most closely correlated with lung virus titer 24 h after administration. A previous study conducted in a similar way, in mice infected with A/WSN/33 (H1N1) and treated with baloxavir acid, had also shown that the Cτ was the best PK parameter to predict virus titers [11] (Supplementary Table S3).
The mean virus titer achieved with the in vivo administration of oseltamivir phosphate was 3.17 log10 TCID50/mL in this model. The corresponding Cτ value of baloxavir acid that achieved a virus titer of 3.17 log10 TCID50/mL in rgH1N1.PA/I38T-infected mice was 31.6 ng/mL ( Figure 5).
PK/PD Analysis in Infected Mice
We performed a PK/PD analysis in a non-lethal mouse model infected with the rgH1N1.PA/I38T and treated it with baloxavir acid suspension.
PK parameters for the 0.25, 1, 4, 16, or 64 mg/kg doses of baloxavir acid in mice infected with rgH1N1.PA/I38T were calculated from the observed mean plasma concentration. Those for the 0.125, 0.5, 2, 8, and 32 mg/kg doses were mathematically scaled, assuming a linear relationship between the dose and PK parameters. These were correlated with the PD data on antiviral activity of baloxavir acid, determined from the virus titer data, using the linear model. Model fitness was evaluated by the coefficient of determination R 2 adjusted for degrees of freedom. The adjusted R 2 values were 0.633, 0.606, 0.627, and 0.672 for AUC 0-24 , C max , C 24 , and C τ , respectively. The C τ was the parameter that demonstrated the highest R 2 , and it most closely correlated with lung virus titer 24 h after administration. A previous study conducted in a similar way, in mice infected with A/WSN/33 (H1N1) and treated with baloxavir acid, had also shown that the C τ was the best PK parameter to predict virus titers [11] (Supplementary Table S3).
The mean virus titer achieved with the in vivo administration of oseltamivir phosphate was 3.17 log 10 TCID 50 /mL in this model. The corresponding C τ value of baloxavir acid that achieved a virus titer of 3.17 log 10 TCID 50 /mL in rgH1N1.PA/I38T-infected mice was 31.6 ng/mL ( Figure 5).
Inhibitory Effect of Baloxavir Acid on Weight Loss Due to Viral Infection in Murine Model
The sub-lethal infectious dose of 300 TCID50 did not cause any loss in body weight in rgH1N1.PA/I38T-infected mice. A dose to examine the inhibitory effect of baloxavir acid on weight loss due to viral infection was established by testing higher inoculation titers. The 10,000 TCID50 infectious dose resulted in 12.7% loss of body weight-but not lethality-at day 6 post-infection in the PA/I38T strain, and it was considered an appropriate dose for this experiment (Supplementary Figure S6). Viral growth and loss in body weight, in mice with this dose, was tested for the H1N1 strains. Infection with the 10,000 TCID50 dose resulted in higher peak titers, across all three strains, than those noted with the 300 TCID50 dose (Supplementary Figures S5A and S7). The mutant strains seemed less pathogenic, as they resulted in lower body weight loss (rgH1N1.PA/I38T, maximum of 13.9% at 6 days post-infection and the rgH1N1.NA/H275Y, maximum of 17.0% at 7 days post-infection) than the rgH1N1.WT (maximum of 27.1% at 9 days post-infection). Furthermore, all mice infected with the mutant strains survived, whereas some mice infected with the WT strain died (or reached the weight of the humane endpoint [70% of the final body weight on the day of the infection]) (Supplementary Figure S8).
Next, mice infected with rgH1N1.WT, rgH1N1.PA/I38T, or rgH1N1.NA/H275Y (10,000 TCID50) were treated at 5 days post-infection. Body weight was observed for up to Lung virus titers were measured, and the linear model was applied to evaluate the relationship between the pharmacokinetic parameters and virus titers. The blue dotted line shows the mean virus titers recorded after treatment with oseltamivir phosphate (3.17 log 10 TCID 50 /mL). The red dotted line is the line of best fit. AUC 0-24 , the area under the curve from 0 to 24 h; bid, twice daily; C τ , plasma concentration at the end of the dosing interval after the first dosing; C 24 , plasma concentration at 24 h after the first dosing; C max , maximum plasma concentration; qd, once daily; qid, four times daily.
Inhibitory Effect of Baloxavir Acid on Weight Loss Due to Viral Infection in Murine Model
The sub-lethal infectious dose of 300 TCID 50 did not cause any loss in body weight in rgH1N1.PA/I38T-infected mice. A dose to examine the inhibitory effect of baloxavir acid on weight loss due to viral infection was established by testing higher inoculation titers. The 10,000 TCID 50 infectious dose resulted in 12.7% loss of body weight-but not lethality-at day 6 post-infection in the PA/I38T strain, and it was considered an appropriate dose for this experiment (Supplementary Figure S6). Viral growth and loss in body weight, in mice with this dose, was tested for the H1N1 strains. Infection with the 10,000 TCID 50 dose resulted in higher peak titers, across all three strains, than those noted with the 300 TCID 50 dose ( Supplementary Figures S5A and S7). The mutant strains seemed less pathogenic, as they resulted in lower body weight loss (rgH1N1.PA/I38T, maximum of 13.9% at 6 days post-infection and the rgH1N1.NA/H275Y, maximum of 17.0% at 7 days post-infection) than the rgH1N1.WT (maximum of 27.1% at 9 days post-infection). Furthermore, all mice infected with the mutant strains survived, whereas some mice infected with the WT strain died (or reached the weight of the humane endpoint [70% of the final body weight on the day of the infection]) (Supplementary Figure S8).
Next, mice infected with rgH1N1.WT, rgH1N1.PA/I38T, or rgH1N1.NA/H275Y (10,000 TCID 50 ) were treated at 5 days post-infection. Body weight was observed for up to 10 days post-infection. Treatment with baloxavir acid demonstrated a significant inhibitory effect on loss of body weight, compared with vehicle (p < 0.05), on different days on 10 mg/kg and 30mg/kg doses. This effect was observed in rgH1N1.WT-infected mice on days 9 and 10 (10 mg/kg and 30 mg/kg doses), in rgH1N1.PA/I38T-infected mice on days 6 and 7 (30 mg/kg dose), and in rgH1N1.NA/H275Y-infected mice on days 6-9 (10 mg/kg dose) and days 6-10 (30 mg/kg dose). The inhibitory effect on loss of body weight was significantly greater with baloxavir acid than with oseltamivir phosphate in rgH1N1.NA/H275Y-infected mice on day 6 (10 mg/kg dose) and on days 6-8 (30 mg/kg dose) (Supplementary Figure S9).
Discussion
This is the first report investigating the in vivo antiviral effect of baloxavir acid at doses simulating human plasma concentrations in animal (mouse and hamster) models infected with H1N1, H1N1pdm09, and H3N2 virus subtypes with PA/I38T substitution. These mutant strains exhibited a similar level of in vivo viral replication as their respective WT strains in the non-lethal animal model used in this and previous studies [11]. Baloxavir acid resulted in a modest reduction in lung virus titers in animals infected with PA/I38T strains compared with those infected with the WT strains. This effect was similar to that observed with oseltamivir phosphate. Since very little weight loss and no lethality were observed in mice infected with PA/I38T strains, we were unable to establish a clear effect of baloxavir acid treatment in reducing clinical signs of infection in these mice.
We noted that the antiviral effect of baloxavir acid was numerically lower in mice infected with the PA/I38T strains compared with the WT and NA/H275Y strains. Baloxavir acid reduced the virus titers in mice infected with the WT strains, at doses as low as 1 mg/kg, but not in those infected with the PA/I38T strains.
It was demonstrated that the viral strains used in this study proliferate under the test condition. It is therefore noteworthy that, under these conditions, even with reduced efficacy against PA/I38T strains, the effect of baloxavir acid, at doses equivalent to clinical administration (10 mg/kg and 30 mg/kg) [27], was significantly greater than that seen with vehicle, as well as comparable to that of oseltamivir phosphate at clinically relevant doses (5 mg twice daily).
No viral rebound was observed in mice treated with baloxavir acid for up to 14 and 8 days post-infection with rgH1N1.PA/I38T and H1N1pdm09.PA/I38T, respectively. These findings suggest that a single dose of 30 mg/kg baloxavir acid was sufficient to reduce the virus titers in mice infected with PA/I38T strains at 6 dpi, leading to viral elimination.
We conducted a PK/PD analysis on PA/I38T infections in mice to strengthen the validity of the findings in the animal model and to facilitate translation into clinical situations. At doses simulating human plasma concentrations, baloxavir acid demonstrated a dose-dependent effect against PA/I38T infections, supporting its potential efficacy against PA/I38T mutant strains. The C τ value was found to be a strong predictive PK parameter for antiviral activity of baloxavir acid against both rgH1N1.WT and rgH1N1.PA/I38T infections [11]. The C τ value of baloxavir acid, which showed a similar in vivo antiviral effect against rgH1N1.PA/I38T infection as oseltamivir phosphate, was 31.6 ng/mL. We found that this plasma concentration was almost achieved in humans at 24 h in a non-Asian population and at 48-60 h in an Asian population, as determined in a previous study [27].
The pathogenicity of the PA/I38T strain was assessed by inoculating mice with a high infectious dose of the PA/I38T strain. The loss of body weight was observed at doses of 3000 TCID 50 and higher. To compare the pathogenicity of the three strains, the mice were inoculated with 10,000 TCID 50 of each strain. While the WT strain demonstrated body weight loss and lethality in infected mice, the loss in body weight and lethality was limited with the same dose of mutant viruses (NA/H275Y and PA/I38T), demonstrating lower pathogenicity. Even though baloxavir acid demonstrated a marginal inhibitory effect on weight loss in mice infected with the mutant strains, this effect may have been limited due to the low pathogenicity of the mutant viruses. Therefore, no clear effect of baloxavir acid on pathogenesis could be evaluated.
Previous experiments in mice, hamsters, and ferrets have shown that variants carrying the PA/I38T mutation demonstrate pathogenicity and replicative abilities that are similar to their respective WT strains, with a minor loss in fitness, and they have a lower transmission ability when mixed with the WT strains [17,18]. This relative fitness cost has been shown to be greater in H1N1pdm09 viruses than in H3N2 viruses. Using reverse genetic mutant strains, as opposed to clinical isolates, for a consistent genetic background, the PA/I38T mutation demonstrated a similar impact in this study. The growth of the mutant strain compared with the respective WT strains was not significantly different at the sub-lethal dose of 300 TCID 50 , confirming the replicative fitness and pathogenicity of the rgH1N1.PA/I38T mutant strain.
Our study has several limitations. First, owing to differences in the EC 50 assay methodology, it is not possible to directly compare the EC 50 of the mutant strains tested in this study with that of strains reported in other studies. The mutant isolates used in this study have shown an EC 50 -based fold change (FC) in baloxavir acid susceptibility comparable to those reported to date [14]. However, a study that used clinical isolates revealed higher EC 50 -based FC values (65-155) [24] than our current results (57). Therefore, further investigation to determine whether baloxavir acid is effective against clinical isolates of different strains is recommended.
Second, in this study, we have only tested the PA/I38T strains. Surveillance data have identified other H3N2 and H1N1pdm09 strains with PA amino acid substitutions, such as E23K/G/R, A36V, A37T, I38M/F/L, E119D, and E199G, resulting from amino acid substitutions in the PA [14]. However, as these strains demonstrate a lower EC 50 -based FC than the PA/I38T strains, in susceptibility to baloxavir acid, we expect baloxavir acid to be substantially more effective against these mutant strains than the PA/I38T strain.
Third, our findings from this study are based on baloxavir acid treatment 5 days post-infection, followed by lung virus titer determination at 6 dpi. Additional experiments are needed to evaluate baloxavir acid efficacy earlier in the therapeutic regimen, as well as explore the prophylaxis effect when used pre-infection. In addition, we suggest that future experiments should be conducted to measure the virus spread in the lungs of infected mice, at different dpi, to further evaluate the drug efficacy.
Finally, the replication efficiency of influenza virus variants in the lungs can differ from drug-susceptible WT strains. We therefore propose that a 50% lethal dose should be determined in mice for each virus (a) to fully evaluate the antiviral efficacy of baloxavir acid in animal models, (b) to achieve comparable virus titers, and (c) to observe the clinical signs of infection caused by drug-resistant and drug-susceptible viruses.
In summary, to the best of our knowledge, this is the first study reporting the effects of baloxavir on infections caused by the PA/I38T mutant strain in mouse and hamster models. Our study showed that, upon clinically equivalent drug exposure in mice, baloxavir acid demonstrated minor, dose-dependent in vivo antiviral activity against PA/I38T mutant strains. Even though the degree of lung virus titer reduction with baloxavir acid was diminished in animal models infected with PA/I38T mutant strains, the observed antiviral effect was comparable to that of oseltamivir phosphate.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v15051154/s1, Figure S1: Time course of virus titers in mice infected with H1N1 PA/I38T strain; Figure S2: Dose-dependent pharmacokinetics of baloxavir acid in non-infected mice and hamsters; Figure S3: Comparison of plasma concentrations of baloxavir acid in humans, mice and hamsters; Figure S4: Virus growth curves in mice infected with H1N1 PA/I38T strain; Figure S5: Viral growth curves in mice infected with influenza A at non-lethal doses; Figure S6: Body weight change in mice infected with different doses of H1N1 PA/I38T strain; Figure S7: Virus growth curves in mice infected with H1N1 strains; Figure S8: Body weight loss in virus-infected mice; Figure S9: Effect of baloxavir acid treatment on body weight change in mice infected with H1N1 WT, PA/I38T or H275Y strains; Table S1: List of primer sequences used for RT-PCR and sequencing; Table S2: Mean values of the parameters used in the PK/PD analysis of rgH1N1.PA/I38T-infected mice; Table S3: Analysis of pharmacokinetic parameters of baloxavir acid in the linear model. Funding: This study was sponsored by Shionogi & Co., Ltd., manufacturer/licensee of baloxavir marboxil. Medical writing assistance was also funded by Shionogi.
Informed Consent Statement: Not applicable.
Data Availability Statement: We will share the data upon reviewing the request. Requests of data sharing from researchers will be reviewed with regard to the validity and feasibility of the research intended. | 2023-05-13T15:13:23.438Z | 2023-05-01T00:00:00.000 | {
"year": 2023,
"sha1": "cfd7437a899b8dd65d27b33c626395a0b53965b0",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.3390/v15051154",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "500c190923136a0b21d1cc4f138d211169099ffd",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
168919075 | pes2o/s2orc | v3-fos-license | Effect of Integrated Nutrient Management on Productivity and Profitability of Maize
Maize (Zea mays L.) is the third most important cereal crop next to wheat and rice belonging to family Poaceae. In India, it is cultivated in an area of 9.43 m ha with a production of 24.35 m t and productivity of 2583 kg ha -1 (GOI, 2012). It is used as a source of carbohydrate to humans and animals due to its high feeding value (Undie et al., 2012). Maize is used as food, feed and nowadays it is gaining tremendous importance on account of its potential uses in manufacturing starch, rayon, adhesives, resins, dye, boot polish etc. and due to this it is rightly called “miracle crop” and also known as “queen of cereals”. Intensive cultivation, use of unbalanced and inadequate fertilizers accompanied by restricted use of manures have made the soils not only deficient in the nutrients but also deteriorated soil health resulting in decline in crop response to recommended dose of fertilizers. Thus, Integrated Nutrient Management (INM) has assumed a great importance and has vital significance in maintaining soil productivity. Organic manures viz. FYM, vermicompost etc. not only supply macronutrients, meet the requirements of micronutrients, improve soil health and also increases water holding capacity of the soils. The organic fertilizers in addition to nutrients contain microbial load International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 3878-3882 Journal homepage: http://www.ijcmas.com
Introduction
Maize (Zea mays L.) is the third most important cereal crop next to wheat and rice belonging to family Poaceae. In India, it is cultivated in an area of 9.43 m ha with a production of 24.35 m t and productivity of 2583 kg ha -1 (GOI, 2012). It is used as a source of carbohydrate to humans and animals due to its high feeding value (Undie et al., 2012). Maize is used as food, feed and nowadays it is gaining tremendous importance on account of its potential uses in manufacturing starch, rayon, adhesives, resins, dye, boot polish etc. and due to this it is rightly called "miracle crop" and also known as "queen of cereals". Intensive cultivation, use of unbalanced and inadequate fertilizers accompanied by restricted use of manures have made the soils not only deficient in the nutrients but also deteriorated soil health resulting in decline in crop response to recommended dose of fertilizers. Thus, Integrated Nutrient Management (INM) has assumed a great importance and has vital significance in maintaining soil productivity. Organic manures viz. FYM, vermicompost etc. not only supply macronutrients, meet the requirements of micronutrients, improve soil health and also increases water holding capacity of the soils. The organic fertilizers in addition to nutrients contain microbial load -100% RDF, T 2 -125% RDF, T 3 -150% RDF, T4-75% RDF + Azotobacter + PSB, T 5 -RDF + 5 tons FYM/ha, T 6 -RDF + 2.5 tons FYM/ha, T 7 -Farmers practice and T 8 -control and each replicated thrice. The RDF used was 120:60:40 NPK/ha. The seed rate, spacing and variety taken in the experiment was 20 kg/ha, 60 cm x 25 cm and HQPM-1 respectively. The pooled results revealed that treatment T 3 (150% RDF) recorded significantly higher growth parameters and yield attributes viz. plant height(201.90 cm), number of grains/cob (393.20), test weight (223.25 g) and grain yield (52.05 q ha -1 ) which was closely followed by treatment T 5 (RDF +5 tons FYM/ha and recorded plant height (200.30 cm), number of grains/cob (391.95), test weight (223.15 g) and grain yield (51.70 q ha -1 ) and was found to be at par to treatment T 3 . The pooled table 2 shows that maximum net return and BC ratio was computed in treatment T 3 having values as Rs.35914.00 and 1.41 respectively followed by treatment T 5. and growth promoting substances which help in boosting plant growth, metabolic activity and resistance to pest and diseases. Boosting yield, reducing cost of production and improving soil health are three interlinked components of sustainable triangle (Singh et al., 2016). Before green revolution era organic manures were the primary source of nutrients for crop production. In recent years there has been renewed interest in use of FYM which is attributed to concerns for maximizing sustainable agricultural production besides preserving the environment. Fertilizers are able to increase crop yields additionally produce enough residues for soil fertility management, while organic sources rehabilitate less responsive soils and makes them responsive to fertilizers (Vanlauwe et al., 2010). In INM, all the possible source of plant nutrients are applied based on economic consideration and the balance required for the crop is supplemented with chemical fertilizers. The INM approach not only enhances production and profitability of crop like maize but also aids in maintaining the permanent fertility status of the soil.
With this background, the present investigation entitled "Effect of Integrated Nutrient Management on productivity and profitability in maize (Zea mays L.)" was designed and conducted during kharif season of 2014 and 2015 at Zonal Research Station, Darisai, under Birsa Agricultural University, Ranchi, Jharkhand with the aim to assess the response of INM on productivity and profitability of maize in this region.
Materials and Methods
An experiment was conducted during kharif season of 2014 and 2015 at Zonal research Station, Darisai, East Singhbhum under Birsa Agricultural University, Ranchi, Jharkhand, for sub-agro climatic zone-VI, which comes under South Eastern plateau and situated at 22 o 41 ' North latitude, 86 o 23 ' East longitude and at an altitude of 521 m above mean sea level. The zone is characterized by humid to sub-tropical type of climate. The annual rainfall varies between 1200-1500 mm. The soil was sandy loam in texture with p H 5.6. The experimental field soil was low in organic carbon, nitrogen and phosphorus but medium in potash. The experiment was designed in Randomized Block Design having 8 treatments viz. T 1 -100% RDF, T 2 -125% RDF, T 3 -150% RDF, T 4 -75% RDF + Azotobacter + PSB, T 5 -RDF + 5 tons FYM/ha, T 6 -RDF + 2.5 tons FYM/ha, T 7 -Farmers practice and T 8 -control and each replicated thrice. The RDF used was 120:60:40 NPK/ha. The seed rate, spacing and variety taken in the experiment was 20 kg/ha, 60 cm x 25 cm and HQPM-1 respectively. Half dose of nitrogen and full dose of phosphorus and potassium was applied as basal treatment wise. The sowing was done on 2 nd July in both the years of experiment. Pre-harvest and post-harvest i.e. growth and yield components and grain yield were recorded as and when required.
Results and Discussion
The result of the experiment entitled "Effect of Integrated Nutrient Management on Productivity and Profitability of Maize (Zea mays L.)". The pooled data pertaining to various components used for treatment evaluation were analyzed statistically to test their significance.
Effect of INM on growth components and development of maize
A perusal of table 1 clearly revealed that growth parameters were significantly influenced by integrated nutrient management. The maximum plant height (201.90 cm), dry matter accumulation (213.45g), number of cobs plant -1 (1.40), number of grains cob -1 (393.75) and test weight (223.25g) was recorded in treatment T3 (150 % RDF) followed by treatment T5 (RDF + 5 tons FYM ha -1 ) which recorded plant height, dry matter accumulation, number of cobs plant -1 , number of grains cob -1 and test weight as 200.30 cm, 212.90 g, 1.40, 391.95 and 223.15 g respectively. A critical review of the table also revealed that treatment T 5 was found to be at par to treatment T 3 with regards to above mentioned parameters. However, minimum growth parameters was recorded in treatment T 8 (Control) followed by treatment T 7 (Farmers practice). The probable reason for recording maximum growth attributes may be due to better availability of nutrients in balanced form during vegetative phase (Makinde, 2007 andRajeshwari et al., (2007).
The other reason might be due to more number of leaves per plant in these treatments which led to more leaf area which is a measure of size of assimilatory system of plant and is a product of leaf length and width which is important for the accumulation and partitioning of photosynthates to the economic parts of the plant.
Fig.1 Effect of integrated nutrient management on yield
The results so obtained in performances probably due to nutrients were responsible for increased cell division, cell enlargement, growth, photosynthesis and protein synthesis for quantitative increase in plant growth (Panwar, 2008). These findings are in accordance of those reported by Ghafoor and Akthar (1991), Balyan et al., (2006) and Ayoola and Makinde (2009) who stated that application of high doses of nutrients in balanced form has significant effect on growth and development of maize.
Effect of INM on yield of maize
An appraisal of table 2 and figure 1 clearly revealed that yield parameters were significantly influenced by integrated nutrient management. The maximum grain yield (52.05q ha -1 ), stover yield (140.70 q ha -1 ) and stone yield (16.20 q ha -1 ) was recorded in treatment T 3 (150 % RDF) followed by treatment T 5 (RDF + 5 tons FYM ha -1 ) which recorded grain yield, stover yield and stone yield as 51.70 q ha -1 , 140.10 q ha -1 and 15.50 q ha -1 respectively. A close examine of the table 2 also revealed that treatment T 5 was found to be at par to treatment T 3 with regards to grain yield, stover yield and stone yield while minimum yield was recorded in T 8 (Control).Crop yield depends on the accumulation of photos assimilates during the growing period and the way they partitioned between desired storage organs of the plant.
In the research carried out during kharif season of 2014 and 2015, grain yield, stover yield and stone yield was significantly influenced by treatment T 3 and treatment T 5. The both treatments recorded better yield over rest of the treatments. The probable reason for significant improvement in grain yield was due to superiority of yield attributes viz. Cob weight, grains cob -1 and test weight. The higher growth attributing traits could also be attributed to sustained availability of macro and micro nutrients which may have taken part in nourishment of the crop and also enhanced the activity of meristematic cells and cell elongation where FYM providing micronutrients has favourable effect on metabolic process, enzymatic activity and growth hormones which in turn lead to better vegetative growth in terms of higher plant height and number of leaves eventually contributed to increased dry matter production. These results are in accordance with those reported by Shanmugam and Veeraputhran (2000), Iman et al., (2002) and Ranjbar and Bahmaniar (2007).
Economic analysis of maize indicated significantly higher net return (Rs.35914.00) and B: C ratio (1.41) in treatment T 3 followed by treatment T 5 which recorded net return and B: C ratio as Rs.35597.00 and 1.40 respectively on account of significantly higher grain, stover and stone yield. Similar findings were also observed by Singh et al., (2005) and Gohawale and Dahipale (2007). | 2019-05-30T13:21:47.369Z | 2017-12-20T00:00:00.000 | {
"year": 2017,
"sha1": "648a20148727dbd8365f84de81e3453a547c0706",
"oa_license": null,
"oa_url": "https://www.ijcmas.com/6-12-2017/Shambhu%20Sharan%20Kumar,%20et%20al.pdf",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "eab21076eba8960007d34fa3dfbaa210e952abb9",
"s2fieldsofstudy": [
"Agricultural And Food Sciences"
],
"extfieldsofstudy": [
"Business"
]
} |
76666167 | pes2o/s2orc | v3-fos-license | Perspectives on Acceptance and Use of a Mobile Health Intervention for the Prevention of Atherosclerotic Cardiovascular Disease in Singapore: Mixed-Methods Study
Background Cardiovascular disease, including atherosclerotic cardiovascular disease (ASCVD), is a growing public health threat globally and many individuals remain undiagnosed, untreated, and uncontrolled. Simultaneously, mobile health (mHealth) interventions using short messaging service (SMS) have gained popularity globally. There is an opportunity for innovative approaches such as mHealth to encourage and enable adherence to medications for ASCVD and its risk factors. Objective This study aimed to understand mobile technology acceptance, use, and facilitating conditions among the study population ahead of the design of an mHealth intervention. Methods Using data from a mixed-methods study conducted in Singapore, we conducted a cross-sectional survey with 100 participants and in-depth, semistructured interviews with 20 patients. All participants were over the age of 40 years with ASCVD or its risk factors. Interviews were conducted in English and Mandarin and if needed translated to English. Nvivo 11 (QSR International) was used for analyses. Results Participants reported their perspectives on technology use and preferences, including low or sporadic mobile phone use and usability concerns including small screen and text size, among others; the benefit of previous mHealth use in creating a favorable opinion of SMS for health information; trust in both the source of mHealth SMS, as well as in treatment; the formation of habits; and fear of sequelae or death for facilitating intention to use an mHealth intervention and adhere to medication. We also highlighted a case that underscored the importance of the period after diagnosis in habit forming as an opportunity for an mHealth intervention. Conclusions We explored both technology- and adherence-related factors that influence a patient’s intention to use an mHealth intervention for adherence to ASCVD medication in Singapore. We highlighted the importance of identifying the right opportunity to engage with patients and promote an mHealth intervention for adherence, such as immediately following diagnosis when patients are establishing medication-taking habits.
Background
Mobile health (mHealth) interventions using short messaging service (SMS) have gained popularity as the number of people using mobile technology increases globally [1].Singapore has one of the highest mobile phone penetration and usage rates in the world, thus providing an interesting case study for such interventions [2].SMS-based interventions have shown promise because of affordability and wide outreach and have been applied to many aspects of health including health promotion and to enable medication adherence [3][4][5].Although mHealth has provided many opportunities to reach patients, especially vulnerable groups, it is important that contextually appropriate patient preferences, usability, and acceptance of technology are considered when undertaking these interventions.
Patient preferences and acceptance are of particular importance when considering mHealth interventions for chronic conditions such as cardiovascular disease (CVD).CVD, including atherosclerotic cardiovascular disease (ASCVD), is a growing public health threat globally, with mortality rates estimated to reach 23.3 million by 2030 [6].Importantly, many individuals with risk factors for ASCVD remain undiagnosed, untreated, and uncontrolled [7,8].This points to the need for greater attention to factors impacting adherence in these patients and innovative approaches, such as mHealth, to encourage and enable adherence to medications for ASCVD and its risk factors in this patient population.
A systematic review demonstrated evidence of the feasibility of mHealth for adherence to medications for CVD.All 10 completed trials included in the review showed improved medication adherence for patients with CVD; all studies also reported positive responses to mHealth use from patients, however, the authors highlighted the paucity of data on studies including user input on the design of the intervention [9].Qualitative studies have also shown the potential of mHealth to provide education, optimize resources, and improve use of health care for CVD management [10].Yet, there is a need for more contextual evidence on patient acceptance and use of mHealth interventions for adherence, specifically exploring the unique factors that influence the use and acceptance of mHealth adherence support for medications to treat chronic conditions.For example, elderly patients managing chronic conditions may face unique usability concerns including functional difficulties using the device, limitations in vision and hearing, as well as the need for appropriate message content [11].To explore the needs of those with or at risk for ASCVD in Singapore, our study population comprised patients over the age of 40 years identified as having ASCVD, or risk factors for ASCVD including hypertension or hyperlipidemia.
This study is the development phase of a proposed mHealth intervention, the txt2heart trial, to support patient adherence to medications for ASCVD.The txt2heart trial is an international collaboration evaluating the efficacy and safety of SMS on clinical outcomes and adherence in different countries including Colombia, Ghana, India, and Singapore.This study sought to explore mobile technology acceptance, use, and facilitating conditions, as well as adherence factors, among the Singapore study population ahead of the design of the mHealth intervention.
The Study Setting: Singapore
To better contextualize our findings, it is necessary to consider larger health system factors relevant to Singapore, primarily the ubiquity of affordable and accessible health care.Singaporeans have access to largely subsidized care offered in polyclinics (government subsidized general practice clinics), private general practitioners, and tertiary care facilities for primary care and chronic condition management.Furthermore, within the primary care setting, doctors regularly prescribe months' worth of medications, thus enabling ease of access to medications.The government also provides subsidies and various schemes for the management of chronic conditions; the majority of our participants reported availing these schemes to pay for their health care and most reported being able to afford and access their CVD medications.
Theoretical Framework
Using a deductive approach, we used the unified theory of acceptance and use of technology (UTAUT) model to analyze our qualitative findings.This was then complemented by emergent themes on medication adherence factors, which prompted us to apply the World Health Organization (WHO) adherence model to our data [12].We then applied the modified and condensed UTAUT model to understand technology acceptance and use among participants with ASCVD and ASCVD risk factors (Figure 1).The UTAUT has been widely used to understand information technology (IT) adoption in general and has also been applied to health IT [13][14][15][16][17].
The UTAUT model employs 4 constructs through which to explore user factors; these being (1) performance expectancy, (2) effort expectancy, (3) social influence, and (4) facilitating conditions.Performance expectancy includes the perceived usefulness and personal outcome expectations associated with technology use.Effort expectancy is the perceived ease of use and complexity of the technology.Social influence includes subjective norms and technology use within a user's social milieu, whereas facilitating conditions include perceived behavioral control and wider contextual circumstances that support the use of technology.We have labeled these as technology-oriented factors as they explore the users' experience and preferences as oriented to their use and acceptance of technology.An important modification necessary to understand use and acceptance of an mHealth intervention for adherence is the adherence factor, which may contribute to the behavioral intention and use of the intervention [18].For our purposes, we have adapted the WHO framework to focus on the condition-related and therapy-related factors impacting adherence.Condition-related factors are the illness-related demands faced by the patient, which ultimately impact a patient's risk perception, treatment beliefs, and the priority they place on adherence [18].Therapy-related factors include side effects, treatment duration, treatment failures, and experience of side effects [18].Although an intervention may meet the technology-oriented needs of a patient, the lived experience of their condition(s) and treatments could present barriers to their behavioral intention, both to adhere to treatment as well as to use an mHealth intervention to support treatment adherence.
In this study, we explore in detail the technology-oriented aspects of UTAUT as well as therapy-and condition-related adherence factors acting upon a patient's behavioral intention.Thus, we use the modified and condensed UTAUT model and key elements of the WHO framework as a way to better understand the factors impacting elderly Singaporeans' acceptance and use of mobile technology for an intervention to support adherence to ASCVD medication.
Sampling (Survey)
This cross-sectional component utilized data from a brief locally adapted survey created for this study, which sought to explore patient technology use.The survey used purposive sampling of an existing patient pool from the Singapore Population Health Study-Community Health Study to recruit those aged 40 years and above with established ASCVD or risk factors who fulfilled inclusion criteria (Textbox 1).
Sampling (Qualitative)
The study took place in Singapore.We used purposive sampling from an existing patient list to recruit those aged 40 years and above with established ASCVD or risk factors for ASCVD (for complete inclusion and exclusion criteria, see Textbox 1).
Survey, Interviews, and Interview Guide
Trained research staff from the National University of Singapore (NUS) conducted the interviews or semistructured in-depth interviews in the participant's preferred language by staff fluent in that language.Interviewer training included description of research protocol, qualitative methods, and research ethics in practice.
Participants
The qualitative component involved semistructured in-depth interviews with 20 participants.Out of the 20 participants interviewed, 19 agreed to audio recording and 1 participant declined.For the latter, detailed field notes and an extensive memo were taken for inclusion in data analysis.
A total of 100 patients met the inclusion criteria for the quantitative survey and were recruited over the telephone.In total, 60 in-person and 40 telephone surveys were completed following informed consent from patients.A total of 100 surveys, inclusive of 1 incomplete survey on mobile phone technology usage section, assessed patients' sociodemographic characteristics, pattern of medication adherence, mobile phone technology usage (ownership, access, and utilization), and interest in mHealth.
Ethical Approval
Ethical approval for the study was obtained from the NUS Institutional Review Board.Informed consent for participation and recording was obtained before the interview started using a participant information sheet and consent form.Participants could refuse to answer any of the questions and/or discontinue their participation in the research at any time.
Statistical Analysis
Statistical analyses were performed using IBM SPSS version 24.0 (IBM Inc).Frequencies (n) and percentages (%) were used to summarize sociodemographic characteristics, clinical characteristics, patterns of mobile phone technology usage, and interest in mHealth.
Qualitative Analysis
A total of 2 research team members coded interviews using Nvivo 11 (QSR International) software applying interpretive and inductive approaches, thematic analysis, and techniques from the constant comparative method, where a line-by-line analysis of early interviews was used on subsequent interviews to test preliminary assumptions [19].Interviews were recorded and transcribed in full.Reviewers agreed on identified codes and themes.To maintain confidentiality, all names reported were pseudonyms and identifying data were excluded.
Participant Characteristics
The mean age of the 100 participants in the analysis sample was 65.3 (SD 9.6) years (Table 1).The largest proportion of participants, 55% (55/100), encompassed those aged greater than or equal to 65 years.The majority of patients (70/100, 70%) were male and 66% (66/100) were of Chinese ethnicity, with women and Malay, Indian, and other ethnicities representing a smaller proportion of the sample.Moreover, the majority of patients, 72% (72/100), reported having hyperlipidemia and 68% (68/100) reported having hypertension.
We conducted 20 in-depth interviews with participants who met the inclusion criteria.The detailed characteristics are presented in
Quantitative Survey Results
A quantitative survey of 100 patients meeting the same criteria outlined above provided our study with descriptive data on our target population.Of the 99 participants who fully completed the survey, 90% (90/99) owned a mobile phone.Of those mobile phone owners, 77% (70/90) reported accessing their mobile phones in general at least once a day, and the same proportion of patients (70/90, 77%) reported using SMS at least once a day.In general, participants predominantly used their phones for phone calls (87/90, 97%), SMS (60/90, 65%), and other text messaging services such as Whatsapp (54/90, 61%; Figure 2).Of those who owned a mobile phone, 53.3% (48/90) indicated their interest in receiving medication information through their mobile phones.
Qualitative Interview Findings
We reported our findings classified into themes based on our modified UTAUT model.In terms of technology-oriented factors, the first theme explored perceived usefulness of the mHealth intervention and personal outcome expectations.The second theme explored effort expectancy, including perceived ease of use, as well as other usability issues experienced by our elderly population.Next, we explored the social influences shaping technology use including the role of family and friends, XSL • FO RenderX as well as the impact of social isolation.Then we looked at facilitating conditions for technology use including trust and previous mHealth use.We next explored adherence factors influencing behavioral intention, including therapy-related factors such as regime complexity, therapy duration, inconvenience, and adverse effects.Finally, we looked at condition-related factors including symptoms and effects of condition on the functional and mental state.See Multimedia Appendix 2 for key themes and examples of evidence.
Perceived Usefulness
Participants reported on their perception of the usefulness of having a reminder system to take their medications.Most participants reported that they saw no need for such a system, as they did not have difficulties remembering.As Yong Liat reported:
I can't forget. Every time I put it on top, and I write down there. So I know. People don't need to tell me. I know myself. [IDI006_M_81-85_Chinese]
However, some participants did agree that hypothetically having reminders may be good for others who forget to take their medications.As Kim Huat explained: They should see the message and it should help.After all the reminder for them to take the medication is actually beneficial for them, and they should heed the reminders.[IDI009_M_71-75_Chinese] Some participants also voiced that low phone usage may inhibit the usefulness of an SMS intervention.As Irene described:
Personal Outcomes Expectations
The majority of participants explained that they personally felt an SMS intervention would not be useful or provide a useful outcome.This is partly because of some participants having low reliance on their phones, meaning the vehicle for delivery may not be aligned with the population's mobile uses.Furthermore, some participants expressed that the intervention does not address what they perceive to be the root causes of nonadherence, which they feel to be beyond the reach of mHealth.For example, an SMS intervention would not help those who intentionally do not take their medication.As Ah Siew explained: If they want to take medication, they will.Some people they're not taking it on purpose.I don't take some of my medication too.We take only the important medicine and don't take those that we feel aren't important.[IDI020_F_71-75_Chinese] Another participant highlighted that regardless of the proposed intervention, a key factor in adherence is patient agency and responsibility in placing importance on their health.Yan Ting explained: I think whatever method you want to use, it's nothing compare to convincing that patient that it is in their own interest and it is important for them to take the medicine regularly according to the schedule...I think if they are serious looking after themselves, it becomes a habit already.[IDI018_M_76-80_Chinese] However, some participants reported on factors that may be modifiable by an mHealth intervention.Irene summed up her perspective of the main factors for nonadherence, forgetfulness, dependency on others, and being busy: Interviewer: How do you think we can use mobile phones to remind people to take their medication?
Participant: When people forget to take their medications it's the following three reasons.First, they're forgetful, like me.I tell myself it's time to take my medication and walk to the kitchen.When I'm in the kitchen, I can't remember why I came into the kitchen.Second, they're dependent.They wait for people to tell them.Third, they are too busy.Just these three types of people.[IDI004_F_66-70_Chinese]
Effort Expectancy
As mHealth interventions predicate on patients being able to interact with SMS, it is important to understand their perceptions of basic mobile technologies as well as their perceptions on their ability to learn how to use such technologies.
Perceived Ease of Use
Some participants described difficulty using a mobile phone, thus preventing them from being interested in an mHealth intervention.However, others were keen to keep up with technology and expressed initiative and interest in learning about their phones.Safiah explained how she previously did not know how to use some features of her phone, but now is able to:
Previously I SMS right--previously aunty don't know how to SMS...So now everything I know. You live in the world of today...Every now and then, you must go out and learn everything. What the youngsters can do, the old also can do. [IDI012_F_71-75_Malay]
Among those who are comfortable using technology, a few reported that SMS is a good and convenient way to convey health information:
Usability Issues
Many participants, however, reported on functional issues preventing them from comfortably using a mobile phone including small screens or font sizes that are barriers to reading SMS messages.As Cheng Han explained: Our eyesight is also failing.Sometimes we see an"8" as an "S."It also makes it difficult to read our SMS.Our senses all deteriorate after 70 years old.
Social Influence
Another important component of technology uptake and use is that of social influence.Those with stronger social networks who use and look favorably on technology may use technology to connect and stay in touch, and these networks may in turn enable them to adopt technology.
Family and Friends' Technology Use
Some participants reported that the lack of a social support system is a reason for not being interested in using a mobile phone, and others reported using the phone less as their friends had passed away.As Wen Cheng explained:
I don't have many friends. I don't need to talk a lot now, or I just use the home telephone. [IDI001_M_71-75_Chinese]
Among those who had stronger social supports, many described that their friends were an important factor in their ability to learn and keep up with technology.Others described how technology had enabled them to keep in touch with friends from abroad and that these groups were helpful to them, thus giving them a favorable impression of mobile technology.One participant described how technology and social support played a role in adherence.As Kavita reported:
Facilitating Conditions
Our participants highlighted the importance of previous exposure to mHealth services and trust as facilitators to mHealth use.
Previous Mobile Health Use
A majority of participants reported positive experiences with previous mHealth apps, namely SMS appointment reminders.Participants felt these were useful and helped them to remember their appointments.Kim Huat explained: They will send SMS to me, and they also give me a paper with the appointment date.They usually remind me via SMS a week before my appointment...It's a good reminder so that you don't forget.
[IDI009_M_76-80_Chinese] One participant, Kamal, highlighted that patients may not be an appropriate audience to target the SMS reminders to and it would be more useful to engage primary caregivers in mHealth interventions: Tell the person looking after the patient to remind them to take their medication...The person cannot remember...so if you tell the children or (caregiver) they will make sure they take the medication, as if the orders come from the doctor the caregiver will scared and make sure they take.[IDI010_M_71-75_Indian]
Trust
An important facilitating factor reported by patients was that of trust.Some patients explained that they do not trust all the information they reiceve on the Web or shared through Whatsapp groups.As Kim Huat said: There are a lot of fake information there (on the phone), not all are true.[IDI009_M_76-80_Chinese] Thus, highlighting the need for an mHealth intervention to come from a trusted and known source.
Kim Huat went on to explain how the underlying trust in treatment is important in an mHealth intervention for adherence to medications: Interviewer: Who do you think this service will help?
Participant: Those that believes that their medication is effective. After all if you face any issues while taking the medication, the doctor and change it for you. [IDI009_M_76-80_Chinese]
This highlighted the need to not only understand user preferences and behaviors, but also to take into consideration the various adherence factors, which influence patient decision making and ability to adhere to medications.
Adherence Factors
Adherence factors include therapy-related and condition-related factors.To design an appropriate mHealth intervention, it is important to understand how both the medication itself and the condition may limit or enable adherence to medication.
Complexity of Medication Regime
Some patients reported adjusting their medication to cope with a complex regime and to facilitate their ability to adhere to their medications.As Sow Tin described: The doctor did tell me to take the blue pills before food.I thought it was troublesome to split them, so I took all my pills before food.So I can remember whether I've taken my medication...The doctor did say I was cheating.But it's okay.It's more time efficient for me.[IDI004_F_66-70_Chinese] Nearly all patients described how using a pillbox and the ritual surrounding taking their medication had enabled them to form habits. Patients also reported that they did not feel the need to
XSL • FO
RenderX have an mHealth intervention to remind them as their current habits and pillbox use enabled them to ensure they had taken medications and prevented them from taking a double dose.
Duration of Therapy
The majority of patients also reported how the long duration of therapy for chronic conditions facilitated their ability to adhere to their medications.Thus, they felt that an mHealth intervention would not be of use, as they had already established habits over the course of managing their chronic conditions.As Karen reported: I just follow...because I have been using it for long, it's no problem to me..
Inconvenience With Lifestyle
Although most patients reported that they largely adhered to their medications and had habits to support their medication taking, some patients reported how lifestyle factors disrupted their routines on occasion.Largely, patients reported how going out of the house or travelling was disruptive.As Ah Siew described:
Adverse Effects
Some patients reported how adverse effects caused them to be nonadherent to their medications.One participant reported that while the doctor wanted to titrate the dose, he decided to stop entirely.As Yong Liat reported: Just recently.Because I told the doctor, I feel giddy then doctor say maybe this one caused...he asked me to take half...so I stopped.
Condition-Related Factors
Patients reported few condition-related factors as impacting their ability to adhere to their medications for ASCVD and its risk factors.One patient reported being prescribed various doses of medication and not taking it unless his blood pressure was high.As Cheng Han described: Yes.I may forget my blood pressure medications...For blood pressure, I usually don't take the medications unless my blood pressure is high.
[IDI014_M_71-75_Chinese]
A few participants also reported how taking medications impacted their mental state, as they felt fearful of sequelae or death.For some participants, this caused them to titrate or change medications.As Sow Tin described: You see, this cholesterol medication was given to me in March, and I still have so much leftover.If I show the doctor, he/she is going to scold me.So I won't tell the doctor...My sister told me that if I take a lot of these cholesterol medication, it will affect my kidneys.So I've stopped taking that one and take this one instead.I'm not as afraid of kidney trouble with this one.[IDI004_F_66-70_Chinese] Whereas, others reported that fear of disability and death motivated them to continue their habits and be adherent to their medications.As Yu Yan explained: I take them right after washing my face as I scared I forget...It's okay to die, it's not okay to be disabled.So I take my medications...Because I'm scared to die, it motivates me to eat my medication, without even any reminder.[IDI008_F_71-75_Chinese]
The Learning Curve and Mobile Technology
One participant highlighted in detail the learning curve after diagnosis and commencement of treatment as a potential opportunity for mHealth interventions for adherence to medications (Textbox 2).This case explored how the uncertainty of a new diagnosis and discharge from hospital care is an opportunity for mHealth to provide adherence support to patients where they have a high intention to uptake adherence behaviors.Patients who have managed their chronic conditions for years may not be as receptive as they have established habits; however, newly diagnosed patients or newly discharged patients may be primed to receive support in their care.
Textbox 2. Case study on the learning curve.
David, a 64-year-old male, had no history of heart disease before having a severe heart attack, which required the insertion of a left ventricular assist device.He was admitted to the intensive care unit for over a month before being discharged.While in hospital, David reported being on 14 different types of medication, which were reduced to 11 upon discharge after he reported adverse side effects.Presently, he is only on 2 medications.David explained the role of his care coordinators, who taught him about his medications before discharge.He also explained how they interceded on his behalf with the doctors to reduce the number of medications he was prescribed.Importantly, he described how they were available to call or SMS when he had questions.When asked about the use of an mHealth intervention to remind patients to take their medications, he explained: "It will be useful for patients who just came out of the ward.But not for long-term patients, because we know what to do." [IDI007_M_61-65_Indian]; David reported that in the days following discharge, the care coordinators would check in daily and ask on his adherence and other required activities (dressing changes, etc); and now they check in every 2 weeks, and he is able to reach out to them should he require their assistance.
Principal Findings
This mixed-methods study explored mobile technology acceptance, use, and facilitating conditions, as well as adherence factors among older Singaporeans ahead of an mHealth intervention to promote adherence to ASCVD medications.
Participants reported variable acceptance of mobile technology.Some participants were tech savvy and used mobile technology regularly and broadly to connect with their social networks, whereas others reported decreasing social connectedness as a reason for not using mobile technology.Participants widely reported usability concerns including reading difficulty because of screen and font size and hearing difficulties limiting their awareness of notifications.This is in line with other research on mobile technology use for health interventions with older participants [20].These technology-oriented factors have an impact on user's behavioral intention to use technology, as well as their intention or interest in using an mHealth intervention for adherence to medications.Indeed, among our study population, whose mean age was 72 years for the qualitative component, participants were largely disinterested in SMS reminders, often citing low mobile phone usage or usability concerns as a barrier to uptake.Suboptimal adherence to medications for ASCVD and its risk factors is well documented [6,[21][22][23]; however, most of our participants reported not needing reminders to take their medication as they have their own established habits and report themselves as being adherent.Yet, some participants reported self-titration and nonadherence when lifestyle factors interfered with their ability to have medications on hand.Thus, caution is warranted in these self-reports of adherence.These reports of adherence may impact patients' behavioral intention to use an mHealth intervention, as many participants reported no interest in an mHealth intervention for adherence to medication, as they did not see personal benefit to such a service.Despite these concerns, some participants did report that SMS was a good avenue through which to receive health information.This is in line with findings from other studies across health research where it has been shown that despite perceived usability issues or dissatisfaction with available options, patients believe in the capacity of mHealth interventions to facilitate better health outcomes [24][25][26][27].In our study, a contributor to a favorable opinion of the proposed intervention was previous mHealth use.Participants who received appointment reminders from health care providers were more open to the idea of health content delivered through SMS, although they were ambivalent on the utility of medication reminders.
Another factor facilitating a positive perception of mHealth was that of trust; both trust in the SMS sender, and trust in the treatment plan.Participants described caution in believing Web-based sources for health information, reported receiving spam SMS messages, and underscored the importance of wanting a trusted source for any mHealth SMS.Other studies have shown that trust is an integral part of intent-to-use technology and predicates on the belief that the other party will not exploit the vulnerability of the user [28,29].Participants also linked the success of any mHealth intervention to whether patients trusted their medications.There is ample evidence on the role of trust, both in treatment and in provider, in medication adherence [30][31][32], and these facets of trust in treatment are important in establishing the behavioral intention to use an mHealth intervention for adherence to medication.
Although technology-oriented factors directly influence a patient's ability and motivation to use and follow an mHealth intervention, adherence factors also play a role in determining if a patient is able to translate the behavioral intention triggered by the SMS into a use behavior.The use of habits and reminders is well documented in facilitating adherence to medications [33][34][35].Our study adds to this evidence showing that patients perceive they are able to cope with treatment complexity by establishing their own habits and rituals and that given the length of treatment required they become accustomed to taking their medications.Importantly, however, patients did report self-titration because of adverse effects, as well as lifestyle factors that disrupted their ability to adhere to medications.These are important factors that may not be modifiable by an mHealth intervention but warrant consideration.
The unique case of David highlights the importance of the learning curve wherein patients develop these habits and rituals for medication taking.After diagnosis, patients may require additional support and be open to receiving that support, as they begin to develop habits and adopt behaviors relating to medication taking and lifestyle adjustment.Although, as is the case with David, this support may taper off over time once adherence behaviors are established, or the perceived utility diminishes.It also presents the opportunity to reintroduce the intervention should new medications be introduced or the patient requests additional support.Although technology-oriented factors, adherence factors, and contextual factors may pose challenges to uptake of an mHealth intervention, this case highlights the importance of identifying opportunities where the barriers to mHealth uptake may be lower as patients are actively seeking support as they take ownership of their care.
Strength and Limitations
A strength of our study is the use of in-depth interviews for the exploration of patients' perspectives on both medication taking, as well as technology use and the opportunities and challenges for mHealth interventions.Furthermore, the inclusion of participants from multiple ethnic backgrounds and older participants adds to the diversity of experiences reported.
A limitation of this study is that it excluded individuals with disabilities, which prevented them from participating in verbal interviews.Also, our participants were all over 60 years of age, thus we are not capturing the perspectives of those middle-aged persons taking medications for ASCVD and its risk factors who may have differing adherence patterns or mobile technology use behaviors.The fact that Singapore has a high mobile phone penetration rate, a small geographical size, and is a 100% urbanized city-state may limit its generalizability to other countries with differences in such characteristics.Furthermore, contextually, health system factors in Singapore, including high accessibility and availability of care may account for some of XSL • FO RenderX the patient accounts of not needing reminders, as they are able to easily access follow-up care.Our study also did not include caregivers, who may be a more appropriate end user for the intervention, particularly among those older persons who rely on caregivers for their medication taking.Furthermore, we relied on self-reported adherence measures, thus some participants may have reported higher adherence to medication.A final limitation is that of desirability bias, whereby participants may be reporting more favorably on their experiences.
Conclusions
Our study had identified several important technology-oriented and adherence-related factors from the patient perspective that warrant consideration in the design of an mHealth intervention to support adherence to medications for ASCVD and its risk factors in Singapore.We also highlighted the importance of finding the right opportunity to engage with patients and promote an mHealth intervention, such as immediately following diagnosis when patients are establishing medication-taking habits.As health care professionals increasingly leverage on innovative approaches such as mHealth to promote adherence to medications for chronic conditions, it will be important to better understand both the technology-related behaviors that impact a patient's intention and ability to use an mHealth intervention, as well as therapy-and condition-related factors that may enable or inhibit successful adoption of such an intervention.
Figure 1 .
Figure 1.Modified unified theory of acceptance and use of technology model for adherence factors adapted from Venkatesh (2003) and World Health Organization (2003).
Figure 2 .
Figure 2. Phone usage activities amongst mobile phone owners.
Figure 3 .
Figure 3. Preferred channel of delivery for mobile health intervention.mHealth: mobile health.
Textbox 1 .
Study inclusion and exclusion criteria.
don't look at my phone...when the phone beeps with an SMS I'll think of checking it later, but I'll forget. | 2019-03-15T02:58:05.560Z | 2019-03-01T00:00:00.000 | {
"year": 2019,
"sha1": "0934b2566c4dc3c4bd09d6d21a011a77907480a5",
"oa_license": "CCBY",
"oa_url": "https://assetapi.jmir.pub/download?alt_file=11108-227607-3-SP.pdf&file=971b2334dad731cd69efd4b7e5d5c5c7.pdf",
"oa_status": "HYBRID",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "1192177e8d285b68ab31c4d284f597e70f78ae97",
"s2fieldsofstudy": [
"Medicine",
"Political Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
267361529 | pes2o/s2orc | v3-fos-license | Neighbourhood environments for a healthy lifestyle among young single-person households experiencing housing poverty in Seoul, South Korea: a spatiotemporal qualitative study protocol
Introduction The number of single-person households is increasing globally—including in South Korea, where they account for over 30% of all households. Young single-person households in South Korea face health problems and housing challenges. Both the perceived and objective aspects of the neighbourhood environment, as a community asset, play a significant role in sustaining a healthy lifestyle. This study aims to explore and describe the meaning, roles and spatiotemporal characteristics of neighbourhood environments for a healthy lifestyle in young single-person households experiencing housing poverty in Seoul, South Korea. Methods and analysis This ongoing study uses an extended qualitative geographic information systems approach to explore a district in the city of Seoul that has the highest population density of young single-person households experiencing housing poverty. The study sample comprises young single-person households aged 19–39 years who are experiencing housing poverty in the study area, with an expected saturation point of approximately 55 participants. We employ online and offline recruitment strategies to ensure the inclusion of diverse perspectives and a multimethod approach that combines descriptive and spatiotemporal data collection techniques (eg, individual in-depth interviews, field observations and mobile global positioning system tracking). The data analysis encompasses thematic and content analyses to understand the neighbourhood environment’s perceived attributes and the spatiotemporal characteristics of healthy lifestyles. In the integrated analysis, we plan to combine the qualitative findings with living space and daily-life patterns using qualitative software and a hybrid relational database. Ethics and dissemination The Institutional Review Board of Seoul National University approved the research protocol on 18 May 2021. The findings will be shared at international conferences and published in academic journals. Additionally, an online seminar will be conducted to share the results with policy-makers, researchers, community organisations and health workers working with young single-person households experiencing housing poverty.
this would enable the circumstances they experience to be more specifically contextualised.2.Again in the Background, the analysis focusses on detailing negative aspects of the neighbourhood environment.As the focus of the paper is on health lifestyle, it would perhaps be more relevant to outline what the elements of a positive aspects of neighbourhood environment are, as this would enable a comparison to be drawn with participants.3. The protocol details many stages for the proposed research.It is not clear, however, the order or timing of these stages.It would be good for the authors to provide a matrix to the order and timings of the stages of data collection 4. The protocol states that is is a qualitative study protocol.However, there are various quantitative stages of data collection.Does this not lean the protocol towards mixed methods? 5.In the limitations, the authors note that the study is time consuming and labour intensive, which is correct.However, they also need to note the expense associated with the study, as per the allocation of all participants with a Garmin Forerunner device.6.The setting chosen for the study would seem to be one that is particularly disadvantaged, even in the Korean context.This needs to be acknowledged in the limitations of the study.7. The study appears to be using stratified sampling methods, but this is not stated as such.This needs to be made clear, as well as the stratification process used.8.The Braun and Clarke reference used is a very old one, there have been more recent updates to their thematic analysis method which could be used.
VERSION 1 -AUTHOR RESPONSE
Reviewer Comments, Author Responses and Manuscript Changes: Reviewer 1 COMMENTS TO THE AUTHOR Thank you for this interesting protocol outlining a novel and important study in the field of neighborhood environments, health and poverty.The text is clear, coherent and cpvers all major aspects required for a protocol paper.However, the following minor issue could be clarified in order to enhance the readability and contribution of the paper:
STENGTHS AND LIMITATIONS OF THIS STUDY
"Strengths and limitations of this study", page 2, lines 13-31: methodological weaknesses e.g.related to quality of data, complexities of combining different sources of data, or inclusion/engagement of the target population, could substitute the statement that relates more to skills in the research team ("This study requires a high level of technical expertise to collect, manage, and analyze data, which can be a barrier for researchers unskilled in GIS technology and qualitative research methods.)
➢
Response: Thank you for taking time to review our manuscript and giving us such insightful comments.We found your comments immensely helpful and have revised accordingly.Detailed corrections are b elow point by point.
➢
This study requires a high level of competence and expertise in managing data quality, addressing the complexities of integrating diverse data sources, and ensuring the active participation of the target population.1 Reviewer Comments, Author Responses and Manuscript Changes: Reviewer 2 COMMENTS TO THE AUTHOR The paper provides a research protocol for a prospective research project.There is clear analysis provided to make this a relevant topic to study.The paper also provides a detailed outline of the research process.The paper is well written and easy to understand.There are a number of improvements that could be made to the paper:
BACKGROUND
1.In the Background section, when talking about young person households in Korea, the relevant comparison should have been with the Korean population as a whole, not the rest of the world, as this would enable the circumstances they experience to be more specifically contextualised.
➢
Response: Thank you for your insightful feedback.We acknowledge the importance of providing a more context-specific comparison for young person households in Korea.In the Background section, we will revise the relevant comparison to focus on the Korean population as a whole rather than a broader comparison with the rest of the world.This adjustment will help to more accurately contextualize the circumstances experienced by young person households in the Korean context.
➢
Changes: (1) BACKGROUND (p3, lines 50-62) Compared to the other demographic groups in Korea, young single-person households face a higher risk of health and housing problems.According to the Korean National Health and Nutrition Examination Survey conducted in 2020, 63.2% of young single-person households aged 19-29 were either obese or overweight; 27.7% showed symptoms of depression, and 29.3% were smokers.These figures were statistically significantly higher than multi-person households (5).In 2021, the Korea Research Institute for Human Settlements (6) reported that 60% of young single-person households spent more than 30% of their monthly income on housing rent, a figure more than five times higher than that of young couple households and young households living with parents.
Additionally, according to the 2020 Korea Housing Survey , 9.6% of single-person households living in houses with less than 14 m 2 of residential space experienced difficulties in soundproofing, ventilation, heating, and kitchen facilities.These housing conditions are imperfect compared to the international minimum housing standards that consider detailed facilities and layouts such as kitchens, bathrooms, bathing facilities, privacy protection, appropriate hot water supply temperatures, and 25 m 2 per person .This suggests that young single-person households in Korea may not have sufficient resources within their homes to manage and protect their health adequately 2. Again in the Background, the analysis focusses on detailing negative aspects of the neighbourhood environment.As the focus of the paper is on health lifestyle, it would perhaps be more relevant to outline what the elements of a positive aspects of neighbourhood environment are, as this would enable a comparison to be drawn with participants.
➢
Response: Thank you for your valuable feedback.We recognize the need to achieve a more balanced perspective in the analysis of this study by including the positive aspects of the neighborhood environment.To address this, we will enhance the focus of our analysis in the Background section by incorporating details about the positive elements of the neighborhood environment.
➢
Changes: (1) BACKGROUND (p3, lines 62-65) Given the challenges faced by these households, namely, insufficient home resources to adequately address health needs, highlighting the health-promoting factors of the neighborhood environment could be a pivotal strategy for improving the quality of life of those living in single-person households.
(2) BACKGROUND (p4, lines 93-94) 3) What positive attributes and characteristics of neighborhood environments are necessary to support healthy lifestyles in young single-person households experiencing housing poverty?
METHODS AND ANALYSIS 3. The protocol details many stages for the proposed research.It is not clear, however, the order or timing of these stages.It would be good for the authors to provide a matrix to the order and timings of the stages of data collection.
➢
Response: Thank you for your valuable feedback on incorporating a time-ordered matrix to address concerns about the clarity of the protocol steps.Your insights have significantly contributed to enhancing the visual representation of the research plan.We appreciate your constructive comments, and the revised Figure 3 now provides a clearer understanding of the sequence and timing of the various stages of the proposed study.
➢
Changes: (1) METHODS AND ANALYSIS (p6, line 158) [Figure 3] 4. The protocol states that is a qualitative study protocol.However, there are various quantitative stages of data collection.Does this not lean the protocol towards mixed methods?➢ Response: Taking note of your observation, we recognize that our research protocol might give the impression of a mixed-methods study, considering the simultaneous collection of descriptive and quantitative information.However, what we want to emphasize is that the research design itself is not based on mixed methods but rather on a qualitative GIS approach that seamlessly integrates spatialtemporal information with descriptive data.Despite incorporating quantitative aspects in the data collection stages, our research fundamentally relies on qualitative inquiry, with a dedicated focus on integrating spatial and narrative information in the research design.To avoid any potential misunderstanding, we have updated the methodology section to explicitly state that our research does not fall under the category of mixed methods.
➢
Changes: (1) METHODS AND ANALYSIS (p5, lines 109-115) Figure 1 shows a schematic diagram of the extended QGIS used in this study.This diagram demonstrates how qualitative, spatial, and temporal data can be combined through simultaneous processing to produce a rich contextual understanding of people's lived experiences in their neighborhoods.This distinctive aspect sets our study's strategy apart from a mixed methods approach (20) as it has a unique sequence and arrangement for collecting both qualitative and quantitative data.Additionally, we take a more qualitative-oriented approach compared to traditional QGIS by interpreting GIS information based on patterns rather than concentrating solely on size, frequency, and density.
5. The study appears to be using stratified sampling methods, but this is not stated as such.This needs to be made clear, as well as the stratification process used.
➢
Response: Thank you for your keen observation.We appreciate your feedback regarding the perceived use of stratified sampling methods in our study.You are correct in noting that this aspect should be explicitly stated for clarity.We will update our methodology section to clearly mention the use of stratified sampling.
6.The Braun and Clarke reference used is a very old one, there have been more recent updates to their thematic analysis method which could be used.
➢
Response: Thank you for your input.In accordance with your feedback, we have updated the reference to Braun and Clarke to a more recent reference, taking into consideration the recent updates to their thematic analysis method.This change is expected to ensure that the discussed content is more contemporary and accurately reflected.
STRENGTHS AND LIMITATIONS OF THIS STUDY
7. In the limitations, the authors note that the study is time consuming and labour intensive, which is correct.However, they also need to note the expense associated with the study, as per the allocation of all participants with a Garmin Forerunner device.
➢
Response: Thank you for bringing attention to the limitations of our study.We appreciate your observation regarding the time-consuming and labor-intensive nature of the research, which is duly noted.Your suggestion to include a mention of the associated expenses, particularly in terms of allocating Garmin Forerunner devices to all participants, is valid.We will incorporate this aspect into the limitations section to provide a more comprehensive understanding of the study's constraints.
➢
This study may be time-consuming, labor-intensive, and costly due to the need to collect and analyze data from multiple sources and devices.
8. The setting chosen for the study would seem to be one that is particularly disadvantaged, even in the Korean context.This needs to be acknowledged in the limitations of the study.
➢
Response: Thank you for highlighting this important consideration.We acknowledge your observation regarding the chosen setting for our study and appreciate your suggestion to explicitly acknowledge this in the limitations section.In line with your feedback, we will emphasize the need for results and interpretations based on the Korean context.This acknowledgment will be incorporated into the limitations section, providing a more transparent understanding of the study's contextual constraints.
➢
Changes: (1) STRENGTHS AND LIMITATIONS OF THIS STUDY (p2, lines 38-39) ➢ The interpretation of the study results should consider the characteristics and context of both the study area and participants, particularly in relation to housing poverty.
➢
Response: Thank you for your valuable feedback.In response to your suggestion, we have incorporated an additional paragraph in the manuscript that provides a comprehensive overview of what a healthy lifestyle entails.This inclusion aims to shift the focus from the negative aspects of individuals to a more balanced perspective, allowing readers to better understand the positive outcomes the research seeks to achieve.If you have any further recommendations or specific points you would like us to address, please feel free to provide additional guidance.We appreciate your thoughtful input and are committed to enhancing the clarity and relevance of our research.
➢
Changes: (1) BACKGROUND (p3, lines 66-74) Recognizing the unique challenges faced by young, single-person households underscores the urgency of prioritizing and cultivating a healthy lifestyle.A healthy lifestyle, intricately intertwined with the surrounding environment, is a holistic concept that transcends physical health, encompassing mental health, nutrition, and overall quality of life (9).The combination of factors such as regular exercise, a balanced diet, mental well-being, and positive social interactions not only contributes to individual well-being but also interfaces with the neighborhood environment (9).This symbiotic relationship operates synergistically within the holistic framework of a healthy lifestyle.Recognizing the interconnectedness of these elements is critical, as together they form the foundation for wellness and fortification against potential health risks, with the neighborhood environment playing a pivotal role in shaping and supporting these health-promoting behaviors (10).
METHODS AND ANALYSIS
2. A minor change to the Figure 3 would be to number the stages in terms of the order that they occur, as this would make the process clearer.
➢
Response: Thank you for your suggestion.We have implemented a minor revision to Figure 3 by numbering the stages in accordance with the order of occurrence.This adjustment aims to enhance the clarity of the process depicted in the figure.If you have any further recommendations or specific details you would like us to address, please feel free to provide additional guidance.We appreciate your attention to detail and constructive feedback in refining the visual presentation of our work.
AUTHOR RESPONSE Reviewer Comments, Author Responses and Manuscript Changes: Reviewer 2 COMMENTS TO THE AUTHOR BACKGROUND 1
. The authors have made a revision to the manuscript in relation healthy lifestyle, but this needs to be elaborated on more.Specifically, the authors could include a paragraph on what a healthy lifestyle looks like.This would enable the reader to understand what the research is aiming to achieve.At the moment, the focus is on the negative aspects of individuals, not on the healthy lifestyles that the research is trying to achieve. | 2024-02-02T05:08:31.380Z | 2024-01-01T00:00:00.000 | {
"year": 2024,
"sha1": "5a7e28df5031beb5b042d3d7700163a25e7d2f14",
"oa_license": "CCBY",
"oa_url": "https://bmjopen.bmj.com/content/bmjopen/14/1/e077234.full.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "5a7e28df5031beb5b042d3d7700163a25e7d2f14",
"s2fieldsofstudy": [
"Environmental Science",
"Sociology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
231598826 | pes2o/s2orc | v3-fos-license | Drug related problems in clinical practice: a cross-sectional study on their prevalence, risk factors and associated pharmaceutical interventions
Drug-related problems (DRP) cause preventable negative health outcomes, especially during hospital admissions. The aim of our study was to examine the prevalence and characteristics of DRP in regular clinical pharmacy, as well as to determine those factors associated with a higher risk of DRP in the hospital setting. We analyzed data from a standardized registry database of regular pharmacy practice (2015- 2016). DRP were classified according to the Pharmaceutical Care Network Europe v6.2 classification. Cross-sectional data were obtained from 1602 adults admitted to medical wards. Crude and adjusted binary logistic regressions were performed to identify associations between potential risk factors and DRP. Overall DRP prevalence was high across medical specialties (45,1%), in a population characterized by advanced age, polypharmacy and multimorbidity. Problems leading to DRP were mainly classified into two domains (effectiveness and adverse reactions), being drug and dose selection the most frequent causes. Interventions were accepted and DRP were totally or partially solved in 74.1% and 4.81% of cases, respectively. In the adjusted model polypharmacy, allergies, BMI > 25 kg/m2 and clearance < 30 mL/min were associated with a higher risk of DRP. The participation of clinical pharmacists into multidisciplinary teams promotes the detection and solution of DRP. Polypharmacy, obesity, renal impairment and allergy are associated with a higher risk of DRP during admission.
Sample procedures. The sample included all adults over 18 years old admitted to the medical wards during the study period: Internal Medicine, Gastroenterology, Geriatrics, Neurology, Pneumology. Cardiology, Oncology and Haematology were excluded due to the presence of transplanted patients and special characteristics in relation to pharmacotherapy.
The study consisted of the assessment of activities and interventions made and registered in regular clinical pharmacy practice. Briefly, three pharmacists performed regular pharmaceutical care activities following their standard workflow (Fig. 1). Firstly, they assessed specific aspects regarding pharmacotherapy at the time of admission and subsequent changes in prescription, including a wide range of activities (i.e. medication reconciliation, allergy check, indication, posology) (Appendix 1). Secondly, certain patients were selected for daily follow-up based on pre-specified criteria (i.e. pharmacokinetics monitoring, risk of adverse effects, potential interaction, renal impairment) or any other clinical criteria (Appendix 1). Additionally, pharmacists received queries raised by physicians, nurses, caregivers or patients. All these activities resulted in specific pharmaceutical interventions.
All patients assessed throughout the aforementioned steps were registered in the pharmacy work database, as part of their standard practice, including selected variables. Pharmaceutical interventions were also registered for those patients with detected DRP. Data collection and variable definitions. Data were obtained from a standardized registry database of regular pharmacy practice. Variables collected in the database were selected aiming to maximize pharmacotherapeutic utility of the data, taking into consideration clinical criteria and the reviews on factors related to DRP by Alomar and Kaufmann et al. 24,30 . Each of the variables described in the reviews was evaluated and those not applicable to the practice context were discarded (i.e. visual impairment, non-adherence; Appendix 2). Additional clinical variables of interest were included by consensus (Appendix 2).
Sociodemographic information included gender, age, country of birth, weight, height, body mass index (BMI). General clinical variables comprised creatinine clearance (Cockcroft-Gault), liver failure, number of chronic conditions, Charlson comorbidity index, allergies and medical specialty. Variables related to the use of health services were the number of last 12-month hospital admissions and coming from a nursing home. Pharmacotherapeutic variables were polypharmacy at admission (number of drugs), lack of information on regular treatment, monitoring drugs (amikacin, carbamazepine, cyclosporine, digoxin, everolimus, gentamicin, lithium, phenytoin, phenobarbital, sirolimus, tacrolimus, valproate, vancomycin), DRP detection, DRP description according to the Statistical analyses. Due to the retrospective nature of the study, a priori sample size estimation was not possible. However, after data collection was finished, we calculated the theoretical needed sample size with the GRANMO calculator to confirm the adequacy of our ulterior analysis. According to the bibliography, DRP in hospitalizations range from 20 to 80% 62 . A minimum sample size over 385 participants was needed considering an infinite population, 5% precision, 95% CI and a DRP prevalence of 50%. Hence, the sample obtained in our study was considered adequate for the analysis. Frequencies, proportions, range, mean, SD, CIs and cross tabulations were applied for descriptive analysis. χ2 Test, Fisher's exact test and t-test were used to measure differences in prevalence of sociodemographic and clinical variables across DRP presence. T test, one-way ANOVA and simple linear regression were used to assess differences in polypharmacy across clinical and socio-demographic variables (age, gender, country, BMI, www.nature.com/scientificreports/ baseline chronic conditions, allergies, last-12 month admissions, creatinine clearance, liver failure, coming from nursing home, estimated 10-year survival, medical department and missing information defined as lack of data regarding chronic treatment in clinical records). Crude and adjusted binary logistic regressions were used to examine the relationship between theoretical factors related with DRP and their presence in our sample. The multivariate logistic regression model included those variables with an association in the bivariate analyses defined as p < 0.1. We fitted additional regression models for combinations of variables to test whether potential theoretical interactions were present with regard to the dependent variable: presence of DRP. Multicollinearity was also tested, defined as a condition index > 20 or VIF > 10. Both the presence of interactions (except number of chronic conditions with renal function) and multicollinearity were rejected. Results are reported as unadjusted and adjusted ORs with 95% CI. We conducted additional sensitivity analyses by age group (< 60 years, 60-69 years, ≥ 70 years). There were no missing data for most of the variables. Information on BMI and renal function were missing in 0.99% and 0.56% of the participants. These rates were considered low. We did not impute these variables as we could not guarantee whether these data were missing at random. Analyses were performed with IBM SPSS statistics V.19.
Description of DRP. DRP were detected in 722 (45.1%) patients and their prevalence differed across BMI, allergy, polypharmacy, multimorbidity, last 12 months admissions and the estimated 10-year survival in (Table 1). DRP were related to medication reconciliation in 38.4% of the cases. www.nature.com/scientificreports/ According to the PCNE V6.2 classification, most of the problems leading to DRP were included into two domains: effectiveness and adverse reactions. The principal problem was "toxic adverse drug-event", followed by "untreated indication" and "effect of treatment not optimal" ( Table 2). Regarding the cause of DRP, the most frequent domains were "drug selection" and "dose selection". The leading causes were "indication for drug-treatment not noticed", "pharmacokinetic problem requiring dose adjustment" and "drug dose too high".
We divided interventions results into prescriber and drug levels. As for the first one, intervention was proposed and approved in two thirds of the cases, while not approved in only 5.8% of cases. Outcome was unknown in 13.9% interventions (for more categories, see Table 2). Pharmaceutical intervention results covered a wide range of aspects, such as: drug dose adjustment in patients with renal or hepatic impairment (e.g.: levofloxacin, simvastatin, paroxetine), treatment changes due to drug-drug interactions (e.g.: valproate with carbapenem antibiotics; high doses of simvastatin with diltiazem), need of dose adjustments (e.g.: low doses of antimicrobials for CNS infections infections in the central nervous system, excessive drug dose leading to potential harms), untreated indications (e.g.: hyperglycaemia, high-blood pressure, ACE Angiotensin-converting enzyme inhibitor induced hyperkalemia), contraindications (e.g.: oral bisphosphonates in patients with dysphagia), medication reconciliation (e.g.: dose/frequency, drugs to be stopped), IV intravenous administration issues (e.g.: excessive infusion rate of vancomycin or electrolytes; wrong serum for dilution, Y-Y incompatibilities), oral administration issues (e.g.: recommendation of available presentations for dysphagia, medicines to be taken on an empty stomach such as alendronic acid), contraindication or excessive duration treatment (e.g.: excessive antibiotic drug length, antihypertensive therapy in patients with low blood pressure), pharmacokinetics monitoring (e.g.: dosage increase/decrease or discontinuation of vancomycin, gentamicin, amikacin, etc.), wrong drug prescribed (e.g.: methimazole for metamizole), cost-efficacy interventions (e.g.: changes of low molecular weight heparins according to the hospital formulary). At a drug level, dosage was changed in 36.6% of cases, a new drug was started in 12.4% and the drug was stopped in 9.2% of the interventions, while 30.04% resulted in no changes. As for the final health outcomes, DRP were considered to be totally solved in 74.1% and partially solved in 4.8% of cases. The outcome was not known in 15.7% of interventions.
Considering pharmacist interventions on DRP as a continuum and clustering categories according to the domains of the PCNE V6.2, certain patterns were observed in the DRP pathway "cause → intervention → outcome" (Fig. 3).
Discussion
To the best of our knowledge, this is one of the few studies to evaluate DRP prevalence and risk factors in real clinical practice during hospital admission in the context of a standardized pharmaceutical care programme, including a validated register of DRP interventions with a global representation of medical specialties. We found a high prevalence of DRP, most being caused by drug or dose selection, in a sample that highlights the worldwide demographical trends of ageing along with multimorbidity and polypharmacy. Pharmacist interventions were accepted in most cases, preventing potential negative health outcomes. As for potential risk factors, Table 3. Correlates of drug-related problems estimated by multivariable logistic regression. Results refer to univariate and multivariable logistic regression for the total sample. Age and polypharmacy were included as continuous variables. AOR adjusted odds ratio, BMI body mass index, CI confidence interval, HA Hospital admissions, NCC number of chronic conditions, NH nursing home, OR odds ratio. www.nature.com/scientificreports/ only polypharmacy, renal impairment, allergies and high BMI were associated with a higher prevalence of DRP.
The main strengths of our study are the large sample size, the standardized procedures of clinical pharmacists throughout the admission in regular clinical pharmacy, the use of a validated DRP classification, the assessment of a comprehensive list of potential DRP risk factors and the inclusion of the most relevant medical specialties. Our study found a great proportion of admissions with DRPs, a 45.1% of the cases. Most of the evidence available at a hospital level have focused on specific medical fields [51][52][53] , being limited to ambulatory patients in numerous occasions [54][55][56] . There are, however, some studies focusing on medical specialties, which found a DRP prevalence ranging from 15 to 81% 26,27,[60][61][62] . Our results fit well into the related literature although it should be noted that literature on geriatrics show the higher results 29,58,63 . In contrast, results from studies which use automatized DRP alerts in computerized prescriber order entry systems have shown lower prevalence in previous studies 26,40 . Thus, these systems should be considered as tools to complement pharmacy practice rather than a substitution of clinical pharmacist functions due to the complexity of hospitalized patients.
Our study highlights the complexity of patients admitted in medical wards, with an average age over 72 years, in line with recent literature 26,27,[60][61][62] . The World Health Organization has recognized demographic transitions as a major priority due to its burden at a health, social and economic levels 64 . Ageing involves a complex set of complications reflected in our results, such as multimorbidity, polypharmacy or functional impairment. Mean number of chronic conditions in our study was six and ranged up to 16 per patient, intimately related to a low expected 1-year survival of 25%. Most of the previous literature focusing on regular clinical pharmacy practice care in medical wards reported minimal data at this level as they had a more drug-centred approach 26,27,[60][61][62] . Polypharmacy stands out as one of the most valuable variables of our study, ranging up to 26 drugs per patient, due to their potential harmful effects. In a context with a high burden of polypharmacy, there is need to move from the classical thresholds of 4+ or 5+ drugs [14][15][16] to a more realistic linear approach, which we used in our analyses. A further complex approach, considering polypharmacy as a qualitative aspect is still being discussed. In the end, these characteristics and those regarding the own health system, such as communication across healthcare levels, will define the objectives for a specific patient and the type of interventions in drug treatment.
Regarding pharmacists' interventions, acceptance accounted for almost 70% cases. This issue has not been properly assessed in many previous studies on DRPs in regular clinical practice 26,27,60,61 . Those studies documenting this data show similar interventions acceptance in regular clinical practice 62 or even higher figures in prospective research studies compared with our results 58 . Also, final outcomes of the interventions showed total or partial resolution of the DRPs in our study, reinforcing the value of our results. Interventions registers are essential, as in other healthcare areas, to document professional activity and to assess the suitability of the approach being taken to eventually improve healthcare outcomes 65 . Despite its possible benefits, registries of clinical pharmacy have normally been present in specific areas, such as those linked to a computerized DRP alert system or nationwide voluntary reporting systems 40,66 .
Finally, one of the most relevant issues regarding DRPs is understanding their potential underlying factors to optimize interventions and preventive measures. As previously stated, there are many possible factors suggested by the literature. Certain theoretical frameworks or reviews have developed lists of risk factors but they may not totally apply to the care of admitted patients 24,30 . For example, issues such as self-medication, visual impairment, civil status or educational level would not be relevant from the acute drug management perspective. In contrast, we found that only polypharmacy, renal impairment, allergies and high BMI were associated with DRPs. A review of the literature on this topic at a hospital level used definitions not totally comparable to ours, i.e. medication errors, but polypharmacy and renal function also resulted to be risk factors 29 . We hypothesize that the idiosyncrasy and complexity of inpatients in our or similar environments may diminish the impact of alternative factors.
Our study has limitations. First, its cross-sectional nature identifies associations but does not allow causal relationships to be determined. Second, our results may differ from geographical areas with different healthcare characteristics, especially in terms of pharmaceutical care implementation. Nor can they be extrapolated to other specialties or primary care. Third, this study refers to DRPs, which by definition have a potential nature. Interventions where made in all detected DRPs, so that it is not possible to quantify the real negative health outcomes. As regular practice, it is not considered ethical to stop performing clinical activities for this reason. Fourth, administration errors, if not notified by nursing staff or detected by the pharmacist staff, were not registered. This limitation has been described in other studies previously. Fifth, underdetection may have happened to some extent, especially for infrequent or unfamiliar DRPs. This problem is partly covered by using a complete, standardized and validated classification system. Another factor that can influence underreporting is professional experience. Also, we did not include potential factors leading to DRP which were considered not applicable to practice context (e.g.: education level, visual impairment). These are relevant factors in primary care and could potentially impact on the results of specific hospitalizations. Moreover, our analyses have shown similar tendencies in the global analysis compared with the sensitivity age group analyses. However, some results did not achieve statistical significance in the younger groups, probably because sample sizes were smaller. Further research should focus on possible differences across age groups. Also, verbal interventions have been associated with higher acceptance rates compared with written interventions. In our study, most of them were verbal but this variable was not recorded and its impact could not be analyzed. Finally, the clinical relevance of interventions was not assessed as it is not collected routinely in regular clinical practice but future studies should address this issue.
DRPs have become a major challenge for health care systems due to their clinical and economic impact, especially in the context of the current demographical ageing trends that imply a high burden of multimorbidity and polypharmacy. The prevalence of DRP in hospitalized patients admitted to medical wards is high regardless of the specialty, which highlights the need of providing pharmaceutical care to prevent negative health outcomes during hospitalization. Problems leading to DRPs are mainly related to effectiveness and adverse reactions, while the most frequent causes are drug and dose selection. Thus, these domains should be prioritized in both pharmaceutical care programmes and general educational activities. The participation of clinical pharmacists www.nature.com/scientificreports/ into the multidisciplinary team promotes the detection and solution of DRP in the majority of cases, and should be considered as a rule in general clinical practice. Finally, only a limited number of factors may be associated with a higher risk of developing DRPs in the hospital setting, such as polypharmacy, allergies, BMI > 25 kg/m2 and renal function < 30 mL/min, which could be useful to prioritize actions. Better understanding of these issues may facilitate the implementation of general approaches in diverse settings and the study of these interactions in the future.
Ethics statement. The study was approved by the Clinical Research Ethics Committee of the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. This Ethics Committee waived the need to obtain informed consent due to the retrospective nature of the data, coming from a standardized regular practice database, and the anonymized analysis. All investigators worked according to the principles expressed in the Declaration of Helsinki.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request. | 2021-01-14T14:25:30.823Z | 2021-01-13T00:00:00.000 | {
"year": 2021,
"sha1": "1b27387a08b8061169572267e2f964818b6b0d36",
"oa_license": "CCBY",
"oa_url": "https://www.nature.com/articles/s41598-020-80560-2.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "122fe907ebc932df9f67561d22ce88b0ea987993",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
268494092 | pes2o/s2orc | v3-fos-license | Endoscopic Features of Gastric Mucosa-Associated Lymphoid Tissue Lymphoma without Helicobacter pylori
Although gastric mucosa-associated lymphoid tissue (MALT) lymphoma without Helicobacter pylori (HP) has increased recently, a specific endoscopic classification has not been established; its endoscopic characteristics have not been investigated. In this study, we retrospectively investigated gastric MALT lymphoma without HP in our hospital and assessed differences in the endoscopic findings according to HP infection status. Fifty-seven patients with gastric MALT lymphoma Lugano stage I, diagnosed between January 2013 and March 2023, were divided into three groups (currently HP infected, previously infected, and uninfected), wherein their endoscopic findings were evaluated. Furthermore, the superficial type, as per the classification of Sano et al., was independently subdivided based on the endoscopic differential diagnoses, as follows: atrophic gastritis-like, angiodysplasia-like, superficial gastritis-like, and undifferentiated carcinoma-like. Compared with the currently infected group, the HP-uninfected group tended to have more small lesions without erosion and more discolored, undifferentiated carcinoma-like depressed lesions. In addition, the positive rate of the tree-like appearance (TLA) and ballooning characteristics of gastric MALT lymphoma in magnified findings was lower in the HP-uninfected group. In patients without HP infection, MALT lymphoma should be excluded, even in the absence of suspicious magnifying findings such as TLA or ballooning.
Introduction
Mucosa-associated lymphoid tissue (MALT) lymphoma was proposed by Isaacson et al. [1].It is a low-grade lymphoma originating from B cells in the marginal region of lymphoid tissue in extra nodal organs such as the gastrointestinal tract, thyroid gland, and lungs, against a background of chronic inflammation [2,3].
Gastric malignant lymphoma often shows various endoscopic findings.The Sano classification [4] (superficial, ulcer, polypoid, fungated, and giant fold types) or that of Yao et al. [5] (superficial spreading, mass-forming, and giant fold types) are often used.However, there is no specific classification for the endoscopic images of gastric MALT lymphoma.Nakamura and Iida [6] classified the disease into four types, as follows: superficial (including gastritis-like, IIc-like depression, and multiple ulcer type), mass (including ulcer and submucosal tumor-like protuberance), diffuse infiltration, and mixed.Moreover, Oda et al. [7] classified the disease into redness/erosion, edema, IIc-like depression, ulceration, cobblestone appearance, protrusion, and white spot.
Approximately 80-90% of gastric MALT lymphomas are reportedly infected with Helicobacter pylori (HP) [8].However, with the recent decline in HP infection rates, there has been an increase in the encountered cases of HP-negative gastric MALT lymphoma, leading to further diversification of their morphology.Therefore, this study aimed to determine the endoscopic features of gastric MALT lymphoma by comparing HP-uninfected cases with current HP-infection and previous HP-infection cases.This study advocated for original subdivisions that could be easily classified at endoscopy, discussing their characteristics.We suggest that the clarification of noteworthy endoscopic findings would greatly contribute to the diagnosis of MALT lymphoma in the future.
Study Design
Fifty-seven cases of gastric MALT lymphoma Lugano stage I [9], diagnosed at our hospital between January 2013 and March 2023, were included.A definitive diagnosis was established based on endoscopic biopsy, followed by histopathological examination, including immunostaining.Blood antibody tests and at least one of the following tests were performed to determine HP infection status: microscopy, urea breath, stool antigen, urinary antibody, and rapid urease tests.If two or more of these tests were positive, the patient was classified as currently infected.The patients were classified as uninfected if there was no atrophic gastritis on endoscopy and if two or more of the above HP infection tests, including the blood antibody test, were negative.Regarding the history of its eradication, cases of confirmed successful eradication plus one or more negative test results, including blood HP antibody test, were classified as previously infected.Even if two or more of the HP infection tests were negative, cases of clear endoscopic atrophic changes were classified as spontaneously eradicated cases in the previously infected group.At this time, we also tested for antibodies related to autoimmune gastritis and confirmed them to be negative.Furthermore, pathological examination confirmed the absence of parietal cell decrease.Cases of inadequate testing for HP infection status and cases of Lugano 2 or higher were excluded.
In each group, endoscopic findings, clinical course, and background information were retrieved from medical records, retrospectively reviewed, and compared.Informed consent for the study protocol was disclosed in the form of an opt-out page on the website available to all participants.The study was approved by the Ethics Committee of Tokyo Women's Medical University Hospital (protocol code: 2022-0158).
Evaluation of Endoscopic Findings
The endoscopic images were examined at the time of diagnosis confirmation.The lesion site, number of lesions, presence or absence of erosions, and gross type were retrospectively assessed, and the gross type was classified according to the Sano classification [4].These evaluations were performed by three Board Certified Gastroenterologists of The Japanese Society of Gastroenterology.
The magnified endoscopic findings were also evaluated using images combined with narrow-band imaging.The presence or absence of a tree-like appearance (TLA) and ballooning, which are considered characteristic endoscopic findings in patients with MALT lymphoma, was investigated (Figure 1).
Subdivision of Superficial Type Cases
Gastric MALT lymphoma has a relatively slow growth of tumor cells and many cases reportedly exhibit lesions corresponding to the superficial type according to the Sano [4] or Nakamura [6] classifications.The superficial type in this classification includes many endoscopic images.In this study, the superficial type was subdivided independently according to differential disease diagnoses, with reference to the existing classifications and their characteristics.
Subdivision of Superficial Type Cases
Gastric MALT lymphoma has a relatively slow growth of tumor cells and many cases reportedly exhibit lesions corresponding to the superficial type according to the Sano [4] or Nakamura [6] classifications.The superficial type in this classification includes many endoscopic images.In this study, the superficial type was subdivided independently according to differential disease diagnoses, with reference to the existing classifications and their characteristics.
Statistical Analysis
All statistical data were analyzed using JMP Pro ® 16 (SAS Institute, Tokyo, Japan).Chi-square and Fisher's exact tests were performed to compare categorical data and t-tests were employed for continuous data.In all cases, p < 0.05 was considered significant.
Comparative Study of the Background and Endoscopic Findings
Of the 57 cases of MALT lymphoma diagnosed in this study, upper endoscopy was performed for the following indications: investigation of symptoms in 14 cases, screening in 33 cases, and other indications in 10 cases.The results of the currently infected, previously infected, and uninfected groups are presented in Table 1; overall, 28, 5, and 24 cases were classified in the currently infected, previously infected, and uninfected groups, respectively.
Table 1 presents the patients' background characteristics and endoscopic image results by HP infection status.
The median ages of the currently infected, previously infected, and uninfected groups were 61, 60, and 62 years, respectively.There were no significant differences in the proportion of male patients among these groups.
Similarly, there were no significant differences in the site of the primary lesion among the three groups.Classifying the size of the primary lesion into large (≥30 mm), medium (10 ≤ x < 30 mm), and small (<10 mm), the uninfected group had the highest proportion of small lesions, at 41%, while the currently infected group had the highest proportion of large lesions, at 64% (p = 0.0039).
Regarding the number of lesions, 85.7% of the currently infected group comprised solitary cases.Similar to the currently infected group, there were more solitary cases rather than multiple cases in the uninfected group.However, the percentage remained at approximately 58.3%, lower than that in the currently infected group.In addition, there was a significant difference between the currently infected and uninfected groups in terms of erosions, with the currently infected group showing a higher incidence of erosions compared to the uninfected group, while no erosions were observed in the previously infected group (p = 0.0047).
When the primary lesions were classified according to the Sano classification, 82.4% were of the superficial type.Furthermore, when classified based on HP infection status, the superficial type was the most common in all groups and all cases in the previously infected group had this type.The ulcer and polypoid types accounted for 8.8% of all cases.The fungated and the giant fold types were observed in lesions ≥Lugano 2, and none were found in the Lugano 1 lesions, which were the subject of this study.
Regarding the magnified endoscopy findings, we examined the occurrence of TLA and ballooning, as shown in Figure 1.A TLA was observed in approximately 25 of the 40 cases (62.5%), wherein the presence could be determined endoscopically.
Table 2 details the results of the magnified endoscopic images by H. pylori infection status.A TLA was observed in 93.8%, 40%, and 42% of the currently infected, previously infected, and uninfected groups, respectively.The TLA-and ballooning-positive rates were significantly lower in the uninfected group than in the currently infected group.
Comparative Study by Subdivision of Superficial Type Cases
There were 47 cases wherein the primary lesion was superficial, as follows: 21, 5, and 21 in the currently infected, previously infected, and uninfected groups, respectively.Atrophic gastritis-like lesions were defined as discolored, depressed lesions that were large, diffuse, and resembled atrophic gastritis, as shown in Figure 2a.TLA: tree-like appearance.
TLA
A TLA was observed in 93.8%, 40%, and 42% of the currently infected, previously infected, and uninfected groups, respectively.The TLA-and ballooning-positive rates were significantly lower in the uninfected group than in the currently infected group.
Comparative Study by Subdivision of Superficial Type Cases
There were 47 cases wherein the primary lesion was superficial, as follows: 21, 5, and 21 in the currently infected, previously infected, and uninfected groups, respectively.Atrophic gastritis-like lesions were defined as discolored, depressed lesions that were large, diffuse, and resembled atrophic gastritis, as shown in Figure 2a.In addition, as shown in Figure 2, lesions that were primarily reddish with conspicuous vasodilatation were classified as angiodysplasia-like (Figure 2b); those with mixed discoloration, redness, and uneven irregularities were defined as lesions that could be a differential diagnosis for superficial gastritis owing to inflammation, regardless of size, and were classified as superficial gastritis-like (Figure 2c); and discolored depressed small lesions suspected to be undifferentiated carcinoma were classified as undifferentiated carcinoma-like (Figure 2d).Finally, we decided that the case status was not significant.The results are summarized in Table 3 and are shown in Figure 3.In addition, as shown in Figure 2, lesions that were primarily reddish with conspicuous vasodilatation were classified as angiodysplasia-like (Figure 2b); those with mixed discoloration, redness, and uneven irregularities were defined as lesions that could be a differential diagnosis for superficial gastritis owing to inflammation, regardless of size, and were classified as superficial gastritis-like (Figure 2c); and discolored depressed small lesions suspected to be undifferentiated carcinoma were classified as undifferentiated carcinoma-like (Figure 2d).Finally, we decided that the case status was not significant.The results are summarized in Table 3 and are shown in Figure 3.The HP-uninfected and previously infected groups were most likely to have undifferentiated carcinoma-like features, while the currently infected HP group was most likely to have superficial gastritis-like features.
This table shows the proportions of groups subdivided into superficial types by H. pylori infection status.As mentioned earlier, when we subdivided the surface layer type into four, the previously infected and uninfected groups had a higher proportion of undifferentiated carcinoma-like lesions than the currently infected group; angiodysplasia-like lesions were observed only in the uninfected group.In addition, the currently infected group had more superficial gastritis-like lesions than the uninfected group.
Furthermore, magnified endoscopic images were examined in each subgroup (Table 4).The TLA positivity rates were 87.5% and 42.1% in the superficial gastritis-like subtype (most prevalent in the currently infected group) and the undifferentiated carcinoma-like subtype (most prevalent in the uninfected group), respectively.The ballooning positivity rates were 71.4% and 21.1%, respectively.This table shows the proportions of groups subdivided into superficial types by H. pylori infection status.
As mentioned earlier, when we subdivided the surface layer type into four, the previously infected and uninfected groups had a higher proportion of undifferentiated carcinoma-like lesions than the currently infected group; angiodysplasia-like lesions were observed only in the uninfected group.In addition, the currently infected group had more superficial gastritis-like lesions than the uninfected group.
Furthermore, magnified endoscopic images were examined in each subgroup (Table 4).The TLA positivity rates were 87.5% and 42.1% in the superficial gastritis-like subtype (most prevalent in the currently infected group) and the undifferentiated carcinoma-like subtype (most prevalent in the uninfected group), respectively.The ballooning positivity rates were 71.4% and 21.1%, respectively.Figure 4 shows examples of the superficial gastritis-like cases frequently observed in the HP-infected group.
The patient was a 74-year-old woman wherein an abnormality was detected during an upper endoscopy performed for a detailed examination of acid regurgitation symptoms.The background was an HP-infected stomach with diffuse redness and blood HP was antibody-positive.From the antrum to the lesser curvature of the upper body, a reddish and discolored irregular mucous membrane was observed, accompanied by multiple erosions.Magnifying endoscopy revealed shiny mucosa without glands, confirming a TLA.In addition, ballooning and duct dilatation were detected in the lesion.Histopathological findings indicated that CD20-positive lymphocytes were present under the epithelium, accompanied by many neutrophils.Furthermore, fibrin and inflammatory cell infiltration with debris were observed.Here, an image of a lymphoepithelial lesion (LEL) where lymphoma cells invaded the gland was observed (Figure 4e) and a MALT lymphoma diagnosis was made.Figure 4 shows examples of the superficial gastritis-like cases frequently observed in the HP-infected group.The patient was a 74-year-old woman wherein an abnormality was detected during an upper endoscopy performed for a detailed examination of acid regurgitation symptoms.The background was an HP-infected stomach with diffuse redness and blood HP was antibody-positive.From the antrum to the lesser curvature of the upper body, a reddish and discolored irregular mucous membrane was observed, accompanied by multiple erosions.Magnifying endoscopy revealed shiny mucosa without glands, confirming a TLA.In addition, ballooning and duct dilatation were detected in the lesion.Histopathological findings indicated that CD20-positive lymphocytes were present under the epithelium, accompanied by many neutrophils.Furthermore, fibrin and inflammatory cell infiltration with debris were observed.Here, an image of a lymphoepithelial lesion (LEL) where lymphoma cells invaded the gland was observed (Figure 4e) and a MALT lymphoma diagnosis was made.
Figure 5 shows the features of a case of a 58-year-old man with abnormalities detected during an upper endoscopy during a health checkup.Figure 5 shows the features of a case of a 58-year-old man with abnormalities detected during an upper endoscopy during a health checkup.
The background was an uninfected stomach without atrophy and an approximately 5 mm depressed lesion with a discolored tone was observed on the lesser curvature of the lower body, which required differentiation from undifferentiated carcinoma.The demarcation was ill-defined and there was no evidence of encroachment; therefore, MALT lymphoma was suspected rather than undifferentiated carcinoma, although it was a solitary lesion.Magnifying endoscopy findings did not reveal any abnormal blood vessels or ballooning suggestive of a TLA in the bleached area of the lesion.Furthermore, according to the pathological examination findings, unlike the case in Figure 4, where most of the specimen was filled with lymphocytes, lymphoma cells were partially aggregated and almost no inflammatory cells, such as neutrophils, were observed in the lesions.Most of the lymphocytes were CD20-positive B cells, and LEL was found in some, leading to the diagnosis of MALT lymphoma.The background was an uninfected stomach without atrophy and an approximately 5 mm depressed lesion with a discolored tone was observed on the lesser curvature of the lower body, which required differentiation from undifferentiated carcinoma.The demarcation was ill-defined and there was no evidence of encroachment; therefore, MALT lymphoma was suspected rather than undifferentiated carcinoma, although it was a solitary lesion.Magnifying endoscopy findings did not reveal any abnormal blood vessels or ballooning suggestive of a TLA in the bleached area of the lesion.Furthermore, according to the pathological examination findings, unlike the case in Figure 4, where most of the specimen was filled with lymphocytes, lymphoma cells were partially aggregated and almost no inflammatory cells, such as neutrophils, were observed in the lesions.Most of the lymphocytes were CD20-positive B cells, and LEL was found in some, leading to the diagnosis of MALT lymphoma.
Discussion
In this study, the uninfected group tended to have more lesions without erosion and fewer lesions ≥30 mm than the currently infected group have.In addition, although solitary cases were the most frequent, the proportion of cases of multiple lesions was higher than that in the currently infected group.The superficial type was the most common in every group; the uninfected group had many discolored and depressed lesions requiring differentiation from undifferentiated carcinoma.One of the characteristics was the many cases of no characteristic magnifying endoscopy findings, such as TLA and ballooning.
According to the literature in Japan, the percentage of HP-negative MALT has recently been reported to be 10.5-54.3%[10] and is gradually increasing [11].In our study, the combined ratio of the previously infected and uninfected groups accounted for approximately half of the total cases.It would be beneficial to clarify the endoscopic features of the HP-uninfected group, which may increase in the future and account for most gastric MALT lymphomas.
In addition, in previous endoscopic reports of HP-negative MALT lymphoma, the total number of cases of gastric MALT lymphoma at a single institution ranged between
Discussion
In this study, the uninfected group tended to have more lesions without erosion and fewer lesions ≥30 mm than the currently infected group have.In addition, although solitary cases were the most frequent, the proportion of cases of multiple lesions was higher than that in the currently infected group.The superficial type was the most common in every group; the uninfected group had many discolored and depressed lesions requiring differentiation from undifferentiated carcinoma.One of the characteristics was the many cases of no characteristic magnifying endoscopy findings, such as TLA and ballooning.
According to the literature in Japan, the percentage of HP-negative MALT has recently been reported to be 10.5-54.3%[10] and is gradually increasing [11].In our study, the combined ratio of the previously infected and uninfected groups accounted for approximately half of the total cases.It would be beneficial to clarify the endoscopic features of the HP-uninfected group, which may increase in the future and account for most gastric MALT lymphomas.
In addition, in previous endoscopic reports of HP-negative MALT lymphoma, the total number of cases of gastric MALT lymphoma at a single institution ranged between 17 and 158 [11][12][13][14][15].The 57 patients in the present study represent a relatively large number of cases in a single center.
In addition, many previous reports treated the previously infected group of HPnegative MALT lymphoma similarly to the HP-uninfected group; there are few studies comparing the previously infected and uninfected groups.In this study, the previously infected group was treated as an independent group and compared with the uninfected group, and the treatment of the previously infected group was examined.The previously infected group showed a tendency similar to that of the uninfected group (e.g., many undifferentiated carcinoma-like lesions and few cases of erosion).However, the lesions in the previously infected group tended to concentrate in the middle and upper parts of the body-a finding different from those of the other groups.Owing to the limited sample size, accumulating more cases to determine whether the previously infected group could be treated as HP-negative is essential.The results may vary depending on the percentage of previously infected patients in the HP-negative group.
In a previous report, Tajika et al. [13] studied 120 cases of gastric MALT lymphoma and found that, in contrast to HP-positive cases with numerous eroded, ulcerative, and discolored regions, many HP-negative cases had cobblestone mucosa.Furthermore, they reported no differences between the two groups regarding lesion sites.Conversely, Nakamura et al. [14] reported that localization in the upper stomach and the macroscopic nonsuperficial type are common, infiltrates deeper than the submucosa, and LEL and lymphoid follicles are rare.In addition, Ono et al. [16] cited discolored areas and cobblestone-like mucosa as characteristics of HP-negative MALT lymphoma, consistent with the present study findings.However, Akamatsu et al. [17] reported no evident differences between HP-negative and HP-positive cases; no consensus has been reached.
Tajika et al. [13] considered that the characteristics of HP-negative MALT lymphoma differed from those of previous reports because the API2-MALT1 fusion gene was mixed in HP-negative MALT lymphoma.The API2-MALT1 translocation-negative group was nonresponsive to eradication and had exophytic features.They reported that the API2-MALT1 translocation-positive group was non-responsive to and was characterized by cobblestone mucosa.
Non-Helicobacter pylori Helicobacter (NHPH) infections, such as those due to Helicobacter suis and Helicobacter heilmannii, were recently reported to cause HP-negative MALT lymphoma.For example, NHPH infection was found in 19.6% of HP-negative cases [18] and 11.9% of HP-positive cases [19].In addition, Takigawa et al. [20] reported that NHPH-infected gastric MALT lymphoma presenting with granular mucosa was a nodular gastritis-like MALT lymphoma, with relatively high specificity.In this study, superficial gastritis-like symptoms corresponding to the granular mucosa were found in approximately 20% of the infected and uninfected groups.However, in the HP-uninfected group of this study, discolored, depressed lesions were most frequent.The infection rates of NHPH and genetic testing were not available in this study, and the possibility that these and other unknown causes may be involved in a complex way cannot be ruled out.Further accumulation of cases and investigation of the causes are warranted.
In this study, each macroscopic type showed erosions, making it challenging to group the cases as a single classification.Previous reports included various treatments for erosions and the lack of consistency (e.g., ulcers and erosions being classified as one category or ulcers being classified separately) was a reason for the inconsistent results.In this study, ulcerative lesions were no longer classified as superficial and were excluded from the subdivision.Moreover, the currently infected group had a higher proportion of lesions with erosions compared to that in the uninfected and previously infected groups in this study.This may be attributed to a stronger infiltration of inflammatory cells, as observed in the cases depicted in Figure 4. Furthermore, the tendency for lesions with redness to be more prevalent in the currently infected group may also be attributed to similar reasons.On the contrary, differences in the positivity rates of a TLA and ballooning are noted as factors that inflammatory cell infiltration alone cannot fully explain.
Characteristic magnifying endoscopy findings of gastric MALT lymphoma include TLA and ballooning.Nonaka et al. [21] reported TLA as "abnormal branching blood vessels found in lustrous mucous membranes with loss of glandular structures," a crucial finding in magnifying endoscopy for gastric MALT lymphoma.The authors reported that, in shiny mucosa without glandular structures, swelling of the intervening part between crypts was observed, depending on the degree of infiltration of the lamina propria by lymphoma cells.An intense infiltration causes the gland structure to disappear completely, restricting its confirmation.In addition, the appearance of abnormal, tree-like blood vessels is recognized at that site and, as the abnormal blood vessels and the glandular structure of the background mucosa tend to disappear, it aids in the differentiation from gastritis and venules in which branching blood vessels are observed [22].
Ono et al. [23][24][25] identified three features, as follows: (1) nonstructural area, (2) ballooning, and (3) abnormal vessels.Similar to a TLA, this ballooning is thought to be based on the degree of infiltration of the lamina propria by lymphoma cells.
In this study, the positivity rates of TLA and ballooning were significantly lower in the uninfected group than in the currently infected group.The undifferentiated carcinoma-like lesions, which were more common in the HP-uninfected group, tended to be less positive for TLA and ballooning.In addition to the fact that many undifferentiated carcinoma-like lesions are small, since the amount of lymphoma cell infiltration into the lamina propria is small, as shown in Figure 5c-e, detecting TLA and ballooning may be challenging.However, it may depend on the biopsy site of the lesion.In comparison, in the superficial gastritislike lesion shown in Figure 4, lymphoma cells fill the lamina propria and both TLA and ballooning are observed in magnified endoscopic images.Moreover, Nonaka et al. reported the presence of abnormal micro vessels just beneath the mucosal epithelium in specimens from MALT lymphoma cases that underwent endoscopic mucosal resection [26].We inferred this as abnormal blood vessels identical to a tree branch on NBI magnified findings.Nakamura et al. [27,28] reported that vascular endothelial growth factor (VEGF) activity was observed in gastric MALT lymphomas, leading to angiogenesis and the proliferation of microvascular networks, resulting in the development of a thick vascular system compared with healthy areas.VEGF activity in the fibroblasts surrounding tumor cells is reportedly associated with lymphoma growth [29].Thus, confirming abnormal blood vessels may be challenging in lesions where lymphoma invasion is weak and relatively small.
A TLA is reportedly observed in 75% of gastric MALT lymphomas [30].In this study, it was observed in 63% of cases.As mentioned above, since a TLA and ballooning were significantly observed in the currently infected group, it is suggested that the TLA-positivity rate may decrease as the uninfected group increases in the future.
In this study, undifferentiated carcinoma-like lesions were most prevalent in the HPuninfected group.When discolored, depressed lesions are observed, it becomes important to differentiate between undifferentiated carcinoma and undifferentiated carcinoma-like gastric MALT lymphoma.Past reports have characterized undifferentiated carcinoma by solitary lesions, clear demarcation, evidence of encroachment, and distinctive vascular patterns such as wavy micro-vessels or a corkscrew pattern.On the contrary, MALT lymphoma is characterized by ill-demarcation, sometimes multiple lesions, and abnormal vessels such as those having a TLA [31].These features aid in the differentiation between the two.However, distinguishing between them is not always straightforward.Particularly in this study, a TLA was observed in only approximately 42% of undifferentiated carcinomalike lesions and the absence of characteristic expansive endoscopic findings does not negate the presence of MALT lymphoma.Therefore, a comprehensive consideration of other findings is necessary for a definitive diagnosis.
This study has some limitations.The first limitation is the bias in the assessment of endoscopic findings by the assessors.Gastric MALT lymphoma presents with a very diverse endoscopic picture, which makes classifying some cases difficult.In this study, endoscopic images were assessed and subdivided by three or more Board Certified Gastroenterologists of The Japanese Society of Gastroenterology to reduce this assessment bias.Another limitation is that the study was conducted at a single center.Although this study enrolled a relatively large number of cases for a single center study, it is still a small number overall.The target diseases were rare and further subdivision limited the number of cases in each group.Indeed, the very small number of angiodysplasia-like lesions made a comparison with other classifications difficult.A further study with more cases will confirm the findings of this study.Furthermore, HP infection status varies across regions and a further multicenter accumulation of cases is warranted in the future.In addition, in this study, the infection rate of NHPH and genetic testing was largely unperformed due to the limited availability of testing facilities domestically.However, as mentioned earlier, there is a possibility that these factors could be intricately involved as causes of HP-negative gastric MALT lymphoma.Further classification of these factors may lead to new discoveries regarding endoscopic features and treatment strategies.
Diagnostics 2024 , 13 Figure 1 .
Figure 1.Magnified endoscopic findings of gastric mucosa-associated lymphoid tissue lymphoma.(a) Tree-like appearance: abnormal blood vessels branching like tree branches found in shiny mucous membranes without glandular structures.(b) Ballooning: swelling of surface ducts.
Figure 1 .
Figure 1.Magnified endoscopic findings of gastric mucosa-associated lymphoid tissue lymphoma.(a) Tree-like appearance: abnormal blood vessels branching like tree branches found in shiny mucous membranes without glandular structures.(b) Ballooning: swelling of surface ducts.
Figure 3 .
Figure 3. Relationship with Helicobacter pylori (HP) infection status in superficial type subdivisions.The HP-uninfected and previously infected groups were most likely to have undifferentiated carcinoma-like features, while the currently infected HP group was most likely to have superficial gastritis-like features.
Figure 3 .
Figure 3. Relationship with Helicobacter pylori (HP) infection status in superficial type subdivisions.The HP-uninfected and previously infected groups were most likely to have undifferentiated carcinoma-like features, while the currently infected HP group was most likely to have superficial gastritis-like features.
Figure 4 .
Figure 4. Example of the superficial gastritis-like subtype in those currently infected with Helicobacter pylori.(a) Rough and irregular mucosa with redness and discoloration is observed from the antrum to the lesser curvature of the upper body, along with multiple erosions.(b) Narrow band imaging-magnified observation revealed ductal dilatation and dendritic vessels in the lesion.(c) Aggregation of fibrin and inflammatory cells, which may be partly debris, is observed.(d) Hematoxylin-eosin staining shows lymphoma cells filling most of the specimen.(e) Lymphoepithelial lesion with lymphoma cells infiltrating the glands.(f) Most of the lymphocytes were CD20 positive.
Figure 4 .
Figure 4. Example of the superficial gastritis-like subtype in those currently infected with Helicobacter pylori.(a) Rough and irregular mucosa with redness and discoloration is observed from the antrum to the lesser curvature of the upper body, along with multiple erosions.(b) Narrow band imagingmagnified observation revealed ductal dilatation and dendritic vessels in the lesion.(c) Aggregation of fibrin and inflammatory cells, which may be partly debris, is observed.(d) Hematoxylin-eosin staining shows lymphoma cells filling most of the specimen.(e) Lymphoepithelial lesion with lymphoma cells infiltrating the glands.(f) Most of the lymphocytes were CD20 positive.
Figure 5 .
Figure 5. Example of the undifferentiated carcinoma-like subtype in a Helicobacter pylori uninfected patient.(a) A discolored, depressed lesion of approximately 5 mm was observed on the lesser curvature of the lower body of the stomach.Undifferentiated carcinoma was a differential diagnosis.(b) Narrow band imaging-magnified observation could not detect the tree-like appearance in the discolored depression.(c) Hematoxylin-eosin (HE) staining shows proliferation of lymphocytes just below the mucosa, but it is limited to the superficial layer of the lamina propria.(d) Proliferating lymphoma cells can be seen infiltrating the glands.(e) Many lymphocytes were CD20 positive.
Figure 5 .
Figure 5. Example of the undifferentiated carcinoma-like subtype in a Helicobacter pylori uninfected patient.(a) A discolored, depressed lesion of approximately 5 mm was observed on the lesser curvature of the lower body of the stomach.Undifferentiated carcinoma was a differential diagnosis.(b) Narrow band imaging-magnified observation could not detect the tree-like appearance in the discolored depression.(c) Hematoxylin-eosin (HE) staining shows proliferation of lymphocytes just below the mucosa, but it is limited to the superficial layer of the lamina propria.(d) Proliferating lymphoma cells can be seen infiltrating the glands.(e) Many lymphocytes were CD20 positive.
Table 1 .
Results by H. pylori infection status.
Table 2 .
Results of magnified endoscopic images by H. pylori infection status.
Table 3 .
Percentage of subdivision groups by Helicobacter pylori infection status.
Table 3 .
Percentage of subdivision groups by Helicobacter pylori infection status.
Table 4 .
Magnified endoscopic findings by superficial type subtypes. | 2024-03-17T16:17:51.367Z | 2024-03-01T00:00:00.000 | {
"year": 2024,
"sha1": "d3b9c9a63c87e0bdef7c50430bb537a87ecbb2a5",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2075-4418/14/6/607/pdf?version=1710313334",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "092132574c12f6973340b9840fac5b99cb041fae",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
132373058 | pes2o/s2orc | v3-fos-license | Photonic-based integrated sources and antenna arrays for broadband wireless links in terahertz communications
This paper analyzes integrated components for ultra-broadband millimeter-wave wireless transmitters enabling the 5 G objective to increase the wireless data rates 10× to 100×. We have pursued the photonic-based approach to generate the millimeter-wave carrier (≈97 GHz in this paper) through photomixing. We have achieved up to 10 Gb s−1 data rate using an OOK modulation format (to reduce latency) and either direct detection (DD) or coherent detection. We show that coherent detection enables a sensitivity improvement of 17 dB over DD. We also demonstrate in this work that such improvement can be achieved using as the transmitter a novel integrated antenna array—the self-complementary chessboard array. This avoids the use of complex coherent schemes at the receiver, enabling simple DD for ultra-broadband links.
Introduction
One of the motivations behind the development of 5 G is to reduce the latency and increase the bandwidth of wireless communication links up to 100 Gb s −1 in order to enable a seamless integration of wired and wireless communication links [1]. This is particularly required for point-to-point links connecting to the network over a range of densely distributed wireless pico-cells positioned closer to the end user to enhance the mobile broadband performance in areas with high traffic [2]. In order to reduce the latency, the modulation schemes should be as simple as possible, On-Off Keying being the extreme case. This is not possible at microwave frequencies simply because there is no spectrum allocation that can provide the required bandwidth. So the carrier frequencies have been pushed up to the millimeter-wave (MMW, 30-300 GHz) and terahertz (THz, 0.3-3 THz) ranges [3]. For example, recent experiments demonstrated data rates up to 48 Gb s −1 on a 300 GHz carrier frequency [3]. One of the bottlenecks for these systems is the generation of carriers at these frequencies having satisfactory power and signal quality. Photonic-based generation requires a laser source and an ultrafast photodiode (or photoconductive device). The optical source depends on the photonic technique used to generate the MMW or THz frequencies, the two most common being cw photomixing and pulsed photoconductive switching.
Pulsed sources generally utilize a mode locked laser (MLL) [4] and have two major advantages over cw techniques. The first advantage is ∼7 dB higher average power levels than photomixing schemes [5]. The second is that MLLs inherently have very stable pulse repetition frequency (PRF) and low pulse-to-pulse jitter [6]. However, MLLs have one major disadvantage: the PRF is fixed by the laser resonator cavity length so cannot be modulated. Therefore, photomixing is the preferred technique when frequency tuning is desired. It requires a laser (or lasers) having two different optical wavelengths, the simplest approach being separated distributed feedback (DFB) lasers. These optical wavelengths are mixed in the photodiode or photoconductive device, generating an electrical beat-note at the difference frequency. Its main drawback is that when the two wavelengths are generated from independent sources, the beat-note exhibits large phase-noise fluctuations associated with the linewidth of the lasers and to the relative wavelength fluctuation between them.
This problem can be addressed using different approaches. The most common has been to stabilize the wavelengths using optical injection or optical phase-locked loops [7]. More recently, the monolithic integration of two DFBs sideby-side has improved the stability by having the two lasers experience the same environmental conditions. Using long quantum-dash cavities and combining the wavelengths using a Y-junction, researchers have demonstrated tuning ranges from 2 to 20 GHz and optical line widths around 1 MHz [8].
Finally, in order to increase the transmit power level, a novel integrated array has been developed and is reported here for the first time. It is based on a planar array of photomixers, each coupled to a self-complementary antenna. We call this new transmitter approach the 'chessboard' array.
The paper is organized as follows. The following section, section 2, presents the radio link powered by an integrated photonic source, section 3 describes the chessboard array to increase the power level, and section 4, presents the conclusion. Figure 1 shows the block diagram and a photograph of the fabricated photonic integrated circuit (PIC) dual-wavelength laser source designed for the MMW range [9].
Photonic integrated optical heterodyne source
For the present purposes, we use only the left-hand side of the chip including two single-mode, 1 mm long DFB lasers combined into a single output waveguide with a 2 × 1 multimode interference (MMI) coupler. This optical port provides an optical output for the dual-wavelength lasers and is designed to allow phase noise reduction through optical injection locking [10]. This chip is especially suited for photomixing, being grown on a semi-insulating InP wafer in order to reduce the parasitic capacitance using butt-joint active/passive integration. The active layers consist of 6 InGaAsP quantum wells, and are the same for the DFB lasers, the semiconductor optical amplifiers (SOAs) and electroabsorption modulation sections. As the DFB emission wavelength and power both vary with the injection current, the SOAs are utilized to equalize the optical power after the MMI.
The wavelength of each of the DFB lasers on this PIC can be continuously tuned varying their injection current [11], which allows varying the generated beat-note frequency. This characteristic is demonstrated in the optical domain in figure 2(a). While maintaining DFB1 current level fixed at 75 mA, the current injected in DFB2 is varied between 50 and 90 mA in 10 mA current steps. At the initial current combination (DFB1=75 mA, DFB2=50 mA), the two lasers emit the same optical output power, and the frequency spacing between their wavelengths is 105 GHz. As we increase the current level in DFB2, its wavelength shifts to longer values, getting closer to DFB1. The wavelength difference between the two DFB lasers decreases and the beat-note frequency decreases accordingly. In figure 2(b), we show the beat-note measured by probing the integrated photodiode with a WR08 waveguide output probe, followed by an F-band subharmonic mixer with a LO frequency of 45 GHz. The photodiode was biased at −2.5 V, so that the maximum generated photocurrent level was below 6 mA. The beat-note frequency varies from 105 to 92 GHz when the current in DFB2 is varied from 50 to 90 mA. The DFB2 wavelength change is due to the temperature increase when the injection current is increased, by thermal expansion of the DFB grating. However, the increase in DFB2 current causes two unwanted side effects. The first is due to thermal crosstalk between the two DFBs, which results in a shift of both wavelengths. The second is that increasing DFB2 current also increases its emitted optical power, causing a growing amplitude difference between the two wavelengths, and increasing the DC component of the photocurrent.
The dual-wavelength laser source PIC was then included into a coherent wireless data transmission experiment, composed of the building blocks shown in figure 3(a). The function for the PIC was to provide the photonic signal to generate the millimeter-wave carrier frequency. For the coherent detection, the two lasers must be phased-locked. This is achieved through optical injection into both lasers the output from a separate optical frequency comb (OFC) generator source. This stabilizes the wavelengths of the two lasers, DFB1 and DFB1, and creates high-spectral-purity signals with phase noise spectral density below −90 dBc Hz −1 at 10 kHz offsets from the carrier [10]. The optical comb source signal is generated by the cascaded phase modulation of a tunable 1550 nm seed laser as previously described [3]. The frequency difference of the two DFBs was set at 97.16 GHz. A continuous-wave generator was set at the fourth subharmonic (97.16/4=24.29 GHz) and then used as an OFC generator in the transmitter as well as the LO in the receiver. An optical circulator at the output port of the PIC allowed us to separate the OFC optical injection signal from the dual-wavelength output of the PIC. A photograph of all the experimental components is shown in figure 3(b). Figure 4 shows the most relevant optical signals in this system. The black trace shows the optical spectrum at the PIC Optical signals in the coherent wireless transmission system: (black) the optical output from the PIC when the two DFB lasers are biased simultaneously; (red), PIC output when the OFC signal is injected; (green), output of the intensity modulator. Note that the black curve is superimposed on the red in the two wavelength peaks. output port when the two lasers are biased with current, and their wavelengths combined. The currents were adjusted to achieve a wavelength spacing of 97.16 GHz, and the side mode suppression ratio (SMSR) is shown to be higher than 35 dB. This is the free-running operating condition, in which the two lasers are independent oscillators with uncorrelated noise processes. In the locked operating condition (red trace), the two wavelengths are phase locked through optical injection of the OFC signal at a power of −6.2 dBm. Stray optical wavelengths from the OFC are visible, and the SMSR level is 25 dB. When this optical signal is intensity modulated, at 10 Gb/s, the optical spectrum is given by the green trace. It is shown that all the optical modes widen due to the data bandwidth. After the modulation, the optical signal was amplified with an erbium-doped fiber amplifier (EDFA). The W-band signal was generated from the EDFA output using an Uni-Traveling Carrier Photodiode (UTC-PD). The total output into free space was measured to be −23 dBm. Figure 5 shows the beat note at 97.16 GHz on a 100 kHz resolution bandwidth. The signal under free-running conditions is shown in figure 5(a), with ∼10 MHz full-width half maximum linewidth. Figure 5(b) shows the linewidth under optical injection, when it is significantly reduced.
At the receiver side, we used two detection schemes. The first is direct detection (DD), using a quasi-optical Schottky barrier diode (VDI QOD, with 100-1000 GHz RF bandwidth and 40 GHz video bandwidth) acting as an envelope detector. The second scheme is coherent detection, using a harmonic mixer (VDI WR8.0ZBD, with WR08 RF input and 19.8 GHz video bandwidth), in which the use of a local oscillator (LO) signal and narrow intermediate-frequency bandwidth provides higher sensitivity.
The bit error rate was measured as a function of the radiation power under injection locked condition of the DFB lasers and coherent detection. The results are plotted in figure 6(a). We used a pseudo random (2 15 -1) length bit stream which increases the bit rate from 10 to 14 Gbit s −1 . We observe that up to 10 Gbit s −1 data-rate, we are able to achieve error-free transmission (BER<10 −11 ), but not with higher data rate increases. The reason is the aliasing between the modulation side-bands on both DFB sub-carriers (figure 4) caused by the OFC injection. Then we compared the BER in the locked and free-running condition (figure 6(b)) with DD, and compared those results versus the best-case scenario-DFB lasers locked with coherent detection. With DD there is no advantage of stabilizing the lasers (o points) versus using them in the free-running state (x points). The real difference is achieved when the coherent scheme is used, with error-free transmission (BER of 9.9×10 −12 ) and 17 dB sensitivity improvement over the DD. Figure 7 shows the recorded eye pattern in the error-free condition. According to Friis' formula, coherent detection thus enables about 7 times longer transmission range than DD. This improvement in the sensitivity is at the cost of increasing the complexity of the transmitter (OFC) and receiver (heterodyne conversion). This has a high impact on the cost of the system. If the transmitted power were increased significantly, the simpler and cost-effective DD scheme would be enabled, avoiding the phasing issues in both transmitter and receiver sides.
Antenna array
The above experiment demonstrates the possibility of achieving a data rate up to 10 Gbps with an integrated photonic source. However, from figure 3(b) it can be seen that the antenna used for the link is a 20 dBi conical horn antenna that precludes a fully integrated solution and is bandwidth limited (75-110 GHz due to the single waveguide-mode configuration). Also, the transmitter as configured cannot provide a power larger than that associated with a single PIC.
To overcome these drawbacks an array configuration for increasing the emitted power is demonstrated in this section. In array configurations, the sources must have mutually coherent properties. This is possible by using the same laser sources for all photomixers. This approach was first demonstrated by Preu et al [12,13]. In [13] the authors used two photomixing sources in a Young's experiment setup for measuring the coherence length. In [12], it is shown how N sources can be combined for increasing the generated THz power. An intensity improvement of N 2 is achieved in the direction of the maximum of the radiation pattern [12,14], although the total radiated power increases only N-fold as expected from classical electromagnetics. This power increase should yield either a BER reduction at a given range, or a range enhancement at a given BER. Figure 8 shows the side view of the proposed array approach. A Gaussian optical beam (blue) is focused on a 2D rectangular array of photomixing sources (dark-red) by using a fused silica microlens array. This preserves the illumination efficiency since it avoids delivering power to non-active areas. The generated THz signal is radiated into the InP substrate (light-gray) by bow tie antennas (gold). A hyperhemispherical silicon lens (dark-gray) is placed below the substrate for radiating the THz power into the air. A detailed top-view of the 2D array configuration is shown in figure 9, showing the antennageometry. It is based on the planar, selfcomplementary bowtie whichcanprovide stable radiation patterns and behavior over the required broadband. Also, in this array configuration, the mutual coupling can be controlled. For example, the optimum pitch size between the elements has been carefully selected to be significantly less than the wavelength at the lower frequencies which will purposefully increase the mutual coupling to constructively enhance the performanceat these frequencies.
Experimental set-up
The bow-tie antennas are laid out in a cartesian grid such that when rotated 45°the array resembles a 'chessboard' pattern on the InP substrate. It is also easy to see that the interconnected self-complementary structure provides an equal and constant RF impedance in all the driving ports (i.e. The generated THz signal (light-red) is radiated into the substrate (light-gray) by bow ties antennas (gold) and, finally, into the air by using a silicon lens (dark-gray). Figure 9. A 3×3 photomixers chessboard array. The devices (red) are placed in the gaps of bow tie antennas (gold) on a 300 μm centerto-center pitch. Each photomixer has an active area of 10×10 μm 2 . The central element is marked with a green box. The inset shows a fabricated device before adding the bias connections. photomixers). In addition, if the photomixers are mutually coherent and synchronous, there should be a stable radiation pattern in the broadband direction. Figure 9 shows a 3×3 element array with 300 μm pitch in both dimensions. Two extra rows and columns of passive antennas have been added to increase the array size and avoid truncation effects. The 300 μm pitch is quite convenient for assembly purposes [15] due to the availability of rectangular-grid microlens arrays, such as the commercial MLA300-14AR. One of the main advantages of the proposed design is the biasing set-up. Each row of the array is given the same voltage V, and the row-torow difference is the desired photomixer bias voltage. Hence, all devices will have the same bias. Another advantage is its polarization stability in the whole band. Being very broadband and dipole-like in nature, the bowtie antennas radiate E and H plane polarizations that are nominally orthogonal with around −10 dB of cross-pol level. This orthogonal polarization could provide another benefit for a communication link by using polarization diversity.
The devices have been manufactured by HHI using not UTC-PDs but rather an ultrafast InGaAs photoconductive photomixer having an interdigitated-electrode structure. The central element of the array is aligned to the Si-lens optical axis by using a microscope with a reticle (see figure 9 inset) and a 2-axis micro-positioner having alignment precision below ±10 μm. The fully assembled chessboard array and bias scheme are displayed in figure 10.
The alignment of the microlens array to the input laser is the most critical step in the assembly process. It can be done by using a 5-axis mechanically controlled micro-positioner (see figure 11). By adjusting the distance between the microlens and the photomixer planes, it is possible to modify the spot sizes, which can be used to trade-off the alignment accuracy with illumination inefficiency.
Simulation results
The concept has been validated for different array sizes through full-wave simulations. The array pitch can be tailored to specific antenna sizes in order to cover different THz bands. In this Section two designs are presented, one working up to 500 GHz and the second one working up to 2 THz. A study of each array configuration together with different Sihyper-hemisphere designs is presented in terms of directivity. Figure 12 shows the achieved directivity for a design working up to 500 GHz. It has a pitch P x =P y of 227 μm. Two different commercial hyper-hemispherical lenses were considered for the simulation: the first one has a diameter of 24 mm and a thickness of 15.3 mm (D24, blue); and the second one has a diameter of 25 mm and a thickness of 15.8 mm (D25, red). The lowest frequency considered for this Figure 10. Assembly of the 3×3 chessboard array shown in figure 9 inset. The array is aligned and then glued onto the backside of a silicon lens. Next, a PCB for doing the bias connections is added. Figure 11. Full assembly. An optical system is added for controlling the laser spot size and collimation. A 5-axis micropositioner places the microlens array between the chessboard array and the laser optical system. design is 50 GHz. Figure 13 shows the 2D radiation pattern of the array when using the 25 mm diameter lens.
A second design was developed working up to 2 THz by reducing the pitch down to P x =P y =57 μm. Its directivity (see figure 14) was obtained for three different commercial Si hyper-hemispherical lenses: a bullet lens with a diameter of 14 mm and a thickness of 9.1 mm (D14 T9.1, blue); a bullet lens with a diameter of 8 mm and a thickness of 5.3 mm (D8 T5.3, red); and a wider lens with a diameter of 15 mm and a thickness of 10.1 mm (D15 T10.13, pink). Figure 14 compares the directivity of all three in the 200-2000 GHz frequency band. figure 15 displays the 2D radiation pattern for the (D8 T5.3, red) bullet lens.
The THz emitted power has been measured for the second design with first lens (14 mm diam bullet lens) up to 500 GHz (figure 16) The radiated cw power in the 100-200 GHz band has been measured with a 13 Hz chopped Golay cell having a filter to block IR but pass THz, and a lock-in amplifier for synchronous demodulation. The maximum radiated cw power was approximately 40-50 μW at frequencies around 150 GHz. This is 8-10 times greater than that of the UTC-PD near 97 GHz in the above communications demonstration. In addition, the chessboard array enables a modular transmitter where the DFB lasers and modulation components are integrated into one PIC, and the photomixers are integrated into a separate monolithic chip. The improvement in power made possible by the chessboard array will allow us to realize, according to figure 6, excellent communications-link performance using a simple DD receiver, and even better performance if a coherent receiver is justified.
Conclusions
In this paper we present key advancements for the deployment of 5 G wireless systems. On one hand, we demonstrate a PIC for generation, optical-frequency-comb stabilization, and modulation of carrier waves in the millimeter-wave range (Wband) through photomixing. This technique enables the seamless integration of wired and wireless links. Error-free real time data transmission at 10 Gb s −1 is achieved for direct and coherent detection. The direct-detection data rate was limited only by the frequency spacing of the OFC. Coherent detection enabled a sensitivity improvement of about 17 dB, but at the expense of significantly increased receiver complexity. Direct detection can be maintained through an increase of the transmitted power. To this end, we have also presented a novel photomixing power-combining schemethe chessboard array. Since the number of elements can be increased with no extra assembly complexity, the chessboard array can potentially become an appealing high-power optoelectronic terahertz cw coherent source for many applications besides communications. | 2019-04-26T13:59:16.385Z | 2019-03-29T00:00:00.000 | {
"year": 2019,
"sha1": "fdde2f6aa85770692e722e908490afb4317b6840",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1088/1361-6641/aaf8f2",
"oa_status": "HYBRID",
"pdf_src": "IOP",
"pdf_hash": "909293d0f15368ab881c0da33e342d83a03e09b6",
"s2fieldsofstudy": [
"Engineering",
"Physics"
],
"extfieldsofstudy": [
"Materials Science",
"Physics"
]
} |
4573216 | pes2o/s2orc | v3-fos-license | Correlation analysis of mesenchymal–epithelial transition factor protein and human epidermal growth receptor 2 protein expression in 1479 cases of lung adenocarcinoma in China
Background To investigate the correlation between mesenchymal–epithelial transition factor (C‐Met) and human epidermal growth receptor 2 (HER2) protein expression in primary lung adenocarcinoma tissues. Method A total of 1479 resected primary lung adenocarcinoma patients were enrolled in the present study for detecting of C‐Met and HER2 protein by immunohistochemistry, and correlation analysis was made between the above two biomarkers and related clinicopathological features. Result Both C‐Met and HER2 proteins were found to stain highly positive in lung adenocarcinomas, and a positive correlation was found between them (χ2 = 118.5, P = 2.707 × 10−21). In addition, HER2 protein expression was correlated with sex, pathological stage, lymph node metastasis, and major subtypes; and C‐Met was correlated with sex (P < 0.05). Conclusion The expression of C‐Met and HER2 protein in lung adenocarcinoma is highly correlated, and whether it is synergistic in the targeted therapy of lung adenocarcinoma deserves further study.
Introduction
In recent years, the incidence of lung cancer has risen in China and worldwide. 1 Lung adenocarcinoma accounts for more than half of non-small cell lung cancer. 2 The evolution of detecting molecular abnormalities, such as epidermal growth factor receptor (EGFR) mutation, ALK fusion and the development of corresponding targeted drugs, has led lung adenocarcinoma to an era of individualized precise treatment. 3,4 However, some cases have been found to be resistant to EGFR inhibitors (EGFR-TKI), so it is necessary to study the mechanism of drug resistance and to explore new therapeutic targets. 5 Human epidermal growth receptor 2 (HER2) and mesenchymal-epithelial transition factor (C-Met) have been found to be mutated and amplified in lung adenocarcinoma, which are associated with EGFR-TKI resistance. 5 There are many studies on HER2 gene mutation and C-Met gene amplification or exon 14 mutation in the literature, but the abnormal expression of protein and the relationship between the two genes are only reported in small samples, and the conclusions are not consistent with clinical pathological features. [6][7][8] The present study intended to detect C-Met and HER2 protein expression in a large sample of lung adenocarcinoma cases from the National Cancer Center in China, and to explore the correlation of the two biomarkers with clinicopathological factors.
Case selection and histological analysis
This study was a retrospective study, which had been approved by the hospital ethics committee to exempt patients' informed consent. All pathologically diagnosed pulmonary adenocarcinoma surgical resection specimens were consecutively collected from the Department of Pathology of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences from July 2013 to September 2014. Clinicopathological data were extracted from medical archives, including the patient's age, sex, AJCC 7th pathological stage, and so on. Histological subtypes were reviewed and distinguished by experienced pathologists according to the 2015 World Health Organization classification of lung tumors (Kappa = 0.72). 9 A total of 1479 cases of lung adenocarcinoma were retrospectively classified according to predominant components, which included invasive lepidic adenocarcinoma (n = 157; 10.6%), papillary adenocarcinoma (n = 228; 15.4%), acinar adenocarcinoma (n = 843; 57.0%), solid adenocarcinoma with mucin produced (n = 177; 12.0%), mucinous adenocarcinoma (n = 37; 2.5%), micropapillary adenocarcinoma (n = 13; 0.9%), adenocarcinoma in situ (n = 8; 0.5%), minimally invasive adenocarcinoma (n = 9; 0.6%), and enteric adenocarcinoma (n = 7; 0.5%). For statistical convenience, we combined those items <1% into a special subgroup including a total of 37 cases, which were 13 cases of micropapillary adenocarcinomas, eight cases of adenocarcinoma in situ, nine cases of minimally invasive adenocarcinoma, and seven cases of enteric adenocarcinomas.
Methods
Preprocessing procedures of specimens were as follows: surgically resected tissues were fixed in 10% neutral formalin for 6-48 hours at 10-fold the volume of the tissue liquid, and then embedded in paraffin. Four consecutive 4-μm thick sections were used for HER2 and C-Met immunohistochemical staining, and the others for routine negative control staining for a matched rabbit monoclonal negative immunoglobulin antibody. All sections were heated to 62 C, and then subjected to the fully automated immunohistochemical assay on the Benchmark XT stainer (Roche Company, Basel, Switzerland). According to the manufacturer's scoring algorithm, a four-level scoring system was used for the evaluation of staining results. HER2 protein was deemed positive with the presence of strong granular cytoplasmic and/or membrane staining. C-Met protein staining was deemed positive in cytoplasm (Fig 1a-c). In addition to that, the staining intensity and percentage of dyed cells were also included in interpretation. Negative quality control sections were first evaluated to remain unstained before evaluation for immunostaining on every case. A four-level classification was applied into interpretation, including negative (0), no staining or <5% dying; weakly positive (1+), 5~25% tumor cells stained; moderately positive (2+), 25~50% tumor cells stained; and strongly positive (3+), >50% tumor cells stained.
Statistical analysis
We used independent χ 2 -test to compare the frequency of clinicopathological characters between HER2/C-Met high expression and low expression groups, and a correlation analysis was also made between HER2 and C-Met protein results. The statistical analyses were conducted using SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA), and statistical significance was set as P < 0.05.
Patient characteristics
A total of 1479 primary lung adenocarcinoma patients were available for analysis, including 658 men (44.5%) and 821 women (55.5%), with the average and median age of 59 years. Other clinicopathological features are listed in Table 1, including pathological stage, lymph node metastasis, and predominant histological subtypes.
Discussion
Molecular targeted therapy of lung adenocarcinoma is the representative of current cancer precise medicine. With the decrease of the cost of gene sequencing and data analysis technology, the search for abnormal gene targets for cancer tissue genomics and proteomics has become the mainstream of targeted drug research and development. 10 At the same time, the application of machine learning in multiple dimension analysis for correlation of different types of proteins also showed great power in the design process of targeting drugs. 11,12 HER2 and C-Met protein are closely related with the EGFR receptor tyrosine kinase, and HER2 protein shows a high similarity with EGFR. In the present †For statistical convenience, a special group was created including those subtype lower than 15 cases, including a total of 37 cases, which were 13 cases of micropapillary adenocarcinomas, 8 cases of adenocarcinoma in situ, 9 cases of minimally invasive adenocarcinoma, and 7 cases of enteric adenocarcinomas. 4) †For statistical convenience, a special group was created including those subtypes lower than 15 cases, including a total of 37 cases, which were 13 cases of micropapillary adenocarcinomas, 8 cases of adenocarcinoma in situ, 9 cases of minimally invasive adenocarcinoma, and 7 cases of enteric adenocarcinomas. study, we retrospectively analyzed lung adenocarcinoma cases from the National Cancer Center within 14 months for expression of C-Met and HER2 proteins, and revealed that they were closely correlated with some clinicopathological factors, such as sex, pathological stage, lymph node metastasis, and so on; also, we found there was no significant correlation either with ALK fusion protein or within mutated EGFR cases (data not shown). As far as we know, this is the first large-scale analysis for C-Met and HER2 proteins in lung adenocarcinomas.
HER2/neu (EBRR-2) gene is located on chromosome 17q21, encoding a transmembrane protein that shows protein tyrosine kinase activity. It is highly homogeneous with the human epidermal growth factor receptor (EGFR HER1) gene, which plays a role in signal transduction. HER2 mutation or overexpression was found in approximately 3% of non-small cell lung cancer. HER2 overexpression increases the sensitivity of lung cancer cells by inhibiting EGFR-TKI two heterologous dimer formation, and may benefit from trastuzumab. 13 C-Met belongs to a tyrosine kinase receptor (receptor tyrosine kinases) superfamily, which is an oncogene for encoding a class of self phosphorylation activity of the transmembrane receptor, located in 7q31, with a size of approximately 110 kb, including 21 exons. It was found that C-Met gene amplification or high expression was one of the important mechanisms of acquired drug resistance against EGFR-TKI for lung adenocarcinoma, and it could benefit from ALK inhibitors. 14,15 In this study, we found that HER2 protein and C-Met protein were highly expressed in lung adenocarcinoma, and there was a significant correlation between the two proteins, which could help to explain some molecular characteristics of lung adenocarcinoma from the protein level. Wei et al. found that the expression of HER2 protein was related to age and the degree of differentiation in the EGFR mutant of advanced lung adenocarcinoma. 8 In a small sample study, it was found that HER2 and C-Met protein could be expressed in lung adenocarcinoma, and showed a significant positive correlation trend. 7 The present study is consistent with the above results. Similar results were found in carcinomas other than lung adenocarcinoma. C-Met has been reported to be associated with HER2 in breast cancer, and it was an adverse prognostic factor independent of HER2 status in patients with lymph node metastasis. 16,17 Approximately 54% of patients with gastric cancer showed the activation of HER1, HER2, HER3, C-Met, or IGF1R, the prognosis was poorer than those with no activation of receptor tyrosine kinases 18 in advanced gastric cancer patients, a significant difference was found between C-Met-positive and -negative patients in differentiation degree, liver metastasis, and alkaline phosphatase levels; and the positive rate of C-Met was found different in HER2-positive and HER2-negative patients (56% vs. 38%, P < 0.05). The above findings showed some correlation of C-Met and HER2 genes in gastric adenocarcinoma. [18][19][20] Study on the mechanism of tumor molecular biology identified that both C-Met and HER2 genes showed a potential trend of co-expression, and both of them showed a definite correlation with EGFR mutation. [21][22][23] The HER2 gene could promote the rapid proliferation and inhibit apoptosis of tumors cells by activating the PI3K-Akt pathway, thereby increasing the metastatic ability of cancer cells. 24 Meanwhile, C-Met could activate Ras-ERK and PI3K-Akt pathways, triggering epithelial mesenchymal transformation, and promoting invasiveness and migration of cancer cells. 25 In conclusion, based on the large sample from the National Cancer Center, we demonstrated from the protein level that the expression of HER2 and C-Met in lung adenocarcinoma were synergistic and statistically correlated with some clinicopathological features. The limitation of this study is that the selected cases were mostly from patients who underwent curative-intent surgery, and there was not enough long time to evaluate protein expression with EGFR target therapy and survival analysis, which will be the direction for further research. | 2018-04-03T00:33:58.223Z | 2018-02-04T00:00:00.000 | {
"year": 2018,
"sha1": "bcf1fc0dc5a726503f39b55a4fabad80c3a07025",
"oa_license": "CCBYNC",
"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1759-7714.12595",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "bcf1fc0dc5a726503f39b55a4fabad80c3a07025",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
259675384 | pes2o/s2orc | v3-fos-license | Farmers’ Acceptance of a Micro-irrigation System: A Focus Group Study
Despite water scarcity and lots of benefits, implementation of micro-irrigation systems on potato crops in the Bekaa Valley of Lebanon is notably low. This could be related to farmers’ acceptance to use this technique. The objective of this study is to investigate the factors that can affect or not the adoption and the investment in a new micro-irrigation system. For this aim, the unified theory of acceptance and use of technology (UTAUT) served as the conceptual framework. A qualitative approach using focus group discussion was applied. A total of 34 farmers in six focus groups were conducted in the three main districts of the Bekaa Valley. From the analysis, performance expectancy, effort expectancy and facilitating conditions emerged as the three most prominent factors to understand farmers’ acceptance and adoption of micro-irrigation systems. The focus
Introduction
Climate change is having a huge detrimental impact on freshwater availability on a worldwide scale, affecting water resources quantitively and qualitatively (Field & Barros, 2014). Water scarcity is one of the most dangerous threats which has already resulted in catastrophic losses, notably in the arid regions. High temperatures, increased evaporation and fluctuations in precipitation are altering water availability and reducing crop yields (Arbuckle et al., 2013;Niles & Mueller, 2016).
These factors affect the management of farms, especially in arid and semi-arid regions (Scoville-Simonds et al., 2020). Moreover, climate change is endangering the agricultural sector presenting risks for developed and developing countries (Field & Barros, 2014;Niles & Mueller, 2016).
Lebanon is a small mountainous country on the Mediterranean Sea's eastern coast, covering a total area of 10,452 Km². From a climatic point, Lebanon is dominated by a Mediterranean climate with a cold rainy winter and a semi-hot dry summer. Lebanon experiences water shortages during the dry season which reaches out from July through October, with about 60 percent of the country's territory undermined by desertification (MoA, 2003). This situation is relied upon to turn out to become more severe in the future due to the impact of climate change (Bank, 2014). As LARI (2019) stated, water scarcity rather than land resources is actually the constraining factor in the country's expansion of agricultural production. In Lebanon, groundwater sources are increasingly stressed by climate change as well by the increased demand from agriculture, the inadequate utilization of underground water, the population growth and the industrial development (UNDP & UNHCR, 2021).
Further, recent results (Halwani & Halwani, 2022) showed that from 1930 to 2019, the average temperature in Lebanon has increased between 1 to 3 ºC and a recent report from USAID (USAID, Middle East and North Africa (MENA) region, in Lebanon and beyond indicated that the use of micro-irrigation in potato cultivation could have promoting results in terms of water savings of up to 40% (Darwish et al., 2003;Darwish et al., 2006), and allowing for energy savings associated with higher crop quality and yields (Karam & Karaa, 2000;Varma & Namara, 2006;Shah, 2011(Rouzaneh et al., 2021. Given the lack of information available on the performances of innovative technologies, farmers may evaluate these new systems through their experience and knowledge. This study aimed to analyze the indirect non observed factors such as farmers' motivations, attitudes and socioeconomic factors which may influence theirs' perceptions and behaviours in affecting their investment in and adoption of a new micro-irrigation system. By disentangling these factors, effective strategies, and support systems for promoting the use of micro-irrigation systems in the area could be designed. To this end, the Unified Theory of Acceptance and Use of Technology (UTAUT) model (Venkatesh et al., 2003) was adopted. The UTAUT model (Venkatesh et al., 2003) is a tool that is used to analyse the individual acceptance and the use of new technology by evaluating the influencing factors. Previous studies utilized the UTAUT model to investigate factors affecting the adoption of pressurized irrigation technology among olive farmer (Nejadrezaei et al., 2018), the acceptance of e-agriculture (Eweoya et al., 2021), farmers' use of communication technologies (Mahamood et al., 2016) as well the acceptance of water saving technologies (Sabbagh & Gutierrez, 2022).
A qualitative study that utilized focus group discussion (FGD) approach was employed. In this study, FGD could be an appropriate tool because it can allow for drawing upon the respondent's knowledge, views, and experiences about the specific topic of introducing micro-irrigation systems.
To the best of our knowledge, this is the first study to use the UTAUT model combined with a Focus Group Discussion approach to shed light on the impact and importance of behavioural factors in influencing the adoption and use of a micro-irrigation system. Hence, the research question is: "what behavioural factors could affect the intention to adopt and invest in a micro-irrigation system by the potato farmers in the Bekaa Valley of Lebanon?
The remainder of this paper is organised as follows. Section two briefly analyses the UTAUT model. Section three explains the methodological approach employed in this study to explore the acceptance of a new micro-irrigation system. Section four presents the results of focus groups conducted with potato farmers in three main districts of the Bekaa valley. Section five discusses the main findings providing insights about policies that government could implement to encourage potato farmers in adopting a micro-irrigation system. In section 6, the main conclusions are presented, and section 7 is related to the study's limitations.
Research behavioural model and Research's questions
A number of theories have been put forward to explain the individual behavioural intention to introduce a new technology. The current study employed as technology adoption model, the Unified Theory of Acceptance and Use of Technology (UTAUT) (Venkatesh et al., 2003) which integrated previous technology acceptance models. Thus, UTAUT is basically a synthesis through unifying at least eight existing technology acceptance and use models and specifically i) the Theory of Reasoned Action (TRA) (Fishbein & Ajzen, 1975); ii) the Theory of Planned Behaviour (TPB) (Ajzen, 1985); iii) the Technology Acceptance Model (TAM) (Venkatesh & Davis, 2000); iv) the Combined TAM and TPB (C-TAM-TPB) (S. ; v) the Innovation Diffusion Theory (IDT) (Moore & Benbasat, 1991); vi) the Motivation Model (MM) (Davis et al., 1992); vii) the Social Cognitive Theory (SCT) (Bandura, 1994;Compeau et al., 1999;Compeau & Higgins, 1995) and finally viii) the Model of PC Utilization (MPCU) (Thompson et al., 1991). According to UTAUT, an individual's perspectives about the technology impact his or her behavioural intent to use and actual use of the technology. Based on the integration of the eight models, UTAUT suggested four major determinants that have an effect on a person's "use behaviour" to adopt a technology: performance expectancy (PE), effort expectancy (EE), social influence (SI), and facilitating conditions (FC). The first three constructs influence use behaviour through a behavioural intention variable, while the fourth construct directly impacts the use behaviour. These constructs can be affected by four moderators a) age, b) gender, c) experience with similar technology, and d) voluntariness of use. Fig.1 presents the model. (Venkatesh et al., 2003); Adapted with permission from Viswanath Venkatesh, MIS Quarterly, 2003. 1 The Performance Expectancy (PE) represents the user's level of belief in how much advantageous a system usage will be and how it will help out to attain benefits (Venkatesh et al., 2003). PE aggregated all job performance related aspects, like usefulness (adapted from TAM/TAM2 and C-TAM-TBP) (S. Venkatesh & Davis, 2000), job fit (from MPCU) (Thompson et al., 1991), relative advantage (from IDT) (Moore & Benbasat, 1991), extrinsic motivation (from MM) (Davis et al., 1992) and outcome expectations which are related to the consequences of the behaviour (from SCT) (Bandura, 1994;Compeau et al., 1999;Compeau & Higgins, 1995). Based on the findings of the old models, PE will significantly and positively influence behavioural intention and technology acceptance (AbuShanab & Pearson, 2007;Venkatesh et al., 2003). Persons with high PE had high intentions to use a new technology (AbuShanab & Pearson, 2007). Additionally, the influence of performance expectancy on behavioural intention is suggested to be impacted by the moderating effects of gender and age (Venkatesh et al., 2003).
The Effort Expectancy (EE) construct suggests that the level of ease of use affiliated with the user's adoption of a system is an important component in the adoption of a new technology (Venkatesh et al., 2003). In this case, it is composed by three constructs that are: perceived ease of use (TAM/TAM2) (Venkatesh & Davis, 2000), complexity (MPCU) (Thompson et al., 1991) and ease of use (IDT) (Moore & Benbasat, 1991). Previous research concluded that EE is a positive predictor of behavioural intention so that the higher the perceived ease of use of a new technology, the higher the intention to adopt it (Bandyopadhyay & Fraccastoro, 2007;Kallaya et al., 2009;Nassuora, 2012;Venkatesh et al., 2003). According to (Venkatesh et al., 2003), the influence of effort expectancy on behavioural intentions is moderated by gender, age, and experience.
The social influence determinant (SI) refers to the magnitude to which individuals perceive they should adopt a technology based on inputs from persons who carry significant positions in their life (Venkatesh et al., 2003). It also consists of "the degree to which peers influence use of the system" (Slade et al., 2015;Šumak & Šorgo, 2016;Venkatesh et al., 2003). Social influence (SI) consists of three variables: a) subjective norms which relate to the person's perception that people who are important to her or him think that they should or should not execute the particular behaviour (Ajzen, 1991;Davis, 1989;Fishbein & Ajzen, 1975;, b) social factors which connects to the interpersonal arrangements that the individual has made with others as with coworkers (Thompson et al., 1991) and c) image which is the extent to which the use of a new technology is seen to enhance one's image or status in one's social system (Moore & Benbasat, 1991). Based on the review of the literature, it is expected that social influence positively influences the behavioural intention to use a new technology (Bandyopadhyay & Fraccastoro, 2007;Im et al., 2011;Kallaya et al., 2009;Slade et al., 2015;Šumak & Šorgo, 2016;Venkatesh et al., 2003). As well, (Venkatesh et al., 2003) hypothesized that the influence of social influences on behavioural intentions is moderated by gender, age, voluntariness and experience.
At the end, facilitating conditions (FC) represent the organizational and technical conditions or infrastructure that the individual believes would encourage the use of the system and make it simpler for him to use it (Venkatesh et al., 2003). The facilitating conditions determinant consists of three distinct constructs: a) perceived behavioural control (Ajzen, 1991; which are the possible internal and external limitations on behaviour related to resources, b) facilitating conditions adapted from (Thompson et al., 1991) which relate to objective factors that persons agree make an act easy to realize, and c) compatibility from (Moore & Benbasat, 1991) which indicates the extent to which a new technology is perceived as being consistent with the current needs and capabilities of potential adopters. Each one of these constructs is operationalized to incorporate technological and/or organizational aspects that are intended to eliminate obstacles to use. Facilitating conditions are found to positively influence use behaviour (de Veer et al., 2011)[19]. According to [19], the influence of facilitating conditions on usage is hypothesized to be moderated by age and experience.
As mentioned above, UTAUT hypothesized that gender, age, voluntariness and experience would moderate the relationships depicted in the model. These variables have been shown to moderate the intention to adopt new technologies in several studies (Al-Gahtani, 2004;Pearson et al., 2002;Venkatesh et al., 2003).
Overall, the present study proposed several research questions to discover how the adoption of a new micro-irrigation system on potatoes in the Bekaa Valley could be accepted and introduced.
Specifically, the research questions are the following: What are the reasons behind using the use of current sprinkler irrigation on potato fields? How do potato farmers perceive a micro-irrigation system and its implementation on their field? How do they observe the opinion of persons holding important positions in their lives and other farmers' successes? What are the difficulties and barriers that they face that prohibit them from adopting a micro-irrigation system? What strategies or policies could be used to encourage potato farmers towards using micro-irrigation?
Materials and Methods
The objective of this study was to explore via focus group discussion how socioeconomic and psychological factors influence the adoption of a micro-irrigation system as a mean to save water and avert the water scarcity crises among potato farmers in the Bekaa Valley of Lebanon. The focus groups discussed the behavioural aspects related to the possible shifting from the current irrigation technique (ordinary sprinkler) to micro-irrigation (drip or mini-sprinkler) that saves more water, induces higher production and better quality in the cultivation of potato crops.
The focus group protocol
The focus group research protocol was divided into three sections. The first section had the scope of warming up the discussion introducing the research theme and to collect information about gender, age, education, type of land management, farm size and the annual irrigation water used. Participants also received explanations of the role undertaken by the facilitator and that audio recordings would have only been used for the purpose of this study reasserting the significance of privacy of all participants. It was explained that all participants were free to reveal their opinions related to the discussion and that all answers were to be accepted.
Section two aimed at providing information regarding the potato cultivation, the status of underground water in the Bekaa region as well as the differences between the sprinkler irrigation system and the micro-irrigation system delivering by that the advantages that could be obtained implementing a micro-irrigation system.
Section three contained open ended questions related to the UTAUT model that the moderator asked to participants of the three main districts of the Bekaa Valley. To trigger the discussion around the behavioural elements of the UTAUT model, section three was opened asking participants about their knowledge of the micro-irrigation system and the reasons behind using the ordinary sprinklers.
This allowed the moderator to explore the degree to which each farmer believes that using the micro-irrigation system will help him or her to attain gains exposing by that the performance expectancy determinant. The moderator then asked about their perceptions of easiness of tasks related to the implementation and operation of the micro irrigation system and how do they perceive the related technical operations. This permitted the moderator to explore their effort expectancy towards micro-irrigation systems. Further, participants were asked to list people whose judgment is important to them that they would approve or disapprove their adoption of a microirrigation system and the effect of personal moral obligation norms to adopt a micro-irrigation system for the sake of protecting the environment by preserving water resources. This revealed the social influence construct. To measure the facilitating conditions, the moderator explored their opinion of being able or not to access required resources, as well as to obtain trainings and the necessary support needed to use micro-irrigation systems. Following the UTAUT model variables, questions related to the moderating variables were raised in the focus groups. The moderator asked participants if they believed that the age of the farmers affect their incentive to adopt new irrigation practices. Experience was tested by the familiarity of the farmers of the functioning of the microirrigation system either by their own trial on their crops or by observing others using it on potatoes or on other crops. For the voluntariness of use, farmers were asked about their tendency to adopt a micro-irrigation system in the case of the presence of external obligations as well as in the case of subsidies offered by the government. Bekaa, Central Bekaa and West Bekaa, respectively. Random sample selection is particularly appropriate when inferences are made to a large population because of the assumption that opinions, attitudes or whatever is being studied will be normally distributed within that population. And since the goal is to select a sample that will yield rich data to understand the phenomenon studied (Hennink et al., 2019), data were randomly collected from a total of 34 farmers in six focus groups consisting of five or six farmers each. Two focus groups in each of the three main districts of the Bekaa valley were made to help ensure a variety of points of views amongst participants and to test their likeliness or unlikeliness to adopt a micro-irrigation system in their farms. The farmers, with whom the focus groups were made, were the ones involved in the decisions regarding the agricultural practices, type of crops, and irrigation strategies to be implemented in their farms.; interviewees were chosen from different ranges of age, different educational levels, having different types of land management, and different farm sizes. The proportion of males among the participants was 100% since there were no women running a farm in the area since potato cultivation fields are largely male owned while female participation is more significant across industries in the region (Konishi, 2017)
4.
All focus groups were audio-recorded and then manually transcribed and analysed qualitatively using NVivo12 software.
Participants characteristics
In Table 1 the demographic characteristics are presented. The focus groups were held among a total of 34 farmers from which 11 participants from the West Bekaa, 11 others from the North of the Bekaa and 12 farmers from the Central Bekaa. Unfortunately, all participants were males due to the fact that there is no women running a farm in the area. In the West Bekaa, the average age was 55 years ranging from 45 to 60 years old for most of the N farmers (N=11). In the North and Central Bekaa most of them were aged having a mean age of 46 (N=11) and 52 (N=12) years, respectively.
In the cited 3 regions, the percentage of farmers who were older than 60 years was somehow equal (36% for both West and North Bekaa while 33% in the Central Bekaa). In regard to the educational level, the minority had a primary level (28%) in the West Bekaa, while the majority had a university diploma (64%) in the North Bekaa. However, in the region of Central Bekaa most of participants had a secondary educational level (42%).
As shown also in the Table 1, in each focus group, there was a diversity in the farms' size in order to gather the maximum possible point of views. In the West Bekaa the average farm was 146 hectares (SD=208), whereas in the North Bekaa, the mean farm size was 590 hectares (SD=1,55). In the region of Central Bekaa was 663 hectares (SD=1,55).
Unfortunately, almost all of the participants were not aware of the quantity of water used in the irrigation of their potato crops which is an alarming problem. 2 (18%) 6 (55%) 6 (50%) Results of the focus groups 4.2.1. This section has as aim to present the findings from the six conducted focus groups. After being transcribed from Arabic language to English, text files were imported into Nvivo12 to first begin with the codings and finding core themes that reflect what participants were discussing indicating the frequency of each core theme (Allsop et al., 2022). The results are categorized into the investigated determinants affecting the acceptance of the micro-irrigation system in potato farming and three key moderators.
To further emphasize and distinguish statements analysis from quotes, all direct quotes given by the participants, within the following findings part, will be highlighted in italics.
Performance Expectancy
Performance expectancy was measured by the perceptions of using a micro-irrigation system in terms of providing benefits. At first, participants were asked about their knowledge of the micro-irrigation system and the reasons behind using the ordinary sprinklers. All the participants showed a basic technological knowledge of the micro-irrigation system stating that it incorporates drip irrigation and mini-sprinklers irrigation. Concerning the reasons of the adoption of the current irrigation system, which is the ordinary sprinklers, the top answer was that sprinklers are less expensive (53%), and changing the ordinary sprinkler network that they have from many years will cost them a fortune. One of the respondents said: The most relevant statements that underpin this construct are the ones that relate to the general benefits associated with micro-irrigation use. Therefore, participants were asked about their perceptions about the possible advantages deriving from the adoption of micro irrigation systems.
Based on the content analysis, the most important benefit mentioned by the respondents was water saving. This pattern is evident from the word cloud in Fig. 3 which depicts the most frequently occurring words emerging from focus group discussions.
In Fig. 3, central words with larger font are the most frequent, while distant words with smaller fonts are the less frequent. Thus, the most recurrent words (water, distribution, saving, control, etc.) are important advantages in the opinion of the farmers. Participants highlighted that microirrigation is a water saving technique since it supplies water directly to the soil surface close to the plant roots, rather than the land around. As well, they believe that micro-irrigation ensures uniform distribution of water by delivering water only wherever necessary and evenly over the whole land despite the presence of wind. Moreover, farmers consider that micro-irrigation enhances the financial benefits by increasing yield, productivity, and therefore, farm profits. They suppose, as well, that the micro-irrigation is a way to reduce operational costs in terms of reducing energy (less energy for water supply/ low pumping needs) and saving pesticides and fertilizers. Overall, it was shown that farmers perceived the micro-irrigation as a system having many key advantages in potato farming from saving water, labor, and pesticides to increasing profits.
Therefore, we expect that ''performance expectancy'' will be positively associated with the intention of using micro-irrigation technology.
Effort Expectancy
Regarding participants perception of the easiness of use of a micro-irrigation system, and if they will be skillful in using it, 62% of them considered micro-irrigation easy to be extended over the field. Half of the 62% said that it saves labor amount and effort because it is installed once at the beginning of the season and no need to worry about moving it. Moreover, the other half believed that micro-irrigation helps saving time. Hence, the farmer can gain more time to take care of other profitable agricultural operations. Accordingly, many participants claimed that
Micro irrigation is easier than sprinkler irrigation, and it is installed only once per season;
therefore, the farmer will not worry about moving the network from one place to another such as the case of the sprinklers. Thus, micro irrigation saves labor.
Micro irrigation does not require significant time and effort to extend and remove the network. It is easier than sprinklers, because the network is extended once at the beginning of the season and does not need to be moved from one part to another part of the land as in the case of sprinklers.
On the other hand, 38% of the participants perceived a high difficulty in extending the network of the micro-irrigation system on large fields and especially in the case of potato farming.
They believed that, once extended, it decreases the efficacy of some agricultural operations.
To highlight this problem some respondents commented The micro irrigation is very difficult to install and needs a lot of time since the technical process to extend the network takes about a week and more. There is a difficulty in the tasks related to micro irrigation because we can't apply pesticides and do all the mechanical agricultural practices when it is installed.
Other than that, they also argued that the installation of the micro-irrigation system needs a lot of attention and a specialized work force which induces a huge effort due to the complexity of the network equipment that should be implemented precisely. Additionally, third of the respondents, who perceived a difficulty in the use of micro-irrigation, claimed that micro-irrigation is time consuming. Furthermore, another third of them considered micro-irrigation as labor consuming because the system needs constant attention in order to prevent damage of the hoses. Furthermore, the effort expectancy construct is relevant to the question asking participants whether they think they will become skillful in using micro-irrigation on potato crops.
Some participants said
On one hand, 88% claimed that they will be skillful in using micro-irrigation. Approximately one third of respondents believed they will do their best to develop their knowledge in order to improve the yield, and possibly to increase their profits; they will get used on any new agricultural practices that give positive results. One-fifth of the 88% participants described the micro-irrigation as an easy technique and it is not difficult to be implemented on potatoes. These responses can be summarized with the following comment Of course, it can be used in a successful way on potato and personally I will use it in a great way since it's not difficult to manage.
Moreover, another fifth thought they will surely become skillful in micro-irrigation after getting appropriate training and guidance. Further, approximately one fifth of the 88% of the participants assumed that they would improve their skills in every new technique and incite themselves to adopt it properly because it may improve their personal skills, thus their productivity. A respondent said: As farmers, we are most interested in developing our agricultural practices and noticing their positive results, and we therefore do our utmost to strengthen our skills in any new agricultural technology we adopt.
On the other hand, 12% of the participants thought they will not become skillful in using microirrigation technology on potatoes. Half of those participants were not convinced in the technology and believed it has no benefits on potato cultivation at all.
No, since I see that it has no benefit in growing potatoes, obviously I don't improve my skills in using it.
The other half considered micro-irrigation difficult and exhausting to be implemented in potato cultivation.
In sum, we find that ''effort expectancy" plays a positive role in user's intention to use microirrigation technology.
Social Influence
In the context of this construct, participants were asked to list people whose judgment is important to them that they would approve and disapprove their adoption of a micro-irrigation On the other hand, 6% of the participants weren't interested in the experience exchange, because they believed that each farmer has his own individual specific agricultural practices and requirements. As per example,
Each farmer has his own technologies and the specification of his land which differ from the other.
Some farmers may give agricultural information that can't be adopted in the same way in my farm.
Further, getting a better sense of farmers' views on climate change (CC) and water scarcity was also related to this construct. Participants were asked to define what do these two terms mean for them.
Firstly, half of the farmers believed that CC and water scarcity lead to loss in yield, thus in profits.
According to them, the scarcity of water resulting from climate change is compelling so that cultivated areas are minimized, resulting in huge losses. They also stated that climate change and water scarcity have negative consequences on agriculture in terms of the quality of yields.
Moreover, 16% argued that CC and water scarcity affect potato farming in particular because potato crops are very sensitive to high temperatures and to low precipitations. This group of farmers confirmed that CC directly and negatively affects the cultivation, especially potato crops, because it makes it vulnerable to climatic fluctuations. That may force them on some point to move from growing potatoes to rain-fed agriculture. Further, 16% of the participants claimed that CC and water scarcity put agriculture continuity at risk, because they lead to disasters that negatively affect agriculture. Furthermore, 9% defined CC as a fluctuation of precipitation and temperature during seasons. According to them, CC lead to changing temperatures during seasons, therefore to low precipitation rates, and consequently water scarcity. They also believed that CC induced the reduction of groundwater. Finally, 3% of the participants argued that CC and/or water scarcity do not exist because they still find water in abundance.
In the same context of social influence, 91% of the respondents affirmed that a farmer should have moral norms and personal obligation of preserving water for the environment, the future generations and for continuing appropriate agricultural practices.
They stated that
It is compulsory to have ethical and personal values to be forced to save water in order to preserve nature, water wealth and to keep the water resource to our children as well as to ensure the natural and continuous development of agriculture.
Personally, as I'm worried about climate change, if the government or a non-profit
organization will support us, I will adopt a micro-irrigation technique to conserve water for the ecosystem's well-being and to maintain a normal life-sustaining atmosphere.
Overall, it seemed that social influence may not influence on the farmers' intention to use a microirrigation system.
Facilitating Conditions
This construct is relevant to the question about the guidance role of the agricultural/irrigation extension services in the area. 79% claimed that there was no presence, neither of agricultural guidance and extension nor of training courses. They assured that the agricultural sector is marginalized and neglected; therefore, the farmers had to rely on their personal experiences or the experiences of other farmers in the surrounding. They added that the non-presence of extension services made them unaware of the existence of new agricultural practices. They stated that The agricultural sector is marginalized, there are no agricultural policies, not even agricultural extension, and we have become used to relying on ourselves, our individual information, and the information we take from each other.
Ministry of Agriculture which does not provide any guidance. Every farmer in this area depends on himself and on his personal experience.
The other 21% of the participants stated that there was limited agricultural extension from some companies and institutions for the purpose of marketing. That is why they do not trust that type of companies and they rely on their personal experience. This common feeling can be summarized from the word of participants: There is no appropriate agricultural extension role, there are some agricultural companies that deal with pesticides, they do some extension courses related only to the subject of insects so as to sell and market their products not more. So I only rely on my personal information and experiences.
We have some agricultural guidance from some agricultural associations and institutions; they are doing all they can for agricultural extension. I take into account the information they provide, because agricultural guidance is necessary and sometimes it is a memory refresh for things I know, but I do not remember.
In the same context of facilitating conditions, participants were asked about the barriers they thought might prevent them from implementing a micro-irrigation system. Participants had the possibility of multiple choices. Several barriers were mentioned by each participant and results are illustrated in Fig.4. All participants considered the most important barrier as the high initial expenses for installing the system: 53% stated they have a lack of capital in order to cover the whole area; 53% believed they need trainings to raise awareness about the benefits of the system; 44% consider the system needs attention and time for minor repairs; 38% emphasized that microirrigation is effort consuming; 38% thought that they need credit facilities as farmers; 35% assured that subsidies are necessary so they can implement this new technique of high cost; 26% they don't have the technical knowledge; 21% perceived that micro-irrigation is not feasible on large fields; 18% find it technologically complicated; 12% stated that they want the spirit among farmers because if they cooperate they can support each other's. However, only 3% need motivation from the family and friends in order to implement micro-irrigation, and another 3% believed that their land is very scattered which impedes the system installation.
Overall, facilitating conditions could improve a farmer's use behaviour of a micro-irrigation system.
Figure 41
Barriers of implementation of a micro-irrigation system
Key Moderators
In addition to the previously mentioned four main determinants, the UTAUT model included four
Experience
Experience was tested by the familiarity of the farmers in micro-irrigation system either by their own trial on their crops or by observing others using it on potatoes or on other crops. Based on the analysis of the focus group discussion, some participants assumed that adopting micro-irrigation is not difficult for them as they witnessed its usage by other farmers on potato cultivation or on other crops. Therefore, they have the know-how which increases their incentive to implement it on potato cultivation if they have the capital for the investment. In the same context, a participant stated
Voluntariness of use
Moreover, "voluntariness of use" was measured by the tendency to adopt a micro-irrigation system in a situation where there is no external obligation to adopt the technology. External obligations can be defined for example as limited quantity of water usage imposed by the responsible authorities in the region. Almost half of the participants (53%) stated that they can adopt micro-irrigation without external obligations, in order to induce good results and to ensure the continuity of their land cultivation: Yes, I will move to a micro irrigation system in order to improve the quality of potatoes and produce more quantities, and the most important thing is to reduce water waste.
However, it is worth mentioning that only one participant asserted that he will gradually adopt micro-irrigation regardless its high initial cost, because he believed that it greatly will improve the quality and quantity of potato yield: Yes, I move to the micro irrigation system, but in stages, due to the high cost.
On the other hand, the other half of the participants (47%) have no tendency to adopt microirrigation spontaneously without external obligations: half of them consider it an expensive technology and they do not have the financial resources. The other approximate half does not perceive any benefit from adopting it on potatoes, and only very few have abundance of water so they don't need a saving-water irrigation technology. Some comments were No, because I am convinced that the sprinklers are better than the micro irrigation on potato crops, and I don't have the financial resources to try and attempt the micro irrigation even on a small part of my land.
No, because I have enough water and I pay careful attention to the amount of water that the plant needs (manual soil testing) so that I don't waste water and therefore micro irrigation
won't help me.
No, I am not convinced that micro irrigation would be better than sprinklers on my land, so I won't implement it.
Furthermore, participants were asked about the possibility of them adopting micro-irrigation if the government decides to subsidize the use of water-saving irrigation systems. It was stated from 85% of the participants that they tend to adopt micro-irrigation system if there were subsidies from the government. According to them, subsidies reduce the financial burden on them at the beginning of the investment, and encourage them to take the first step toward the total adoption of the micro-irrigation system: Yes, if the government provides subsidies, conducts training courses and supports us to export our production, of course I will adopt it.
Yes, I agree, because the state and the government have an obligation to take care of the farmer, who is the core of the Lebanese economy. Hence, micro irrigation is essential and necessary in improving the quality of potatoes to become competing with potatoes from other countries.
Nonetheless, 15% of the participants insisted on not moving to micro-irrigation system even if there is support, because they do not perceive any benefit from it: No, I don't agree… At the end, the productivity will be identical to that of the sprinklers.
No, although this technique provides large quantities of crop production, however, it does not match with the large areas I cultivate, and thus the moth will surely appear resulting in high losses.
In this section, it is important to mention that those who first had tendency to adopt micro-irrigation without external obligations tend as well to adopt it if subsidies are introduced because it lessens the financial burden. Further, participants who said they would not use micro-irrigation because of its expensive cost changed their mind when the interviewer mentioned the subsidies. The most notable change in intentions was that of the participants who had no tendency to adopt the system claiming that it has no benefits. However, 50% of them changed their answers when the question of subsidies was raised. They stated in this section that they will move to micro-irrigation gradually by applying it at first on a small part of the land to test its advantages. For example: Yes, it will be possible for me to start adopting it on only one hectare. If my results are positive and there are no diseases, then I will gradually adopt it year after year until I have thoroughly checked its benefits.
The Direct Determinant: The Behavioural Intention
The measurement of behavioural intention in this study included the intention and predicted use of micro-irrigation system. The behavioural intention was measured by addressing questions whether the participants have a possible plan for the adoption of a micro-irrigation system in the following 12 to 24 months as well as the major concerns related to this system. 59% of the participants said that they do not have any plan for the adoption of micro-irrigation in the next 12-24 months. This group of participants was divided into 3 groups according to the reason behind not having a plan for adoption: a) the unstable economic conditions in Lebanon that does not encourage farmers to invest high capitals (the majority); b) the lack of micro-irrigation usefulness in terms of profits and feasibility (the quarter of them); c) lack of financial means (only 10%). The following quotes revealed the participants answers: No, because the sprinklers irrigation is more comfortable for the farmer and does not require much effort, and I am satisfied from the quality and productivity that I get.
No, if the government does not support me, I will not adopt the micro-irrigation system.
On the other hand, 41% of the participants stated that a plan to adopt the micro-irrigation system is possible in the near future. This group also was divided into several groups in terms of Yes when necessary, and that means if the water runs out on my land, I will adopt a micro irrigation system." Fig.5 below shows the different answers obtained when investigating the concerns of the participants over the micro-irrigation systems. Each participant had the possibility to mention multiple concerns. As clear, the top concern was the high cost of initial equipment and the possibility of financial losses (47%). In addition, 15% confirmed that micro-irrigation is labor intensive technique that requires a lot of effort, time and attention. Further, 29% have no concerns at all. The remaining concerns differ in little percentages from the frequent maintenance to the emergence of diseases (fungal and moth), short lifespan, feasibility on large areas, no wind resistance.
Figure 5
The concerns related to the micro-irrigation system Further, when asked about their willingness to adopt a new micro-irrigation system, 82% of the participants said yes and 18% said that they are not willing to.
At the end, in order to recapitulate the main results of each construct, the following table summarize the findings:
Effort Expectancy
A. Perception of the easiness of use of a micro-irrigation system.
Easy extension over the field, labor and effort saving, time saving. B. Skillfulness in using micro-irrigation. 88% of farmers claimed that they will be skillful in using micro-irrigation.
Social Influence
A. List people whose judgment is important to farmers and that they would approve and disapprove their adoption of a micro-irrigation system.
47% stated that they don't care to others' opinions. 21% considered the opinion of "other farmers" or "nearby farmers" important. 20% highlighted the importance of their family members' opinion such as fathers, sons and/or cousins. 12% of the farmers were interested in NGO's judgment and advice, as well as agricultural association, organizations and engineers. B. The importance of collecting information from other farmers and observing their possible successes before adopting a new irrigation system.
94% of farmers were very interested to have access to the experiences and suggestions of other farmers.
Facilitating Conditions
A. The guidance role of the agricultural/irrigation extension services in the area. 79% claimed that there was no presence, neither of agricultural guidance and extension nor of training courses. They assured that the agricultural sector is marginalized and neglected; therefore, the farmers had to rely on their personal experiences or the experiences of other farmers in the surrounding. B. Barriers that farmers thought might prevent them from implementing a micro-irrigation system.
The most important barrier was the high initial expenses for installing the system.
Age
A. The age of the farmers affects their incentive to adopt new irrigation practices.
62% of the participants considered that age had no influence on the intention of use of a new agricultural technology because no matter his age, a farmer remains enthusiastic and encouraged to adopt new technologies, thus developing himself and his land.
Experience
A. The familiarity of the farmers in micro-irrigation system either by their own trial on their crops or by observing others using it on potatoes or on other crops Participants assumed that adopting microirrigation is not difficult for them as they witnessed its usage by other farmers on potato cultivation or on other crops. Therefore, they have the know-how which increases their incentive to implement it on potato cultivation if they have the capital for the investment. B. The tendency to adopt a microirrigation system in a situation where there is no external obligation to adopt the technology.
Half of the participants (53%) stated that they can adopt micro-irrigation without external obligations, in order to induce good results and to ensure the continuity of their land cultivation.
Voluntariness of use
A. The possibility of adopting microirrigation if the government decides to subsidize the use of water-saving irrigation systems.
85% of the participants stated that they tend to adopt micro-irrigation system if there were subsidies from the government. According to them, subsidies reduce the financial burden on them at the beginning of the investment and encourage them to take the first step toward the total adoption of the microirrigation system.
Behavioural Intention
A. A plan for the adoption of a microirrigation system in the following 12 to 24 months as well as the major concerns related to this system. 59% of the participants said that they do not have any plan for the adoption of micro-irrigation in the next 12-24 months due to the unstable economic conditions in Lebanon that does not encourage farmers to invest high capitals; the lack of micro-irrigation usefulness in terms of profits and feasibility and the lack of financial means. 41% of the participants stated that a plan to adopt the micro-irrigation system is possible in the near future if there is the presence of subsidies by the government. B. Concerns regarding the microirrigation system. the top concern was the high cost of initial equipment and the possibility of financial losses (47%). 15% confirmed that micro-irrigation is labor intensive technique that requires a lot of effort, time and attention. 29% have no concerns at all. The remaining concerns differ in little percentages from the frequent maintenance to the emergence of diseases (fungal and moth), short lifespan, feasibility on large areas, no wind resistance.
Discussion
As initially mentioned, the purpose of this study was to get a deeper understanding of the influential determinants for potato farmers' adoption of micro-irrigation technology on their lands in the Bekaa region in Lebanon. This research further examined which factors seem to influence the farmers and their willingness to use a micro-irrigation system.
Based on the focus group analyses performed, performance expectancy, effort expectancy and facilitating conditions could play a significant effect on the acceptance of micro-irrigation technology while the social influence could not.
The effect of performance expectancy on behavioural intention was found to be relevant for many participants, which reflects the perceived benefits obtained using micro-irrigation system. The benefits were identified as saving water, reducing labor effort and time, saving energy, increasing yield, improving crop quality and improving the agricultural operations. The farmers' performance expectancy might increase by focusing on the usefulness of micro-irrigation systems. That means if the advantages of micro-irrigation systems were presented in meetings made by specialists, this probably would increase the acceptance and adoption for people who were against this method, and who preferred the ordinary sprinklers. Almost all participants declared that generation of good results and water saving were the top advantages of micro-irrigation system. However, they were very anxious about losing the financial investments in case they would not be able to apply this method without professional guidance. This asserts the idea of the essentiality to establish an agricultural guidance, in order to promote the advantages of micro-irrigation system and its usage.
The effort expectancy was measured by the perception of ease of learning and using the system, as well as how much effort should be spent to use the micro-irrigation system on potatoes. From the focus group analysis, it seemed that farmers preferred to adopt an easy way to use system which required less effort and time than ordinary sprinklers on potato crops. Furthermore, almost all participants, including a part of those who showed a high effort and attention concerns in extending the micro-irrigation system on their potato lands, demonstrated their willingness to learn about the micro-irrigation functions. By that, organizing trainings and pilot studies could be a way for farmers to decrease their level of doubt. During on-field trainings, farmers discover how micro-irrigation functions, and the adequate way of its installation over the potato fields. Similar with other research (Birch & Irvine, 2009;Im et al., 2011;Louho et al., 2006;Venkatesh et al., 2003(Nkandu & Phiri, 2022), effort expectancy could have an effect on behavioural intention.
The third determinant, the social influence, seemed to have an insignificant impact on behavioural intention to use micro-irrigation. This result was consistent with (Venkatesh et al., 2003) and (Rosen, 2005(Yang et al., 2020). In his research, (Venkatesh et al., 2003) had found that the adoption of a new system depends on the user's beliefs and not others' opinion. Social influence was found not affecting potato farmers to adopt a micro-irrigation system since the vast majority does not care about the opinion of nearby farmers, family members, NGOs, engineers, agricultural associations and organizations. This is why promoting the importance of agricultural associations and farmers' gatherings, will revitalize the spirit among farmers and the cooperation between them.
Lastly, the facilitating conditions determinant was measured by evaluating the available resources and support to use micro-irrigation systems. The study results clearly depicted the direct effect of facilitating conditions on use behaviour of using micro-irrigation systems consistently with (Hung et al., 2006;Im et al., 2011;Venkatesh et al., 2003;Wang & Shih, 2009). Guidance departments at the Ministry of Agriculture, NGOs working in agricultural extensions especially on the climate change subject, advertising on social media raising awareness on new ways of saving water, in addition to any other available services to assist individuals to adopt and use microirrigation systems could be an essential way to enhance the adoption of a micro-irrigation system. Nevertheless, all farmers confirmed that these conditions are unavailable in Lebanon, and there is no guidance on agricultural features in whole country, which means that they cannot know about the benefits of micro-irrigation, or its right usage.
With respect to the moderating effect of age, it emerged that it was an important moderator in the context of adopting a micro-irrigation system among potato farmers. The younger group affirmed that it would be more difficult to persuade the older generation who doesn't have initiative to try new technologies, contrary to what the elderly said. In fact, the moderation by the age impact was reported in several studies (Morris et al., 2005;Venkatesh & Morris, 2000;Venkatesh et al., 2003).
Secondly, experience, was considered by (Venkatesh et al., 2003) as one of the important factors that affect behaviour intention. In this study, it was shown that the effect of effort expectancy on behaviour intention was in fact moderated by experience. The findings of this study revealed that, in terms of micro-irrigation usage, experienced farmers were more likely to accept and use micro-irrigation than inexperienced farmers.
However, it appeared that experience was not a moderator of the effect of the facilitating conditions construct on use behaviour because farmers of different levels of experience have almost the same perceptions towards the resources supporting the use of micro-irrigation. This result is not consistent with the study of (Alshehri et al., 2013) who claimed that experience moderates the effect of facilitating conditions on use behaviour.
At the last, voluntariness of use had moderated the effect of social influence on behaviour intention. It was measured on the basis of not using external obligations or incentives in order to implement the new irrigation system. The results confirmed that in the case of subsidies, the level of adoption will increase and farmers will definitively implement the system. That is, if the micro-irrigation system was financially subsidized, almost all farmers in Lebanon will adopt it.
Furthermore, the study findings showed that almost half of the participants had not the tendency to adopt a micro-irrigation if there is no external obligation which is consistent with what (Venkatesh et al., 2003) had reported. In this case, if the government grants subsidies to support the implementation of a micro-irrigation system, the vast majority will adopt it gradually or immediately.
Conclusions
The aim of this study was to investigate the potato farmers' behaviour in adopting a micro-irrigation system. To achieve this objective, we adapted the unified theory of acceptance and use of technology (UTAUT) model.
The outcomes offer visions for the policymakers to encourage potato farmers' in adopting a new micro-irrigation system. Firstly, farmers are willing to accept micro-irrigation technology when they can make gain and reduce task uncertainty on their farming activities. Secondly, they are keen to adopt a micro-irrigation system if they find that it reduces effort and time of their farming activities.
Finally, it is relevant to encourage farmers to adopt it through financial aids or subsidies which provide opportunities for farmers to decrease the financial burdens on them. As well agricultural extensions, field trainings, pilot area studies are also important in increasing the farmers' intention to adopt a micro-irrigation system.
Limitations
Legal restrictions and safety measures linked to the COVID19 pandemic were a reason of the limited sample size. Also, the sample used lacked gender differentiation since no females operated farms in the study area. Thus, it would be useful to repeat the analysis with a larger sample for focus group discussions incorporating female participation and extending the study to other countries. | 2023-07-12T08:15:57.675Z | 2023-05-15T00:00:00.000 | {
"year": 2023,
"sha1": "aabfff9ee6c548d7b6796e019fb1c2755fef15fd",
"oa_license": "CCBY",
"oa_url": "https://oaj.fupress.net/index.php/bae/article/download/13464/11779",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "2c15dfe4584ef9ea737f2b30717a5ce5ef7a992c",
"s2fieldsofstudy": [
"Economics"
],
"extfieldsofstudy": []
} |
266826249 | pes2o/s2orc | v3-fos-license | SWOT Based Service Quality Improvement Strategy at PT. FOKUS KUALITAS UTAMA
. In a highly competitive world, companies face numerous challenges. For service-based companies, success is determined by the quality of their services. PT. FOKUS KUALITAS UTAMA is a business consulting firm that assists in enhancing the performance of human resources within a company or organization. The objective of this research is to identify the internal and external factors that can improve the service quality in PT. FOKUS KUALITAS UTAMA. This study utilizes qualitative methods and collects primary and secondary data through observation, interviews, literature review, and SWOT analysis using IFAS and EFAS tables. The research findings indicate that PT. FOKUS KUALITAS UTAMA services fall within Quadrant I, implying an aggressive strategy.
INTRODUCTION
In this increasingly competitive business era, the quality of services provided by service-based companies, retailers, technology firms, or any other industry plays a central role in determining the level of success that can be achieved.Customers nowadays have high expectations for the experiences offered by companies.For instance, there are numerous customers who actively seek services of high quality and are willing to pay a premium to receive such services (Chaudhary & Gupta, 1999).To ensure the growth and development of a company, it is crucial for the company to anticipate the increasingly competitive economic landscape by implementing appropriate strategies to avoid being left behind in the competition.(Fernos & Putra, 2019).PT.FOKUS KUALITAS UTAMA is a company engaged in consulting services that assist other companies or organizations.In this study, the researcher aims to analyze the internal and external factors and the strategies that should be used to enhance the service quality at PT. FOKUS KUALITAS UTAMA.Dengan metode analisis Strenght, Weakness, Opportunities, Threath (SWOT) untuk mengetahui mengidentifikasi kekuatan internal yang dapat meningkatkan kualitas By using the SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis method, we can identify internal strengths that can enhance service quality, weaknesses that need to be addressed, opportunities to improve existing services, and threats that may affect the quality of services offered.By understanding the SWOT factors within PT.FOKUS KUALITAS UTAMA, the company can develop comprehensive and effective enhancement strategies.This structured and comprehensive approach is expected to assist the company in meeting customer expectations, strengthening the brand image, and gaining a competitive advantage in the increasingly competitive market.
THEORETICAL STUDY SERVICE QUALITY
Service quality is a key indicator used by customers to measure the performance and success of a company or organization in meeting their expectations and needs, shaping their satisfaction, loyalty, and overall perception.Tjiptono (1996:54) "Service quality is closely related to customer satisfaction, as it provides a strong foundation for building customer relationships and fostering customer loyalty.High-quality service creates a positive experience for customers, encouraging them to establish a strong and enduring connection with the company.".Fundamental principle in business development involves prioritizing the improvement of service quality as a key factor to be considered.According to (Annisa, 2020) if the received or perceived service aligns with customer expectations, the service quality is perceived as good and satisfactory.However, if the received service exceeds customer expectations, then the service quality is perceived as excellent and of high quality.Service quality is the ability to match the desires or demands of service recipients with the service provided by the service provider in the form of products or services, in accordance with predetermined criteria.(Kepegawaian et al., 2021).
STRATEGIC MANAGEMENT
Strategic management is an approach that involves the systematic planning, development, and implementation of long-term strategies with the aim of achieving the vision and goals of an organization or company through a structured process.by consumer customers in the future.(Ardiansyah et al., 2021).In the context of a company, strategy is a necessity to guide and manage the operations of the organization.The design of an effective strategy involves active participation from all stakeholders within the company's scope.A good strategy always begins with the easiest to implement.(Sri Anugerah Natalina & Arif Zunaidi, 2021).Through the implementation of strategic management, a company can have a clear direction in achieving its predetermined goals, as well as monitor and control the processes involved in achieving those goals.Menurut (Siregar et al., 2020) In the perspective of the future, where paradigm shifts are expected to occur in various fields, it is important for every organization to continuously evaluate their strategic management, both in terms of ongoing strategies and those to be planned.
SWOT ANALYSIS
SWOT analysis involves efforts to identify the strengths, weaknesses, opportunities, and threats that determine the performance of a company.(Nisak, 2004).In this analysis process, it is important to identify internal and external factors that have a significant impact on the success of a company or organization.The performance of a company or organization is determined by the interaction between internal and external factors.SWOT analysis (Strengths, Weaknesses, Opportunities, Threats) is a method used in the business world to evaluate internal and external factors that can impact the performance and strategies of a company.When applied accurately, this simple assumption has a significant impact on the design of successful strategies and business environment analysis, providing the necessary information to identify opportunities and threats within the company (Astuti & Ratnawati, 2020).Therefore, a strategic planner must analyze the strategic factors of the company (strengths, weaknesses, opportunities, and threats) in the current conditions (Jannah et al., 2017).Accord to (F Reza, et al., 2020) If SWOT analysis is conducted accurately by incorporating these four elements, there is a great opportunity for the success of a pre-planned company, ensuring a smooth and improved operation with optimal and maximum results.
According to Pearce and Robbinson (2011:156), the following are the definitions of Strengths, Weaknesses, Opportunities, and Threats:
b. Weakness
It refers to the limitations or deficiencies in one or more resources of a company compared to its competitors, which become barriers in effectively meeting customer needs.
c. Opportunities
It refers to the primary favorable conditions in the environment of a company.These conditions can provide opportunities for the company to grow, succeed, or gain a competitive advantage.
d. Threats
It refers to the primary unfavorable conditions in the environment of a company.These conditions can pose challenges, risks, or threats to the company's operations, growth, or competitive position.
RESEARCH METHODS
The research was conducted from August to December 2023 at PT. FOKUS KUALITAS UTAMA.The study utilized a qualitative descriptive research approach.
According to Sugiyono (2004), descriptive research is a type of research conducted to determine the values of independent variables, either one or more, without making comparisons or linking them to other variables.Qualitative research methods focus on gaining in-depth understanding of the meanings given by individuals or groups in the context they experience, with the aim of exploring richer and deeper insights into social phenomena.According to (Firmansyah et al., 2021), in qualitative research, the theory is not predetermined from the beginning.Instead, the theory can be sought and developed during fieldwork, allowing for a comparison between existing theories and the phenomena observed in the field.This process enables researchers to refine and complement existing theories based on the data collected, without compromising the authenticity and originality of the research.
The data collection in this study utilized primary data, which was obtained directly by the researcher from the primary source or the location where the research object is situated.The c. Determine ratings on a scale of 1 (Poor) to 4 (Outstanding) based on the impact of each factor on the company's performance.For example, a rating of 1 may be assigned to a low opportunity, while a rating of 4 may be assigned to a high opportunity.Conversely, for threat factors, a rating of 1 may be given for a low threat, while a rating of 4 may be given for a significant threat.
d.
Calculate the weighted score by multiplying the weight and rating values, using a range of 1.0 (poor) to 4.0 (outstanding).
Conceptual Framework
The next step involves conducting a SWOT analysis on PT FOKUS KUALITAS UTAMA.
Through the SWOT analysis, the researcher explores the internal and external factors of the company, including strengths, weaknesses, opportunities, and threats.The data processing in the SWOT analysis will result in a comprehensive overview of the current condition of PT FOKUS KUALITAS UTAMA, indicating its strengths, weaknesses, opportunities, and threats.
Based on this assessment, strategies can be formulated for PT FOKUS KUALITAS UTAMA.
Picture 1 represents the conceptual framework of this study.
RESULTS AND DISCUSSION
In this research, the SWOT analysis is utilized with the IFAS (Internal Factor Analysis Summary) and EFAS (External Factor Analysis Summary) matrix approaches as the chosen method.This analysis is then presented in the form of a SWOT diagram to evaluate the company's position in terms of strengths, weaknesses, opportunities, and threats.The results of the SWOT analysis are subsequently used as a foundation for designing appropriate strategies through the use of the SWOT matrix.
Matriks IFAS
The matrix is constructed by formulating strategic factors within the internal PT.FOKUS KUALITAS UTAMA, specifically in the Strengths and Weaknesses categories, based on the quality of its services.Source: Processed Data From Table 1, it can be observed that having a professional workforce is a strength in PT.
FOKUS KUALITAS UTAMA, as indicated by the highest score of 0.64.On the other hand, a weakness is the limitation in technology in service provision, with a score of 0.13.
External Strategy Factor Matrix
The matrix is constructed by formulating strategic factors within the internal PT.FOKUS KUALITAS UTAMA specifically in the Opportunities and Threats categories based on the quality of its services.This strategy is also effective in addressing weaknesses and mitigating potential threats.
It is important for the company to conduct regular evaluations to ensure its effectiveness and alignment with long-term goals..
4.
Based on the calculations and analysis conducted at FOKUS KUALITAS UTAMA, which indicate that the company is positioned in Quadrant 1, the following strategies can be considered: the resources controlled by or available to a company that make it relatively superior to its competitors in meeting the needs of its served customers.JIMEK-Vol.4,No.1 Maret 2024 primary source of data in this study was obtained through interviews conducted with the President Director, General Manager, and Staff at PT. FOKUS KUALITAS UTAMA.These interviews aimed to gather firsthand information from them regarding the research topic and gain deeper insights into the company.Secondary data refers to information collected from existing sources.These sources of secondary data include company records or documentation, industry analyses by media outlets, websites, the internet, and so on.Secondary data is gathered from sources that have already compiled and documented information related to the research p-ISSN:2809-9427; e-ISSN: 2809-9893-0143, Hal 208-219 212 Jurnal Ilmu Manajemen, Ekonomi dan Kewirausahaan (JIMEK)-Vol.3,No.2 Juli 2023 topic.Researchers can analyze and interpret this existing data to gain insights and support their research findings.This method involves analyzing the internal environment (strengths and weaknesses) and external environment (opportunities and threats) of the company being subjected to SWOT analysis.SWOT analysis is conducted through the use of the IFAS matrix (Internal Factor Analysis Strategy), which outlines the major strengths and weaknesses of the company, and the EFAS matrix (External Factor Analysis Strategy), which outlines the opportunities and threats faced by the company.Both matrices provide insights into the current position of the company by considering the internal and external factors that influence its performance.(Astuti & Ratnawati, 2020).According to Raflah (2021), the steps involved in creating the IFE (Internal Factor Evaluation) matrix and EFE (External Factor Evaluation) matrix are as follows: a. Identify internal and external factors using the IFAS and EFAS matrices.The Internal Factor Analysis Strategy (IFAS) is used to identify internal factors and classify them as strengths (S) and weaknesses (W), while the External Factor Analysis Strategy (EFAS) is used to identify external factors that include influential opportunities (O) and threats (T) to the company.b.Assign weights to the factors using a scale ranging from 0.0 (not important) to 1.0 (very important).
customers and assuring them that the company adheres to appropriate opertiomal standards b.Providing high-quality services to meet the need of evert client c.Enchacing trust in services among potential clients and providing evidence of the company's high quality in online meet client expectations and innovating in other industries to enchance satisfaction anda maintain customer loyalty, thereby remaining competitive with competitors b.Leveraging a strong portfolio to build a wider network deliver excellent service quality to creat a positive image.c.Instiling trust in clients by ensureing compliacne with legal regulations p-ISSN:2809-9427; e-ISSN: 2809-9893-0143, Hal 208-219 218 Jurnal Ilmu Manajemen, Ekonomi dan Kewirausahaan (JIMEK)-Vol.3,No.among all employees to improve service delivery and minimize threats from tecnological advancements b.Deepening knowledge about online promotion and introducing company services c. nSeeking investors to maintain financial stability and continue providing excellent services while minimizing the impact of unforeseen economic conditions Table 1 represents the IFAS (Internal Factor Analysis Summary) matrix..
Table 2 .
Table 2 represents the EFAS (External Factor Analysis Summary) matrix.EFAS Matrix analysis results | 2024-01-08T16:45:41.998Z | 2024-01-04T00:00:00.000 | {
"year": 2024,
"sha1": "b86b87fd3c33e6be187c9153671f6226f5e9bcf0",
"oa_license": "CCBYSA",
"oa_url": "https://journal.amikveteran.ac.id/index.php/jimek/article/download/2690/2111",
"oa_status": "HYBRID",
"pdf_src": "Anansi",
"pdf_hash": "1f389e4daf09a641c21ea4478556559c4bc65995",
"s2fieldsofstudy": [
"Business"
],
"extfieldsofstudy": []
} |
197538098 | pes2o/s2orc | v3-fos-license | Impact of the Time to Initiation of Parenteral Nutrition on Patient Outcomes in Critically Ill Adults
Background: The optimal time to initiate parenteral nutrition (PN) in critically ill adults in whom enteral nutrition is not feasible is controversial. Objective: The objectives were to compare in-hospital mortality and hospital length of stay in patients initiated on PN within 7 days or after 7 days of poor nutrient intake. Methods: This single-center, retrospective study included critically ill adult patients who received at least 2 consecutive days of PN during hospitalization from May 2014 to July 2016. Results: The median duration of PN (interquartile range) was 8 (5-13) days. In total, 110 patients received PN within 7 days of poor nutrient intake while 49 patients received PN after 7 days of poor nutrient intake. There was no statistically significant difference in in-hospital mortality between groups (29.09% vs 18.37%, P = .1535). Patients initiated within 7 days had a significantly shorter median hospital length of stay than patients initiated after 7 days (20 days vs 27 days, P = .0013). There were 69 patients who were classified as obese. Obese patients initiated within 7 days had a significantly shorter median hospital length of stay than obese patients initiated after 7 days (17 days vs 33 days, P = .0007). Conclusions: Time to initiation of PN did not impact in-hospital mortality. However, there was an association between early initiation of PN and a shorter hospital length of stay that was most pronounced among obese patients.
Introduction
Nutritional deficits are a complication of critical illness. Enteral nutrition (EN) is associated with fewer adverse effects but is not feasible to correct nutritional deficits in all cases. 1 Consequences of not meeting nutritional targets include weakness, infection, an increased duration of mechanical ventilation and death. 2 Furthermore, the optimal time to initiate parenteral nutrition (PN) in critically ill adults in whom EN is not feasible is controversial. Guidelines for the Provision and Assessment of Nutrition Support Therapy in the Adult Critically Ill Patient published by the American Society for Parenteral and Enteral Nutrition (ASPEN) and the Society of Critical Care Medicine (SCCM) recommend withholding PN over the first 7 days if the critically ill patient is at low nutrition risk (eg, NRS 2002 [Nutrition Risk Screening] ⩽ 3 or NUTRIC [NUTrition Risk in Critically Ill] score ⩽ 5), but initiating as soon as possible if at a high nutrition risk (eg, NRS 2002 ⩾ 5 or NUTRIC score ⩾ 5) or severely malnourished and early EN is not feasible. 3 Conversely, the European Society for Clinical Nutrition and Metabolism guidelines recommend that clinicians initiate PN in all patients within 24 to 48 h after the patient is admitted to the intensive care unit (ICU) if EN is contraindicated and the patient is not expected to receive normal nutrition within 3 days. 1 The When Is PN Appropriate? Consensus Recommendations suggest initiating PN after 7 days for well-nourished stable patients, within 3 to 5 days in those who are nutritionally atrisk, and as soon as feasible in those with baseline moderate or severe malnutrition if oral intake or EN is not possible or sufficient. 4 Investigation is warranted in light of the inconsistency among guideline recommendations and other literature. The purpose of this study was to investigate in-hospital mortality and hospital length of stay based on initiation of PN within 7 days or after 7 days of poor nutrient intake in critically ill adult patients.
Methods
This study was conducted at Cooper University Hospital, a 600-bed urban academic medical center. Adult patients admitted to this institution and initiated on PN for at least 2 consecutive days from May 2014 to July 2016 were retrospectively evaluated for study inclusion. Patients were excluded if they 2 Nutrition and Metabolic Insights were <18 years of age, pregnant, received concomitant EN, were initiated on PN prior to admission or were not admitted to an ICU.
At Cooper University Hospital, a dietitian consult service in conjunction with the multidisciplinary support of physicians, pharmacists, and nurses manages PN without a formal nutrition support team. For individualized PN orders, dietitians advise appropriate macronutrient provision using a published predictive equation or a simplistic weight-based estimate before PN is initiated. 3 Indirect calorimetry is unavailable at this institution. Generally, on day 1 of PN, 50% of goal intake is provided and if tolerated patients are advanced to goal intake on day 2 of PN. At this institution, lipid injectable emulsion (ILE) is provided from the time of PN initiation unless contraindicated. During the study period, exclusively soy-oil based ILE was used at this institution. Suggested default electrolytes in the PN order set and pharmacist recommendations support physicians in ordering appropriate micronutrients. The institution's guideline suggests weaning PN when enteral intake achieves 50% to 75% of requirements for energy, protein, and micronutrients.
The objectives of this study were to compare in-hospital mortality and hospital length of stay in critically ill adult patients initiated on PN within 7 days of poor nutrient intake and after 7 days of poor nutrient intake. Seven days was used for categorization in this study since guideline authors incorporated that timeframe into current recommendations. 3 Poor nutrient intake was defined as less than 50% of daily nutritional requirement. A pre-determined subgroup analysis stratified patients based on nutritional classification at the time of PN initiation. This study described nutritional status at presentation based on percentage of ideal body weight (IBW). Actual body weight (ABW) represented the admission weight or pre-hospitalization usual weight if documented from the dietitian's interview with the patient or their caregiver. Patients were identified for the underweight subgroup if their ABW was less than 89% of their IBW. Patients were identified for the normal weight subgroup if their ABW was within the range of 90% to 129% of their IBW. Patients in the obese subgroup had an ABW greater than 130% of their IBW. This subgroup analysis compared in-hospital mortality and hospital length of stay in critically ill patients initiated on PN within 7 days of poor nutrient intake and after 7 days of poor nutrient intake.
Baseline patient demographics and PN characteristics were collected retrospectively using the institution's Electronic Medical Record (EMR). Baseline demographics were determined at the time of PN initiation. Investigators of this study used baseline demographics documented in the EMR to calculate morbidity and mortality scores, which include the Charlson Comorbidity Index (CCI), Acute Physiology and Chronic Health Evaluation II (APACHE II) score, and the Sequential Organ Failure Assessment (SOFA) score. [5][6][7] Investigators also retrospectively calculated a modified NUTRIC (mNUTRIC) score, excluding interleukin-6. 8 The number of co-morbidities for the mNUTRIC score was determined from the CCI.
All statistical analyses were conducted using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). Quantitative variables were compared using Student t-test if normally distributed or Wilcoxon Rank-Sum, otherwise. In addition, hospital length of stay was graphically represented using Kaplan-Meier curves and analyzed using forward selection Cox Regression model to control for any significant confounder. Categorical variables were analyzed using χ 2 test or Fisher's exact test. A multivariate logistic regression with forward selection was completed to determine the effect of the days of poor nutrient intake on in-hospital mortality, controlling for any significant confounders identified through the bivariate analyses. Statistical significance was defined as a P < .05.
Results
There were 546 patients who were screened for inclusion. Of these patients, 375 were excluded as they were not critically ill at the time of PN initiation and 12 patients were excluded given the unclear duration of poor nutrient intake. A total of 159 patients were included in this analysis. In the total study population, the mean age was 61.6 years and the mean body mass index (BMI) was 29 kg/m 2 . The median duration (interquartile range [IQR]) of PN was 8 (5-13) days and 98% received central PN. The median number of days of poor nutrition intake prior to PN initiation in those who received PN within 7 days and those who received PN after 7 days was 4.00 (3.00-6.00) and 10.00 (9.00-14.00), respectively. The mNU-TRIC, SOFA score, and APACHE II score were similar between groups (Table 1).
Results comparing in-hospital mortality and hospital length of stay for patients who were initiated on PN within 7 days of poor nutrient intake (n = 110) vs patients who were initiated on PN after 7 days of poor nutrient intake (n = 49) are reported in Table 2. While there was no statistically significant difference in in-hospital mortality rates based on PN initiation within 7 days or after 7 days (29.09% vs 18.37%, P = .1535), patients who were initiated on PN within 7 days had a significantly shorter median hospital length of stay compared with those initiated on PN after 7 days (20 days vs 27 days, P = .0013). Patients were more likely to have a shorter duration of hospitalization if they were initiated on PN within 7 days of poor nutrient intake (hazard ratio [HR] = 1.65, 95% CI [1.17-2.33], P-value = .0042; controlling for age, location at initiation, and total APACHE II score) ( Figure 1).
A subgroup analysis consisting of underweight patients (n = 12), normal weight patients (n = 78), and obese patients (n = 69) compared in-hospital mortality and hospital length of stay in patients initiated on PN within 7 days of poor nutrient intake and after 7 days of poor nutrient intake (Table 3). Underweight, normal weight, and obese patients initiated on 3 A multivariate logistic regression with forward selection found no statistically significant effect of number of days of poor nutrient intake on in-hospital mortality after controlling for the following statistically significant confounders: baseline liver disease, CCI, and the SOFA score (Table 4). Although there was a non-statistically significant trend toward improved mortality in patients initiated on PN after 7 days, there was a shorter length of stay among patients discharged live from the hospital who were initiated on PN within 7 days (Table 5).
Discussion
To date, there were no studies identified comparing outcomes of critically ill patients initiated on PN within 7 days and after 7 days of poor nutrient intake. This study found no statistically significant difference on in-hospital mortality rates between critically ill patients who were initiated on PN within 7 days and after 7 days of poor nutrient intake, but patients initiated on PN within 7 days had a significantly shorter median hospital length of stay. A key methodologic strength is that results of this study are not subject to immortal time bias. This is evident since investigators of this study accounted for how long patients were without adequate nutritional provision before starting PN both during and prior to hospital admission. Given the median mNUTRIC score for both study groups reflecting high nutrition risk, these findings in the overall patient population in this study support the less restricted use of PN that emerged in recent guidelines and consensus recommendations for patients at high nutrition risk. 3,4 Pre viously, 2009 guidelines recommended PN not be given to any patients unable to receive EN within the first 7 days regardless of nutrition status and disease severity. 9 Historically, data were limited for PN use in high nutrition risk or malnourished patients, and suboptimal PN management practices may have contributed to unfavorable outcomes in early studies. 4 Patient outcomes in studies comparing early and late initiation of PN in critically ill patients have been variable, but generally favor delaying PN initiation in patients who are not at high nutrition risk or malnourished. Of note, these studies have often used predictive equations to determine patients' nutritional requirements. While the use of predictive equations is common in clinical practice and recommended by the guidelines in the absence of indirect calorimetry, studies have demonstrated that nutritional goals derived from this approach are often incorrect. 10,11 An early, randomized trial assigned patients to receive either PN or prolonged glucose administration (250-300 g/day) for up to 15 days after surgery. 12 Providing no nutrition after 14 days of hospitalization resulted in higher mortality and longer hospital length of stay; however, withholding PN in the initial postoperative period did not negatively impact outcomes for most patients. 12 An unblinded, multicenter, randomized study compared the outcomes of critically ill patients initiated on PN within 48 h of ICU admission and on day 8 of ICU admission. 13 In the subset of patients who had an absolute contraindication to EN and exclusively received PN, patients initiated early had a higher rate of infections and were less likely to be discharged alive than patients initiated on PN after 8 days of ICU admission. However, the median ICU length of stay for patients in that study was only 3 to 4 days and many were admitted for elective procedures indicating the severity of illness was questionable. 13 Conversely, other studies have reported benefits of early initiation of PN in critically ill patients. A multicenter, randomized, single-blind study evaluated early PN in critically ill 15 Heidegger and colleagues conducted a randomized controlled trial at two centers in Switzerland to assess whether delivery of 100% of the energy target from days 4 to 8 in the ICU with supplemental PN could optimize clinical outcomes. 16 Supplemental PN 4 days after ICU admission reduced nosocomial infections. 16 In a follow-up study, investigators determined that providing supplemental PN from days 4 to 8 in critically ill patients is associated with improved immunity and less systemic inflammation. 17 In alignment with these findings from Switzerland, it has been reported that surgical ICU patients with appropriate energy and protein provision are more likely to be discharged home. 18 Since findings in heterogeneous critically ill patients have been variable and current guideline and consensus recommendations provide recommendations based on nutritional risk, 3,4 the investigators conducted a pre-determined subgroup analysis stratifying patients based on nutritional classification at the time of PN initiation. In this study, the time to initiation of PN did not impact in-hospital mortality within each nutritional classification. This finding may be due to high mNUTRIC scores in underweight, normal weight, and obese groups. However, this study suggests that critically ill obese patients initiated on PN within 7 days of poor nutrient intake will have a shorter duration of hospitalization compared to obese patients initiated on PN after 7 days of poor nutrient intake. Since mNUTRIC scores were high in underweight, normal weight, and obese groups, these results may indicate that obesity adds an additional element of nutritional risk that should be considered and studied further for potential earlier initiation of PN. In fact, protein turnover and catabolism rate are higher for patients with obesity suggesting that lack of nutrition therapy may impact outcomes. 19 Current consensus recommendations suggest initiating PN within 3 to 5 days in adult patients who are at nutritionally-at-risk based on factors such as weight loss, BMI < 18.5 kg/m 2 , and altered or inadequate intake for more than 7 days, but obesity is not incorporated into this definition. 4 Data regarding the impact of obesity on morbidity and mortality are conflicting. [19][20][21][22][23] Meta-analyses have associated obesity during critical illness with an increased ICU length of stay without an increase in mortality. 20,21 As a consequence of our results and other literature, obesity should be studied further as a potential indicator of patients who are nutritionally-at-risk warranting earlier PN initiation.
As another area for future study, the provision of energy and protein within 7 days of poor nutrient intake and after 7 days of poor nutrient intake should be considered. In this study, patients were advanced to goal in a median of 2 days and energy and protein provision at goal were similar for groups initiated on PN within or after 7 days of poor nutrient intake for underweight, normal weight, and obese patients. Of note, caloric provision was not as conservative as cited in the literature, especially for obese patients. Based on a low quality of evidence, guidelines suggest that feeding with ⩽20 kcal/kg/day or 80% of estimated energy needs, but adequate protein (⩾1.2 g/kg/day) may be appropriate in high risk or severely malnourished patients requiring PN in the first week of hospitalization to reduce infectious complications, duration of mechanical ventilation, and hospital length of stay. 3,24 In addition, a significant percentage of this study's patient population was obese, but the hypocaloric feeding approach that is recommended for critically ill obese patients to improve nitrogen balance and shorten length of stay in the ICU had not been consistently implemented at the institution in the time period of retrospective review. 3,25 Beyond patient outcomes, shortening the median hospital length of stay by 7 days for critically ill patients initiated on PN within 7 days has remarkable cost savings implications. This effect was more pronounced for critically ill, obese patients in which the median hospital length of stay was 16 days shorter for those initiated on PN within 7 days. Hospital length of stay, the development of subsequent infectious complications, and costs are often interrelated. Pradelli and colleagues demonstrated that optimizing energy provision with supplemental PN on days 4 to 8 if EN is insufficient decreases the cumulative energy deficit, reduces the risk of nosocomial infection by 10%, and results in lower costs. 26 Doig and colleagues conducted an economic analysis of cost implications of early PN to critically ill patients with short-term relative contraindications to EN. 27 Early PN reduced the need for mechanical ventilation and decreased the ICU length of stay resulting in a significant reduction in hospital costs per patient. 27 Several limitations were identified in this study. First, as a retrospective, observational single-center study, the results of this study may not be generalizable to other institutions or patient populations. The investigators relied on available documentation to determine the number of days of poor nutrient intake prior to PN initiation. Also, the nutritional classification in this study was based on ABW relative to IBW as opposed to the recommended NUTRIC or NRS scores. The institution at which this study was conducted had not implemented a scoring system for nutritional risk. Investigators found ABW relative to IBW to be appropriate for the subgroup analysis as it can be easily applied in clinical practice. The NRS score classifies nutritional risk based on several factors including the amount of time to develop a >5% weight loss, a decrease in nutrient intake, BMI, age, and co-morbidities. While the NRS score has demonstrated a relation between positive outcomes and nutrition support in patients with a score greater than or equal to 3, it is impossible to accurately calculate retrospectively. However, investigators found it feasible to capture a baseline mNUTRIC score that does not include interleukin-6 and uses the CCI as a substitute for the standard co-morbidities included in the NUTRIC score. 15 Finally, an a priori power analysis calculation was not conducted and the small sample size may have resulted in a type II error for evaluation of in-hospital mortality.
Furthermore, the small sample size of underweight patients may have limited our ability to find a statistically significant difference in patient outcomes for this high nutrition risk patient population; however, previous meta-analyses have suggested benefits of PN in malnourished critically ill patients. 28,29 Conclusions Time to initiation of PN did not have a significant impact on in-hospital mortality in this study, but warrants further investigation. However, patients who received PN within 7 days of poor nutrient intake had a shorter hospital length of stay compared with patients who received PN after 7 days of poor nutrient intake. A subgroup analysis found that obese patients who received PN within 7 days of poor nutrient intake had a shorter hospital length of stay compared with obese patients who received PN after 7 days of poor nutrient intake. Future studies should confirm these results that suggest critically ill obese patients may be a nutritionally at-risk population warranting earlier PN initiation and investigate optimal provision of energy and protein based on days of poor nutrient intake and time in the ICU. | 2019-07-20T13:04:17.352Z | 2019-01-01T00:00:00.000 | {
"year": 2019,
"sha1": "71cc818e2cda8cd0f9bb8b39622bbf96f403204f",
"oa_license": "CCBYNC",
"oa_url": "https://journals.sagepub.com/doi/pdf/10.1177/1178638819859315",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "71cc818e2cda8cd0f9bb8b39622bbf96f403204f",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
247603884 | pes2o/s2orc | v3-fos-license | Influence of digital services of infographics on effectiveness of mnemonics when teaching foreign language vocabulary
The problem and the aim. Modern linguodidactics pays special attention to identifying the role of vocabulary in learning a foreign language, determining strategies for learning foreign language vocabulary, finding methods for testing vocabulary, mastering the vocabulary of scientific discourse, using mnemonics in memorizing lexical norms. New training formats necessitate the use of information visualization technologies and digital means in the study of a foreign language. The purpose of the study is to identify and confirm effectiveness of using digital services of infographics in mnemonics to improve the quality of teaching foreign language vocabulary. Research methods. Digital tools for data visualization are used in the system of mnemonic operations (grouping, classification, structuring, systematization, analogy, association, repetition) and methods (rhyme, association, mnemonics, chain method, memory cards, mnemonic cards). Infographic services support semantisation of lexical material and its activation, explanation of rules, situational illustration, and foreign language communication. A special test is developed, it includes questions on English vocabulary and assignments based on the methodology of A.R. Luria. Auxiliary visualization methods are used: data design in the form of mind maps, tables, charts, graphs. The experiment involved 30 students of the training program Pedagogical education (Bachelor's programme) of Vyatka State University. WordArt is used as a digital service of infographics. Statistical processing of the results is performed using the nonparametric method - Wilcoxon's T-test. Results. Students learn digital infographic services, use them to study new lexical material, memorize and present concepts/terms, set expressions, and combine familiar lexical elements in a variety of contexts. Statistical assessment of the reliability of positive dynamics of students' skills to recognize words, understand lexical units, to construct new phrases was carried out T emp <T crit0.05 (45<107). In conclusion rules and conditions, implementation of which ensures effectiveness of using digital services of infographics in mnemonics to improve quality of teaching a foreign language, are summarized.
Introduction
O ne of the initiatives of UNESCO, regulated by the 2003 Convention, is the program dedicated to the preservation and development of the language [1]. The two main components of any language are grammar and vocabulary. Special attention is paid to studying vocabulary in modern international linguodidactics. In the comprehensive analysis of scientific and methodological works on teaching vocabulary F. Boers generalizes that researchers identify the following topical areas: role of vocabulary when studying a foreign language, strategies for learning vocabulary of a foreign language, methods of testing vocabulary of foreign language learners, mastering the vocabulary of scientific discourse, using mnemonics when studying vocabulary, using innovative technologies in teaching [2]. For example, R. Karimian Shirejini, A. H. Derakhshan note that there is a contradiction between the number of words of the target language that a student can master in a limited time of study and those communicative tasks that he/she must solve [3]. In addition, most students are faced with the problem of memorizing and reproducing foreign words. A way out of this situation can be the use of innovative pedagogical technologies and various methods of memorizing, storing and reproducing information. One of the methods of enhancing the cognitive activity of students in the opinion of I. J. Chen is mnemonics [4].
Thus, on the one hand, when studying foreign languages various intensive methods are used that activate students' thinking processes. Many language schools and online courses offer their own mnemonic techniques: the use of musical devices, storytelling, etc.
At the same time, the COVID-19 pandemic has had a significant impact on the traditional teaching methodology of academic disciplines in university and school subjects. Since there is a transition of learning to the online space, the degree of assimilation and memorization of the material changes. For example, due to the lack of experience in learning in the network environment using computer platforms and programs. L. Tarkhova et al. come to the conclusion that the efficiency of assimilation of theoretical facts and concepts can be increased by introducing new technologies into linguodidactics that can be used not only in the online space, but also in the classroom [5].
Infographic resources can be used when implementing such innovative technologies. Infographics are a visual presentation of a variety of textual and statistical information. According to A. V. Makulin, integration of visual aids into the process of teaching languages is a promising direction [6]. Such tools not only meet modern requirements of language teaching, but are also multifaceted in relation to types of tasks where they can be used (project activities, organization of research activities).
In conditions of development of the digital educational space in the arsenal of modern teachers interactive services appear, they support collecting information, comprehension, highlighting the important and the secondary, processing and transferring it from one type to another. Such educational and cognitive activities also fully comply with the requirements of the current federal educational standards.
At the moment there are some studies on the inclusion of digital services of infographics in teaching students of pedagogical training programs (for example, when preparing future physics teachers [7]). However, teaching foreign language vocabulary has its own fundamental methodological principles, techniques and methods of memorizing new words, techniques for formation of lexical skills, a system of exercises [8]. Thus, there is an objective need for additional study of the issues of including digital services of infographics in teaching foreign language vocabulary as an innovative pedagogical technology of higher education in order to: 1. include new methods and means of storing information in formation of foreign language competence in general and lexical competence in particular; 2. use digital services of infographics in educational, cognitive, professional, communicative activities of future highly qualified specialists. The research hypothesis -the use of digital tools for visual presentation of information, memorization and presentation of concepts/terms when teaching a foreign language will provide additional conditions in situations of: • introduction, semantization of a new word and its primary repeating; • training and creating strong speech connections; • creating dynamic lexical speech connections, that is learning how to combine familiar lexical elements in a variety of contexts. The purpose of the study is to identify and confirm effectiveness of using digital services of infographics in mnemonics to improve the quality of teaching foreign language vocabulary.
Materials and methods
The following methods were used in the work: theoretical analysis and generalization of literature when describing conditions for effective teaching of foreign language vocabulary, techniques of mnemonics in linguodidactics, and the didactic potential of digital means of visual presentation of information.
The main element of infographics as a technology of cognitive visualization is the shift in emphasis from the illustrative function in teaching to development of cognitive abilities, critical thinking of students.
Infographics are used as a means for the semantisation of lexical material and its activation, explanation of rules, situational illustration, and foreign language communication. The study used static (graphic images, mind maps) and dynamic (graphic images with added animation and possibility to transform) infographics.
Creating infographics by students is carried out taking into account principles of the system-activity approach to learning: understanding of relationships, principles and algorithms of word formation happens when compiling a set of words; attention and memory are activated.
When organizing practical work with digital infographic services, WordArt was used (https://wordart.com/). This resource is for creating tag (word) clouds. It is helpful for users with no prior knowledge of graphic design. WordArt includes tools for visualizing concepts you need to focus on.
To obtain up-to-date information on effectiveness of using digital services of infographics in mnemonics when teaching foreign language vocabulary empirical methods are used: monitoring communication of all participants of interaction; analysis of answers, results of working with the digital infographic service (the number of words in the cloud and the connections between them); the number of attempts to build a word cloud; time to study theoretical material; the volume and correspondence of functional capabilities used for cognitive visualization, etc.
To assess the input conditions testing was used, which includes 20 tasks on the topics of the discipline "Foreign language" and a task based on the methodology of A. R. Luria. The time to do the tasks is 100 minutes, since the chosen technique includes delayed (in 50-60 minutes) repeating. Also, auxiliary methods of computer data processing were used: data design in the form of mind maps, tables, diagrams, graphs.
The experimental study was carried out on the basis of Vyatka State University while studying the courses "Digital Technologies in Education" and "Foreign Language". 30 first year students of the training program Pedagogical education (Bachelor's programme) were involved. The software is the WordArt (https://wordart.com/). The average age of the respondents was 19 (78% of girls and 12% of young men). Classes in both disciplines were conducted by the same teacher. The choice of only one group was justified by the following circumstance: the work program for the chosen training program was changed. As a consequence, it was not possible to maintain the same conditions for data comparison. Since there was a comparison of two dependent samples, statistical processing of the results was performed using a nonparametric method -Wilcoxon's T-test.
Literature review
Analyzing various approaches that foreign specialists in linguodidactics use in the practice of teaching English, F. Boers notes that the modern methodology considers vocabulary as one of the integral components of formation and development of speech skills [2]. Vocabulary in the works of D. Özmat and N. Senemoğlu is understood as a set of words that are part of the language [9]. R. Karimian Shirejini and A. Derakhshan define that knowledge of foreign languages has recently become an integral part of the professional competence of many specialists [3]. D. J. Nishonova et al. understand lexical competence as knowledge of vocabulary of a language, which includes elements of vocabulary and grammar and the ability to use them in speech [10]. Thus, according to the provisions of the work of T. Miyatsu and M. A. McDaniel, mastering vocabulary allows: to create necessary conditions for communication in a foreign language; correctly, clearly formulate thoughts; develop cognitive abilities; develop a linguistic personality as a culture-bearer [11]. The authors conclude that when studying a foreign language, all linguistic material can be divided into 3 large groups: words, grammar and fixed expressions.
O. M. Osiyanova and V. D. Demina conclude that the process of memorizing new words is the most laborious. At the word level the pronunciation of English words is mastere; the ability to perceive words by ear, the ability to read, and then the ability to write in English are practiced [12]. G. A. Shor, N. E. Korn believe that the introduction of new training components is not only a clarification of the content of training, but also the search and development of adequate teaching aids, which are a prerequisite for effectiveness of the process of teaching a foreign language [13]. Although didactic means do not have a decisive influence on results of educational work, they nevertheless facilitate the acquisition of knowledge. They just have to be correctly selected; skillfully incorporated into the methods and organizational forms of teaching used by the teacher. This can enrich the use of teaching methods and increase their effectiveness.
In international practice various intensive methods are used when studying foreign languages. For example, J. Chen describes learning outcomes, where memorization of new words occurs through songs and music videos without subtitles [4]. A comprehensive study by F. Ciaramella, E. A. Lorè, A. Rega is also devoted to the search for new methods and techniques for memorizing foreign language vocabulary [14]. When analyzing the literature, the authors conclude that many language schools and online courses offer their own techniques for using mnemonics. In schools and universities teachers rely on the accumulated theoretical material and practical teaching experience and develop and use their own methods.
Based on experimental data T. Miyatsu, M. A. McDaniel argue that techniques of mnemonics are quite effective, they should be used for memorizing foreign language vocabulary [11]. Mnemonics, in their opinion, is a system of various techniques and methods that make it easier to memorize a large amount of information, most often it happens by creating associations and images. Domestic specialists in didactics, whose authority is recognized in the world community, A. V. Makulin, M. I. Korzat conclude: the traditional process of memorizing five unknown words by a modern schoolchild is associated with torment, with graphics it becomes fascinating [6].
So, according to E. E. Naumenko et al. mnemonics is a promising direction in teaching, which allows not only to learn foreign languages, but also to activate reserves of memory and thinking [15]. Its essence lies in the fact that it is usually difficult to memorize disparate, unrelated facts and phenomena. When there is a connection between new information and existing information, memorization, which is proved in the work of O. Yu. Muller, is much easier and faster. In many respects it happens thanks to the logical associative connections between objects and images [16].
The result of training in a modern university should be formation of the bilingual linguistic personality capable of using own language and speech competences in order to be competitive in the labor market [17]. F. M. Hamid et al. prove that the active inclusion of working with various types of infographics in the curriculum will make development of communicative norms of a language more effective [18]. They study various effects of such classroom work. Infographic tools allow students to focus on the correct use of endings in various grammatical forms. In addition, the effect of visualization should be considered. From the cognitive point of view in the basis of work with infographics cognitive mechanisms of perception and processing of information, activation of associative (subject-logical) connections are involved; the connections arise during the work of images that require verbalization [19].
In addition, "at the level of verbal-logical reflection-display of information (written speech) the visual series also plays an important role in development and support of the person's speech and thinking abilities". According to D. Roy, if habitual visualization is a representation of a phenomenon or process in a form convenient for visual perception, then cognitive visualization is a more multifaceted concept [20]. Cognitive visualization also refers to a certain illustration of the studied subject, but at the same time it involves its subsequent rethinking.
Infographics is an alternative way of presenting information, combining illustrations and verbal and logical content of the text. The use of educational technologies based on infographics increases effectiveness of language teaching, which is expressed in a qualitative increase in the level of foreign communicative competence [7].
S. Simakova believes that infographics is a cognitive visualization tool; it is characterized as a graphical way of presenting certain information. The main goal is to present complex information clearly and quickly [21]. A. V. Makulin, M. I. Basket note that infographics as a methodological device allows to combine visual elements and the logical content of text fragments that explain them [6]. Infographics can be used in general to practice communication skills in the digital space; for critical analysis of certain information; for development of visual thinking [22]. All of the listed characteristic features of infographics correspond to modern tasks of teaching a foreign language [23].
D. Roy notes possibilities of multimedia technologies for implementation of infographics in teaching a foreign language. Typology of infographics can be different: from the position of the presentation object (statistical infographics, timeline, map, diagram, hierarchy, matrix, algorithm, photo, comparison); from the standpoint of technology static, dynamic, videoinfographics are distinguished [20].
On the one hand, mnemonics as a set of techniques that increase the amount of memory and make it easier to memorize information is an effective technology in teaching foreign language vocabulary. On the other hand, infographics includes a wide range of functionalities for creating associations and images. In addition, there is an active development of computer technologies for presentation of infographics in the digital space.
Thus, there is an objective need for additional study of the issues of including computer services of infographics in teaching foreign language vocabulary as an innovative pedagogical technology of the digital school.
Research program
The main goal of the experiment was to assess the impact of digital infographic services on effectiveness of mnemonics in teaching foreign language vocabulary. The experimental study was carried out on the basis of Vyatka State University while studying the courses "Digital Technologies in Education" and "Foreign Language". 30 first year students of the training program Pedagogical education (Bachelor's programme) were involved. The software is the WordArt (https://wordart.com/). The average age of the respondents was 19 (78% of girls and 12% of young men). Classes in both disciplines were conducted by the same teacher.
At the preparatory stage of the experiment the teacher considered various techniques of mnemonics, which are actively used in teaching foreign language vocabulary. The following mnemonic operations were analyzed and selected: grouping, classification, structuring, systematization, schematization, analogy, recoding, association, repetition. It was decided to use the following methods to support cognitive visualization: rhyme, association, chain method, memory cards, mind maps. For example, the chain method and analogy were used when memorizing national holidays, cities. Flash cards were used in the study of modern professions.
Various digital services to support infographics were analyzed: http://www.visual.ly, www.dailyinfographic.com, www.coolinfographics.com, https://wordart.com/, https:// www.canva.com/ru_ru/, https://www.gloster.com/ and others. The following criteria were used as selection criteria: type of technology (cloud/online or offline), financial basis (free/ commercial), functionality (adding illustrations/graphic images, work with animation, audio import (music/sound), interface and design. Based on the analytical work the WordArt service (https://wordart.com/) was chosen. Its advantages over the others are: it is easy to use, it allows load word lists, fonts and surfaces, it has various forms of export and it is free. There are only two exceptions: 1) if you want to use word clouds obtained using the service for commercial purposes; 2) free download of clouds in high quality is not available (these are images that the average user will never need).
Testing was developed to assess the input conditions; it includes 20 tasks on the topics of the discipline "Foreign language" and a task based on the methodology of A. R. Luria. The time to do the tasks is 100 minutes, since the chosen technique includes delayed (in 50-60 minutes) repeating. The materials of the fund of assessment tools are compiled according to the working program, take into account the theoretical and practical nature of activities when teaching foreign language vocabulary. Examples of tasks: 1 (to test the ability to critically evaluate information, the ability to find errors in the use of word formation methods). Students are invited to listen/read word forms and express their opinion (Can you say so? How to say it correctly?). For example, butter is in the "butter course" (butter dish); salt is in the "salt keeper" (salt shaker).
2 "Palindromes". Students write down words (on a specific topic or from the available vocabulary) backwards. Modification of the task: Students decipher the word. If this is a noun, then the article is also called.
Modification of the task: by analogy with the game "Cities". Continue the list: Makarenko, Owen, Newton, etc.
(knowledge). "
In what year did the UN start holding International Women's Day?" or "What day do unmarried girls make wild guesses?" For each completed task the student received 1 point. It was necessary to choose the appropriate method for diagnosis since the use of mnemonics involves activation of memory and attention. The following methods were analyzed: "Memorizing 10 words" (A. R. Luria), "Mediated memorization" (A. N. Leontyev), "Pictogram" (L. S. Vygotsky, A. R. Luria, S. V. Loginova), "Reproduction of stories" and express methods ("Cross out the named pictures", "Numbers and letters" and others). The methodology of A. R. Luria "Memorizing 10 words" was selected and adapted for the study, since it is used in a complex manner. It was used to assess the state of memory, fatigue and attention activity. The teacher prepared a protocol with nine short monosyllabic and twosyllable words that had no connection with each other. They were nouns in the singular nominative case, not related to each other. For example, at the preparatory stage the study used the option "Number, chorus, stone, mushroom, cinema, umbrella, sea, bumblebee, lamp, lynx". The words were repeated 5 times. Normally, after the first presentation the student must reproduce from three to five words, after the fifth -from eight to ten. After delayed repeating it is seven to nine words.
Based on the results of the work the student scores points according to the algorithm: • 4 points (remembered 9-10 words after the fifth presentation, 8-9 words after delayed repeating); • 3 points (remembered 6-8 words after the fifth presentation, 5-7 words after delayed repeating); • 2 points (remembered 3-5 words after the fifth presentation, 3-4 words after delayed repeating); • 1 point (remembered 0-2 words after the fifth presentation, 0-2 after delayed repeating). Thus, students could receive from 1 to 24 points for the control testing. Thus, it was possible to collect data on 30 students.
The second stage of the experiment was devoted to changing the structure of the sections for studying vocabulary of a foreign language in accordance with the purpose of the study. Firstly the teacher in the classroom during "Digital Technologies in Education" class studied the digital service to support infographics. Then, in foreign language classes the topics "Traveling", "Food", "Education" were mastered.
The third stage of the study. Further, when organizing practical work, the research and creative activity students were offered the studied concepts, new words and stable expressions to organize in the form of a cloud of words. The student could use WordArt as a computer program. But the student was free to choose and use a different digital service.
Research results
In the process of analysis and generalization of scientific literature the authors' positions on the key concepts of the study were determined: • mnemonics is a system of methods and techniques that ensure effective memorization, storing and reproduction of information, and support development of speech; • infographics is one of the ways to form students' written competence. Infographics is a fusion of the use of information and communication technologies and information visualization. This is not just any graph or diagram built on the basis of a certain amount of data, but a metaphorical formation of visual information; • main functions of infographics: informative, analytical, constructive, adaptive, expressive, aesthetic; • connection between mnemonics and infographics is that the latter supports a combined effect on the organs of sight and hearing. This happens with the help of audiovisual, multimedia tools that affect long-term memory and ensure the processing and assimilation of information; • digital infographic services are applications that due to built-in tools and templates allow to automate processing and structuring of statistical information (presented in the form of a graph, table, animation). One group is involved in the experimental study, therefore, before studying digital infographic services, including them in mnemonics when teaching foreign language vocabulary, an entry test was carried out. Students were asked to answer 20 questions and complete tasks according to the method of A. R. Luria. The measurement results are presented in Table 1 1.
2. To learn to use the WordArt service when solving educational and cognitive tasks. The actual result is a report on the work done, a text document WordArt_ <Group_ Surname>.docx ". Tasks marked with * required any real answers (screenshot, explanation, solution) to be included in the report. Tasks not marked with * are compulsory to do, but they did not require additional data to be entered into the report. The research activities of students were carried out according to the specific plan.
1. Sign up for WordArt. 2. Explore the interface and functionality of WordArt. 3. *Create a word cloud using the example provided by the teacher in the WordArt service.
Homework 4. * Create your own word cloud that reflects the specifics of your subject. 5. *Write a review (8-10 sentences) about using WordArt. Record the merits and demerits of WordArt that you noted. Write whether you would use WordArt in your teaching activities or not.
Every word cloud, WordArt project has a name. Word cloud management contains the following groups: Words, Shapes, Fonts, Display, Colors, Export. It is possible to add words one at a time (to enter into the window or to manually add a new line using the button). Alternatively, a list of words can be imported at once. For each word, the size, Color, Angle, Font, Link can be specified. In the course of work students used the following tools: rename the project, save the project as, print the word cloud, delete the project; change the terms of privacy, links to methodological developments for creating word clouds in English, to pictograms.
Specific practical results that marked the end of practical activities in the infographic service: 1. Theoretical knowledge about visualization tools in modern education. 2. Formation of the ability to create interactive word clouds using the WordArt. Understanding the practical value of the products of this service.
3. An interactive word cloud that reflects the specifics of the subject. Figure 1 shows one of the results of the students' work in WordArt.
Figure 1
The result of work with the digital infographic service Further the students were asked to do a creative task (project) on using digital infographics service when studying a foreign language. For example, students modeled the infographics "Algorithm of registration on the site". Working with it involved the following subtasks: a. Write down the algorithm using present tense verbs in the third person plural. b. Change the mood of the verbs in the text. Use verbs in the imperative ("do"), indicate forms of verbs. c. Write out all material nouns from the infographics. d. Write out all cardinal numbers from the infographics. e. Using the infographics, fill in gaps in the text using cardinal numbers. f. Create text based on the infographics.
Further in a foreign language classroom, when studying the topics "Traveling", "Food", "Education" students did tasks that integrate infographics into the techniques of mnemonics. Example 1. Mnemonics in English make it easy to remember the quantity of days in months: Thirty days has September, April, June and November. In February, as you know, there are 28 or 29 days, and in the rest of the months there are 31 days. Develop a "word cloud" for visualizing the mnemonics (own or from the Internet).
Example 2. Using a chain of associative images you can remember the PIN code of your bank card. Let's say you need to remember the number "1850". Then it is necessary to assign a bright image to each number in a certain sequence. The number "1" looks like a crane. The number "8" looks like the symbol of infinity or cloud. The number "5" can be associated with a five-storeyed building or the mark "5" (excellent, first-class). The number "0" is like a circle, like a round window. Then you can imagine and remember the following picture: a crane raises its boom into endless clouds and builds a five-storeyed first-class house with round windows. Develop an infographics for computer visualization of this image.
Example 3. The teacher selects a set of static images, rules on the topic "How to defeat a jet lag". Students need to design dynamic infographics to automatically resize it based on location and travel goals. Next the reverse task is. Share the received interactive posters with each other. Translate the text of the infographics into Russian.
Next, it is necessary to show the statistical significance of the obtained result. The number of points the student receives for testing is a measurable criterion. By the value of this criterion the level of the student's lexical competence can be judged. This criterion as well as the difference between the values obtained in the second and first tests (the socalled shifts) have the following properties: connectivity (if a ≠ b, then either a <b or a> b), asymmetry (if a <b, then a ≠ b), transitivity (if a <b and b <c, then a <c). This is a sign that this criterion sets a scale of order for measuring the studied quality (the ability to perform tasks on foreign language vocabulary). In such a situation, takin into account that the samples are dependent, it is possible to prove the statistical significance of the differences using the Wilcoxon nonparametric T-test. In this case, the hypothesis is formulated as follows: H0: the observed shifts in the direction of increasing the scores of the test results can be explained by the influence of purely random factors and are not statistically significant.
H1: The observed shifts in the test results are not accidental and are statistically significant.
For a shift in an atypical direction a negative value is taken (i.e., a shift towards a decrease in the number of tasks solved in the test) because most of the shifts are in the positive direction. Zero shift is dropped. So, N = 27.
The value of the Wilcoxon T-test is equal to the sum of the shift ranks in the atypical direction: T emp = Σ (Ri) = 45. According to the tables (https://medstatistic.ru/methods/ methods3.html), the critical values for N = 27 of this criterion are as follows: T crit (p = 0.05) =107, T crit (p = 0.01) =83. We get that T emp <T crit(0.05) (45<107). So, hypothesis H0 is rejected and the observed typical shifts are not accidental. The recorded shifts are due to the use of digital services of infographics in mnemonics when teaching foreign language vocabulary.
Discussion of the results
In the course of the pedagogical experiment it was possible to find out that the typical shifts that occurred can only be due to factors influencing the student in the period between the first and second testing. It is most likely that the ability of students to do tasks, exercises on foreign language vocabulary was influenced by working with the digital service of infographics as a means of mnemonics. Indeed, the tasks on the topics were carried out only according to the described system. There were no other pedagogical influences on students when studying foreign language vocabulary. Therefore, the positive dynamics recorded in terms of the ability of students to recognize words, understand lexical units, carry out transformation at the level of words/phrases, construct and combine new phrases can be only due to the use of digital infographic services in mnemonics.
Of the other functional capabilities that have a positive didactic effect on cognitive interest, memory, attention, the participants in the experiment noted: • using accounts of worldwide social networks (Facebook, G + or Twitter); • ability to repeat words, the presence of hyperlinks; • creating own form for word clouds. Also in the discussion the following advantages of using infographic elements when teaching a foreign language were highlighted: work with a large amount of theoretical information; rich lexical material, active vocabulary; solving a variety of tasks (transformation of both content and form); development of information literacy; training to critically think of network sources, etc.
The latter advantage is of particular importance in the context of training highly qualified specialists of the future. Working with infographics only at the first stage is a reflection (What is it? What is this image associated with? What famous/unknown words are used?, etc.). Further, the work process is based on identifying information hidden in the infographics about the hierarchy, importance/relevance, cause-and-effect relationships in foreign language vocabulary. On the basis of observations carried out after the experimental teaching a decrease in the number of spelling mistakes in English words, a quick mastering abstract concepts, improvement in memory and attention indicators in students were noted.
As directions for improving the proposed option for using digital means for visual presentation of information, memorizing and presenting concepts/terms when teaching a foreign language the following were proposed: to expand the range of infographics with clouds of foreign words to other disciplines (for example, "Pedagogy", "Age anatomy, physiology and hygiene"); to add tasks to establish a connection between vocabulary and grammar (for example, when studying the topic "Education" to talk about past school habit using 'used to').
The positive influence of infographics on the teaching of grammar was proven in foreign works [24]. However, the research materials justify that this is also true for another subsystem of the language -vocabulary. The research materials confirm the conclusions of F. Boers about the potential of mnemonics for teaching foreign language vocabulary [1] and expand the ideas of O. Kalugina, N. Tarasevich about the potential of digital services for training highly qualified specialists of the future [ 7].
The obtained results develop and complement the conclusions of Yu. Radchenko to the level of innovative pedagogical technologies and digital means [25]. Moreover, they fully correspond to the data of V. Pisarenko, M. Bondarev that visual technology activates cognition, stimulates intellectual activity [26].
Conclusion
The most important principle of modern education is optimization of learning through the use of innovative educational psychological and pedagogical technologies. One of them is mnemonics as a system of effective methods for supporting assimilation of large volumes of theoretical material by students and preventing information overload.
For foreign language teachers the use of mnemonic operations, techniques is of particular importance when learning new words (memorization); converting words into images (encoding); formation of connections between support images and memorized images (memorization); exclusion of intermediate images through formation of a direct connection word/pronunciation -image/meaning of the word (consolidation of connections in the brain).
In the course of analytical activities it was found that the use of infographics in teaching a foreign language contributes to: increasing the degree of assimilation of information due to visual images with the help of which this information is presented; lowering the level of information noise in the process of foreign language communication; successful conceptualization of the topic on which the information is presented.
The research materials allow us to reasonably assert that the use of digital tools for visual presentation of information, memorization and presentation of concepts/terms when teaching a foreign language will provide additional conditions for introduction, semantization of a new word and its primary reproduction; training and creating strong speech connections; creating dynamic lexical speech connections.
When summarizing the results of the pedagogical experiment the rules, implementation of which ensures effectiveness of using digital services of infographics in mnemonics to improve the quality of teaching foreign language vocabulary, were formulated: • creating an information resource in the program environment should be preceded by work on the analysis of the corresponding mnemonics (for example, to consider various mnemonics on the New Year, highlight key words in them, analyze lexical norms); • behind each word in the interactive "word cloud" (interactive poster) there should be a hyperlink to a specific theoretical material; • infographics must have at least 15 words; • the shape of the cloud should reflect the topic of vocabulary. • when you hover over a word, the word should change color/style, but not the relationship with other words (lexical norms); • displaying words in the cloud should not only be horizontal; • organization of oral discussion of digital resources, ensuring the relationship of visual and speech material with the specific language situation. If there is enough time, a class discussion on the topic of infographics can be organized. For example, discussion of students' personal experience, their advice can be used while traveling or when choosing a profession. Mnemonic exercises, supported by digital resources, contribute to development of reading and translation skills as well as the ability to debate on the specific topic and speak out loud. Besides, infographics can be used as a form of control.
Dynamic images in infographics have a positive effect on formation of the skill of language guessing, a significant component of compensatory competence and activating the chain of mental operations. This fits the essence of infographics as a cognitive visualization tool.
Thus, the inclusion of digital services of infographics in mnemonics and for visual presentation of information is an effective method of teaching foreign language vocabulary.
The proposed technique can be applied variably for organizing the learning process and perceiving information in the online space and in other disciplines, since it uses the universal properties of memory. | 2022-03-23T15:09:19.481Z | 2022-03-01T00:00:00.000 | {
"year": 2022,
"sha1": "6a37fffb5d3d477a98ca54f5271c949aae6eb97c",
"oa_license": null,
"oa_url": "https://pnojournal.files.wordpress.com/2022/03/pdf_220115.pdf",
"oa_status": "GOLD",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "e471321404ea3ace1a7601c7bf3183c1849a6c6d",
"s2fieldsofstudy": [
"Education",
"Computer Science",
"Linguistics"
],
"extfieldsofstudy": []
} |
262222886 | pes2o/s2orc | v3-fos-license | RBX2660 (REBYOTA®) in preventing recurrence of Clostridioides difficile infection: a profile of its use in the USA
RBX2660 (fecal microbiota, live-jslm; REBYOTA ® ) is an emerging option for the prevention of recurrent Clostridioides difficile infection (CDI) following standard of care (SOC) antibiotics. RBX2660 is a first-in-class, live biotherapeutic product available as a single-dose microbiota suspension for rectal administration. RBX2660 was effective in reducing recurrent CDI following SOC antibiotic therapy in the pivotal, phase 3 PUNCH CD3 trial. In a Bayesian analysis model, RBX2660 was superior to placebo in terms of treatment success, defined as the absence of CDI diarrhea within 8 weeks of study treat-ment. Most patients with treatment success at 8 weeks remained free of CDI recurrence at 6 months. The effectiveness of RBX2660 has also been demonstrated in the real-world setting. RBX2660 was well tolerated in the PUNCH CD3 trial, with a manageable adverse event (AE) profile. The most common AEs with RBX2660 were gastrointestinal in nature. Most AEs occurred during the first 2 weeks after treatment and were of mild or moderate severity.
. Severe complications of CDI can include sepsis, toxic megacolon, bowel perforation, and death [1].The Infectious Disease Society of America and Society for Healthcare Epidemiology of America [4] and the American College of Gastroenterology [5] guidelines for the management of CDI recommend the use of antibiotics such as vancomycin, fidaxomicin, and metronidazole as initial therapy.However, the antibiotic selected to treat CDI can also be a contributing factor to the cycle of recurrence [6].It is estimated that ≈ 20-30% of patients experience CDI recurrence within 1-2 months of the first infection, with the risk of recurrence increasing after each episode [3,7,8].Therefore, new strategies to prevent CDI recurrence are needed.
Fecal microbiota transplantation (FMT), which involves the transplantation of feces from a healthy donor to a recipient, has been used to treat recurrent CDI [3,6,8].FMT is currently recommended as a therapeutic option for second or subsequent
Adis evaluation of RBX2660 (REBYOTA®) in preventing recurrence of CDI
First-in-class, fecal microbiota-based live biotherapeutic product Supplied as a 150 mL suspension for rectal administration Restores the intestinal microbiome Reduces recurrent CDI following SOC antibiotic therapy, with a sustained response through 6 months Well tolerated, with a manageable AE profile CDI recurrence [4,5].However, the procedure is not standardized, associated data for FMT is very heterogeneous, and improper donor screening can result in the transfer of pathogens and multidrug-resistant bacteria [2,6].This has led to the development of standardized live biotherapeutic products that are regulated as drugs by the FDA [2].One such product is RBX2660 (fecal microbiota, live-jslm; REBYOTA ® ), a first-in-class, rectally administered fecal microbiota suspension approved in the USA for the prevention of recurrence of CDI in individuals 18 years of age and older, following antibiotic treatment for recurrent CDI [9].A summary of the US prescribing information for RBX2660 is provided in Table 1.
How does RBX2660 (REBYOTA®) work?
RBX2660 is a live fecal microbiota suspension for rectal use [9].It is manufactured from donated human fecal matter that has been screened for transmissible pathogens.The exact mechanism by which RBX2660 exerts its efficacy in preventing recurrence of CDI is unknown [9].
Prevention of recurrent CDI with RBX2660 was associated with restoration of the intestinal microbiome, with responders' microbiomes showing greater microbiome diversity and becoming more similar to RBX2660 [10,11].Administration of RBX2660 was correlated with taxonomic and functional pathway composition convergence of patients' microbiota to the donor microbiota [11,12].Specifically, Bacteroidia and Clostridia increased after treatment, while Gammaproteobacteria and Bacilli decreased [10,13].RBX2660 also reduced the abundance of antibiotic-resistant organisms and antibiotic-resistance genes [11,12], with fecal antibiotic resistance gene carriage decreasing in direct relationship to the degree to which donor microbiota engrafted [12].Introduction of some strains of antibiotic-resistant organisms was observed during the transplantation process; however, most of these were Escherichia coli commonly found in healthy populations, and none were correlated with clinical infection [11].Of note, this trial enrolled patients from December 2014 to November 2015, prior to recognition of extended-spectrum beta-lactamase (ESBL) as an important aspect of donor screening.During this time, donor stools were screened for carbapenem-resistant Enterobacteriaceae (CRE) but not ESBL, whereas all donor stools are now screened for both CRE and ESBL [11].To date, there have been no adverse infection events due to bacterial transmission from RBX2660 in any clinical trials [11,14].
Accumulation of primary bile acids (i.e., cholic acid and chenodeoxycholic acid) due to the disruption of intestinal microbiota may promote recurrence of CDI, while secondary bile acids (i.e., lithocholic acid and deoxycholic acid) dominate the healthy fecal bile acid profile [15].RBX2660 altered bile acid composition by significantly reducing fecal levels of primary bile acids and concurrently increasing fecal levels of secondary bile acids [15].
Clinical response to RBX2660 in a phase 3 trial (PUNCH CD3) was associated with clonal engraftment of species into the patients' microbiome [16] and restoration of bile acid compositions from less to more healthy [17].
PUNCH CD3
Patients eligible for enrolment in PUNCH CD3 were adults aged ≥ 18 years with documented recurrent CDI (defined as one or more recurrences after a primary episode) who had completed one or more courses of SOC antibiotics or had two or more episodes of severe CDI resulting in hospitalization within the previous year [18].Within the previous 30 days, patients were required to have a positive stool test for the presence of C. difficile with the capability to produce toxins assessed by polymerase chain reaction, enzyme immunoassay, or other assays.All patients were taking within the first 8 weeks were eligible to receive a second treatment course with open-label RBX2660 [18].
Of the 289 patients who were randomized to treatment, 267 were treated with RBX2660 (n = 180) or placebo (n = 87) and comprised the intention-to-treat (ITT) population [18].Baseline characteristics were generally comparable between treatment groups, although the proportion of patients aged < 65 years was higher in the placebo group than in the RBX2660 group (62 vs 51%).Patients had a median age of 63 years and a median ATLAS (age, treatment with systemic antibiotics, leukocyte count, albumin, and serum creatinine [23]) score for the qualifying CDI episode of 3.0 Table 1 Prescribing summary of RBX2660 (REBYOTA ® ) in preventing recurrence of Clostridioides difficile infection in the USA [9].Consult local prescribing information for further details
What is the approved indication of RBX2660?
The prevention of recurrence of CDI in individuals 18 years of age and older, following antibiotic treatment for recurrent CDI How is RBX2660 supplied?Pre-packaged carton containing suspension bag of a single 150 mL dose of fecal microbiota, live-jslm Each dose contains between 1 × 10 8 and 5 × 10 10 CFU/mL of fecal microbes including > 1 × 10 (scores range from 0 to 10, with higher scores correlating with a lower cure rate [23]).The most common antibiotic used to treat the qualifying CDI episode was vancomycin (88%).The primary endpoint was treatment success, defined as the absence of CDI diarrhea within 8 weeks of study treatment.The primary analysis population [i.e., the modified ITT (mITT) population] comprised all randomized patients who successfully completed treatment and did not discontinue the trial during the first 8 weeks for reasons unrelated to CDI (n = 262).The primary endpoint was analyzed using a Bayesian hierarchical model that formally integrated information about the treatment effect from the earlier phase 2b PUNCH CD2 trial.This model provides estimates of the treatment success rates for each treatment group in PUNCH CD3 as well as the estimated treatment effect and the associated posterior probability of superiority [18].RBX2660 was effective at reducing recurrent CDI, as evidenced by a significantly higher treatment success rate with RBX2660 than with placebo in the mITT population (Table 2) [18].Similar results were seen in the ITT and perprotocol populations (Table 2).Prespecified subgroup analyses of the primary endpoint demonstrated consistent efficacy of RBX2660 across subgroups based on sex, age (< 65 vs ≥ 65 years), race (white vs non-white), and number of previous episodes of CDI (≤ 3 vs > 3) [18].In post hoc analyses, RBX2660 treatment success rates were consistent regardless of comorbidities [i.e., baseline Charlson Comorbidity Index score of 0-2 (mild), 3-4 (moderate), or 5+ (severe)] [24,25] and the presence of underlying cardiac, renal, or gastrointestinal (GI) disorders [24].Among patients with a first CDI recurrence (n = 86), 79% of RBX2660 recipients and 61% of placebo recipients achieved treatment success at week 8 [26].
The proportion of patients with treatment success at 8 weeks who remained free of CDI recurrence at 6 months was 92% with RBX2660 and 91% with placebo (mITT population) [18].Of the 41 RBX2660 recipients with confirmed treatment failure who received a second treatment course with open-label RBX2660, 22 (54%) achieved treatment success within 8 weeks; of these, 19 (86%) had a sustained response through 6 months.Of the 24 placebo recipients with confirmed treatment failure who were subsequently treated with open-label RBX2660, 15 (63%) achieved treatment success within 8 weeks and all 15 had sustained response through 6 months [18].
RBX2660 improved health-related quality of life (HR-QoL) in patients with recurrent CDI, as assessed using the 32-item C. difficile HR-QoL (Cdiff32) instrument (total scores range from 0 to 100, with 100 being the best possible score) [27].Cdiff32 total scores improved significantly from baseline at all time points (i.e., weeks 1, 4, and 8) in both treatment groups (all p < 0.001).At week 8, RBX2660 was associated with a significantly (p < 0.05) greater improvement in Cdiff32 mental domain score than placebo.In multivariable adjusted analyses, there were statistically significant differences between RBX2660 and placebo at week 8 for Cdiff32 total score, physical domain score, and mental domain score (all p < 0.05).Among responders (i.e., no recurrence of CDI), improvements from baseline to week 8 were statistically significant (all p < 0.001) for Cdiff32 total score and all three domain scores (i.e., physical, mental, and social), for both RBX2660 and placebo.Non-responders showed numerical improvements from baseline with RBX2600 but not placebo [27].
Table 2 Efficacy of RBX2660 (REBYOTA ® ) for the prevention of recurrent Clostridioides difficile infection in the phase 3 PUNCH CD3 trial [18] (m)ITT (modified) intention to treat, PP per protocol a Matched populations from the phase 2 PUNCH CD2 trial [19] were used to generate Bayesian model-estimated treatment success rates b Primary endpoint, defined as the absence of C. difficile infection diarrhea within 8 weeks of study treatment c Threshold of > 0.975 (selected to control the nominal type I error rate without borrowing at one-sided 0.025) provides evidence of a statistically significant phase
In the real-world setting
Real-world experience with RBX2660 supports the efficacy results observed during the PUNCH CD3 trial [30].Because an enforcement discretion policy was in place for FMT, RBX2660 was administered off-study via this mechanism for patients who were ineligible/unable to participate in a clinical trial or who required additional treatment beyond a clinical trial.Patient experience was then assessed in a retrospective study of 94 patients aged ≥ 18 years with recurrent CDI who received one or two doses of RBX2660.The mean age of patients was 60 years, and 45% of patients were aged ≥ 65 years.Comorbid conditions included IBS (22%), microscopic colitis (11%), Crohn's disease (8%), and ulcerative colitis (6%).The overall rate of treatment success (i.e., absence of CDI recurrence within 8 weeks of treatment) was 83% in the primary safety set (PSS; n = 64) and 70% in the full analysis set (FAS; n = 94).Among patients who achieved treatment success at week 8, 89% of those in the PSS and 88% of those in the FAS had a sustained clinical response through 6 months.In both study populations, treatment success rates were similar in patients who received one or two doses of RBX2660 [30].
What is the tolerability of RBX2660 (REBYOTA®) in preventing recurrence of CDI?
RBX2660 is well tolerated with a manageable adverse event (AE) profile, based on data from the pivotal PUNCH CD3 trial [18].Through 6 months after blinded treatment, AEs occurred in 56% of RBX2660 recipients and 45% of placebo recipients.The most common (incidence ≥ 5%) AEs with RBX2660 were diarrhea (20% vs 19% with placebo), abdominal pain (19% vs 9%), nausea (11 vs 5%), and abdominal distension (6 vs 5%).Most AEs occurred during the first 2 weeks after treatment and were mild or moderate in severity.Serious AEs occurred in 4% of RBX2660 recipients and 2% of placebo recipients.One patient in the RBX2660 group discontinued because of an AE [18].
The safety of RBX2660 was demonstrated in an integrated analysis of data from three phase 2 trials (PUNCH CD, PUNCH CD2, and PUNCH CD open-label) and two phase 3 trials (PUNCH CD3 and PUNCH CD3 OLS) [14].In the safety population (n = 1061), treatment-emergent AEs (TEAEs) through 6 months were reported in 507/763 (66%) patients who received RBX2660 only and 50/83 (60%) patients who received placebo only.Most TEAEs were of mild or moderate severity and were related to pre-existing conditions.The most common (incidence ≥ 5%) TEAEs associated with RBX2660 were diarrhea (21% vs 18% with placebo), abdominal pain (15 vs 8%), nausea (8 vs 4%), flatulence (7 vs 1%), abdominal distension (7 vs 4%), and urinary tract infection (6 vs 5%).Serious TEAEs occurred in 12% of RBX2660 recipients and 7% of placebo recipients, most of which were considered related to CDI and preexisting conditions.There were no unexpected TEAEs and the incidence of potentially life-threatening TEAEs was low.TEAEs leading to death within 6 months after treatment occurred in 18 (2%) RBX2660 recipients.One death (severe CDI recurrence) was considered related to CDI and cardiovascular comorbidities and possibly related to RBX2660; however, the event was subsequently determined to not be a product-related safety concern [14].
What is the current clinical position of RBX2660 (REBYOTA®) in preventing recurrence of CDI?
RBX2660, the first fecal microbiota-based live biotherapeutic, is an emerging option for the prevention of recurrent CDI following SOC antibiotic therapy.In the pivotal PUNCH CD3 trial, a single, rectally administered dose of RBX2660 effectively reduced recurrent CDI, with a sustained response through 6 months [18].One limitation of PUNCH CD3 is the exclusion of participants with IBS and IBD [18].However, RBX2660 also consistently reduced CDI recurrence in PUNCH CD3-OLS [21,22] and in a retrospective real-world study [30], both of which enrolled more diverse populations, including patients with underlying GI comorbidities such as IBS and IBD.These populations are likely to be more representative of the general recurrent CDI population.
As per current treatment guidelines, FMT is recommended as a therapeutic option for patients with two or more recurrences of CDI [4,5].However, for patients with demographic, pharmacologic, and environmental risk factors for CDI recurrence, earlier usage (i.e., for a first recurrence) should be considered [31].Indeed, results of a post hoc analysis of PUNCH CD3 support the use of RBX2660 in patients with a first CDI recurrence [26].
Despite its efficacy and inclusion in treatment guidelines, conventional FMT is limited by the lack of standardized manufacturing processes [14].To ensure patient safety, RBX2660 is subject to standardized screening procedures and testing protocols in accordance with US FDA requirements [14].As part of the manufacturing process, RBX2660 stool donors are required to undergo thorough screening and routine testing for a wide range of pathogens, including viruses, bacteria, and parasites [18].Moreover, administration of RBX2660 is well tolerated in patients with recurrent CDI.The AE profile of RBX2660 is manageable, with most AEs occurring during the first 2 weeks after treatment and being of mild or moderate severity.
To date, no randomized clinical trials have directly compared the efficacy of RBX2660 with other approved pharmacological therapies for prevention of CDI recurrence.For example, SER-109 (fecal microbiota spores, live-brpk) is an orally administered microbiota-based therapeutic indicated to prevent the recurrence of CDI following antibiotic treatment for recurrent CDI [32].A recent systematic review and meta-analysis (up to May 2021) comparing interventions added to antibiotic therapy found that RBX2660, bezlotoxumab/actoxumab, and bezlotoxumab were all more effective than placebo at reducing CDI recurrence, with odds ratios of 0.47 (95% CI 0.22-0.99),0.47 (95% CI 0.37-0.60),and 0.53 (95% CI 0.42-0.68),respectively [33].Actoxumab and SER-109 were not superior to placebo [33].Results of such indirect comparisons should be interpreted cautiously.Headto-head clinical trials comparing the efficacy and tolerability of RBX2660 relative to other agents would be of interest.A multicentre, single-arm, phase 3 trial (CDI-SCOPE) is currently underway to assess the safety and efficacy of RBX2660 when delivered by colonoscopy to adults with recurrent CDI [34].
As the most common healthcare-associated infection in the USA [35], CDI carries a substantial clinical and economic burden [36,37].Moreover, recurrent CDI can considerably increase the use of medical resources and related expenses, with direct medical costs related to recurrent CDI in the USA estimated to be $2.8 billion per year [35].In a recent cost-effectiveness analysis using a Markov model with a lifetime horizon, RBX2660 was found to be cost effective relative to SOC from a US third-party payer perspective, with an incremental cost-effectiveness ratio of $US18,727 per quality-adjusted life-year gained [37].In a budget impact analysis, RBX2660 was demonstrated to be cost saving from a US third-party payer perspective, with higher initial drug costs being offset by savings in direct medical costs through prevention of CDI recurrence [36].
should RBX2660 be stored?
5 CFU/mL of Bacteroides and ≤ 5.97 g of polyethylene glycol 3350 in saline How Upon receipt Store carton in ultracold freezer (− 76 °F to − 130 °F) or refrigerator (36 °F to 46 °F) for up to 5 days (including thaw time) Store administration set at 50 °C to 93 °F; do not store in freezer Prior to use Thaw product completely in refrigerator (36 °F to 46 °F) for ≈ 24 h; do not thaw using heat source; do not refreeze How should RBX2660 be prepared?Remove carton from refrigerator; remove bag containing thawed product from carton Open administration set and close pinch clamp Remove tab from spike port of bag and remove cap from administration tube spike; insert administration tube spike through spike port of bag How
should RBX2660 be administered?
Administer product 24-72 h after last dose of antibiotics for CDI Instruct pt to empty bladder and bowel if possible; place pt in left-side or knee-chest position Apply water-soluble lubricant to administration tube tip; gently insert tube ≈ 12 cm into rectum in direction of umbilicus Open pinch clamp on administration tube; gradually raise bag to allow delivery via gravity flow; do not allow tube to sag or loop; do not squeeze bag; do not hang bag from a stand After delivery, close pinch clamp and slowly withdraw tube; keep pt in left-side or knee-chest position for up to 15 min to minimize cramps What
are the contraindications to the use of RBX2660?
Pts with history of severe allergic reaction (e.g., anaphylaxis) to any known product components How
other special warnings and precautions pertain to the use of RBX2660?
(vancomycin alone, vancomycin plus another antibiotic, fidaxomicin alone, or other).Both study treatments were administered rectally as per instructions for use and standard site procedures.Patients who experienced treatment failure | 2023-09-25T15:08:23.460Z | 2023-09-23T00:00:00.000 | {
"year": 2023,
"sha1": "cf9ae46d1f9aedb83a0f077cd5cb11316d48a54c",
"oa_license": "CCBYNC",
"oa_url": "https://figshare.com/articles/online_resource/RBX2660_REBYOTA_in_preventing_recurrence_of_Clostridioides_difficile_infection_a_profile_of_its_use_in_the_USA/24069618/1/files/42266424.pdf",
"oa_status": "GREEN",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "d4a88fc10943788536a8378ddf66d9838a305241",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
100361238 | pes2o/s2orc | v3-fos-license | Time resolved spectroscopic investigation of SiD2 + D2: kinetic study
Silylenes (silanediyls) have made an important impact on organosilicon chemistry even if it is of more recent foundation than carbenes in organic chemistry and much less complete. These species are highly reactive intermediates. They play a central role in the chemical vapour deposition (CVD) of various silicon-containing thin films which have a technological importance in microelectronics as well as in the dry etching processes of silicon wafers. Spectroscopic methods have been developed to observe these species, a necessary pre-requisite to their direct monitoring. In this work, deuterated phenylsilane precursor, PhSiD3 was chosen for SiD2 because its analogue phenylsilane, PhSiH3 proved to be a good precursor for SiH2 and the high quality decay signals observed revealed that SiD2 be readily detected from PhSiD3 and that if other decomposition pathways (e.g. PhSiD + D2) are occurring, they do not effect measurements of the rate constants for SiD2. The absorption spectrum of SiD2 formed from the flash photolysis of a mixture of PhSiD3 and SF6 at 193nm were found in the region 17384-17391 cm with strong band at 17387.07 cm. This single rotational line of Q1 was chosen to monitor SiD2 removal. Time-resolved studies of SiD2 have been carried out to obtain rate constants for its bimolecular reactions with D2. The reactions were studied over the pressure range 5-100 Torr (in SF6 bath gas) at four temperatures in the range 298-498K. Single decay from 10 photolysis laser shots were averaged and found to give reasonable first-order kinetics fits. Second order kinetics were obtained by pressure dependence of the pseudo first order decay constants and substance D2 pressures within experimental error. The reaction was found to be weakly pressure dependent at all temperatures, consistent with a third-body mediated association process. In addition, SiH2+ H2 reaction is approximately ca. 60% faster than SiD2+D2 reaction. Theoretical extrapolations (using Lindemann-Hinshelwood model and Rice, Ramsperger, Kassel and Marcus (RRKM) theory) were also carried out and obtained data fitted the Arrhenius
Introduction
The desire to understand the mechanistic details of the reactions of silylene, SiH 2 prompted us to extend our studies to include isotope effect experiments. [1,2]. The only kinetic information available for deuterated silylene, SiD 2 was that for the reaction with H 2 . Mason et al. [3] reported a rate constant of 3.8±0.2x10 -12 cm 3 molecule -1 s -1 at 5 Torr total pressure in which the reaction found to be pressure independent in the range 2-100 Torr. The possibility of studies with SiD 2 offers the opportunity to give new insights into reaction system previously studied with SiH 2 .
For the reaction of SiH 2 with D 2 at room temperature,Jasinski reported a rate constant of 2.6x10 -12 cm 3 molecule -1 s -1 [4]. This was the first reported measurement for this reaction. It compares with a value of 3.2x10 -12 cm 3 molecule -1 s -1 for the rate constant for the reaction of SiH 2 +H 2 obtained by extrapolation to infinite pressure of a pressure dependent process [5,6]. These high values suggest only a small isotope effect and that the energy barrier to insertion was d 1 kcal mol -1 [4], this provides experimental support for the higher values of heat of formation at room temperature. This suggestion has been supported by ab initio calculations carried out by Gordon and Gano [7]. The calculations for the potential energy surface of the silylene reaction with hydrogen gave an activation energy of 1.7 kcal mol -1 .
Subsequently Baggott et al. [8] extended Jasinski's room temperature study to cover a range of 298-333K and found k to be in good agreement with Jasinski's reported value [4,5]. The authors found an almost temperature independent rate constants of 1.88x10 -12 cm 3 molecule -1 s -1 . The uncertainties in the rate constants were ca. 7%. This indicated that any activation energy was very small and certainly <1.57 kcal mol -1 . A slightly negative activation energy cannot be ruled out.
In this work we report the first kinetic studies for the reaction of SiD 2 + D 2 at a wide range of pressures and temperatures. Kinetic studies of SiD 2 are of particular interest in two respects, viz (i) a comparison with SiH 2 data will help elucidate reaction mechanisms, (ii) many SiH 2 reactions exhibit pressure dependence often making it problematical to obtain high pressure limiting rate constants. Isotopic scrambling effects make it possible to circumvent this problem with SiD 2 studies. PhSiD 3 was prepared by reduction of phenyltrichlorosilane (PhSiCl 3 ) with LiAlD 4 in dry ether [3]. The prepared PhSiD 3 was purified by distillation and found to be 99.8% pure (isotopic purity >92.6%). The PhSiD 3 precursor was chosen because its analogue phenylsilane, PhSiH 3 proved to be a good precursor for SiH 2 . Although SiH 2 formation is not the major product of PhSiH 3 photolysis [9], nevertheless, its shown observation, suggest that SiD 2 should be readily detected from PhSiD 3 . The high quality decay signals observed in the current work reveals that this is true and that if other decomposition pathways (eg to PhSiD + D 2 ) are occurring, they do not effect measurements of the rate constants for SiD 2 . A typical averaged decay trace is shown in figure 1. The absorption spectrum of SiD 2 , formed from the flash photolysis of a mixture of SiD 4 and D 2 , was first investigated by Dubois et al. in the region 16175-19200 cm -1 [10]. They recorded a visible spectrum of the ( 1 B 1 )m( 1 A 1 ) band and found the bending vibrational frequency in the excited state to be 610 nm. However a more detailed spectroscopic study of SiD 2 radicals was reported by other researchers such as Fukushima et al. and Muramoto et al. [11][12][13]. Fukushima et al. [11] photolysed PhSiD 3 at 193nm in the supersonic free jet and observed 11 vibronic bands of the SiD 2 A ( 1 B 1 ) m X ( 1 A 1 ) transition in the wavelength region between 21739.13 and 15625 cm -1 .
SiD precursor and visible absorption spectrum
Because little is known about the SiD 2 radical, any fresh spectroscopic data (as well as kinetic data) are of practical and theoretical interest. Laser spectroscopic investigation of SiD 2 at higher resolution than earlier studies has reached similar conclusions in our lab [3]. Mason et al. [3] recorded the absorption spectrum of SiD 2 in the gas phase, using the flash photolysis kinetic absorption technique. SiD 2 was generated by photolysing 10mTorr of PhSiD 3 in 5Torr of SF 6 using a series of oneshot photolysis experiments. The authors observed an absorption spectrum in the 17384-17391 cm -1 region (the laser linewidth was approximately 3x10 -5 cm -1 ) with a strong band at 17387.07cm -1 , which was attributed to an unknown rotational line A( 1 B 1 )(0,3,0)mX( 1 A 1 )(0,0,0) vibronic transition of SiD 2 . This assignment was based on the original work of Fukushima et al. [11]. A 6 cm -1 composite spectrum of vibronic transition of SiD 2 is shown in figure 2 (a). Figure 2 (b) also shows the absorption spectrum of SiH 2 for comparison reason. Approximately 8 peaks were displayed for SiD 2 absorption spectrum. The 17387.07 cm -1 was found to be the most intense peak. Therefore this peak was chosen to observe the SiD 2 species in this kinetic study. It has been estimated that the intensity of this peak is approximately a quarter of that for the transition used to monitor the SiH 2 species in kinetic studies under similar conditions [3].
Experimental Setup
Laser flash photolysis/laser absorption apparatus is diagrammatically represented in figure bellow (figure 3). A mercury-free, glass vacuum line was connected to purpose built variable photolysis cell fitted with a high quality crown glass end windows at Brewster's angle backed by a rotary pump. SiD 2 was produced by the 193 nm flash photolysis of phenylsilane (PhSiD 3 ) using an Oxford KX2, ArF excimer laser. The emitted pulses have 0.1-1J of energy delivered in a 10 ns light pulse with a repetition rate of 1 Hz. SiD 2 concentrations were monitored in real time by means of a Coherent 699-21 single-mode dye laser pumped by an Innova 90-5 Ar + laser using Rhodamine 6G in solution as an active medium, which provides tunable radiation in the range 560-610nm. The monitoring laser beam was multipassed between 24-48 times through the reaction zone to give an effective path length of between 0.6-6 m. The monitoring laser was tuned to 17387.07 cm -1 . A differential amplifier was connected to two photodiodes and then fed into Datalab Dl910 transient recorder (20 MHz resolution). The transient recorder's external trigger was activated by radiofrequency noise produced when the excimer laser was fired. Traces were observed during runs and interfaced to a computer via a commercial interface (Camplus) where the signal could be displayed and analyzed.
Results
The SiD 2 exponential decay curves were recorded and averaged for 10 excimer laser shots. The degree of depletion of the photolysis mixture was negligible. Experiments were carried out with gas mixtures containing a few mTorr of PhSiD 3 , varying quantities of D 2 up to 9.5 Torr, and inert diluent bath gas (SF 6 ) at total pressures from 5 to 100 Torr to test for pressure dependence. For each pressure, a series of runs (7 points) was performed at different substrate concentrations, as been illustrated in figure 4.
Only second order plots for data recorded at a total pressure above 20 Torr in SF 6 were linear. Plots for total pressures below this were curved. The quadratic procedure using the polynomial regression was applied for these cases in order to obtain the second order rate constants as gradients of the plots. The intercept corresponds to loss of silylene in the absence of substrate i.e. by reaction with PhSiD 3 . The correlation of fitting constants means that the rate constants are somewhat less reliable by this procedure. Second order rate constants were calculated using the following expression: The conversion factor, F, was obtained from the ideal gas equation (PV=nRT where R=8.3145 J K -1 mol -1 ). Its value is F= 9.657×10 18 .
The second order rate constants for these experiments, representing over 100 measurements of individual rate constants are shown in figure 5 and listed in table 1.
The uncertainties in individual second order rate constants are about ca. ± 20% for total pressures <20 Torr and ± 10% for total pressure >20 Torr, but are not shown in the figures to avoid complicating them. Comparison of absolute rate constants can only be made at 300K between the reactions of SiH 2 +H 2 and SiD 2 +D 2 due to big differences of temperatures used in these studies, see figure 6. The graph shows that the SiH 2 + H 2 reaction is approximately ca. 60% faster than SiD 2 +D 2 reaction at high pressure. The measured rate constants demonstrate the reaction is relatively slow and rate constants are weakly pressure dependent at all temperatures, consistent with a third-body mediated association process. In addition, SiH 2 + H 2 reaction is approximately ca. 60-70% faster than SiD 2 +D 2 reaction at higher total pressure.
Extrapolation by empirical procedures
Since SiD 2 +D 2 study lies in the fall-off region at accessible pressures, a reliable method of extrapolation to infinite pressure must be employed. A number of methods have been suggested and used by various workers.
The earliest extrapolation procedure employed plots of k -1 versus P -1 . This plot follows from the Hinshelwood-Lindemann model. Recently Oref and Rabinovitch [14] have suggested another empirical procedure which theoretically should yield more accurate infinite pressure rate constants since the precise nature of the extrapolation is determined by the actual data. They suggest a plot of k -1 versus P α where α lies between 0 and 1 and is optimised to give the best linear plot. Values of α on either side of the optimum value will give curved plots which would lead to an overestimate and underestimate of k f resp. It is important to state that there is no absolute theoretical justification for either of the methods described above. They are simply empirical procedures which have been found to give reasonable linear plots. In the present work the latter method of extrapolation (where α= 0.5 and 1) has been tried and the results are compared.
When data are only available at pressures well into the fall off region it becomes clear that the choice of method of extrapolation becomes crucial, since different methods give different answers. In the current work it was extremely difficult to judge the quality of the fits because of data scatter, values of k f (represented by (intercept) -1 ) had a large uncertainty. Therefore a modified procedure was adopted here in which a number of points were selected from interpolated curves (drawn by hand). These interpolated fall-off curves were used to represent the data in order to obtain k f with minimum error (see figure 7). We find that the plot is not absolutely linear. Consequently some error in the infinite pressure rate constants obtained using a least-squares line must be expected. If we consider the k -1 versus P -0.5 we find that such plots suffer from marked curvature making extrapolation difficult and leading to an underestimated of k f . On the other hand the k -1 versus P -1 plots are much less curved. It must be noted, however, that scatter in the experimental data can make calculation of an optimum α difficult and may limit the applicability of this method.
The temperature dependence for the reaction may be expressed in Arrhenius form. The derived Arrhenius parameters are shown in table 5. Figure 8 shows a graph of the Arrhenius plot for both SiH 2 +H 2 [15] and SiD 2 +D 2 reactions. These data show a number of interesting features. First the reaction has a negative activation energies E= -0.82±0.05 kcal mol -1 . Secondly there is a significant difference between SiH 2 +H 2 and SiD 2 +D 2 . The former reaction is ca. 60% faster than the latter.
RRKM modelling of experiments
In order to try to fit the observed pressure dependence of the insertion of SiD 2 + D 2 , RRKM calculations were carried out on the pressure dependence of the inverse silane, SiD 4 decomposition in the conventional manner.
<ΔE> down were based on the assumption of a fixed energy removed on each collision (stepladder model). However we did not include some other correction factors (eg F rot ) which could have some effect. For a small molecule like silane (highly excited), the anharmonicity effect was found to be important [16,17]. This correction factor influences the density of states of the reactant at an internal energy equal to the critical energy. The low pressure limiting rate constant is proportional to the anharmonicity factor, by increasing the value of this factor better agreement with the data was obtained [17][18][19]. It has been suggested that increasing the value of this factor has roughly the same effect as increasing the "looseness" of the transition state.
The fall-off predictions are very sensitive to some of the parameters and not very sensitive to others. The exact assignments of reactant frequencies, transition state frequencies, and reactant path degeneracy are not very important as long as these assignments reproduce the required high pressure A-factor. On the other hand, assignment of bath gas collision efficiencies and their temperature dependencies can be very important, particularly when the reaction is well into its pressuredependent region and the bath gas molecules are weak colliders.
The effect of varying the energy level grain size (GRAIN) is rather unpredictable and its choice affects the position of the fall-off curves for both strong and weak collision situations. This parameter is used to define the energy gap in the quasi-quantised energy scale covering the energy range of interest. It is clear that the smaller its value, the more closely packed are the levels. Therefore in principle smaller grain sizes are best (although this increases the size of programme storage required and decreases the speed of calculations). However the calculation is also sensitive to the positioning of the critical energy, E o with the quantised energy levels. The best situation appears to be when E o lies in the center of a grain. The choice of minimum energy value, E min and grain size, determines this. In practice there were chosen to get closer to this condition. The energy range starts from just below the initial energy and goes up to 2-3 times its value. In the present calculation for silylene reaction with hydrogen this parameter was set to be 50 cm -1 . The vibration frequencies assigned to the SiD 4 complex were based on those from ab initio calculations of Gordon et al. [20]. The details of the model and parameters used are shown in tables 2, and 3. 14.38 (298K) Z LJ / 10 -10 cm 3 molec -1 s - 1 4.354 (SF 6 , 298K) The calculations were carried out using both a strong and a weak collision model to generate fall-off curves at four different temperatures. A weak collision (<ΔE> down = 2.3 kcal mol -1 ) was fixed to fit the rate constants at all four temperatures and is reasonably consistent with collisional efficiencies found with similar systems [20]. However these calculations were carried out without including the F an factor which was assumed to have a small effect.
The results are plotted in figure 9 with experimentally determined rate constants. The high pressure limit rate constants were derived from the isotope effect calculations combined with SiH 2 + D 2 rate data [15]. Reasonable agreement was obtained with the data when compared to that of the previous reaction, except for T=357K where the theoretical fit and data were in disagreement. However there are probably other choices of parameters that would have produced equally good fits.
Discussion
These studies proved to be more difficult than earlier ones for silylene with olefins [21]. This arose because the rate constants were smaller and a significant amount of hydrogen was required to observe a reaction. This meant that at low total pressures, reaction of silylene with precursor was always a substantial fraction of the total decay. The magnitude of the intercept of the second order plots were kept as small as possible. This was done by reducing the partial pressure of SiD 4 to the minimum consistent with signal to noise constraints. This restricted the range of possible substrate pressures and temperatures over which reactions could be studied. At high partial pressures of hydrogen necessary to obtain reaction, SF 6 is present in insufficient quantity to ensure that it is the dominant bath gas collider. This causes curvature in the second order plots (at fixed total pressures). By using a polynomial procedure (quadratic), we are assuming that the slope at [D 2 /Torr]=0 will provide a reliable estimate of the firstorder rate coefficients, k obs , for the removal of SiD 2 in the presence of hydrogen. However this leads to higher uncertainties in this case compared to those of other studies (eg SiH 2 + C 2 H 4 ) [21].
A high pressure limit rate comparison is shown in table 5 using different extrapolation procedures. A comparison is also made for the activation energies and the A-factors for silylene reaction with hydrogen and its isotopic analogues in table 6 (obtained from extrapolation of experimental data to the high pressure limit using empirical procedure). [15] b . Based on isotope effect calculations [15] c . P T = 10 Torr [15], d . P T = 10 Torr [3] The following figures can be drawn from this table. Arrhenius parameters of E a = -0.48 ± 0.33 kcal mol -1 and A-factor= 0.80±0.35u10 -12 cm 3 molecule -1 s -1 can be averaged. The activation energy for the reaction (SiH 2 +H 2 ) is approximately a factor of 1.5 smaller than that obtained from reaction (SiD 2 +D 2 ) by extrapolation to the high pressure limit.
A reasonable agreement was seen between theory and experiment for SiD 4 decomposition. However there are probably other choices of parameters that would have produced equally good fits. Parameters such as the average energy removal, <ΔE> down , and the high pressure limit rate constant, k f , can be adjusted to obtain a better fit (see table 5).
Summary and future work
Time-resolved study of SiD 2 generated by laser flash photolysis of (PhSiD 3 ) has been carried out to obtain rate constants for its bimolecular reactions with D 2 . The reaction was studied over the pressure range 5-100 Torr (in SF 6 bath gas) at four temperatures in the range 298-498K. The reaction was found to be pressure dependent at all temperatures, consistent with a third-body mediated association process. The reactions were modelled using empirical and theoretical procedures to obtain the high pressure limit rate constants. The empirical procedure employed the Hinshelwood-Lindemann model. The data obtained using these extrapolations were fitted to the Arrhenius equation. RRKM modelling, based on the transition states (SiD 4 ) of Gordon et al, gave a reasonable fit to the experiments. Further work is required in the following areas; (i) the RRKM calculation described in this work can be improved by considering some other correction factors in addition to that of F an . (ii) the pressure dependence of the pyrolysis of silane also needs to be reinvestigated over a wider pressure range in order to obtain an improved value for k f for SiD 4 o SiD 2 + D 2 . This would then improve the value for the heat of formation of silylene. | 2018-08-31T20:52:57.732Z | 2017-03-01T00:00:00.000 | {
"year": 2017,
"sha1": "6ad2d906716132ce3b92a4a5e52ff4886106e801",
"oa_license": "CCBY",
"oa_url": "https://www.epj-conferences.org/articles/epjconf/pdf/2017/08/epjconf_nanop2017_00004.pdf",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "999e7a5d0a49216fe70a4b19ef1f11721abc3072",
"s2fieldsofstudy": [
"Chemistry"
],
"extfieldsofstudy": [
"Chemistry"
]
} |
204937157 | pes2o/s2orc | v3-fos-license | Hybrid architecture for satellite data processing workflow management
The ever growing demand for remote sensing data products by user community has resulted in many Indian and foreign remote sensing satellites being launched. The diversity in the remote sensing sensors has resulted in heterogeneous software and hardware environments for generating geospatial data products. The workflow automation software knows as information management system is in place at National Remote Sensing Centre (NRSC) catering to the needs of the data processing and data dissemination. The software components of workflow are interfaced in different heterogeneous environments that get executed at data processing software in automated and semi automated modes. For every new satellite being launched, the software is modified or upgraded if new business processes are introduced. In this study, we propose a software architecture that gives more flexible automation with very less manageable code. The study also addresses utilization and extraction of useful information from historic production and customer details. A comparison of the current workflow software architecture with existing practices in industry like Service Oriented Architecture (SOA), Extensible Markup Languages (XML), and Event based architectures has been made. A new hybrid approach based on the industry practices is proposed to improve the existing workflow.
I. INTRODUCTION
D ATA processing area of National Remote Sensing Centre (NRSC) is responsible for generation of remote sensing data products for Indian (IRS) and foreign remote sensing satellites.Data product generation involves interfacing different business processes at User request processing, Data processing, Value addition, Film generation, Photo processing and Quality control.These processes have been converted to information technology processes by the information management system (IMS) software.The software identifies every process as a work center or group of work centers.The product generation is based on the type of satellite and processing level that is used to route the products to different work centers based on the concept of work order or job order.The contents of work order are available virtually as and when required to different nodes of work centers to process in an automated mode or through human interaction.It is noted that during the life time of a product generation, the contents of the work order change, i.e., the contents are dynamic.The IMS application is based on three tier software architecture with database layer under Oracle 8i environment, business layer and presentation layers that are implemented using J2EE technologies as shown in Figure 1 providing the M. Naresh Kumar at National Remote Snsing Centre, Hyderabad, Telangana, 500 037 India e-mail: (nareshkumar m@nrsc.gov.in).flexibility to replace any layer with out affecting the other layers [5].The IMS design has been accomplished using object oriented methodologies (which provides the mechanism of capturing the functionality and the data associated with real object [12], [14] and is implemented in Java.Exchange of data between presentation layer and business layer is through business objects.The data is formatted for presentation before displaying it on to the browser, wherever human intervention or monitoring is involved. The data is also provided to automated processes like data processing schedulers that generate data products by ingesting user inputs that are a part of the work order, and ancillary data of the satellite as shown (ADIF) in Figure 2.
A transfer of data to presentation layer and data processing system clients is accomplished by data marshaling and unmarshaling using java remote method invocation [13].These interactions are tightly coupled resulting in high maintenance of code for every change in a business process.The work order flow from system to system is based either on interactions through a browser or requests from and automated process i.e., the scheduler.The transfer of data is highly synchronous and happens as blocking and unblocking calls in the client and server processes resulting in slowing down of the system during heavy data transfers.The IMS software stores online transactions between the work-centers in an operational environment and historic transactions in a lightly summarized manner.Being a centralized repository of data, management looks for information for decision making from database managed by IMS software.Therefore building a dataware house environment would help to build and realize the decision making tools.
The paper is divided in to the following sections.In Section 2 we highlight the adaptation of service oriented architecture including event based process communication for IMS is highlighted.In Section 3 XML based data exchanges between the different layers are discussed.In Section 4 data warehouse schemes are discussed.In Section 5 hybrid architecture for workflow management is proposed and Section 6 conclusions are presented that summarizes the benefits of proposed hybrid architecture for workflow automation of data products generation.
COMMUNICATION
The aim of service oriented Framework is to provide a loose coupling between operating systems, programming languages and other technologies which underlie applications [11].SOA separates functions into distinct units, or services [1], which are made accessible over a network in order that they can be combined and reused in the production of business applications [6].SOA is an architecture that relies on serviceorientation as its fundamental design principle.In a SOA environment, independent services can be accessed without knowledge of their underlying platform implementation [4].SOA relies on services exposing their functionality via interfaces which other applications and services read to understand how the service can be utilized.
Workflow automation requires functionality like work-order generation, updating and dispatch which are similar for different work centers.The work-centers interface with these software interfaces for automating the tasks in the production chain.Also the work-centers involved may be generating data products heterogeneous systems.To continue the usage of the existing and new systems in the production these software interfaces can be coined as services and service orientation will permit the usage of different production hardware and software environment to coexist.Also the development and maintenance of the software modules is reduced by adopting the service oriented architecture.
The communication between the software modules across the data products generation systems is done using object oriented messaging.This has resulted in a tightly coupled software communication interfaces.The present industry standard to reduce the coupling between the software modules Fig. 3. Messaging passing between the sender and receiver using message queue on the messaging server is the Event-driven architecture (EDA).The EDA is a software architecture pattern promoting the production, detection, consumption of, and reaction to events.The architectural pattern is applied for design of applications and systems which communicate using loosely coupled software components and services.An event-driven system typically consists of event emitters (or agents) and event consumers (or sinks).Sinks have the responsibility of applying a reaction as soon as an event is presented.The reaction might or might not be completely provided by the sink itself.For instance, the sink might just have the responsibility to filter, transform and forward the event to another component or it might provide a self contained reaction to such event.The first category of sinks can be based upon traditional components such as message oriented middleware while the second category of sinks (self contained online reaction) might require a more appropriate transactional executive framework.Event-driven architecture can complement service-oriented architecture (SOA) because services can be activated by triggers fired on incoming events [3], [8].This paradigm is particularly useful whenever the sink does not provide any self-contained executive.
There are two types of control namely centralized and event based which determines the ordering and rules for execution of the component.There are three types of coupling in the centralized control referential, communication and execution.The referential coupling requires the sender to know the receiver in advance before communication.The communication coupling requires the sender and receiver to activate at the same time.The execution coupling the sender must wait for the receiver to complete the message processing.The event based communication provides the features like dependency inversion, concurrency and message queuing.The dependency inversion reverses the referential coupling, concurrency removes the execution coupling and message queuing removes the communication coupling.The temporal decoupling between the sender and the receiver can be achieved by implementing a messaging server Figure 3.
III. XML BASED DATA EXCHANGE MECHANISM
The present design of data exchange is based on sending serialized objects and ASCII file based mechanisms to different automated and non automated work-centers.The serialization and de-serialization in the remote procedure calls (RPC) results in increased communication between the client and server systems.Also, the change in the object would results in a different version of the software.The ASCII based mechanism provides cross platform support but the volume of data to be transferred would increase and leads to communication overheads.The present industry standard for data exchange is extensible markup language (XML).
XML is a user-driven, open standard for exchanging data both over corporate networks and between different enterprises, notably over the Internet.XML's biggest potential lies in its ability to mark up mission-critical document elements self-descriptively.XML transports the metadata (the information about the data) together with the relevant data, thus allowing its meaning to be easily interpreted.In addition, XML enables suitably coded documents to be read and understood without difficulty by both humans and machines.XML as a data interchange format is compelling, primarily because it gives developers, a language with which to more easily identify interoperability problems and a common syntax and tool set with which to fix them.The XML related standards like eXtensible Stylesheet Language (XSL), XSL transformations (XSLT) and document type definition (DTD), provide the mechanisms for styling the web pages, validating the data and verifying the well-formed ness of the data respectively.The styling using XSL is external to data and is independent of any software as is being currently done using Servlets/JSP generating HTML tags.Different look and feel can be provided with out any additional software just by configuring the XSL files for report generation.
The data from databases can be easily retrieved from the databases like Oracle using XML SQL Utility (XSU).The XSU uses a schematic mapping that defines how to map tables and views, including object-relational features, to XML documents.Oracle translates the chain of object references from the database into the hierarchical structure of XML elements.The XSU can also be used for executing queries in a Java environment and retrieve XML from the database.XML file exchange mechanism is platform independent and also has different mechanism for styling, validating and other features making it attractive approach for data exchanges across platforms and loose coupling of software modules.
The present file based using client/server based software involves lot of development and maintenance cost.Also, the presentation (look and feel) of the data for report generation requires addition software to be developed.Adopting XML based exchange mechanism would reduce the maintenance costs of the software modules.
IV. DATA WAREHOUSE ENVIRONMENT
IMS software uses Oracle 8i database which is an operational database optimized for transaction management.NRSA data users and other work-center users involved in the data products generation constantly require historic information in the form of reports like turn around time (TAT) for each work center, products wise TAT , completed products between any two dates, pending analysis reports etc.,.The present database being fine tuned for transaction management and normalized Fig. 4. A snowflake schema model for product sales to a high degree resulting in overheads on the database server.Analysis on the production data can be better exploited using a data warehouse environment.A Data warehouse environment provides drill down and roll up kind of analysis which is the crux for applications like customer relation management.
A data warehouse is the repository of the entire organizational data and is designed to optimize reporting and analysis of the archived data [7].The operational databases are optimized for data integrity and speed of recording the business transactions with the use of relational models and normalization.The relational databases are efficient in managing the relationships between the tables and they are fast in inserts and updates when dealing with less volume of data.However, they become slower as the data size increase.
In a data warehouse, data is gathered from operational systems and retained even after the same has been purged from operational systems.The operational data is lightly summarized and modeled as facts and dimension table.Data is often stored multiple times -in their most granular form and their summarized forms are called aggregates.Further, the data is de-normalized by dimensional data modeling [9] that in turn facilitates a very quick retrieval of the same.
The data warehouse can be designed in two ways knows as star schema and snowflake schema.The snowflake schema the dimension tables are further normalized which is not the case in star schema.The snow flake schema reflect the way in which users think about data therefore the drill down and roll up analysis can be easily performed [10].The snowflake models are intuitive and easy to understand, amenable to query optimization since arbitrary n-way joins with the fact table can be evaluated by a single pass through the fact table, can accommodate for aggregate data, and are easily extensible by adding new attributes to the fact table or to one or more of the dimension tables and new dimension tables to the schema without interfering with existing database programs.
An example of data ware modeling would be a snowflake schema for remote sensing data product sales.The remote sensing data user product sales can be modeled as a fact and dimensions could be customer, satellite, sensor, product type, level of correction.The schema shown in the Figure 4 optimizes the queries for finding the summary of total amount of sales in different dimensions.If the management wants to know the total sales and total amount for each dimension can be easily retrieved from the data ware house because the total amount and sales are already pre computed for different dimensions.The star schema is easily convertible to snow flake schema by normalizing all the dimension tables.The Oracle database supports the snowflake schema which would improve the time required for data analysis.The snow flake schema design is user specific, i.e. data analysis is optimized from the perspective of the management.The data warehouse building design is to be undertaken in planned and phased manner.A corporate data model is developed keeping in view the requirements of an analyst.The extract, transform and load programs (ETL) are to be executed as when the wrinkle time (the time data will not be used further updated in the database) of the data is reached in the operational database which is normally 24 hours for standard products.These are some of the overheads for building the data ware house environment.For ever changing business orientation the analysis can be easily done using the data ware house environment than the operational databases.
V. PROPOSED HYBRID ARCHITECTURE
The data products generation at NRSC involves many heterogeneous system with operating system platform generating data products of all the remote sensing satellite right from IRS 1A to Cartosat-2.The data products for IRS1A to IRS-P6 satellite are being generated on SGI systems on IRIX operating system.The IRS P6 AWIFS products are generated on PC based Linux OS platform.The IRS P5 and Cartosat-2 products are being generated on PC based Linux platforms.In addition, there are two value additions work-centers supporting more than ten systems for value addition to the remote sensing data products.The IMS software interfaces provide services like ADIF and work-order to all data products generation and value addition systems.The other work-center users' interact with IMS software for product entry and monitoring using reports using Browser based services.The IMS software currently handles more than 100 products per day in the production chain.Considering the heterogeneity of the platforms, operating systems, complexity of the application domains and interfaces to associated processes, a hybrid approach to software architecture simplifies realization of interoperable machine independent interfaces and provides extensibility for future requirements.
Considering the advantages of technologies discussed in above sections, we propose a hybrid software architecture that is services oriented, facilitating data exchanges using XML and message exchanges between the modules using event mechanisms, as refinements to the existing IMS architecture.These approaches would enable loose coupling between the software modules.The software maintenance and deployment The hybrid architecture (Figure 5) also supports a simultaneous operation of existing computationally intensive data products generation systems with an option to reengineer the processes with state of art plug and play technologies.
A new layer known as service layer is introduced between the business layer and the presentation layer.The service layer provides necessary abstraction of underlying mechanisms for providing workflow operations such as work-orders, product routing etc., A data exchange mechanism using XML removes nativity of the data associated with the system i.e. it becomes platform independent and also de-links IMS software implementation modules from data processing scheduler software.XSU is supported in Oracle which would enable retrieval and storage of the data in to a database as an XML file.Report formatting and information content are subjected to change as per requirements of work centers resulting in software version change.Usage of XSL and DTD would result in easier formatting and validation of data and new requirements would not effect software level changes.
The present software modules interact across systems based on RMI calls that are executed synchronously.This results in communication, execution coupling between the sender and the receiver.Event based architectures decouple sender and receiver message exchanges using events which are asynchronous rather than message calls.Implementation of event based architectures requires a messaging server and maintenance of a message queue.Each sender and receiver would have to register for events to be received or raised.An event router would enable messages to be transmitted across geographical boundaries.Messages from other centers can be received and logged in a centralized server for further transmission or analysis.
The ever growing needs of management to improve data product sales requires knowledge of past data products being sold or generated.To undertake such analysis historic production data has to be archived.Data ware house technology provides a platform for storing historic in lightly summarized form.Building a data warehouse is undertaken in a controlled environment.A data model is evolved from different perspectives of analysts for storing the data warehouse data.The implementation also requires ETL programs to be developed for populating data warehouse data.Parameters for storing data in data warehouse like wrinkle time to be evolved.A data warehouse technology provides a mechanism for drill down and roll up analysis suitable for extracting knowledge from the data.The mining and extraction of knowledge from data stored in data warehouse would facilitate improved and timely managerial decisions.
VI. CONCLUSION
As has been noted earlier, software modules in existing architecture are tightly coupled.Asynchronous message passing mechanism would increase throughput for bulk data products generation as against existing synchronous message passing mechanism.Implementation of XML technologies through databases such as Oracle facilitates better and improved production chain monitoring and realization of interoperable machine independent interfaces with data products generation software.A hybrid architecture encompassing the above technologies streamlines workflow and software management at process level for ever changing business requirements.The benefits of the proposed hybrid architecture are summarized as under: 1) Service orientation facilitates loose coupling of processes between operating systems, programming languages and other technologies including middleware.2) Use of event based message exchange mechanism results in decoupling of the sender and receiver messages.Event based architectures also facilitates transmission and processing of data across geographical boundaries.This architecture also facilitates state of art event routing information security mechanisms through subscriberpublisher models.3) XML based data exchange facilitates platform independence and automated checks on well formed ness of a document and styling for report generation.4) Support for plug and play processes ensures designer would focus on interfacing and integrating different processes for each activity, thereby eliminating or reducing development time to support new missions.5) A realization of interoperable machine independent interfaces facilitates design and development and realization of reengineered processes.6) Replacing existing legacy processes with state of art reengineered processes in a plug and play environment facilitates and streamlines software maintenance activities.7) Usage of XSU technology would enable data to stored and retrieved in XML.The report generation software module maintenance is reduced using XSL.8) Implementations of data warehouse facilitates roll up and drill down analysis of production data that in turn enables business process reengineering leading to improved product generation and customer satisfaction levels by.9) The present study facilitates further study in related areas like service discovery, knowledge discovering and event routing.%appendices
Fig. 5 .
Fig. 5. Proposed hybrid architecture for work flow automation software for monitoring data product generation | 2015-09-25T17:25:03.000Z | 2015-09-25T00:00:00.000 | {
"year": 2015,
"sha1": "b167614208964a28ce871447d75f11ef9c7ec77a",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Arxiv",
"pdf_hash": "b167614208964a28ce871447d75f11ef9c7ec77a",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Computer Science"
]
} |
15171480 | pes2o/s2orc | v3-fos-license | Evaluation and Comparison of the Antibacterial Activity against Streptococcus mutans of Grape Seed Extract at Different Concentrations with Chlorhexidine Gluconate: An in vitro Study
Introduction Streptococcus mutans has been implicated as primary microorganisms which cause dental caries in humans. There has been an increased interest in the therapeutic properties of some medicinal plants and natural compounds which have demonstrated antibacterial activities. Grape is one of the plants of this group which contains tannin and polyphenolic compound. Aim To evaluate and compare antibacterial activity of grape seed extract at different concentrations with chlorhexidine gluconate against S. mutans. Materials and methods Grape seeds were extracted with ethanol/water ratio of 70:30 volume/volume. The extracts were filtered through Whatman No. 1 filter paper until it becomes colorless. Streptococcus mutans strains were taken. To check the antimicrobial properties of grape seed extract at different concentration and chlorhexidine gluconate, they were added to S. mutans strain and incubated for 48 hours than colony-forming units/mL were checked. Results Grape seed extract at higher concentration were found to be more potent against S. mutans. Chlorhexidine gluconate was found to have most potent antibacterial action compared to all different concentrations of grape seed extract. Conclusion Grape seed extract as a natural antimicrobial compound has inhibitory effect against S. mutans. How to cite this article Swadas M, Dave B, Vyas SM, Shah N. Evaluation and Comparison of the Antibacterial Activity against Streptococcus mutans of Grape Seed Extract at Different Concentrations with Chlorhexidine Gluconate: An in vitro Study. Int J Clin Pediatr Dent 2016;9(3):181-185.
INTRODUCTION
Dental caries is a chronic infectious disease that produces widespread lesions throughout the world. 1 It is a multifactorial disease in which there is interplay of three principle factors: The host (primarily the saliva and teeth), the microflora, and the substrate or diet. In evaluating the caries risk of a patient, a number of factors must be taken into consideration. Salivary counts of mutans streptococci, combined with the measurement of salivary flow rate and buffer effect and sugar consumption, are frequently used for diagnostic and predictive purpose in cariology. 2 Mutans streptococci (MS) group has been implicated as primary microorganisms which cause dental caries in humans and experimental animal models. Relying on sampling, this finding aims to isolate colony-forming units (CFU) from dental plaque and saliva. 3 Salivary MS counts rarely exceed 10 7 CFU/mL, and a highly significant correlation has been demonstrated between the salivary numbers of MS and caries prevalence. 4 In the recent past, there has been an increased interest in the therapeutic properties of some medicinal plants and natural compounds which have demonstrated anticariogenic activities in both in vitro and in vivo conditions. Among these phytoconstituents, several polyphenolic compounds like tannins (catechins) and flavonoids seem to be the most promising biomolecules. Remarkable anticariogenic potency has been observed for alkaloids. 5 Tannins are naturally occurring plant metabolites which can prevent bacterial dental plaque 6 and can enhance remineralization of dental enamel. 7 Grape is one of the plants of this group which contains tannin and polyphenolic compound. So, grape seed extract also have remarkable antibacterial properties. 5 Grape seed extract at lower concentration is ineffective against S. mutans bacterial strain. 8 The purpose of this study was to check in vitro antibacterial effect of grape seed extract against S. mutans at different concentrations and with chlorhexidine gluconate.
MATERIALS AND METHODS
The study was conducted in the Department of Paedodontics and Preventive Dentistry. Prior permission was taken from the Department of Microbiology of the university, and ethical approval was obtained from Ethics Committee of the University.
The following materials were used for the conduction of the study: • Bacterial strains: S. mutans (ATCC 25175) • Incubator (Fig. 1) • Mitis salivarius bacitracin agar medium • Disposable Petri dishes • Inoculation loop for counting CFU.
Procedure of preparing Grape Seed Extract (Vitis vinifera L.)
Grape seeds were collected from red grapes which are available in market (Fig. 2). These seeds were dried under sunlight for 2 days, and then these seeds were grounded with cold pressure technique (Fig. 3). Ground grape seeds (100 gm) was extracted with ethanol/water ratio of 70:30, vol/vol, by maceration method under stirring at 45°C for 2 hours. The extract was filtered through Whatman No. 1 filter paper. 8 It was sterilized by Millipore filter and final concentrations of grape seed extract were prepared. Grape seed extract was divided into three different experimental groups along with one positive control and one negative control group.
Study Groups
Group I: Vehicle ultrapure water (negative control) Group II: Grape seed extract (500 mg/mL) Group III: Grape seed extract Experimental groups (250 mg/mL) Group IV: Grape seed extract (125 mg/mL) Group V: Chlorhexidine gluconate (positive control).
Thirty-five tubes (5 groups) were prepared containing test compounds dilutions and incubated at 37°C for 48 hours. Each concentration (500, 250, and 125 mg/mL) of the extracts was tested. The vehicle (ultrapure water) was used as negative control, and chlorhexidine gluconate was used as positive control as a 20% (w/v) aqueous solution. Each group has 7 Petri dishes. Numbering for these Petri dishes were done like I1 to I7 for the 1st group followed by other groups.
Bacterial Strains and Growth Conditions
The tested bacterial strain was included Gram-positive strain S. mutans ATCC 25175. The study samples were inoculated on Mitis salivarius bacitracin agar medium (MSB agar), which is a highly selective medium for S. mutans, for counting CFU (Fig. 4). The tested bacterial inoculum
Antimicrobial Activity
Grape seed extract and chlorhexidine gluconate were decided by counting S. mutans CFU/mL after 48-hour incubation (Fig. 5).
STATISTICAL METHODS
The data obtained was compiled systematically, transformed from a precoded proforma to a computer, and a master table was prepared. The total data was distributed meaningfully and presented as individual tables along with graphs. Analysis of the data was made by descriptive statistics (percentages, frequency distribution) and analytical statistics using Statistical Package for the Social Sciences (SPSS) 20.0 software. Observations were subjected to statistical analysis using Krushkal-Wallis test and Wilcoxon rank sum test.
RESULTS
As Graph 1 shows, the distribution of S. mutans count was achieved after 48 hours in different groups. In vehicle ultrapure water group, maximum grade of mean value was seen which shows that it has not any bactericidal effect on S. mutans. While grape seed extract with 500 mg/mL concentration shows great amount of antibacterial effect, marked effect as antibacterial agent was noted with 2% chlorhexidine gluconate group. Table 1 shows that while vehicle ultrapure water group was compared with grape seed extract at different concentrations after 48 hours, there was statistically significant difference observed with 500 and 250 mg/mL concentrations of grape seed extract; however, no statistical significance was seen with 125 mg/mL concentration of grape seed extract group. This shows that 125 mg/mL have not any significant antibacterial activity against S. mutans bacterial strain. Marked antibacterial activity against S. mutans bacteria was seen with the group of 2% chlorhexidine gluconate which shows to be a potential antibacterial agent for S. mutans bacteria. Table 2 shows that there was no statistically significant difference seen between any concentrations of grape seed extracts. But based on mean rank value, it shows that while compared grape seed extract 500 mg/mL with 125 mg/mL, there was a marked difference between mean rank though no significant statistical difference was observed. However, based on these mean rank value 500 mg/mL concentration have more antibacterial effect than 125 mg/mL concentration of grape seed extract group. Table 3 shows the comparison of grape seed extract of different concentrations with 2% chlorhexidine gluconate as antibacterial agent for S. mutans. There were statistically significant difference seen between each group of grape seed extract (different concentrations) and 2% chlorhexidine gluconate group. But here also, based on mean rank values, maximum significant difference was noted with 125 mg/mL concentration of grape seed extract group, which shows that this group has less antibacterial effect compared to other 250 and 500 mg/mL concentration groups of grape seed extract.
DISCUSSION
In this study, the effect of grape seed extract (Vitis vinifera) with different concentrations has been evaluated against the most important bacterial strain in dental pathologies. To check their antibacterial effect, two other groups were also taken in this study, which were vehicle ultrapure water and 2% chlorhexidine gluconate.
Streptococcus mutans is the primary causal agent for dental caries specially in the initiation and development stages, 9 and this microbe was first described in 1924. 10 Grape (Vitis vinifera) seeds are considered rich source of polyphenolic compounds, mainly monomeric catechin and epicatechin, gallic acid and polymeric, and oligomeric procyanidins. 11 Grape phenolics are simple molecules, such as hydroquinone, pyrocatechol, caffeic acid, ferulic acid, p-coumaric acid, gallic acid, ellagic acid, and resveratrol. 12 Furthermore, grape seed extract is a rich source of diverse bioflavonoids, collectively known as grape seed proanthocyanidins extract. 13 Polyphenols are well documented to have microbicidal activities against a huge number of pathogenic bacteria. 14,15 The mechanism of polyphenols toxicity against microbes may be related to inhibition of hydrolytic enzymes (proteases and carbohydrolases) or other interactions to inactive microbial adhesions, cell envelope transport proteins, and nonspecific interactions with carbohydrates. 15 In present study, chlorhexidine gluconate was taken as a positive effective agent against S. mutans because, as per Addy et al, 16 chlorhexidine gluconate has significantly antibacterial effect against S. mutans, which was in accordance with present study. The different concentrations of grape seed extract (500, 250, and 125 mg/mL) were significantly less effective than 2% chlorhexidine gluconate.
Grape seed extract at lower concentration (125 mg/mL) was not having any significant antibacterial effect while comparing with negative control. It was supported by the study by Mirkarimi et al,8 which focused that grape seed extract with lower concentration has not showed any bactericidal or bacteriostatic effects against S. mutans.
Thimothe et al 17 found phenolic compounds of grapes inhibit the biological activity of S. mutans which supported that grape seed extract with 500 and 250 mg/mL concentrations has an antibacterial effect.
In the present study, we have used the macrodilution broth method to evaluating the antimicrobial activity. This
IJCPD
difference between methods may have an important role in making variation in results. Therefore, based on all these findings it has been proved that grape seed extract at higher concentration acts as a natural antimicrobial compound, derived from V. vinifera, which has specifically inhibitory effect against S. mutans bacteria.
CONCLUSION
• Grape seed extract has antibacterial effect at 250 and 500 mg/mL concentrations, but it was significantly less compared to chlorhexidine gluconate. • Grape seed extract was not that much effective as an antibacterial agent compared to chlorhexidine gluconate. | 2018-04-03T00:55:59.404Z | 2016-07-01T00:00:00.000 | {
"year": 2016,
"sha1": "f482ee204655658f1184b3dd4efc7a96851ea1c7",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.5005/jp-journals-10005-1360",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "f482ee204655658f1184b3dd4efc7a96851ea1c7",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
15568673 | pes2o/s2orc | v3-fos-license | Exploring Treebank Transformations in Dependency Parsing
This paper presents a set of experiments performed on parsing the Basque Dependency Treebank. We have concentrated on treebank transformations, maintaining the same basic parsing algorithm across the experiments. The experiments can be classified in two groups: 1) feature optimization, which is important mainly due to the fact that Basque is an agglutinative language, with a rich set of morphosyntactic features attached to each word, 2) graph transformations, ranging from language independent methods, such as projectivization, to language specific approaches, as coordination and subordinated sentences, where syntactic properties of Basque have been used to reshape the dependency trees used for training the system. The transformations have been tested independently and also in combination, showing that their order of application is relevant. The experiments were performed using a freely available state of the art data-driven dependency parser [11].
Introduction
This work presents several experiments performed on dependency parsing of the Basque Dependency Treebank (BDT) [1]. Several syntactic analyzers based on dependencies have been developed, with proposals ranging from systems that directly construct dependency structures [9] to other systems based on the more traditional constituency structures that allow the extraction of dependencies [2]. The present work has been developed in the context of dependency parsing exemplified by the CoNLL 1 shared task on dependency parsing in years 2006 and 2007 [12], where several systems had to compete analyzing data from a typologically varied range of 11 languages. The treebanks for all languages were standardized using a previously agreed CONLL-X format (see Figure 1). BDT was one of the evaluated treebanks, which will allow us to make a direct comparison of results.
Many works on treebank parsing have dedicated an effort to the task of pre-processing training trees [4,13]. This paper extends these works, applying treebank 1 CoNLL: Computational Natural Language Learning. transformations [7,10] to a morphologically rich, agglutinative language.
The rest of the paper is organized as follows. Section 2 presents the main resources used in this work, including the BDT and a data-driven open source parser. Section 3 presents the different proposals for Treebank transformation that have been devised in order to improve the parser's accuracy. Next, section 4 will evaluate the results of each transformation. Section 5 examines related work, and the last section outlines the main conclusions.
Resources
This section will describe the main elements that have been used in the experiments. First, subsection 2.1 will present the Basque Treebank data, while subsection 2.2 will describe the main characteristics of Maltparser, a state of the art and data-driven dependency parser.
The Basque Dependency Treebank
BDT [2] can be considered a pure dependency treebank, as its initial design considered that all the dependency arcs would connect sentence tokens. Although this decision had consequences on the annotation process, its simplicity is also an advantage when applying several of the most efficient parsing algorithms. The treebank consists of 55,469 tokens forming 3,700 sentences, 334 of which were used as test data 2 .
(1) Etorri de-la eta joan de-la esan zien. come has-that and go has-that tell he-to-them He told them that he has come and he has gone. Figure 1 contains an example of a sentence (1), annotated in the CONLL-X format. The text is organized in eight tab-separated columns: word-number, form, lemma, category (coarse POS), fine-grained POS, morphosyntactic features, and the dependency relation (headword + dependency). Basque is an agglutinative language, and it presents a high power to generate inflected word-forms.. Verbs offer a lot of grammatical information, as each verb form conveys information about the subject, the two objects, as well as the tense and aspect. As a result of this wealth of information contained within word-forms, complex structures have to be built to represent complete morphological information at word level. The information in Figure 1 has been simplified due to space reasons, as typically the Features column will contain lots of morphosyntactic features, which are relevant for parsing.
Maltparser
Maltparser [11] is a state of the art dependency parser that has been successfully applied to typologically different languages and treebanks. While several variants of the base parser have been implemented, we will use one of its standard versions (Maltparser version 0.4).
The parser is based on two basic data-structures. A stack stores the dependency-graph that is formed by linking the input sentence's words, while an input sequence contains the elements that have not yet been examined. The basic algorithm applies a set of four parsing actions (shift into the stack, reduce, left-arc, or right-arc) and obtains deterministically a dependency tree in linear-time in a single pass over the input. To determine which is the best action at each step, the parser uses history-based feature models and discriminative machine learning. In all the following experiments, we made use of a SVM 3 classifier. The specification of the features used by the classifier, allows to select the number of elements of both stack and input to be considered during learning, and also indicates the kind of information for each element, which can in principle be any kind of data described in Figure 1 (such as word-form, lemma, category or morphosyntactic features).
Experiments
We have performed two classes of experiments. First, we have tested the effect of simplifying morphosyntactic features. Second, we have applied three different tree transformations to the treebank.
Feature optimization
Basque is an agglutinative and morphologically rich language, and this opens the way to experiment with many combinations of morphological features. The original annotation of the BDT contained 359 different 3 We used SVM with a polinomial kernel of degree 2 (LIVSM parameters: -s 0 -t 1 -d 2 -g 0.2 -c 0.4 -r 0 -e 0.1 -S 0) morphosyntactic feature values. This led us to experiment with several modifications: • Grouping complex features into a set of simpler ones. For example, complex case suffixes were simplified, as in DAT_INS (a complex case suffix that is internally formed by the dative case followed by the instrumental case), which was changed to INS(trumental), as the last case suffix is syntactically more relevant. • Deletion of several features that were interesting in the description of the internal morphology of a word but were not relevant for syntactic analysis.
• The original annotation of 359 values marked them as totally unrelated values, without indicating which feature (say, case) each value was an instance of. We added a label prefix to each value, which allowed us to experiment the inclusion of a feature. For example, ABS(olutive) was transformed to CASE:ABS.
Graph transformations
Algorithms for dependency-tree transformations are applied in a black box manner in four steps: 1) apply the transformation to the training data, 2) train a parser on the transformed data, 3) parse the test set, and 4) apply the inverse transformation to the parse output, so that the final evaluation is carried over the original tree representations.
We will experiment with three different tree transformations, ranging from a language independent method in one extreme, like projectivization, to a pure language specific approach on the other, going through a transformation on coordinated structures, which lies in the middle, as coordination is present in all languages but needs an adaptation depending on each language and parser.
Projectivization (T P )
Several parsing algorithms are unable to deal with nonprojective arcs, that is, arcs that cross each other. The solution can be either to design a modified algorithm (e.g., Covington's, see [11]) or transform the tree into a projective one. This option is more attractive if the original algorithm is simple, efficient and accurate, as is the case with Nivre's transition-based algorithm [11]. This transformation is totally language independent, and can be considered a standard transformation. We include it because: • We want to test the effect of consecutive transformations against the base treebank. • Its performance on BDT has been already tested [13]. This is in accordance with BDT having a 2.9% of non-projective arcs.
[10] proposes three types of projective transformations: path, head, and head+path. After testing them we found that the head transformation gave the best results, so this will be the one used in the following work.
Subordinated sentences (T S )
Subordinated sentences are formed in Basque by attaching the corresponding morphemes to verbs, either the main verb (non-finite verbs) or the auxiliary verb (finite verbs). However, in BDT the verbal elements are organized around the main verb (semantic head) while the syntactic head corresponds to the subordination morpheme, which appears usually attached to the auxiliary. Its main consequence for parsing is that the elements bearing the relevant information for parsing are situated far in the tree with respect to their head. In Figure 2, we can see that the morpheme -la, indicating the presence of a subordinated completive sentence, appears down in the tree, and this could affect their correct attachment of the two coordinated verbs to the conjunction (eta), as conjunctions should link elements showing similar grammatical features (-la in this example). Similarly, it could affect the decision about the dependency type of eta with respect to the main verb esan (to say), as the dependency relation ccomp_obj is defined by means of the -la (completive) morpheme, far down in the tree. Figure 3 shows the effect of transforming the original tree given in Figure 2. The subordination morpheme (-la) is separated from the auxiliary verb (da), and is "promoted" as the syntactic head of the subordinated sentence. New arcs are created from the main verbs (etorri and joan) to the morpheme (which is now the head), also adding a new dependency relation (SUB). Figure 3 shows that the tree suffers important transformations. However, as the order of sentence elements is maintained, the transformation does not so greatly affect the annotated treebank (see Figure 1), and the transformations can be described by changes in dependency links and splitting of words together with each morpheme's morphological features.
A similar solution was proposed by [6] when parsing the Prague Dependency Treebank, where relative clauses are annotated introducing an additional level with a new category (SBAR), that helps distinguish simple VPs from relative subordinated sentences. We have extended this idea to most types of subordinated sentences, as relative clauses, temporal clauses and completive, indirect interrogative, causal, adversative and modal clauses. An important difference with respect to this work is that in [4] the change is performed on the shape of the (constituency) trees, not affecting the input sequence of words, while in our case the morphemes are detached from the root words. Transformations on finite verbs are similar to those in Figure 3 (e.g., dela is transformed to da(AUXV) + -la(COMPletive)). Non-finite verbs are transformed separating the suffix from the main verb (so, etortzea is transformed to etorri(V) + -tzea(COMPletive)).
Coordination (T C )
This transformation can be considered general but it is also language dependent, as it depends on the specific configurations present in each different language, mainly the set of coordination conjunctions and also the types of elements that can be coordinated, together with their morphosyntactic properties (such as head initial or head final). Basque is considered a head final language, where many important syntactic features, like case or subordinating conjunction are located at the end of constituents. Coordination in BDT has been annotated in the so called Prague Style (PS, see Figure 4), where the conjunction (represented as S in Fig. 4) is taken as the head, and the conjuncts depend on it. [10] advocates the Mel´cuk style (MS) for parsing Czech, taking the first conjunct as the head, and creating a chain where each element depends on the preceding one (they also test its effectiveness with Arabic and Slovene). Being Basque head-final, we propose two symmetric variations of MS.
In the first one (v(ersion)1 in Figure 4) the coordinated elements will all be dependents of the last conjunct (which will be the head), going from left to right. In the second version (v2), the final conjunct is again the head, and the coordination conjunction dependent on it, while the rest of the dependents attach to the conjunction. Figure 5 shows the effect of applying the v1 transformation to the tree in Figure 3. Figure 5 shows that an important number of arcs can be modified. A negative consequence could be that the original tree structure could be lost. This would have the effect that the expected improvement could be compensated by the noise introduced by the algorithms. In this regard, we have evaluated that the transformations can be recovered with more than 97% precision.
Evaluation
Training and testing of the system have been performed on the same datasets presented at the CoNLL 2007 shared task, which will allow for direct comparison of the results (see Table 1). The best system obtained a score of 76.94% on Labeled Attachment Score (LAS). This system combined six different variants of a base parser (Maltparser), being the first system in 5 (out of 11) languages, competing with 19 systems in the case of Basque.
Our work will consist in applying different treebank transformations using the same treebank and the same base parser, so we can consider the last system in Table 1 as our baseline. The singlemalt parser described in [8] obtained the fifth position at CoNLL 2007. This system tried to optimize Maltparser's results on BDT by tuning parameters and selecting different training configurations. This system applied the projectivization transformation (T P ).
Evaluation was performed dividing the treebank in two sets: training set (50,000 tokens, using 10-fold cross validation) and test set (5,000 tokens). Figure 5: Transformed tree (T S + T C(v1) ).
gives 66.89% LAS. The second row shows the results using the full set of morphological features, which we take as the baseline, as it presents a system optimized on the basic BDT version (regarding coordination, this version contained the original Prague Style annotation). The second and third rows in Table 2 can be considered a strong baseline, as the CoNLL systems tested many variants of training and parse configurations, mainly taking into account morphological features, that are crucial when dealing with morphologically rich languages. The table shows the LAS scores calculated on several of the multiple combinations that were experimented. Rows 5, 6, and 7 show the effect of transforming coordinate structures, compared to the baseline (PS, row 2). MS presents the worst results (-4.53 lower than PS on the test set). They also shows that v1 and v2 transformations are more suitable than PS as the target representation. A partial explanation can be found in the effect of "short-dependency preference", as MS presents the longest average dependency-length, followed by PS, v2 and v1. The rest of the tests were performed using the best transformation (v1).
The results show how the application of all kinds of transformations improves significantly the results, giving a best score of 76.80% (14 th row) on the test set, which is near the best CoNLL 2007 (combined) system.
The table also shows how the order of application of the tree transformation affects the overall results in both cross validation and test set. For example, T S is dependent on T P , as the results vary changing their relative order of application. We corroborated this result when examining the transformed treebanks, and found that T S leads to loss of projectivity, adding a new set of non-projective arcs. This implies that the results are better if T S precedes T P . We made a study of the relations involved between subordinated sentences and their heads, such as cmod (clausal modifier) or xcomp_subj (clausal complement acting as subject), and found that T S maintained recall on the set of subordinating dependency relations and also augmented precision significantly (for dependencies that link subordinate and main sentences, recall and precision increase 3.05% and 4.13%, respectively).
Related work
Collins [4] applied his parser to Czech, a highly-inflected language, which shares several characteristics with Basque. [6] applies Collin's parser to Spanish, concluding that morphological information improves the analyzer.
[7] experiments the use of several types of morphosyntactic information in the analysis of Turkish, showing how the richest the information improves precision. In a related work, Eryiğit and Oflazer (2006) also show that using morphemes as the unit of analysis (instead of words) gets better results, in line with T S results. [6] conclude that an integrated model of morphological disambiguation and syntactic parsing in Hebrew Treebank parsing, improves the results of a pipelined approach. Dividing words into morphemes fits into this idea, as we postpone the treatment of subordination morphemes from morphology to syntax. [9,10] present the application of pseudoprojective and coordination transformations to several languages using maltparser, showing that they improves the results. As for coordination, they only test the PS and MS variants.
Conclusions
We have tested a number of transformations in the Basque Dependency Treebank, such as: • Feature optimization. Basque is a morphologically rich language and presents many opportunities to tune the set of morphosyntactic features, adding, deleting, generalizing or specializing features. • Projectivization. This is a language independent transformation already tested in several languages. • We also tested two language specific transformations, such as coordination and modification of subordinated sentences. They cause important changes in the trees, but also help to improve results. In the case of coordination, we have shown that it is dependent on the specific features of each language. • We also have found that the order of transformations can be relevant. This effect opens the study of which factors affect the order of transformations, as the creation of non projective arcs or the average length of dependency arcs.
Overall, one of the applied transformations is totally language-independent (projectivization, T P ). T C (coordination) can be considered in the middle, as it depends on the general characteristics of the language. Finally, feature optimization, and the transformation of subordinated sentences (T S ) are specific to the treebank and intrinsecally linked to the agglutinative nature of Basque. The transformations affect a considerable number of dependencies (between 5.94% and 11.97% of all arcs). The best system, after applying all the transformations, obtains a 76.80% LAS (2.24% improvement over the baseline) on the test set, which is the best reported result for Basque dependency parsing using a single parser, and close to the better published result for a combined parser (76.94%).
The results on feature optimization do not allow us to extract a definite conclusion, as it does not help on development data but gives an improvement on test data. [7] argues that "adding inflectional features as atomic values was better than taking certain subsets with linguistic intuition …" due to the ability of SVMs to do this successfully. However, Table 2 shows that feature optimization slightly increases LAS when transformations are combined (see the improvement in T S + T P + T C with and without F+).
T S + T P shows how the use of morphological information gives a substantial improvement in accuracy, even when the number of modified dependency links is modest in relation with the full size of the treebank (this transformation affects 5.94% of all arcs). Another interesting result is that when applying several types of transformations, the order of application is significant, as earlier transformations can condition the following ones. This has been demonstrated in the case of T S , which introduces a new set of non-projective arcs, and does not give an improvement unless it is combined with T P . The relations among the rest of the transformations deserve future examination, as the actual results do not allow us to extract a precise conclusion. For example, T C seems to be independent of the rest of transformations. | 2014-10-01T00:00:00.000Z | 2009-09-01T00:00:00.000 | {
"year": 2009,
"sha1": "f3201e7b0de3bcfc416f964afc6b62f64c7d5c54",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "ACL",
"pdf_hash": "7a4ea5520711fe7b829a4db46864c7562d91b7d7",
"s2fieldsofstudy": [
"Computer Science",
"Linguistics"
],
"extfieldsofstudy": [
"Computer Science"
]
} |
232405712 | pes2o/s2orc | v3-fos-license | Mechanical, Wear and Thermal Behavior of Polyethylene Blended with Graphite Treated in Ball Milling
Additive manufacturing, civil, and biomechanical applications are among the most important sectors, where the filler’s presence can significantly improve the quality of polymeric products blends. The high market demand of new low-cost material to be used as shock absorbers and mechanical joints arouses our curiosity to study a relatively common commercial polymer and filler. The possible improvement by blending high-density polyethylene (HDPE) and graphite was investigated for these sectors. To achieve this objective, we have prepared HDPE/graphite nanocomposites following mechanical treatment to understand which parameter provides the researched properties. As widely reported in the literature, milling treatment leads to the decrease of the particle size and the exfoliation of graphitic layers. Therefore, graphite has been previously treated with a ball mill for different times (1–16 h) to enhance its lubricating action. We checked an improvement in stiffness, yielding strength, thermal stability, and, in particularly, wear resistance that increased by 65% with respect to that of polyethylene (PE). A treatment time of eight hours in ball milling could be enough to give an appreciable improvement. The wear behavior of HDPE with treated graphite has not been deeply investigated so far, and it could be important because HDPE is considered a “carrier polymer” for different low-friction applications.
Introduction
Polyethylene (PE) is a chemically inert material with a low coefficient of friction, which is flexible, ductile, mechanically resistant, tough, nontoxic and sterilizable with gas or disinfectants [1,2]. It is easily workable with the normal technologies of transformation of thermoplastic materials [3]. Among all the various types of PE, high-density polyethylene (HDPE) is white, translucent and very crystalline. Thanks to its remarkable properties of chemical inertia, HDPE is used to produce biomaterials (from containers for chemical or pharmaceutical substances, bags, drainage tubes, disposable gloves, catheters, etc.) that come into direct contact with both cells and tissues [4].
To further improve the physical and mechanical features of polymers, it is possible to add carbonaceous fillers such as graphite, carbon fibers, carbon nanotubes, and graphene [5][6][7]. It is known that carbon nanofibers and graphite, although they do not offer the same mechanical and physical improvement compared to nanotubes and graphene, significantly reduce high costs and complex processing associated with the production of carbon nanotubes and graphene [8]. Naturally unmodified graphite (NG) is an allotropic form of carbon organized in hexagonal planes made up of carbon atoms, each covalently bonded to three neighboring atoms. These planes are sheets that are only one atom thick and make up the layers of graphene [9,10]. Graphite is, hence, the accumulation of monatomic layers of graphene arranged with one on top of the other, joined by weak Van der Walls bonds. Through the chemical path, these bonds can be altered by inserting acidic components into the solution that catalyze each other and form a pressure difference such as breaking these surfaces (Hummer method) [11,12]. In this way, an exfoliated or expanded graphite (EG) layer can be produced. Similarly, stacks of nanosheets can be obtained by rapid heating of a Graphite Intercalation Compound (GIC) [13]. Exfoliated or EG is also called as graphite nanoplatelets (GNPs), graphite nanosheets (GNs) or graphite nanoflakes (GNFs), being a type of graphitic nanofillers composed of stacked two-dimensional (2D) graphene sheets and having an outstanding electrical, thermal, and mechanical properties [14].
In addition to chemical or thermal methods, the exfoliation of graphite can take place with a mechanical procedure, such as the micromechanical exfoliation of graphite. These procedures are based on the application of a force to the surface of oriented graphite crystals, so that the attractive Van der Waals force which binds one of graphene planes above the other is contrasted to unfold the crystalline layers until a single layer is obtained [11,15]. The "Scotch method" (Nobel Prize, 2004) tries to open these planes through an adhesive action from the contact between graphite with some materials with the consequent unfolding of the graphene along the contact surface between the two materials. This method demonstrates that, directly from graphite, it is possible to exfoliate graphene or to unfold it in planes with a monatomic thickness of carbon sp2 simply by applying the tearing action of one layer of a scotch tape on a graphite plane and exerting a breakout force [15].
Graphite is usually added in several different polymers, both thermosets (such as epoxy resin, and polyester resin) and thermoplastics (such as polystyrene, polymethyl methacrylate, ethylene vinyl acetate, polyurethane, and polyvinylidene fluoride). The common result is that the treated graphite positively changes the polymer's mechanical and insulating features [8,9].
Some authors have specifically studied the effect of graphite fillers on the tensile mechanical and electrical features of HDPE-based composites [16][17][18][19][20][21]. They were usually obtained by melt blending. The authors employed different weight amounts of graphite fillers (type: NG, EG, or GNPs) [9,14]. Graphite fillers were of different sizes, different distributions of particle size, and different specific surface areas. They checked the changes in electrical, thermal, and tensile mechanical properties. The results suggested that these features have been generally improved compared to those of neat HDPE. It was also pointed out that the worsening of other properties (or the lower-than-expected result) could be attributed to the poor affinity between the polar graphite and the nonpolar matrix during the realization of the nanocomposite [17]. Thus, particle volume fractions, aspect ratio, number, and distribution of particles, type of the boundary between the filler and the polymeric matrix, and particle's shape have great effects on the resulting nanocomposite properties [22][23][24].
In addition, Jiang and Drzal investigated the flexural features and the impact strength of HDPE reinforced with exfoliated GNPs [25]. The authors highlighted the importance of filler dispersion in the polymer matrix and the filler's adhesion to the matrix in order to have improved flexural features. Graphene can be functionalized to obtain graphene oxide and to decrease the difference in the polarity of components in the polar matrix.
Graphite could be also considered to improve the wear resistance of polymers, since it is an additive capable of increasing tribological properties [26][27][28]. This occurs thanks to the known lubricating properties of carbonaceous layers [29].
Thus, in this paper we prepared a nanocomposite blend made by HDPE and ball milled graphite (obtained by mechanical treatment in a ball milling machine for different times and with reduced particle's size). The nanocomposites were characterized by torque, tensile, wear, and calorimetric analysis. To the best of our knowledge, the improvement of the wear resistance property of HDPE with BMG has not been deeply investigated so far. Applications of low-friction HDPE are, for instance, to produce water pipelines with improved fluo-dynamic [30] or work as a "carrier-polymer" which is easily mixable with a specific filler and then dispersible in a higher-viscosity and "difficult-to be-processed" polymer, such as the ultrahigh-molecular-weight polyethylene (UHMWPE). Then, the blend-filled HDPE/UHMWPE results in a higher-wear-resistance PE for biomedical use [26,[31][32][33] or for use in civil engineering. An example application in civil engineering is the pendulum isolator, a seismic isolation device used in buildings and bridges for the earthquake protection [34,35].
Materials and Methods
Graphite powder (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany); pureness: 99.9%; code: 282863) was previously ground by a ball mill (Retsch, model: v5001) at a frequency of 20 Hz for varied times (i.e., 1, 2, 4, 6, 8, and 16 h), to give shear and compression forces also useful to separate graphene layers from the graphite bulk and to crush the graphite powder in smaller size (Figure 1a). The ball mill was composed of two jars, of which the volume was 25 mL. The spherical stainless-steel ball (inserted in each jar) had a diameter of 15 mm and a volume of 14.1 mL. Furthermore, 1 g of graphite was inserted into each jar for its mechanical treatment. Ball milling treatments were set for a maximum consecutive time of 1 h, always followed by a rest period of 15 min before restarting the next treatment (Figure 1b). Graphite particle size was evaluated by dynamic light scattering (DLS) measurements using a Malvern Zetasizer Nano S instrument equipped with 4 mW He-Ne laser operating at a wavelength of 633 nm. Measurements were performed on samples dispersed in ethanol at 20 • C, with a detection angle of 90 • and processed with Zetasizer software in z-average mode. Graphite's morphology was observed by a scanning electron microscope (FEI Quanta FEG450 microscope). The scanning electron microscope was operated at an accelerating voltage of 15 kV and in low-vacuum mode. The samples adhered to aluminum holders by means of a graphitic adhesive. Images were taken at magnifications of 15 and 150 Kx. provement of the wear resistance property of HDPE with BMG has not been deeply investigated so far. Applications of low-friction HDPE are, for instance, to produce water pipelines with improved fluo-dynamic [30] or work as a "carrier-polymer" which is easily mixable with a specific filler and then dispersible in a higher-viscosity and "difficult-to be-processed" polymer, such as the ultrahigh-molecular-weight polyethylene (UHMWPE). Then, the blend-filled HDPE/UHMWPE results in a higher-wear-resistance PE for biomedical use [26,[31][32][33] or for use in civil engineering. An example application in civil engineering is the pendulum isolator, a seismic isolation device used in buildings and bridges for the earthquake protection [34,35].
Materials and Methods
Graphite powder (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany); pureness: 99.9%; code: 282863) was previously ground by a ball mill (Retsch, model: v5001) at a frequency of 20 Hz for varied times (i.e., 1, 2, 4, 6, 8, and 16 h), to give shear and compression forces also useful to separate graphene layers from the graphite bulk and to crush the graphite powder in smaller size (Figure 1a). The ball mill was composed of two jars, of which the volume was 25 mL. The spherical stainless-steel ball (inserted in each jar) had a diameter of 15 mm and a volume of 14.1 mL. Furthermore, 1 g of graphite was inserted into each jar for its mechanical treatment. Ball milling treatments were set for a maximum consecutive time of 1 h, always followed by a rest period of 15 min before restarting the next treatment (Figure 1b). Graphite particle size was evaluated by dynamic light scattering (DLS) measurements using a Malvern Zetasizer Nano S instrument equipped with 4 mW He-Ne laser operating at a wavelength of 633 nm. Measurements were performed on samples dispersed in ethanol at 20 °C, with a detection angle of 90° and processed with Zetasizer software in z-average mode. Graphite's morphology was observed by a scanning electron microscope (FEI Quanta FEG450 microscope). The scanning electron microscope was operated at an accelerating voltage of 15 kV and in low-vacuum mode. The samples adhered to aluminum holders by means of a graphitic adhesive. Images were taken at magnifications of 15 and 150 Kx. Then, HDPE in pellets (supplied by Versalis; code: ERACLENE-MP90) and the graphite powder ball-milled at the different times were put inside the static mixer chamber of a Brabender Plasticorder (model PL2000) at 180 °C at a speed rate of 30 rpm and for Then, HDPE in pellets (supplied by Versalis; code: ERACLENE-MP90) and the graphite powder ball-milled at the different times were put inside the static mixer chamber of a Brabender Plasticorder (model PL2000) at 180 • C at a speed rate of 30 rpm and for a mixing time of 15 min. The ball-milled graphite amount was fixed at a 0.3 weight percentage in all the blends to have the best improvement in mechanical behavior, according to the experimental evidences of [36,37]. These authors highlighted in their studies that this filler amount was optimal to improve the polymer's mechanical behavior (tensile strength, fracture strain, and yield strength). The obtained blends were codified as "PE-G" followed by a number, which represented the time in the ball milling treatment of graphite, as resumed in Table 1.
The torque value (expressed in Nm) of each material during the mixing time was recorded. The torque (that was the index of the viscosity variation of the blended materials in a molten state) was measured at predetermined time intervals (i.e., one minute) with a transducer interfaced with the specific Brabender software operated on a PC.
The materials obtained from the mixing process were placed in a stainless-steel mold. The materials with a length of 12 cm, a width of 12 cm, and a thickness of 1 mm were used for the tensile test, and the materials with a length of 2 cm, a width of 2 cm, and a thickness of 2 mm were employed for the wear test. Subsequently, they were compressed at 180 • C and 100 bar for 10 min by means of a hot press machine (DGTS srl, Verduggio, Monza Brianza, Italy), with Teflon release films (thickness: 300 µm) and subsequently water-cooled. Dog bone samples for the tensile test were obtained from a Ray-Ran cutter (according to the ASTM D-638 standard).
The static tensile tests of the pure HDPE and the one blended with ball-milled graphite at different times (from 0 to 16 h which was the highest ball milling time) were performed using a Lloyd LR10K universal machine. Specimens used had the type V geometry, according to the ASTM D638-10. The crosshead speed was 10 mm/min. Tensile mechanical parameters, obtained from the resulting stress-strain curves, were as following: Young's modulus (E, MPa), yield stress (σ y , MPa), yield strain (ε y , %), stress at break (σ b , MPa), strain at break (ε b , %), work at rupture (W b , J), and maximum load (Load, N). Resulting values were the average of 10 samples for each test.
Wear resistance measurements were performed in a pin-on-disc wear tester in air and at room temperature by using a Ti-6Al-4V alloy with 2 mm in diameter. The pin-on-disc system gave a circular-shape wear trajectory with a testing load of 30 N, speeds of 0.25, 0.50, and 1.00 rad/s, and a test duration of 2500 turns for each measurement. For each sample, the specific wear rate Wsp (mm 3 /Nm) was calculated as: where ∆m (mg) is the mass loss of the specimen, ρ(g/mL) is the density, F n (N) is the normal load, and L (m) is the total sliding distance. The final value of the specific wear rate Wsp was determined by the average of the Wsp values of n.3 polymeric samples (for each nanocomposite obtained) [29,30]. The mass loss and the density were evaluated by a highsensitivity electronic weighing balance (Explorer pro, OHAUS Corporation, Parsippany, NJ-USA, EP 214C) with an accuracy of 10 4 g. The density of pure PE was 0.9604 g/cm 3 , and that of the nanocomposite with 0.3 wt % of ball milled graphite was 0.965 g/cm 3 . Thermal analyses were conducted by means of differential scanning calorimetry (DSC) using a TA Instruments DSC Q100, from room temperature to 200 • C, with a heating rate of 10 • C/min and water cooling. The crystalline degree of HDPE, χ, was calculated using the following equation: where ∆H m (J/g) is the melting enthalpy, ϕ is the weight fraction of the studied material and ∆H 0 of 293 (J/g) is the theoretical enthalpy of fusion of a polymer crystal with infinite extension [38].
The TGA of the graphite samples was performed by an SDT Q600 thermogravimetric analyzer, in argon from 50 to 900 • C at a rate of 20 • C/min. by a high-sensitivity electronic weighing balance (Explorer pro, OHAUS Corpo Parsippany, NJ-USA, EP 214C) with an accuracy of 10 4 g. The density of pure P 0.9604 g/cm 3 , and that of the nanocomposite with 0.3 wt % of ball milled graph 0.965 g/cm 3 . Thermal analyses were conducted by means of differential scanning calor (DSC) using a TA Instruments DSC Q100, from room temperature to 200 °C, with ing rate of 10 °C/min and water cooling. The crystalline degree of HDPE, χ, was lated using the following equation:
Results and Discussion
where ΔHm (J/g) is the melting enthalpy, φ is the weight fraction of the studied material a ΔH0 of 293 (J/g]) is the theoretical enthalpy of fusion of a polymer crystal with extension [38].
The TGA of the graphite samples was performed by an SDT Q600 thermograv analyzer, in argon from 50 to 900 °C at a rate of 20 °C/min. SEM morphological investigations were performed on the pure and ball-milled graphite at different times. In Figure 3a,b (sample PE-G0) and in Figure 3c,d (sample PE-G1), we can see large planes of graphite, which were nearly comparable in width or slightly less after one hour of graphite ball milling treatment. From these images, it is evident that the mechanical treatment did not show significant variations after one hour compared to the pure graphite, since the graphitic planes, wide and extended (greater than 200 nm) were always observed. A similar situation was repeated after four hours of the treatment. To highlight the significant fragmentation of graphene planes, a mechanical treatment for longer times, at least 8 h (PE-G8 sample in Figure 3g,h) or even better still after 16 h (PE-G16 sample in Figure 3i,l), was required. The fragmentation into very small particles was very evident after 16 h of mechanical treatment, as the large graphitic layers disappeared in favour of small clusters smaller than 200 nm (as discussed below). To verify the correlation between the mechanical, calorimetric, and wear properties of PE and PE-BMG composites with the effective size of the filler particles, a DLS analysis was conducted. It allows measuring the average particle size of graphite as the mechanical treatment time increased. The pure sample had an average particle size of approximately 340 nm. Then, the size decreased progressively to about 322, 295, 255, 250, 220, and 190 nm after 1, 2, 4, 6, 8, and 16h, respectively, (see Figure 4a). The DLS analysis showed a drastic reduction in size after 8 h of treatment (−40%) compared to that in the pure sample, but only a further small variation after 16 h (−51%). As shown in Figure 4b, the higher the mechanical treatment time, the lower the average diameter of the particle size. The DLS results are numerically in agreement with what was observed through the SEM analysis. The morphological analysis with SEM has in fact highlighted a drastic reduction of the graphite particles thanks to the mechanical treatment, both after 8 h and even more after 16. was conducted. It allows measuring the average particle size of graphite as the mechanical treatment time increased. The pure sample had an average particle size of approximately 340 nm. Then, the size decreased progressively to about 322, 295, 255, 250, 220, and 190 nm after 1, 2, 4, 6, 8, and 16h, respectively, (see Figure 4a). The DLS analysis showed a drastic reduction in size after 8 h of treatment (−40%) compared to that in the pure sample, but only a further small variation after 16 h (−51%). As shown in Figure 4b, the higher the mechanical treatment time, the lower the average diameter of the particle size. The DLS results are numerically in agreement with what was observed through the SEM analysis. The morphological analysis with SEM has in fact highlighted a drastic reduction of the graphite particles thanks to the mechanical treatment, both after 8 h and even more after 16. Finally, the TGA analysis (Figure 4c) showed an increase in weight loss for the ballmilled graphite (ca. 35 wt %) with respect to the untreated one (ca. 2 wt %), suggesting that an oxidation reaction occurred during the ball milling treatment. It can be seen that the degree of oxidation was independent of the ball milling time and the treatment for a longer time (16 h) did not improve the oxidation with respect to the lower-one treatment (2 h). Therefore, the quiet period of 15 min that we used among the one-hour ball milling treatments of graphite powder described in Section 2 (Materials and Methods) was useful to avoid unwarranted progressive graphite's oxidation with the increase of milling time. Finally, the TGA analysis (Figure 4c) showed an increase in weight loss for the ballmilled graphite (ca. 35 wt %) with respect to the untreated one (ca. 2 wt %), suggesting that an oxidation reaction occurred during the ball milling treatment. It can be seen that the degree of oxidation was independent of the ball milling time and the treatment for a longer time (16 h) did not improve the oxidation with respect to the lower-one treatment (2 h). Therefore, the quiet period of 15 min that we used among the one-hour ball milling treatments of graphite powder described in Section 2 (Materials and Methods) was useful to avoid unwarranted progressive graphite's oxidation with the increase of milling time.
Results and Discussion
The stress-strain graphs of the pure PE and all the mixtures (PE-G0, PE-G1, PE-G2, PE-G4, PE-G8, and PE-G16) are shown in Figure 5. The PE-G0 sample (PE mixed with pure graphite, not mechanically treated) showed a different stress-strain curve than the pure PE. In fact, an increase in the slope of the initial section and an increase in stiffness were observed (Young's elastic modulus increased from about 500 to about 900 MPa), as visible in the magnified graph in the frame of Figure 5. The mechanical strength, both at yield and at break, decreased from approximately 28 to approximately 25 MPa and from approximately 16 to approximately 12 MPa, respectively. Strain at break decreased considerably from approximately 1200% to approximately 22% with a corresponding collapse of the work at break from approximately 24 to approximately 7 J. The stress-strain graphs of the pure PE and all the mixtures (PE-G0, PE-G1, PE-G2, PE-G4, PE-G8, and PE-G16) are shown in Figure 5. The PE-G0 sample (PE mixed with pure graphite, not mechanically treated) showed a different stress-strain curve than the pure PE. In fact, an increase in the slope of the initial section and an increase in stiffness were observed (Young's elastic modulus increased from about 500 to about 900 MPa), as visible in the magnified graph in the frame of Figure 5. The mechanical strength, both at yield and at break, decreased from approximately 28 to approximately 25 MPa and from approximately 16 to approximately 12 MPa, respectively. Strain at break decreased considerably from approximately 1200% to approximately 22% with a corresponding collapse of the work at break from approximately 24 to approximately 7 J. The results indicated that the presence of graphite added to HDPE, even with a small quantity (0.3 wt %), caused a significant alteration of the polymeric structural organization. It acted as an inclusion, making the material very fragile. This involved the marked worsening of all the mechanical properties of the PE, which became brittle from a ductile material. The mechanical behavior of the nanocomposite mixture changed considerably, if the HDPE was mixed with the same weight of previously ball-milled graphite. In fact, mechanical stress reduced its size, and it managed to distribute itself in a homogeneous and uniform manner, intercalating itself within the macromolecular structure. The experimental evidence of what has been described revealed the reduction of modulus to approximately 630-640 MPa and the rise of the other mechanical parameters, especially after long times (8-16 h) of mechanical treatment.
Some parameters of the nanocomposite mixture became even higher than those of the pure PE. For example, in the PE-G16 sample, the mechanical strength at yield increased to a value of approximately 35 MPa, the stress at break increased up to a value of approximately 21MPa, the maximum load reached approximately 77 N. The work at break was approximately 20 J close to that of the pure PE, and the deformation at break (i.e., approximately 200%) decreased by an order of magnitude. The results indicated that the presence of graphite added to HDPE, even with a small quantity (0.3 wt %), caused a significant alteration of the polymeric structural organization. It acted as an inclusion, making the material very fragile. This involved the marked worsening of all the mechanical properties of the PE, which became brittle from a ductile material. The mechanical behavior of the nanocomposite mixture changed considerably, if the HDPE was mixed with the same weight of previously ball-milled graphite. In fact, mechanical stress reduced its size, and it managed to distribute itself in a homogeneous and uniform manner, intercalating itself within the macromolecular structure. The experimental evidence of what has been described revealed the reduction of modulus to approximately 630-640 MPa and the rise of the other mechanical parameters, especially after long times (8-16 h) of mechanical treatment.
Some parameters of the nanocomposite mixture became even higher than those of the pure PE. For example, in the PE-G16 sample, the mechanical strength at yield increased to a value of approximately 35 MPa, the stress at break increased up to a value of approximately 21 MPa, the maximum load reached approximately 77 N. The work at break was approximately 20 J close to that of the pure PE, and the deformation at break (i.e., approximately 200%) decreased by an order of magnitude.
As the graphite mechanical treatment time increased, there were increases in the yield stress and in the elongation at break. Therefore, the addition of graphite ball-milled for all the time periods was beneficial for increasing the mechanical properties of the pure HDPE.
The mechanical parameters vs. the median diameter particle size is shown in Figure 6. We can observe the decreases of the stress at break and at yield (Figure 6a), the decreases in strain at break and at yield (Figure 6b), the increase in Young modulus (Figure 6c), and the decrease in work at break (Figure 6d), with the increasing of the mean diameter particle size. Figure 7a shows the DSC curves, while the relationships of the melting temperature and that of the degree of crystallinity with the median particle diameter are shown in Figure 7b,c, respectively. How the melting peaks of nanocomposites shifted toward right to higher vales were observed (as indicated by the row in Figure 7a), with respect to for the pure PE. Hence, the melting temperature decreased with increasing mean diameter particle size (Figure 7b), while the degree of crystallinity and the melting enthalpy increased (Figure 7c,d). Observing the values detailed in Table 2 If we compared the literature results in similar HDPE materials reinforced with the same graphite amount (0.3 wt %), we can observe that: the results in [20] showed a greater improvement in stiffness (55%), a significant decrease in deformability (greater than two magnitude orders), and a similar improvement in break strength (40%), while the results in [25] showed a greater improvement in stiffness (66%, from 600 to 1000 MPa) and a decrease in impact strength (−22%, from 450 to 350 J/m), compared to the results in the present paper. Thus, our results reflected a better filler interaction within the polymeric matrix compared to the other similar materials, since the brittleness of our nanocomposites was lower. Finally, Figure 6 highlights that there was a small difference in the mechanical behaviors of PE-G8 and PE-G16 samples. Figure 7a shows the DSC curves, while the relationships of the melting temperature and that of the degree of crystallinity with the median particle diameter are shown in Figure 7b,c, respectively. How the melting peaks of nanocomposites shifted toward right to higher vales were observed (as indicated by the row in Figure 7a), with respect to for the pure PE. Hence, the melting temperature decreased with increasing mean diameter particle size (Figure 7b), while the degree of crystallinity and the melting enthalpy increased (Figure 7c,d). Compared to in the pure PE, increases in all parameters were identified for all the composites, as shown by the details of the values shown in Table 3. The Tm value increased by 4.6%, and ∆H and X both increased by 11%. Figure 7. Differential scanning calorimetry (DSC) analysis of the pure HDPE and its composites with graphite ball milled at different times: (a) calorimetric curves; (b) melting temperature vs. median particle diameter; (c) crystalline degree vs. median particle diameter; (d) melting enthalpy vs. median particle diameter.
Compared to in the pure PE, increases in all parameters were identified for all the composites, as shown by the details of the values shown in Table 3. The T m value increased by 4.6%, and ∆H and X both increased by 11%. These results are partially in agreement with what was reported in [20], which considered as a reference a value of ∆H = 288 (J/g) and had an increase of ∆H of 5% (in a mixture of HDPE with 60 wt % graphite EG for electrical conductivity measurements and an average size of 40 µm). Instead, the melting temperature of the same composition was almost unchanged compared to for the pure HDPE. The calorimetric data, therefore, indicated a general increase in the thermal properties of the nanocomposite, which grew as the median particle size decreased, suggesting that this improved the dispersion of the filler within the polymer matrix and therefore managed the better interaction between the filler and the matrix due to large surface area.
According to mechanical results, the DSC data in Table 3 highlighted that there was a small difference in the thermal behaviors of PE-G8 and PE-G16 samples.
The specific wear rate decreased compared to that of the pure PE (during 2500 cycles), as the ball milling time increased (regardless of the used speed ω; Figure 8a). The value changed from approximately 1.1 × 10 −3 to approximately 0.4 × 10 −3 mm 3 /Nm, with a maximum improvement of 65% in wear resistance (Figure 8b). The specific wear rate decreased compared to that of the pure PE (during 2500 cycles), as the ball milling time increased (regardless of the used speed ω; Figure 8a). The value changed from approximately 1.1 × 10 −3 to approximately 0.4 × 10 −3 mm 3 /Nm, with a maximum improvement of 65% in wear resistance (Figure 8b). The wear rate decrease agreed with the torque behavior decrease observed in Figure 1. We highlighted that the presence of ball-milled graphite decreased the torque value and hence the friction of the HDPE inside the mixing chamber. The more the graphite, the The wear rate decrease agreed with the torque behavior decrease observed in Figure 1. We highlighted that the presence of ball-milled graphite decreased the torque value and hence the friction of the HDPE inside the mixing chamber. The more the graphite, the longer the ball milling time. The wear rate, likewise, decreased, as the ball milling time increased. Further expanding the wear action, only two samples (PE-G8 and PE-G16) showed the best wear resistance, and then extending from 2500 to 10,000 cycles, no significant change in the wear rate value was observed (Figure 8c). This confirmed that these samples managed to have an appreciable wear resistance even after longer wear stress times.
Therefore, the mechanical action of the flaking of the graphitic planes managed to break up the graphite, which was smaller in size and better distributed within the polymeric structure, managing to give a good intercalation. In this way, the graphitic layers can act as a lubricant that improved the wear resistance of HDPE. More in-depth observations of the degree of intercalation of graphite within the polymeric matrix and its degree of exfoliation will be the subject of a future study.
Finally, the effect of the median diameter particle size on the percentage of improvement in wear resistance is plotted in Figure 8d; here, we can see that the highest wear resistance was obtained with the lowest mean diameter of the graphitic particles.
As seen through the mechanical, thermal, and wear results described above, it is evident that the eight-hour treatment could be considered enough to obtain a good reduction in particle size (19 µm). In fact, there was a good mechanical performance thanks to a sufficient distribution within the polymeric matrix and a positive interaction between the filler and the matrix. The further mechanical treatment (up to 16 h) still produced a reduction effect compared to the eight-hour treatment (16 µm), but the effect was smaller than the expected. The use of mechanical milling for a long time, such as 16 h, resulted in high mechanical forces concentrated upon the graphite layers. We should avoid the oxidation of graphite during the ball milling treatment that could occur after a long-time mechanical stress. This aspect suggests again that the treatment time of eight hours can be considered as optimal, in agreement with the mechanical tensile results.
Conclusions
In this paper, we studied PE-G nanocomposites, made with HDPE and ball-milled graphite (G). Graphite was both pure and mechanically treated in a ball mill for different times (in the range 0-16 h). Mechanical treatment was performed to reduce the size of graphite layers, which had a significant lubricating action. The aim of this work was, therefore, to verify the change in the physical-mechanical characteristics of PE-G nanocomposites, with particular attention to the variations in wear resistance. Graphite before and after the mechanical treatment has been studied by means of SEM morphological observation and the DLS analysis.
The nanocomposites were characterized with numerous investigations: during the mixing of HDPE and G, the torque was checked. Then, the static tensile, wear, and calorimetric analyses of both the pure PE and PE-G nanocomposites have been performed.
Experimental results showed that the mechanical treatment can reduce the size of the graphite particles from about 340 to 160 and 190 nm, after 8 or 16 h of mechanical ball milling treatment, respectively. The improvement in ball milling time progressively reduced the torque of the mixtures compared to in the pure PE to an ever-greater extent, when the ball milling time of the graphite was increased. This suggested an interaction between the small particles of ball-milled graphite and the polymer matrix.
From a mechanical point of view, we have verified improvements in stiffness and yield strength. The nanocomposites have also shown a progressive growth in wear resistance. In particular, the maximum improvement in wear resistance was 65%. Thermal resistance improved as well, although in little amount.
A treatment time of eight hours in ball grinding was sufficient to give an appreciable upgrade and to avoid the possible oxidative phenomena of graphite that could occur after excessive mechanical stress. The wear behavior of HDPE with ball-milled graphite has not yet been thoroughly investigated and could be important because HDPE is considered a "carrier polymer" for various low-friction applications, from biomedical use to civil engineering.
In a future work, we will focus on the optimization of the nanocomposite: the amount of mechanical treatment time (ball milling) will be fixed at eight hours, and the percentage by weight of graphite will be changed, thus testing quantities lower but also higher than 0.3%. In addition, HDPE loaded with an appropriate amount of G will be mixed with UHMWPE to verify the effect of this "carrier polymer" in the antiwear properties of PE for biomedical use or in civil engineering. | 2021-03-30T05:11:14.017Z | 2021-03-01T00:00:00.000 | {
"year": 2021,
"sha1": "4e57b60687b10a636c3ec3a71e3f0d38f037809d",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2073-4360/13/6/975/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "4e57b60687b10a636c3ec3a71e3f0d38f037809d",
"s2fieldsofstudy": [
"Materials Science",
"Engineering"
],
"extfieldsofstudy": [
"Medicine"
]
} |
166744630 | pes2o/s2orc | v3-fos-license | Ideo-Cultural and Lexical Challenges Encountered in Translating Qur'anic Metaphoric Expression into English: With Reference to Three Translations of the Meaning of the Holy Quran
The current study aims to investigate the possible inconsistencies and incongruities that exist in the translation of metaphoric expressions of Qur'anic verses in the English. The analysis has been conducted through the comparison of prominent English translators of Quran such as Mohammed A.S Abdel Haleem, Mohammed. M Pickthall and Mohammed Khan and Mohammed Taj Al-Din Al-Hilal. Also, the study aims to explore how the three translators deal with the metaphoric expressions in their renditions of the Holy Quran. This endeavor is crucial for a comprehensive understanding of English translations of the Quran, particularly in the context of English readers for non-Arabic Muslims in particular, where euphemisms are concerned. The primary purpose of the present study is to examine the extent to which the three translators are accurate in translating the Qur'anic metaphors into English based upon the contexts and the interpretive meaning. It is hypothesized in this paper that Qu'ranic metaphors are rhetorical, aesthetic devices, but have unfortunately remained unattended by the majority of Holy Quran translators. The study has revealed that metaphors are used profusely throughout the Holy Quran, although translating these metaphors accurately is a difficult process owing to cultural and linguistic barriers that exist between Arabic and English cultures. It is expected that the study will cast light on two important ideas. The first is that the translators are fully aware of the existence of metaphoric expressions in the Qur’an. Secondly, it is essential that translators of the Holy Qur’an must translate these metaphors accurately to preserve the original meaning of the Qur’anic text.
INTRODUCTION
It is commonly assumed that the translators of the Holy Quran encounter many cultural and lexical problems and constraints in rendering this Holy Book. These problems emerge when the translators discover that there are some expressions in the Qur'anic text that is virtually untranslatable. It is observed that different versions of Qur'anic translations lead to different levels of comprehension and understanding on the part of the lay reader, especially when metaphoric expressions are considered.
The Holy Quran is rich in metaphoric expressions on various sensitive issues and topics such as sex, divorce, death and so on that need to be translated and examined. Sacredness and beauty of the Holy Quran make translation of the Qur'anic euphemisms problematic and challenging for the translators. To convey the Islamic values and beliefs accurately, the translator is forced to use different strategies such as paraphrasing, partial equivalents, literal translation, functional translation, among other techniques to address the challenge of correctly translating Qur'anic euphemisms.
There is a common belief that the translations of the Holy Quran can never achieve the same semantic and linguistic value that the original version of text contains. This problem arises for the following reasons: a. English and Arabic are never sufficiently similar to express the same realities. b. The Arabic language is richer not only in vocabulary but also in meaning, for example, metaphor is an obvious semantic problem that translators usually encounter when translating the text of the Holy Quran into English. c. The beautiful and eloquent style of the Holy Quran.(In Arabic) d. Metaphoric meanings of words. e. The lack of equivalence at word levels.
Research Questions
To meet the issues mentioned above the following research questions are raised to find out to what extent: 1. does metaphor constitute one of the main components of translating the Holy Qur'an? 2. does metaphor offer one of the most effective parameters according to which both the literary competence of the translator and religious cultural awareness of the reader of the translation of the Holy Qur'an are revealed and gauged? between languages. It is best seen as a communication process where the transfer of a message/written content from one language into a new language takes place. Translation is the general term refer to the transfer of thoughts and ideas from one language SL to another TL, whether the languages are in written or oral form, whether the languages have established orthographies or do not have such standardization or whether one or both languages is based on signs ,as with sign languages of the deaf. Translation can be defined as :''The placement of textual material in one language by equivalent textual material in another language''. (Catford,1990,p.78) This definition vaguely refers to the textual material. It does not, however, clearly indicate as to whether the significance is more on the meaning or style or the linguistic elements like the words and sentence structures. Catford's work'' A Linguistic Theory of Translation( LTT)' ' (p.7) primarily focuses around various processes of translation with special emphasis on the linguistic elements like phonetic, phonological, grammatical and lexical, graphalogical and other kinds of translation like complete vs. partial, total vs. restricted and the like. He also deals about transliteration. The argument of Catford cannot be underestimated, but the point of concern is that the outlook of Catford is very restricted and narrow and does not satisfactory fulfill the requirements of translation. More important in the process of translation in the conveying of message from one language to the other and the linguistic equivalence is secondary to the thematic equivalence. According to Newmark (1998, p.45): Translation is such an art wherein the message conveyed through one language is replaced by the same message in the other language.
The examination of various definitions leads us to conclude that translation is such an art whereby the message in the text in one language is transferred into the text of another language. The limitations and constrains involved in the process of translation are of serious concern. ( Halliday,1994,p.34), on the other hand, considers translation as ''the relation between the text in the two languages involved,''. According to him, the texts accomplish the same task under the same circumstances through the two different languages. He has no doubt aptly emphasized upon the significance of meaning. The aspects emerge out of a detailed examination of the viewpoints of various scholars of translation: Translation is a linguistic exercise that takes place between two languages. The language of the original text is called the source language and the language into which the translation is made is called the target language. The text in the target language is called the translated text. The process of transfer or re-establishment of the meaning from the source language into the target language is the essence of the art of translation. The expression between the SL and TL become synonymous. In other words, they convey the same meaning without distortion. Several aspects figure in the process of translation. They include the linguistic aspects, socio-cultural aspects and contextual aspects. A unique combination of all these aspects could result in a successful and meaningful translation. The sole aim of the translator is to successfully transfer the essence of the original text in the translated text. Understanding of ' translation' could be considered in two contexts: In an extended context and In a restricted context. Translation in the extended context is considered as the transfer of meaning in one symbolic constitution into the other symbolic constitution. "Symbolic constitution'' refers to the structural nuances of the two languages. On the other hand, translation in the restricted sense is considered as the process that takes place between two languages. This primarily refers to the linguistic aspects and the applications of principles to the art of translation. In fact, translation becomes meaningful if any and only if it is considered both in the restricted sense as well as the extended sense. Translation is the process of replacing an original text, known as the source, with a substitute one, known as the target text.
The process is usually an interlingual translation (translation proper) in that the message in the source language text is rendered as a target text in a different language, and it is in this sense that we have referred to translation so far. But sometimes the term is also used to refer to an intralingual translation (rewording), a process whereby a text in one variety of the language is reworded into another. This would be the case where the message of a text in, say, Old English (OE) is reworded into a text in modern English, or a text in one dialect or style is reworded into another. And we can speak of 'translation' when the replacement involves not another language but another, non-linguistic, means of expression, in other words a different semiotic systems. In this sense we can say for instance that a poem is ' translated' into a dance or a picture, a novel into an operator a film. Such transmutations are examples of intersemiotic translation. (Jakobson, 1959(Jakobson, /1990. What all these three processes have in common is that they involve the replacement of one expression of a message or unit of meaningful content by another in a different form.
The term 'translation' is also sometimes used to describe linguistic activities such as summarizing or paraphrasing. Although such activities resemble translation in that they replace a message that already exists, they differ in that they are designed not to reproduce the original as a whole but to reduce it to its essential parts, or adapt it for different groups of people with different needs and expectations.
The term "translation" is the mental term used for all tasks where the meaning of an expression in one language (the 'source' language) is turned into the meaning of another (the 'target' language), whether the medium is spoken, written or signaled. It is an operation performed on languages through the replacement of textual material in one language (source language) by equivalent textual material in another language (target language). By replacement of textual material, we mean a replacement of source language graphology, grammar and lexis by equivalent target language graphology, grammar and lexis. Source language (SL) means the form from which the translation is made and target Language/ receptor language (TL/RL) is the form into which the SL is to be changed. By equivalence we mean text in different languages can be equivalent in different degrees(fully/partially equivalent), in respect of different levels of presentation (equivalent in respect of context of semantics, grammar, and lexis ) and at different ranks (word-for-word, phrase-for-phrase, sentence-forsentence).Since meaning is central in translation, it will not be acceptable to say that translation only involves changing of form of the first language to the form of the second language. Translation consists of transferring the meaning of the SL into the TL which is done by going from the form of SL to the form of TL by way of semantic structure.
There have been a number of theories of translation that have been debated about. They include: i. Linguistic Theory. ii.
Relativist Theory . While Catford(1991) is the proponent and authority on the linguistic theory of translation, Steiner(1992) has proposed the universal and relativist theories. The theory of translation primarily deals with the linguistic aspects like the structural and lexical equivalences, formal correspondence, transference, transliteration, several types of translation like partial and total translation, phonological and graphalogical translation, translation shifts and the limits of translatability. (Kelly p.1997, p.60) as the name of the theory is indicative, the linguistic theory of translation is mostly concerned about the structure and less about the content/theme. Thus, the linguistic theory of translation fails to take care of the content aspect which is a serious setback to the art of translation. The reason is that the primary purpose of translation is to convey the content/theme from one language to the other without loss or distortion of the theme in the source language; thereby the significance of the thematic accuracy over-rides the linguistic accuracy. There is no exaggeration if it is argued that the linguistic accuracy in translation plays a secondary role. However, the linguistic aspects should not be made insignificant. It means that while utmost importance is given to the thematic accuracy, linguistic accuracy and correspondence between the SL and TL need to be maintained to the maximum possible extent. It follows that a good translation necessitates an ideal integration of the thematic transfer and linguistic transfer from the SL to TL. (Kelly, p.1997, p.61) The Universalist theory according to Hewson and Martin is based on an extension of the economic concept of contractual transaction. (Kelly, p.1997, p.68) .The term 'contract' refers to the act of translation. The term 'transaction' refers to the act of conciliation between the two languages and the unification established between them by the process of translation.( By unification is meant the establishment of one to one correspondence between SL text and the TL text not merely from the linguistic aspect but from the thematic aspect as well. According to Martin and Mason (1997,p.45): Translation, as a particular form of contract, is an agreement between the two LC is involved to transfer signification on a common convertibility basis in so far it is not detrimental to the specific differences between cultures. The fundamental notion both on the economic and on the translational planes is compromise; i.e., the agreement to remain separate in order to achieve a common goal.
A good translation or an ideal contract is possible within the scope of a single culture. Therefore, the Universalist conversion envisages the relationship between cultures as possible. But, necessarily, such relationship is only partial or flawed. ( Cohen.1990,p.34). Some scholars, however, argue that in spite of diversity of cultures, there exists reasonable quantum of universals based on which the transaction or translation could be considered as reasonable and sufficient though the transaction/ translation excludes the total correspondence or one to one correspondence. They agree that the transfer of the deeper and wider interpretations between the two cultures gets precluded. (Cohen.1990, p.36) In believing that cultural relationships are contractual transactions, translation can be conceived of as a process of transference based on the criterion of equivalence. Practically, this view boils down to the argument that a sound and reasonablecompromise between the structural and thematic equivalences has to be ensured in good translation. Departing a little bit from this view, scholars like Hewson and Martin have preferred to argue that transference is necessarily partial and therefore, translation necessarily involves some loss. They, however, give a word of caution that this possible loss should be kept to the minimum and to the extent possible; it is to be compensated with the normalization of the common core.They conclude that translation consists in constantly perfecting the fundamentally uncontestable compromise. (Devey,1990,p.77).
The relativist theory is concerned with the concept of production within an interactive structure. From this point of view, common core or the universals are not only compressive as non-existence, but they contribute to ''denaturing'' of communication. The most important aspect in the art of translation is that the essential of signification lies in particulars and differences which can never be Universalist any way, but only exist in proportion to their specificity. It follows that signification can never be repealed, duplicated or transferred; it can only be reformulated and adapted to the ever changing conditions of meaning definition. The point to be taken note of in this context is that ''meaning'' is the most significant aspect in the art of translation. The act of producing the meaning intact in the language translated into besides the alterations in the factors involved in communication i.e., the medium or language adopted is essential. It is for this reason that constant adaptation is extremely significant and important in the context of translation. (Cohen.1990,p.67) As stated above, perfect integration and correlation between the structure and content, expression and reality needs to be ensured which is associated with the concept of signification in the context of translation. Drawing the attention to these factors, Steiner (1992, p.89) argues that; The role of translation is determining in this process of cultural cross determination, since in translation the dialectic of unison and plurality is dramatically at work Martin and Mason (1997)call this process as ''hermeneutic". Meschonnic conceives of translation as a unique combination of ''rapport and tension''. While rapport refers to a close correspondence between the SL and TL expressions, ''tension'' refers to the idiosyncrasies particularly in the context of aspects relating to culture. These aspects could be taken care of suitably to make the translation as perfect as possible only through exemplification and explanations of typical and unique cultural and social aspects etc. The exemplifications and explanations so required are called ''complexification of perspective'' Martin and Mason (1997, p.38).
Research Approach
In the current study, the researcher used descriptive qualitative methods. Secondly, text analysis design was used to find metaphoric expressions in the English translation of the meanings of the Holy Quran. The Qur'anic translations explored in the study are:
Data Collection Procedure
The current research aims at describing, analyzing and evaluating the principles, methods, and procedures of translating the text of the Holy Quran, and particularly, explaining the problems of translating metaphoric expressions in the three selected translations of the Holy Quran.The objective of the current research is the establishment of the basic and secondary meaning of e metaphoric xpressions and their derivations in the translation of the meaning of Holy Quran. The most vital and crucial research instrument is reading, analyzing and comparing the translated text of selected Surrah by the three different translators. This study is an eclectic study where three popular translations of the Holy Qur'an have been analyzed and identified as the different kinds of translation, i.e., semantic translation, communicative translation, etc. When analyzing the three translations, the researcher followed the following procedures: a. quoting the Arabic versions of Quranic ayat in which metaphoric expressions under investigation occur, enumerating metaphoric expressions in both versions Arabic and English, and then putting the three translations of the same ayah into a table directly under each one of the three translators. b. Studying metaphoric expressions in terms of the problems of meaning and textual problems based on (strong/mid/weak) connotation. c. Analyzing metaphoric expressions aspects of meaning focusing on some selected ayahs in which metaphoric expressions appear in the Holy Quran, here the researcher chooses some examples for the context particularly in which either metaphoric expressions appear. d. Analyzing Abdel-Haleem, Khan and Hilali and Pickthal's translations and identifying their accuracy, effectiveness, and then giving comments on the three translations.
RESULTS AND DISCUSSIONS
Regarding the analysis of the collected data, some Quranic have been selected by the researcher. The selected ayahs contain some metaphoric expressions .The analysis of the of the data was carried out by utilizing comprehensive tables displaying: SL texts, TL text, meaning, types of methods, Furthermore, the analysis of the data has been devoted to metaphoric expressions and the way each selected translator used them. However, all ayahs have not been analyzed in this thesis due to the limitations of the current study. Then, only the translations that appear give different meanings from what has established have been explored. Finally, in case of having an effective rendering, which coincides with the religious interpretation, it will be chosen as a proposed rendering; otherwise, a new rendering will be suggested.
Why do their rabbis and scholars not forbid them to speak sinfully and consume what is unlawful? How evil their deeds are! Why do not the rabbis and the religious learned men forbid them from uttering sinful words and from eating illegal things. Evil indeed is that which they have been performing.
Why do not the rabbis and the priests forbid their evil-speaking and their devouring of illicit gain? Verily evil is their handiwork.
Discussions
The meaning of this ayah is: had it been but that the Rabbaniyyun and the Ahbar forbid them from committing such evil acts The Rabbaniyyun are the religious learned men having the authority, ''Evil indeed is that which they have been performing ''in reference it is to their abstaining from forbidding of committing evils. It is to their abstaining from forbidding of committing evils. It is recorded that Ibn Ibbas said:''No verse in the Qur'an heaps reproaches than this verse:'' Why do not the rabbis and the religious learned men forbid them''," Similar it is to what Adhahak said:'' There is no verse in the Qur'an that I'm admonished there with than this verse. One upon a time, Ali Abi-Talib delivered a speech after praising Allah and thanking Him saying,'' O 'people ! What perished those who were before you was that they used to committed evils and the Rabbaniyyun and the Ahbar amongst them didn't forbid them against committing such evils. When they insisted on committing sins. They were seized with punishments. Therefore, enjoin righteousness and forbid evil before the like of what had fallen them strikes you and know that commanding what is righteous and forbidding what is evil does not cease provision nor does it shorten one's fixed term of life.'' It is narrated in the Hadith; "No people who have amongst them those who commit what is prohibited while they themselves are of more power and might than the sinners and they didn't stop them,but that they will be afficted with totutre from Allah"( narrated by Imam Ahmed) In (Abdel-Haleem's rendition of the metaphoric expression l-suḥ'ta َ`ْaﱡ c^ا he gives the intended meaning of the metaphor " l-suḥ'ta َ`ْaﱡ c^ا '' which is '' consume what is unlawful ''and ignores the metaphoric expression " lsuḥ'ta َ`ْaﱡ c^ا '' whose meaning is less embarrassing than the meaning of bribe. Moreover, Khan and Hilali rendered the same metaphoric expression into'' eating illegal things'' but their translation is literal and they have been used literal translation strategy to come up with exact or better meaning of the metaphor " l-suḥ'ta َ`ْaﱡ c^ا but he ignores intended meaning of I-suḥ'ta َ`ْaﱡ c^ا which is bribes, Furthermore, Pickthall has used the same strategy when translated the metaphor l-suḥ'ta َ`ْaﱡ c^ا into English'' devouring of illicit gain ''. He also, ignores intended meaning of l-suḥ'ta َ`ْaﱡ c^ا which is bribes.
However, the three translators' rendering for the metaphoric expression l-suḥ'ta َ`ْaﱡ c^ا is inappropriate because it does not convey the given meaning of'' l-suḥ'ta َ`ْaﱡ c^ا which is bribes and the receptor may be confused or frustrated. Moreover, Hilali and Khan used the translation explanation and addition strategies to render the lexeme more appropriately and accurately but their rendition does not convey the accurate meaning. Moreover, the three translators used the communicative method which hits high degree of translational coincidences with the interpretation in Khan and Hilali's renderings, but it hits average in Abdel-Haleem's and Pickthall's renderings. We assuredly had taken him by the right hand
Discussions:
The meaning of this ayah is: " we would seize him by the right hand because it is more severer in grabbing .Abdel-Haleem and Pickthall used literal translation to render the metaphoric expression lqِ zَ q ْ^W ِ sbilyamīni into '' We would certainly have seized his right hand'','' We assuredly had taken him by the right hand'' respectively. Semotactically, Abdel-Haleem Khan and Hilali's rendering(seized his right hand) is ambiguous; hence, their rendition is inaccurate and inappropriate and unnatural to the receptor's expectations and definitely confusing, but, Khan and Hilali's rendering hence his rendition ranks the best, because they clarified the ambiguity by adding (with power and might). Also Hilali & Khan combining their translation with an explanatory note, telling the receptors directly about the intended meaning of the text at hand(a translation method that combines two procedures to deal with a single problem "literal translation + explanation").
All the three translators used the Communicative translation which hits high degree of translational coincidences with the interpretation for translating render the metaphoric expression lqِ zَ q ْ^W ِ sbil-yamīni into ' ' To conclude, the tendency of the translators to reduce the ST informativity factor widens the gap among the textual cohesive links'' which feeds into the overall textuality and textness of Quran discourse '' (Abdul-Raof,2004,160). The TT could be judged as not being as informative as required in particular the continuity of It is made lawful for you to have sexual relations with your wives on the night of As-Saum (the fasts) It is made lawful for you to go in unto your wives on the night of the fast
Discussions
The meaning of this ayah is: (It is made lawful for you to have sexual relations with your wives on the night of As-Saum),Allah made it lawful for Muslims what was unlawful and hard to bear at the beginning of Islam when it was lawful for a Muslim to eat, drink and copulate only from the time he beaks his fast till the time of praying the 'Isha., and that if he slept or prayed the 'Isha prayer, it is forbidden for him then to eat or drink or copulate till the next set of the sun."( Tafsir Ibn Kathir volume. I,2007,p.108) According to Al-Esfahani (1997,p.359) ُ …َ ﱠ † t^اl-rafathu is a word that has an implied meaning used to refer to sexual intercourse. It is a metonym for sexual intercourse. Considering the above translations, it can be noted that Abdel-Haleem rendered the metaphoric lexeme ُ …َ ﱠ † t^اl-rafathu into "lie with your wives'' but Khan and Hilali rendered the same lexeme into'' have sexual relations with your wives'. Therefore, they ignored the metaphoric expression which is used to reduce the direct meaning of sexual intercourse and mentioned only the interpretive meaning of (have sexual relations). Pikthal rendered the metaphoric lexeme ُ …َ ﱠ † t^اl-rafathu into "to go unto"; hence he did not translate the intended and the interpretive meanings of the lexeme. According to (Hosni, 2007,p.626 ) In this verse, the word [Ɂarrafaθ] ,i.e. going in unto wives' is a euphemism for the sex-act or copulation . Instead of the explicit mention of having sex with one's wife on the night of the fast , the word [Ɂarrafaθ] is used so as to gloss over the down-to-earth direct expression of copulation . The context within which the word [Ɂarrafaθ] occurs necessitates a sort of mollification .This , however , is due to the devotion of votaries who are supposed to go in unto their wives only after they break their fast. Breaking one's fast does occur only at dusk time and into the night . Only then are fast-breakers allowed to have intimacy with their wives . The whole situation has to do with the ritual of fast and the admissible acts involved . The word [Ɂarrafaθ] , therefore , is being used as a euphemism for "having sex with" or "copulating with" or "having sexual intercourse with" (one's wife) , etc.
On their turn, Al-Zamakhshari( 1998,p.389) and Al-Baydawi(1999,p.10) agree that the word rafath (…َ †َ )ر sounds more negative than some other expressions used as euphemisms for the same purpose such as afḍā ila . They both agree that the reason for using such a negative word is disapproval of the actions of some Ṣaḥābah who had sexual relations with their wives when it was prohibited. To approach the meaning of metaphoric expression ُ …َ ﱠ † t^اl-rafathu ( based on Tafsir Al-Jalalayn and Tafisir Ibn Kahir) in the ayah, Khan and Hilali as well as Abdel-Haleem used the semantic method which hits average degree of translational coincidences with the interpretation .However, Pickthal used Communicative method which hits low degree of translational coincidences with the interpretation.
Type Semantic translation Communicative translation No. of Translation High Average Low High Average Low T1)permitted to lie with your wives + T2)have sexual relations with your wives + T3)to go in unto your wives + Khan and Hilali's rendition is semantic translation producing an exposed sort of euphemism by using the expression "sexual relations''. Hence, their rendering has mild connotation In other words, using the word 'relation' in Khan and Hilali's rendition, it is obvious that the two translators have attempted to produce a metaphoric translation However, studies failed to produce evidence that the phrase' sexual relations' can be understood as metaphor in English . Pikthal rendered the metaphoric lexeme ُ …َ ﱠ † t^اl-rafathu into "to go unto"; hence he did not convey metaphoric meanings of the lexeme , hence his rendition has weak connotation. But, Abdel-Haleem rendered the metaphoric lexeme ُ …َ ﱠ † t^اl-rafathu into "lie with your wives'', and his translation is accurate and clearer , hence, his rendition for the metaphoric lexeme ُ …َ ﱠ † t^اl-rafathu has strong connotation.
Method of Translation
Strong connotation Mild connotation Weak connotation T1 Semantic method + T2 Semantic method + T3 Communicative method + To conclude that, metaphor has come to be realized not only as a matter of language, but also as an important aspect of cognition. Thus, according to cognitive linguists, metaphor is not merely a figure of speech; it is also a way of thinking and conceptualizing It is who created you all from one soul, and from it made its mate so that he might find comfort in her It is He Who has created you from a single person (Adam), and (then) He has created from him his wife [Hawwa (Eve)], in order that he might enjoy the pleasure of living with her He it is Who did create you from a single soul, and therefrom did make his mate that he might take rest in her
Discussions
In the above ayah, the word ' َl ُ •ْ cَ qِ liyaskuna (lit that he might live.) in expressions like 'liyaskuna ilayha َmُ Oْ Sَ [ِ @ > َ nْ [ َ @ ِ إ ' in the ayah above is a metaphor that expresses the utmost kind of love and intimacy through sexual relation between husband and wife or couples since they are described as one soul (Al-Tabari, 2000,). This ayayh in was contextually revealed to remind humans of Allah's signs among which are that He created them from a single soul, namely, Adam, and made his spouse, Eve, out of him. The purpose behind this creation is that Adam might live in ultimate peace with his wife through being intimate with her and to bring offspring as a result.
(http://www.altafsir.com). The metaphoric expression 'liyaskuna ilayha> َ nْ [ َ @ ِ إ َmُ Oْ Sَ [ِ @ ', is translated into ' he might enjoy the pleasure of living with her by Khan and Hilali, and such a literal translation is more general and convey non-sexual connotation because the phrase ' enjoy the pleasure ' has never been used to express any sexrelated meaning (Rawson, 1981,). Further, '' enjoy the pleasure'' does not even convey the general meaning of the said expression' 'liyaskuna ilayha' in the ST which entails the ultimate level of living in peace and satisfaction with a wife (Qutb, 2003, p.37). In contrast, the ST is transferred into 'he might rest in her' by Pickthall and this expression is also more general and, as metaphor, is more associated with ''the relaxation that is obtained through death, as in eternal rest, called to heavenly rest, go to rest, and laid to rest " (Rawson, 1981, p.236). As for Abdel-Haleem, the ST is rendered into a more illustrative , accurate and clearer expression He might find comfort in her '', which indicates the translator's awareness of the ST intention.. The use of 'comfort in her' as equivalence to ' liyaskuna ilayha' is a real success since 'comfort' is metaphoric expression for intimate copulation especially when sought by a male (Holder, 2008, p.1) To approach the meaning of metaphoric expression is' َl ُ •ْ cَ qِ liyaskuna ( ( based on Tafsir Al-Jalalayn and Tafisir Ibn Kahir) in the ayah, Abdel-Haleem used the semantic method which hits high degree of translational coincidences with the interpretation . Khan and Hilali used communicative method which hits average degree of translational coincidence with the interpretation , whereas Pickthall used the same method which hits low degree of translational coincidence with the interpretation,
Type
Semantic translation Communicative translation No. of Translation High Average low High Average Low T 1)he might find comfort in her + T 2) might enjoy the pleasure of living with her + T3) he might take rest in her + Abdel-Haleem is adequate in rendering the intended connotative and euphuistic meaning َl ُ •ْ cَ qِ liyaskuna when he renders it as (find comfort in her). Hence, his translation has strong convocation. Unfortunately, Khan and Hilali as well as Pickthall's renditions for the same connotative and metaphoric meaning are inaccurate and out of context. Hence, their translations have weak connotation.
How could you take it when you have lain with each other and they have taken a solemn pledge from you?
And how could you take it (back) while you have gone in unto each other, and they have taken from you a firm and strong covenant?
How can ye take it (back) after one of you hath gone in unto the other, and they have taken a strong pledge from you ?:
Discussions
"How can ye take it (back) after one of you hath gone in unto the other, and they have taken a strong pledge from you ?" (Pickthall). And how could you take it (back) while you have gone in unto each other) how can you take back the dowry from the woman with whom you had sexual relations and she had sexual relations with you Ibn `Abbas, Mujahid, As-Suddi and several others said that this means sexual intercourse. (And how could you take it (back) while you have gone in unto each other and they have taken from you a firm and strong covenant) (Be kind with women, for you have taken them by Allah's covenant and earned the right to have sexual relations with them by Allah's Word.). In this example the metaphor was translated perfectly into English This verse tackles a significant matter that has to do with the husband's commitment to the rituals of marriage . It is incumbent upon him to live up to the promise he had made to be noble and chivalrous . This means that there is a knot of matrimony being effected and a dowry is paid out to the bride , and this alreadypaid dowry should not be taken back in the wake of the consummation (of marriage) . This , in matter of fact , is an admonition addressed to the husband so as to act on it and never to overstep his limits . In other words , when the husband consummates the marriage , he has no right Literal translation of some words, idioms and fixed expressions in the Quran fails to render the intended meaning of the original ones clearly into English. This is because in some cases, the translators, especially Arberry, render only the denotative meanings of these linguistic features. They transfer them as they were found in the dictionary. Since the connotative meaning of a word in Arabic is often not the same as that of an English word almost having almost the same denotative meaning, the literally rendered meaning neither transfers the genuine meaning nor does it match the general content of the text. The words are English but they do not relay an obvious message. Consequently, the reader fails to grasp the meaning. Dickins et al. (2002: 97) warn that "in translation, lexical loss is very common, and this arises from the fact that exact synonymy between ST words and TT words, is relatively rare". They (ibid) add that "meanings are not found exclusively in the words listed individually in the dictionary". Lexical loss then results in a text that lacks clear meaning, badly affecting intelligibility.
The 'firm covenant' in this verse refers to marriage. For marriage is a firm covenant of fidelity. It is only because a woman has faith in the firmness of this covenant that she entrusts herself to a man. If a man decides of his own will to break it, he has no right to withdraw the amount he offered his wife by way of bridal-due at the (1) is stated as follows: Metaphoric constitutes one of the main components of translating the Holy Qur'an. This hypothesis addresses Research question which aims to find out to what extent metaphor constitutes as one of the main components of translating the Holy Qur'an into English. The analysis of the verses proved that translator has to deal with quite distinctive cultures with temporal and spatial differences in rendering metaphor into English. Also the translator has to face multifarious difficulties of rendering metaphor through a balanced approach .For example, the translator will essentially provide all inevitable introductory knowledge about Qura'nic metaphoric and its style. Hypothesis Two: Hypothesis (2) is stated as follows: Metaphoric offers one of the most effective parameters according to which both the literary competence of the translator and religious cultural awareness of the reader of the translation of the Holy Qur'an are revealed and gauged.
This hypothesis arises from Research Question(2) which aims to find out to what extent metaphor offers one of the most effective parameters according to which both the literary competence of the translator and religious cultural awareness of the reader of the translation of the Holy Qur'an are revealed and gauged. metaphor involves the semantic structure of both individual words and texts. It, therefore, deals with complex semantic relations working at the level of microsemantics, and it is for this reason, strongly related to literary in general and religious text in particular . metaphor in words, expressions and texts expounds both the expressive and the emotive aspects of language and as such it seems that all connotative words ,metaphoric expressions and connotation thus constitutes one of the main components of translating Holy Quran, and by virtue of its suggestive power as an emotive and expressive vehicle, it offers one of the most effective parameters according to which both the literary competence of the translator and the cultural awareness of the reader are revealed and gauged.
Khan and Hilali and Abulhaleem's translations of the Quranic metaphoric expressions are the best examples of both the extremes, where Pickthall's rendering for the Quranic metaphoric expressions seems complex being too literal, which thwarts comprehension , , and on the contrary, Khan and Hilali and Abulhaleem's dynamic rendering with abrupt turns in the form of lexical expansion has been appreciated by the majority of readers either Muslim or non Muslim. Finally, the Quranic metaphoric expressions need to be rendered figuratively or reworded into effective metaphoric and connotative with footnotes. Hypothesis Three: Hypothesis (3) is stated as follows: This hypothesis arises from Research Question(3) which aims to find out the strategies are adopt by the three translators to ensure interaction between the translated texts and the Arabic socio-cultural contexts. A number of translation strategies are found to be applied by the three translators in their attempt to render the Qur'anic metaphoric expressions into English. These strategies include the following: a. Khan and Hilali's translation of the Holy Quran is an example of an approach that attempts to be most'' faithful'' to the source; being text-centered. This explains their frequent use of footnotes to explicate ambiguous terms and expression. Moreover, footnotes are one of the most common strategies used by translators for explication terms and phrases that do not have an equivalent in the TL, or whose direct equivalent results in a drastic loss of meaning. Sometimes footnotes are also used to refer to other ayahs related to the term or expression to help explain the meaning( see the examples) b. Pickthall does not provide his translation with footnotes or commentary to enable the readers of his translation to gain sufficient information for a proper understanding. Moreover, he does not support his translation with Hadiths and exegeses, which can help in reinforcing the elements of the suras ( see the examples ) c. Abdul-Halemm often uses footnotes in his translation. According to him (2005, p.87)'' footnotes are meant to be minimal, and to explain allusions, references, and cultural background only when it was felt these were absolutely necessary to clarify meaning and context( see the examples). d. Khan and Hilali and Abdul-Haleem have adopted an explanatory approach a long with transliteration (e.g. examples from 1-20). e. Translation of the Holy Quran texts often tends to include additional statements and phrases for the purpose of explication. Addition (expansion strategy) takes more than one form and used for different purposes depending on the context and style of the translator. Abdul-Haleem uses addition strategy in his translation( see the examples) f. Khan and Hilali as well as Abdul-Haleem sometimes uses cultural substitution strategies in their rendering and by explaining cultural items through meaning of sense in the ST, therefore, give only a literal translation which may lead to ambiguity (see the examples) g. All the three translators sometimes use the communicative translation strategies which aim at rendering the Qur'anic euphemistic expressions into English and producing for its readers the closet effect that of the ST.All the three translators sometimes use the semantic translation strategies which aims at rendering and producing, as closely as the structures and nature of the SL, besides allowing the exact meaning of the SL message. (see the examples).
CONCLUSION 5.1 Summary of Findings
On the bases of the theoretical part and data analysis, the current study has come up with the following conclusions: 1. Translating Qur'anic metaphor is an even more demanding task than translating metaphor in other subjects owing to the fact that religious text, such as The Holy Quran, contains more complex connotative meanings and therefore, no universality exists in the terms used. This is in stark to contrast to the domain of science where each term and definition has a universal acceptance. 2. Translating metaphor meanings in the Quranic texts is not quite easy. This is because Qur'anic metaphor involves very subtle differences in meaning that are difficult to grasp. 3. There are various instances where cultural details are given in the Quran. These expressions are usually rendered by literal rendering or performing transliteration. The translators are often unable to analyze these cultural terms and aspects of the Holy Quran and neither are they able to find the best and closest expressions to convey the same meaning and images. 4. It is extremely difficult to translate the Qur'an literally because the Arabic terms, expressions, and lexemes often have multiple literal meanings and are often used figuratively. In addition, many forms of Arabic lexical structures contain nuances of meaning that cannot be translated into another language owing to linguistic barriers. Therefore, the translations of the Holy Quran are largely based on interpretation, paraphrasing, and explanation of the source text.
Implications and Recommendations for Future Research
This study and other similar studies can play a role in enhancing the translational knowledge, understanding, and performance of students. Moreover, the study can support teaching Arabic to English translations in Arab universities. Students can potentially benefit from this study in the application of the knowledge of translational techniques and strategies to holy texts such as Qur'an.
The results of the current study call for future research on analyzing the problems involving translating, collocations, euphemisms and lexical ambiguity in the Holy Quran. This future research could be applied not only to Arabic and English, but also to other languages, which are genetically unrelated. Additional research is needed to explore, metaphor in reference to two translations of the meaning of the Hadith. | 2019-05-28T13:10:14.190Z | 2019-01-01T00:00:00.000 | {
"year": 2019,
"sha1": "88e9a6b80cd92219781d4c8778086c62dd0935c8",
"oa_license": "CCBY",
"oa_url": "https://www.iiste.org/Journals/index.php/JLLL/article/download/46530/48046",
"oa_status": "HYBRID",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "b5d77d329a3129b03463e44d4d0d8a4d435e5f0c",
"s2fieldsofstudy": [
"Linguistics"
],
"extfieldsofstudy": [
"History"
]
} |
258021164 | pes2o/s2orc | v3-fos-license | A Retained Foreign Body as a Rare Cause of Small Bowel Obstruction (Gossypiboma): A Case Report
A retained foreign body (RFB) is a rare but possible complication of surgery. Among the most common retained foreign bodies are sponges, which may include lap pads and gauze pieces. Surgical never events are errors in medical care that are identifiable and preventable but have serious consequences for the patient, making it an important problem in terms of the safety and credibility of a healthcare facility. They also pose a major medicolegal threat to healthcare organizations and a diagnostic challenge for surgeons. Herein, we present the case of a 35-year-old woman who presented with signs and symptoms of acute intestinal obstruction. She revealed a history of Caesarean section 11 months prior. She had a stormy postoperative course then and had to undergo a diagnostic laparoscopy for pus aspiration three months after surgery, where no finding other than pus was reported. Upon presentation at our tertiary care center, she was examined and found to have an RFB for 11 months. She was managed surgically with successful laparoscopic removal of the gossypiboma and consequent resolution of all her symptoms. Though rare, the possibility of an RFB, especially after open surgery, should be kept in mind when diagnosing patients who present with pain, mass in the abdomen, or symptoms of an infection. Laparotomy is the mainstay of treatment for gossypiboma, but successful laparoscopic removal of the RFB provides a definite treatment with the super-added benefits of laparoscopy. Laparoscopic removal of gossypiboma has been reported in the literature and demonstrated in our tertiary care center.
Introduction
Gossypiboma, textiloma, or cottonoid are the common terms used to describe a mass in the body caused by a retained surgical sponge surrounded by a foreign body reaction. A surgical sponge is the most common type of retained foreign body (RFB) [1]. The exact incidence rate of gossypiboma in clinical practice is difficult to estimate, as cases are underreported. In the abdomen, a retained sponge can invaginate a hollow viscus or cause a mass effect due to fibrous encapsulation. It can go undetected for years if asymptomatic, and when it becomes symptomatic, it causes a great deal of physical agony and mental stress for the patient and monetary compensation, embarrassment, or even loss of job for the surgeon.
We present the case of a 35-year-old female patient who presented with signs and symptoms of acute intestinal obstruction. She revealed a history of Caesarean section 11 months prior and had been more or less asymptomatic since then.
Case Presentation
A 35-year-old woman presented to the gastroenterology outpatient department with complaints of pain in her abdomen and inability to pass stools for five to six days. The pain was dominantly in the infraumbilical region, acute in onset, dull in nature, nonradiating, not associated with food intake or movement, and nonresponsive to oral pain medications. Occasionally, she also felt a slight discomfort in the periumbilical region. She denied any episodes of vomiting or fever and mentioned that she was able to pass flatus.
In early 2021, the patient underwent a laparoscopic myomectomy for a large uterine fibroid. Following that, she received a trial of in vitro fertilization, which resulted in the implantation of three fetuses, of which two were in the uterus and one was tubal ectopic, for which she underwent a second laparoscopy. She carried the uterine twin pregnancy to term and delivered via Caesarean section in January 2022. Her postoperative course was complicated by abdominal pain and nonpassage of flatus, but she was managed conservatively for subacute intestinal obstruction.
In March 2022, she experienced vague abdominal pain and underwent ultrasonography. Free fluid was observed in the pelvis; therefore, the patient underwent a diagnostic laparoscopy, during which pus was aspirated from the pelvis, with no other significant findings.
The patient presented to our tertiary care center with the aforementioned complaints. On examination, she was tachycardic with a heart rate of 105 beats per minute, and the rest of her general examination results were unremarkable. Her abdomen was nondistended and soft but tender in the left iliac fossa and infraumbilical region. A nasogastric tube was inserted, and the patient kept nil per oral and was managed in the ward with fluids and adequate analgesia.
Investigations
None of the hematological investigations revealed any abnormalities. Erect radiography of the abdomen revealed air-fluid levels. An ultrasonography study was reported to show multiple dilated jejunal loops and an echogenic foreign body of approximately 8 cm, with posterior acoustic shadowing in the lower midline above the bladder. The findings from a contrast-enhanced computed tomography (CT) scan of the abdomen and pelvis resonated with the ultrasonography findings, showing 40-mm wide jejunal loops and jejunoileal junction narrowing. Radiologists also reported a 10 cm × 5 cm oblong low-density structure with thick walls closely adherent to the bowel loops and adjacent uterus ( Figure 1).
FIGURE 1: CECT of the abdomen and pelvis showing an
oblong low-density structure (orange arrow) encapsulated between the bladder and the uterus, adhering to the bowel.
CECT, contrast-enhanced computed tomography
Performing a diagnostic laparoscopy with removal of the foreign body with resection and anastomosis of any damaged bowel was decided. All four quadrants of the abdomen were examined. The right and left upper quadrants and the right lower quadrant were normal. In the left lower quadrant, a firm lump was found. The vesicouterine pouch was opened via a hydrodissection, exposing a cotton gauze measuring 10 cm × 15 cm, encapsulated by fibrous tissue, stained with stool, and surrounded by pus ( Figure 2). Along with this lump was an adhered loop of jejunum, which the gauze had invaginated into, causing a perforation. The foreign body was removed laparoscopically, and the perforated bowel loop was resected out. Eight centimeters of distal jejunum was resected, and a side-to-side jejunoileal anastomosis was performed. The retained gauze, about 10 cm × 6 cm, after extraction is shown in Figure 3.
Discussion
Gossypiboma is derived from the Latin word Gossypium (meaning cotton) and the Swahili word boma (meaning place of concealment). An RFB discovered by a fresh set of surgeons poses a professional and personal dilemma. For this reason, gossypiboma cases remain grossly underreported. However, the incidence of gossypiboma was reported to be 1 in 100-5,000 surgical procedures and one in 1,000-1,500 intra-abdominal operations [2]. Gawande et al. identified three risk factors of gossypiboma: emergency procedures, an unplanned change during the procedure, and the patient's body mass index [3]. Gossypiboma is also more common in female patients who have undergone gynecological procedures owing to the discrepancy between the size of the incision and the area explored [4], which is consistent with our case, where the patient had a lower segment Caesarean section 11 months before the presentation.
An RFB can lie dormant in the body for long periods, and the body deals with it in two ways: first, through an exudative, infectious response that leads to abscesses and fistulas, and second, an aseptic fibrinous response that leads to encapsulation and subsequent mass effect [5,6]. A patient with gossypiboma can present with fever, vomiting, obstruction, intra-abdominal sepsis, fistula formation, and gastrointestinal bleeding. In our case, a laparoscopy performed three months after a Caesarean section revealed a purulent collection in the pelvis, but the sponge was most probably missed because of its location, which was deep in the vesicouterine pouch, and it was covered with adhesions. A retained sponge can also migrate into an adjacent hollow viscus and lead to perforations, fistulae, or obstruction. The most common site for invagination is the small intestine because of its large surface area and thin wall [6]. This is consistent with the finding of jejunal perforation in our case.
A gossypiboma can be difficult to diagnose. Theoretically, all radiological investigations such as ultrasonography, radiography, CT, and MRI can identify RFBs but may be challenging. In the case of radiography, the sponge may be hidden, like in our case, where it was in a pelvic pouch, or the skiagram may be complicated by gas shadows or incidental findings such as renal stones. Our patient's radiograph only showed features of small bowel obstruction. The RFB may not be detected on MRI because the radiopaque marker is not magnetic. For all these cases, a CT examination is the investigation of choice. The characteristic appearance is that of a spongiform pattern with gas bubbles [2].
When a gossypiboma is found, surgical removal of the foreign body is mandatory. The two available options for dealing with a gossypiboma are exploratory laparotomy and diagnostic laparoscopy, both followed by the removal of the RFB and needful surgery. While an open method can be quicker and easier and provide better exposure, laparoscopy has advantages, including a less painful postoperative course, smaller scars, early ambulation, early oral intake, lesser risk of hernia, and overall quicker recovery and return to daily life activities [5,7]. However, laparoscopy may involve a longer operating time because of adhesiolysis and the surgical technique and poses a risk of bowel injury during entry to the abdomen. Reports on laparoscopy used for the retrieval of an RFB are limited, but in appropriately selected patients, it can provide faster recovery and decrease the anxiety of undergoing an operation due to a previous poor experience.
A gossypiboma is classified as a surgical never event. According to the definition of the US National Quality Forum, never events are errors in medical care that are identifiable, preventable, and serious in their consequences for patients, making it an important problem in terms of the safety and credibility of a healthcare facility [8]. To prevent this event, various guidelines are followed, such as the frequency of performing the sponge count and the World Health Organization surgical safety checklist. The decision to use and update the safety checklists regularly depends upon the institution and its resources, but the recommendation is to use and update them frequently.
Conclusions
Though rare, an RFB should not be completely ruled out when making a differential diagnosis. To avoid RFBs, multiple checks at multiple levels should be ensured and the nursing staff should be given proper education about safety checklists and the importance of instrument and gauze counts. Although laparotomy is the go-to procedure for RFB removal, successful laparoscopic removal allows for diagnostic exploration, with the added advantages of early ambulation, decreased postoperative pain, early diet tolerance, and overall early return to normal life.
Disclosures
Human subjects: Consent was obtained or waived 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. | 2023-04-08T15:14:24.556Z | 2023-04-01T00:00:00.000 | {
"year": 2023,
"sha1": "48657c0a583d8affccb2b37ba18c1d569f6a5dd8",
"oa_license": "CCBY",
"oa_url": "https://assets.cureus.com/uploads/case_report/pdf/149038/20230406-13102-1xp0qmu.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "eb634e63e9ebc5b3b65d4b148a9a5c8722871e12",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
233185310 | pes2o/s2orc | v3-fos-license | Prevalence and coprevalence of modifiable risk factors for upper digestive tract cancer among residents aged 40–69 years in Yangzhong city, China: a cross-sectional study
Objectives To describe the prevalence of modifiable risk factors for upper digestive tract cancer (UDTC) and its coprevalence, and investigate relevant influencing factors of modifiable UDTC risk factors coprevalence among residents aged 40–69 years in Yangzhong city, China. Design Cross-sectional study. Participants A total of 21 175 participants aged 40–69 years were enrolled in the study. 1962 subjects were excluded due to missing age, marital status or some other selected information. Eventually, 19 213 participants were available for the present analysis. Main outcomes measures Prevalence and coprevalence of eight modifiable UDTC risk factors (overweight or obesity, current smoking, excessive alcohol consumption, insufficient vegetables intake, insufficient fruit intake and the consumption of pickled, fried and hot food) were analysed. Results The prevalence of overweight/obesity, current smoking, excessive alcohol consumption, insufficient vegetables intake, insufficient fruit intake and the consumption of pickled, fried and hot food in this study was 45.3%, 24.1%, 16.2%, 66.1%, 94.5%, 68.1%, 36.0% and 88.4%, respectively. Nearly all (99.9%) participants showed one or more UDTC risk factors, 98.6% of the participants showed at least two risk factors, 92.2% of the participants had at least three risk factors and 69.7% of the participants had four or more risk factors. Multivariate logistic regression analysis revealed that men, younger age, single, higher education, higher annual family income and smaller household size were more likely to present modifiable UDTC risk factors coprevalence. Conclusions The prevalence and coprevalence of modifiable UDTC risk factors are high among participants in Yangzhong city. Extra attention must be paid to these groups who are susceptible to risk factors coprevalence during screening progress. Relative departments also need to make significant public health programmes that aim to decrease modifiable UDTC risk factors coprevalence among residents aged 40–69 years from high-risk areas of UDTC.
INTRODUCTION
According to WHO, more than 70% of the total deaths worldwide were related to non-communicable diseases (NCDs) in 2016. 1 Cancer is the second cause of NCDs, accounting for 22% of total global deaths related to NCDs. Globally, in 2018, an estimated 572 034 individuals were diagnosed with oesophageal cancer (OC), and 1 033 701 individuals were diagnosed with stomach cancer (SC), with approximately 50% of new cases occurring in China. There are an estimated 508 585 cancer deaths of OC and 782 685 cancer deaths of SC in 2018, accounting for 5.3% and 8.2% of cancer-cause deaths. 2 Apparently, upper digestive tract cancer (UDTC) (oesophagus, stomach) has become a significant morbidity and mortality source
Strengths and limitations of this study
► This is the first study examining the prevalence and coprevalence of modifiable upper digestive tract cancer (UDTC) risk factors and investigating relevant influencing factors in Yangzhong city, with large and representative residents from southeast China. ► Participants who volunteered to participate in our study are considered to be the high-risk group for UDTC, which is significantly important for the prevention and control of UDTC in China. ► A cross-section study cannot exam the causality or temporal relationship. ► The modifiable UDTC risk factors included in our study were self-reported by participants, which may contribute to recall and reporting bias, except body mass index. ► We only focus on the eight modifiable UDTC risk factors, which may underestimate the average number of modifiable UDTC risk factors among participants.
Open access related to cancer. According to the National Cancer Center, 3 OC has been the sixth most common cancer and the fourth most common cancer cause of cancer-related death. SC has been the second most common cancer and the third most common cancer cause of cancer-related death. The incidence rate of OC and SC was 17.87/100 000 and 29.31/100 000, the mortality rate of them was 13.68/100 000 and 21.16/100 000, respectively, in China in 2015. 3 Hence, UDTC has become a major public health challenge in China, and the disease burden of it is also considerable. 4 Due to the population health-seeking behaviour and the diseases' character, UDTC is mostly diagnosed at a late stage, which is leading to a low survival rate. It was estimated that its 5-year survival rate was less than 20% if diagnosed at an advanced stage but is as high as 95% if detected at an earlier stage. [5][6][7] Although the cause of UDTC is not clear, it is believed by researchers that the epidemic of UDTC in China is attributed to the multiplicity of demographic factors, diet, lifestyle, family health, environment, gastrointestinal history and genetic factors. 8 It is well known that tobacco, alcohol consumption, overweight or obesity, thermal irritation (drinking scalding liquids) and insufficient intake of vegetables and fruit, consumption of pickled and fried food are eight risk factors that can be altered by a tangible action for UDTC control. [9][10][11][12][13] Although parts of these risk factors have decreased because of a set of interventions implemented by the Chinese government, 14 15 the others have increased and will continue to grow in the next decades because of the rapid transition of urbanisation, industrialisation and ageing. 16 17 Furthermore, several studies have indicated that these risk factors coprevalence was common in the population which would further increase the risk of UDTC. 18 19 A comprehensive assessment of the distribution and the status of UDTC risk factors coprevalence is significant for cancer prevention and control. Once we have such data, interventions can be planned and implemented efficiently to minimise these modifiable risk factors, thereby minimising the health risks of increasing UDTC-related morbidity and mortality.
The Yangzhong city of Jiangsu Province is one of the high-risk areas of UDTC, especially in rural areas. 8 In 2015, the incidence rate of OC was 69.2/100 000, the mortality rate of OC and SC was 70.24/100 0000 and 81.89/100 0000, respectively, in Yangzhong city, which is higher compared with the average of the nation. 20 21 Hence, Yangzhong city had been one of the project sites of the Upper Digestive Tract Cancer Early Diagnosis and Treatment (UDTCEDAT) since the 2006. 8 Many studies have estimated the risk factors for UDTC in different areas worldwide. [9][10][11][12][13] The results reveal that risk factors for UDTC are widespread. The modifiable risk factors are significantly crucial for the prevention and control of UDTC because these factors can be changed by some healthy education or other interventions implemented by doctors and government and improved with the increase of personal health awareness. However, the evidence on the coprevalence of these modifiable risk factors in highrisk areas is still limited. Thus, we aimed to report the prevalence and coprevalence of modifiable UDTC risk factors and analyse the relevant factors influencing modifiable UDTC risk factor coprevalence among residents aged 40-69 years in Yangzhong city, which is a high-risk area of UDTC.
Study population
For the present study, we used secondary data collected from the screening of UDTC, focusing on the early diagnosis and treatment of UDTC among high-risk populations (aged 40-69 years) in Yangzhong city, China, from 2006 to 2017. 22 We use the method of multistage stratified cluster sampling to select the study sample. In the first stage, we stratified Yangzhong city into six regions (Sanmao, Baqiao, Youfang, Xinglong, Xilai and Xinba), covering the whole Yangzhong. In the second stage, we randomly selected clusters of three regions (Baqiao, Youfang and Xinglong) by region distribution and economic level based on Yangzhong Yearbook data. In the third stage, administration villages or neighbourhood communities in each chosen regions were randomly selected with probability proportional to size. In the fourth stage, each resident group or village group was selected from chosen administration villages or neighbourhood communities. In the fifth stage, all man or woman eligible from each household in the sites mentioned above were invited for cancer screening, unless they met the following exclusion criteria: (1) history of UDTC or mental disorder; (2) contraindications for endoscopic examinations and (3) inability to complete the whole interview or informed consent. Inclusion criteria for participants were as following: (1) aged 40-69 years; (2) permanent residents in Yangzhong City and (3) willing to accept endoscopic examination.
Before the screening, we obtained written informed consent from all participants after informing them about the backgrounds, objectives, procedures, benefits, confidentiality agreement of personal information and possible consequences of the whole programme. Then questionnaire-based interview, physical examinations, laboratory tests were performed by professional investigators. At last, the endoscopic examinations, pathological diagnosis and necessary therapy for participants were conducted by well-trained doctors in People's Hospital of Yangzhong city. The screening procedure follows China's cancer screening and early diagnosis and treatment technology programme strictly. 22 The data used in this study derived mainly from the questionnaire and physical examinations. Finally, a total of 21 175 individuals were surveyed, with a response rate of 60.9% (21 175/34 743), 1962 residents were excluded due to missing age, marital status or some other factors, leaving 19 213 participants available for the present survey. The sample size accounted for about 17.3% of the total target population Open access of Yangzhong city. We provided health education about UDTC and the potential role of modifiable risk factors related to UDTC to all eligible participants after collecting information relating to risk factors with the questionnaire. Besides, we combined active and passive follow-ups to collect outcome information for participants diagnosed with UDTC or precancerous lesions. We also performed a regular re-examination for patients according to the diagnosis.
Questionnaire data collection Before implementing data collection, training sessions organised by the expert group on UDTCEDAT were provided for all staff. The aim of this study, the standard measurement methods, how to perform questionnaires properly and the concrete study procedure were included in the training contents. At the end of the training sessions, all staff participated in the assessment and proved to be qualified.
We use uniformly structured questionnaires to collect information through face-to-face interviews. Each questionnaire took approximately 25 mins to complete. The questionnaire information included demographic factors (gender, birthday, address, ID, marital status and household size), socioeconomic characteristic (education and annual family income), behavioural factors (excessive alcohol consumption, current smoking), dietary habits (insufficient fresh vegetables intake and insufficient fruit intake, consumption of pickled, fried and hot food) and body mass index (BMI).
Physical examination
Physical examination included height and weight. Height and weight measurements were taken by height scale and digital weight, respectively, with the help of trained examiners based on a standardised programme. All subjects were asked to remove any footwear, hats, and heavy clothing before height and weight were measured. Height was measured to the nearest 0.1 cm, while weight was measured to the nearest 0.1 kg. BMI was calculated by dividing body weight (in kilograms) by the square of height (in metres).
Assessment criteria
Definitions of UDTC modifiable risk factors and the coprevalence of these risk factors Eight modifiable UDTC risk factors were defined based on current national guidelines or related references. Overweight/obesity was defined as BMI ≥24.0 kg/m 2 . 23-25 Current smoking was defined as self-reported having used any tobacco products, including cigarettes, cigars or pipes daily continuously. 26 Excessive alcohol consumption, insufficient fresh vegetables and fruit intake were defined according to the Dietary Guidelines for Chinese residents (2016). 27 Accordingly, excessive alcohol consumption was defined as consumption of more than 25 g (for males) or 15 g (for females) alcohol drinks per day after calculating pure alcohol based on the type of alcohol they chose, insufficient vegetables intake as self-reported consumption of vegetables less than seven times per week and insufficient fruit intake as self-reported consumption of fruit less than seven times per week. 27 Besides, self-reported consumption of pickled, fried or hot food at least once a week were classified as 'yes' in dietary habits, respectively.
Based on considering the literature and the average number of risk factors in the research population, coprevalence of modifiable UDTC risk factors was defined as presenting at least four related risk factors in one individual. 24 28 Covariates Covariates included in this study were demographic and socioeconomic information ascertained by questionnaire, including age (40-44 years, 45-49 years, 50-54 years, 55-59 years, 60-64 years, 65-69 years), gender (male and female), marital status (single, currently married, divorced/widowed/separated), educational status (no institutional education, primary school, junior high school, senior high school and higher), household size (0-3, 4-5, ≥6) and annual family income (tertiles: lower, middle and higher). 29
Statistical analysis
Descriptive statistics were applied to describe the sociodemographic characteristics of the sampled population. The difference in continuous variables was analysed by student's t-test and by χ² test to assess the differentials in the prevalence values among categorical variables. Differences in men and women, the prevalence of each modifiable UDTC risk factor and the distribution of modifiable UDTC risk factors coprevalence (0, 1, 2, 3 and ≥4) in sociodemographic and other characteristics were described in the overall population, respectively. Multiple logistic regression models were adopted to explore the association between relevant characteristics and UDTC risk factors coprevalence. Only the variables that we found statistically significant at p<0.05 in the univariate analysis were included in the multiple logistic regression models. The result of multiple logistic regression analyses was presented in terms of adjusted ORs and their respective 95% CIs. All statistical analyses were performed by SPSS software V.17.0. A two-sided p<0.05 was considered to be statistically significant.
Patient and public involvement
No participants or public were included in the design phase of this study. No participants were asked to advise on interpretation or writing up of results. Dissemination of the result of the research to participants and relevant participants community was prohibited. All the participants had the right to receive the result of health check if they wanted.
Sociodemographic and other characteristics of participants
The description of sociodemographic and other characteristics of 19 213 participants are presented in table 1.
Prevalence of modifiable UDTC risk factors
The prevalence of overweight or obesity, current smoking and excessive alcohol consumption in this study was 45.3%, 24.1% and 16.2%, respectively. The prevalence of current smoking and excessive alcohol consumption in men was significantly higher than in women (all p<0.001). In addition, insufficient vegetables intake, insufficient fruit intake and the consumption of pickled, fried and hot food in participants accounted for 66.1%, 94.5%, 68.1%, 36.0% and 88.4%, respectively. The prevalence of consumption of pickled and hot food was higher in women than in men (all p<0.05) (table 2). As shown in table 3, there were significant differences in age, marriage status, education level, and annual family income in the eight UDTC risk factors (all p<0.05). The prevalence of these eight modifiable UDTC risk factors tends to be higher in single participants, except for overweight or obesity (all p<0.001). Moreover, the prevalence of excessive alcohol consumption, insufficient vegetables intake, insufficient fruit intake and the consumption of pickled, fried and hot food varied significantly with the household size (all p<0.001) (table 3).
Coprevalence of modifiable UDTC risk factors
The prevalence of zero to eight modifiable UDTC risk factors participants had simultaneously in the study (overweight/obesity, current smoking, excessive alcohol consumption, insufficient vegetables intake, insufficient fruit intake, the consumption of pickled, fried and hot . Participants who were married (OR 0.511, 95% CI 0.330 to 0.792) were less likely to have four or more modifiable UDTC risk factors than those who were single. In addition, we divided the education into four groups, which showed that increasing the level of education was a risk factor for modifiable UDTC risk factors coprevalence. Compared with participants who had a higher level of annual family income, those who had middle (OR 0.218, 95% CI 0.197 to 0.241) and lower (OR 0.223, 95% CI 0.201 to 0.247) level of annual family income were less likely to have four or more modifiable UDTC risk factors. Modifiable UDTC risk factors coprevalence were less common among participants who had more than six family members than those who had less than three ones (OR 0.598, 95% CI 0.527 to 0.678) (table 5).
DISCUSSION
As far as we know, this is the first large population-based survey investigated the prevalence and coprevalence of eight modifiable UDTC risk factors and described the sociodemographic and socioeconomic factors associated
Open access
with these among Yangzhong city residents aged 40-69 from southeast China. The present study revealed that the prevalence and coprevalence of overweight or obesity, current smoking, excessive alcohol consumption, insufficient vegetables intake, insufficient fruit intake and consumption of pickled, fried and hot food were high, which implied the health risk of UDTC residents have in Yangzhong city. We found that the prevalence of insufficient fruit intake and hot and pickled food consumption were the top three modifiable UDTC risk factors in the population surveyed. Besides, 69.7% of the participants presented at least four UDTC risk factors.
Open access
The prevalence of overweight or obesity (45.3%) in our study was higher than that observed in Nanjing (35.6%), 23 and nationwide population (42.0%), 30 but our findings were closed to that observed in some other regional. 24 31 The prevalence of current smoking in this population (24.1%) was much higher than that in a cross-sectional study in Shenzhen (10.5%) 24 and Barbados (9.2%), 32 which was consistent with a survey in Nanjing (24.5%). 23 However, the rate was not as high as reported (28.1%) in the China national nutrition and chronic disease survey (2015). 30 Our findings showed a high prevalence of excessive alcohol consumption in the Yangzhong population (16.2%) relative to the national average of 11.1% in men and 2.0% in women. 30 The rate of excessive alcohol consumption we found was similar to Barbados, Nanbu and the prospective study of China Kadoorie Biobank, where excessive alcohol consumption rates were around 14.5%, 32 16.7% 19 and 14.9%, 33 respectively. We found higher levels of insufficient intake of vegetables (66.1%) in this population than those observed in the Tanzania 34 and Hubei Province 35 where the insufficient intake of vegetables or fruit is 55.8% and 29.7%, respectively, while the levels of insufficient intake of fruit (94.5%) in our study were also much higher than that observed in the region mentioned above. 34 35 Moreover, the proportion of the Yangzhong population had dietary habits of consumption of pickled, fried and hot food were greater than the levels in Huaian (22.7%, 7.1%, and 10.9%), 36 as well as in Nanbu (28.63%, 1.95%, and 6.11%), 19 both of these region mentioned above are high-risk areas of UDTC in China. The co-prevalence of risk factors for chronic diseases is widespread. 19 Several previous studies reported the coprevalence of chronic diseases in the Chinese population. For example, among 49 247 Chinese aged 15-69 years from the 2007 China Chronic Disease and Risk Factor Surveillance, the prevalence of having zero, one, two and at least three chronic disease risk factors were 9.1%, 33.9%, 32.4% and 24.6%, respectively. 37 Also, other regional studies have examined the coprevalence of some specific chronic diseases in residents. Hong et al 23 reported that 30.1% and 35.2% of the Nanjing population presented one and at least two cardiovascular diseases (CVD) risk factors. Conversely, a much higher rate of CVD risk factors coprevalence was noticed by Ni et al in Shenzhen city. 24
Open access
In our present study, it was observed that 0.1%, 1.3%, 6.4%, 22.6% and 69.7% of participants had zero, one, two, three and at least four modified UDTC risk factors, respectively, among residents aged 40-69 years. The modified UDTC risk factors coprevalence was prevalent considerably in Yangzhong city. Different estimates of the risk factors coprevalence for UDTC were found in the literature. He et al 19 showed that among residents aged 40-69 years in UDTC high-risk areas, 33.08%, 35.99%, 16.76% and 11.93% of participants had one, two, three and at least four OC risk factors, respectively. In another casecontrol study of 2 266 Chinese adults, 32.5% and 41.1% of the participants presented three and four or more risk factors, respectively, for OC or SC. 18 Compared with these two studies mentioned above, 18 19 a much higher coprevalence of risk factors was noticed in our present study. The variations could change likely due to the difference in diagnostic criteria, the number and kind of risk factors included in the research and the participants' age group. Overall, there are some other national, and worldwide studies on the coprevalence of some common or specific chronic diseases risk factors. Still, the study on modifiable UDTC risk factors is limited.
The factors associated with modifiable UDTC risk factors coprevalence included gender, age, marriage status, education, annual family income and household size. We found the prevalence of modifiable UDTC risk factors coprevalence was lower in women compared with men, which was consistent with findings from other settings. 19 23 37 The possible reason could be Chinese men are less aware of self-protect for chronic diseases and have worse health-seeking behaviour, and may also attend more social occasions, tend to consume more tobacco/cigarette high-salt, high-fat and high-calorie food compared with women. 23 31 In addition, this study revealed that the prevalence of modifiable UDTC risk factors coprevalence was increased with age, which was consistent with previous studies. 19 23 24 37 Studies showed that in Nanbu, China, the coprevalence of OC risk factors increased with age may attribute to the lower level of awareness, practice and willingness for health among the elder. 19 Meanwhile, it is also shown in table 5 that being over 65 protects against having more than four risk factors. A possible reason for this difference is as follows: with the ageing of the body and the deterioration of organ function, an elder individual possesses a higher risk of health disorder and has a greater demand for medical care. As a result, this creates more opportunities to get diagnosed with some health screening, including UDTC. Correspondingly, the elderly have more chance to get a healthy education from physicians than the younger. 38 Our study also showed that single participants had more prevalence of modifiable UDTC risk factors coprevalence compared with participants who were currently married, following a previous study. 19 A possible explanation is that being a single older resident comes with its own economic and emotional challenges, contributing to the unhealthy habit of lifestyle and diet. 39 40 Our study demonstrated that the level of socioeconomic status (SES, education, annual family income) was positively associated with modifiable UDTC risk factors coprevalence, which was inconsistent with other reports. 19 23 37 Residents with a higher level of SES are more aware of control and prevention of chronic disease and have better health-seeking behaviour compared with those with a lower level of SES. 19 23 41 Moreover, the poor or lower education participants may have relatively more inaccessibility and unaffordability to medical services. 38 This paradox may be due to most of the participants enrolled in our study were from rural areas, and their SES was generally low. However, it may also imply that the higher income may contribute to unhealthy lifestyles, 42 and knowledge alone may not be sufficient to change unhealthy lifestyles. Therefore, the level of education and income are two essential SES factors for modifiable UDTC risk factors coprevalence.
It is, however, important to note that participants who have more than six family members had a lower prevalence of modifiable UDTC risk factors coprevalence compared with those who have less than three ones. Changes in household size are bound to affect the adjustment of the family diet. As the household size increase, it is more likely to increase dietary diversity (eg, fruits, vegetables and milk) every day. 43 Besides, the affection, information and economic support among family members will also increase significantly, which can adjust and correct the unhealthy lifestyle of individuals. 44 45 Our study explored the prevalence and influencing factors of modifiable UDTC risk factors coprevalence in the UDTC high-risk area, Yangzhong City, based on the community-based project for UDTC screening with a large sample size. Additionally, the physical measurement and the data collection implemented by trained interviewers strictly according to standard protocol and instrument increase the validity of our results. In order to reduce the prevalence and coprevalence rate of UTDC risk factors, the screening teams should focus on individuals with coprevalence of risk factors in screening and improve their unhealthy lifestyles continually through a range of methods such as post-screening health education, personalised interventions and disease follow-up. The social impact of screening should be expanded to improve the compliance of high-risk groups, thereby increasing the output of screening health benefits. Meanwhile, the government should also help high-risk groups (especially the older and male groups) to improve their health literacy and awareness of UTDC prevention through diversified education, motivation and publicity methods, such as health education, health talks and mass media campaigns. By guiding the culture of smoking, drinking and other food culture, promote high-cultural groups to transform their cognitive and economic advantages into advantages in UTDC prevention and healthcare, and effectively change unhealthy habits. Besides, the government should focus on single and residents with small household size in the high-risk groups in the Open access process of health education and the development of prevention strategies. The findings may also provide the reference for departments in charge of the prevention and control of UDTC in Yangzhong city, Jiangsu province and relevant departments in other UDTC high-risk areas (eg, Linzhou, Feicheng, Yanting).
There were also several possible limitations to our study. First, a cross-section study cannot exam the causality or temporal relationship between the coprevalence of modifiable UDTC risk factors and its influencing factors. Second, the modifiable UDTC risk factors included in our study were self-reported by participants, which may contribute to recall and reporting bias, except BMI. Our study results were from Yangzhong city only, and cannot be generalised to the other high-risk areas and the whole of southeast China. Additionally, the study response rate was relatively low, particularly among males, which may affect the results' representativeness. Finally, our study only focused on the eight modifiable UDTC risk factors, but there are far more than eight risk factors for UDTC. Hence, further studies are needed.
CONCLUSION
In summary, this cross-sectional study shows that the prevalence and coprevalence of modifiable UDTC risk factors are high among participants in Yangzhong city. Our analyses indicate that men, younger adults, single adults and participants with higher SES or smaller household size are susceptible to modifiable UDTC risk factors coprevalence. Policies to prevent UDTC have already been developed in the strategic plan and operational plan, however, the accuracy and validity of implementing the undertaken policies are still insufficient. Consequently, extra attention is required to pay to these high-risk groups during the progress of screening. Relative departments also need to make effective public health programmes targeting modifiable UDTC risk factors that aim to decrease UDTC risk factors coprevalence in high-risk groups from highrisk areas of UDTC.
Author affiliations 1 | 2021-04-09T06:19:10.323Z | 2021-04-01T00:00:00.000 | {
"year": 2021,
"sha1": "281bf7c7c1b6e60a1a42fffb0e25b9f8b8ec7e17",
"oa_license": "CCBYNC",
"oa_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031018",
"oa_status": "GREEN",
"pdf_src": "PubMedCentral",
"pdf_hash": "1ee28e5dafab078241414fb06af0f1cb357e909a",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
196542364 | pes2o/s2orc | v3-fos-license | Musculoskeletal Outcomes from Chronic High-Speed High-Impact Resistive Exercise
Abstract Subjects (n=13) did 30 workouts with their left leg on an Inertial Exercise Trainer (IET), while their right leg served as an untreated control. Before and after the 30 workouts, they underwent isokinetic strength tests (knee and ankle extensors of both legs) whose peak torque (PT), time to PT (TTPT), and rate of torque development (RTD) values were each analyzed with 2(leg)×2(time)×3(velocity) analysis of variances (ANOVAs), with repeated measures per independent variable. Peak force (PF) and total work (TW) data were measured from each IET workout, and they represent time course strength changes produced by our exercise intervention. PF and TW values for the three IET exercises that comprised each workout were each analyzed with one-way ANOVAs with time as the independent variable. Results included significant ankle and knee extensor PT increases, whereby the left leg achieved higher values at posttesting, but there were no significant TTPT changes and a time effect for ankle extensor RTD. Our data show that PF and TW each had significant increases over time, with the latter exhibiting greater gains over the 30-workout intervention. Our results imply that the IET yields strength gains over time comparable to standard resistive exercise hardware.
INTRODUCTION
Musculoskeletal changes to chronic exercise offer a temporal framework for which to anticipate strength improvements and/or attenuation of in-flight losses from exercise hardware and/or protocols. Temporal changes refer to the rate and magnitude of adaptations over time, which help formulate training strategies to limit in-flight strength losses and thereby optimize long-term musculoskeletal health for long-term exposure to microgravity (Fleck and Kraemer, 2014). Two 60-day bed rest studies assessed temporal adaptations to concurrent exercise countermeasures that sought to abate musculoskeletal losses (Kramer et al., 2017;Trappe et al., 2007). Those countermeasures included flywheel-based resistive exercise (Trappe et al., 2007) and high-intensity jump training (Kramer et al., 2017). Results showed that concurrent exercise reduced muscle mass and strength losses in those who received the experimental treatment as compared to bed-rested controls, yet neither countermeasure addressed bone losses, which is an important in-and postflight concern (Kramer et al, 2017; Trappe et al., 2007). However, a recent study with ambulatory subjects examined a 30-workout highspeed high-impact resistive exercise intervention on muscle and bone adaptations (Caruso et al., 2018). Subjects trained their left knee and ankle extensors on the high-speed high-impact device while the same muscle groups on their right legs served as untreated controls. Strength data obtained before and after the 30-workout intervention showed significant gains in left leg's knee and ankle extensor strength (Caruso et al., 2018). Yet, unlike the aforementioned bed rest studies, 30 workouts on the high-speed high-impulse device evoked large and significant gains in left leg's calcaneal bone mineral content (+29%) and density (+33%), as well as a significant decline in osteoclast activity (Caruso et al., 2018). Such results hold much promise, as the calcaneus incurs among the largest in-flight losses that are exacerbated by heightened osteoclast activity (Heer et al. 2005;LeBlanc et al., 2007). The high-speed high-impact device, called an Inertial Exercise Trainer (IET; Impulse Technologies, Newnan, GA), has a mass sled that traverses its 1.9-m track parallel to the Earth's surface and is thus not impacted by gravitational forces. The high-speed nature of its repetitions is created by the IET's low-friction track in which the sled glides upon with little force exertion (Davison et al., 2010). The IET has low mass, area, power needs and is easy to stow and operate, allowing it to conform to in-flight hardware guidelines (Smith et al., 2014). Impact forces are incurred as the sled changes direction with each new repetition, which is also when the peak force (PF) for each repetition occurs. With is a muscle's maximal ability to exert force (Brown 2000). Despite differences in workout and test modalities, the use of PT to assess performance changes was extended to training studies whereby exercise occurred against heavy loads with standard resistive exercise equipment (Gentil et al., 2006). Due to the high-speed nature of IET repetitions, perhaps better variables to test performance changes from its chronic intervention are time to PT (TTPT) and rate of torque development (RTD), which are instantaneous variables often cited as key determinants of success in performance tasks where reaction time is crucial (Brown et al., 2005). Since IET repetitions occur at high rates of speed, TTPT and RTD changes may prove valuable to monitor. Understanding temporal changes is essential to aid in the development of successful exercise prescriptions. Chronic high-speed IET training may yield unique neuromuscular adaptations that also evoke improvements in PT, TTPT, and RTD values over time (Cormie et al., 2009). Our study quantifies adaptations to strength gains from a chronic training intervention, whereby PF and total work (TW) values from IET workouts are monitored over time, for their impact on PT, TTPT, and RTD changes measured by strength tests on an isokinetic dynamometer. We use IET workout data from the aforementioned investigation (Caruso et al., 2018) as part of our study's analyses. We hypothesize in healthy subjects, whereby one leg undergoes the IET intervention and the other serves as an untrained control, that their trained leg's adaptations over time will yield significant PT, TTPT, and RTD improvements from the IET intervention.
Experimental Approach to the Problem
Subjects did 30 IET workouts with their left leg, at an average rate of one every 2.3 days, while their right leg served as an 2.0-2.5 repetitions done per second over 1-min sets, as well as an average PF of 900 N for one of its exercises, the IET is indeed a high-speed high-impact training device (Caruso et al., 2018). With a small mass added to its sled and rapid force exertion, its repetitions are inherently ballistic. Impulses (D force/D time) from each repetition impart far different mechanical loading stimuli than standard exercise hardware. Thus, the IET may yield temporal adaptations to strengthbased variables unlike those seen previously which, to date, have not been examined. Figures 1 and 2 show the overhead and side view illustrations of the IET, respectively. Studies that charted temporal strength changes used variables such as peak torque (PT) to assess improvements caused by exercise interventions (Caruso et al., 1997). PT done with a velcro cuff around their distal left shank. As the knee extended ~10-15°, the sled traveled rapidly to the end of the track. As it traveled, the knee flexed back to its initial joint angle. Before the sled reached the end of the track, the next repetition occurred, which accelerated the sled to the track's opposite end. These high-speed movements were repeated over successive repetitions until subjects were proficient in the exercise. Changes in sled direction created an impact force, which was high due to the sled's rate of movement (Davison et al., 2010). Per impact force, high PF and impulse (D force/D time) values were also generated. Figure 4a and b depicts the standing knee extension exercise. With a velcro strap wrapped around the arch of subject's left foot, the standing hip extension exercise was done in a similar fashion. Figure 5a and b depicts hip extension repetitions. Unlike the knee and hip extension exercises, the seated calf press was done over a shorter range of motion. Given the limited sled displacement for the seated calf press, high rates of movement were more difficult to attain. For seated calf presses, the strap was wrapped around the metatarsal heads of subjects' left feet. Figure 6a and b depicts seated calf presses on the IET. Like the standing knee extension exercise, per standing hip extension and seated calf press repetition high PF and impulse (D force/D time) values were generated. The principal investigator was present to ensure that, by the end of familiarization sessions, subjects properly executed repetitions per exercise.
Workouts
All 30 workouts that comprised the IET intervention were carried out in the same manner. They commenced with a 5-min bilateral warm-up on a cycle ergometer (Ergotest, Stockholm, untreated control. The IET was instrumented with software that allowed real-time capture of data. Variables from IET workouts were recorded and examined for changes over time. Before and after the 30-workout intervention, subjects also underwent isokinetic strength tests, at three angular velocities, for the knee and ankle extensors of both legs, to provide PT and TTPT values. PT and TTPT changes in the left leg may be attributed to IET workouts. Per subject, Figure 3 depicts a timeline with an overview of their involvement.
Subjects
Before admittance into our study, the University of Louisville's Institutional Review Board approved all procedures. Subjects (mean ± standard error of the mean [SEM]: 29.4±5 years; two men, 11 women) gave informed written consent and filled out a medical questionnaire, prior to their participation. They were free of the following conditions: diabetes, asthma, hypertension, tachycardia, ischemia, arrhythmias, hyperthyroidism, and convulsive disorders. Aside from the physical requirements of their normal daily activities, our subjects did not engage in exercise during their participation. Their body mass and body fat percentage were 69.2±3 kg and 26.9±0.20%, respectively. They were well versed in various forms of exercise, but none had prior experience with IET workouts. Thus, prior to the intervention, each subject did two familiarization sessions so repetitions were done correctly.
Familiarization sessions
Subjects practiced repetitions with their left leg for the following exercises: standing knee extension, standing hip extension, and seated calf press. Each was done with 3.4 kg of mass added to the 1.0 kg IET sled. Knee extension was periods (for workouts 1-6, 7-12, 13-18, 19-24, 25-30), which allowed a comparative examination of each training variable's temporal changes.
Isokinetic strength testing
Left and right leg knee extensor strength tests, followed by those for the ankle extensors, occurred before and after the 30-workout intervention. Tests occurred at three (0, 1.62, and 4.86 rad./second) angular velocities. For each test, we aligned subject's knee or ankle joint with the dynamometer's (System 3 Biodex, Shirley, NY) axis of rotation and were held constant across test sessions. Velcro straps limited extraneous body movement. Tests began with five submaximal isometric contractions at 90° of knee flexion separated by 30-s rest periods. After a 90-s rest, the isometric protocol was repeated with maximal contractions. They then repeated the paradigm at 1.62 rad./second, and then 4.86 rad./second over a 90° range of motion. The ankle extensor protocol then commenced. With the dynamometer configured for ankle testing, repetitions occurred over a 45° range of motion, from a dorsi-to a full plantar-flexed position. Ankle isometrics occurred at a 0° (neutral) angle. Otherwise, its protocol was identical that of the knee extensors. PT and TTPT values, from maximal effort contractions, were obtained per velocity. We also calculated RTD as the D torque/D time ratio.
Statistical Analyses
Our sample size was based on a power analysis conducted prior to data collection. Ten subjects offered >90% power to detect a 15% change from our 30-workout intervention with a two-factor repeated-measures design with leg and Sweden) against 1 kp of resistance at a self-selected velocity. Subjects then performed IET exercises in the following order with their left leg: standing knee extension, standing hip extension, and seated calf press. Per exercise, they did three 60-s sets separated by 90-s rests. They were instructed to exert maximal effort and were verbally encouraged during sets to perform repetitions as rapidly as possible as they maintained proper form. Our exercise protocol's rationale stems from the idea that osteogenic exercise should be nontraditional and/or nonsteady state in nature (Mittag et al., 2015;Yang et al., 2015, Yang et al., 2014. To evoke greater osteogenesis, we attempted to elicit high bone strains over short durations (Lanyon et al., 1986;Lanyon 1992;Nguyen et al., 2008). Three sets per exercise were done since bone strain stimuli reached a saturation point after only a few cycles (Lanyon et al., 1986;Lanyon 1992). IET instrumentation entailed a calibrated TLL-2K load cell (Transducer Techniques, Temecula, CA) and position sensor (Model CX3-AP-1A, Automationdirect.com) anchored to the center of the track, directly below the path of the mass sled. Load cell and position sensor data were continuously sent to a DI-158U signal conditioner (DATAQ Instruments, Akron, OH) and assessed on separate channels at 4000 Hz. Our instrumentation methods provide reproducible data (Caruso et al., 2008). Workouts were 25 min in duration. Per set and exercise, we derived a PF and TW value, which represents instantaneous and cumulative measures of exercise performance, respectively. Increases to PF and TW values over the 30-workout intervention represent adaptations over time produced from training. Per exercise, PF and TW values were averaged and pooled across six consecutive time five time periods were as follows: workouts 1-6 87.6±3.7, workouts 7-12 88.6±3.5, workouts 13-18 90.1±3.1, workouts 19-24 93.8±3.0, workouts 25-30 91.4±3.3. Post hoc analysis of standing knee extension TW results was as follows: 19-24>25-30, 13-18>7-12, 1-6. PT, TTPT, and RTD results are summarized in Tables 1-6, with knee and ankle extensor data presented for the left (trained) and right (untrained) leg per time point and angular velocity examined. Despite the high speed nature of the IET, Table 1 only shows significant knee extensor PT increases at 0 rad./ second. Post hoc analyses showed that left knee extensor PT posttest values were the interaction source. Pre-post percentage gains in knee extensor PT were higher for the left (+11%), than the right (+4), leg. Yet, Table 2 shows significant two-way ankle extensor PT gains per velocity examined. Left ankle extensor posttesting values were the source of each two-way interaction. Pre-post percentage ankle extensor PT increases were higher for the left, than the right, leg at 0 (+21% vs +7%), 1.62 (+19% vs +5%), and 4.86 (+18% vs +7%) rad./second. Tables 3 and 4 display nonsignificant TTPT changes for the knee and ankle extensors, respectively. TTPT data show more variability than PT values, which accounts for the lack of statistical significance. RTD results show nonsignificant knee extensor (Table 5) changes, while Table 6 shows ankle extensor data elicited a time effect (post-intervention>pre-intervention).
DISCUSSION
Our results include significant two-way ankle and knee extensor PT increases. With different modalities for workouts and strength tests, our time course changes suggest an IET intervention that evokes large increases in TW (+30-42%), but not PF (+7-9%) and yields significant gains in isokinetic PT, but not TTPT and RTD, over time. Our IET intervention averaged 10 weeks to complete 30 workouts. Some studies that also examined high-speed exercise employed training interventions of comparable durations. It is of interest to compare our results to those studies. A 10-week total body resistive exercise program evoked similar elbow flexor PT gains among men (+11.6%) and women (+11.8%) which concur with the magnitude of our study's knee extensor PT increases when tested at 0 rad./second (Gentil et al., 2016). Men were assigned to a strength, power, or a nonexercise group with no crossover for 10 weeks (Cormie et al., 2009). Strength subjects did back squats at 75-90% of 1RM loads, while the power group did jump squats at 0-30% of 1RM loads. There were comparable vertical jump (+17%) and sprint (+2%) gains after 10 weeks among the trained groups, yet strength-trained subjects had greater squat 1RM gains (+31.2%) than those who were power trained (+4.5%). Strength training enhanced time as within-subjects factors (Fritton et al. 2008). Thus, our sample (n=13) should exceed these power and change thresholds for our three-factor repeated-measures PT, TTPT, and RTD analyses. Our data were examined for compliance with analysis of variance (ANOVA) assumptions (normality, independence, equal variances). PF and TW values per exercise were examined with one-way ANOVAs from average values for the five time periods. Those five periods were as follows: workouts 1-6, workouts 7-12, workouts 13-18, workouts 19-24, and workouts 25-30. PT, TTPT, and RTD values were each analyzed with 2(leg) × 2(time) × 3(velocity) ANOVAs, with repeated measures for leg, time, and velocity. An alpha level of 0.05 denoted significance for all analyses. Tukey's HSD test identified the source of the differences.
The effects of an 8-week submaximal eccentric ankle extensor protocol were examined in participants either trained 2 days/week at 0.35 rad/s, or served as untrained controls with no crossover (Barrué-Belou et al., 2016). Training loads were adjusted every 2 weeks to maintain a 75% maximum of eccentric torque for workouts. After 8 weeks, muscle activation (+23-29%) and force measurements (+18-21%) rose significantly in the eccentrically trained group. Results suggest that force increases produced by training were at least in part due to enhanced neural drive (Barrué-Belou et al., 2016). The magnitude of PT gains from ballistic and eccentric training studies concurred with the improvements in ankle extensor PT seen with our study's intervention (Balshaw et al., 2016;Barrué-Belou et al., 2016). Like our study, a prior investigation monitored strength improvements over time, albeit its repetitions occurred at far slower rates (Caruso et al., 1997). Temporal knee extensor strength changes were assessed in subjects randomized to a concurrent oral albuterol or placebo treatment with no crossover (Caruso et al., 1997). Subjects trained on an isokinetic dynamometer, which imparted concentric and eccentric resistance, 2 days/week for 10 weeks at 0.79 rad./ second. After 10 weeks, four dependent variables showed time effects (+8-18%) and six others had two-way interactions (+9-21%). The variables that exhibited time effects were concentric measures that elicited significant gains generally within the first 2-3 weeks of training. Five of the six variables with two-way interactions were eccentric and produced significant intergroup differences after 3 weeks of training. It was implied that relatively greater eccentric gains were from the higher forces exerted as muscles lengthen and/or faster rates of motor unit synchronization (Caruso et al., 1997).
Despite differences in exercise modes and repetition rates, the magnitude of improvements for the knee extensor study is like those from our study (Caruso et al., 1997). The current PT, TTPT, and RTD results produced small nonsignificant gains to the untrained leg over time, which implies a potential cross-education effect from the IET intervention. Yet, the magnitude of these gains is less than those reported in other papers, in which a comparable degree of improvement was achieved over a shorter time period (Green, Gabriel 2018; Hunter 2017). Nonetheless, our study's PT, TTPT, and RTD changes to the untrained leg may be the result of heightened central drive, which occurred in a prior study but has yet to be affirmed from IET workouts and thus warrant future inquiry (Green, Gabriel 2018). With our workouts geared toward bone growth, we achieved high strains over very short lengths of time, but to do so our absolute knee extensor range of motion per repetition was small. In contrast, a larger range of motion was used for knee extensor testing. This discrepancy represents a potential source of error for our knee extensor test results. Despite the discrepancy, our results include a significant twoway interaction for knee extensor PT at 0 rad./second, with left leg posttest values as the source of the differences. In contrast, knee extensor tests at 1.62 and 4.86 rad./second entailed dynamic movement, and discrepancies in the range of motion for knee extensor training and testing are possible reasons for those dependent variables that did not reach statistical significance. It is also of interest that ankle extensor ranges of motion for training and testing were similar, and analyses of test data for that muscle group produced two-way interactions for each of velocity examined. Our IET intervention produced disparate results, as strength tests showed more benefit to ankle, as compared to knee, extensors, and significant improvements in PT, but less for TTPT and RTD. In similar fashion, IET workouts yielded relatively greater TW gains over time than PF for each exercise. It is important to explain possible reasons for these outcomes, as they may impact IET future training prescriptions. Aside from differences in the range of motion for testing the knee and ankle extensors, as well as inherent differences in PF and TW measurements, whereby the former is an instantaneous, and the latter a comprehensive, index of workout performance, there are other factors that account for our disparate results. In particular, the length of the Achilles tendon impacts the level of series elastic element activity from successive high-speed repetitions that likely has a major influence on ankle extensor strength test performance (Hunter et al., 2015).
Our IET workouts sought to provide an osteogenic benefit, for which nontraditional and/or nonsteady state forms of activity composed of high bone strains applied briefly over few loading cycles are recommended (Lanyon et al., 1986;Lanyon 1992;Mittag et al., 2015;Nguyen et al., 2008;Yang et al., 2015, Yang et al., 2014. Unloading-induced skeletal losses are highest in areas with weight-bearing responsibility and large amounts of trabecular bone, such as the calcaneus ). Astronauts on Skylab 3 (59 days) and 4 (84 days) had significant calcaneal bone mineral density (BMD) losses that ranged from 4.5% to 8% and also produced large increases in bone resorption, as did many longer flights (Leblanc et al., 2007). Cosmonauts incurred calcaneal BMD losses that varied from 1% to 20% for missions of 75-184 days (Leblanc et al., 2007). Seventeen-week bed rest studies showed that the highest (9-10%) density losses in humans were to the calcaneus (Le Blanc et al., 2002;Shackelford et al., 2004).
Since bones adapt to mechanical stimuli, treatments to abate to in-flight losses should include large muscle forces (Rittweger et al., 2010). Some think that skeletal remodeling is the sum total of muscular force exertion (Schoenau et al., 2002;Rittweger et al., 2000;Rubin & Lanyon 1987). The largest strains are produced by muscle forces (Schiessl et al., 1998). Given the results of the recent ambulatory study (Caruso et al., 2018), the IET holds considerable promise as an in-flight countermeasure to musculoskeletal losses. Our hypothesis was partially affirmed as IET workouts yielded significant gains in PT, but not TTPT and RTD, over time.
The current results may aid in the development of in-flight protocols with the IET, as the knee and ankle extensors incur significant strength losses in microgravity (Fitts et al., 2000;Trappe et al., 2009). Since both muscle groups typically maintain posture against Earth's gravity, their adaptation to space flight sees losses in PF and TW (Fitts et al., 2000). Vast musculoskeletal losses impair in-flight procedural tasks, mission goals, and astronaut health upon their return to Earth (Baldwin et al., 1996;Fitts et al., 2000). Similar changes occur to ground-based analogs. The knee and ankle extensors incur among the largest losses (Belavy et al., 2009;Riley et al., 2002;Widrick et al., 1998). A 14-day bed rest led to a 9% knee extensor torque loss (Bamman et al., 1998). A 30day bed rest elicited knee extensor strength losses (-20%) like those of a 28-day space flight (Convertino et al., 1989). Simulated microgravity for 20-25 days evoked knee extensor torque deficits of 17-21% (Berg et al., 1991;Berg et al., 2007;Gogia et al., 1988;Schultze et al., 2002). Forty days of unloading (-17-22%) and 90 days of bed rest (-31-60%) led to larger knee extensor losses (Alkner et al., 2004;Caruso et al., 2004). Unloading for 14-17 days caused significant (9-18%) ankle extensor strength loss (Adams et al., 1994;Bamman et al., 1997;Widrick et al., 1998). Five weeks of bed rest evoked a 26% loss in ankle extensor strength (LeBlanc et al., 1988). After 40 days, research showed that ankle extensor strength losses were 25% less than preunloading values (LeBlanc et al., 1988). Resistance exercise, which imparts more intense mechanical loading stimuli than aerobic activity, preserved ankle extensor strength during short-term (14-21 days) unloading (Akima et al., 2003;Bamman et al., 1998;Schultze et al., 2002). Yet, despite in-flight aerobic and resistive exercise done 5 and 3 days/week, respectively, 6 months aboard the International Space Station still led to significant ankle extensor mass and strength losses and shifts to faster myosin isoforms to create a more fatiguable muscle (Trappe et al., 2009). It was concluded that hardware that offers a different mechanical loading stimulus was required for long-term manned space travel (Trappe et al., 2009). An absence of in-flight mechanical loading not only adversely impacts musculoskeletal health and performance, but inevitably compromises other body systems. NASA goals of long-term manned space exploration cannot be met unless the issue of in-flight musculoskeletal and strength losses is addressed. Thus, exercise countermeasures to these adverse effects must be a priority. Given our results, whereby workouts produced time course gains in PF and TW that led to gains in PT at multiple angular velocities, the IET warrants continued inquiry. Future research on temporal adaptations to chronic IET interventions may include continued examination of some of our findings; they include 1) significant PT increases that coincided with relatively larger TW gains, as compared to those for PF achieved from workouts and 2) mechanisms that identify how cross-education effects are imparted by this unique exercise modality. Since the IET operates independent of gravity, and its design conforms to requirements for in-flight hardware, new research should assess musculoskeletal changes to chronic exercise in a space flight analog, such as human bed rest subjects (Smith et al., 2014). | 2019-07-15T22:29:34.672Z | 2019-06-17T00:00:00.000 | {
"year": 2019,
"sha1": "e39c0f36e6dbe336b8c2581fd1917a282aea2d6e",
"oa_license": null,
"oa_url": "https://doi.org/10.2478/gsr-2019-0004",
"oa_status": "GOLD",
"pdf_src": "DeGruyter",
"pdf_hash": "084bbada08227325d54ec10819078cd24f1aa52c",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
119361530 | pes2o/s2orc | v3-fos-license | Strong Correlations in a nutshell
We present the phase diagram of clusters made of two, three and four coupled Anderson impurities. All three clusters share qualitatively similar phase diagrams that include Kondo screened and unscreened regimes separated by almost critical crossover regions reflecting the proximity to barely avoided critical points. This suggests the emergence of universal paradigms that apply to clusters of arbitrary size. We discuss how these crossover regions of the impurity models might affect the approach to the Mott transition within a cluster extension of dynamical mean field theory.
Appendix: CFT at work 35 Table 1. Acronyms and main notations used in the text.
Non-interacting electron bandwidth U
Hubbard on-site repulsion T * F Quasi-particle effective Fermi temperature Γ Impurity hybridization width T K Kondo temperature ρ(ǫ) Impurity density of states Σ(iω) Impurity self-energy in Matsubara frequencies MIT Mott metal-to-insulator transition DMFT Dynamical mean-field theory NRG Wilson's numerical renormalization group CFT Conformal field theory DOS Single-particle density of states
Introduction
The Mott metal-to-insulator transition (1; 2) emerges out of the competition between the opposite tendencies of the electrons to delocalize throughout the lattice in order to maximize the band-energy gain and their mutual Coulomb repulsion which, on the contrary, tends to suppress valence fluctuations by localizing the carriers. If the bandenergy gain, which can be identified with the "bare" bandwidth W , is small enough with respect to the short-range Coulomb repulsion, commonly parametrized by an onsite Hubbard U, and the average electron-density per site is integer, charge gets localized and the system is a Mott insulator; otherwise it remains metallic.
Despite its intuitive nature, the Mott phenomenon is extremely difficult to study because it is inherently non-perturbative and because it escapes any simple singleparticle description. Those can only deal with band-insulators, characterized by an energy gap separating totally filled from unfilled bands. The simplest example of a Mott insulator is provided by the single-band Hubbard model at half-filling, which always has an insulating phase at sufficiently large repulsion. Yet, in order to make this phase appear for instance in Hartree-Fock theory, one is obliged to assume an antiferromagnetic order parameter that doubles the unit cell so as to fulfill the necessary requirement for a band-insulator -an even number of electrons per unit cell. With this trick, the Mott transition is effectively turned into a metal to band-insulator transition driven by magnetism. In reality, local moments form and eventually order as a consequence of charge localization by the Mott phenomenon. This distinction might look pedantic since the ground state is anyway both insulating and magnetic, but in fact it is not, as we are going to argue by the qualitative behavior of the entropy in the Mott insulator and in the contiguous metal.
In Fig. 1, we sketch the typical temperature dependence of the entropy deep inside a Mott insulating phase, U ≫ W . At high temperature, T U, valence fluctuations are suppressed and the local charge gets locked to some fixed value n 0 . Yet, all local electronic configurations with n 0 electrons are thermally occupied with equal probability, leading to a constant entropy regime that could be identified as the ideal Mott insulator, where charge degrees of freedom are frozen while all other degrees of freedom, in particular spin, are completely free. However, at some lower temperature, other energy scales come into play whose role is to lock these additional degrees of freedom, namely to favor one or several among all the local electronic configurations with n 0 electrons. These energy scales may include for instance the on-site Coulomb exchange, responsible for the Hund's rules, the inter-site direct-or super-exchange, the crystal field, the coupling to the lattice, etc. We will collectively denote these energy scales by J, which may be identified as the temperature below which the entropy of the residual degrees of freedom of the ideal Mott insulator starts to be suppressed. Consequently, at low temperature, a realistic insulating phase is established, which is commonly accompanied by a symmetry breaking phase transition at T = T c ≤ J, for instance a magnetic ordering, a collective Jahn-Teller distortion, etc. Below T c , the entropy decreases to zero as T → 0, generically faster than linearly. For instance, in the half-filled single-band Hubbard model, the ideal Mott insulator corresponds to a regime in which each site is singly occupied but its spin can be with equal probability either up or down, leading to an entropy ln 2 per site. However, below a temperature of the order of the inter-site spin-exchange, the ln 2 entropy decreases until the system crosses a magnetic phase transition, below which its entropy vanishes according to the dimensionality of the system and to the dispersion relation of the spin waves.
Recently, an amount of research activity has focused on the possibility that different symmetry broken phases may compete in the insulator, leading to exotic low temperature phenomena (3). Here, we will completely discard this event and concentrate on a different competition which emerges in the metallic phase adjacent the Mott insulator.
In Fig. 2, we draw how the entropy versus temperature might look like for a stronglycorrelated Fermi-liquid-like metal, assuming that no symmetry breaking intervenes down to zero temperature. As before, we expect that the charge entropy is, this time only partially, reduced at some high temperature of order U ≃ W . The rest of it, as well as the entropy of the other degrees of freedom, are instead suppressed by the formation of the degenerate quasi-particle gas. This occurs below a temperature T * F , that can be identified as the effective quasi-particle Fermi temperature. Since quasi-particles carry the same quantum numbers as the electrons, the entropy quenching involves all degrees of freedom at once, including the charge. Below T * F the entropy vanishes linearly, S(T ) ≃ γ * T , with a specific heat coefficient γ * usually larger than its non-interacting value γ ∼ 1/W .
Let us suppose that the Mott transition (MIT) were continuous and try to guess how that might happen from the point of view of the entropy. Obviously, since quasi-particles disappear in the Mott insulator, T * F has to vanish at the MIT. Therefore, sufficiently close to the MIT, the quasi-particle Fermi temperature T * F must become smaller than J. When this happens we should expect, by continuity with the insulating side, that part of the spin entropy gets suppressed already at temperatures of order J, above the onset of Fermi-degeneracy. This amounts to some kind of pseudo-gap opening above T * F , which is at odds with the conventional Landau-Fermi-liquid theory. One way out, apart from a first-order MIT, is that something new occurs when T * F ≃ J. Indeed, the presence of J provides the metallic phase with an alternative mechanism to freeze spin degrees of freedom independently of the charge ones, a mechanism that becomes competitive with the onset of a degenerate quasi-particle gas when T * F ≃ J. This competition is likely to lead to an instability of the Landau-Fermi-liquid towards a low-temperature symmetry-broken phase (this happens in the insulating side), but may also signal a real break-down of Fermi-liquid theory.
Notice that, unlike the competition between different symmetry broken Mottinsulating phases, which requires a fine tuning of the Hamiltonian parameters that may only accidentally occur in real materials, this other type of competition -whose effects have not been discussed in the literature before to the extent we believe they deserveshould be encountered whenever it is possible to move gradually from a Mott insulator into a metallic phase, for instance by doping or by pressure. We also know several examples where this competition is argued to be at the origin of interesting phenomena. For instance, in heavy fermion materials the Kondo effect, favoring the formation of a coherent band of heavy quasi-particles, competes with the RKKY interaction (for a comprehensive review see e.g. Ref. (4)). Here this competition is supposedly the key to understand the anomalies which appear at the transition between the heavy fermion paramagnet and the magnetically ordered phase (5; 6; 7).
Competing screening mechanisms in Anderson impurity models
The heavy-fermion example is a particularly pertinent one to introduce the subject of this Topical Review. Indeed, the competition between Kondo effect and RKKY coupling has interesting consequences not only in the periodic Anderson model but already at the level of Anderson impurity models.
For instance, the phase diagram of two spin-1/2 impurities coupled to a conduction bath and mutually by a direct antiferromagnetic exchange has two limiting regimes: one where each impurity is independently Kondo screened by the conduction electrons; and another where the exchange locks the impurity spins into a singlet state, now transparent to the conduction electrons. Under particular circumstances -the two involved scattering channels must be independently coupled each to one impuritythese two regimes are separated by a quantum critical point, at which non-Fermi liquid behavior emerges (8; 9; 10; 11; 12).
The phase diagram grows richer when one consider three antiferromagnetically coupled spin-1/2 impurities (13; 14). Here, besides a Kondo screened regime, there are other phases where the direct exchange prevails, but is unable to fully quench all impurity degrees of freedom. This leads to stable non-Fermi liquid phases analogous to overscreened multi-channel Kondo models (15; 16). These impurity-cluster models are interesting not only as simple attempts towards an understanding of the fully periodic Anderson model, but also because compact cluster of impurities are achievable experimentally by adsorbing atoms on metallic surfaces. Trimers of Cr atoms have already been realized on gold surfaces (17), which has actually motivated the most recent theoretical activity on impurity trimers (18; 19; 20; 14). In this context, the major task is to identify those phases which are stable towards perturbations generically present on metallic surfaces. Therefore the quantum critical points that separate stable phases are of minor interest from an experimental point of view, as they require such a fine tuning that is extremely unlikely to occur in physical systems.
Impurity models and Dynamical Mean Field Theory
Unstable critical points arise when the competition between Kondo screening and RKKY coupling is maximum. This is nothing but the impurity counterpart of the situation in which T * F ≃ J we previously met in connection with the Mott transition. This weak analogy turns into an actual equivalence within the so-called dynamical meanfield theory (DMFT) (21), the quantum analogue of classical mean-field theory which, like the latter, is exact for infinite coordination lattices. In this limit, the single-particle self-energy becomes fully local but maintains a non-trivial time-dependence, obtained within DMFT by solving an auxiliary single-impurity Anderson model that is designed so as to have an impurity self-energy that coincides with the local self-energy of the lattice model. This requirement translates into an impurity model identified by the same local interaction as the lattice model and by a coupling to a conduction bath that must be self-consistently determined. The single-site formulation of DMFT has provided a lot of useful information about the Mott transition per se, disentangled from magnetism or whatever symmetry breaking occurs in the insulating state. However, even though single-site DMFT can account in a Hartree-Fock manner for simple magnetic phases on bipartite lattice, it is inadequate to study our anticipated competition. For instance, it misses precursor effects in the paramagnetic phase close to the magnetic phase transition, caused by inter-site processes which disappear in infinite coordination lattices. For this reason, several extensions of DMFT have been recently proposed to include short-range spatial correlations (22; 23; 24; 25; 26). In these novel versions, the lattice model is mapped onto a cluster of Anderson impurities, subject to self-consistency conditions.
The physics of the Anderson impurity model turned out to be a precious guideline to interpret single-site DMFT results (21). Similarly, we expect that the preliminary knowledge of the general properties of impurity clusters is useful, perhaps even necessary, in connection with any cluster-extension of DMFT. However, apart from few exceptions (8; 13), little is known about impurity clusters. In addition, since models of impurity clusters involve many intra-and inter-impurity energy scales, it is not a priori evident that there should be a common interpreting framework like the Kondo physics in the single-impurity case. This is actually the purpose of this Topical Review. Specifically, we are going to present the phase diagram of the simplest among impurity clusters, namely dimers, trimers and tetramers, that could be used to implement a cluster DMFT calculation on strongly correlated models. Besides our main objective to identify the common features among different clusters, which should presumably play the most significant role in a DMFT approach, we will also try to argue how much of the impurity cluster physics might survive the DMFT self-consistency, hence the possible consequences on the phase diagram of the lattice models. Needless to say, the interest in impurity clusters goes beyond its possible relevance to strongly correlated models near a Mott transition. As we previously mentioned, these clusters may be experimentally realized on metallic surfaces or, eventually, by arranging quantum dots in proper geometries. Moreover, these models represent a theoretical challenge by themselves, which requires the full machinery of Wilson's numerical renormalization group (NRG) (27; 28; 29) and conformal field theory (CFT) (30) for a detailed comprehension.
Before entering into the details of our calculations, it is worth briefly presenting the physical idea that guided this work. First of all, let us recall some basic facts of the single-site DMFT mapping onto impurity models. Within this mapping, the quasiparticle effective Fermi temperature, T * F , translates into the Kondo temperature, T K , of the impurity model. The self-consistency condition causes T K to vanish at a finite value of U, which signals, in the lattice counterpart, the onset of the Mott transition. This also implies that the metallic phase just prior to the Mott transition translates into an Anderson impurity model deep inside the Kondo regime, with a very narrow Kondo resonance and pre-formed Hubbard side-bands (21). The same behavior should occur even when dealing with a cluster of impurities, which should therefore translate into a cluster of Kondo impurities. The novelty stems from the other energy scales which we collectively denoted as J, and that take care of quenching in the Mott insulator the degrees of freedom other than the charge. Indeed, near the Mott transition, J translates into additional processes, like for instance a direct exchange between the impurity-spins, which tend to remove, completely or partially, the degeneracy of the cluster. Consequently, J competes with the Kondo effect, an agent that takes more advantage the more degenerate the impurity-cluster ground state.
We notice that this competition is always active in impurity clusters, while it is commonly absent in single-impurity models except in multi-orbital cases (31; 32) whose physics is in fact close to clusters. We believe that this additional ingredient is precisely the common denominator of all impurity-cluster models, which endows them with the capability of providing a more faithful description of a realistic Mott transition within DMFT.
Indeed, in the presence of the intra-cluster coupling J, the approach to the Mott transition changes as qualitatively shown in Fig. 3, with a Kondo temperature smoothly decreasing from its initial value W as U/W increases and becoming of order J just before the transition. Analogously, see also Fig. 3, starting from the Mott insulator and doping it, T K will smoothly increase from its value T K = 0 at zero doping, until it will again cross a value of order J. In other words, any impurity cluster should experience, within DMFT, two different regimes. The first, when T K ≫ J, in which full Kondo screening takes place and the impurity density of states displays the usual Kondo-resonance. In the lattice model, this regime translates into a conventional correlated metal. The second, when T K ≪ J, particularly close to the Mott transition, in which no or only partial Kondo screening occurs. Here the impurity density of states is pseudo-gaped at the chemical potential. As we will show, these two regimes of the impurity cluster are generically separated by an almost critical crossover-region that reflects the proximity to a true quantum critical point. How do the unscreened phase and the almost critical crossover-region of the impurity cluster translate in the lattice model? The answer to this question would be simple if a true impurity critical point existed, as discussed in Refs. (33) and (34). Indeed, near a critical point, the impurity model displays strongly enhanced local susceptibilities, equivalently enhanced local irreducible four-leg vertices, in several instability channels. Within DMFT, the irreducible four-leg vertices, which enter the Bethe-Salpeter equations, coincide with the local ones (21). Therefore, it is reasonable to argue that, after full DMFT self-consistency is carried out, these local instabilities may turn into symmetry-breaking bulk-instabilities that correspond to the same instability channels of the impurity critical point. However, establishing which one of these symmetry breakings really occurs requires full DMFT calculations, as it depends on other details, like for instance the nesting of the Fermi surface. These speculations have been tested with success by a DMFT analysis of a two-orbital Hubbard model (32). Although these criticalities are, rigorously speaking, avoided in impurity-cluster models pertinent to DMFT, still we believe that these models approach a critical point so closely that the physics does not change qualitatively.
In the following, we will describe in succession the case of dimer, trimer and tetramer clusters, and, in spite of their obvious differences, we will identify the universal aspects of the competition discussed above.
The impurity dimer
The simplest impurity cluster that is relevant to DMFT is a dimer described by the Hamiltonian where c † a kσ creates a conduction electron in channel a = 1, 2 with momentum k, energy ǫ k , measured with respect to the chemical potential, and spin σ, while d † a σ is the creation operator at the impurity site a = 1, 2 with spin σ, n a = σ d † a σ d a σ being the occupation number. This model describes two Anderson impurities, each hybridized with its own conduction bath and in turn coupled to the other impurity by a single-particle hopping t ⊥ . Since within cluster DMFT the self-consistent baths must mimic the effects of the rest of the lattice on the two sites of the dimer, also the two baths are coupled by a hybridization t ⊥ k . The role of the inter-bath hybridization is to generate a frequencydependent contribution to the inter-impurity hopping that, together with t ⊥ , produce off-diagonal elements a = b to the impurity Green's function In fact, any coupling among the baths transfers into a coupling among the impurities and vice-versa, apart from the frequency dependence that can be anyway neglected in the asymptotic low-frequency regime. For this reason, in what follows we will indifferently refer to inter-bath or to inter-site depending upon the context. Close to the Mott transition the effective impurity model resides well inside the Kondo regime, where U ≫ V k , t ⊥ . Here, the model can be mapped via a Schrieffer-Wolff transformation onto two spin-1/2 impurities that, up to order 1/U, are coupled to the two conduction baths by a Kondo exchange and together by an antiferromagnetic J = 4t 2 ⊥ /U. This means that the spectral distribution of the inter-impurity hybridization is transferred to high energy, and what remains at low energy is mainly the exchange J.
Within the DMFT self-consistency scheme, we should then expect that also the direct hybridization among the baths, t ⊥ k , behaves similarly, which suggests that one could start the analysis with the large U-limit of the Hamiltonian where S a is the spin-density operator of impurity a = 1, 2, plus a weak inter-bath hybridization to be considered as a perturbation. This does not at all imply that the latter is irrelevant. Rather, as we are going to discuss, this hybridization turns out to be a relevant perturbation. It only means that this perturbation becomes influential at energy scales much smaller than those at which the main effects caused by the competition between J and J K start to appear, as we shall discuss later. For the time being, let us consider the Hamiltonian (3). This model was originally studied with NRG by Jones and Varma (8; 9; 10). They found that the phase diagram includes two stable phases. When the Kondo temperature, T K , is much larger than J, each impurity is Kondo screened by its conduction bath. On the contrary, when J ≫ T K , the two impurities lock into a singlet and no Kondo screening is required anymore. These two stable phases were found to be separated by a critical point with non-Fermi liquid properties (10). We notice that, since T K is a decreasing function of U, the phase diagram at fixed J/U ≪ 1 as a function of U/Γ, where Γ = Γ(0) is the hybridization width defined through for large U, as (4) one expects the critical point to occur approximately around In other words, perturbation theory breaks down at a finite value of the interaction within model (3), which is per se an interesting situation uncommon in interacting Fermi systems. The detailed properties of the critical point were later unraveled in Refs. (11) and (12) by means of conformal field theory (CFT). The use of CFT to study impurity models relies on the fact that only a finite number of conduction electron scattering channels are hybridized with the impurity. This implies that, when Γ(ǫ), Eq. (4), is smooth around the chemical potential, ǫ = 0, on a scale larger than the Kondo temperature, the asymptotic low temperature/frequency behavior is similar to a conventional onedimensional semi-infinite chain of non-interacting electrons, the impurity sitting at the edge. It is known that non-interacting electrons in one dimension can be mapped through bosonization (35) onto a 1+1 critical field theory -the criticality corresponding to the fermionic spectrum being gapless -that is not only scale but also conformal invariant. This allows to fully identify and classify all critical properties: thermodynamic quantities, correlation functions and finite-size energy spectra (36; 37; 38; 30). In impurity models, the effective one-dimensional chain turns out to be semi-infinite, implying that one has actually to deal with a boundary CFT (39). Since a single impurity can not induce any gap in the bulk spectrum, the conformal invariance of the free electrons remains intact; the only effect of the impurity is to change the boundary conditions (BCs) among the conformally invariant ones. A crucial step to determine the allowed BCs is the so-called conformal embedding (30), which amounts to identifying the conformal field theories corresponding to the symmetry groups under which the Hamiltonian of the conduction electrons plus the impurity stays invariant. Note that the number of gapless degrees of freedom must be conserved, which corresponds, within CFT, to the fact that the CFTs in the absence and presence of the impurity have the same total central charge (30). A conformal embedding can be justified rigorously by identifying the partition function of free electrons with that obtained by combining the partition functions of the CFTs that emerge out of the embedding. Some simple applications of this very powerful method are given in the Appendix. In the most favorable cases, the proper BCs correspond to conformally invariant BCs only within one of the different CFTs of the embedding. The next useful information is that the conformally invariant BCs within each sector can be obtained by the so-called fusion hypothesis (39; 40), according to which, starting from the spectrum of a known BC, one can obtain all the others upon fusion with the proper scaling fields, called primary fields, of the CFT, see the Appendix. Fusion is just the technical word to denote the process in which the impurity with its quantum numbers dissolves into the conduction electron Fermi sea.
Let us consider for instance model (3), and assume, for simplicity, that the two baths are particle-hole invariant. As a consequence, the baths, in the absence of the impurities, can be described by a CFT (40) which includes independent spin SU(2) 1 and charge isospin SU(2) 1 for each bath, see Appendix, namely an overall can be regarded here as the number of copies of spin-or isospin-1/2 electrons participating to the SU(2) algebra (35), while the superscript refers to the bath. Since the charge isospin generators commute with the Hamiltonian even when the coupling to the impurities is switched on, the charge sector can still be represented by two independent isospin SU(2) 1 CFTs. On the other hand, only the total spin SU(2) transformations leave the Hamiltonian invariant, which translates into an SU(2) 2 (two copies of electrons) CFT. As a result the proper embedding in the spin sector is (11), see also the Appendix, where Z 2 denotes an Ising CFT which reflects the symmetry under permutation of the two baths. The Ising CFT has three primary fields, the identity I, with dimension 0, ǫ (the thermal operator), with dimension 1/2, and σ (the Ising order parameter), with dimension 1/16. Each state and operator can then be identified by the quantum numbers (I 1 , I 2 , S, Ising) where I 1 and I 2 refer to the charge isospin of each channel, S to the total spin and Ising to the Z 2 sector. Affleck and Ludwig (11;12) realized that the different fixed points found by NRG, see Fig. 4, correspond to the three different BCs of the Ising CFT, namely two fixed BCs, the stable phases -where one Ising-spin orientation is prohibited at the boundary -and one free BC, the unstable fixed point -where both orientations are allowed. Starting from the unscreened phase, the Kondo screened phase is obtained by fusion with the Ising primary field ǫ, while the unstable critical point is obtained by fusion with the Ising order parameter σ. Furthermore, the critical point is identified by a finite residual entropy S(T = 0) = 1 2 ln 2, showing that part of the impurity degrees of freedom remains unscreened.
CFT also determines, by the so-called double-fusion (40), the dimensions of the relevant operators, see the Appendix. It turns out that there are three equally relevant (i.e. with dimension less than one) symmetry breaking perturbations which can destabilize the unstable fixed point, identified by two asterisks in Table A4 of the Appendix. They all have the same dimension 1/2 as the invariant operator, single asterisk in Table A4 of the Appendix, which moves away from the fixed point and corresponds to a deviation of J from its fixed point value J * at fixed T K , or, vice versa, a deviation of T K at fixed J. The first symmetry breaking operator is an opposite spin magnetization for the two baths, namely a local operator of the form and corresponds in Table A4 of the Appendix to the operator with quantum numbers (I 1 , I 2 , S, Ising) = (0, 0, 1, 0). The second is a BCS term in the inter-bath Cooper singlet channel: The last perturbation is a direct hybridization between the two baths, which breaks the O(2) channel symmetry. Both (8) and (9) correspond in Table A4 of the Appendix to the spin-singlet operator with quantum numbers (I 1 , I 2 , S, Ising) = (1/2, 1/2, 0, 0). On the contrary, both a chemical potential shift that moves away from particle hole symmetry, or a perturbation that splits the two conduction channels, do not destabilize the critical point. Indeed, if the position of the impurity levels is modified, so that the average number of electrons on each impurity moves away from n a = 1, a = 1, 2, the critical point is still encountered, although it will shift to larger U/Γ at fixed J/U ≪ 1 (34), see Fig. 5.
Dynamical behavior of the impurity dimer
The instability channel (9) is very important since, as we mentioned, the inter-bath as well as the inter-impurity hybridization are always present. Therefore the relevant issue becomes whether this hybridization completely washes out the critical behavior of the underneath critical point, or whether a critical region still remains. In order to answer this question, it is convenient to analyze the impurity spectral function, which is also the key ingredient of the DMFT self-consistency procedure.
We start by noticing that, in spite of the fact that both Kondo screened and unscreened phases of the Hamiltonian (3) off a structure-less impurity potential, infinite in the Kondo screened phase and zero in the unscreened one), the dynamical properties of the impurities are completely different. Indeed, the conduction-electron scattering S-matrix at the chemical potential should satisfy where ρ(0) is the actual density of states (DOS) of the impurity at the chemical potential, while ρ 0 = 1/(πΓ) is its non interacting U = J = 0 value. Since both stable phases are Fermi-liquid like, it follows that the S matrix is unitary, hence can be written as S = e 2iδ , where δ is the phase-shift at the chemical potential. The Kondo screened phase is identified by a phase-shift δ = π/2, that implies S = −1 hence ρ(0) = ρ 0 ; the DOS at the chemical potential is unaffected by the interaction. On the contrary, in the unscreened phase δ = 0, thus S = 1 and ρ(0) = 0. In other words, while in the Kondo screened phase the DOS is peaked at the chemical potential -the conventional Kondo resonance behavior -it vanishes in the unscreened one. Furthermore, according to CFT, right at the critical point S = 0, namely ρ(0) = ρ 0 /2. These results are actually reproduced by NRG, see e.g. Ref. (34). In Fig. 6, we draw our NRG results for the impurity DOS of the dimer model (3) for U = 8, J = 0.00125, in units of half the conduction bandwidth, and for various values of Γ across the critical point Γ * , which lies between 0.42 and 0.44. The upper inset shows that, on large scales, the DOSs in the screened and unscreened phases are practically indistinguishable. The differences emerge at very low energies. Apart from the value of the DOS at the chemical potential, which, as mentioned, can be anticipated by general scattering theory arguments, other useful information can be extracted from the whole low-energy behavior. As was realized in Ref. (34), the DOS is controlled by two energy scales. In the Kondo screened phase, there is a narrow Kondo peak on top of a broad resonance. The Kondo peak shrinks as the critical point is approached, while the width of the large resonance remains practically constant. Indeed, right at the critical point, only the latter survives. On the contrary, inside the unscreened phase, the Kondo peak turns into a narrow pseudo-gap within the broad resonance, leading to a low-energy DOS ρ(ǫ) ∼ ǫ 2 . This behavior has been found to be well reproduced by the following model-DOS at low energy (34): where the + sign refers to the screened phase and the -one to the unscreened. T + is the width of the broad resonance, T − the one of the narrow peak in the screened phase and the amplitude of the pseudo-gap in the unscreened regime. As the critical point is approached on both sides, T − ∼ |Γ − Γ * | 2 → 0 (34), in accordance with the CFT prediction that the relevant operator has dimension 1/2. Away from particle-hole symmetry, n a = 1, the two stable phases are still identified by a relative π/2-shift of δ, although the unscreened phase value, δ 0 , is different from zero. In this case, the following model-DOS was found to reproduce well the NRG data (34): with µ ± = ±T + sin 2δ 0 . This formula shows that, in the unscreened phase, the pseudogap remains pinned at the chemical potential, even if, since the broad resonance shifts, the pseudo-gap fills in of an amount proportional to the "doping", i.e. | n a − 1|. By the model DOS (11), one can extract a model self-energy, Σ(iω) in Matsubara frequencies, which, for low ω > 0, behaves as in the Kondo screened phase, as in the unscreened one, and finally as exactly at the critical point, T − = 0, or in the range T − ≪ ω ≪ T + . The model self-energy reproduces well the actual NRG results, shown in Fig. 7. Only in the screened phase, panels (a) and (b) in Fig. 7, the self-energy has the standard perturbative behavior, Σ(iω) ∼ 1 − 1/Z iω, with Z the quasiparticle residue, which breaks down at the critical point, where Σ(iω) goes to a constant value for ω → 0, and even more in the unscreened regime where, as shown on different frequency ranges in panels (a), (c) and (d) of Fig. 7, Σ(iω) diverges as ω → 0. Let us now consider the original dimer model (1). In order not to deal with too many Hamiltonian parameters, we consider the case in which the inter-bath hybridization is zero, t ⊥ k = 0, and take U = 8, as before, and t ⊥ = 0.05, such that J = 4t 2 ⊥ /U = 0.00125, the same value used previously. In this model t ⊥ has a double role: on one side it generates a spin-exchange able to drive the model across the critical point, but at the same time it also breaks the relevant O(2) channel symmetry thus making the critical point inaccessible. Indeed, as shown by the behavior of the impurity DOS in Fig. 8, a crossover now joins the Kondo screened phase and the unscreened one. There are still quite distinct DOSs deep inside the screened and unscreened phases, the former characterized by a Kondo resonance, the latter by a pseudo-gap. However, the transition from the two limiting behaviors is now just a crossover, although quite sharp.
More interesting are the changes that intervene in the impurity self-energy with respect to the model (3). In presence of t ⊥ , the self-energy, besides diagonal elements, Σ 11 (iω) = Σ 22 (iω), which are imaginary, also acquires off-diagonal components, Σ 12 (iω) = Σ 21 (iω) * , which turn out to be purely real. In Fig. 9, we plot Im and Re Σ 12 (iω) versus ω for the same values of Γ's as in Fig. 8, on two different energy ranges. We notice that the gross features of Σ 11 (iω) remain intact, namely, as the model moves from the screened regime towards the unscreened one, the diagonal self-energy increases quite fast in absolute value. However, a linearly-vanishing "Fermi-liquid" behavior is eventually recovered at very low energies, as shown in the left-bottom panel of Fig. 9. In fact, the model in the presence of t ⊥ does not need to develop a singular self-energy anymore to open up a pseudo-gap at the chemical potential as in model (3). It is the off-diagonal self-energy, Σ 12 , that accomplishes the job in this case. Indeed, as shown in the right panels of Fig. 9, Σ 12 becomes so large at low energy to open up an appreciable hybridization gap, not explainable by the tiny value of t ⊥ as compared to the hybridization width Γ. This result could be justified simply by stating that the strong repulsion U enhances the effective strength of t ⊥ . However, the previous results on the dimer-model (3) and the strong energy-dependence of the self-energy, see Fig. 9, suggest that this anomalous behavior reflects rather the properties of the avoided critical point which exists in the presence of J at t ⊥ = 0. In other words, we believe that our results testify that a sizable critical region still exits and largely explains the physical behavior.
Obviously, as in any other case of avoided criticality, the width of the critical region depends on the actual value of t ⊥ with respect to the other parameters U and Γ. Since both t ⊥ and Γ are self-consistently determined within DMFT as function of U and the bare bandwidth, we cannot establish with certainty what might happen in a DMFT simulation of a Hubbard model using a dimer as a representative cluster. However, because the exchange J derives from high-energy processes and survives even inside the Mott insulator, while the coherent hopping dies out at the Mott transition, we believe that a sizable critical region should exist in the effective impurity-cluster model and plays an influential role in determining the bulk properties after the DMFT self-consistency.
The impurity trimer
An important lesson of the impurity dimer was that, in order to identify in all details the properties of the critical region, it is more convenient to study a model in which the impurities are coupled together by an antiferromagnetic exchange rather than by a hopping as would be the case in reality. In the end, we will discover that, just like in the dimer example, the hopping is a relevant perturbation, and yet a critical region survives. Therefore, let us consider the next simple cluster, which is the impurity trimer drawn in Fig. 10 with the Hamiltonian where H K a has been defined in (3). This model describes three spin-1/2 impurities, coupled together by antiferromagnetic J and J ′ and each of them hybridized to a conduction bath. As before, we assume that the baths are degenerate and particlehole invariant. This model, although simplified by the absence of any inter-impurity hopping, is much more complicated than the dimer model (3). Therefore, in order to unravel its phase diagram, we need to combine the NRG analysis, which provides the low-energy spectra of the various fixed points, with CFT, which allows to identify each fixed point with a particular boundary CFT, whose properties can be determined exactly. For this reason, we cannot start our analysis before introducing some CFT preliminaries.
CFT preliminaries for the trimer
As in the dimer example, also in the trimer model (16) at particle-hole symmetry the charge degrees of freedom can be described by three independent isospin SU(2) (a) 1 CFTs, a = 1, 2, 3. For the spin degrees of freedom, the expression of the inter-impurity exchange suggests naturally that we must first couple the spin sectors of baths 1 and 3 into an overall SU(2) 2 via the embedding and finally couple the SU(2) 2 to the bath 2 into an SU(2) 3 , according to φ Figure 11. Phase diagram of the trimer model (16). All different phases are discussed in the text, and their properties briefly summarized in Table 2.
As we previously mentioned, the possible conformally invariant boundary conditions can be classified by means of the fusion hypothesis (15; 16; 12). Namely, starting from the spectrum of a simple BC, one can obtain the spectra of other allowed BCs upon fusion with primary fields of the CFTs. By comparing the low-energy spectra determined in this way with those obtained by NRG, one can identify and characterize all fixed points of the model.
Fixed points in the trimer phase diagram
In Fig. 11, we draw the phase diagram of the trimer as obtained by NRG (27; 28; 29). In order to have a classification scheme which works equally well for Fermi-liquid and non-Fermi-liquid phases, the fixed points are identified through the zero-frequency values of the scattering S-matrices of the baths, (S 1 , S 2 , S 3 ), which can be obtained by CFT (44; 45) through the modular S-matrix (30). Note that, through Eq. (10), the values of the scattering S-matrices give direct access to the values of the DOS at the chemical potential of each impurity. We just recall that S a = −1 means that the impurity-a DOS has a Kondo resonance, S a = 1 that it has a pseudo-gap, ρ a (ǫ) ∼ ǫ 2 , while any intermediate value implies a non-Fermi liquid behavior. The physical properties of the different phases are furthermore summarized in Table 2. Table 2. Summary of the main physical properties of the different phases in Fig. 11, including the behavior of the zero-frequency DOSs for the three impurities, ρ i (0) with i = 1, 2, 3, with respect to the non-interacting value ρ 0 , and the dimension of the relevant symmetry-breaking single-particle operators. "NOT" means that the operator is not relevant, i.e. has dimension not smaller than one. φ is the golden ratio.
a These particle-hole and particle-particle operators are spin-singlets. b These operators are not relevant in the sense that their dimension is not smaller than one. However, they do generate in perturbation theory one of the truly relevant perturbations, with dimension smaller than one, so that in reality they are relevant too.
Let us now present briefly the features of each fixed point. This fixed point, that describes a conventional perfectly Kondo-screened phase, will be used as the ancestor BC which, upon fusion with primary fields, will provide all other BCs. It is quite obvious that this phase exists and extends in a whole region around the origin J = J ′ = 0 in Fig. 11. Indeed, when J = J ′ = 0, each impurity is independently Kondo screened by its own conduction bath and this perfect screening cannot be affected by finite J and J ′ much smaller than the Kondo temperature. It is far less obvious that this fixed point remains stable for large J ≃ J ′ . When J ′ = J ≫ T K , the impurities lock into two degenerate S=1/2 configurations. In the first, sites 1 and 3 are coupled into a triplet which in turn is coupled with site 2 into an overall spin-1/2 configuration. Since this is even by interchanging 1 with 3, we denote it as |e . The other configuration, which we denote as |o as it is odd under 1 ↔ 3, corresponds to sites 1 and 3 coupled into a singlet, leaving behind the free spin-1/2 moment of site 2. The Kondo exchange projected onto this subspace reads J K 3 |e e| S · (2J 1 (0) − J 2 (0) + 2J 3 (0)) where S describes the effective S=1/2 of the trimer, while J a (0) is the spin density of bath a = 1, 2, 3 at the impurity site, assumed to be the origin. All the above screening channels flow to strong coupling within a simple one-loop calculation. Since it can be readily shown that the impurity can be perfectly screened, both in the spin and in the even-odd channels, one has to conclude that the whole line J = J ′ at finite J K corresponds to the Kondo screened fixed point (−1, −1, −1), as in Fig. 11. A small deviation from J = J ′ is an irrelevant perturbation that splits the degeneracy between |e and |o . Only a finite deviation eventually destabilizes this fixed point, the faster the smaller J K .
2.
(S 1 , S 2 , S 3 ) = (0, 1, 0) This fixed point occurs for J ≫ T K , J ′ , see Fig. 11. The NRG spectrum is compatible with that obtained by fusing the (−1, −1, −1) fixed point with the field σ ′ of the TIM. It is not difficult to realize that this fixed point is equivalent to the non-Fermi liquid phase of the S=1/2 two-channel Kondo model (15; 16). Indeed if J ′ = 0 and J is large, the trimer locks into the S=1/2 configuration which we previously denoted as |e , to indicate the even parity upon 1 ↔ 3. The Kondo exchange projected onto this configuration is, see Eq. (19), Hence, while baths 1 and 3 are still antiferromagnetically coupled, the coupling with bath 2 turns effectively ferromagnetic. The ordinary one-loop renormalization group calculation would predict that the Kondo exchanges J (1) K > 0 flow towards strong coupling, while J K > 0 should behave asymptotically as (20). If J (2) K = 0 this is just the two-channel spin-1/2 impurity model (15; 16), which is non Fermi-liquid with S-matrices S 1 = S 3 = 0 (44; 45). It is easy to show that the small ferromagnetic J (2) K transforms into an antiferromagnetic exchange of the form J 2 (0) · (J 1 (0) + J 3 (0)), which is irrelevant. Consequently, we expect that this phase should remain non-Fermi liquid and identified by the S-matrices (S 1 , S 2 , S 3 ) = (0, 1, 0), as indeed confirmed by CFT. In addition, through the modular S-matrix, one can show that the zero-temperature entropy S(0) = 1/2 ln 2 is finite and coincides with that of the S=1/2 two channel Kondo model. Since σ ′ × σ ′ = I + ǫ ′′ , with the latter having dimension 3/2 > 1, this fixed point is stable to symmetry-preserving perturbations. Yet, there are several symmetry-breaking relevant perturbations of dimension 1/2. One of them corresponds to the staggered magnetization All the other relevant operators break the degeneracy between bath 1 and 3, as for instance the spin-singlet operator and the direct hopping or singlet-pairing between baths 1 and 3, both known to be relevant perturbations at the overscreened non-Fermi-liquid fixed point (46). Note that, although the hopping/pairing operators between baths 1 and 2 as well as 3 and 2 have dimension one, they do induce indirectly a symmetry-breaking coupling among baths 1 and 3 of dimension 1/2, hence they are effectively relevant, specifically marginally relevant. This phase with S-matrices (S 1 , S 2 , S 3 ) = (0, 1, 0) extends at finite J ′ just because J ′ does not generate any symmetry-breaking relevant perturbation. The approach to the fixed point is controlled by two leading irrelevant operators of dimension 3/2: ǫ ′′ and the scalar product of the staggered magnetization (21) with the first spin descendant. Similarly to the overscreened two-channel Kondo model (15; 16), these operators produce logarithmic singularities in the impurity contribution to the specific heat coefficient and to the magnetic susceptibility, C imp /T ∼ χ imp ∼ ln(1/T ).
(S
Since the Kondo screened phase, (−1, −1, −1) and the non-Fermi liquid one, (0, 1, 0), are essentially different, it is clear that an unstable critical line separates the two, see Fig. 11. We find that the NRG spectrum can be reproduced by fusing the (−1, −1, −1) fixed point with ǫ of the TIM. The S-matrices are φ −2 (−1, 1, −1) and the residual entropy is S(0) = ln φ, where φ = (1 + √ 5)/2 is the golden ratio. Since ǫ × ǫ = I + t, the operator which moves away from the critical line has dimension 3/5. The most relevant symmetry breaking operator is still the staggered magnetization (21), which has now dimension 2/5. Once more, the approach to this fixed point is controlled by the scalar product of the staggered magnetization with the first Kac-Moody descendant of the SU(2) 3 CFT, which has dimension 1 + 2/5. Analogously to the multichannel Kondo (15; 16), this operator produces impurity contributions to the specific-heat coefficient and magnetic susceptibility that diverge like T −1/5 .
The spin-singlet operator (22) is also relevant, although with a larger dimension 3/5. In addition, there is a new class of dimension-3/5 operators which correspond to coupling into a spin-singlet two particles, or one hole and one particle, belonging to bath 2 and either bath 1 or 3. Finally, this critical line is stable towards moving away from particle-hole symmetry, as it was the case in the dimer. These two fixed points occur when J ′ > J is larger or comparable with the Kondo temperature. They have a very simple explanation. Indeed, when J = 0, site 2 is only coupled to bath 2 with a Kondo exchange, leading to a full screening, i.e. S 2 = −1. Sites 1 and 3 plus their own baths realize a two-impurity Kondo model which, as discussed before, has two stable regimes. One is Kondo screened, Fig. 11, and the other unscreened for J ′ ≫ T K , (S 1 , S 2 , S 3 ) = (1, −1, 1) in Fig. 11. These two regimes are stable towards switching on a small J ≪ J ′ . When J = 0, we also know that an unstable fixed point at J ′ = J ′ * ∼ T K separates these two stable phases, which is identified by S 1 = S 3 = 0, hence the label (0,-1,0) in Fig. 11. Since site 2 is tightly bound into a singlet state with bath 2, a finite but small J ≪ T K will simply generate a ferromagnetic exchange of order −J 2 /T K by virtually exciting the singlet state. The net effect is that the unstable fixed point at J = 0 is just the endpoint of another critical line which, for J ≪ T K , moves to larger values of J ′ . From the CFT viewpoint, the (1, −1, 1) and (0, −1, 0) fixed points can be obtained by fusing with ǫ ′′ of the TIM or σ I of the Ising CFT, respectively. The properties of the unstable (0, −1, 0) critical line are the same as those of the dimer critical point. In particular there is a relevant operator in the singlet Cooper channel that now involves pairing among baths 1 and 3, as well as an equally relevant operator which corresponds to an opposite magnetization of bath 1 and 3, i.e. J 1 − J 3 .
Dynamical properties of the trimer
In the J-J ′ parameter-space of the trimer model (16), the J ′ = 0 line is actually pertinent to a Hubbard model on a bipartite lattice and with nearest neighbor hopping simulated within cluster-DMFT. In this case, the phase diagram is qualitatively similar to that of the dimer model, see Fig. 5, apart from the fact that the unscreened phase corresponds now to the (S 1 , S 2 , S 3 ) = (0, 1, 0) non-Fermi liquid phase, while the critical line represents the (S 1 , S 2 , S 3 ) = φ −2 (−1, 1, −1) unstable fixed point.
Both the critical line and the non-Fermi liquid phase are unstable to a nearestneighbor hopping. If, instead of three impurities coupled by J, we consider three impurities coupled by a hopping, the transition from the Kondo screened phase to the non-Fermi liquid one transforms into a crossover between Fermi-liquid regimes, exactly like in the dimer. In Fig. 12, we draw the self-energies in Matsubara frequencies of the 3 impurities coupled by the spin-exchange J (panels (a) and (b)), as well as of the impurities coupled by a hopping t ⊥ along the bonds 1-2 and 2-3 (panels (c) to (f)). The Hamiltonian parameters are U = 8, J = 0.00125 and t ⊥ = 0.05, like in Figs. 7 and 9, while Γ = 0.4, 0.44, 0.5. In the absence of t ⊥ , the self-energies, which are diagonal and imaginary, behave as ℑmΣ aa (iω) ∼ −ω in the Kondo screened phase, Γ = 0.5, and tend to a constant at the fixed point, Γ ≃ 0.44. In the unscreened phase, Γ = 0.4, ℑmΣ 11 (iω) = ℑmΣ 33 (iω) → const. while ℑmΣ 22 (iω) ∼ −1/ω, in accordance with the values of the scattering S-matrices. Similarly to the dimer, when the impurities are instead coupled by a single-particle hopping, a Fermi-liquid behavior ℑmΣ aa (iω) ∼ −ω is eventually recovered at very low energy, see panels (c) and (d), although the DOS at site 2 may still display a pseudo-gaped behavior due to the large value of ℜeΣ 12 (iω) = ℜeΣ 32 (iω).
In conclusion, the dynamical behavior with J or in the presence of t ⊥ is qualitatively similar to the 2-impurity cluster.
The impurity tetramer
Let us move finally to the last type of cluster investigated, the tetramer drawn in Fig. 13 with the Hamiltonian This model now describes four spin-1/2 impurities, coupled together by nearest J, and next-nearest neighbor J ′ , antiferromagnetic exchanges. In addition, each spin is Kondocoupled to a conduction bath by J K > 0. The four baths are once more assumed to be degenerate and particle-hole invariant. As before, the impurities are for convenience only coupled through a spin-exchange and not by hopping terms, which we will take into account as perturbations. Figure 13. The 4-impurity cluster freedom, the way in which the impurities are exchange-coupled naturally leads to the following conformal embedding scheme
CFT preliminaries for the tetramer
where c = 1 CFT stands for the Z 2 orbifold of a free bosonic CFT (the bosonic field φ and −φ must be identified) with compactification radius R = √ 2p ′ and p ′ = 6 (30; 47).
The two step process represented by 24 corresponds to the coupling of the SU(2) 1 spin sectors of the impurities on the diagonals into SU(2) 2 , followed by the coupling of these two new sectors into an SU(2) 4 . The resulting cosets are the two Ising sectors and the c = 1 CFT. The embedding can again be rigorously proven through the character decomposition, but the proof is very technical so we prefer not to give any detail. It is also convenient to represent the two Ising sectors as a single c = 1 free bosonic CFT, now with compactification radius R = √ 4, i.e. p ′ = 2. The Z 2 orbifold of a c = 1 CFT with compactification radius R = √ 2p ′ includes, besides the identity, the following primary fields (30, pg. 785): (i) p ′ − 1 fields, φ h , with dimensions h = λ 2 /4p ′ , with λ = 1, . . . , p ′ − 1; (ii) a doubly-degenerate field, φ Table 3. Since the calculation is numerically heavy, we had to use a large Λ = 10, which is sufficient to characterize the low energy spectra, hence the various phases, but not adequate to provide accurate estimates of the critical points (for instance, when J = 0, we find (J ′ /T k ) * ≃ 0.24 instead of the two-impurity value ≃ 2 (10)). However, even though the absolute values of the critical J/T K and J ′ /T K are underestimated, we still believe that the overall shape of the phase diagram should be representative. For this reason, we have decided to plot the NRG data rescaled in such a way that, when J = 0, the critical point has the value found in the two-impurity model. (10) (iv) the dimension-1 operator θ.
The fusion rules among the primary fields can be found for instance in Ref. (30, pg. 786).
The embedding has already been discussed in Ref. (48) in the context of the 2-impurity 2-channel Kondo model. We will see in what follows that some of the anomalies found by those authors do also appear in our model.
Fixed points in the tetramer phase diagram
In Fig. 14, we draw the phase diagram of (23) as obtained by NRG. As before, each fixed point is identified by the S-matrices (S 1 , S 2 , S 3 , S 4 ) (n) , where the superscript (n) is introduced to distinguish between different fixed points with the same S-matrices. In Table 3 the main physical properties of the different phases are summarized. Table 3. Summary of the main physical properties of the different phases in Fig. 14. ρ(0) is the zero-frequency DOS of any of the four impurities, ρ 0 being its non-interacting value. As in Table 2, several single-particle operators are considered, specifying whether they are relevant, in which case their dimension is indicated, or not. d † i d i+1 and d † i d † i+1 denotes hopping and singlet-pairing, respectively, along the sides of the plaquette, have the same meaning but along the diagonals, i.e. i − i + 2 stems for 1 − 3 and 2 − 4. x is a parameter (not to be confused with the coordinate x that is used in the first row to identify the different phases, according to Fig. 14) that changes continuously along the critical lines I and II, reaching at the first order point along the diagonal J = J ′ the value x = 1 for line I and x = 2 for line II.
This fixed point corresponds to a perfectly Kondo screened phase. It occurs when T K is large compared with both J and J ′ . Once again, we will use the Kondo screened (−1, −1, −1, −1) fixed point as the ancestor BC to generate all the others through fusion.
2. (S 1 , S 2 , S 3 , S 4 ) = (1, 1, 1, 1) (1) If J ′ = 0 and J ≫ T K , the tetramer locks into a non-degenerate singlet state which is obtained by coupling sites 1 and 3 into a triplet, as well as sites 2 and 4, and coupling the two triplets into an overall singlet. This configuration transforms like the function x 2 − y 2 under the C 4 point-group of the plaquette, hence the label in Fig. 14. The singlet decouples from the conduction electrons which do not feel the presence of the impurities anymore, resulting in a phase-shift δ = 0 in every channel. This phase is Fermi-liquid-like and remains stable even in the presence of a finite J ′ , provided the lowest excitation gap from the ground state of the isolated tetramer is much larger than T K . Within CFT, there are several possible fusions which lead the (−1, −1, −1, −1) fixed point to this new one. One is for instance the fusion with the primary field θ of the c = 1 CFT with p ′ = 6.
3.
(S 1 , S 2 , S 3 , S 4 ) = (1, 1, 1, 1) (2) If J = 0, sites 1 and 3 are decoupled from sites 2 and 4, hence the tetramer reduces to two independent Kondo dimers. If J ′ ≫ T K each pair of impurities, 1 and 3 or 2 and 4, is strongly bound into a singlet which decouples from the conduction electrons. This impurity configuration transforms like xy under the C 4 point group, which explains the label in Fig. 14. This fixed point is obviously stable to a small J being turned on, hence, in analogy with the dimer, it should be obtainable by the (−1, −1, −1, −1) fixed point upon fusion with ǫ (1) I , a = 1, 2, are the energy operator of the two Ising CFTs. This is also the dimension-1 primary field θ of the c = 1 CFT with p ′ = 2. The NRG spectrum agrees with this prediction not only for small J, but for the whole region J ′ > J with J ′ ≫ T K , see Fig. 14.
First order line
If J = J ′ ≫ T K , the tetramer locks into a doubly degenerate spin-singlet, the states with symmetry x 2 − y 2 and xy previously mentioned. The Kondo exchange provides a coupling between these two configurations only at second order, namely via a quartic conduction-electron operator, which is therefore irrelevant. Hence the tetramer decouples asymptotically from the conduction baths, and its degeneracy remains untouched. This is confirmed by the NRG calculation, which shows the same Fermi liquid spectrum as in the absence of the impurity-cluster apart from each state being doubly degenerate. This phase is the analogous of a first order line, hence its name in Fig. 13, with a relevant operator of dimension 0 that describes the splitting of the double degeneracy of the tetramer.
5.
(S 1 , S 2 , S 3 , S 4 ) = (0, 0, 0, 0) (1) and (0, 0, 0, 0) (2) The Kondo screened phase at small J and J ′ is essentially different from the two unscreened phases at large J > J ′ and large J ′ > J, respectively. Hence there are two critical lines that start from the J ′ = 0 axis as well as from the J = 0 one, see Fig. 14, and finally merge with the first order line at large J = J ′ . This might happen either through a multicritical point or by a gradual evolution of each line into a first order critical point. The latter scenario is actually realized, since, unlike in the trimer model, the NRG low-energy spectra along both critical lines varies continuously, signaling the existence of marginal perturbations. For the same reason, a precise identification of these critical lines with appropriate boundary CFTs is not a simple task. Let us start from the simplest case at J = 0, which corresponds to two independent dimers, sites 1 plus 3 and sites 2 plus 4. The fixed point which separates the Kondo screened phase from the unscreened one is obviously the superposition of the fixed points of each dimer, discussed previously. It is obtained by the Kondo screened fixed point upon fusion with the product σ (1) I σ (2) I of the two Ising CFTs (11), and is identified by zero scattering matrices, hence (0, 0, 0, 0) (2) in Fig. 14, as well as by a residual ln 2 entropy. We already showed that the fixed point of a dimer can be destabilized only by the symmetry-breaking operators (7)-(9), which are not generated by a small J. Therefore, a finite J ≪ J ′ does not spoil the unstable fixed point (0, 0, 0, 0) (2) , but only moves its position to larger J ′ , as it generates a weak ferromagnetic exchange along each dimer. We notice that, upon double fusion, where the last expression on the right-hand side is written in terms of the corresponding fields of the p ′ = 2, c = 1 CFT. In agreement with NRG, the operator content includes the marginal operator θ, besides the dimension-1/2 relevant operator that moves away from the fixed point. Since for two independent dimers we do know that there is no such marginal operator at the unstable fixed point, we must conclude that θ acquires a finite coupling-constant only for J = 0. This situation, which is quite exceptional in impurity models, resembles that found in Ref. (48) in the 2-impurity 2-channel Kondo model. An important discovery of Ref. (48) was that this marginal operator not only influences the spectrum but also the operator content. Specifically, Georges and Sengupta recognized, by abelian bosonization of the model, that the fixed point Hamiltonian in the presence of the marginal operator is similar to an X-ray edge problem in bosonization language (49). Therefore any operator which involves creation or annihilation of the corresponding "core-hole" acquires an additional dimension. It is not difficult to realize that the same happens in our case. Indeed the dimension-1/2 relevant operator can be mapped within bosonization (35) into the operator ǫ (1) which represents the creation (annihilation) of a core-electron, d † (d), and the contemporary annihilation (creation) of a conduction electron at the core-hole site, Ψ(0) Ψ(0) † . Analogously, the marginal operator transforms like , which corresponds to the interaction between the core-hole and the conduction electrons.
In the presence of this term, the dimension of the relevant operator (26) changes according to where x parametrizes the critical line, and is actually related to the phase-shift induced by the core-hole in the equivalent X-ray edge problem. Since the end-point at J = J ′ is expected to be a first order one, we conclude that x moves from 0, J = 0, to 1, J = J ′ , along the critical line. The dimensions of all the other dimension-1/2 symmetry-breaking operators changes instead as so that they all become marginal at J = J ′ . Notice that, since the twist operators are not affected by the marginal perturbation, the S-matrices do not change along the line.
Concerning the other critical line, (S 1 , S 2 , S 3 , S 4 ) = (0, 0, 0, 0) (1) in Fig. 14, we find that, when J ′ = 0, the NRG spectrum is reproduced with a good approximation by fusing the (−1, −1, −1, −1) BC with the primary field φ 1/6 of the p ′ = 6, c = 1 CFT. Once again, since φ 1/6 × φ 1/6 = I + θ + φ 2/3 , the operator content includes, besides the relevant operator of dimension 2/3 that moves away from criticality, the marginal operator θ of the p ′ = 6, c = 1 CFT, which explains the continuous evolution of the NRG spectrum from J ′ = 0 to J ′ = J. The role of this marginal operator should be similar to its analogous on the other critical line. Therefore, following the previous analysis and in accordance with Ref. (48), we expect the critical line to be parametrized by a "phase-shift" x that modifies not only the spectrum but also the operator content. In particular, the dimension of the operator φ 2/3 that moves away from the critical line changes according to Since this operator eventually acquires vanishing dimension at J ′ → J, we conclude that x → 2 at the end-point. The precise determination of x along the line is however difficult to extract from our NRG spectra. At x = 0, the most relevant symmetry breaking operator would correspond to the staggered magnetization with dimension 1/3, which, at finite x, changes into hence becomes marginal at J = J ′ . This is physically sound, since at J = J ′ there is maximum spin frustration. Besides the staggered magnetization, there are other less relevant symmetry-breaking operators of dimension 2/3 at x = 0. One of them is the singlet four-fermion operator whose dimension at x = 0 is 1/2 + (2 + 2x) 2 /24, thus becoming soon irrelevant. The other symmetry-breaking operators of dimension 2/3 correspond actually to all possible mean-field decoupling schemes of the exchange term into inter-bath single-particle operators. Among them, we just mention the inter-bath hopping, as well as the d-wave Cooper pairing, They are all degenerate and, at x = 0, have dimension At x = 2 they become marginal, but, interestingly enough, their dimension is nonmonotonic in x, although always greater than the staggered magnetization (27). Along this line, too, the S-matrices and the residual entropy remain constant and equal to (S 1 , S 2 , S 3 , S 4 ) = (0, 0, 0, 0) and ln 2, respectively. Like in the dimer and trimer examples, the relevance of the inter-bath hopping implies that both critical lines are no more accessible if, instead of four impurities coupled by a spin-exchange, one considers four impurities coupled by a single particle hopping, which is the actual situation within cluster-DMFT. Unfortunately, in this case we cannot obtain reliable spectral functions by NRG because of numerical limitations. Therefore, we cannot verify whether, in spite of the fact that the critical point is washed out, a sizable critical region still survives. However we tend to believe that it is the case, just like in the previous examples. Finally, since both critical lines are stable towards the conventional particle-hole symmetry breaking, the phase diagram for J = J ′ , as function of U/Γ and of the average impurity-occupancy, still looks like the dimer one, see Fig. 5.
Discussion and conclusions
In this Topical Review, we attempted to uncover the key features that distinguish Anderson impurity clusters from single-impurity models and that could play an important role within cluster dynamical mean-field theory as opposed to its original single-site formulation. All the examples that we have studied, namely 2-, 3-and 4impurity clusters, share very similar properties.
In particular, if the impurities within the cluster are coupled to one another by a two-body spin-exchange while each of them is hybridized with its own separate conduction bath, the phase diagrams as function of the average impurity occupancy and of the Hubbard U are practically the same, see Fig. 5. For U/Γ less than a critical value, perfect Kondo screening occurs and the impurity-cluster spectral functions show the conventional Kondo resonance. Above that critical value, the inter-impurity exchange prevails instead and takes care of freezing out the impurity degrees of freedom. Here, the impurity spectral functions develop a pseudo-gap at the chemical potential, which is gradually filled in by "doping", i.e., by moving the average impurity occupancy away from half-filling. These two regimes are separated by a critical line that is identified by several instability channels. In all cases, the instability channels correspond to all possible mean-field decoupling schemes of the spin-exchange into bilinear operators. They include the intra-bath magnetization, staggered according to the signs and relative strengths of the spin-exchange constants, and all singlet inter-bath bilinear operators, like the inter-bath hoppings or singlet Cooper pairs. The trend from the dimer towards the tetramer is towards a prevailing instability in the staggered magnetization channel.
The dynamics across the critical point is basically controlled by two separate energy scales. One of them, which we denoted as T + , is finite across the transition and roughly of the order of the spin-exchange. The other, T − , is the scale generated by the deviation X from the critical line. It vanishes as T − ∼ |X| α , where, in the most interesting case of the tetramer, the exponent 1 ≤ α ≤ 3, is non-universal and depends on the frustration. From the point of view of the impurity spectral functions, see e.g. Fig. 6, T + is the width of a broad incoherent peak within the Hubbard side-bands, smooth across the critical point. On the contrary, T − is the width of the Kondo-like resonance that develops on top of the broader one, in the Kondo screened phase. As the critical point is approached, the Kondo resonance becomes narrower, and eventually disappears right at the critical point, leaving behind only the incoherent peak. In the unscreened phase, T − controls the width of the pseudo-gap that opens inside the incoherent part.
If the impurities inside a cluster are coupled one another by a single-particle hopping, t ⊥ , instead of a spin-exchange, the transition turns into a crossover from the Kondo-resonance behavior to the pseudo-gaped one. Indeed, the inter-impurity hopping plays a double role. On the one hand it generates, for large U, a spinexchange, J = 4t 2 ⊥ /U, that might drive the model across the critical point. On the other hand, it also induces a small inter-bath hybridization, V ∼ J K t ⊥ /U, that is a relevant perturbation and destabilizes the critical point. Specifically, V cuts off all critical point singularities below an energy scale E cut−of f ∼ V β , where β ≥ 3 in the tetramer and β = 2 in the dimer. Since V is small and β large, we expect that, in spite of the critical point being no longer accessible, a "critical region" should still survive if E cut−of f ≪ T + ∼ J. We indeed found evidences in favor of this scenario both in the dimer as well as in the trimer, where the impurity spectral functions are numerically accessible.
Coming back to our original scope, let us imagine that we implement a cluster-DMFT simulation of a Hubbard model using a dimer, a trimer or a tetramer as representative clusters. As U increases driving the model towards the Mott transition, the effective impurity cluster must necessarily go through the above mentioned critical region (even if a true criticality is, rigorously speaking, not accessible since the impurities as well as the baths are coupled by a single-particle hopping). In this region, the instability channels of the avoided critical point will be amplified and, after the DMFT self-consistency, can induce a true bulk instability in the lattice model. At half-filling, our results on the impurity clusters suggest that most likely magnetism appears, even in the presence of frustration. However, since instabilities in particle-hole channels are weakened or removed by doping away from commensurate fillings, while that weakening does not happen in particle-particle channels, a superconducting dome may emerge near half-filling. Indeed, recent cluster DMFT simulations of the Hubbard model on a square lattice (50; 51) found evidence of a d-wave superconducting phase away from half-filling, in close analogy with the phase diagram of high-T c superconductors.
Nevertheless, irrespectively of which symmetry-broken phase actually occurs at low temperatures, the physics of impurity-clusters suggests that, in the normal phase above a critical temperature, the transition to the Mott insulator is accompanied by the gradual opening of a pseudo-gap in the single-particle spectral function. In this pseudogaped region, Fermi-liquid behavior, i.e. Σ(iω) ∼ iω, is recovered only at extremely low energies, suggesting the existence of a finite temperature non-Fermi liquid behavior. Evidence in favor of this scenario has been found in a recent cluster-DMFT simulation of a paramagnetic two-dimensional Hubbard model. (52) Another aspect worth emphasizing concerns the behavior of the Drude weight in the metal away from half-filling across the symmetry-breaking phase-transition. From the point of view of the effective impurity model, a symmetry breaking in the conduction baths opens up new screening channels that can rid the impurity of its residual entropy at the critical point. This in turns leads to an increase of screening energy gain that, translated back into the lattice model, implies an increase of band-energy gain, i.e. of the Drude weight. This behavior is actually the fingerprint of this kind of instability that reflects the underlying impurity critical point, as opposed to the conventional Stoneror BCS-instability that are accompanied by a decrease of Drude weight. The increase of Drude weight has been indeed observed in DMFT simulations of the two-dimensional Hubbard model (50) as well as of a two-band Hubbard model (32) that maps, within DMFT, onto the impurity dimer.
n being integer. The Hamiltonian in momentum space reads where v F has to be identified with the Fermi velocity of the original tight-binding model. Let us define, for positive k, α k = c k , β k = c † −k , so that, apart from an (actually infinite) constant, the Hamiltonian becomes The partition function at temperature T is simply where conventionally (30) q is defined as One can show that where θ 3 is the third Jacobi theta-function and ϕ the Euler function On the other hand, it is known by bosonization (35) that, for positive p = 2πn/L > 0, the operators satisfy bosonic commutation relations. In addition their equation of motion can be reproduced by the Hamiltonian where it is assumed that ρ(p = 0) is the variation ∆ N of the electron number with respect to a reference value. The partition function of this bosonic model is the product of the free-boson term plus the contribution of ∆ N which is, assuming an infinite reference number, One immediately recognizes that the bosonic partition function coincides with the fermionic one.
We can proceed further on, and consider spinful Dirac fermions. Obviously, the partition function is the square of Z Dirac in Eq. (A.4). As the simplest example of conformal embedding and character decomposition, we consider a perturbation that only preserves independently the spin SU(2) symmetry and the charge isospin SU(2), defined through the generators I, which are the q = 0 components of the so-called isospin current operators The spin SU(2) current operators are instead where σ are the Pauli matrices. In real space these current operators, J a = I a , S a , satisfy the commutation relations with k = 1. For generic k ≥ 1, the above commutation relations identify an SU(2) k CFT (30), where the label k may be regarded as the number of channels which are used to build up the generators. An SU(2) k CFT has primary fields φ (k) 2j with spin quantum numbers j, such that 2j = 0, 1, . . . , k. Their dimensions are x j = j(j + 1)/(k + 2). The product of two primary fields with spin j and j ′ yields all primary fields with spin between |j − j ′ | and min(k − j − j ′ , j + j ′ ). The Hilbert space of the theory is obtained by applying the primary fields on the reference vacuum state and, from this ancestor state, by generating all descendant states applying the current operators with q < 0. This is what is called a conformal tower (30). The energy difference between the descendant states and their ancestor is an integer multiple of the fundamental level spacing ∆ = 2πv F /L. The character χ (k) 2j represents the contribution to the partition function of the conformal tower generated by the primary field φ (k) 2j (30). This construction may look abstruse but actually has a simple physical interpretation. Let us consider again a single spinful fermion, k = 1. Let us further assume that on average the number of electrons is equal to the number of sites, and the latter is even. In this case the ground state is obtained by filling with two electrons of opposite spin all single-particle levels below the chemical potential, which lies in the middle of two consecutive single-particle levels separated by the fundamental spacing ∆, from now on our energy unit. With this definition, the Hilbert space can be constructed as follows. One can start from the vacuum and act on it with particle-hole excitations, namely with I z (q) or S(q) with q < 0. In addition, one can apply the operators I + (q) or I − (q), again with q < 0, to change by an even multiple the number of electrons, and then consider all particle-hole excitations on top of these states. In this way, one obtains all states which have even number of electrons, like the vacuum state. This is nothing but the conformal towers obtained by the ancestor fields φ The rest of the Hilbert space includes all states with odd number of electrons. Since a single electron carries isospin and spin 1/2, all these states have half-odd integer values of I z and S z . One realizes that they can all be obtained by applying the product of the isospin and spin primary fields φ 1 spin , which is nothing but the single electron operator, and construct out of it all descendant states. Their contribution to the partition function should then be χ where η(q) = q 1/24 ϕ(q), is the Dedekind function. In the specific case of k = 1, where θ 4 (q) = which, apart from the vacuum polarization contribution q −1/12 , is exactly the partition function of two species of Dirac fermions. The spectrum, and correspondingly the partition function, can be represented as in Table A1. In that and all forthcoming tables, we identify each conformal tower by the quantum numbers of the primary fields which generate the ancestor states.
x is the energy in units of ∆ of the ancestor state with respect to the chemical potential. The descendant levels of an ancestor have energies x + n, with n a positive integer. Notice that an important consequence of conformal invariance is that the energy of each state in units of ∆ coincide with the dimension of the operator which, applied to the vacuum, yields that state. Following the same reasoning, one can easily show that the table of the energy spectrum in the case of odd chains at half-filling (i.e. odd average number of electrons) is the one of Table A2. The ground state is fourfold degenerate, the chemical potential Table A2. The spectrum of spinful electrons when the ground state contains an odd number of particles. coinciding with a single-particle level. One can readily realize that the even and odd chain spectra can be turned one into the other by fusion with a charge or a spin primary field φ 1 . This is the physics of the single-channel spin-1/2 Kondo impurity model. Indeed, when Kondo effect is established, the impurity site becomes effectively a new site of the chain, thus changing the parity of the number of sites, i.e. the boundary conditions. The two channel model Let us consider a more involved conformal embedding in the case of two channels of spinful fermions. The partition function is the square of the partition function (A.7) of a single channel, and can be written for even chains as Z = n 1 ,n 2 =0,1 These expressions manifestly show that the two-channel free conduction electrons are invariant under independent spin or isospin SU(2) transformations within each channel, namely under a large symmetry SU(2) × SU(2) × SU(2) × SU (2). Let us suppose that the impurity couples only to the spin-current operators in such a way that only the overall SU(2) spin symmetry is preserved. Therefore, while the charge degrees of freedom can still be represented by two SU(2) 1 CFT's, the appropriate conformal embedding for the spin sectors should involve an SU(2) 2 CFT, since the total spin current is made up of two channels, times the coset CFT, namely Since the central charge is conserved and each SU(2) k has a central charge 3k/(k + 2), the coset theory should have c = 1/2, which corresponds to the central charge of an Ising CFT. This can be proved rigorously by the character decomposition.
The Ising CFT has three primary fields, the identity I, with dimension 0, the energy field ǫ, with dimension 1/2, and the spin field σ with dimension 1/16. The fusion rules are (30) I ×I = I, ǫ×ǫ = I, σ×σ = I +ǫ, I ×ǫ = ǫ, I ×σ = σ, ǫ×σ = σ.(A.10) The characters χ I x , where x is the dimension of the primary field, are given by (all functions are assumed to depend on the variable q, even when not indicated) where the second Jacobi function is defined by One can show that the product of characters of two SU(2) 1 CFTs, χ 2j χ (1) 2j ′ , can be related to the product of characters of an SU(2) 2 and an Ising CFTs, χ (2) 2l χ I x , by χ 2 χ I 0 . By means of the above decomposition one can write the tables of the spectra for even and odd chains, as given in Table A3. By these tables one easily realizes that the spectrum of even chains can be turned into that of odd chains, and vice versa, either by fusion with the SU(2) 2 primary field of spin 1, or by fusion with the Ising field ǫ. As we Table A3. The spectra of two channels on even chains, left table, and odd chains, right table. I 1 and I 2 are the isospin value of each channel, S the value of the total spin and Ising refers to the Ising primary fields. mention in Section 2, the unstable fixed point of the two spin-1/2 Kondo impurity model was found by Affleck and Ludwig (11;12) to correspond to fusion with the Ising field σ.
If one performs such a fusion in the spectra of Table A3, one obtains, by means of the fusion rules (A.10), a new spectrum that was shown to reproduce the NRG spectrum Table A4. Their physical meaning is discussed in Section 2. | 2019-04-14T02:13:48.696Z | 2007-02-27T00:00:00.000 | {
"year": 2007,
"sha1": "9578bd183c6beedeb8b13b4684ac06566c260567",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/cond-mat/0702629",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "25d1d48a69cac0c6828852bf244072cca7196232",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
266175981 | pes2o/s2orc | v3-fos-license | Investigating the ability of deep learning-based structure prediction to extrapolate and/or enrich the set of antibody CDR canonical forms
Deep learning models have been shown to accurately predict protein structure from sequence, allowing researchers to explore protein space from the structural viewpoint. In this paper we explore whether “novel” features, such as distinct loop conformations can arise from these predictions despite not being present in the training data. Here we have used ABodyBuilder2, a deep learning antibody structure predictor, to predict the structures of ~1.5M paired antibody sequences. We examined the predicted structures of the canonical CDR loops and found that most of these predictions fall into the already described CDR canonical form structural space. We also found a small number of “new” canonical clusters composed of heterogeneous sequences united by a common sequence motif and loop conformation. Analysis of these novel clusters showed their origins to be either shapes seen in the training data at very low frequency or shapes seen at high frequency but at a shorter sequence length. To evaluate explicitly the ability of ABodyBuilder2 to extrapolate, we retrained several models whilst withholding all antibody structures of a specific CDR loop length or canonical form. These “starved” models showed evidence of generalisation across CDRs of different lengths, but they did not extrapolate to loop conformations which were highly distinct from those present in the training data. However, the models were able to accurately predict a canonical form even if only a very small number of examples of that shape were in the training data. Our results suggest that deep learning protein structure prediction methods are unable to make completely out-of-domain predictions for CDR loops. However, in our analysis we also found that even minimal amounts of data of a structural shape allow the method to recover its original predictive abilities. We have made the ~1.5 M predicted structures used in this study available to download at https://doi.org/10.5281/zenodo.10280181.
Introduction
Deep learning has revolutionised the field of structural biology with tools such as AlphaFold2 (AF2) (1), RosettaFold (2) and ESMFold (3) that can accurately predict protein tertiary structure from primary sequence.These tools are all trained on the known protein structure landscape derived from the PDB (4) and have been shown to generalise well to proteins that were not seen during training.Several studies have used these models to enrich the existing protein structure landscape by making extensive predictions from the larger available sequence space.Analysis of these predictions revealed many examples of structures that are very different from the closest available match in experimentally defined data (3,5).
By analysing over 365,000 high confidence structures predicted by AF2, Bordin et al. were able to define 25 novel superfamilies which did not cluster into any existing CATH classifications using their CATH-Assign protocol (5).A second example of new knowledge arising from structural predictions was provided by ESMFold (3).Here, Lin et al. predicted the structures of over 600M metagenomic sequences isolated from diverse environmental and clinical samples.The use of these metagenomic sequences increased the probability of finding examples that were highly distant from the sequence and structural data used to train ESM2 and ESMFold respectively (3).Within a sample of 1M modelled structures defined as high confidence (predicted local distance difference test score, pLDDT > 0.7 and predicted template modelling score, pTM > 0.7), the authors found over 125,000 predictions with no close match in the PDB [defined as pTM > 0.5 carried out using Foldseek (6)] and in close alignment to the corresponding predictions from AF2.While both studies demonstrate that structure prediction tools can confidently generate novel structures, X-ray crystallography data was not obtained to conclusively validate the predictions.It is also not clear if the novel structures generated are composites of large substructural fragments present in the training data.
To attempt to explicitly address whether models can generalise to unseen regions of structural space, Ahdritz et al. carried out 'outof-domain' experiments using OpenFold (7).In particular, examining if OpenFold can generalise from limited data to accurately predict alpha helices or beta sheets despite their omission from training datasets.However, they were not able to completely remove all signal of these secondary structures from their training data, and hence the models were likely still learning from a much-reduced set of examples, rather than extrapolating to a completely unknown structure based on their induction of biophysical rules.
These analyses raise the question of whether current deep learning-based models are truly capable of predicting conformations which are never present in training data.While extrapolation by deep neural networks is theoretically plausible (8,9) searching for evidence of this is difficult and requires extensive classification of training data and the resulting predictions.
One limitation of deep learning based protein structure predictors is their poor performance on stretches of sequence that are intrinsically disordered (10,11) or explore diverse conformational space (12).The loops of adaptive immune receptors, antibodies, and T cell receptors, fall into the latter category.These loops form the majority of the binding site (paratope) of these proteins and are termed complementarity determining regions (CDRs) (13).
The protein sequences that make up the CDR loops arise from two genetic mechanisms, termed V(D)J recombination (14,15) and somatic hypermutation (16).In antibodies, the process of V(D)J recombination randomly pairs V-, D-and J-genes (VJ genes for light chains and VDJ genes for heavy chains) and introduces junctional diversity through insertion and deletion of nucleotides.Further diversity is introduced to the V-gene region of the antibody by somatic hypermutation, where point mutations that modify the amino acid sequence and improve binding affinity are positively selected and progressively dominate the immune response to a pathogen.These mechanisms create high levels of sequence diversity and are evolutionarily advantageous as they combine to provide a nearly limitless potential of binding solutions which allow antibodies to neutralise the correspondingly limitless diversity of pathogens to which humans can be exposed (17).Of the six CDR loops on an antibody, diversity is highest within the CDRH3, the residues of which often disproportionally govern paratope-epitope interactions (18).Structure predictors have been found to perform poorly on this region of antibodies, for example with average RMSD values between predictions and ground truth that exceed 2.5 Å for state-of-the-art models such as AlphaFold-Multimer (AFM) (19) and ABodyBuilder2 (ABB2) (20).
The predictive performance on the remaining five loops, CDRL1-3 and CDRH1-2, is far better (average RMSD <1Å), despite these being subject to the genetic process of somatic hypermutation and being influenced by neighbouring hypervariable loops (21).The ability to accurately model these can be explained by canonical forms, the term given to sets of CDR loops of the same length that adopt similar backbone conformations and share a sequence motif (22).These canonical forms were first observed in crystallographic datasets of available antibody structures before 1986 (23).With the deposition of more structural data both the number of canonical conformations and the sequences that could be assigned to each were continuously expanded and redefined (24)(25)(26)(27)(28)(29).This information linked diverse sets of sequences to distinct loop conformations and thus was increasingly useful to antibody researchers, by providing a form of sequence-to-structure prediction that could be automated by template search and homology modelling tools (27,30).
The latest CDR structure and sequence pairings harvested from antibody structural data are defined in PyIgClassify2 (28).The definitions were released as the 'penultimate classification of canonical forms' in reference to the breakthroughs in structure prediction research that may soon render the predictive power of this relationship obsolete.While structure prediction methods are still being evaluated, especially in the domain of adaptive immune receptors, PyIgClassify2 can serve as a map of the known conformational space explored by antibody CDRs.
Using the rigorous definitions from PyIgClassify2 as a reference point in structural space, we set out to test whether the predicted structures from all available paired antibody sequences in observed antibody space, OAS, (31, 32) reveal novel canonical clusters or highlight conformations not explored by existing experimental data.We then assess these new areas to determine whether they represent evidence of extrapolation or had direct origins in the training data.
ABodyBuilder2 (ABB2) is a structure prediction tool specific to antibodies (20).It uses an ensemble of four deep learning models trained on the structures of over 3500 antibodies as well as a fast minimisation in the AMBER14 forcefield (33,34) to make predictions with comparable accuracy to AFM in a fraction of the time.We used ABB2 to predict the structures of ~1.5M paired antibody sequences.We mapped the conformational space of the CDRL1-3 and CDRH1-2 loops and used existing classifications of the canonical forms in experimental data as reference points.By comparing the loop conformations of canonical clusters to clusters found in predictions derived from the ~1.5M heterogeneous sequences we were able to redefine and identify new canonical clusters.
These new clusters (potential canonical forms) were defined by unique sequence motifs and shared loop conformations and typically arose from enrichment of a small number of examples in the experimental data.We also observed apparently novel clusters (canonical forms) that derived from similar shapes (and sequences) of a different loop length, a phenomenon that has been previously described within the structural dataset (26), termed length independence.
Using our mapping of structural space and the definitions of canonical forms we designed out-of-domain retraining experiments which explicitly tested the capability of ABB2 to both generalise and extrapolate.We found that with zero examples of a given CDR shape ABB2 was consistently unable to predict it.However, with the introduction of very small numbers of a shape, the predictive ability was restored.Overall, these analyses exemplify the power of augmenting experimental data with predictions and provide simple tests for extrapolation and effective data recapitulation that may help inform the next generation of structure predictors.
Selection of paired antibodies sequences for ABodyBuilder2
Paired antibody sequences were retrieved from observed antibody space (OAS) (31, 32) (1-March-2023, https:// opig.stats.ox.ac.uk/webapps/oas/).Non-redundant sequence pairs were filtered by minimum lengths defined in the ABB2 workflow such as minimum residue length of 70, starting IMGT residue number less than 8, and end residue number greater than 120, (see sequence checks https://github.com/oxpig/ImmuneBuilder). Sequences containing any gaps or ambiguities were removed to leave 1,492,044 pairs for processing.ABB2 was run on all sequences and 1,492,031 structures were successfully predicted.
Annotation of CDR loops in paired antibody sequences
To group the relevant modelled structures for conformational analyses, the CDR loops of all input sequences were annotated with information on their sequence composition and length.The CDRs were defined according to the IMGT numbering scheme (CDR1: 27-38, CDR2: 56-65, CDR3: 105-117) (35).This numbering system was chosen as the anchor residues and CDR locations are consistent for both heavy and light chains, as well as being the standard reference point for V(D)J gene annotation.Each sequence was IMGT numbered using ANARCI (36) and the CDR sequences checked against corresponding information from IgBLAST annotations (37).For a given heavy or light chain, if there was any discrepancy between IgBLAST and ANARCI CDR definitions then this chain was not taken forward for conformational analysis (resulting in the exclusion of 6414 light chains and 9822 heavy chains from the structures predicted above).
Retrieval and selection of experimental structures from SAbDab
The structural antibody database (SAbDab) (38,39) is a curated database which contains all antibody, single chain variable fragment (SCFV) and nanobody structures available in the PDB (4).IMGT numbered structures used in ABB2 test, train and validation datasets were downloaded from SAbDab.These structures were derived by X-ray crystallography or cryogenic electron microscopy (cryo-EM) and with a resolution better than 3.5 Å (full list given in SI of 20).
Annotation of SAbDab structures with PyIgClassify2 information
The information on CDR loop canonical forms was obtained from the pyig_cdr_data.txtfile downloaded from the PyIgClassify2 website (http://dunbrack2.fccc.edu/PyIgClassify2/,21-Feb-2023).This provides complete information for all CDR loops on each structure within a given PDB file, including information on sequence identical but structurally distinct members of the asymmetric unit which are distinguished by their PDB chain identifier.For each loop the relevant information includes the length, sequence, canonical form assignment (both with and without an electron density confidence cut off), PDB identifier of the parent chain and information on whether the CDR is structurally complete or is missing any backbone coordinates.This data was used to filter the relevant structures and their CDR loops for each analysis, and to annotate the experimental data points by canonical cluster membership.
Alignment of IMGT and AHo numbering systems
PyIgClassify2 CDR lengths are defined according to the AHo numbering scheme (40) which symmetrically places insertions and deletions around positions defined as key residues in each CDR.This deviates from the IMGT numbering scheme (35) which places insertions centrally within each CDR at fixed positions.The different approaches to defining the CDRs mean that IMGT defined lengths for CDRL1-2 and CDRH1-2 are shorter than those defined in the AHo numbering scheme used in PyIgClassify2.Therefore, for each CDR and length combination analysed in this study, we have listed the corresponding AHo CDR lengths in Table 1, along with the PyIgClassify2 defined canonical forms and sequence motifs described in (28).
Pre-processing of SAbDab datasets
SAbDab files included SCFV structures where the heavy and light chain are part of a single continuous sequence, as well as datasets with multiple sequence-identical copies in the asymmetric For each CDR, the IMGT lengths used in these analyses are presented alongside the corresponding AHo lengths and PyIgClassify2 defined canonical forms for that CDR.PyIgClassify2 canonical forms are named according to the CDR (e.g.L1) followed by the Aho length (e.g.-6), and the form number (e.g.-1) to form a unique identifier for each form (e.g.L1-11-6).For each canonical form the consensus sequence motif, as defined in (28) is given in brackets.Uppercase letters of the consensus sequence indicate highly conserved amino acids at a given position, while lowercase letters indicate a less conserved amino acid that was still observed in the cluster of loops found in the PyIgClassify2 analyses.Information is only provided for the loops analysed in this study, i.e., those which corresponded to the most dominant non-redundant sequences in OAS, (see Figure 1B).
unit.To ensure all CDR loops could be correctly identified and consistently aligned, the relevant chains in each dataset were isolated, IMGT numbered and then saved as individual files linked to the corresponding PyIgClassify2 meta data.For SCFV structures the continuous sequence was broken and each fragment treated as an individual chain.If a chain within a dataset could not be numbered with ANARCI, or a specific CDR loop was missing residues (as indicated by the PyIgClassify2 'cdr_ordered' flag), they were not included in structural analyses.This processing resulted in 11821 heavy and light chains from 3355 PDB files which were used for further analyses.As ABB2 predicted structures were all correctly numbered and contained only a single copy in the asymmetric unit, they did not require any pre-processing for downstream analyses.
Structural analysis of CDR loops of the same length
To perform structural analysis, CDR loops of predicted structures were grouped according to their CDR type (i.e., CDRL1 or CDRH2), amino acid length and sequence composition (non-redundant sequences only).These were analysed alongside all relevant loop structures from SAbDab, for these experimental data points redundant sequences were included as they may contain alternate conformations of the same sequence.
To provide a consistent frame of reference for each CDR length, a loop template was chosen from the highest resolution PDB structure available, this structure also had to be classified as representative of a PyIgClassify2 defined canonical form ('is_representative' flag) and thus was not likely to be an outlier or exhibit any structural features that set it apart.All CDR loops in the analysis were aligned to this template by superimposition of the alpha carbon atoms of the 10 framework residues either side of the loop (CDR1: 22-26 & 39-43, CDR2: 51-55 & 66-70, CDR3: 100-104 & 118-122).If superimposition resulted in RMSD values greater than 1.5 Å then these were not taken forward for loop comparisons.For predicted structures, the framework regions were highly consistent and less than 5 loops per analysis were eliminated.For experimental data points the number eliminated due to framework misalignments ranged from 0 to a maximum of 31 for CDRL1-Len-6 (out of 2499 chains), with a median number of 3 data points eliminated across all analyses.
The carbon and nitrogen backbone atom coordinates of the aligned loops were extracted and saved (CDR1: 27-38, CDR2: 56-65, CDR3: 105-117).Atom counts were checked and then all pairwise RMSD values calculated.This resulted in an N-by-N pairwise distance matrix of RMSD values including both predicted and experimental datapoints for each CDR loop type at every length.To limit the size of pairwise matrices, loops of predicted structures were analysed in batches of 42,000.Where a CDR and length had more than this number of non-redundant sequences (see Table 2) subsequent batches of a maximum size of 42,000 were run until all relevant loop structures were analysed.All batches were run through the clustering and visualisation pipeline (see sections below) and then inspected to ensure results were consistent across all analyses.For multi-batch CDRs, graphs of the first batch are shown in main figures and graphs of subsequent batches are provided in the Supplementary Information (Supplementary Figure 7).
Analysis of CDRH3 loops
We do not present CDRH3 analyses due to the comparatively poor prediction accuracy in this region by ABB2, and other tools such as AFM (20).This uncertainty meant had we found "novel" canonical forms or observations in the CDRH3 region, we could not have confidence that they reflected real loop conformations.
Furthermore, when we performed CDRH3 clustering on the high frequency shorter sequences (CDRH3 lengths 12, 13, 14, Supplementary Figure 8), there was a lack of high-density clusters.This was likely related to the more evenly distributed occupancy of structural space as seen from the multidimensional scaling plots.When density-based clusters were found, the logo plots were uninformative with no apparent motif present in the middle of the CDRH3, and enrichments localised to the beginning and end of the loops (Supplementary Figure 8).Given the heterogeneity of CDRH3 we felt these results were to be expected and this region did not warrant further exploration for novel canonical forms.
Structural analysis of CDR loops of different length
For later analyses aimed at discovering length independent conformations, we also calculated the distance between loops of different lengths.The normalised dynamic time warping (DTW) scores were used to quantify the relative distance in groups of loops that included both length matched and mismatched pairs.Loops were aligned as described above to a high-resolution template, then raw DTW scores calculated between the coordinates using the 'dtaidistance' library in python (41).Normalisation was applied by squaring the score, dividing by the number of atoms being compared, and taking the square root (this resulted in a DTW score that is equivalent to RMSD when lengths are matched).The squared score was divided by the maximum number of atoms (i.e., for length 9 versus 8, the score was divided by 27 to account for 9 residues, each comprised of two carbon and one nitrogen backbone atoms).
Density based clustering
The pairwise distance matrices of RMSD or DTW values contain information that represents the structural relationships between all loop conformations in an analysis.To identify clusters of loops with similar conformations within this highdimensional data, we employed density-based clustering, using the density-based spatial clustering of applications with noise (DBSCAN) function from the scikit-learn library (SK learn) (42).The DBSCAN function takes the input distance matrix and two parameters: the minimum number of data points required to form a cluster (min points) and the minimum distance between any two points in the same cluster (epsilon).The number and size of clusters identified in each analysis is highly sensitive to these values and must be optimised based on the input data.Therefore, we systematically calculated these values in a consistent manner for all analyses, allowing us to find the maximum number of clusters within high-dimensional space and assess whether any cluster related to a novel canonical form.Min point values were calculated by taking the square root of either the number of loops being compared, or the same number divided by 2. For epsilon values we performed a K-nearest neighbours (KNN) analysis on the distance matrix for values of K ranging from 2 to 5. For each value of K, the elbow point was taken from a scree plot of all KNN distances, and this elbow point value used as epsilon in subsequent DBSCAN analyses.This resulted in four separate DBSCAN analyses (one for each value of K from 2 to 5).To select the most appropriate analysis for cluster inspection and visualisation, we matched the K value used to determine epsilon with the number of clusters identified by DBSCAN.If there was no match, then the next closest match was taken for the highest value of K.While dominant clusters were often evident and easily found using multiple values of epsilon, the impact of optimising these parameters was most apparent when identifying smaller or overlapping clusters.We call the clusters generated in this way DBS clusters (short for DBSCAN-based selection).Code is available at: https://github.com/oxpig/OAS-CanonicalForms
Inspection of density-based cluster structures and sequences
We manually inspected the loops comprising each DBS cluster by visualisation of both structures and sequences.This allowed us to assess the structural difference between each cluster and relate the sequence logos back to the defined sequences of PyIgClassify2 canonical clusters.The aligned 3D loops that were assigned to each DBS cluster were visualised using PyMol (43).We selected random samples of up to 20 loops from the predicted structures, all of which belonged to a specific DBS cluster.Samples were coloured according to cluster membership and viewed in the same frame.These loops were presented in multiple orientations to highlight backbone differences that led to distinct cluster assignment.For sequence logo plots, sequences from the predicted structures which had been assigned to a DBS cluster were plotted in R using the ggseqlogo package (44).Logo plots are shown in the bitwise format (opposed to the proportion format) to maximise identification of For each CDR and IMGT length explored in this study, details of the number of non-redundant sequences (and hence loops structurally analysed) and the corresponding results of structural analyses are given.The new information arising from analysis of the predicted structures could be defined as either the identification of a new canonical cluster, or the sub-division of an existing cluster of loops that had previously been defined as belonging to a single canonical form.Further details are given on whether these clusters arose from length independent conformations and/or existing experimental data points classified as unassigned by PyIgClassify2 (28).N/A, not applicable.
the dominant amino acid enrichments and motifs specific to each cluster.
Multidimensional scaling visualisation
To simplify the complex high-dimensional pairwise distance matrix and allow for easy visualisation, we applied multidimensional scaling (MDS) to create a 2D representation.The axes of these plots are labelled as MDS1 and MDS2 and represent unitless scales that capture the spatial differences between data points.We used the parallelised MDS function from the 'lmds' R package, before processing and plotting the output data using tidyverse packages (45).These plots were then annotated according to the DBS cluster membership of each data point, or the canonical cluster assignment of only the experimentally derived data points.
ABodyBuilder2 out-of-domain experiments
Out-of-domain experiments involved the removal of all data points related to a specific CDR length, or a specific canonical cluster, from both the training and test datasets of ABB2.The model was then trained on this modified dataset from scratch.The criteria for removing data from ABB2 training samples involved dividing the MDS map into quadrants and selecting the quadrant with the most distinct canonical cluster, i.e. clearly separated from other data points.All experimental data points in this quadrant were excluded.To ensure the removal of all relevant data points from the training data, any samples defined as the excluded canonical form by PyIgClassify2 without an electron density cutoff (using the 'cluster_nocutoff' flag) were also eliminated.
Data inclusion experiments
For out-of-domain experiments where small numbers of the excluded canonical form were reintroduced into the training data, we first added the highest-resolution datasets identified as representative of the missing canonical form (determined by the 'is_representative' flag in PyIgClassify2).Subsequently, we progressively reintroduced the next highest-resolution datasets not designated as representative but still belonging to the highconfidence canonical cluster.
Retraining of ABodyBuilder2
The original ABodyBuilder2 model consists of an ensemble of four models each trained independently.To make a prediction the outputs from each model are averaged and the prediction closest to average is selected as the final output.Of the four models, one utilised a 128-dimensional embedding and three utilised a 256-dimensional embedding.Models were trained until no further improvement was seen in the validation loss after 100 epochs.
To facilitate the training of multiple models, for the initial experiments in this study we retrained a single model with a 128dimensional embedding (not an ensemble).Each model was trained on either a Nvidia GeForce GTX-1080 Ti GPU or Quadro RTX 6000/8000 GPUs for 150-340 epochs for each training stage (see training methods 20), continuing until no further improvement in validation loss was observed after 50 epochs (half the number used to train original ABB2 models).For specific retraining experiments (those used to confirm data inclusion thresholds important for prediction), an ensemble of models was created, each consisting of one model with a 128-dimensional embedding and three models with a 256-dimensional embedding.Each model followed the original ABB2 protocol (training until no improvement after 100 epochs).This process allowed us to carry out a larger number of experimental runs and only build full models when we had identified the data cutoffs that significantly affected prediction accuracy (assessed by RMSD between predictions and experimental data points).
Dominant CDR lengths in paired sequence space are matched by comparable distributions in structural data
We predicted the structures of ~1.5M paired antibody sequences from OAS (31, 32) using ABodyBuilder2 (ABB2) (20), a state of the art deep learning antibody structure predictor.We examined this structural space for evidence of novel canonical forms.
We analysed the length distributions of the CDRL1, CDRL2, CDRL3, CDRH1 and CDRH2 loops in this dataset by both absolute frequency (Figure 1A) and by non-redundant CDR sequence frequency (Figure 1B).This revealed that many loops belonging to a specific length were dominated by a smaller number of unique sequences.For example, CDRL1 IMGT length 6 had a frequency of 753,690 in 1.49M, of which only 21,700 were unique.Therefore, we decided to focus our structural analysis on the CDR loops and length combinations (e.g.CDRL3 loops of length 9, after this point referred to as CDRL3-Len-9) which had the highest number of nonredundant sequences within the dataset (bars marked with an asterisk in Figure 1B, details and numbers given in Table 2).
The length distributions of the experimentally derived SAbDab (38,39) structures used to develop ABB2 are shown in Figure 1C.Only structures from the train, test and validation datasets which had information on canonical forms detailed in PyIgClassify2 were analysed (28) (38 datasets used in development of ABB2 were not categorised in PyIgClassify2).The CDR loop and length combinations taken forward for further analysis were also enriched in the structural units used to train ABB2 (Figure 1C), with the minimum number of examples seen by ABB2 during training being 268 for CDRH1-Len-9.
We next analysed the high confidence PyIgClassify2 canonical form assignments of each CDR loop length marked for further investigation by plotting the proportions of each canonical form within all experimental units (Figure 1D, an experimental unit refers to the fact that one PDB file may have multiple copies in the asymmetric unit).This analysis demonstrated a similar bias, with a single canonical cluster dominating over 50% of assignments for 13 out of the 17 CDR loop and length combinations.Some canonical forms had a very small proportion of examples contained in the ABB2 development data, with the minimum number being 8 examples for CDRH1-Len-8 (Figure 1D).The biases in both the length and canonical clusters distributions of the experimental data indicate that some data-poor areas may benefit from augmentation with predicted structures.
Having selected the CDRs and lengths which dominated our predicted structures, we next built structural clusters for these and explored whether the predicted structures gave rise to new canonical forms or provided insights which were not evident from experimental data alone.
Predicted structures fall into dense regions of conformational space defined by existing canonical forms
We created a map of the structural space for each of the dominant CDR loop and length combinations, identified above, using the predicted antibody structural data.Each map was analysed to find clusters of CDR loops that shared the same backbone conformation.If a SAbDab structure belonged to a cluster this allowed us to annotate the clusters canonical form according to PyIgClassify2.These annotated maps of canonical form structural space enabled us to navigate the predicted structural space and identify highly occupied regions of space not currently defined by a canonical form.
A full description of how these structural space maps were generated is given in the methods.In brief, each map is a 2D representation of the 3D clustered space of a CDR type at a given length.Data points representing loops from both experimental and predicted structures are coloured by their DBS cluster membership.All data points which are not assigned to a DBS cluster are coloured black.For canonical form annotation the experimental data points are coloured according to their PyIgClassify2 high confidence canonical cluster assignment.Any loops that do not belong to a high confidence PyIgClassify2 cluster (defined by an asterisk in the canonical cluster label) are coloured in red.
Figures 2A, B show the structural space map for CDRL1-Len-6.The projections are overlaid with either DBS cluster membership information (Figure 2A), or canonical cluster classifications from PyIgClassify2 (Figure 2B).The sequences of the loops which comprised these clusters are visualised using logo plots (Figure 2C) to identify the motifs and amino acid enrichments which should match to the canonical sequence motifs described in PyIgClassify2 (Table 1).Samples of loops were also inspected in 3D to assess differences in backbone conformations that give rise to the distinct clusters (Figure 2D).
For many of the CDR and length combinations analysed in this way, the clusters arising from predicted structures aligned well with the dominant clusters of experimentally defined canonical forms and did not highlight any new areas of density without canonical cluster assignment (Figure 2; Supplementary Figure 1; Table 2).Inspection of loop alignments revealed that our RMSD and DBS analysis method could distinguish between backbone kinks, peptide flips and minor variations that equated to less than 1 Å RMSD between data points (Supplementary Figures 2A-C).These minor differences in conformation were also detected by the dihedral angle metric used to compile PyIgClassify2 clusters resulting in similar global divisions of structural space.Before exploring areas that were not accounted for by existing definitions of canonical forms, we next inspected any inconsistencies between our RMSD/DBS analysis and PyIgClassify2.
Differences between PyIgClassify2 definitions and density based structural clusters
While most DBS clusters detected in our analysis could be mapped to experimental data points that adhered to a high confidence canonical form, several of the more subtle PyIgClassify2 definitions were assimilated into a single DBS cluster.We investigated these assimilated data points to assess whether our method was missing important conformational differences.
The loop which best exemplified this was CDRL3-Len-8 (Supplementary Figure 1A).Our analysis pipeline identified two DBS clusters, the centroids of which were 1.45 Å apart and had distinct sequence motifs (Supplementary Figure 1A).Inspection of the PyIgClassify2 canonical clusters demonstrated that cluster 1 (coloured blue in Supplementary Figure 1A) defined by the logo motif of QQYysxxT was subdivided into two canonical clusters, of which one had a proline at position 7 (see Table 1, canonical forms L3-8-1 and L3-8-3).We reran our DBS clustering method using an alternate min points term (square root of N data points, opposed to square root of N/2) and found this was able to subdivide the major cluster into two distinct clusters (Supplementary Figure 2E) distinguished by the proline at position 7 (Supplementary Figure 2F).These two clusters are only 0.4 Å apart and exhibited a large degree of overlap in conformational space (Supplementary Figure 2G).We found additional examples of this within the clusters of loops for CDRL1-Len-6 (Figure 2B) and CDRH2-Len-8 (Supplementary Figure 1C) where the effect was apparent from the smaller number of DBS clusters annotated by a larger number of canonical cluster labels.However, these were often canonical forms assigned to a comparatively low proportion of experimental data points, with more dominant canonical clusters showing greater overlap with our analyses (see later figures).We reasoned that such subtle shifts in conformation would not serve as strong evidence of extrapolation, despite being valid definitions of canonical forms from the dihedral angle perspective.Therefore, their detection was not crucial to our exploratory search for new knowledge arising from structure prediction tools.
Predicted structures enrich the experimental landscape revealing subdivisions of existing classes with defined sequence motifs
Having confirmed that our clustering pipeline was able to pick out major differences in loop conformations across large datasets, we next investigated the structural clusters and sequence logos which did not sit within with PyIgClassify2 defined canonical forms.These ambiguous clusters could be divided into two categories, those which contained experimental data points defined by a canonical form but could be further subdivided into new clusters with distinct sequence motifs and loop conformations, and those which contained experimental data points that were not assigned to a canonical form.
Firstly, the enrichment of the existing structural space with predicted structures led to subdivisions of existing canonical clusters.For example, in CDRH1-Len-8 (Figures 3A, B) and CDRH1-Len-10 (Figures 3C, D) as well as CDRL1-Len-11 and CDRH2-Len-7 (Supplementary Figures 3A, B), we observed DBS clusters with distinct amino acid motifs and loop conformations.These were resolved from the increased structural dataset.The four subclusters observed in CDRH1-Len-8 (Figure 3A) and derived from the PyIgClassify2 canonical form H1-13-5 (Figure 3B, for motifs and length comparisons see Table 1) showed different amino acid patterns at position 2, 4 and 5 of the loops.These subclusters had conformations which differed by RMSD of between 0.79-1.43Å for cluster centroids.Meanwhile the two subclusters identified from the canonical form H1-15-2 (Figure 3D; Table 1) in CDRH1-Len-10 loops exhibited a difference of 1.09 Å and had sequence patterns that differed at six of the ten positions (Figure 3C).
For all four CDR loops where predictions gave rise to sub clusters, the RMSD between new cluster centroids were often close to the mean value of each analysis (range of mean values from all pairwise comparisons: 0.96 -1.03 Å, range of distances between cluster centroids: 0.27 -1.64 Å) and originated from examples present in the training data.Hence the novel canonical classes arising here did not come from generalisation or extrapolation, but simply by statistical power -the increased sensitivity of densitybased clustering on the far larger structural set (number of experimental data units versus predicted structures for CDRL1-Len-11: 768 vs 6,992, CDRH1-Len-8: 5,196 vs 61,617, CDRH1-Len-10: 314 vs 2,500 and CDRH2-Len-7: 1298 vs 27,769).
Enrichment of unassigned areas of structural space defines new canonical forms within heterogeneous sequences
The second set of novel clusters identified related to dense areas of predicted structural space where a smaller number of experimental structures existed but were defined as "unassigned" to any canonical form in PyIgClassify2.For example, for CDRL1-Len-7 a cluster made up of 909 predicted data points (and 32 experimental data points) was identified which was distinct from the centroid of two existing canonical clusters by RMSD values of 3.94 and 4.31 Å respectively (Figure 4A).This "new" canonical form has a sequence motif with a strong preference for SGH at positions 1-3 of the loop, in contrast to QSV in both existing forms (see logo plot in Figure 4A, PyIgClassify2 annotations in Figure 4B and corresponding motifs in Table 1).A second example, CDRL1-Len-9 (Figure 4C), had fewer experimental structures within that area (13 were present in training data) and comprised of 673 predictions.The central motif of INV at positions 3-5 showed no overlap with the enriched residues at the same positions within the three existing canonical forms (Figure 4D, see Table 1), with RMSD values between the corresponding cluster centroids of 2.99-3.51Å.Both observations were enabled by increased population of structural space with ABB2 predictions from heterogeneous sequences, however they are not evidence of extrapolation given their origins in the training data.
New forms exemplify length independent canonical classes arising from somatic hypermutation
Within the CDRL3 loops we found two further examples of highly populated DBS clusters that did not fit with any PyIgClassify2 definitions.These clusters were identified in the analyses of CDRL3 loops of lengths 10 and 11.Both areas of density contained experimental structures that were classified as unassigned to any high confidence canonical cluster by PyIgClassify2 (CDRL3-10 Figures 5A,B, and CDRL3-11 Figures 5D, E).However, these loop shapes are potentially derived from somatic hypermutation (SHM) insertions into CDR loop sequences classified as canonical forms at a shorter length (Supplementary Figures 4A-C).These were evident from inspection of logo plots of CDRL3-Len-10 cluster 3 position 8 (Figure 5C), and CDRL3-Len-11 clusters 1 and 5 at position 9 (Figure 5F), where the motif was nearly identical to that of a highly populated cluster in the CDR one amino acid length below (PyIgClassify2 canonical forms of L3-9-2 and L-10-cis78-1, see Table 1).The SHM insertions were clearly visible on each logo plot as they resulted in no consensus amino acid enrichment at a fixed position in the loop (positions are marked by an arrow in Figures 5C, F respectively).
Given we could identify the corresponding canonical cluster at the shorter length (we termed this the 'origin cluster') (Supplementary Figure 4), we decided to quantify and compare the conformation differences between the two sets of loops.To obtain distance scores that could be used for comparison, both for loops of the same length and differing lengths, we substituted pairwise RMSD calculations with dynamic time warping (DTW) calculations which can be performed on coordinate arrays of differing dimensions (see methods for details).
We represented the structural relationships between CDRL3 loops of length 9 and 10 (Supplementary Figure 5A), and CDRL3 loops of length 10 and 11 (Supplementary Figure 5B) using DTW.The cluster of loops representing the novel conformation in CDRL3-10 (cluster 4: QQYxxxPxxT, coloured yellow in Supplementary Figure 5A) was closest in 3D space (DTW distance between cluster centroids of 0.68 Å) to a cluster composed of CDRL3-Len-9 loops (cluster 1: QQYysxxxT, coloured blue in Supplementary Figure 5A), and only 1.21 Å away from the proposed origin cluster (CDRL3-Len-9 cluster 2: QQyxxxPxT, coloured red in Supplementary Figure 5A).These distances were less than, or comparable to both clusters present at the same length of 10 (distances of 1.83 Å and 1.12 Å apart respectively).
The same effect was more pronounced for the novel conformation in CDRL3-Len-11 (cluster 5: QQYxxxPPxxT, coloured pink in Supplementary Figure 5B).Here the centroids of clusters found at the same length were 2.48 Å and 2.94 Å away, while the cluster at the shorter length was only 2.07 Å away.Visual inspection of the loop backbones from different clusters shows how similar conformations of mismatched lengths (Figures 5H, J) can be closer in 3D space than matched lengths from different DBS clusters (Figures 5G, I).
These observations fit with the previously described idea of length independence in canonical forms (26), where the closest partner of a structural cluster, or CDR loop, is another cluster, or loop, present at a different length.We hypothesise that the high frequency of predicted structures derived from heterogenous sequences in OAS altered by SHM, helped to reveal these length independent patterns.
ABB2 can generalise across CDRL3 loops which differ in length by one amino acid
To explicitly test whether the training and test data points with a specific CDR length influence the predictions of CDR loops of a different length we performed several out-of-domain experiments.These involved modifying the ABB2 training and test data to remove all data points containing CDRL3 loops length of 8, 9 or 10 (one length per experiment).In each case a new instance of the ABB2 model was trained on a reduced dataset.The resulting models were used to make predictions from sequences of the withheld length which could be assessed individually for accuracy, and together for occupancy of structural space.
The first model tested was trained in the absence of all 392 datapoints (referring to all copies in the asymmetric unit of each PDB file) that had CDRL3 length 8 loops.Prediction accuracy was poor (Supplementary Figures 6A, B), with median RMSD values (prediction versus ground truth) of 1.41 Å compared to 0.46 Å for the fully trained ABB2 model (Figure 5K).There was no clear separation of canonical clusters in conformational space, with median values for each form above 1 Å from the ground truth structure (Supplementary Figure 6B).
In contrast the model trained in the absence of CDRL3-Len-9 data had better prediction accuracy on the withheld structures (3865 datapoints, median RMSD 1.05 Å) but still worse than the fully trained ABB2 ensemble (median RMSD 0.46 Å).However, the MDS representation of conformational space showed early separation of data points defined by similar canonical clusters (Figure 5L; Supplementary Figures 6C, D), indicating some rationalisation of the sequence to structure relationship via length offset data.Accuracy for the CDRL3-Len-10 model was the worst (median RMSD 1.66 Å Figure 5K), however a higher standard deviation reflected the correct separation of global conformational space for data points of some canonical forms where loops were close to 1 Å RMSD from the ground truth structure (Figure 5M; Supplementary Figures 6E, F).There are only 9 data points of CDRL3 length 7, and this may explain why all predictions of CDRL3 length 8 for the starved model fell into a single cluster.In contrast, models starved of CDRL3 lengths 9 and 10 were able to separate some predictions into areas of conformational space close their ground truth structure.This may have been due the abundance of data points either side of the missing length in training data.These experiments, in addition to the structural overlap of predictions of different lengths, provided evidence of generalisation by ABB2 with origins in CDR length independence.Furthermore, these out-of-domain experiments serve as a powerful method to further explore the ability of deep learning-based structure prediction methods to extrapolate and find evidence of truly novel predictions.
Retraining whilst withholding canonical conformations highlights limited ability to extrapolate
Our analyses so far have not found any evidence of structural clusters representing novel conformations within the CDR loops of predicted antibody structures.Therefore, we set out to explicitly test whether ABB2 could predict a loop conformation not seen in the training data and without a parallel example at a different length.We ran out-of-domain experiments to train models in the absence of all examples of a specific canonical cluster and any close conformations (see methods).We focused on CDRL1 lengths 6-9 as these analyses showed the clearest cluster separation and the smallest proportion of data points that did not fall into a DBS cluster.This helped to avoid any ambiguity in the contents of the training data.
Separate models were trained for each withheld canonical class (numbers and training details given in Table 3).Each model was analysed as before, by predicting the structures of sequences in the withheld data and assessing the individual prediction accuracy as well as total occupancy of structural space.
For the 'starved' model trained in the absence of CDRL1-Len-6 canonical cluster L1-11-3 (blue dots in Figure 6A, for sequence motif see Table 1), all predictions failed to match the ground truth conformation (Figure 6A) with mean (SD) RMSD difference of 1.54 (0.51) Å.The high standard deviation reflects how some predictions were closer (less than 0.5 Å) to the ground truth structure, however the majority adopted a similar conformation to the closest canonical form L1-11-4 (pink dots in Figure 6A, cluster centroid distance of 1.81 Å) rather than the more distant forms of L1-11-1 or L1-11-2 (green and mustard dots Figure 6A, combined into one cluster by our method, centroid distance: 2.75 Å).
A more pronounced loss of the ability to extrapolate was observed for models starved of a canonical form in the remaining experiments using CDRL1-Len-7 (Figure 6B) and CDRL1-Len-9 (Figure 6D).Here all withheld data points fell into a more distant region of conformation space associated with mean prediction accuracies that were much lower, at mean (SD) RMSD values of 1.98 (0.09) Å RMSD for length 7, and 3.34 (0.19) Å for length 9.
For CDRL1-Len-7 the low prediction accuracy may have been due to very similar sequence motifs (DBS cluster: QSVSSSY, corresponding PyIgClassify2 cluster L1-12-1: RASqSVSSSYLa, versus DBS: QSVSSNY, PyIgClassify2 cluster L1-12-2: RASQSVSSNYLA, see Table 1), as well as a small number of examples within the training data (63 examples of the withheld canonical form).In the case of CDRL1-Len-8, the model made predictions of L1-13-3 (purple dots Figure 6C) that were in a similar region of structural space, however they were still 2.43 (0.19) Å away from ground truth values (Figure 6C).
These out-of-domain tests clearly demonstrated that the models trained in the absence of a canonical class were unable to recapitulate the correct conformations which could be predicted by the fully trained ABB2 ensemble.The extent of the distance between predictions and ground truth differed for each starved model and related to both sequence similarity as well as structural deviation.This led us to investigate whether prediction accuracy could be recovered by adding very small amounts of training examples back into the model.
Inclusion of a small number of examples in training is sufficient to recover predictive capacity
We set out to assess whether limited amounts of training data could recover missing predictions and thus quantify the level of representation needed to produce more accurate models.We chose CDRL1-Len-6 and CDRL1-Len-7 as these had the largest standard deviations in prediction accuracy (see box plots Figure 6).For each we progressively added increasing numbers of data points back into the initial out-of-domain tests resulting in separate models for each addition of data (CDRL1-Len-6 Figures 7A, B and CDRL1-Len-7 Figures 7C, D).
pt?>As there were different total numbers of examples of CDRL1-Len-6 and CDRL1-Len-7 loops in training data (Table 3), the absolute number of PDB structures added back for each experiment did not represent the same proportion of datapoints.Therefore, we calculated the included loops as a percentage of all CDRL1 loops seen for each model (Table 4).Using these values (Table 4) and inspection of the corresponding model performance (Figure 7), we could see that very small percentages of training data (less than 1%) were enough to allow the models to accurately Data points that make up less than 1% of training data are sufficient to recover predictive capacity.Out-of-domain experiments were performed as in Figure 6, however for each model a specified number of experimental data points relating to the withheld canonical form were added into the training data (left to right for each panel, graphs are labelled with the percentages of data points added).Separate models were trained for each of the incrementally increasing data additions until the prediction accuracy came close to the fully trained ABB2 ensemble predictions and the ground truth experimental data.MDS plots for CDRL1 length 6 models (A) and CDRL1 length 7 (C).The amount of data being included is labelled at the top of each panel as a percentage of the total number of unique structures out of all data points seen in training (see Table 4).The corresponding ABB2 predictions and ground truth data are shown in the far-right MDS plots in each panel.RMSD distance values are plotted in (B, D) for the predicted data points relating to the dropped canonical form from each model relative to the ABB2 ensemble predictions (top graph) or ground truth data (bottom graph).For each model, the mean RMSD is given above the box plots, with standard deviation given in brackets.For (B, D), the top, far-right graph RMSD values are zero as the ABB2 model predictions are being compared to itself, with the comparison of ABB2 to ground truth data shown below.
recapitulate the cluster corresponding to the missing canonical form.
To check that these small proportions of data were enough to facilitate accurate predictions within the original ensemble architecture of ABB2, instead of a single model, we trained ensembles on two inclusion proportions (one above and one below the proportion where we first saw improvement ~0.6%, see Table 4) and analysed the resulting final output (the average structure from all four predictions).This demonstrated that the ensemble models still failed to recapitulate at the lower percentage, while the higher percentages were sufficient for the ensemble to progressively populate the missing structural space despite still being below 1% of total examples of that CDR loop (RMSD plots marked as 'ensemble' in Figures 7B, D).
These analyses underline the importance of sufficient data representation and suggest that even small numbers of datapoints can influence the predictive capacity drawn from large datasets.Ultimately the inability of models to truly extrapolate from physical principles means researchers must pay close attention to the contents of their datasets and continue to collect experimental data that explores lesser studied areas of structural space.
Discussion
In this study we analysed the predicted structures for paired sequences present in OAS generated by ABodyBuilder2 (ABB2) (20).Our data driven approach allowed identification of structural clusters within CDR loops of the same length and subsequent linkage to existing definitions of canonical forms.Our analyses aimed to explore the ability of structure predictors to enrich the experimentally defined landscape of canonical forms and identify novel conformations that would reflect generalisation or extrapolation arising from a deep learning method.
The augmentation of existing data with predicted structures enabled us to define new canonical clusters composed of heterogeneous CDR sequences which were united by the same loop backbone conformation and a sequence motif.These arose from both subdivision of areas of conformational space with uniform PyIgClassify2 annotation, as well as within highly populated areas of conformational space not assigned to any existing canonical form definition. Novel clusters of predicted loop conformations were also produced via the phenomenon of length independence (26).We observed areas of new density which had sequence enrichments identical to those of canonical forms at a shorter CDR length but contained a positionally fixed high entropy residue likely indicative of somatic hypermutation insertion.We analysed the distances between loop conformations at both the shorter and longer lengths by dynamic time warping.This revealed that different length loop clusters were indeed closer in conformational space than any of those of the same length.Outof-domain experiments confirmed that ABB2 was able to generalise across loops of different lengths and suggested predictions were influenced by high frequency experimental datapoints seen in shorter CDR loops.
However, our analyses could not find new clusters which had no origin in training data and thus represented true extrapolation.Therefore, we performed further out-of-domain experiments by retraining our ABB2 structure predictor whilst withholding data points which belonged to specific canonical forms.These 'starved' models were then challenged with correctly predicting the unseen CDR loop conformation.We found that ABB2 retrained in this way was unable to predict conformations not seen during training, but this inability could be resolved by inclusion of a small number of examples representing between 0.5-1.0% of the total data used in development.This suggests that effective prediction accuracy by structure predictors can be achieved for conformations even when they have very poor representation in the dataset.
Our study highlights important limitations regarding the current capabilities of deep learning structure prediction tools specific to the domain of immune receptor CDR loops.Whilst numerous studies have performed out-of-domain experiments and exploratory analysis on protein folds, the conformational space of CDR loops may offer greater challenges, particularly in regions that are inherently flexible or adopt distinct structures in bound and unbound states.
These challenges emphasise that if we wish to predict outside current known structure space, new structure prediction tools that have learnt the underlying rules which govern tertiary structure, instead of just patterns in the training data, will be required.While AlphaFold2 was heralded as a huge advance in structural biology and machine learning, the higher goal of building models that can capture biophysical laws has still not been reached (46,47).In the For inclusion experiments a specific number of PDB structures were gradually reintroduced to training data for a series of models.As PDB files may contain more than one structure in the asymmetric unit, the number of exact structural units is given for each experiment, as well as the corresponding percentage of all CDRL1 data points present in each training run.
absence of architectures that can extrapolate, greater amounts of training data, particularly in regions of structure space with poor coverage, may help improve predictive accuracy.Our demonstration that a small number of examples can address gaps means that a critical mass of data could be achieved to overcome the limitations of current models.However, this does place a large burden on experimental researchers to collect more data.Finally, our results focused on an area of structural immunology relatively abundant with data and analyses, that of antibodies.As T cell receptors become more important in both immunotherapy research and the clinic, a need to better classify and understand this protein for the purpose of structure prediction may supersede that of antibodies.Therefore, the questions posed in this study may take on more relevance in a field with a relative paucity of structure and paired sequencing data (48), as well as several unanswered questions on TCR loop flexibility and comparative conformational freedom (29).
The original purpose of canonical forms was their ability to predict structure from sequence, however these use cases have been superseded by the improved performance of ML methods for structure prediction.For experimental techniques such as X-ray crystallography to be rendered redundant in immunology we must have confidence that structure prediction algorithms faithfully replicate the most important region of immune receptors, the CDR loops.
1
FIGURE 1 Dominant CDR lengths in paired sequence space are matched by comparable distributions in structural data.Frequency distributions of sequences present in OAS by CDR and loop length, for the total number, including all redundant sequences (A), and unique sequences only (B).Asterisks above certain bars in (B) indicate the lengths with the most unique sequences, the predicted structures of which were analysed.(C, D) show data for the antibodies used to develop ABB2.The loop length frequency for each CDR including all structural units (all copies in the asymmetric unit) which have information in PyIgClassify2 (28) are shown (C).Breakdown of canonical form assignments for corresponding CDR loops present in structures of ABB2 training data (D).Each colour within a bar represents a distinct canonical form, red portions indicate loops that could not be assigned to any canonical form with high confidence in PyIgClassify2 analyses (not labelled with a number in the colour legend).
2 Predicted
FIGURE 2 Predicted Structures fall into dense regions of conformational space defined by existing canonical classes.Analysis of CDRL1 length 6 loops.Multidimensional scaling plots derived from pairwise RMSD data (A, B).Data points in (A) are coloured according to density-based clustering (DBS) membership, with the cluster centroid marked by an X.Those data points which do not belong to a DBS cluster in (A) are coloured black.In (B) all experimental data points present in the MDS analysis are coloured according to their high confidence PyIgClassify2 canonical form annotation. Any loops that do not belong to a high confidence PyIgClassify2 cluster (defined by an asterisk in the label, e.g.L1-11-*) are coloured in red.The predicted data points are coloured in black and underly the experimental annotations.Logo plots are shown for all sequences in each DBS cluster (C).For a sample of 20 loops in each cluster, the framework aligned backbone conformations are shown in three different orientations (D).The logo plots (C) and backbone (D) colours of blue, green and red correspond to the numbered DBS cluster colours in (A).
3
FIGURE 3 Subdivisions of existing canonical classes into clusters with distinct sequence motifs and backbone conformations.For CDR loops of CDRH1 length 8 (A, B) and CDRH1 length 10 (C, D), distinct DBS clusters contained multiple experimental data points sharing the same canonical form.(A, C) show colour coded DBS cluster logo plots, MDS plots coloured by DBS cluster membership and framework aligned backbone conformations of a sample of 20 loops from each cluster in three different orientations.Data points in the MDS plots which do not belong to a DBS cluster are coloured black.(B, D) show all experimental data points present in the MDS analysis coloured according to their high confidence PyIgClassify2 canonical form annotation. Any loops that do not belong to a high confidence PyIgClassify2 cluster (defined by an asterisk in the label, e.g., H1-13-* and H-15-*) are coloured in red.The predicted data points are coloured in black and underly the experimental annotations.
4 5 ABB2
FIGURE 4 Novel canonical classes revealed by structure prediction.Breakdown of analyses where regions of structural space populated by *unassigned experimental data (denoted by red data points with an asterisk in the PyIgClassify2 annotations) resolved to DBS clusters with distinct sequence logo plots for CDRL1 length 7 (A, B) and CDRL1 length 9 (C, D). (A, C) show colour coded DBS cluster logo plots, MDS plots coloured by DBS cluster membership and framework aligned backbone conformations of a sample of 20 loops from each cluster in three different orientations.Data points in the MDS plots which do not belong to a DBS cluster are coloured black.(B, D) show all experimental data points present in the MDS analysis coloured according to their high confidence PyIgClassify2 canonical form annotation. Any loops that do not belong to a high confidence PyIgClassify2 cluster (defined by an asterisk in the label, e.g., L1-12-* and L-14-*) are coloured in red.The predicted data points are coloured in black and underly the experimental annotations.
FIGURE 6Retraining whilst withholding canonical clusters highlights limited ability to extrapolate.Results of out-of-domain experiments where ABB2 was retrained in the absence of all experimental data points assigned to a specific canonical form in PyIgClassify2 (both high and low confidence) for CDRL1 lengths 6-9 (A-D).MDS plots show experimental data points coloured according to their high confidence PyIgClassify2 canonical form annotation. Any loops that do not belong to a high confidence PyIgClassify2 cluster (defined by an asterisk in the label, e.g., L1-11-*) are coloured in red.CDRL1 length 6 was retrained in the absence of all data points assigned to the PyIgClassify2 cluster 'L1-11-3' [coloured blue in (A)].For CDRL1 length 7 'L1-12-2' was dropped [coloured blue in (B)].For CDRL1 length 8 cluster 'L1-13-3' was dropped [coloured purple in (C)], and for CDRL1 length 9 cluster 'L1-14-1' was dropped [coloured green in (D)].For each panel, the MDS of experimental data points found in SAbDab are shown in the far-left panel.The MDS plot of ABB2 model predictions of the corresponding sequences are shown in the middle panel (labelled 'ABB2'), and predictions of the starved model are shown in the right panel (labelled 'Starved').The area of structural space investigated through exclusion during training is highlighted in a red box.The boxplots (median and upper and lower quartiles) and dot plots on the far right of each panel indicate the RMSD values for each predicted data point from the starved model from its target structure in predictions from the fully trained ABB2 ensemble (top graph) or the ground truth structure (bottom graph) with each sub graph labelled accordingly.
TABLE 1
Alignment of IGMT CDR numbering, AHo CDR numbering and PyIgClassify2 Canonical Forms.
TABLE 2
Results of RMSD and DBS cluster analysis in high frequency CDR lengths.
TABLE 3
Details of retraining in the absence of canonical clusters.
TABLE 4
Inclusion data points as a proportion of total CDRL1 data units in training. | 2023-12-13T14:13:43.177Z | 2023-12-09T00:00:00.000 | {
"year": 2024,
"sha1": "eaa8c2627f2c594d4f8cb3afcd03c0787f226fc0",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1352703/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "b5580c2ed9865da9d8b38c62f4b5cf4aca6e1bd1",
"s2fieldsofstudy": [
"Computer Science",
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Biology"
]
} |
249544051 | pes2o/s2orc | v3-fos-license | Underwater wireless communication via TENG-generated Maxwell’s displacement current
Underwater communication is a critical and challenging issue, on account of the complex underwater environment. This study introduces an underwater wireless communication approach via Maxwell’s displacement current generated by a triboelectric nanogenerator. Underwater electric field can be generated through a wire connected to a triboelectric nanogenerator, while current signal can be inducted in an underwater receiver certain distance away. The received current signals are basically immune to disturbances from salinity, turbidity and submerged obstacles. Even after passing through a 100 m long spiral water pipe, the electric signals are not distorted in waveform. By modulating and demodulating the current signals generated by a sound driven triboelectric nanogenerator, texts and images can be transmitted in a water tank at 16 bits/s. An underwater lighting system is operated by the triboelectric nanogenerator-based voice-activated controller wirelessly. This triboelectric nanogenerator-based approach can form the basis for an alternative wireless communication in complex underwater environments.
Results
Working principle of the underwater electric field communication. A conceptual diagram of the application of the studied underwater communication is shown in Fig. 1. The TENG converts sound (i.e. sonic waves in air) to electric signals in water (Fig. 1a), and the electric signals carrying the voice information can be transmitted in water and received by the diver. In this way, an underwater wireless communication is established via Maxwell's displacement current generated by the TENG. Figure 1b is the flow chart of the underwater communication. It needs to be noted that this method is different from the electric field communication generated from a pair of electric dipoles (see Supplementary Note 1).
The working principle of the underwater communication can be understood, approximately, with a capacitance model (Fig. 1c). The propagation of underwater electric field is analyzed from the perspective of displacement current. The transmitting and receiving electrodes form the positive and negative electrodes of the capacitor, while the water solution is the dielectric. With the presence of electric field E, the dielectric can be polarized where a polarization electric field P can be generated. It should be noted that the polarization electric field P is originated from the negative polarization charge to the positive polarization charge. E 0 is the combined electric field of E and P, If the relative permittivity is defined as ε r ¼ E=E 0 , the relationship between the polarization charge and the charge Q on the transmitting electrode is Q 0 ¼ ð1 À 1=ε r ÞQ. Due to the attenuation of the electric field during through propagation medium, the received charge Q 00 at the receiving electrode is <Q 0 .
The underwater communication can be demonstrated theoretically with the Maxwell's equation. Remind that the Gauss's law of the Maxwell's equations is where ρ is the distribution of free charges in space, and D is the electric displacement vector, which can be expressed as where permittivity in vacuum is ε 0 . The Maxwell's displacement current density can be defined as From Eq. (3), the first term ε 0 ∂E=∂t gives rise to electromagnetic wave. Studies of Prof. Zhonglin Wang reveal that the second term (∂P/∂t) in the Maxwell's displacement current can be directly related to the output electric current of the TENG 15 .
It is worth mentioning that the internal circuit in the TENG is dominated by the displacement current, and the observed current in the external circuit is the capacitive conduction current (see Supplementary Note 2). The research on the underwater electric field propagation is inspired by the built-in electric field of the TENG. Comparing the TENG-based underwater electric field with electromagnetic waves, the propagation of electromagnetic waves does not require a medium, and the propagation effect is best in vacuum. At this time, ∂E/∂t reaches the maximum value, and ∂P/∂t is 0. The propagation of the polarization electric field requires a medium (see Supplementary Note 3). As ∂E/∂t gets significantly reduced in water, the propagation effect of ∂P/∂t gets improved.
To examine the performance of the underwater communication, an acoustic-driven TENG is applied to convert sound in air to electrical signals in water. The output performance of the acoustic-driven TENG has been investigated systematically in our previous study 27 . As shown in Fig. 2a, the TENG consists of a Helmholtz resonant cavity, an aluminum film with evenly distributed acoustic holes, and a fluorinated ethylene propylene (FEP) film with a conductive ink-printed electrode (details about the TENG is shown in Supplementary Notes 4 and 5). The transmitting electrode in water is connected to the aluminum electrode of the TENG, while the other electrode of the TENG is grounded so that the TENG operates in the single-electrode mode. In reaching an electrostatic equilibrium state, higher electrical output can be obtained by acquiring ground charges 28 . It is worth noting that one piece of conduct materials, such as metal and salt water, can serve as a charge reservoir for the TENG. Figure 2b shows the working principle of the underwater communication, which is based on the interface polarization from the Maxwell-Wagner effect. The electrical output is generated from the variation of the built-in electric field in the TENG, which is directly related to the second term (∂P/∂t) in the Maxwell's displacement current 15 . A transmitting electrode is connected to one electrode of the TENG, thus an electric field E 0 is induced in water as the TENG works (see Fig. 2b). Corresponding to the variation of electric field E 0 , the positive and negative ions in the water move reciprocally, generating a polarization electric field P 0 . The current in the receiving electrode induced by the polarization electric field can be measured with an electrometer. The electric field E 0 generated by the TENG is related to the charge density ρ 0 in the transmitting electrode in the following form: P 0 is the polarization electric field generated from the electric field E 0 , which is Therefore, the second term J p in the Maxwell's displacement current (generated by the polarization electric field) is For the acoustic driven HR-TENG, when the FEP film contacts with the aluminum film, the electron clouds on the surfaces of the two films overlap, and some of the electrons from the aluminum film enter the deeper potential well of the FEP film. Due to the much higher electronegativity of FEP than aluminum, the free electrons on the surface of the aluminum film transfer to the lowest unoccupied molecular orbital of the FEP interface. So the aluminum film becomes positively charged ( Supplementary Fig. 1a). Since the transmitting electrode is connected to the aluminum film, positive charges are also distributed on the surface of the transmitting electrode. Negatively charged ions in the water are attracted by the transmitting electrode, while positively charged ions are repelled to the surroundings. When positive ions contact with the receiving electrode, electrons in the receiving circuit flow to the receiving electrode, so the electrometer detects a positive current. Due to the change in the acoustic pressure difference, the FEP film is separated from the aluminum electrode. At the moment, electrons flow from the ground to the conductive ink electrode to balance the electric field between the FEP film and the conductive ink electrode. Due to the negative charge distributed on the surface of the FEP film, the free electrons on the aluminum film are repelled, so the electrons flow from the aluminum film to the transmitting electrode. Opposite to before, positive charged ions in the water are attracted by the transmitting electrode, while negative charged ions are repelled to the surroundings ( Supplementary Fig. 1b). When negative ions c Schematic diagram of the capacitance model, ε r is relative permittivity, E is the original electric field, P is the polarization electric field, E′ is the combined electric field of (E) and (P), and all about Q are amount of charge.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31042-8 ARTICLE contact with the receiving electrode, electrons in the receiving electrode flow to the receiving circuit, so the electrometer detects a negative current. Figure 2c, d compares the electric signals in air with those in ordinary water. Under acoustic waves (80 Hz, 80 dB), the corresponding periodic output short-circuit current signals yield the peak value of 14.9 μA (Fig. 2b), which is directly measured with the electrometer connected to the aluminum electrode. When the (electrometer-connected) receiving electrode is two meters away from the submerged transmitting electrode, the peak value of the current decreases slightly to 14.5 μA while the waveform of electric signals remain constant (Fig. 2c). The peak value of open-circuit voltage output of the TENG decreases from 28.5 V in air to 13 V in water (see Supplementary Fig. 2). When the water tank is grounded by a wire, the output current decreases significantly, but the waveform of electric signals stay consistent with the original signal ( Supplementary Fig. 3). This can be explained by the tendency that charges from ground would balance the electrical potential field in water. Furthermore, the current signal could still be measured even when the transmitting electrode is insulated from water by a Kapton tape (Supplementary Fig. 4), and the electric field generated by the TENG can propagate across both gas and liquid media ( Supplementary Fig. 5). These prove that the transmission of the signals depends on the electric field radiated by the TENG rather than the direct exchange of electrons between water and electrode plates. Both theoretical analysis and experiments have shown that for the whole system, the propagation of underwater electric field has demonstrated the characteristic of displacement current (see Supplementary Note 6). Previous study 29 proved that when the electric field propagates in a medium, conduction current dominates when σ=ω > ε and displacement current dominates when ε > σ=ω (σ is conductivity, ω is angular frequency, and ε is permittivity). The Rayleigh distance of the TENG generated electric field can be calculated by R ¼ 2D 2 =λ, where R is Rayleigh distance, D is the maximum size of the transmitting electrode, and λ is the wave length. As a result, R is so small that it can be neglected. According to these theories, for the TENG-based underwater electric field communication, ε > σ=ω and the transmitting distance is larger than the Rayleigh distance. So the displacement current domains the underwater electric field while conduction current only appears in a very short distance.
It is worth mentioning that underwater communication can also be realized by various types of TENGs, such as the TENGs that harvest wave energy and vibration energy (see Supplementary Fig. 6, and the first and second items in Supplementary Table 1). The development of the electric field communication depends on the development of TENG technology. The techniques for designing TENGs with high frequency and good output performance (see Supplementary Table 1) provides good potential for the application of the TENG in underwater communication [30][31][32][33][34][35][36][37] .
Transmission performance of the underwater electric field. The characteristics of the underwater electric field under different parameters such as water volume, electrode position/size, salinity, water turbidity, and underwater obstacles, have been studied. Figure 3 shows the attenuation of the underwater electric field. In a 3 m × 2 m × 0.4 m water tank (Fig. 3a), the receiving electrode is placed certain distance away from the transmitting electrode. As the distance increases from 1 m to 3 m, the current signals remain almost unchanged (Fig. 3b). This is also verified by the simulation of underwater polarization electric field shown in Supplementary Fig. 7. Furthermore, the complete signals output by the TENG and received signals underwater, and their Fourier transforms are shown in Supplementary Figs. 8-9, showing that the frequencydomain features of the signals remain unchanged underwater. As more water is added into the tank, the peak value of the current decreases. Actually, the peak value decreases by 30% from the original signals as the water volume reaches 6 m 3 (Fig. 3c). This can be understood, as the electric field energy W in water is According to Eq. (7), the energy density of the electric field decreases with the water volume V while using a TENG with constant power output. Therefore, the peak value of the current decreases with larger water volume. As shown in Fig. 3d-f, the current can be enhanced by using a receiving electrode plate of larger area. With a 10 cm × 5 cm electrode plate, the peak value of the current signal increases by 18%, compared to that with a thin electric wire. This means more ions in water contacting with the electrode, which is verified by the underwater polarization electric field simulation shown in Supplementary Fig. 10. In addition, the peak value of the current is subject to the angle between the transmitting and receiving electrode plates, but the effect caused by the angle is very small in the water tank (see Supplementary Figs. 11-12), which is quite different from the case in a bipolar electric field 38 .
By comparing the distribution of the polarization electric fields without and with the receiving electrode (Fig. 3g, h), it is found that the receiving electrode can change the distribution of the polarization electric field. This can be explained by the fact that the electrode is equivalent to a terminal with a potential of zero voltage. The variation of the polarization electric field and the variation of the terminal charges in the receiving electrode are shown in Fig. 3i. A 2D simulation is performed and the polarization electric field distribution corresponding to Fig. 3i is shown in Supplementary Figs. 13 and 14. The attenuation of the polarization electric field and terminal charges at the receiving electrode with distance can be fitted respectively The dependence of the underwater electric field on disturbances in water is shown in Fig. 4. It is found that the peak value of the current signal in salty water (5 g L −1 , adjusted by adding salt to water) increases by 40% on top of that in pure water (Fig. 4a). This indicates that the ions in salt water can enhance the polarization electric field. However, water salinity increase beyond 15 g L −1 will not further promote the current signals. Similarly, when acid or alkali is added to the pure water to change the pH, the ion concentration in the water will change, indicating that as the pH of water deviates from 7, the received current signals will increase (Supplementary Fig. 19). This may be attributed to the improved relative permittivity of the aqueous solution. Figure 4b, c reveal that the waveform of the received signals identical to original ones, regardless of obstacles or turbidity in the water tank. In this sense, the polarization electric field has shown robustness to obstacles and water turbidity.
As achieving reliable communication across the pipe is very important for the pipeline robot system 39,40 , the performance of the polarization electric field in liquid pipes is investigated (Fig. 4d-f). Figure 4d is a schematic diagram of the drilling platform with the oil pipeline. In a pipe filled with salt water, the peak value of the current is also found to decreases with the distance between the transmitting electrode and receiving electrode. In fact, the value decreases by 66% when the distance in-between is 100 m (Fig. 4e), which is consistent with the simulation result of the polarization electric field in the pipe (as shown in Supplementary Fig. 20).
In fact, the collision between ions and water molecules may influence the performance of the electric field. In addition, it is interesting to find that the received signals in a spiral pipe are the same with those in a straight pipe (Fig. 4f). From Supplementary Fig. 21, it is found that independent of the flow status, the current signals can also be obtained in the mixture of oil and water, which means the polarization electric field communication can be applied in complex pipelines.
It is worth noting that the electric field communication is also insensitive to water temperature and ambient lightness (Supplementary Fig. 22). Further comparisons between acoustic, optical, and electromagnetic waves methods are shown in Supplementary Table 2. What's more, by studying the effect of the ground on the electric field, it is theoretically proved that this system may work in open water area as shown in Supplementary Note 7.
Modulation and demodulation of the underwater electric field communication. The modulation and demodulation process of current signals for data transmission in water is shown in Fig. 5. The current signals converted from sound waves by the TENG can be modulated to digital signals containing the information of texts or images in water via the electric field communication (Fig. 5a). The signal modulation method is based on the on-off keying (OOK), in which longer signals with time intervals of 50 ms is set as "1", and shorter signals with time intervals of 25 ms is set as "0". A 25 ms interval is inserted between each digital signal to separate adjacent digital signals. After transmission in water, the modulated digital signals can be received by the electrometer (Fig. 5b). The fundamental frequency of the signals generated by the TENG is 80 Hz, and the frequency of the modulated digital signals is 16 Hz. Alternatively, the digital signals can be modulated with other frequencies or other methods (see Supplementary Fig. 23a, b). Higher frequency yields a fast information transmission rate, while lower frequency yields a strong anti-interference ability. By demodulating the received signal (with the MATLAB codes), the signals of "0" and "1" can be identified accurately ( Fig. 5b and Supplementary Fig. 23c). The current signals can be modulated into text by the standard encoding. The received signals can be accurately demodulated into the original text (Fig. 5c). Supplementary Movie 1 shows that the real-time current signals generated by the TENG is modulated, transmitted, and demodulated, and the text obtained after demodulation is displayed on a computer screen. This electric field communication can also be used for image transmission. Figure 5d shows part of the received current signals, and the complete signals are shown in Supplementary Fig. 23d. A 2.7-KB image is transmitted within 1353 s at 16 bits/s (owing to the low fundamental frequency of the TENG). There is no error signal in the continuous transmission of~20,000 digital signals (100,000 working cycles of the TENG). In addition, by applying an external alternating current on the dielectric material's electrode, the electric signals with higher frequencies (up to kilohertz) can be modulated and transmitted in water, demonstrating that this approach can be used for high frequencies communication (see Supplementary Note 8).
It should be noted that the current signals output by the TENG and received signals underwater at a range of 60-200 Hz are compared. It turns out that the signals received underwater are always consistent in waveforms with the signals output by the TENG (see Supplementary Fig. 24). What's more, the power spectrum is obtained by performing Fourier transform to the modulated digital signals and noise. The power spectrum shows that energy is evenly distributed in the frequency range from 40 kHz to 85 kHz (see Supplementary Fig. 25), proving that the bandwidth of the system with water channel is greater than 85 kHz.
Realization of wireless control using the underwater electric field communication. To further study the ability of the underwater wireless communication, a demo voice control of an underwater lighting system is performed (Fig. 6). A microphone-style TENG that converts voice to electrical signals is to control the underwater lighting system wirelessly (Fig. 6a). The signals containing the voice information (e.g. "red" and "green") are transmitted in water and received by the electrometer (Fig. 6b). By performing short-time Fourier transform to the signals, the words "red" and "green" can be distinguished with a neural network algorithm (see Fig. 6c and Supplementary Fig. 26). Subsequently, the words are converted into digital signals to control the lights. This approach can be applied in the real-time voice control of underwater lights (Fig. 6d and Supplementary Movie 2). It is worth mentioning that the entire underwater communication realized by the TENG is self-powered.
At the same time, the signals receiving and controlling device in the water can be designed independently. By touching a (contact-separation mode) button-type TENG, people can use the electric to control the independent working system in water (see Fig. 6e, f and Supplementary Movie 3). The independent working system consists of a weak current acquisition board, a single-chip microcomputer, batteries, a relay, and an underwater working light. The pulse signals generated by the TENG are collected by the weak current acquisition board, and the analog signals are converted into digital signals sent to the microcontroller. The single-chip microcomputer processed the digital signals and controlled the underwater working light.
In another demo experiment, a sandwich-like TENG (S-TENG) with an output current of 60 μA is deployed in a 50 m × 30 m × 5 m basin (with all boundaries connected to the ground, see Fig. 6g, h. When the S-TENG is shaken, the current signals outputted by the S-TENG can be transmitted in water and received by the receiving electrode 5 m away from the transmitting electrode. The signals are detected by a current acquisition board (Supplementary Note 9), which sends the signals to a computer through WiFi and then the waveforms are displayed on the screen (Fig. 6i and Supplementary Movie 4).
Discussion
In summary, an underwater communication via Maxwell's displacement current is proposed and investigated. In the Maxwell's displacement current, the first term ∂E/∂t gives rise to electromagnetic waves. However, in underwater environments, the high- ARTICLE frequency electromagnetic waves can be easily absorbed, and the low-frequency electromagnetic waves can only be transmitted through an antenna of several kilometers. In this study, the second term (∂P/∂t) in the Maxwell's displacement current is utilized for underwater communication. An acoustic-driven TENG connected to a transmitting electrode is applied to generate alternating electric field in water, so that the sound in air can be converted into underwater electrical signals, which can be measured with a receiving electrode connected to an electrometer. Through a salt water pipe of 100 m length, the peak value of the current signal decreases by 66% compared to the original signal, while the electric signals are not distorted in waveform during transmission.
Based on the on-off keying method, texts and images have been successfully transmitted by modulated current signals in a water tank at 16 bits/s. Throughout the continuous transmission of about 20,000 digital signals, no error appears. By successfully converting voices into current signals, the TENG is capable of controlling an underwater lighting system wirelessly. What's more, the current signals output by a sandwich-like TENG can be transmitted in a 50 m × 30 m × 5 m basin with the signals displayed on screen in real time. Compared to traditional sonic, optical, and electromagnetic communications, the underwater communication via Maxwell's displacement current appears to be less vulnerable to disturbances, which exhibits considerable potential for applications in complex underwater environments. length of 32 mm are fixed on the resonant cavity. The aluminum film with 440 uniformly distributed acoustic holes acts as the electropositive triboelectric layer. The length, width, and thickness of the film are 45 mm, 45 mm, and 0.1 mm, respectively and the diameter of the holes is 0.5 mm. The FEP film is used as the electronegative triboelectric layer on observation of its strong electronegativity and good flexibility. It has a thickness of 12.5 μm and a working area of 45 mm × 45 mm. Given that the FEP material is insulated, a conductive ink electrode with a micron thickness is attached to the other side of the FEP film to transfer electrons. A screen printing device (Tou) is used to print the conductive ink (CH-8(MOD2)) on the FEP film (WitLan). The shell is printed by a 3D printer with PLA material. The TENG to recognize voice is similar to the HR-TENG, except that it has no dule-tube structure but has a 45 mm × 45 mm opening on one side of the resonance cavity. The contact separation distance between the FEP and aluminum film is 0.2 mm. The membrane structure of the button-type TENG is the same as HR-TENG without a cavity. The acrylic plate of a single layer S-TENG is of 5 mm thickness and 10 cm diameter. Two aluminum electrodes with a thickness of 50 µm and an area of 6 cm × 4 cm are parallel attached onto two sides of the acrylic plate. PTFE balls with 10.5 mm diameter are filled between two acrylic plates and they are produced by 3 M company. Each S-TENG unit consists of 10 layers stacked S-TENG in parallel connection and acrylic block shell. The acrylic block shell has 10 cm diameter and 20 cm height. There are four AC output copper ends in an S-TENG unit, one pair at the top and the other pair at the bottom. The buoy consists of 5 S-TENG units as the power module and an acrylic shell as the frame structure. The S-TENG units integrated inside are in parallel connection to make the AC electrical output in-phase and the they are fixed through packing tape.
Methods
Experimental process and measuring equipment. The output signals are measured with a Keithley 6514 electrometer. The HR-TENG is mounted on an optical plate with a loudspeaker (JBL), driven by sinusoidal waves from a function generator (YE1311). One electrode of the TENG is grounded and the other electrode is immersed in water. The wires used for electrodes has a 0.3 mm copper core, and the electrode plates are copper films with the size of 100 × 50 × 0.06 mm. The water pipe used in the experiment is an ordinary PVC rubber water pipe with an inner diameter of 13 mm. A 12 V DC motor is used to control the flow of liquid in the pipe. The signal modulator consists of a microcontroller development board (STM32F7) and a relay. The MATLAB interface in LABVIEW is used to demodulate and display the real-time signals measured with the electrometer. The signals generated by the voice-driven TENG need to be filtered at 50 Hz and its harmonics after being received.
The transmitting electrode in water and the aluminum electrode of the TENG is connected by an ordinary copper wire. The HR-TENG is applied to convert sound in air to electrical signals in water. In this way, acoustic waves with specific frequencies can be got by controlling the signal generator and a loudspeaker. Furthermore, under the excitation of the acoustic waves, electrical signals with specific frequencies can be generated by the HR-TENG to examine the performance of the underwater communication. Button type TENG is a basic and commonly used TENG with the simplest structure. The application of the button type TENG prove that underwater communication can be realized by general TENGs, demonstrating the application potential of this approach.
Numerical simulations. In order to verify the accuracy of the derived theory and experimental results, COMSOL Multiphysics software has been used for numerical simulations. The AD/DC modules, electrostatic interfaces, and transient state analysis are used in simulations. The distribution of the polarization electric field and terminal charge of the receiving electrode has been simulated. As the element size influences the calculation results, the ultra-fineness meshing option has been adopted in the simulation. For 3-D simulations, the size of the model is limited to 15 m. The 2-D simulation is used to analyze the attenuation of the electric field through longer distance. The error of the 3D simulation depends on the mesh size and model scale.
Data availability
The data supporting this study are available within the article and the Supporting Information. Source data are provided with this paper. | 2022-06-11T06:16:26.141Z | 2022-06-09T00:00:00.000 | {
"year": 2022,
"sha1": "ca5174d189e9a4a6675032455c7a9dffef70f908",
"oa_license": "CCBY",
"oa_url": null,
"oa_status": null,
"pdf_src": "PubMedCentral",
"pdf_hash": "871ec09e97a14a6db5b16182d5d113da7ce33215",
"s2fieldsofstudy": [
"Engineering"
],
"extfieldsofstudy": [
"Medicine"
]
} |
4478278 | pes2o/s2orc | v3-fos-license | SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load
Restriction of HIV-1 in myeloid-lineage cells is attributed in part to the nucleotidase activity of the SAM-domain and HD-domain containing protein (SAMHD1), which depletes free nucleotides, blocking reverse transcription. In the same cells, the Vpx protein of HIV-2 and most SIVs counteracts SAMHD1. Both Type I and II interferons may stimulate SAMHD1 transcription. The contributions of SAMHD1 to retroviral restriction in the central nervous system (CNS) have been the subject of limited study. We hypothesized that SAMHD1 would respond to interferon in the SIV-infected CNS but would not control virus due to SIV Vpx. Accordingly, we investigated SAMHD1 transcript abundance and association with the Type I interferon response in an SIV model. SAMHD1 transcript levels were IFN responsive, increasing during acute phase infection and decreasing during a more quiescent phase, but generally remaining elevated at all post-infection time points. In vitro, SAMHD1 transcript was abundant in macaque astrocytes and further induced by Type I interferon, while IFN produced a weaker response in the more permissive environment of the macrophage. We cannot rule out a contribution of SAMHD1 to retroviral restriction in relatively non-permissive CNS cell types. We encourage additional research in this area, particularly in the context of HIV-1 infection.
has also been presented 20,24,25 . At the same time, in some cells, SAMHD1 may be part of a feedback system, as it has been reported to antagonize the expression of certain Type I IFN-stimulated genes 26,27 , strikingly in monocyte-derived dendritic cells 28 . However, some myeloid-lineage cells and resting CD4+ T-cells from individuals with SAMHD1-inactivating mutations supported robust infection in vitro 12,29 , suggesting that the observed anti-IFN activity of SAMHD1 is cell type specific or that its ablation is insufficient to allow the interferon response to protect against HIV-1 infection. Differential transcript-level expression of SAMHD1 has been reported in cells of one cohort of HIV-1 elite suppressors 30 , but not in two others 31,32 .
In contrast with the periphery, relatively little is known about SAMHD1 levels during retroviral infection of the brain and progression to the HIV-associated neurocognitive disorders (HAND). Neurologic correlates of HIV infection have been observed since the earliest days of the pandemic [33][34][35] and may even occur despite successful virologic control [36][37][38] . Some pharmacologic agents may contribute to HAND 39 by inducing oxidative stress. Infiltrating macrophages and resident microglia are productively infected 40 . Astrocytes, the most abundant cells in the brain, are also infected 41 , albeit rarely productively in vivo. In one study of SAMHD1 in CNS cells, interferon alpha transiently increased SAMHD1 production in immortalized human stem cell-derived astrocytes 23 . In another, astrocytes were found to have higher levels of SAMHD1 than microglia, which are relatively permissive to infection 42 . Host miR-181a was found to regulate SAMHD1 in vitro 42 , although miRNA association with SAMHD1 levels was not apparent in vivo 43 .
Since SAMHD1 transcription and Type I interferon responsiveness in the retrovirus-infected CNS are unknown, we hypothesized that SAMHD1 transcript levels might respond to SIV infection, but without affecting viral replication due to the action of SIV Vpx. We further hypothesized that macaque astrocytes, which are relatively refractory to infection and host only low levels of viral replication, would display high levels of SAMHD1 transcription (as now also reported for human astrocytes 42 ). We therefore examined transcription of SAMHD1 in thalamus in an SIV model 44 in which an acute infection phase of ten to 14 days includes peak cytokine responses 45 and is followed by a latent or asymptomatic phase. Recapitulation of markers of human disease progression is accompanied by AIDS and CNS disease by three months post-infection in > 90% of cases, with sustained inflammation and other factors contributing to neurodegenerative pathologies 46 . SAMHD1 transcript was also measured in primary macaque astrocytes infected with SIV and/or treated with interferon beta, the Type I interferon most important in the CNS response to viral replication.
Our results indicate that SAMHD1 mRNA expression in thalamus mirrors the rise of proinflammatory cytokine expression during acute infection and appears to remain elevated during asymptomatic infection and into disease progression. In vitro, primary macrophages and astrocytes did not undergo significant changes in SAMHD1 expression during the course of long-term infection with HIV or SIV, although early changes were observed in SIV-infected astrocytes. However, treatment of macrophages and especially astrocytes with exogenous interferon-beta resulted in significant fold changes in SAMHD1.
Results
Regulation of SAMHD1 transcript in CNS during retroviral infection. Thalamus RNA samples from 58 subjects (Table 1) were obtained and subjected to quantitative PCR. With reference to the average of two housekeeping genes, GAPDH and beta actin, and compared with levels in six uninfected control animals, SAMHD1 was significantly increased during acute infection (4, 7, and 10 dpi) (Fig. 1). Indeed, an increase over baseline was maintained at later time points in all but a small number of subjects, even during the "latent" or "asymptomatic" phase that includes 14 and 21 dpi. This increase was nominally significant at 56 dpi, but this should be viewed in the context of the broader trend towards increased expression. SAMHD1 and the Type I interferon response. Based upon reports in the literature and our experience with measuring host antiviral responses 45 , we postulated that the observed increases in SAMHD1 are part of the Type 1 interferon antiviral program. To investigate this possibility, we first used a bioinformatics approach to confirm that the macaque SAMHD1 promoter sequence, like that of human, contains interferon stimulated response elements (ISREs). MatInspector predicted ISREs in both human and animal sequences ( Table 2). Next, we compared expression of SAMHD1 and the interferon stimulated gene Mx1 ( Fig. 2A). A strong correlation between the two was observed. This finding is consistent with Type I interferon responsiveness of both genes. SAMHD1 transcript during lentiviral brain disease development. In the rapid model, disease develops by the 42 and 56 day post-inoculation time points. While Mx1 was correlated with CNS disease severity (Fig. 2B), SAMHD1 displayed a weaker correlation with disease ( Fig. 2C) at these time points. A trend towards greater expression of SAMHD1 in severe disease was partially offset by several "non-responsive" animals with lower SAMHD1 expression despite high MX1 response (Fig. 2C). As we hypothesized, however, higher levels of SAMHD1 expression did not associate overall with lower viral loads in brain tissue (Fig. 3). Both MX1 and SAMHD1 expression were positively correlated with severity of CNS disease, consistent with a runaway immune response that, in acute phase infection, might be protective, but at later stages becomes damaging.
SAMHD1 regulation in monocyte-derived macrophages.
To follow up on the in vivo findings consistent with Type I interferon responsiveness of SAMHD1 in brain, we next sought to determine which cell types might be responsible for SAMHD1 regulation in the CNS. We assayed SAMHD1 expression in several relevant cell types in vitro, in response to interferon beta (IFNB1) treatment or infection. The first cell type assayed was the human monocyte-derived macrophage. We studied human macrophages because of their relative availability at the time of these experiments. Following infection, monocyte-lineage cells enter the brain within days, seeding CNS viral infection. During progression to disease, perivascular and parenchymal macrophages support productive infection and release damaging proinflammatory cytokines. In human monocyte-derived macrophages, differentiated by response to macrophage colony stimulating factor (M-CSF), Mx1 was upregulated rapidly and significantly as early as 2 hours post-treatment with recombinant IFNB1 (Fig. 4A). In contrast, TNFalpha did not trigger significant changes in Mx1. Interferon beta resulted in later upregulation of SAMHD1, beginning between 8 and 12 hours post-treatment (Fig. 4B). This increase was significant at 12 and 24 hours. It is unclear why the effect was delayed for SAMHD1 compared with Mx1. Possibly, the interferon effect is indirect, requiring initiation of a secondary signaling cascade. TNFalpha treatment resulted in apparently lower SAMHD1 expression. However, this trend did not reach significance.
In parallel, we assessed the effect of HIV infection on macrophages. Cells were infected at a multiplicity of infection of 0.05, and samples were harvested at time points including two, seven, nine, 14, and 21 days post-infection. Productive infection was confirmed by p24 ELISA (not shown). Despite an apparent trend towards increased SAMHD1 abundance at seven and nine dpi, none of the changes was significant (Fig. 4C). Given the significant increase in SAMHD1 in interferon beta-treated cells, this result might seem counterintuitive. Would infected or exposed cells not generate interferon and thus render themselves and surrounding cells less susceptible to infection or replication? There are several explanations we can offer. First, macrophage innate responses to HIV infection may be turned off or subverted by viral products or specific cellular responses to viral products 47 . As cited above, according to several reports, SAMHD1 itself can dampen IFN responses under certain circumstances. Second, the timing of the experimental exposure to recombinant interferon may have led to negative feedback by cellular factors such as miRNAs 48 , influencing results. Finally, replicating in vitro the microenvironment of the brain during SIV infection may be a considerable challenge.
SAMHD1 regulation in primary astrocytes.
The second cell type we tested was the primary astrocyte.
To take advantage of a unique resource-rhesus adult primary astrocytes-this experiment was performed with SIV/17E-Fr infection, not HIV. The most abundant cell type in the brain, astrocytes, are readily infected in vitro 49 . They are also infected in vivo, but incompletely characterized blocks to replication usually prevent productive infection. By sheer numbers, however, astrocytes comprise the most abundant, if largely inactive, lentiviral reservoir in the CNS. Astrocytes infected or not with SIV/17E-Fr at a multiplicity of infection of 0.1 were then exposed or not to 100 units of recombinant macaque IFNB1 per milliliter of culture medium, creating four experimental conditions.
Figure 2. Relationship of SAMHD1 transcription, Type I interferon response, and disease severity.
There was a significant positive correlation of SAMHD1 transcript and level of MX1, an indicator of the Type I interferon response (A). Comparing Mx1 (B) and SAMHD1 (C) transcript with disease severity as assigned by pathology (none, mild, moderate, or severe), the association of Mx1 with disease appeared stronger than the association of SAMHD1 and disease. Transcript abundance was normalized to the average of beta actin and GAPDH; hence, the smaller the "dCq" value, the greater the abundance. We compared the change from pre-infection at post-treatment time points of two, seven, 14, and 21 days, with the untreated, uninfected condition as the reference at each time point. At two days post-treatment, SAMHD1 levels were significantly upregulated in all interferon and infection conditions, including an approximately eight-fold upregulation in response to interferon (Fig. 5). Thereafter, SAMHD1 remained upregulated in the presence of exogenous interferon but not in the SIV alone condition. The large fold change of SAMHD1 in astrocytes compared with macrophages suggests, with the caveats noted above, that astrocytes may contribute a larger portion of the observed upregulation of SAMHD1 transcript in brain. Furthermore, it is interesting to note the comparatively greater response of SAMHD1 in the astrocyte, generally considered a non-permissive cell type, versus the permissive macrophage. However, since the astrocytes used in these in vitro experiments do support viral replication, additional work will be needed to elucidate the details of the role of SAMHD1 in enforcing viral latency in astrocytes in vivo.
Discussion
We report that SAMHD1 expression is significantly upregulated in thalamus during retroviral infection and tracks with the Type I interferon response as represented by MX1. Our in vitro results suggest that, in part, this upregulation of SAMHD1 may be driven by the strong response of astrocyte SAMHD1 to Type I interferon-specifically IFNB1-as well as a comparatively minor and short-lived response to retrovirus exposure. We cannot rule out that this high level of SAMHD1 expression may help to protect astrocytes from retrovirus infection, even in the case of Vpx-expressing SIV. Although the contribution of macrophages is substantially less clear due to contradictory results in the literature, our findings suggest that macrophages may also contribute an augmented complement of SAMHD1 following IFNB1 exposure.
Despite the upregulation of SAMHD1 in thalamus during SIV infection, SAMHD1 transcript levels do not correlate negatively with viral load. Thus, the arms race against the retrovirus is lost overall, likely in part due to SIV counteraction of SAMHD1 protein in infected cells. This contrasts with our results on HIV-1 in the periphery, where, in a study of PBMC of HIV-1 elite suppressors and chronic progressors, a negative correlation of viral load with MX1 and SAMHD1 was observed in viremic individuals 31 . It will be interesting to follow up on these findings in HIV-infected brain.
Strategies to upregulate SAMHD1, for example during "shock and kill" reactivation therapy 50 , could supplement the actions of traditional antiviral drugs and further prevent the spread of any newly produced virus. Transcriptional regulation is of course not the only determinant of protein activity. One form of post-transcriptional regulation is microRNA-mediated repression: regulation of the SAMHD1 transcript may include post-transcriptional blocks such as miRNA-mediated degradation or translational attenuation. As we reported recently 43 and at the 2013 Conference on Retroviruses and Opportunistic Infections 51 , miRNAs with seed sequence matches to SAMHD1 that appear to bind to the transcript include miRs-34a, -125b, -150, -155, and the -181 family. This effect has recently been confirmed for miR-181a and miR-155 42 and may occur for other miRNAs as well 7 . The finding that host miRNAs contribute to SAMHD1 regulation could form the basis of a SAMHD1-enhancing strategy as mentioned above. Protein-level activity is also governed by post-transcriptional factors including phosphorylation state 52,53 and multimerization 54 . Blocking Type I interferon signaling in myeloid and plasmacytoid dendritic cells allowed the cells to become permissive to HIV-1 infection 55 , and the antiviral form of SAMHD1, without phosphorylation on residue Threonine 592, is promoted by interferon 53 . Accordingly, the phosphorylation and multimerization states of SAMHD1 should also be investigated further in the CNS.
Methods
Animal studies and ethics statement. Studies of animal materials were conducted using archived samples from previous, concluded pigtailed macaque studies involving dual-inoculation with an immunosuppressive SIV swarm and a neurotropic clone 56 ; no new animal subjects were involved in this project. Characteristics of the subjects are outlined in Table 1. In addition to uninfected controls, post-infection time points examined were 0, 4, 7, 10, 14, 21, 42, 56, and 84 dpi. All animal studies were approved by the Johns Hopkins University Institutional Animal Care and Use Committee and conducted in accordance with the Weatherall Report, the Guide for the Care and Use of Laboratory Animals, and the USDA Animal Welfare Act.
Macrophage differentiation. Peripheral blood mononuclear cells (PBMCs) were isolated from de-identified blood products by standard Percoll protocol as described previously 57,58 . PBMCs were cultured at a density of 4 × 10 6 cells per well in 12-well plates or 2 × 10 6 cells per well in 24-well plates in macrophage differentiation medium (MDM) with 20% serum [MDM20: Dulbecco's modified Eagle's medium (DMEM, Invitrogen), 20% human serum (GemCell), 2 mM L-Glutamine (Sigma), 10 mM HEPES (Gibco), 10 mM sodium pyruvate (Sigma), 50 ng/ml M-CSF (R&D), and 2 mg/ml Gentamicin (Gibco)] for one week, with half-volume re-feeding at three days post-plating. Note that human serum lots were selected for minimal pyrogen activity and tested in cell culture before use. After seven days, supernatants were removed and adherent macrophages were washed with PBS. Medium was replaced with MDM with 10% human serum.
Macrophage infection, harvesting and RNA isolation. Differentiated macrophages in 12-well plates were infected with HIV-1-BaL (50 ng p24) for 4-6 hours, and virus was removed with extensive washes with PBS. Infected and uninfected cells were harvested in mirVana RNA lysis buffer (Ambion/Life Technologies) day 0 and at 2, 7, 9, 14, and 21 dpi and stored at − 80 °C. Supernatants were also collected and stored at − 80 °C. Short-term macrophage cytokine treatment. Macrophages were sham treated or treated with 100 units/mL of recombinant human IFN-beta or 20 ng/mL TNF-alpha (PBL Interferon Source, now PBL Assay Science, Piscataway, NJ). Lysates were collected at 2, 4, 8, 12, and 24 hours post-treatment as described above.
Astrocyte infection with SIV. Primary macaque astrocytes were purchased from Cambrex and cultured in astrocyte growth media with supplements (Lonza) as described previously 41 . Twenty-four hours prior to infection, astrocyte medium was changed to DMEM with 10% fetal bovine serum. Cells were infected at moi = 0.05 with SIV/17E-Fr 59 . At six hours post-infection, cells were washed five times with PBS and re-fed with fresh medium. The next day, cells were treated or not with 100 units/mL of recombinant macaque IFN-beta (PBL Assay Scientific RepoRts | 6:22629 | DOI: 10.1038/srep22629 Science, Piscataway, NJ). Cells were half re-fed twice weekly with medium containing the cytokine as appropriate. Astrocyte lysates at the specified time points were collected in mirVana lysis buffer and stored at − 80 °C until RNA purification. RNA isolation from tissue and cell culture. Thalamus tissue from the rapid Macaca nemestrina model of HIV CNS disease was obtained after long-term maintenance at − 80 C. RNA was extracted with a modified Trizol protocol with column clean-up as previously described 60 . 30-50 mg frozen tissue was placed into 1 mL Trizol (Invitrogen, Grand Island, NY) in 2 mL screw-cap tubes, and one tube of Lysing matrix D (MP Biomedicals, Solon, OH) was added. Tissue was disrupted for 2 × 30 s using a desktop bead beater with five minutes on ice interspersed. Tubes were spun to collect beads and homogenate, and 200 μl chloroform was added. Tubes were firmly capped and shaken vigorously for 1 min, then centrifuged at 12,000 × g in an Eppendorf C2415 centrifuge for 15 min at 4 °C. The aqueous phase was transferred to a new tube, with care taken not to disturb the interphase. 1.25 volumes 100% ethanol was added. Sample was applied to mirVana miRNA isolation kit columns for cleanup (Ambion/Life Technologies).
Total RNA was extracted from frozen cell culture samples using the mirVana miRNA isolation kit (Ambion/ Life Technologies), following the manufacturer's protocol. RNA for each experiment was isolated together to avoid batch effects. RNA concentration of all samples was assessed by NanoDrop, and integrity of astrocyte RNA and several additional samples was assessed by BioAnalyzer. All RNA was stored at − 80 °C.
Data analysis, statistical methods, and figures. Data processing and analysis were conducted using tools from Microsoft Excel, GraphPad Prism, and XLStat. Analysis of Variance (ANOVA) was used to calculate p values. Significance of correlations was calculated as the two-sided probability value of the Pearson correlation coefficient. | 2016-05-04T20:20:58.661Z | 2016-03-03T00:00:00.000 | {
"year": 2016,
"sha1": "2e05925929ed3ea803a2035d21c5bf86f3902739",
"oa_license": "CCBY",
"oa_url": "https://www.nature.com/articles/srep22629.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "2e05925929ed3ea803a2035d21c5bf86f3902739",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
53082532 | pes2o/s2orc | v3-fos-license | It’s going to be okay: Measuring Access to Support in Online Communities
People use online platforms to seek out support for their informational and emotional needs. Here, we ask what effect does revealing one’s gender have on receiving support. To answer this, we create (i) a new dataset and method for identifying supportive replies and (ii) new methods for inferring gender from text and name. We apply these methods to create a new massive corpus of 102M online interactions with gender-labeled users, each rated by degree of supportiveness. Our analysis shows wide-spread and consistent disparity in support: identifying as a woman is associated with higher rates of support - but also higher rates of disparagement.
Introduction
Despite substantial efforts to reduce gender disparities in online social contexts, gender gaps persist and, increasingly, negatively affect women through online harassment (Duggan, 2017). Online social platforms still serve a critical role for individuals as they seek to fill informational and emotional needs, frequently by interacting with others (Goswami et al., 2010;Chuang and Yang, 2012;Hether et al., 2016). The supportive replies of others help promote personal well-being (Mac-George et al., 2011), yet unsupportive replies can not only lead to distress but discourage online engagement altogether. Given gender disparity in the receipt of anti-social behavior, to what degree does this disparity persist in individuals' receipt of support? We answer this question, illustrated in Figure 1, by examining supportive and unsupportive message rates across millions of online interactions, using a new computational model of support. Our work is motivated by an agenda of promoting supportive online platforms where people can participate equally.
This work connects with the growing body of Comment: KatieZ22: I'm nervous about my differential calculus exam next week. My current idea is work through problems on previous exams. But yiiiikes. Reply: PizzaMagic: You can ace that test! Your plan seems smart and you have plenty of time to prepare. :) Figure 1: In this fictitious example, KatieZ22 receives a supportive reply from PizzaMagic. In choosing their names, each user has chosen a particular gender performance, signaling female and genderanonymity, respectively. In online settings, such gender performances evoke stereotypes that affect how others interact and provide access to online resources. Our study asks what effect does this gender signaling have on individuals receiving support and disparagement? computational studies of gender disparity in online behavior (e.g., Lam et al., 2011;Magno and Weber, 2014;Garimella and Mihalcea, 2016;Li et al., 2018); our work here examines this disparity along a new dimension, support, and unlike prior work, examines disparity along the full spectrum of both pro-social (supportive) and anti-social (unsupportive) behaviors. Prior works have also examined the language of support in online support forums for health-related issues (Biyani et al., 2014;De Choudhury and De, 2014;Althoff et al., 2016;De Choudhury and Kiciman, 2017), often with the aim of improving people's access. Here, we aim to study support in general, everyday interactions, drawing upon theories of how support is expressed in language (Cutrona and Suhr, 1992;Wright et al., 2003).
Our investigation provides four main contributions. First, we introduce a new task of rating the supportiveness of a message and provide an accompanying dataset of 9,032 post-reply pairs with annotations ( §2). Second, using this data, we develop a new computational model for automatically identifying supportive and unsupportive replies ( §3), using theory-based features that operationalize linguistic strategies for giving support. Third, we develop a new state-of-the-art sys-tem for classifying the gender of a username ( §4) and construct a massive dataset of over 102M postreply pairs from three online platforms ( §5), where participants are labeled by gender. Further, the text of each post is rated for its gender predictiveness, enabling studying gender performance at the name and textual levels. Finally, we apply our support classifier to our social interaction dataset to reveal wide-spread disparity on the basis of gender ( §6). Our results show that when gender is performed, female performances are associated both with higher rates of supportive comments and with higher rates of unsupportive comments, highlighting that gender disparity is not just for negative behaviors online.
The Language of Support
Individuals engage in online platforms for a variety of reasons and supportive responses to this engagement can take many forms (Shumaker and Brownell, 1984;Vaux, 1985), from informational support like advice to emotional support like expressions of sympathy. Responders may choose from different linguistic support strategies depending on the speaker and context (Cutrona and Suhr, 1992). For example, given an individual commenting on a Wikipedia talk page about their idea for adding new content, a responder may point to an additional resource they can use, whereas given an individual posting to Reddit for relationship advice, a responder may express sympathy. Our goal is to study the language and behavior of everyday supportive or unsupportive interactions as they occur on three large social platforms: Reddit, StackExchange, and Wikipedia. These platforms represent common settings people seek out to engage in discussions and ask for help. Therefore, we annotate a dataset by degrees of support and analyze how linguistic expectations of support manifest in online interactions. Data and Annotation Post and reply pairs were selected from the three platforms. Many of these interactions are short and therefore to increase diversity, annotated pairs were sampled by balancing by platform and the lengths of posts and replies seen in each.
As a social activity, support is often expressed by drawing upon other social strategies such as politeness (Feng et al., 2013). To help focus annotators' attention on support specifically and disentangle related social cues, we pair our sup- port annotation with contrastive annotation questions for three other related phenomena: agreement (with the post's message), politeness, and offensiveness. Annotators were asked to rate support on a five-point Likert scale from very unsupportive and very supportive, with analogous questions each for agreement and politeness; offensiveness was rated on a five point scale from inoffensive to very offensive. All data was annotated using CrowdFlower with detailed instructions and example replies for each level of the Likert scale. Each task presented five post-reply pairs and with detailed instructions that ask annotators to focus on rating each reply along these four dimensions. Annotators were required to pass a training phase where they had at least 70% agreement with a gold standard annotation on 10 items. After training, each task included one control question, which was used to remove annotators whose agreement with the gold standard fell below 70%.
In total, 9,032 instances were annotated by three annotators, who had a Krippendorff's α of 0.766, indicating substantial agreement on the data (Artstein and Poesio, 2008). Figure 2 shows the distribution of ratings. Support is positively correlated with agreement (r=0.71) and politeness (r=0.51), though annotators rated replies as having more politeness than support on average. Support annotation examples are shown in Table 1 Table 2: Support strategies and their presence in our data, as shown through the mean supportiveness rating (-2 to 2) for replies using that strategy and the percentage of posts in the top 25% of the most-supportive that employ the strategy. For supportiveness, posts are compared with all others not employing that strategy, with significance measured using the Mann-Whitney U test. Throughout the paper, *** denotes p<0.001, ** p<0.01, and * p<0.05. and context. Cutrona and Suhr (1992) proposed a broad taxonomy of support strategies based on inperson interactions, such as offering an appraisal of the current situation or seeking to relieve the other person of blame. We examine to what degree are these strategies employed in online, relativelyanonymous settings and whether their usage online is associated with higher perceived support.
To test this, support strategies were automatically identified using a combination of regular expressions for lexical patterns and dependency-parsed trees, together with specialized lexicons matching each strategy and rules for detecting negation. For example, suggestions were detected by identifying a second-person subject with a modal verb indicating possibility (Quirk et al., 1985, p. 219).
Many of the strategies suggested by Cutrona and Suhr (1992) for expressing support in person were also observed online; Table 2 shows the average supportiveness rating for replies containing each strategy, where supportiveness is centered to [−2, 2]. Further, their effect on perception of supportiveness, while small, is significant and positive for many. However, we do observe two notable negative trends. First informational support strategies of aiding a person by reassessing their situation (e.g., offering a new perspective) and by teaching were considered less supportive. We observed that in several cases these strategies were employed when an individual was not seeking support, in which case unrequested new information can appear condescending. Indeed, replies employing these two support strategies were still rated polite, 0.30 and 0.31 mean politeness respectively, suggesting the context in which new information is given weighs heavily on whether it is treated as supporting the individual. Support can also be conveyed implicitly through unconscious stylistic choices. Rains (2016) notes linguistic accommodation is frequently observed in supportive responses, where individuals match the function word frequency of the original communication (Bucholtz and Hall, 2005;Danescu-Niculescu-Mizil et al., 2011). Shown in the bottom of Table 2, we observe only a weak non-significant positive association between support and accommodation (as measured using Danescu-Niculescu-Mizil et al. (2011)), though the single post-reply unit of analysis limits our ability to detect long-term accommodation across multiple dialog turns. Liviatan et al. (2008) and Li and Feng (2014) found that supportive replies often to contain more emotional language, which evokes a personal connection and intensity (Spottswood et al., 2013;Braithwaite et al., 1999). Shown in the bottom of Table 2, we find a significant and positive association where more emotive posts are viewed as more supportive.
Finally, we note that a few strategies suggested by Cutrona and Suhr (1992) were not seen in our annotated dataset. Strategies of offering to participate, using physical affection, assurances of confidentiality, material and financial loans, and prayer were not seen; a broader scan of our unannotated data did find these attested but rare in practice. We attribute this rareness to the public, online nature of the interactions, in contrast to the interpersonal setting studied by Cutrona and Suhr (1992).
Computational Model of Support
Our primary objective is to measure the relationship between identity and support in online social systems. Therefore, we next develop a classifier to automatically label replies with support. Features As a social activity, the language of support draws upon multiple lexical and stylistic cues. We base on classifier on theory-inspired and datadriven features. The first set consists of the operationalized linguistic strategies for expressing support, shown in Table 2. Further, in constructing our feature set, we build upon past linguistic analyses of related social-situated language. Wellman and Wortley (1990) note that the availability of support is related to social distance, which is in part expressed linguistically through the degree of formality (Hovy, 1987;Sigley, 1997). Therefore, we include features from Pavlick and Tetreault (2016), which examined linguistic markers of formality. Advice giving is a core component of many theories of support (MacGeorge et al., 2011) and such advice is frequently wrapped in politeness language (Feng et al., 2013), e.g., hedging suggestions rather than imposing direction, which provides face-saving opportunities for the person receiving support (Clark and Schunk, 1980). Therefore, we include the feature set of Danescu-Niculescu-Mizil et al. (2013), which though focused on requests, provides many general lexical patterns for politeness.
Beyond these, we include features motivated by observational studies of support. In analyzing online support groups, Alpers et al. (2005) found that LIWC (Pennebaker and Stone, 2003) was valid as a construct for analyzing messages and compared similarly human judgments about the categories. To capture emotional language (Li and Feng, 2014;Liviatan et al., 2008), we include the NRC emotion lexicons of Mohammad and Turney (2013). Given the informational support strategy, we include lexicons from argumentation for capturing explanatory replies (Teufel, 2000). Support may be given in response to stressors, which change in nature throughout a person's lifetime (Vaux, 1985;Segrin, 2003). To potentially capture variation in the language of support based on the posting individuals, we include features known to be associated with age such as elongation and capitalization (Goswami et al., 2009;Barbieri, 2008), grammatical differences in sentence construction and length (Hovy and Søgaard, 2015), and a lexicon for age of acquisition (Kuperman et al., 2012).
Data-driven features include (1) lexical features capturing the presence of n-grams, their relative frequency, (2) grammatical features from dependency-parsed triples, which are also backed off to parts of speech, (3) word lexicons for formality, sentiment, and subjectivity, (4) style features such as word and sentence length, complexity, and use of contractions, and (5) the average word vector for the sentence.
In total, our model includes 23,903 features, the bulk of which are n-grams and dependency triples. A detailed listing of all features is provided in Supplemental §1.
One notable feature that we did not include was the presence of self-disclosure in a reply, which has been linked to high-social support as a way of conveying connection and empathy (Wright et al., 2003). While computational models for selfdisclosure have been proposed (Bak et al., 2014;De Choudhury and De, 2014), we were unable to scale these methods to the size of our analysis. Task Setup Support ratings are discretized to create a ternary classification task with labels {−1, 0, 1}, denoting unsupportive, neutral, and supportive comments. Ratings were discretized by treating all those ratings ≤-0.67 as negative and those with a rating ≥0.67 as positive.
A Random Forest classifier was trained on all 23,903 features; random forests are robust to overfitting even with large numbers of features, making them suitable for this high-dimensional feature space (Fernández-Delgado et al., 2014). Furthermore, random forests are able to learn conjunctive features, allowing us to learn how combinations of strategies are employed to yield support. As the majority of posts are neutral, we mitigate the class imbalance using SMOTE (Chawla et al., 2002) Table 3: Support classification performance training fold using the 5 nearest neighbors, taking care to avoid contamination of the test set. The classifier is implemented using Scikit-learn (Pedregosa et al., 2011) and syntactic processing was done using spaCy (Honnibal and Johnson, 2015). Word vectors are the publicly released Google-News word2vec vectors (Mikolov et al., 2013). Three works have examined related tasks where Biyani et al. (2014) and Khanpour et al. (2018) classify posts in online cancer support groups as providing informational or emotional support and classify the degree of support along these dimensions. Here, we solve a more general task that includes unsupportive comments and is in the general domain. Evaluation We compare our full model for predicting support against three models: our 14 features for detecting support strategies from Table 2, a model trained on the subset of unigram features (4,352), and a model trained on bigram features (8,897), the latter of which is known to be a strong lexical baseline (Wang and Manning, 2012). All models were tested using five-fold cross-validation with Macro-F1 for evaluation and including baselines for labeling instances at random or choosing the most frequent.
Our full model obtains substantial improvements over all baselines and models, as shown in Table 3. Further, the simple support strategy features provide a large and statically-significant improvement over the two baselines. The model using support strategy features performs similarity to the unigram model, despite having two orders of magnitude fewer features. A follow-up analysis on cross-platform performance, described in Supplemental §1.2, showed that while within-platform performance was relatively high (0.54 Macro F1 for Reddit and 0.53 for Wikipedia), performance for the more technical StackExchange site was lower both within (0.44) and across (0.40 when trained on Reddit and 0.42 when on Wikipedia). Examining our full model's most important features showed that the two support strategies for validation and compliments (cf. Table 2) were the most important features, followed closely by lexicons for emotion: Anger in LIWC, Disgust in NRC, and the positive sentiment in Liu et al. (2005), all of which were motivated by theory. These results confirm that our theory-inspired features are both salient for supportiveness and effective as features.
Inferring Gender
As a part of interacting, individuals present a view of themselves as an interlocutor, revealing aspects about themselves such as gender through explicit means (Marwick, 2013; Allen and Wiles, 2016), e.g., profile pictures, or through implicit-and potentially unconscious-cues such as stylistic choices in language (e.g., Eckert and McConnell-Ginet, 2003;Bamman et al., 2014). In the relatively anonymous and deindividuated online setting, these identity cues can have a profound impact on how other perceive and interact with them (e.g., Mickelson et al., 1995;Herring, 2003;Ammari et al., 2014;Megarry, 2014) and these minimal gender cues give rise to full-fledged social stereotypes and, potentially, the negative behavior that comes when treating someone as a stereotype (Kiesler et al., 1984;Lea and Spears, 1991;Postmes et al., 1998;Wang et al., 2009). Here, we develop methods for inferring gender from two signals: (1) names that users chose; and (2) implicit cues conveyed by linguistic features.
Gender from Names
Prior work has developed models for inferring gender from username alone (e.g., Tang et al., 2011;Liu and Ruths, 2013;Jaech and Ostendorf, 2015;Knowles et al., 2016). Here, we develop a new character-based neural model that incorporates rich gender-labeled username information for identifying additional gender-salience cues in usernames from roles and attributes, e.g., SuperDad1 or AspiringActress99. Data Individuals convey their gender in multiple ways beyond using gender-associated names. Therefore, to capture this variety, we collect usernames from two online platforms where users have self-declared their perceived gender. First, Twitter usernames and screen names were collected from a 10% sample from 2014 to 2017. Here, we identify usernames whose biography contains an explicit mention of their gender, e.g., by stating a gendered role "mom to two kids" or spec-ifying pronoun preferences "he/him/his." Gendered profiles were collected for 4,900,250 individuals using a selection of lexical patterns with aggressive filtering to remove false positives. Second, we collect 283,427 usernames from Reddit identified through self-declarations of gender in /R/RELATIONSHIPS, e.g., "I [23F] need to talk to my boyfriend [27M]", and 84,068 usernames where the user has chosen a gender-indicating flair (a visual icon displayed within the subreddit). These two sources provide much-needed variation for gender in usernames beyond those mirroring full names. Model Given a username, we infer gender using a character-based encoder consisting of three stacked LSTM networks (Hochreiter and Schmidhuber, 1997). Following platform restrictions on usernames, character sequences are restricted to being in ascii range and are embedded into 16 dimensional vectors as input. Adopting best practices (Ioffe and Szegedy, 2015), batch normalization is applied prior to the dense layer used to compute the gender prediction. LSTMs were sized at 256 after limited hyperparameter tuning on development data. We optimize with Adam (Kingma and Ba, 2014) with a learning rate of 0.002. Training and Evaluation All data is partitioned into 80% train, 10% development, and 10% test splits. As some usernames are repeated in different communities, we keep only one unique instance prior to partitioning to avoid leakage between partitions.
Training mini-batches were balanced for both genders, which yielded better performance in tests on the development data. We compare the performance of our model on the test set against two current state-of-art systems available off the shelf for inferring gender from usernames, demographer (Knowles et al., 2016) and Jaech and Ostendorf (2015). Demographer is trained on names from the Social Security Administration and the method of Jaech and Ostendorf (2015) is trained on usernames from OkCupid and uses 3.5M Snapchat usernames for self-learning to improve accuracy. Table 4, our model outperforms both systems by substantial margins for both Twitter and Reddit data. In tests on data from both papers reported in Supplemental §3, our model also outperforms their systems. High accuracy is not expected for these models in most domains, as many usernames do not signal gender.
Gender from Text
Gender can also manifest through more subtle, stylistic cues (e.g., Schnoebelen, 2012;Flekova and Gurevych, 2013;Bamman et al., 2014;Volkova et al., 2015;Garimella and Mihalcea, 2016;Carpenter et al., 2016). Thus, even when a person chooses a neutral username, their linguistic style may reveal their gender. Therefore, we construct a regression model to infer the degree to which either gender is expressed through text. Data and Model Gender-labeled post data was constructed using held-out data from our three platforms where posts were authored by a user with a high-confidence gender prediction. Posts were randomly sampled across forums (e.g., subreddits) from the held-out data to achieve gender parity with 555K posts for Wikipedia, and 58K for StackExchange; Reddit was subsampled to 1M posts total due to its size. Features were selected by drawing upon prior work: (i) stylistic features like punctuation and number frequencies, casing, word length and (ii) content features including n-grams, sentiment, and specialized lexicons like LIWC. A full listing of features is reported in Supplemental §1.
Following prior work (Bamman et al., 2014), a logistic regression model was trained for each platform using L2 regression; we adopt separate models for each to better adapt to any platformspecific gender variation. Evaluation Models were evaluated using AUC with five-fold cross validation, with 0.661 AUC for StackExchange, 0.700 for Wikipedia, and 0.661 for Reddit. The models perform substantially better than random choice (0.5) for the challenging task of inferring gender from a single post, as many posts contain no signal of gender. Additional analyses are reported in Supplemental §1.2.
Gender-Salient Interaction Data
To quantify the social support people receive online, we examine communications from three major online communities: Reddit, StackExchange, and Wikipedia. We refer to a communication between individuals as a post with a reply, defining these for each platform next.
Reddit Reddit data was selected using a longitudinal sample of one month (July) per year, from 2006 to 2017, and a continuous sample of one full year's data in 2017. Our initial data consists of the top 10,000 subreddits ordered by the total number of posts in the data. From these communities we restrict our analysis to a comment and its first reply, which reduces confounds from multiparty communication. These post-reply pairs were further filtered to remove non-English posts using Google CLD2 (McCandless, 2010), yielding 434.29M candidate communications. StackExchange StackExchange (SE) contains substantial social interaction in the comment to posts and replies (Ahn et al., 2013;Danescu-Niculescu-Mizil et al., 2013). These communications often expand beyond the immediate topic. Directed communication within these comments is frequently signaled using an explicit mention starting with an " ," which we use to identify pairs. In total, we collected post-reply pairs from the full history of all StackExchange, yielding 3.16M pairs across 162 sites. Wikipedia Wikipedia features an active social component in its talk pages, with more personal communication-or even personal attacksduring debates around appropriateness or suggested changes (Bender et al., 2011). Similar to Reddit, we construct post-reply pairs by identifying each comment and its first response on a talk page, yielding 26.7M pairs from 387K talk pages. Assigning Gender All post-reply pairs were labeled using our post classifier ( §4.1). Posts with high-confidence gender predictions (softmax probability > 0.9 or < 0.1) were labeled with the predicted gender. To contrast the effects of having a gendered name, we construct a complementary dataset where the posting user's username is effectively gender neutral, e.g., user1209; these neutral names are chosen from those with near-chance probability in the output softmax 0.45 < p < 0.55. The relative counts of high-confidence and neutral gender names in each platform are shown in Table 5, along with examples in Table 6.
Restricting the dataset to pairs where we have a salient identity, our final dataset for analysis consists of 49.58M, 0.72M, and 3.69M pairs for gender in Reddit,StackExchange,and Wikipedia;and 46.19M,201.7K, and 1.60M for neutral in each, respectively. Where possible, we also record any high-salience identities for replying users.
Gender and Support
The gender cues provided through computer mediated communication provide enough information that a person will fill in the result with a stereotype (Lea and Spears, 1991;Spears and Lea, 1992). What effect might this stereotyping have for access to support? While establishing full causality for an answer is infeasible in our current observational study, we take the first step by quantifying whether disparity in support exists and examine what contextual factors may affect support giving. Using our classifier, we label the 102M post-reply pairs from our dataset ( §5), which includes both high-confidence and gender-neutral users.
the use of a gender-conveying name is associated with both higher rates of supportive comments and unsupportive comments. Our results agree with those from the small scale study of Feng et al. (2013) who found that accounts with human pictures and person-sounding usernames receive higher social support. Indeed, while several studies have touted the benefits of anonymity online for discussing sensitive topics (Campbell and Wright, 2002;Wright, 2002a,b), our results suggest that selecting a gender neutral name may lead to lower support overall. Our findings also reinforce the observation that the personal-anonymity online does not lead to equal support due to cues about identity (Postmes and Spears, 2002).
Second, the rates of supportive and unsupportive comments are significantly associated with both kinds of gender performances (i.e., names and writing style). These results suggest that online audiences are sensitive to both kinds of overt and implicit gender displays and that even innocuous choices such as gendered usernames can shape our online interactions.
Third, when gender is performed in together name and writing, female performances are consistently associated across all three platforms with higher rates of receiving supportive replies and unsupportive replies. Note that this trend is seen in the cumulative effect on support after combining coefficients for the interactions term with the coefficients for writing and name. We illustrate this cumulative effect for two types of gender performances in Reddit, shown as separate axes in Figure 3. Indeed, when a user has a gendered username, the cumulative effect of male writing performance is consistently associated with fewer supportive replies. In small-scale interpersonal studies, Abbey et al. (1991) and Barbee et al. (1993) portive comments; however, we did not observe this disparity in our online setting. Does the replier's gender matter? Mickelson et al. (1995) note that men and women differ in how they receive support, with the gender composition of the interacting pair driving the kind of supportive behavior. Here, we examine whether men and women differ in the rates they give support to one another, using gendered names as a proxy for identity. Because only Reddit has sufficient data, we construct a mixed-effect regression model for Reddit using the 4.5M post-reply pairs where the replier has a high-confidence or neutral gender and include the replier's gender as a factor with interactions for the poster. The results shown in Table 8 reveal two main conclusions. First, men and women give supportive comments at different rates, with women being far more likely to leave supportive replies and less likely to leave unsupportive replies. Second, the interaction terms show that there is minimal dyadic interaction between the gender identities of the poster and replier with SUP. UNSUP. intercept −2.391 * * * −3.107 * * * P:♀name 0.561 * * * 0.288 * * * P:ǿname 0.450 * * * 0.330 * * * P:♀ 1.263 * * * 0.290 * * * R:♀name 0.249 * * * −0.153 * * * R:ǿname −0.057 * * * −0.036 * * P:♀name ∧ P:♀ 0.914 * * * 0.212 * * P:ǿname ∧ P:♀ 0.082 0.131 P:♀name ∧ R:♀name −0.103 * * * −0. respect to rates of giving supportive or unsupportive comments. We only observe significant interactions indicating (1) replying users with female names are less likely to leave supportive replies to posting users with female names and (2) replying users with male names are i) more likely to leave supportive replies to other users with male names, ii) less likely to leave supportive comments if the writing appears more female, iii) more likely to leave unsupportive comments if the posters name is female, and iv) less likely to leave unsupportive comments if the writing appears more female.
Limitations The observations of our study should still be viewed within its practical limitations, of which we note two. First, in examining the content of replies, we do not control for potential direct or indirect requests for help in text that may ultimately affect the rates of support. This issue could be a potential confound, as the cultural norms for masculinity often promote self reliance (Addis and Mahalik, 2003), ultimately leading to gendered differences in requests. Second, this observational study cannot establish causality between gender displays and support; while the disparity is real, exogenous factors could potentially explain the disparity without finding gender displays as a cause, though the mixed effects still control for some contextual variability in the different support frequencies across communities. In spite of these limitations, we view this work as an important first step for demonstrating gender disparity in support-both positive and negative-and inviting future work to establish a causal explanation.
Ethical Considerations
The use of gender as a variable in NLP requires that we also discuss ethical considerations resulting from this work, as it directly relates to identity and the dignity of persons being studied. Following the guidelines of Larson (2017) for using gender in NLP, our use of gender is intentional and central to this study on gender disparities in received support. We base our notion of gender as one of linguistic performance (DeFrancisco et al., 2013), in which individuals adapt their style and name to emphasize or de-emphasize certain aspects of their gender identity (Eckert, 2008). Accordingly, we have opted represent gender performance along a graded scale, though we recognize that this representation does not capture nonbinary gender identities. The gender inference methods introduced here raise ethical considerations as they ultimately enable automatic identification of gender for any person on the basis of name or writing (Hamidi et al., 2018). Such technology could be used to unfairly identify and target persons of either gender for malicious behavior or may harm through misgendering individuals. Ultimately, we decided that such risk was acceptable given the positive impact of our study on revealing gender disparity. We hope to also use our method to better support privacy-preserving behavior (Allen and Wiles, 2016; Reddy and Knight, 2016) by helping individuals identify and change names or statements that would indicate a particular gender. Further, we hope that when used in combination with our support classifier and a larger context of gendered interactions (Voigt et al., 2018), these technologies can identify healthy communities that are supportive of all people.
Conclusion
Individuals use social media to support their informational and emotional needs. Our study has shown wide-spread disparity in the levels of support individuals receive on the basis of their perceived gender. Our results were made possible through the development of a new massive 102M post-reply dataset tagged with high-salience and neutral gender and the introduction of a new task, annotated dataset, and model for classifying supportive messages. All data, code, and annotation guidelines are publicly released at https: | 2018-10-29T13:07:22.260Z | 2018-01-01T00:00:00.000 | {
"year": 2018,
"sha1": "348b7b6d2bd8012f98791ff22aaec509df0e8e84",
"oa_license": "CCBY",
"oa_url": "https://www.aclweb.org/anthology/D18-1004.pdf",
"oa_status": "HYBRID",
"pdf_src": "ACL",
"pdf_hash": "348b7b6d2bd8012f98791ff22aaec509df0e8e84",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Computer Science"
]
} |
231944288 | pes2o/s2orc | v3-fos-license | Protocol for a gallbladder cancer registry study in China: the Chinese Research Group of Gallbladder Cancer (CRGGC) study
Introduction Gallbladder cancer (GBC), the sixth most common gastrointestinal tract cancer, poses a significant disease burden in China. However, no national representative data are available on the clinical characteristics, treatment and prognosis of GBC in the Chinese population. Methods and analysis The Chinese Research Group of Gallbladder Cancer (CRGGC) study is a multicentre retrospective registry cohort study. Clinically diagnosed patient with GBC will be identified from 1 January 2008 to December, 2019, by reviewing the electronic medical records from 76 tertiary and secondary hospitals across 28 provinces in China. Patients with pathological and radiological diagnoses of malignancy, including cancer in situ, from the gallbladder and cystic duct are eligible, according to the National Comprehensive Cancer Network 2019 guidelines. Patients will be excluded if GBC is the secondary diagnosis in the discharge summary. The demographic characteristics, medical history, physical examination results, surgery information, pathological data, laboratory examination results and radiology reports will be collected in a standardised case report form. By May 2021, approximately 6000 patient with GBC will be included. The clinical follow-up data will be updated until 5 years after the last admission for GBC of each patient. The study aimed (1) to depict the clinical characteristics, including demographics, pathology, treatment and prognosis of patient with GBC in China; (2) to evaluate the adherence to clinical guidelines of GBC and (3) to improve clinical practice for diagnosing and treating GBC and provide references for policy-makers. Ethics and dissemination The protocol of the CRGGC has been approved by the Committee for Ethics of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (SHEC-C-2019–085). All results of this study will be published in peer-reviewed journals and presented at relevant conferences. Trial registration number NCT04140552, Pre-results.
INTRODUCTION
Gallbladder cancer (GBC) is the most common type of biliary tract cancer [1][2][3] and one of the most lethal malignancies, with a 5-year Strengths and limitations of this study ► The Chinese Research Group of Gallbladder Cancer study is the first large-scale registry cohort study of gallbladder cancer (GBC) in China, covering 76 tertiary and secondary hospitals across 28 provinces. ► A standardised quality control and data management plan was designed to ensure the accuracy and reliability of the data. ► The electronic medical record systems are not consistent across hospitals, which may introduce variance in data recording and result in difficulty in systematic data formatting and integration. ► This is a retrospective study using convenience sampling. The study population may not be completely representative of patients with GBC in China. ► There is a lack of biospecimens from involved patients. The survival data are not validated through Chinese death registry.
Open access survival rate of 5%-15%. 1 4 5 Much effort has been made to optimise the treatment of GBC; however, the prognosis remains dismal 4 6 , and the quality of current evidence for GBC is still far from perfect. Due to its relatively low incidence, clinical trials on GBC are difficult to conduct. Most recommendations and guidelines for GBC from the National Comprehensive Cancer Network (NCCN; 2019 V.4) and American Joint Committee on Cancer (AJCC; eighth version) were derived from evidence of moderate quality. 7 8 Most of these studies were single-centre studies with limited sample sizes and generally no more than 300 cases, [9][10][11] which might introduce systematic bias into the conclusion.
On the other hand, common-used coding systems addressed little on GBC. The nomenclature of GBC in the literature is inconsistent. GBC defined by the AJCC eighth staging manual is a primary cancer in the gallbladder and cystic duct (C23.9 and part of C24.0; ICD-O-3 codes). 8 However, many epidemiological studies refer to 'GBC' as 'GBC and extrahepatic cholangiocarcinoma (ECC; C23.9 and C24.0)', leading to confusion in its incidence, mortality and other epidemiological features. 12 13 In addition, cystic duct cancer is undistinguishable from ECC in most cancer registry studies, which means that this specific subset of patients is likely to be omitted. 14 Moreover, a commonly used coding system for surgery, the Facility Oncology Registry Data System, classifies GBC as 'all other sites', making it unlikely to define the extent of surgery and distinguish patients who undergo re-resection after GBC is incidentally found. 15 Regarding regional lymph nodes, the Collaborative Stage (V.0204) system defines coeliac, superior mesenteric and para-aortic lymph nodes as regional nodes, which is not consistent with either the AJCC seventh or AJCC eighth definition. 16 The coding problems in both patient identification and site-specific variables might lead to less stringent interpretation of the conclusions.
China is a high-GBC risk country, but little evidence has been based on the Chinese population. 12 Data from GLOBOCAN show that, taking GBC and ECC together, the number of annual new cases in China accounts for 24.7% of new cases worldwide. 13 Currently, the largest retrospective study of GBC in China was conducted by Zou and Zhang, 17 including 3922 patients from 116 hospitals in 28 provinces of China during 1986-1998. This study described the demographic characteristics of GBC in China, without further data on detailed staging, treatment and prognosis information. Another study of 2379 patient with GBC from five northwestern provinces during 2009-2013 18 reported that 55.1% of patient with GBC had advanced-stage tumours. Other reports were mainly single-centre studies with limited sample sizes. 19 20 The critical characteristics in the diagnosis, treatment and prognosis of GBC in China are unknown.
Therefore, this study aimed to design a GBC cohort, the Chinese Research Group of Gallbladder Cancer (CRGGC) study, (1) to comprehensively evaluate the clinical characteristics, including demographics, pathology, treatment and prognosis of patient with GBC in China; (2) to evaluate adherence to clinical guidelines of GBC and (3) to improve clinical practice and guidelines for GBC and provide references for policy-makers.
Registry design
The CRGGC study is a multicentre retrospective registry cohort study. The project was launched by the Shanghai Key Laboratory of Biliary Tract Disease Research, with collaborators from 76 tertiary and secondary hospitals across 28 provinces in China (until 8 March 2020; see online supplemental file 1). We will review the electronic medical records (EMRs) of all diagnosed patient with GBC from 1 January 2008 to December 2019, and extract the related clinical and treatment information. The clinical follow-up data will be updated until 5 years after the last admission of each patient with GBC.
Patient enrolment
Patients will be identified with various search strategies: (1) ICD-10 code equals C23.9, or C24.0 with 'cystic duct'; (2) discharge diagnosis includes 'gallbladder cancer' (search strategy in Chinese: (("胆囊") AND (" 癌" OR "恶性肿瘤" OR "占位"), which means "gallbladder"/"cystic duct" AND ("cancer" OR "malignancy" OR "space-occupying lesion")); and (3) pathological reports include "gallbladder cancer". All three search strategies will be applied in each centre. The results will be merged for subsequent exclusion. These search strategies are designed to be redundant because some search strategies may not be applicable in specific EMR systems and in specific periods.
All identified admissions to the hospital will be manually filtered according to the diagnostic criteria of the NCCN 2019 V.4 guidelines for hepatobiliary cancer. 7 Patients with a pathological or radiological diagnosis of malignancy, including cancer in situ, from the gallbladder and cystic duct are eligible. Patients will be excluded if GBC is the secondary diagnosis in the discharge summary because patients admitted for other diseases are likely to have obscure cancer traits.
The study will include patients diagnosed before December 2019. According to our preliminary estimation, more than 6000 cases will meet our inclusion criteria. We expect to finish data collection by May 2021. After finishing enrolment of a short-term target of 2000 cases, a primary analysis will be performed. The follow-up will be updated until 5 years after the admission of each patient. More centres are expected to participate in the CRGGC study; thus, the collaborator list may be expanded.
Clinical outcomes and follow-up
The main outcome is the 5-year overall survival (OS). OS is defined as the duration from the date of first diagnosis to the date of death, and it is censored at the date of the last follow-up when the patients are alive. We will also include the following outcomes: progression-free survival (PFS), defined as the duration between the date of first diagnosis and the date of recurrence, and censored at the date of the last follow-up when the patients have no evidence of recurrence; cancer-specific survival defined as the duration between the date of first diagnosis and the date of cancer-caused death, and censored at the date of the last follow-up when the patients are alive or died from other causes; 3-year OS; and 90-day mortality (for patients who undergo surgery), which will be used to indicate perioperative mortality. Clinical follow-up is defined as the routine practice of hospitals of collecting patient data on treatment, tumour recurrence and patient survival, either by outpatient/inpatient records or telephone. We require hospitals to equip such a system and at least one follow-up per year to join our collaboration. Based on these data, we will update patients' follow-up statuses every 12 months. The data being collected from clinical follow-up will include date of recurrence, date of death, date of last contact, whether reresection is performed if the malignancy is found incidentally, and whether the patient receives adjuvant therapy.
Data collection
The workflow of data collection and quality control is shown in figure 1. Before data collection, a group of hepatobiliary specialists designed a structured case report form, aiming to delineate features of patients with GBC and answer corresponding clinical questions. The case report form includes the following information: demographic characteristics, medical history, physical examination results, surgery information, pathological data, laboratory examination results and radiology reports. We have compiled a codebook to standardise the definition of each variable. The data centre will be responsible for training doctors to collect data. Data collection will be carried out by using EpiData (V.4.6.0.2, EpiData Association, Denmark).
Automated logic checks will be applied to prevent outof-range values. Duplicated entry will be required. If any discrepancies are found, a third specialist will be brought in for discussion and make a final decision.
After data entry and quality control in each centre, the data will be anonymised and transferred to the servers in the data centre. The data centre is located at Shanghai Key Laboratory of Biliary Tract Disease Research, which is equipped with data servers and essential firewall and backup systems. The data centre will be responsible for quality assessment, storage, sharing and analysis of the data. A group of researchers in the data centre will manage the database.
The data manager will assess the quality of the data after transfer to the data centre. The assessment is based on the structure of missing data and a comparison to baseline data. First, we will apply a grading system, where variables are classified into essential, important and normal importance. Based on the proportion of missing values in each category, the entries will be graded as level A, B, C or D in quality. Entries of category D quality will be normally excluded from analysis. Second, outliers and inconsistent data will be identified. Third, we will compare baseline characteristics of the new data to previous data, with indicators including sex ratio, mean age, proportion of tumor, node, and metastasis (TNM) stage, and 5-year OS. We will apply χ 2 test, t-test and log-rank test between the two datasets. When a significant difference is found, the data manager will analyse and record suspicious data. The data manager will inquire about the data in question Open access with the data source and ask for confirmation. The desensitised data will be accessible to collaborators after the completion of the database. A research proposal to the CRGGC Scientific Committee will be essential for analysis of the data.
Demographic data and medical history
The EMR data for each patient will be collected for every hospital visit from 1 January 2008. The baseline data will be retrieved, including the following aspects: (1) demographics: age at diagnosis, sex, race and date of diagnosis; (2) medical history: emergency operation, chief complaint, endoscopic retrograde cannulation of the pancreas (ERCP) performed within 30 days before surgery, percutaneous transhepatic cholangial drainage (PTCD) performed within 30 days before surgery, neoadjuvant therapy and method of diagnosis (pathology, radiology or other); (3) medical history: history of gallstone, history of gallbladder polyps, history of other malignancies, hypertension, diabetes mellitus and other comorbidities; (4) social and personal history: marital status, smoking history and use of alcohol; and (5) other aspects: weight, height, family history and total expenditure.
Surgery information
1. The preoperative and intraoperative diagnoses will be recorded. A diagnosis of 'GBC', 'gallbladder tumour', or 'space-occupying lesion in gallbladder' is regarded as the detection of malignancy. 2. Regional lymphadenectomy requires the resection of hilar nodes. 8 Further clearance of lymph nodes is classified as extended lymphadenectomy. 3. The extent of lymphadenectomy includes the cystic duct, common bile duct, portal vein, hepatic artery, common hepatic artery, postsuperior pancreatic, coeliac, superior mesenteric, suprapyloric, left gastric artery and paraaortic lymph nodes. 8 21 22 4. Combined hepatectomy is classified as no hepatectomy, liver wedge resection/partial hepatectomy, IVb+V segmentectomy, hemihepatectomy, extent more than hemihepatectomy, radiofrequency ablation and hepatectomy for other reasons. 5. If the malignancy is diagnosed after surgery, further treatment information may not be available (the patient may turn to a second hospital for reresection). Patients in this case will be categorised as 'simple cholecystectomy performed; further treatment not available'. If reresection is available, its operative reports will be reviewed as previously mentioned. 6. ERCP, PTCD and transarterial chemoembolisation are not defined as surgery but as supportive treatment. 7. Palliative surgery is defined as resection of the primary tumour, reconstruction of the digestive tract, or both when there is evidence of distant metastasis or unresectable tumour.
Other surgery-related variables include date of surgery, laparoscopic surgery, combined bile duct resection, tumour positioned on the hepatic or peritoneal side, perivascular invasion, perforation, porcelain gallbladder, duration of surgery, intraoperative blood loss and American Society of Anesthesiologists score.
Pathological data
Pathological data will be recorded, including size of the tumour (in three dimensions), resection margin, tumour positioned on the hepatic or peritoneal side, tumour positioned on the fundus, body, neck or cystic duct, depth of invasion (carcinoma in situ or lamina propria, muscularis, perimuscular connective tissue, full layer, serosa, adjacent organ or major vascular invasion 8 ), liver invasion, number of nodes examined, positive lymph nodes, number of hilar nodes examined, positive hilar lymph nodes, region of positive nodes, region of nodes examined (with codes the same as those used for the region of lymphadenectomy in surgery), grade, histology type (using ICD-O-3 codes), 23 microvascular invasion and perineural invasion. Tumours will be staged according to the AJCC eighth staging manual according to pathological reports derived from the aforementioned variables. Notably, the description of 'invasion of full layer' for depth of invasion is not suggested in the AJCC eighth manual but is commonly used in China.
Laboratory examination
Laboratory examination results for patients will be collected with the date of examination. Indicators of interest include the following: (1) routine blood tests: white cell count, haemoglobin and platelet count; (2) liver function tests: total bilirubin, direct bilirubin, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transferase; (3) renal function tests: blood urea nitrogen and creatine; (4) lipid indicators: triglycerides and total cholesterol; (5) inflammation indicators: C reactive protein and lactic dehydrogenase; (6) coagulation indicators: international normalised ratio, prothrombin time, activated partial thromboplastin time and fibrinogen; (7) tumour markers: carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 125 and alpha fetoprotein; and (8) other tests: blood type and hepatitis B test. The test method and normal range of each indicator may vary across hospitals. Thus, we will first uniform the units of each indicator according to the first enrolled hospital; then, based on the first enrolled hospital, we will normalise each result of laboratory examination by its normal range across different hospitals.
Radiology reports
Radiological reports will be collected with the date of examination. The following indicators will be collected: the type of examination (ultrasound, CT, MRI and/ or other types) and the conclusion of the examination (inflammation, polyp, tumour, gallstone and/or others).
Statistical analysis
The continuous variables will be described visually by histogram and summarised as mean±SD or median Open access (IQR), for normal-distributed and skewed-distributed values. The discrete variables will be summarised as frequency (percentage). The proportion of missing data will be described, and an indicator of missing will be deployed for analysis. Differences with a two-sided p<0.05 are considered as statistically significant.
We will calculate the average number of GBC diagnosed per year in each hospital, because the volume for patients with cancer showed impact on patients' characteristics, treatment modalities and prognosis. 24 The distribution of hospitals' average patients with GBC per year will be described in bar plot; 1-2 cut-off points will be determined by inspecting the pattern to classify hospitals into low volume and high volume. Correlation between hospital volume and patients' characteristics will be shown by either Pearson's R, Spearman's r, or χ 2 , whichever appropriate. Potential correlated variables includes (but not limited to) sex, age at diagnosis, TNM stage, gallstone, surgery type and adjuvant therapy.
Time trends for age, sex, TNM stage, surgery type, adjuvant therapy and diagnosis time will be shown by scatter plot fitted by linear or locally estimated scatterplot smoothing, whichever appropriate.
The median survival time and lost to follow-up rate will be described. The Kaplan-Meier method will be applied to assess the survival of patients, stratified by TNM stage and by whether surgery performed. Cox proportional hazards regression will be applied to evaluate predictors of prognosis, including (but not limited to) age, sex, T stage, N stage, M stage, adjuvant therapy, extent of resection, gallstone, resection margin, histological grade, perineural invasion and microvascular invasion.
As proposed in both NCCN and Chinese Medical Association guidelines, 7 25 GBC more advance than T1b requires resection of liver bed and regional lymphadenectomy. Moreover, patients with nodal metastasis are recommended to take chemotherapy. These three indicators will be described to evaluate the adherence to clinical guidelines of GBC. Also, their significance in prognosis will be tested by survival analysis.
Patient and public involvement
Patients or the public involvement are not in the plans of this research.
Ethics and dissemination
The protocol of the CRGGC has been approved by the Committee for Ethics of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (SHEC-C-2019-085). All results of this study will be published in peer-reviewed journals and presented at relevant conferences.
DISCUSSION
The CRGGC study is a large multicentre registry cohort study to evaluate the clinical presentation, treatment and prognosis of patients with GBC in China. The focus of CRGGC is to (1) describe the status quo of treatment and survival of patient with GBC in China and (2) improve the standardised treatment of GBC.
On reviewing the published studies on GBC, we found a lack of large observational studies on GBC in China focusing on its clinical features and prognosis. Moreover, international studies on GBC were limited by small sample sizes and inconsistent coding systems for GBC. Our data will establish a collaborative platform for GBC research, providing valuable data from China.
GBC is a relatively rare but lethal malignancy, making it important to address the standardisation of its primary care, treatment and post-treatment follow-up. Researchers have shown unsatisfactory adherence to clinical guidelines. Radical cholecystectomy was recommended for T1b GBC by the NCCN guidelines nearly 10 years ago; however, only 50% of T1b patientswith GBC in the USA received radical cholecystectomy. 14 Bergquist et al 26 reported that from 2004 to 2012, only 28.2% of patients with GBC with positive nodal disease received adjuvant chemotherapy in the National Cancer Database of the USA even though this was the recommended treatment in the NCCN guidelines. Knowing the current situation is essential for future improvement; however, no data have been reported on GBC treatment in China. Well-designed observational studies in China will help to point out weakness in clinical practice and, at the same time, summarise valuable clinical experience in the treatment of GBC and pave the way for further standardised treatment.
GBC cases in China account for nearly 1/4 of cases worldwide; thus, GBC poses a significant disease burden in China. However, few clinical studies of the diagnosis and treatment of GBC have been performed in China, making this significant population under-represented. By launching the CRGGC study, we also expect to boost collaborations among Chinese researchers. We hope this collaboration could induce further translational research and clinical trials in China, providing essential evidence on GBC treatment.
There are several limitations and potential biases in our study design. (1) The retrospective nature is inevitably related to information bias and heterogeneity in the data recording. This will cause difficulty in the standardisation of data and a relatively large proportion of missing data. To overcome such bias, we composed and continue to update a codebook for standardisation of each variable. Researchers responsible for data entry are trained and qualified at the data centre. The missing data are analysed to determine potential bias.
(2) This is a retrospective study using convenience sampling. Thus, the cohort may not be completely representative of patient with GBC in China. However, we attempt to include centres in every province in China. Moreover, most patients with cancer in China are treated in tertiary hospitals. (3) Biospecimens of the involved patients are not collected. Future collaboration on this issue will be considered. (4) As patients with incidental GBC may turn to other hospitals for reresection, resulting in incomplete treatment information. We addressed this problem by defining these patients separately to aid further sensitivity analysis. (5) Currently, we have not made collaboration with Chinese death registry, thus, part of the death information may be lost and the follow-up data might be biased due to lack of validation. On the one hand, the CRGGC study actively seek cooperation with relevant registries; on the other hand, we require collaborated hospitals to equip clinical follow-up system, compare prognosis data in each hospital to identify systematic bias, and update follow-up data yearly. | 2021-02-18T06:17:04.053Z | 2021-02-01T00:00:00.000 | {
"year": 2021,
"sha1": "abfd0385e1f846a188d0d70ac3ebde58203861fd",
"oa_license": "CCBYNC",
"oa_url": "https://bmjopen.bmj.com/content/bmjopen/11/2/e038634.full.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "e142f83795fbf30b469cd41dafa2d4fc49878151",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
261094227 | pes2o/s2orc | v3-fos-license | Normative range of various serum hormonal parameters among Indian women of reproductive age: ICMR-PCOS task force study outcome
Summary Background The hormonal profile varies considerably with age, gender, ethnicity, diet or physiological state of an individual. Limited population-specific studies have studied the variations in hormonal parameters among apparently healthy women. We aimed to analyse the biological reference interval for various hormonal parameters in the reproductive-aged healthy Indian women. Methods Out of 3877 participants that were clinically evaluated, 1441 participants were subjected to laboratory investigations. All participants underwent a detailed clinical, biochemical and hormonal profiling. The hormone analysis was carried out at a single centre using a uniform methodology. Among the participants evaluated for biochemical and hormonal parameters, participants that presented any abnormal profile or had incomplete investigations (n = 593) were excluded for further analysis. Findings The mean age (±SD) of the participants retained in the final analysis (n = 848) was 29.9 (±6.3) years. In the present study, the biological reference interval (2.5th–97.5th centile) observed were: serum T4: μg/dL (5.23–12.31), TSH: μg/mL (0.52–4.16) and serum prolactin: ng/mL (5.13–37.35), LH: mIU/mL (2.75–20.68), FSH: mIU/mL 2.59–15.12), serum total testosterone: ng/mL (0.06–0.68), fasting insulin: mIU/mL (1.92–39.72), morning cortisol: μg/dL (4.71–19.64), DHEAS:μg/dL (50.61–342.6) and SHBG: nmol/L (21.37–117.54). Unlike T4, TSH, LH, and E2, the biological reference interval for prolactin, FSH, testosterone, C-peptide insulin and DHEAS varied when the participants were stratified by age (p < 0.05). The comparative analysis showed marginal differences in the normative ranges for the hormones analysed among different populations. Interpretation Our first large composite data on hormonal measures will benefit future endeavours to define biological reference intervals in reproductive-aged Indian women. Funding The study was financially supported by the grant-in-aid from 10.13039/501100001411ICMR vide file No:5/7/13337/2015-RBMH.
Introduction
Women's health requirements are dynamic and pulsatile not only to their growing age but to their sexual and reproductive functions as well. 1 The reproductive phase is the most critical period in a woman's life that alters the physiology of the body, and wherein any dysregulation can lead to serious health consequences. Menstruation-related illnesses and uterine or ovarian diseases are the specific physiological alterations in women throughout this stage of development. Although, the physiological changes that take place during the reproductive phase usually follow a uniform order, the pace of these changes varies depending on external factors (e.g. food habits, lifestyle, etc). 2 The clinical significance of female reproductive physiology is relevant for a variety of clinical problems, including menopause, pregnancy, infertility, and adolescent entrance into childbearing years.
A panel of blood tests including biochemical and hormonal profiles, routinely prescribed by healthcare professionals, are critical in representing the normal functioning of the body. Of late, with the recent advances in quantitative estimation approaches, hormone profiling is in a critical transition period. 3,4 While the precision in estimation has significantly increased in the recent past, the accuracy of biological reference intervals employed, not only depends upon the assay type but on other indicators as well. 5 Moreover, the hormonal parameters fluctuate significantly depending upon the individual's age, physiological state of the body, gender, ethnicity, nutritional status, lifestyle, and use of medications. Among these factors, age and gender are two pivotal drivers of variations in hormonal parameters. Besides, the biological reference intervals of ready-touse kits provided by different vendors lack comprehensive information on such issues. 5 The biological reference interval for hormones is critical in reproductive endocrinology, particularly in clinical assessments of the presence of any other reproductive or metabolic anomalies in women. A functional assessment of the hypothalamus-pituitary-gonadal axis requires measurements of the hormones, serum testosterone, prolactin, cortisol, estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), dehydroepiandrosterone sulphate (DHEAS) and sex hormone binding globulin (SHBG). The generation of biological reference intervals for hormonal parameters in a country is essential for enhancing the standard of care, because failure to do so could result in wasteful spending or denial of care to those in need. These reference values for hormonal parameters in healthy individuals are subjected to variations due to analytical and pre-analytical factors. For that purpose, their determination for every country, even every region, is of major importance. Health data in India is scarce and its quality is a perennial source of contention. 6 This scarcity can be attributed to India's large heterogenous population, and inaccessibility of the available data. 6 For a normative range of hormonal parameters, India also lacks such data, especially for healthy women of reproductive age. Therefore, in the current large study, we attempted to evaluate the biological reference interval for various hormones in reproductive-aged Indian women.
Study design
The detailed protocol of the study participants is published elsewhere. 7 Briefly, this national cross-sectional study envisioned to estimate the burden, and associated risk factors of polycystic ovary syndrome (PCOS), recruited participants from 2018 to 2022 in six zones (North India, Northeast, South India, Eastern India, Central India, and Western India) spreading across the country, involving 10 regional centres that represent a majority of ethnic groups. The screening of the participants was done employing a uniform methodology using a pre-designed screening questionnaire, designed by the coordinating centre. The questionnaire was pilot tested and then circulated among various study sites. The study was conducted following the guidelines enshrined under the Helsinki 1975 declaration and was
Research in context
Evidence before this study Globally, limited population-specific studies have reported the variations in hormonal parameters among apparently healthy women. However, no such large countrywide study has been carried out in India.
Added value of this study
Our first large composite data on hormonal measures generated by a uniform methodology on a cohort of reproductive-age women truly representative of the reproductive age across the country would provide the biological reference range for the studies hormones.
Implications of all the available evidence
The study is likely to fill the vacuum of biological reference interval data for various hormones among women of reproductive age.
Articles approved by the institute Ethics Committees (IECs) of the respective study institutions. Informed written consent was taken from all the participants before their enrolment. The data on PCOS cases is not reported here.
Study population
The study participants included apparently healthy women aged 18-40 years from the urban and rural areas of the country. Inclusion criteria included those participants who lived in the area for at least a year, were non-pregnant, with regular menstrual cycles, apparently healthy, and were willing to participate. Participants with any previously confirmed disease or disorder, or were on any medication for the same, and those who didn't want to participate in the study were excluded. We used a structured questionnaire to record their socio-demographic characteristics such as place of residence, age, marital status, level of education, occupation and socioeconomic status, 8 religion, and type of family followed by uniform clinical and laboratory assessment.
Clinical and laboratory assessment
Participants underwent a detailed clinical history and physical examination which included measurement of parameters like body weight, height, BMI, and waist circumference measurements by using standard calibrated instruments (SECA 213, Hamburg Germany), and blood pressure measurements (Omron HEM7120). A mean of three readings was taken as the final value for these parameters. All participants were further evaluated for biochemical and hormonal parameters. The oral glucose tolerance test (OGTT) was performed after an overnight fast of 10-12 h. A total of 5 mL of venous blood was drawn in a fasting state between day 2 and day 7 of the menstrual cycle. The samples were immediately separated in a cold centrifuge and aliquoted for hormones and other required laboratory investigations. The aliquots for hormones and other required investigations were shipped to the co-ordinating center in a cold chain (dry ice) and were stored at −80 • C until the assay. The quantification of hormones (including serum total T4, thyroid-stimulating hormone (TSH), total testosterone, 17-OHP, cortisol, prolactin, luteinizing hormone, and follicle-stimulating hormone, E2, C-peptide, insulin, and DHEAS) and SHBG (a transport carrier that binds and regulates the biological activity of oestrogen and androgens) was conducted using electrochemiluminescent immunosorbent assay on Cobas e411 (Roche Diagnostics).
Quality control assessment
The standards (reagents with known concentrations) were run daily to validate the accuracy of the hormone analyser and was calibrated when required as per the manufacturer's instructions.
Data management and statistical analysis
Data were entered into Microsoft Excel database. The biological reference intervals were calculated using nonparametric methods. Besides the median, reference intervals were determined at 2.5th and 97.5th percentiles. Participants were further stratified by age, BMI (WHO-Asian BMI criteria Normal: 18.5-22.9, Overweight: 23-27.5, and Obese: ≥27.5 kg/m 2 ) and WHO general population BMI criteria (Normal: 18-24.9, Overweight: 25-29.9, Obese: ≥30 kg/m 2 ), and residence (rural and urban) for the estimation of the reference intervals. Mean, median and standard deviations were computed for each of the biochemical and hormonal parameters of the study participants. Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS; Version 26, IBM). Kruskal-Wallis test was used to determine the differences in the data after stratification by age, BMI, and place of residence. All tests were considered significant with two-tailed p < 0.05. Moreover, a literature survey for comparative analysis was also performed for comparing the data from the present study with the already published data.
Role of funding source
The study was financially supported by the Indjan Council of Medical Research, Govt of India vide|file No: 5/7I1337/2015-RBMH, It is to certify that authors were not precluded from accessing data in the study, and they accept responsibility to submit for publication.
Results
The details of all the participants recruited in the study are provided in Fig. 1. A total of 7107 apparently healthy controls were enrolled for the present study out of which 3877 were screened for detailed socio-demographic information. Among the screened participants, 1441 participants were assessed for hormonal parameters. After excluding the participants (n = 593) with any underlying hormonal imbalance or comorbidities like subclinical hypo/hyperthyroidism, hyperprolactinemia, premature ovarian failure, or exogenous Cushing syndrome, a total of 848 participants were retained for the analysis. The majority of participants were unemployed (64.8%) and represented the lower and middle socioeconomic class (31%) of the population. Based on the population structure of the country, 9 expectedly, most of the participants were Hindu (71%) followed by Muslim participants (26.1%).
The mean age and BMI (±SD) of the participants recruited in the study were 29.9 (±6.3) and 23.2 (±3.3) respectively. The mean values (±SD) of various hormones analysed are presented in Table 1 and all the centile distribution is presented in Table 3). The biological reference intervals (median and 2.5th-97.5th percentiles) of prolactin, FSH, testosterone, E2, C-peptide, fasting insulin and DHEAS varied when stratified by age (p < 0.05) ( Table 3). The data on biological reference intervals for the studied hormones reported in the relevant published literature across different populations is presented in Table 4. The comparative analysis showed marginal differences in the normative ranges for these hormones among different populations. The biological reference intervals for prolactin, C-peptide, T4, DHEAS, fasting insulin, and SHBG varied when the participants were stratified by BMI (p < 0.05) (Supplementary Tables, S1-S3). The data on anthropometrical and biochemical indicators are not reported here.
Discussion
The present country-wide study was aimed to frame the biological reference values of clinically important hormones among Indian women of reproductive age. To the best of our knowledge, this is the first large pan-India study to report the normative values of the studied hormones. The data on detailed clinical and biochemical and hormonal examination from a total of 1441 apparently healthy women, aged between 18 and 40 years, were analysed for the study. After the exclusion of various comorbidities, a total of 848 healthy participants were further analysed for generating hormonal reference intervals.
Serum levels of hormones in women fluctuate significantly during different stages of life, starting from menarche to menopause. 24 Age and health status are two characteristics that have an impact on the levels of sex hormones, with age being the primary influencer in women. 25 Thyroid dysfunction (clinical or biochemical) is a common clinical condition with its presentation modulated by gender, ethnicity, genetic predisposition, and iodine status. 26 An expanding set of research outlines the negative consequences of subclinical thyroid illness in women, thereby drawing attention to early intervention of T4 therapy. This may progress over a period of time to overt hypothyroidism particularly in females, those with thyroid peroxidase antibody positivity and a TSH of more than 10 mIU/mL. 27 Thyroid function reference intervals for adults have been thoroughly established in various populations including India, where Marwaha et al. reported the TSH level of 2.2 ± 0.9 mIU/L (mean) for adult Indian women. 23 The United States National Health and Nutrition Examination Survey III (NHANES-III) similarly evaluated a disease-free population and observed significantly lower TSH and T4 values as compared to the general population. 21 The results for T4 and TSH from our study are consistent with the findings of NHANES-III report. However, our normative values for T4 and TSH vary from this earlier Indian study by Marwaha et al. 23 Unlike ours, the study by Marwaha et al. 23 Articles serum T4 and TSH didn't change (P > 0.05) suggesting our reference can be applied to all the studied age subgroups. Moreover, unlike a large Chinese study, 28 the median values for T4 were elevated in higher BMI groups in our study. While our study was based on female participants of reproductive age, this earlier Chinese study did not stratify the participants based on gender. 28 The normative ranges for quantifying prolactin secretion show it depends upon multiple biological or clinical factors, including gender, age, BMI, other hormones, nutrition, stress, physical activity, medications and renal disease. 29 In the current study, we found our reference interval for serum prolactin levels was higher in younger women (18)(19)(20)(21)(22)(23)(24)(25) year group) when compared with older women (≥26 years). Earlier studies have also found a decrease in prolactin levels with the increase in the age of women. 30,31 Moreover, in the current study, we did not observe any significant difference in the biological reference range of prolactin with the increase in BMI. More replicative studies are warranted to substantiate these findings.
DHEA plays a pivotal physiological role in the reproductive tissues, bone, mood, cognition, vasculature, breast, muscle, and other systems in women. 32 Nearly all androstenedione produced in the adrenal gland is synthesised from DHEA in humans. 33 Elevated DHEAS is considered a marker for hyperactive adrenal cortex production of androgens. 32 Majority of women with PCOS are reported to have increased DHEAS levels. 34 Similar to the published literature, in the current study we found the median levels of DHEAS decreased with age. 35 Based on the findings of a recent meta-analysis, serum cortisol is believed to have increased circulating levels in the follicular phase of the menstrual cycle. 38 The cause-effect relationship between obesity and cortisol is poorly understood. Moreover, the data on its correlation with BMI and age is varying. 39 Some studies have reported low cortisol levels in obese than normal-weight participants 39,40 while another study shows elevation in the cortisol levels with age. 41 In the current study, we observed a marginal rise in the median cortisol levels with age or BMI but did not attain statistical significance.
In a typical menstrual cycle, E2 levels are lowest during the early follicular phase of monthly bleeding and rise subsequently until ovulation. Just before Articles ovulation, levels of LH and FSH spike, but progesterone (P4) levels rise following ovulation and peak at the midluteal phase. In our study, the median normative serum levels of LH, E2 and SHBG didn't vary across different age groups which is inconsistent with the findings of the studies carried out in different populations. 10,11,13,18 However, the biological reference interval for FSH elevated significantly with age. The increase in the FSH levels has been coupled with a decline in fertility rates typically beginning in the third decade onwards of a woman's life. 42 In women, androgen levels begin to decline with age. 37 In line with the published literature, we also observed a decline in the levels of testosterone when the participants were stratified by age. Moreover, unlike men, serum androgens levels positively correlate with BMI in women, 43 however, in the current study the testosterone levels did not change with the increase in BMI. The difference might be attributed to a smaller sample size in the model after stratification, and needs further validation.
C-peptide is a widely used and useful method of assessing the functioning of the pancreatic β-cells, and its measurements serve as indicators of insulin production. 37,44 C-peptide is secreted in equimolar levels and has a longer half-life than insulin. As a result, C-peptide levels provide a more accurate reflection of the long-term level of insulin than does the actual amount of insulin. High C-peptide levels are related to increased mortality risk in both men and women in the general population between the ages of 40 and 74. 45,46 As expected, our data support homeostatic insulin secretion in glucose normo-tolerant participants following the glucose load. Moreover, similar to earlier studies, in the current study, we observed significantly elevated median levels of fasting insulin and C-peptide in the higher age and BMI groups. 47 However, Tohidi et al. reported a decrease in trend with age in Iranian participants. 48 Unlike ours, the study by Tohidi et al. was based on a smaller sample size, thus warranting further research with a larger sample size to substantiate these findings.
Studies have persistently propounded upon the ethnic differences on the normative reference interval for various biochemical and hormonal indicators. 49 Most of the data on these reference intervals is published from developed countries. Barring the study by Marwaha et al. for T4 and TSH, no comprehensive study has been carried out in India for establishing normative reference intervals. On comparative analysis of our data with the already published literature, we observed marginal differences in the normative ranges for the studied hormones among different populations. However, these differences can be attributed to different estimation methods employed in these studies. 3,50 Our community-based study based on a countrywide reasonably large sample size utilized a robust study design to establish a population-specific normative range of common hormonal parameters in reproductive-aged Indian women and explored its variability with age, residence, and BMI. For establishing normal hormone reference intervals, the current study has employed reliable sensitive and uniform methodology using ECLIA at a single study centre in addition to stringent exclusion criteria.
The data on single-gender within a specific age group, and a selected number of hormone parameters limits the generalisability for reference ranges. Besides a relatively smaller sample size after subsequent stratifications might be a limiting factor of the present study. However, these factors are unlikely to change the main outcome of the study.
In conclusion, this is the first large composite data from India on common hormonal parameters generated using a uniform methodology on a cohort of reproductive-age women representative of the country. Therefore, this is likely to fill the vacuum of normative data among these women in future.
Data sharing statement
Any data generated in this study is available on a reasonable request to the corresponding author.
Declaration of interests
All authors declare that they do not have any conflict of interest/ competing interests. | 2023-08-25T05:13:32.163Z | 2023-05-30T00:00:00.000 | {
"year": 2023,
"sha1": "92fedad9a3bb4072302194be46b79b4c140c899e",
"oa_license": "CCBYNCND",
"oa_url": "https://doi.org/10.1016/j.lansea.2023.100226",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "92fedad9a3bb4072302194be46b79b4c140c899e",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
249545114 | pes2o/s2orc | v3-fos-license | Development of a machine learning-based prediction model for extremely rapid decline in estimated glomerular filtration rate in patients with chronic kidney disease: a retrospective cohort study using a large data set from a hospital in Japan
Objectives Trajectories of estimated glomerular filtration rate (eGFR) decline vary highly among patients with chronic kidney disease (CKD). It is clinically important to identify patients who have high risk for eGFR decline. We aimed to identify clusters of patients with extremely rapid eGFR decline and develop a prediction model using a machine learning approach. Design Retrospective single-centre cohort study. Settings Tertiary referral university hospital in Toyoake city, Japan. Participants A total of 5657 patients with CKD with baseline eGFR of 30 mL/min/1.73 m2 and eGFR decline of ≥30% within 2 years. Primary outcome Our main outcome was extremely rapid eGFR decline. To study-complicated eGFR behaviours, we first applied a variation of group-based trajectory model, which can find trajectory clusters according to the slope of eGFR decline. Our model identified high-level trajectory groups according to baseline eGFR values and simultaneous trajectory clusters. For each group, we developed prediction models that classified the steepest eGFR decline, defined as extremely rapid eGFR decline compared with others in the same group, where we used the random forest algorithm with clinical parameters. Results Our clustering model first identified three high-level groups according to the baseline eGFR (G1, high GFR, 99.7±19.0; G2, intermediate GFR, 62.9±10.3 and G3, low GFR, 43.7±7.8); our model simultaneously found three eGFR trajectory clusters for each group, resulting in nine clusters with different slopes of eGFR decline. The areas under the curve for classifying the extremely rapid eGFR declines in the G1, G2 and G3 groups were 0.69 (95% CI, 0.63 to 0.76), 0.71 (95% CI 0.69 to 0.74) and 0.79 (95% CI 0.75 to 0.83), respectively. The random forest model identified haemoglobin, albumin and C reactive protein as important characteristics. Conclusions The random forest model could be useful in identifying patients with extremely rapid eGFR decline. Trial registration UMIN 000037476; This study was registered with the UMIN Clinical Trials Registry.
INTRODUCTION
The number of patients with chronic kidney disease (CKD) is increasing worldwide, resulting in an increased number of patients requiring dialysis and kidney transplantation and suffering from cardiovascular (CV) events. [1][2][3][4] According to the Global Burden Disease study data, the incidence, prevalence and mortality rate of CKD increased by 89%, 87% and 98%, respectively, between 1990 and 2016. 5 However, many patients with CKD are asymptomatic until the kidney function deteriorates. Therefore, a diagnosis of CKD at an
STRENGTHS AND LIMITATIONS OF THIS STUDY
⇒ We also adapted a unique and novel approach for clustering using the hierarchy of curve steepness and trajectory, which makes the algorithm efficient and fast. ⇒ A key limitation is that training of our model only relies on electronic health record data we have, and, thus, even though our data were collected from a large hospital and we use fair algorithms, our resultant models may incur inherent bias in the data if existing. ⇒ We adopted an intriguing design of prediction model using machine learning, which differentiates the rapid decline group from the other groups using subject's covariates with such groups, which are clustered by the shape of estimated glomerular filtration rate declines using a novel and practical automatic trajectory clustering algorithm.
Open access earlier stage is required. However, trajectories of glomerular filtration rate (GFR) vary between patients and are reported to depend on the primary kidney disease, blood pressure and proteinuria. [6][7][8] We believe that it would be useful if we could accurately predict the extremely rapid estimated GFR (eGFR) decline before the deterioration begins to determine the causes of deterioration, avoid renotoxicities and provide earlier treatment with renoprotective drugs. In addition, there may be crucial causes such as incidence of rapidly progressing glomerulonephritis accompanying original kidney diseases especially in patients with extremely rapid eGFR decline. If causes of kidney function deterioration are identified earlier, we could treat the patient appropriately. Artificial intelligence (AI) has been in development since the 1980s, and investigations using machine learning have been progressing in various fields, including medicine. [9][10][11][12][13] Machine learning can identify irregularities in data and deal with large data sets with complex variables and relationships (ie, 'big data'). Therefore, machine learning can often be used for the prediction of associated phenomena from big data in healthcare. [14][15][16] In nephrology, for instance, AI has enabled a more precise equation for eGFR. 17 18 Our hospital has maintained a large database of more than 900 000 patients who were treated and followed up for various diseases since 2004. We previously demonstrated a prediction model for patients with an eGFR decline of ≥30% within 2 years using machine learning. 19 However, we realised that the patterns of eGFR decline varied even among those patients. A more detailed prediction is crucial in the real-world clinical settings because of higher risks of progression to end-stage kidney disease and the incidence of CV disease. Therefore, we focused on extremely rapid eGFR declines and analysed a much larger population data of 914 280 patients from the single-centre database than in the previous model. In addition, the purpose of the present study was to adopt an intriguing design of prediction model using a novel and practical automatic trajectory clustering algorithm. To the best of our knowledge, no AI-based methods have been proposed to create a prediction model related to the trajectories of eGFR, especially among patients with extremely rapid eGFR decline. Therefore, we aimed to create a model for extremely rapid eGFR decline among patients with CKD with an eGFR decline of ≥30% within 2 years based on a large database and using machine learning.
Data set and samples
We used a database of 914 280 patients from the Fujita Health University Hospital between June 2004 and July 2019. Medical data were available for 286 494 patients with eGFR, of which the findings in 29 466 patients included the following CKD criteria: an eGFR <60 mL/min/1.73 m 2 and/or urine protein ≥1+ on a dipstick for >90 days.
Patients of <20 years of age and those who had undergone kidney transplantation were excluded. When measuring eGFR, we used the average eGFR measurements over the preceding 90 days to avoid temporal spikes in measurements. On detection of different and distinct spans of GFR decline in the same patient, we included the first value in the analyses. Patients with CKD with an eGFR decline of ≥30% within 2 years, defined as rapid eGFR decline according to previous reports, were enrolled. [20][21][22] Overall, there were 7315 such samples in the study. Of these, we only included samples with an initial eGFR of ≥30 mL/ min/1.73 m 2 because we aimed to detect patients with extremely rapid eGFR decline at an earlier stage. In addition, many patients with eGFR <30 mL/min/1.73 m 2 have a clinical course of extremely rapid eGFR decline within a short period because the lower the initial eGFR, the more rapid is the eGFR decline, in general. Hence, we excluded patients with advanced kidney dysfunction. Finally, 5657 unique samples of GFR decline were analysed.
Clustering eGFR decline curves
To automatically cluster the eGFR decline curves, we used an expansion of the group-based trajectory model posited by Nagin. 23 The original algorithm was modified as follows: the curves were hierarchically grouped by the initial eGFR values and curve steepness. We used a single response variable for the eGFR. We sampled 10 points with equal time intervals after applying linear interpolation to the response variables to obtain 10 points with equal time intervals for each patient. To obtain such 10 points by sampling for the entire population, we applied linear interpolation of the response variables on all of them. We also did not assume that those points were on the specific function of the elapsed times.
To modify the existing method for hierarchical grouping, we used the following equation: where m indicates the groups of initial eGFR values and k indicates the groups of curve steepness. The notation y is the response variable consisting of 10 points on the eGFR curve, while the probability that y belongs to class m is denoted by p m , and to class m and k is denoted by p m, k . Note that we hierarchically grouped the curves using p m and p m, k , which are both estimated from the data as model parameters. The notations M and K are the total number of those classes. The function f y (y|C 1 =m,C 2 = k) is the conditional density of the observed data, m is the initial eGFR value of class C 1 and k is the curve steepness of class C 2 .
To form the conditional density function, f y (y|C 1 =m,C 2 = k), we assumed that each point in y is generated from the Gaussian distribution: µ m,k,d,t d = β m,k,d t d , for d = 2, . . . , T, Open access µ m,k,d,t d = β m,k,d t d , for d = 2, ..., T, where β is the estimated distribution parameter. In this study, we did not estimate the variance in the distribution, σ . In our model, y 1 was determined only based on the initial eGFR class of m, whereas the latter points of y were determined by m and k and the timestamp t. Note that, unlike the original model, we assumed that each point is independently generated from the Gaussian distribution.
In our experiments, we used 3 for m and 3 for k, which are set to give a similar number of trajectory groups as Nagin reported. 23 Samples for classification For each type of high-eGFR, middle-eGFR and low-eGFR decline curve, we classified the most acute curves. In the low-GFR decline group, which included 2437 samples, curves were categorised in detail as mild, moderate and acute; we identified 222 positive samples of acute curves and 2215 negative samples of mild and moderate curves. In the middle-GFR decline group, which included 2652 samples, the curves were categorised as mild and moderate (n=2139) and acute (n=513). In the high-GFR decline group, which included 568 samples, acute curves included 103 samples, while moderate and severe curves included 465 samples.
Features used for the classification model
With the aforementioned positive and negative samples of eGFR decline, we extracted the laboratory values, including 15 longitudinal data as well as 5 static data, The former includes transferrin saturation, blood urea nitrogen, serum uric acid, haemoglobin, haemoglobin A1c, ferritin, eGFR, systolic and diastolic blood pressures, C reactive protein (CRP); body mass index, serum total cholesterol, serum creatinine, serum albumin and urine protein. The latter includes sex, age, comorbidity of diabetes mellitus, history of acute kidney injury and prescription of renin-angiotensin system inhibitors. Summarising these longitudinal data to form explanatory variables in the prediction model, we used the average and SD for each variable over 90, 180, 360 days to the beginning of the eGFR decline and the exponentially smoothed average (ESA) of all available past data. The methods are shown in online supplemental table 1). These nine summarisation methods are then applied to all longitudinal data, respectively, and, thus, we have 135 longitudinal features. By adding static data, we use 140 features in total as inputs to our classification models.
Model solving
We used logistic regression and random forest algorithm for classification. To evaluate the proposed model, we tuned hyperparameters, including the number of trees in the forest, the minimum number of samples required at a leaf node and the minimum number of samples required to split an internal node, for example, random forest. To identify the best parameters in the inner four-hold crossvalidation, we evaluated the random forest and logistic regression models using outer five-fold cross-validation. Note that we found the best hyper parameters only using the training data of each fold of the cross-validation, not using the test data. By solving the model, we can compute the probability of a patient to be classified in the group of the most acute curves. The following is the formula to compute the probability on using logistic regression: parameters, x i is a feature value indexed by i, mean i , std i are the mean and SD for ith feature values, and n is the number of features. Actual values for α i , mean i and std i are shown in online supplemental table 2, online supplemental table 3 and online supplemental table 4 to compute the probability. We omitted the computation while using random forest to avoid complexity but the probability can be computed by averaging the results of each decision tree used in the algorithm. We then used the area under the curve (AUC) of the receiver operating characteristic (ROC) curve as representative performance metrics, which is computed as the mean of the results of fivefold. In our experiment setting, validation data sets were created from the data that were not used in training for each fold, which is a common and practical method when evaluating the model even using one cohort. Other statistical parameters (such as sensitivity and specificity) were computed using the fold, which has a median AUC out of five folds. The best cut-off was found at a point of the ROC curve at the minimum distance from the top left corner, which is commonly used when determining the cut-off as well as Youden index, taking the AUC into account. 24 We should note that such derivation of 'best cut-off' could lead to bias 25 26 where bias reduction such as using smoothed ROC curve and others is discussed. 25 In this study, we mostly use AUC for comparing and evaluating prediction performance as suggested in a previous report. 26 For examining the importance of features, the contributions to the eGFR decline were examined according to the weight of each variable in logistic regression and by Gini impurity in random forest model. In this study, we applied logistic regression and random forest using the Python code with scikit-learn library (https://scikit-learn.org/) as well as for classification model solving. For solving the clustering model, we used PyStan (https://pystan.readthedocs.io/en/latest/), which is the Python interface of Stan (https://mc-stan. org/). Stan is a general statistical modelling platform where we declaratively depicted our probabilistic models as we explained before. Note that we used the Markov chain Monte Carlo algorithm to estimate model parameters including p m , p m, k , β m and β m, k, d .
Statistical analysis
We compared the all-cause mortality among the nine subgroups. The data on the outcome were obtained Open access from the medical records. All-cause mortality rates were compared using log rank test with Kaplan-Meier curves.
Patient and public involvement
Patients were not involved at any stage of this research.
Comparison of patient characteristics and laboratory data at reference points between the groups Table 1 summarises the comparisons of patient characteristics and laboratory data at the reference points between the subgroups with high and other rates of eGFR decline. Patients with extremely rapid eGFR decline were significantly older than those in the other groups in G1 and G2. Blood haemoglobin and serum total cholesterol and albumin levels were significantly lower in the extremely rapid eGFR decline subgroups in each group. Serum CRP levels were significantly higher in the extremely rapid eGFR decline subgroups in each group.
Comparison of cumulative all-cause survival rate between the subgroups We compared the cumulative survival rate among the nine groups. Significant differences were observed between them (log-rank test: p<0.001). Figure 2 illustrates the ROC curves and calibration plots with its slope and intercept for prediction of extremely rapid eGFR decline in each group according to the random forest-based model. Table 2 summarises the AUC of the logistic regression and the random forest models for prediction of extremely rapid eGFR decline.
AUC and calibration plot in each group
The AUCs of the G1, G2 and G3 groups according to the logistic regression model were 0.682, 0.647 and 0.754, respectively, and those according to the random forestbased model were 0.694, 0.712 and 0.788, respectively. We conducted the same analysis without including the serum creatinine level because we considered multicollinearity between eGFR and serum creatinine level. Subsequently, the AUCs of the G1, G2 and G3 groups according to the random forest-based model were 0.687, 0.705 and 0.789, respectively. The calibration plot of G1 indicated that the predicted probabilities of the machine learning model were close to the actual probabilities. Meanwhile, in G2 and G3, the higher the predicted probabilities, the higher were the actual probabilities compared with the predicted probabilities.
Features affecting the prediction in the three groups Online supplemental figure 3 illustrates the heatmap for features that affected the random forest-based model. The redder a column, the higher is its effect on extremely rapid eGFR decline; in contrast, the greener a column, the lesser is its effect. Kidney function including eGFR; age; diastolic blood pressure and albumin, cholesterol, haemoglobin and uric acid levels were demonstrated as features potentially useful for distinguishing G3 from G1 and G2. Figure 3 summarises the ranking of the top 10 features in the random forest model. Except for the features of kidney function, including eGFR and creatinine levels, the features related to haemoglobin and cholesterol were ranked high in G1, those related to albumin and haemoglobin were ranked high in G2, and those related to CRP and albumin were ranked high in G3.
DISCUSSION
The prediction model that we created could detect patients with CKD with extremely rapid decline in eGFR using machine learning. The results of the present study have three features. First, the patient data of the present study were obtained from a large-scale database. There have been some reports concerning CKD by analysing big data. [27][28][29] However, most of the studies in this field have included the general population. We believe that the present study is significant, as it examined a large number of patients with various diseases. Second, we used AI to analyse and create different prediction models, including random forests. AI-based disease prediction is progressing in many fields. Electronic medical record systems have been in use for >10 years in many hospitals in Japan. Therefore, a large amount of information, including laboratory data, can be analysed using AI. Machine learning enables the addition of an exponential smoothing average for different variables from a large amount of data. Third, we found that the variables that affect the pattern of eGFR decline vary according to the kidney function at baseline. We were able to classify into three groups automatically according to eGFR at the reference points.
Open access
In general, kidney function in patients with CKD gradually worsens. However, patterns of eGFR decline, which are called trajectories of eGFR, vary between patients. 30 A report from six large-scale, randomised controlled trials revealed that more cases presented with non-linear eGFR decline among diabetes patients. 31 Many reports have demonstrated risk factors related to the decline in eGFR. The Chronic Renal Insufficiency Cohort study, which was conducted in the USA, clarified many factors associated with CKD progression in patients with pre-dialysis CKD. [32][33][34][35] These risk factors included proteinuria, the presence of inflammatory cytokines and elevated serum uric acid levels. In Japan, the Chronic Kidney Disease Japan Cohort revealed that anaemia, blood pressure and albuminuria were independent risk factors of CKD progression. 36 The eGFR of the patients enrolled in the two representative cohorts ranged from 10 mL/min/1.73 m 2 to 70 mL/min/1.73 m 2 . Meanwhile, we decided that an eGFR ≥30 mL/min/m 2 at baseline was the cut-off in the present study because the period before initiating renal replacement therapy was extremely short to classify the patterns of eGFR decline. Furthermore, patients with an eGFR of ≥90 mL/min/m 2 at baseline were enrolled. We created a prediction model for identifying patients with rapid eGFR decline among those with CKD in our previous study. 19 However, we could not adjust the models and stratify them according to eGFR. Machine learning enables the classification of trajectories of eGFR decline into nine patterns using eGFR at baseline and the rate of eGFR decline. Interestingly, we found that different
Open access
clinical parameters, including haemoglobin and CRP levels, were more important in predicting an extremely rapid eGFR decline according to the baseline kidney function. Some prediction models for eGFR decline based on AI have been reported. 27 Raynaud et al recently demonstrated that the donor age, eGFR, proteinuria and pathological findings of the transplanted kidney predicted progression to end-stage kidney disease in kidney transplanted patients who were classified into eight groups. 37 Meanwhile, our model included features that we used as variables, including haemoglobin and CRP levels, which were not used in other models. Factors related to anaemia, such as haemoglobin ESA7 and the 90-day and 180-day averages, were associated with trajectories of eGFR in patients with an eGFR of approximately 90 mL/min/m 2 . Anaemia is often accompanied by CKD because the reduction of functional kidney mass leads to a decrease in the production and secretion of erythropoietin. However, renal anaemia usually develops at an eGFR of <30 mL/min/1.73 m 2 . In the present study, we defined anaemia as a haemoglobin level below its lower normal limit. In other words, anaemia might be detected more strictly in the study than in a clinical setting. Therefore, we found that the management of renal anaemia may be vital in patients in earlier stages of CKD. In contrast, the factors related to serum albumin were associated with the trajectories of eGFR in patients with an eGFR of approximately 60 mL/min/m 2 and 40 mL/min/ m 2 . We believe that the progression of CKD causes not only an increase in the amount of proteinuria but also malnutrition. Interestingly, the serum CRP level ranked high in patients with an eGFR of only 40 mL/min/m 2 , which suggested that inflammation might be more significant in patients in advanced CKD stages. Additionally, eGFR starts decreasing closer to critical points, and larger changes in variables can be observed. It is possible that the predicted probability increases by adding an exponential smoothing average for different variables. We showed good calibration for the G1 group and lesser for the G2 and G3 groups. If nephrologists estimate higher risk in patients in G2 and G3 from the present prediction model, practices for renal protection such as use of renin-angiotensin system blockers, sodium glucose cotransporter-2 inhibitors, erythropoiesis-stimulating agents and protein restriction can be changed. Many studies have indicated that proteinuria or albuminuria is crucial risk factors for a decline in kidney function, incidence of CV disease and all-cause mortality. Contrary to expectations, proteinuria did not significantly influence the prediction, as opposed to our previous report on a prediction model for rapid eGFR decline. 19 We believe that this was because patients with extremely rapid eGFR decline who were at a higher risk were enrolled. A relatively high amount of proteinuria was already recognised in the patients in this study. Some clinical variables have been reported to be risk factors of eGFR decline. [38][39][40][41][42][43] To create a more precise prediction model for trajectory of eGFR decline, more variables related to kidney function should be considered. To accomplish that, it is necessary to conduct a prospective study in the future.
Open access
The present study has some limitations. First, the participants might have suffered from not only kidney diseases but also other diseases. Therefore, the results may not always apply to the general population. Unfortunately, we could not get detailed information about the diseases that the patients suffered from because items included in the data were huge and complicated. We considered that underlying diseases might be an independent risk factor. Second, the intervals between the frequency of examinations, including blood tests, differed between patients. Therefore, we used the average values over periods of 90, 180 and 360 days prior to the reference points. Third, the results of the calibration plots were different among the three groups. This could be because the criteria for CKD in G1 differed from that in G2 and G3. Most patients in G1 were defined to have CKD with only proteinuria because the eGFR values at the start of the decline were approximately 90 mL/min/1.73 m 2 ; therefore, eGFR values of <60 mL/min/1.73 m 2 , which is one of the conditions for CKD, were rare. However, in both G2 and G3, most patients were diagnosed with CKD using both eGFR and proteinuria because the eGFR values at the start of the rapid decline were approximately 60 and 40 mL/ min/1.73 m 2 , respectively. Additionally, in both G2 and G3, the actual observation probabilities were higher than the predicted probabilities by approximately 0.3. Therefore, we may need to estimate the risk of decline in eGFR than the predicted probabilities in patients with relatively high predicted probabilities.
CONCLUSION
We have created a prediction model for extremely rapid eGFR decline using machine learning to identify patients at a high risk. Further statistical research includes exploiting recent deep learning algorithms, which could enable to use more complex and multimodal features and to provide higher classification and calibration performance. Recent generative approaches using deep learning also has the potential to directly predict the shape of eGFR decline curves even in a non-parametric approach. To use this model in a real-world clinical setting in the future, we could intervene by preventing eGFR decline. A typical usage is, thus, by inputting patient's features to the model, showing whether a patient is in the group of the extremely rapid eGFR decline, and also exhibiting the typical shape of eGFR decline. Such an application can be provided by being connected to the electronic health record system of a hospital to obtain features and showing the model results via a personal computer or mobile interfaces.
To this end, validations using external datasets are needed because our models are created from one hospital data as well as prospective studies to confirm the accuracy of the present results. Contributors DI, RY and YY were involved in study design and data interpretation. DI and HH contributed to the writing of the manuscript. AK, TI and MK analysed the data. SF overviewed and criticized the manuscript. All authors were involved in drafting, reviewing, and approving the final manuscript. AK and TI had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. DI acts in as the guarantor.
Funding This study was supported by research funds with no restrictions on publication from Kyowa Kirin Co., Ltd. IBM Research provided support for this study in the form of salaries for A. Koseki, T. Iwamori and M. Kudo. There are no patents, products in development or marketed products associated with this research.
Competing interests Yes, there are competing interests for one or more authors and I have provided a Competing Interests statement in my manuscript and in the box below.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Patient consent for publication Not applicable.
Ethics approval Ethics Committee: Center for research promotion and support, Fujita Health University number ID: approval number: HM19-157. We used only medical information, which was not included identified personal information such as name, ID number of medical records. However, informed consent was obtained in the form of opt-out on the website.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available upon reasonable request. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.
Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. | 2022-06-11T06:16:25.815Z | 2022-06-01T00:00:00.000 | {
"year": 2022,
"sha1": "d8a1b83eb295381f17ac831d6e93745d861f28f1",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "BMJ",
"pdf_hash": "ba34ecc885d590c4653f14590a7e4a656d2a528d",
"s2fieldsofstudy": [
"Medicine",
"Computer Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
24098907 | pes2o/s2orc | v3-fos-license | The Day Dapsone DRESSed as DILI
Dapsone is a diaminodiphenylsulfone drug approved to treat leprosy with off-label use in HIV. It is a rare cause of idiosyncratic liver injury and has been associated with hypersensitivity. The pattern of liver injury is typically cholestatic, and the development of cholestasis due to dapsone has been described in rats. Here we describe a case of intrahepatic cholestasis and hypersensitivity syndrome in a patient with AIDS after initiation of dapsone for Pneumocystis pneumonia. The patient was admitted from clinic for scleral icterus of 3 days duration. He was febrile, hypotensive, and tachycardic with scleral icterus, jaundice, and a patchy erythematous rash on the torso. Laboratory studies showed eosinophilia and cholestatic injury pattern with value of R=0.5. All anti-retroviral medications and dapsone were stopped. Given concern for immune reconstitution in this AIDS patient, a liver biopsy was performed which was consistent with drug induced liver injury. Anti-retroviral therapy was re-started and the patient’s aminotransferases and bilirubin gradually improved over span of 8 weeks following discontinuation of dapsone.
Introduction
Dapsone is a diaminodiphenylsulfone drug approved to treat leprosy with off-label use in HIV for Pneumocystis treatment and prophylaxis in those patients unable to receive standard therapy. It is a rare cause of idiosyncratic liver injury and has been associated with hypersensitivity [1]. The pattern of liver injury is typically cholestatic, and the development of cholestasis due to dapsone has been described in rats [2]. Here we describe a case of intrahepatic cholestasis and hypersensitivity syndrome in a patient with AIDS who presented with scleral icterus after initiation of dapsone for Pneumocystis pneumonia.
Case Report
A 27 year-old African American male with AIDS (CD4 count 70 cells/uL) receiving treatment for Pneumocystis pneumonia was admitted to the hospital with three days of scleral icterus. He had no prior history of icterus, no pre-existing liver disease, and denied abdominal pain, nausea or vomiting. He had been diagnosed with Pneumocystis pneumonia 5 weeks prior to presentation and, due to past history of sulfa allergy (rash and facial swelling), was prescribed a 3 week course of clindamycin, primaquine and prednisone and antiretroviral therapy (ART-darunavir, raltegravir, ritonavir, and zidovudine). This regimen was used in lieu of traditional Pneumocystis treatment with trimethoprim/sulfamethoxazole given patient's sulfa allergy. A test for glucose-6-phosphate dehydrogenase (G6PD) activity in erythrocytes was normal. He completed the treatment course, after which ART therapy was continued and dapsone, 100 mg/day, was prescribed for Pneumocystis prophylaxis.
Two weeks later he presented with jaundice. He denied family history of liver disease. He drank 3 beers per month and did not use recreational drugs or dietary supplements. On physical exam, he was febrile, hypotensive, and tachycardic with scleral icterus, jaundice, and a patchy erythematous rash on the torso. Laboratory studies showed eosinophilia (15%, absolute eosinophil count 1600 /ul), serum aspartate aminotransferase (AST) 68 U/L, alanine aminotransferase (ALT) 145 U/L, alkaline phosphatase (ALP) 909 U/L and total bilirubin 12.4 mg/dl (direct 8.1). The value of R*=0.5, indicating cholestatic injury [3]. Urine drug screen was negative and there was no evidence of hemolysis. Viral hepatitis serologies and antimitochondrial antibodies were negative. Abdominal ultrasound did not demonstrate biliary obstruction. Given concern for likely drug induced liver injury, dapsone and anti-retroviral drugs were stopped. After 2 weeks off of these medications, his total bilirubin and INR continued to rise to a peak of total bilirubin 19.3 mg/dl and INR 1.6, respectively (Table 1). Given concomitant AIDS and recent ART initiation, there was concern for immune reconstitution inflammatory syndrome, and liver biopsy was performed. The liver biopsy showed a cholestatic hepatitis pattern of injury (Figure 1), typified by a predominant component of cholestatic injury with only mild lobular necroinflammatory activity. There was moderate hepatocellular and canalicular cholestasis; the parenchyma also showed foci of lobular inflammation with apoptotic hepatocytes and patchy hepatocyte dropout. Occasional loosely formed granulomas were identified in the hepatic lobules. The portal tracts were expanded by a mixed inflammatory infiltrate with readily identifiable eosinophils. While occasional interlobular bile ducts exhibited degenerative epithelial changes in keeping with bile duct injury, there was no evidence of bile duct loss. Special stains for acidfast bacteria, fungi, cytomegalovirus, and Epstein-Barr virus were negative. The cholestatic hepatitis was favored to be drug-induced, most likely related to dapsone, especially in the context of portal eosinophil infiltrates and lobular granulomas. ART was re-started and symptoms and liver tests gradually improved to normal over a period of 8 weeks. Figure 1C. Also note small, loosely formed granuloma.
Discussion
Dapsone has been linked to cases of idiosyncratic cholestatic liver injury and also hypersensitivity syndrome (a form of drug reaction with eosinophilia and systemic symptoms also known as DRESS) [4]. Review of interactions among the patient's medications does show that ritonavir and darunavir in combination with dapsone can lead to a very minor increase in dapsone levels through decreased CYP3A4 metabolism-this is classified as a "non-significant" or "minor" interaction by Micromedex though it could have contributed to the injury seen in this case. The case reported here is a rare presentation that demonstrates both cholestastic liver injury (R=0.5) and hypersensitivity syndrome (fever, rash, eosinophilia, hepatitis) with a time course, injury pattern, and resolution typical of dapsone [1,3].
Given the presence of a hypersensitivity component in this case, we considered administering corticosteroids, but the patient was already on a steroid taper for treatment of P carinii. There are no randomized controlled data to support steroids in DILI, although there may be benefit in dapsone-related DILI when patients have hypersensitivity features of fever, rash, and eosinophilia [1,3]. The potential benefit from steroids is likely related to the proposed pathogenesis of dapsone-related injury as a hypersensitivity mechanism, perhaps through its metabolism to an antigenic metabolite [1,5]. In summary, there is a potential for idiosyncratic liver injury and hypersensitivity syndrome with dapsone exposure, especially in patients with prior history of hypersensitivity to other drugs. The pattern of liver injury is cholestatic (R<2), and is likely mediated by the host's intrinsic and adaptive immune response. * R is defined as serum ALT/upper limit of normal for ALT divided by serum alkaline phosphatase [AP]/upper limit of normal for AP. By convention, R<2 denotes 'cholestatic' type liver injury. | 2019-01-19T14:13:24.663Z | 2015-06-28T00:00:00.000 | {
"year": 2015,
"sha1": "f4d660d12c7691b5eda1324f57aa49fc25076104",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.4172/2161-0495.1000253",
"oa_status": "HYBRID",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "d36abfb434f2af21dcde041d01402279386dfc7f",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
186200758 | pes2o/s2orc | v3-fos-license | Higher quality quit-date goal setting enhances quit attempts among quitline callers
INTRODUCTION At tobacco quitlines, coaching and cessation medications are commonly structured around setting a date for making a quit attempt. However, limited literature evaluating this practice suggests that callers do not routinely set quit-date goals. High quality goal setting may increase the frequency of caller quit attempts. In this study, we examine the quality of quit-date goal setting and its association with in-program quit attempts and the timing of callers’ first quit attempt. METHODS Using call recordings, we scored the quality of quit-date goal setting among 90 callers enrolled at Arizona Smokers’ Helpline between August and December 2017. The primary exposure was quality of quit-date goal setting assessed using the Lorencatto et al. rating scale. Coding reliability was assessed using Cohen’s kappa. Multivariable logistic regression was used to examine the association between quality of goal setting and in-program quit attempts (>24 h tobacco free). RESULTS The mean quality goal setting score was 3.1 (range: -3 to 7). Sixty-nine callers (77%) set a quit date and 39 (43%) made a quit attempt. Compared to callers who experienced low-quality goal setting, the adjusted odds of in-program quitting for high quality goal setting was AOR=3.98 (95% CI: 1.55–10.20) and for making a quit attempt within two weeks OR=6.23 (95% CI: 1.52–25.49). CONCLUSIONS Quit-date goal setting is an important element of quitline services and callers benefit from high quality quit-date goal setting. Quitlines should establish quality improvement measures to ensure that coaches are trained to provide high quality quit-date goal setting opportunities to all callers.
INTRODUCTION
Tobacco use remains the leading preventable cause of death and disease in the US and globally 1 . Quitting is difficult, and most tobacco users make multiple attempts before they successfully become abstinent 2 . Clinical practice guidelines recommend that cessation services, like those offered by quitlines, provide nicotine replacement medication and coaching to educate and support tobacco users to develop coping skills and make positive behavior changes 3 . Quitlines improve individuals' odds of quitting and have become the standard of care for tobacco cessation 4 .
At quitlines, it is recommended that services are structured around setting a quit date as a goal for callers to become tobacco abstinent 5,6 . Quit dates provide a point of focus for providing behavior change coaching and establishing a schedule to begin using cessation medication. In their first session, coaches should encourage callers to select a day, usually within two weeks, after which they will no longer smoke or use tobacco 7 .
Despite quitlines' emphasis on setting quit dates, many callers do not. In a research trial, only 32% of quitline participants who indicated an intention to quit in the next 30 days set a quit-date goal 8 . Several factors may influence the decision to set a quit date. However, without high quality services to support quit-date goal setting, callers are not likely to do so. To examine the quality of quit-date goal setting, Lorencatto et al. 6 developed a rating scale and scored the quality of setting quit-date goals. They found, on average, that the results were 'low quality' and only 21% of callers made an inprogram quit attempt. However, compared to lowquality goal setting, when high quality goal setting was delivered, callers were more likely to make an in-program quit attempt (OR=2.60, 95% CI: 1.54-4.40). Addressing the quality of goal setting is an important step to improve quitlines' ability to work with callers and clarify potential discrepancies between their desired behavior change and current behavior.
In this study, we examined one primary and two exploratory analyses. In our primary analysis, we sought to advance the Lorencatto et al. 6 work by investigating whether the quality of quitdate goal setting was associated with callers making an in-program quit attempt at the Arizona Smokers' Helpline (ASHLine). We hypothesized that high quality goal setting would be associated with increased odds of making a quit attempt. Recognizing that smokers with mental health conditions (MHC) have historically smoked at higher rates and experienced greater difficulty in quitting 9 , we also examined whether having an MHC modifies the influence of high quality goal setting.
In two exploratory analyses, we further examined whether high quality goal setting is associated with: a) making a quit attempt within two weeks, and b) long-term tobacco cessation. Given that the Lorencatto et al. 6 quality rating scale prioritizes setting quit dates within two weeks of the first coaching session, in our first exploratory analysis we hypothesized that callers who experience high quality goal setting will make a quit attempt sooner than those who receive low-quality goal setting. In the second analysis, to assess if high quality goal setting is associated with long-term quit outcomes, we examined callers' odds of being abstinent at the 7 months follow-up. We hypothesized that high quality quit-date goal setting would be associated with increased odds of being abstinent at follow-up.
Study setting
ASHLine provides behavioral coaching and four weeks of nicotine replacement therapy (NRT) in the form of patches, gum, and lozenges. Callers are assigned a dedicated coach and receive motivational interviewing and elements of cognitive behavioral therapy delivered over seven sessions. Coaches contact enrolled callers weekly to help them identify triggers, set quit dates, develop strategies to manage their urges to smoke, and provide support. In accordance with ASHLine's client-centered service approach, motivational interviewing techniques are used to explore if and when callers are interested in setting a quit-date goal. Once abstinent, check-in calls are made less frequently. Participation ends when callers: 1) complete the program, or 2) indicate they no longer desire coaching calls or are unreachable (early exit). Callers complete the program at 90 days or once they have received seven coaching sessions. Program participation is not contingent upon setting a quit-date goal.
Study sample
We selected callers who completed or exited the program between 1-10 January 2018. Of the 175 callers who completed or exited during this time, 90 met the study's inclusion criteria. Reasons for exclusion are provided in Figure 1. Because our primary dependent variable involved quitting while in-program, this allowed us to ensure that selected callers were no longer active in the program. The sample was also restricted to callers who enrolled at ASHLine between August and December 2017. In August, ASHLine implemented a coaching protocol that included guidelines for delivering quit-date goal setting congruent with the Lorencatto et al. 6 quality goal setting scale.
We conducted a power analysis and identified that we needed a sample of at least 88 callers. This estimate assumed one-tail directionality, alpha error probability of 0.05, beta error probability of 0.20, and an expected odds ratio (OR) of 2.0. The OR estimate was based on results from the Lorencatto et al. 6 study.
Data collected in the enrollment survey and during counseling sessions were de-identified for analysis. We followed STROBE (Strengthening The Reporting of Observational Studies) checklist guidelines 10 and the study was reviewed and approved by the university's Institutional Review Board.
Primary independent variable
Quality of quit-date goal setting is the primary independent variable. We analyzed audio recordings of callers' first coaching session and measured quitdate goal setting quality using the Lorencatto et al. 6 rating scale. The scale comprises 10 components that reflect positive or negative elements of quit-date goal setting (Table 1). One point was awarded or subtracted based on the presence of each element. Points were then totaled into an overall score for each caller with a potential range of -3 to 7. To accommodate ASHLine's process for providing NRT to callers, the criterion for setting a specific quit date was defined as either selecting a date (i.e. dd/mm/yy) or an event (i.e. arrival of NRT). Because NRT sent from ASHLine can take between five and ten days to arrive at callers' place of residence, this coding assured that in the analysis callers were positively awarded for setting a specific timeline for initiating their quit attempt. Upon conclusion of scoring, we found that the quality goal setting was bimodally distributed with a median quality score of 3.0. For analysis, we dichotomized scores and organized callers into low (-3 to 3) and high-quality groups (4 to 7).
In scoring quality goal setting, one reviewer coded all 90 sessions and a second scored a subset of 15 sessions. This process allowed us to audit the data and assess criteria clarity. External audits are useful for confirming agreement and verifying data trustworthiness 11 . Dual coding only a selected portion of the data also reduces the cost and effort of multiple coding entire datasets 12 . To complete Score Quit-date goal setting components 0 Absence of goal setting (no invitation to set a quit-date goal or behavioral support is delivered by the coach) 1 The coach encourages callers to set a quit-date goal 1 *Agreed quit date is a specific date (dd/mm/yy) OR a specific event (e.g. arrival of NRT) 1 Quit date is scheduled within 14 days following the first coaching session 1 The quit date is scheduled to allow time to first obtain NRT or cessation medication 1 Coach discourages callers from beginning to reduce or cut down smoking/provides advice that reducing is less effective than abruptly quitting on a planned date 1 Coach explains that a quit date entails becoming completely abstinent (e.g. NRT replaces cigarettes, no smoking, not even a puff, after the quit date) 1 Coach provides information and examples about effective behavior changes or NRT/cessation medication use strategies to support quitting and remaining abstinent -1 Coach encourages callers to reduce or cut down smoking before a scheduled quit date -1 Set a 'flexible' quit date or a goal that is not clearly defined as a specific day/date -1 The quit date is not scheduled within 14 days following the first coaching session and/or does not allow caller time to obtain NRT or cessation medication Table 1. Lorencatto et al. 6 quality of goal setting rating scale *Twelve codes were used to score the quality of quit-date goal setting. Originally, setting a specific date or a specific event were coded separately. However, they were combined for analysis and callers were awarded a point if they set a specific quit date OR a specific event. This preserved the Lorencatto et al. 6 original score range (-3 to 7). NRT: nicotine replacement therapy. the audit, the first five sessions were coded by two reviewers and compared. Based on observed discordance, criteria were adjusted and the next five sessions were again co-coded and compared. Once the criteria were deemed satisfactory, the primary reviewer scored all remaining sessions. Five additional sessions were randomly selected and cocoded to assess agreement within the full sample. In total, 15 sessions were dual coded. These comprise 180 data points-12 codes for each caller's session. Eleven codes were based on the Lorencatto et al. 6 original components plus an additional code: setting a goal around a specific event. To examine intra-rater reliability, 10 sessions (120 data points) were also repeat-coded by the primary reviewer at different time points. We used Cohen's kappa statistic to compare coding differences 13 . Separate kappa scores are reported for the first five training sessions, the remaining 10 inter-rater audited sessions, and the 10 intra-rater sessions.
Dependent variables
The primary dependent variable was making an inprogram quit attempt. This was defined a priori as being abstinent from tobacco for at least 24 hours, consistent with recommended guidelines 14 . These data were based on caller self-report and were collected by coaches who assessed callers' tobacco use status in each phone session and recorded the date of their in-program quit attempts. In the primary analysis, quit attempts were classified as a dichotomous outcome (yes/no). In the exploratory analyses, we assessed whether quality of quit-date goal setting was associated with time (in days) until making the first quit attempt. We dichotomized days until abstinent (1-14, ≥15) to reflect the two-week, optimal time frame as defined in the Lorencatto et al. 6 quality of goal setting rating scale. We also assessed tobacco abstinence at the 7 months follow-up. This was measured as self-reported, 30-day point prevalence abstinence.
Covariates
To account for potential differences in callers' motivation and ability to quit, we selected control variables prior to analysis, based on literature review. In quitline settings, researchers have found that household smoking bans 15 , not having a mental health condition 16 , and higher confidence to quit 17 are positively associated with quitting. In other settings, living with other smokers 18 , higher nicotine dependence 19 , and lower levels of education 20 , have been found to be negatively associated with cessation outcomes. We also included self-reported age, race, gender and ethnicity, which are known psychosocial determinants of smoking 21 . Because all 90 callers reported an intention to quit smoking in the next 30 days, the variable was not included in the analysis. We measured comorbidities by callers' self-report of ever having been diagnosed by a healthcare professional with: asthma, cancer, COPD, diabetes, heart disease, or hypertension (yes/no). Mental health status was similarly assessed as having ever been diagnosed with: anxiety disorder, depression, bipolar disorder, schizophrenia, or alcohol/drug abuse disorder (yes/no). The Fagerström test was used to measure nicotine dependence (low 0-2, moderate 3-5, heavy 6-10) 22 and home smoking bans were categorized as smoking not allowed anywhere in the home (full ban), smoking allowed in some places (partial ban), and smoking allowed anywhere (no ban). We dichotomized other smokers in the home (yes/no) and callers' confidence in being able to remain abstinent for a 24-h period. Caller confidence was defined as not confident (not or somewhat confident) and confident (confident, very confident, or extremely confident). Finally, we included a categorized count of callers' previous ASHLine enrollments (0, ≥1).
Primary analysis
This study was a retrospective cohort study. We described characteristics for callers who experienced high-quality and low-quality quit-date goal setting ( Table 2). We used chi-squared, Fisher exact and unpaired two-sample t-tests to examine differences between them. We used multivariable logistic regression models to examine the adjusted and unadjusted odds and 95% confidence intervals (95% CI) for making an in-program quit attempt by quality of quit-date goal setting. Our a priori hypothesis was that, compared to low-quality goal setting, highquality goal setting would be associated with higher odds of making a quit attempt. To examine potential moderation from having a mental health condition, we used a likelihood ratio test to assess its interaction with quality of quit-date goal setting. In a multivariable logistic regression model, we included the caller demographic, tobacco history and program use variables listed in Table 2. This represented our full model. To avoid over-fitting the model, we used backwards selection to remove variables not significantly associated with making an in-program quit attempt. Covariates were excluded with a p-value >0.05. Quality of quit-date goal setting and age remained in the model. This represented our reduced model. We used a gamma test to examine the pairwise association among the independent variables 23 . We used a likelihood ratio test to examine differences between the full and reduced models.
Exploratory analyses
In the exploratory analyses, we assessed the relationship between quality of quit-date goal setting and making a quit attempt within two weeks of initiating coaching services. For this, we fit a second logistic regression using a subset of callers from the original sample who made an in-program quit attempt (n=39). To keep the independent variable distinct from the dependent, we re-scored the independent variable, quality of quit-date goal setting, by removing the two elements that penalized and rewarded setting long and short quit-date goals, respectively. Beginning with the same set of covariates used in the primary analysis, we again used backwards selection to fit a reduced model. We forced quality of quit-date goal setting into the model and set a stopping rule to drop all other variables with a p-value >0.05. Only quality of quit-date goal setting remained. We repeated these steps to also examine the odds of smoking abstinence among callers reached at the 7 months follow-up (n=33) by quality of quit-date goal setting. As before, only quality of goal setting remained in the reduced model. The full and reduced models for both of the exploratory analyses were compared using a likelihood ratio test. Statistical tests were performed using Stata 15.1 (StataCorp LLC. 2017 College Station, TX).
RESULTS
We reviewed 90 ASHLine callers' first coaching session delivered by 15 coaches. The inter-rater Cohen's kappa for the five training sessions was 0.59. This is interpreted as 'weak' agreement 13 . Following adjustment to the coding criteria, the additional 10 inter-rater sessions had a kappa of 0.94 and the intrarater Cohen's kappa was 0.92. These represent 'almost perfect' levels of agreement 13 . Forty-three callers experienced high-quality quit-date goal setting and 47 low-quality goal setting. The mean quality score was 3.1. While in-program, 69 callers (77%) set a quit-date goal and 39 (43%) made a quit attempt. Among callers who made a quit attempt, 33 (85%) had recorded a quit-date goal and 25 (64%) experienced high-quality goal setting. Compared to those who experienced low-quality quit goal setting, callers who experienced high-quality goal setting were more likely to have set a quit date in their first coaching session (p<0.001) and to have made an in-program quit attempt (p=0.007). No other demographic, tobacco use history or program engagement characteristics were statistically different between the high-quality and low-quality groups ( Table 2).
In the adjusted model, callers who experienced high-quality goal setting were more likely (AOR=3.98, 95% CI: 1.55-10.20) to make an inprogram quit attempt than those who experienced low-quality goal setting (Table 3). Compared to callers 45-64 years old, those over 65 years were also more likely to make a quit attempt (AOR=3.83, 95% CI: 1.15-12.68). Pairwise association tests showed no association between covariates in the reduced model. We found that MHC did not moderate the association between quality quit-date goal setting and in-program quitting; the likelihood ratio test showed that the interaction term was not significant in the model (p=0.27).
In our first exploratory analysis, using the re- scored, quality of goal setting independent variable, we found that among callers who made an inprogram quit attempt, the odds of making a quit attempt within two weeks of the first coaching session was greater (OR=6.23, 95% CI: 1.52-25.49) for those who experienced high-quality quit-date goal setting compared to low-quality goal setting.
In the second, we explored long-term quit outcomes among 33 callers reached at follow-up (37% response rate). Of those, 14 indicated that they had not smoked in the past 30 days for an intent-to-treat (ITT) quit rate of 16%. The ITT quit rate was 21% for callers who experienced high-quality quit-date goal setting and 11% for those who experienced lowquality goal setting. However, the difference in odds of being abstinent were not statistically significant (OR=2.00, 95% CI: 0.49-8.24) for callers who experienced high-quality compared to low-quality goal setting. As with the primary analysis, we used the reduced model in both exploratory analyses; the likelihood ratio tested showed no difference between full and reduced models for each.
DISCUSSION
Consistent with the Lorencatto et al. 6 study, we found that delivery of high-quality quit-date goal setting was positively associated with callers making an inprogram quit attempt. Higher quality goal setting was also associated with increased odds of callers reporting a quit attempt within two weeks of their first coaching session. Callers' long-term odds of being abstinent were not significantly different for those who experienced high-quality versus low-quality quit-date goal setting. Given that the ITT quit rate for high-quality goal setting was almost double the quit rate for low-quality goal setting (21% vs 11%), this finding is likely due to the small and insufficient sample of callers who completed the follow-up assessment at 7 months. In the Lorencatto et al. 6 pilot study, using the quality of goal setting rating scale, an average quitdate quality goal setting score of 1.6 was described with 21% of the sample initiating a quit attempt. In our sample, both the quality of quit-date goal setting and the prevalence of quit attempts were higher, with an average quality of quit-date goal setting score of 3.1 and 43% of sampled callers reporting an inprogram quit attempt. This difference may reflect ASHLine coaching protocols and routine training that include components from the quality of goal setting rating scale; prior coach training was not described in the Lorencatto et al. 6 study.
These findings highlight the importance of the quality of goal setting with regard to setting a quit date. They suggest that when quality quit-date goal setting is consistently delivered, a higher portion of callers will initiate a quit attempt. The quality of goal setting rating scale stresses the importance of setting a proximal quit date and combining it with NRT or other cessation medications. These stood out as important elements of quit-date goal setting. Setting a proximal quit date orients callers' use of cessation medication and provides a point of focus around which to plan subsequent counseling sessions 24 . Because most quitlines limit callers' number of counseling sessions, timely initiation of this process is important. Early and open discussions around setting quit-date goals may clarify program objectives, normalize callers' expectations, and facilitate coach and callers' ability to articulate potential ambivalence around quitting tobacco.
Limitations and strengths
Strengths of our study include a statistically powered sample size, review of recorded coaching calls in a sample of coaches trained in quit-date quality goal setting, and use of a previously used instrument for assessing the quality of goal setting. While using the quality of goal setting rating scale may introduce bias in scoring, our data suggest this was well-controlled in our sample with high inter-coder and intra-coder reliability kappa scores. Limitations include the selfreported nature of our primary outcomes including quit attempt and timing of quit attempts. This is an observational study based on a small cohort of callers enrolled in a single quitline. Therefore, the results may not translate to other tobacco cessation services or to the general population of tobacco users. The sample may also be biased as 5% of potential participants were excluded due to missing data, although a comparison of demographic and clinical characteristics did not suggest any significant differences.
In interpreting these findings, it is important to recognize that quality of goal setting rating scale rewards elements that relate to coaching and caller characteristics. It captures an experience to which both contribute. For example, the scale accounts for coaches encouraging callers to set a quit-date goal and whether callers agree and select a date. However, the scale and our analyses did not consider all coaching and caller characteristics that may be associated with quit attempts and cessation. Regarding caller characteristics, we did not assess for specific MHCs or NRT use. Related to coaches, our analyses did not account for differences among coaches, including potential variation in coaching skills. While this is less concerning given the large number of coaches included in the study (n=15), it is plausible that if a coach had a lenient attitude toward quit-date goal setting, leniency towards other key coaching tasks may also be demonstrated, such as commitment to applying motivational interviewing techniques. If present, these differences may have influenced the findings, especially among callers whose quit-date goal setting was scored as low quality. Finally, the study was designed to assess the primary research question. The exploratory analyses may have been underpowered due to small sample sizes.
Future research
We found evidence that high-quality quit-date goal setting is associated with an increased likelihood of making a quit attempt. We suggest four areas where future research may examine this relationship further. First, future work should determine if standardized quitline training emphasizing high-quality quit-date goal setting practices would improve the quality of quit-date goal setting and enhance the frequency of caller quit attempts. It has been shown that instruments like structured checklists increase protocol compliance and outcomes in care delivery settings 25 . It will be important to develop similar instruments and a fidelity monitoring process to ensure routine delivery of highquality quit-date goal setting.
Second, to facilitate standardized quit-date goal setting, it may be beneficial to explore ways to improve the quality of goal setting rating scale. Measurement instruments vary in how they aggregate score components 26 . Some dichotomize quality by measuring indicators using an 'all or nothing' logic. Others equally weight scale elements and create a composite score by summing present elements 27 . The Lorencatto et al. 6 scale uses the latter method. An additional approach is to weight elements that are regarded as more important than others 28 . This third option may be better suited for measuring the quality of quit-date goal setting. In our study, we identified a bimodal distribution in the quality of quit-date goal setting scores. This suggests that some elements may 'stick together'. If there are core elements like setting proximal quit dates, future research could assess whether less important elements are dependent upon them. Thus, it may be beneficial to simply construct a scale with fewer components. For example, of the Lorencatto et al. 6 10 components, only a few were independently associated with quit attempts. A reduced scale would likely enhance evaluators' ability to use it to actively monitor program performance. A randomized experimental design and factor analysis may be appropriate to explore this further.
Third, in the Lorencatto et al. 6 scale, it is assumed that reducing or cutting down one's smoking prior to a planned quit attempt is negative. This appears to contradict literature that shows little or no difference in outcomes between smokers who quit abruptly compared to reducing prior to a quit date. Findings that quitting abruptly may be superior to cutting down 29 attenuate when smokers are consciously attempting to reduce. For example, when instructed to reduce, smokers making a quit attempt demonstrate similar 30 or superior 31 abstinence outcomes compared to abrupt quitters. This finding holds among studies examining cutting down while using 32 or prior to using NRT 33 . We do not find a rationale to discourage callers who desire to begin reducing their smoking prior to a quit date. We checked this assumption by repeating the primary analysis using a modified independent variable. The recalculated quality of quit-date goal setting score excluded the components that rewarded and penalized discouraging and encouraging callers to reduce their smoking prior to making a quit attempt, respectively. While still statistically significant, this change (OR=3.07, 95% CI: 1.22-7.70) attenuated the odds of making an in-program quit attempt compared to the original analysis (OR=3.98, 95% CI: 1.55-10.20). To examine this further, future studies should measure callers' intention and progress in reducing at the time of enrollment and as a behavior change strategy in their coach-facilitated quit plan.
Finally, future studies should further explore the association between quality of quit-date goal setting and long-term quitting and relapse. Studies with larger and more diverse samples would allow researchers to investigate additional caller characteristics and their readiness to change. This would also enable a more detailed view of the relationship between MHC and quit-date goal setting. Despite evidence that having an MHC decreases quitline callers' likelihood of quitting tobacco 9,16,34 , we did not find that MHC modified the association between quality of quit-date goal setting and in-program quitting. In a better powered, longterm follow-up analysis, it would be possible to explore if individuals with an MHC, controlling for quality of goal setting, experience greater difficulty in remaining abstinent. Future analysis would also benefit from controlling for type of MHC.
CONCLUSIONS
Quit-date goal setting is an important element of tobacco cessation coaching, though it has not yet been robustly evaluated in the literature. We found that when callers experienced high-quality quit-date goal setting, their odds of making an in-program quit attempt was four times greater than when quit-date goal setting was lower quality. High-quality goal setting was based on the combination of coaches recommending and clients accepting specific goal-setting conditions, like setting a quit date in conjunction with acquiring NRT and adopting additional behavior changes. This approach also translated to higher odds of making a quit attempt within two weeks of the initial coaching call. Future research should explore interventions to promote high-quality quit-date goal setting, including routine monitoring of quit-date coaching and callers' goal setting practices. | 2019-06-08T20:07:31.445Z | 2019-06-06T00:00:00.000 | {
"year": 2019,
"sha1": "e2cc1d6805dac1724dee38c2235f5a7dfa05f4d0",
"oa_license": "CCBY",
"oa_url": "http://www.tobaccopreventioncessation.com/pdf-109537-40351?filename=Higher%20quality%20quit_date.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "e2cc1d6805dac1724dee38c2235f5a7dfa05f4d0",
"s2fieldsofstudy": [
"Medicine",
"Business"
],
"extfieldsofstudy": [
"Psychology",
"Medicine"
]
} |
13171791 | pes2o/s2orc | v3-fos-license | A new approach to evaluate the impact of climate on human mortality.
The objective of this study is to introduce a new procedure to determine the impact of climate on human mortality with the use of a synoptic climatological approach. The holistic nature of synoptic categories allows for the simultaneous evaluation of numerous weather elements as they realistically appear within air masses. In addition, this approach allows for a better distinction between pollution-induced mortality and weather-induced mortality. A synoptic categorization was performed for St. Louis, Missouri, and each category was evaluated in terms of its mean daily mortality. Of 10 summer categories found in St. Louis, one possessed the highest mean mortality by far, and 8 of the top 10 mortality days in St. Louis occurred when this category was present. Further analysis determined that long, consecutive day periods of this hot, oppressive category are associated with a continuing rise in mortality. It was determined that the procedure described here has the potential to be used in a weather/mortality watch-warning system. Finally, it appears that day-to-day mortality fluctuations are much more sensitive to weather than to pollution concentrations, as the oppressive category associated with the greatest mortality possessed levels of six major pollutants that were not noteworthy.
Introduction
The impact ofvariable climate on human health and well-being has been the subject ofnumerous studies, with the majority ofthe work being performed by medical specialists and a small minority by climatologists. With the threat ofan anthropogenic-induced global warming, the interest in climate/human health studies has increased, and at least three comprehensive reports summarizing most of this research have appeared in recent years (1)(2)(3).
The majority of the climate/human health evaluations have concentrated on mortality, and virtually all of the studies correlate a number of climate variables with daily or weekly mortality statistics. For example, medical researchers have noted that mortality attributed to weather seems to vary considerably with age, sex, and race, although there is disagreement in defming the most susceptible population group (4)(5)(6). Some researchers have found that many causes other than heat stroke increase during extreme weather (7Z8). Evaluations have been performed for winter weather impacts as well, and mortality increases have been noted during extreme cold waves (6)(7)(8)(9)(10)(11). More recent studies have concentrated on interregional impacts of weather on human health, and the potential adverse effects ofglobal climate change (12,13). In addition, the sophistication of climatic modeling within these has increased, and "threshold temperatures," which represent the temperature beyond which mortality significantly increases, have been identified for numerous cities (14).
All of these studies suffer from at least two shortcomings. First, the use ofindividual weather elements to evaluate the impact of climate on human health may provide misleading results. Climate affects human health as a holistic unit composed si-*Center for Climatic Research, Department of Geography, University of Delaware, Newark, DE 19716. multaneously of numerous climatic elements that react in concert. Thus, the isolation ofindividual elements can represent an unrealistic situation that does not duplicate climatic reality. Second, the impact of pollution concentration is often ignored in a climate/mortality evaluation. It is possible that the mechanisms responsible for increasing mortality are truly pollution-oriented, but these are imbedded (and ignored) within the climate evaluation. It is not feasible to evaluate individual pollutants (e.g., SO2 or NO2 concentrations) along with individual climate elements (e.g., temperature or dew point) as independent variables within a health study because the climate and pollution variables are somewhat collinear.
The objective ofthis study is to introduce a new procedure to determine the impact ofclimate on human mortality that does not suffer from the problems outlined above. This involves the use of a synoptic climatological approach, which combines weather elements into groups or categories that are representative oftrue meteorological situations at a moment in time (15). Synoptic climatological approaches characterize similarities in general circulation patterns and active meteorological elements within a holistic framework, facilitating analyses ofclimate's impact on a variety of environmental variables such as human mortality. Thus, the impact ofthe totality ofweather on the environmental variable can be determined. Additionally, synoptic categories possess differential abilities to retain high concentrations of pollutants. Thus, certain synoptic categories are usually associated with low pollutant concentrations, while others are almost always highly polluted. Kalkstein and Corrigan (16) determined that the 9 most polluted days in Wilmington, Delaware, during the winters of 1974-1978 were associated with two synoptic categories that occurred only 15 % of the time during the period. It is clear that this approach can distinguish between offensive weather situations and offensive pollution situations.
Methodology
The National Center for Health Statistics (NCHS) provided us with a very detailed mortality database that contains records of every person who died in the United States from 1964 to the present [refer to NCHS (17) for a detailed description of these records]. We selected St. Louis, Missouri, for our test location, as previous research suggests that the area is particularly sensitive to heat-related deaths (14). The data contain cause ofdeath, place of death, age, date of death, and race, and these were extracted for the St. Louis Standard Metropolitan Statistical Area for 11 years: 1964-66, 1972-78, and 1980 (although the data are complete for the entire record, during intervening years the date of death was omitted). The number of deaths for each day was tabulated and divided into categories based on age (all ages and elderly, classified as greater than 65 years old) and race (white and nonwhite).
All mortality data were adjusted to account for changes in the age, racial distribution, and total population ofthe St. Louis area during the period of record. A direct standardization procedure was used, and a standard population was constructed for each of the evaluated mortality categories [refer to Mausner and Bahn (18) and Lilienfield and Lilienfield (19) for a discussion of this standardization procedure].
An automated air-mass-based synoptic climatological index was developed for St. Louis to categorize each day into its particular synoptic category. The synoptic procedure is designed to classify days that are considered to be meteorologically homogeneous. This is accomplished by defining each day in terms of seven readily available meteorological elements, which include air temperature, dew point temperature, visibility, total cloud cover, sea-level air pressure, wind speed, and wind direction. There elements are measured four times daily (0100, 0700, 1300, 1900 hr local standard time), and the developed 28 variables represent the basis for categorization.
The developed temporal synoptic index (TSI) uses principal components analysis (PCA), a factor analysis technique that rewrites the original data matrix into a new set of components that are linearly independent and ordered by the amount of variance they explain [for a detailed explanation of this procedure, refer to Kalkstein et al. (20)]. Component loadings are calculated, which express the correlation between the original 28 variables and the newly formed components. Each day is then expressed by its particular set of "component scores," which are weighted summed values whose magnitudes are dependent on the weather observation for each day and the principal component loading. Thus, days with similar meteorological conditions will tend to exhibit proximate component scores.
A clustering procedure was then used to group those days with similar component scores into meteorologically homogeneous groups. There are numerous clustering methodologies available, but the one deemed most efficient in the development ofa synoptic climatological index is the average linkage method (20). Once the groups have been determined, average values are calculated for the 28 meteorological variables for all days within each particular group. Weather map classification is also possible by reviewing maps for those days within a group and describing general similarities.
The mean daily mortality for each synoptic category, along with the standard deviation, was then determined to ascertain whether particular categories were distinctively high or low. Potential lag times were accounted for by evaluating the daily synoptic category on the day ofthe deaths, as well as 1, 2, and 3 days before the day ofthe deaths. Daily mortality was then sorted from highest to lowest during the period of record to determine whether certain synoptic categories were prevalent near the top or bottom.
Mean daily pollution concentrations were also examined for each synoptic category in an attempt to determine if the synoptic situations associated with the highest mortailty are also the most polluted. The EPA made available mean daily pollution concentrations for six raw pollutants at several automatic recording monitoring stations in the St. Louis area. The monitor with the most complete record was selected for this study, and mean daily values of the following pollutants were extracted: total suspended particulates (TSP), NO., NO2, total oxidants, 03, and S02. It was also noted whether the highest mortality days within each synoptic category were the most polluted.
Synoptic Categorization
Ten major summer synoptic categories were uncovered for St. Louis and were identified based on their mean meteorological characteristics (Table 1). Two hot, oppressive synoptic situations, categories 6 and 9, were identified, with the latter being more extreme. Several anticyclonic categories were identified, which generally possess the most stable atmospheric situations. Two categories (4 and 8) were frequently associated with convective precipitation patterns and represented unstable atmospheric conditions. Mean pollution concentrations varied considerably between categories ( Table 2). The three anticyclonic synoptic patterns (categories 1, 2, and 3) generally possessed the highest concentrations for most of the pollutants. Category 2 exhibited the highest mean TSP, ozone, and NO, concentrations, while category 1 was highest for NO2 and oxidants. The only exception was for SO2, where the highest concentrations were found within maritime tropical category 5. The two hottest synoptic situations, categories 6 and 9, were generally not distinctive, except for a moderately high TSP reading for category 6. The most oppressive synoptic situation, category 9, generally possessed mean pollutant readings near or below the overall norm. A cursory evaluation ofthe very hottest category 9 days indicated that they were no more polluted than any of the other days within the category.
Mortality Associations
An evaluation of mortality for the 10 synoptic categories uncovered some startling results (Table 3). After examining the potential lag-time relationships, it was determined that a 1-day lag existed between the causal synoptic mechanism and the mortality response (the between-category differential in mean daily mortality was greatest for a 1-day lag situation), with mean mortality values up to 52% higher than the other categories. The distinction between category 9 and the other synoptic situations was especially strong for elderly and nonwhite mortality. Interestingly, the second hottest synoptic situation, category 6, possessed mortality means that were almost identical to the other nondistinctive categories. This seems to imply that weatherinduced mortality rises dramatically only after a particular oppressive threshold is exceeded. It appears that only category 9 possesses conditions that exceed this threshold for St. Louis.
It is noteworthy that almost all the highest mortality days in St. Louis occurred when category 9 was present (Table 4). For total mortality, 8 ofthe 10 highest days occurred under the domination of category 9, even though this oppressive category occurred only 7% ofthe time during the period of study. This finding suggests that a vast majority of deaths that can be attributed to weather occur only when category 9 is present. For the 20 highest mortality days, category 9 was present 11 times. Although this is a bit less impressive, this still represents 8 times the expected occurrence of category 9 over a typical 20-day period. Category 6 only occurred once within the top 20 mortality days, further suggesting that the second hottest synoptic category is not oppressive enough to induce any extra deaths. Categories 3 (anticyclonic, marine influence) and 5 (maritime tropical, cloudy) are present among the top 20 days, but their frequencies are not much different from their expected occurrence over a typical 20-day period.
Standard deviations in mortality within the 10 synoptic tyI indicate further that category 9 is distinctive ( Table 5). The stE dard deviations for all the synoptic situations are somewl similar with the very apparent exception of category 9, whi possesses values about three times higher than the other ca gories. This indicates that, although most ofthe highest mortal days are associated with category 9, numerous days within t synoptic type possess much lower mortality totals. An evaluati of all days within category 9 confirms this assumption, a numerous category 9 days exhibit mortality totals at sometimes below the means of the other synoptic types.
In an attempt to understand why daily mortality shows su considerable variation within category 9, a detailed with: category evaluation was performed for total and elderly morta ty. The objective was to determine which meteorological a pollution-oriented aspects of synoptic category 9 are respon ble for the highest mortality. To with the temperature hovering near 95 F for many hours appears to have a greater influence on mortality than a day where the temperature exceeds 100T for a short time but falls rapidly to normore tolerable levels [refer to Kalkstein and Davis (14) for details on the computation]. A final nonmeteorological independent variable was included, "day in sequence," which noted how a particular category 9 day was positioned within a consecutive day my sequence. For example, if the day in question was the third of a category 9 consecutive day sequence, it was assigned a value of pes 3. All single-day category 9 days that were not part of a conan-secutive sequence were assigned a value of 1. hat The results explained much ofthe variance in mortality within ich category 9 ( Table 6). The day in sequence variable explained the te-largest proportion of the variance in both the total and elderly lty mortality models, and the direct relationship indicated that a his category 9 day near the end ofa consecutive day sequence is most ion nd ibble 5 bThis value represents an adjusted R2, which has been adjusted for degrees of freedom in the model.
CThis is the day-in-sequence variable. Refer to text for explanation.
important. Thus, a long consecutive day sequence of category 9 days imposes the greatest amount ofstress as expressed by increasing mortality through the sequence. Afternoon wind speed was the second-ranking independent variable in both models, and low wind speeds seem to exacerbate stressful conditions. This finding runs counter to some studies that indicate that higher wind speeds at very warm temperatures increase stress on the body (21,22). Not surprisingly, minimum temperature was a statistically significant variable and was directly related to mortality within category 9. However, maximum temperature was not significant in either the total or elderly models. Visibility was quite important in the total mortality model, and fell just short of statistical significance at the 0.05 level. Interestingly, visibility was directly related, indicating that visibility-reducing high pollution concentrations are not important contributors to increased mortality. Rather, this relationship might imply that the heat load imposed by a transparent atmosphere that increases insolation might be important by further increasing stress.
Discussion and Conclusion
One of the major findings from this synoptic evaluation is evidence suggesting that fluctuations in daily mortality appear to be much more sensitive to stressful weather thanto high levels of pollution. Those anticyclonic synoptic categories with the highest mean pollution concentrations were not associated with unusually high numbers of deaths. Conversely, category 9, which was associated with the highest daily mortality totals, did not possess high mean pollution concentrations for the six pollutants examined. Further, those days within category 9 that were among the top 20 mortality days through the period of study exhibited pollution concentrations somewhat near (and sometimes below) the mean.
These findings run counter to research involving mortality and air pollution in London (23) and morbidity and air pollution in Los Angeles (24). The London study detected a significant relationship between high mortality and British smoke (particulates), while the Los Angeles study noted that chest discomfort, eye irritation, and headache probabilities were associated with high NO2 and SO2 concentrations.
It is quite possible that pollution levels in London are often higher than those in St. Louis, as stringent U.S. clean air laws might keep pollutant levels below health-damaging thresholds.
To determine ifthis was the case, a cursory evaluation similar to the St. Louis analysis was performed for 9 additional U.S. cities (Table 7), and synoptic indices were developed for each of them. Preliminary results indicated that 7 of the 10 a To have a moderate or high weather/mortality signal, a city must possess a synoptic category that has an unusually high mean daily mortality. To have a moderate or high pollution/mortality signal, a city must possess a synoptic category with an elevated pollution level that has an unusually high mean daily mortality. In this preliminary analysis, this was determined subjectively. Another interesting result is the absence of maximum temperature as a significant variable within the category 9 regression models, a surprising finding considering the importance of minimum temperature within both models. This supports a contention made in a previous nonsynoptic climate/mortality study that suggests that oppressive nighttime conditions offering little relief after a very hot day might be more stressful than the maximum temperature itself (14).
The results ofthis evaluation strongly suggest that the synoptic climatological approach is more robust than traditional climatological procedures in the evaluation ofweather/mortality relationships. It was noteworthy that only 1 of 10 synoptic categories which are present in St. Louis during summer is oppressive enough to raise daily mortality beyond baseline levels. This supports the notion that mortality does not increase linearly as weather becomes increasingly stressful. Rather, threshold conditions exist beyond which mortality significantly increases in abrupt fashion.
It is possible that the procedure described here has the potential to be used in a weather/mortality watch-warning system, designed to warn of impending meteorological conditions that could raise mortality significantly among high-risk groups such as the elderly. For example, if a category 9 synoptic situation is approaching, a within-category evaluation can be performed based on the regression analyses presented in Table 6. According to Box and Wetz (25), a regression model may be considered to be a worthwhile predictor ifthe actual F-ratio is larger than some multiple ofthe critical F The magnitude ofthis multiple is dependent on the degrees of freedom. Using the criteria set forth by Box and Wetz (25), the two models presented in Table 6 are acceptable predictors for summer mortality. At this point in the study, no model validation has yet been performed, and the results ofthis paper should be considered for analytical content rather than predictive capabilites. However, the next stage in this research will involve a detailed evaluation ofthe nine additional cities to add further credence to this methodology. In addition, model validation on a data set not used in model development is neccessary to determine if, in fact, this procedure has predictive value in a weather/mortality watch-warning system. | 2014-10-01T00:00:00.000Z | 1991-12-01T00:00:00.000 | {
"year": 1991,
"sha1": "6e15110322db400c8d646c5d7ab567ff3cb2cc41",
"oa_license": "pd",
"oa_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1568248/pdf/envhper00416-0139.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "6e15110322db400c8d646c5d7ab567ff3cb2cc41",
"s2fieldsofstudy": [
"Environmental Science"
],
"extfieldsofstudy": [
"Geography",
"Medicine"
]
} |
15953626 | pes2o/s2orc | v3-fos-license | Hot and cold gas accretion and feedback in radio-loud active galaxies
We have recently shown that X-ray observations of the population of `low-excitation' radio galaxies, which includes most low-power, Fanaroff-Riley class I sources as well as some more powerful Fanaroff-Riley class II objects, are consistent with a model in which the active nuclei of these objects are not radiatively efficient at any waveband. In another recent paper Allen et al. have shown that Bondi accretion of the hot, X-ray emitting phase of the intergalactic medium (IGM) is sufficient to power the jets of several nearby, low-power radio galaxies at the centres of clusters. In this paper we combine these ideas and suggest that accretion of the hot phase of the IGM is sufficient to power all low-excitation radio sources, while high-excitation sources are powered by accretion of cold gas that is in general unrelated to the hot IGM. This model explains a number of properties of the radio-loud active galaxy population, and has important implications for the energy input of radio-loud active galactic nuclei into the hot phase of the IGM: the energy supply of powerful high-excitation sources does not have a direct connection to the hot phase.
INTRODUCTION
In the conventional picture of active galactic nuclei (AGN), accretion of cold matter on to the central supermassive black hole of a galaxy proceeds by way of a luminous accretion disc: the disc provides the radiation field that photoionizes the broad-line region (BLR) and narrow-line region (NLR) in the optical and gives rise to the X-ray emission via Compton scattering. Without radiatively efficient accretion via the disc, none of these standard features of such an AGN would be observed. Unified models propose that a direct view of the BLR, the optical continuum, and the soft X-rays may be obscured (e.g. in Seyfert 2s) by a dusty 'torus': but in this case the torus re-radiates strongly in the mid-IR band, while the hard X-rays are still detectable, so that the presence of a luminous AGN can still be inferred. Heavily obscured nuclear X-ray emission combined with mid-IR radiation is strong evidence for an obscuring torus.
Although this simple picture is broadly consistent with observations of radio-quiet AGN, it has been known for some time (Hine & Longair 1979) that it is not sufficient to explain the properties of radio-loud objects -the radio galaxies and quasars. By analogy with radio-quiet objects, we would expect that face-on radioloud objects (the broad-line radio galaxies and radio-loud quasars) would show optical continuum emission, unabsorbed X-rays, and broad and narrow optical lines, while edge-on radio-loud objects (narrow-line radio galaxies, NLRG) would show narrow lines only in the optical, would have heavily absorbed nuclear X-rays, and would have a clear mid-infrared signature of the absorbing torus.
This radio-loud unified picture does indeed seem to describe the nuclei of many of the most powerful radio sources (e.g. Barthel 1989;Haas et al. 2004), although an additional jet-related X-ray component must be present to explain the nuclear soft X-ray detections of the radio galaxies (e.g. Hardcastle & Worrall 1999;Belsole et al. 2006). However, Hine & Longair observed, in work more recently confirmed by others (e.g. Laing et al. 1994;Jackson & Rawlings 1997), that many radio galaxies do not have the strong high-excitation narrow-line optical emission that is expected from a conventional AGN. The objects lacking these narrow lines, which we shall refer to here as low-excitation radio galaxies (LERGs) are commonest at low radio luminosities: indeed, almost all low radio power (Fanaroff-Riley class I, or FRI) radio galaxies are LERGs. But in samples of radio galaxies the LERG phenomenon persists to high radio luminosities; many powerful FRII radio sources are LERGs as well. LERGs in general show no evidence in the mid-IR for an obscuring torus, either at low or high luminosities (Whysong & Antonucci 2004;Ogle et al. 2006) and their optical nuclei are consistent with being dominated purely by jet-related emission (Chiaberge et al. 2002). The relation between their emission-line and radio properties is also different (Baum, Zirbel & O'Dea 1995). Most recently, we have argued Hardcastle et al. 2006, hereafter H06) that both low-power FRI and high-power FRII LERGs show no evidence for accretion-related X-ray emission, absorbed or unabsorbed, over and above what is likely to originate in the nuclear (pc-scale) jets. It seems most likely that the LERG population simply does not have a radiatively efficient accretion flow, and so produces none of the optical or X-ray characteristics of a conventional AGN. In other words, we and others have argued that LERGs may be a class of luminous active galaxies that accrete radiatively inefficiently, with almost all the available energy from accretion being channelled into the jets.
In this paper we take this argument a step further. We show that it is possible that these apparently different accretion modes may be a result of a different source for the accreting gas, building on the recent result of Allen et al. (2006), who showed that some low-luminosity radio galaxies in the centres of clusters could be powered by Bondi accretion from the hot, X-ray emitting medium, and supporting arguments about the nature of the accretion mode in low-power radio sources recently made by Best et al. (2006). We propose that the low-excitation objects are fuelled by accretion from the hot phase, while the high-excitation objects require fuelling from cold gas. If different types of radio sources do have different accretion modes, then a number of features of the radioloud AGN population can be explained; there are also important implications for the so-called 'feedback' process in which radioloud AGN do work on their hot-gas environments. We begin (Section 2) by using some simple quantitative tests to show that it is at least plausible that accretion from the hot phase can fuel even powerful low-excitation radio sources, but that it is hard to see how it can power the most powerful FRIIs. In Section 3 we discuss some of the wider implications of this model. We summarize our results and discuss future tests of the model in Section 4.
BONDI ACCRETION AND POWERFUL RADIO SOURCES
Without committing ourselves to a particular model of the way in which the accreted material actually powers the jets, we may use the Bondi accretion rate to estimate the amount of power that the central supermassive black hole can extract from accretion of the hot phase of the IGM, following the analysis of Allen et al. (2006). The Bondi rate is given bẏ where λ is a constant which has the value 0.25 for an adiabatic index 5/3, cs is the sound speed in the medium (cs = ΓkT /µmp), rA is the Bondi accretion radius and ρA is the density at that radius. rA is given by so that, for λ = 0.25, we have the simple forṁ and we assume, again following Allen et al., that all material that accretes across the Bondi radius is accreted on to the black hole, so that the available power for AGN activity, PB = ηṀ c 2 , where η is an efficiency factor. To show that the jet can be powered by accretion of the IGM, we require that the jet power Q < ∼ PB. Allen et al. (2006) were able to show that Bondi accretion could supply enough energy to power the radio sources they studied because they were able to estimate the power in the jets Q from the timescales and energies required to inflate observed cavities in the external medium, and the density at the Bondi radius ρA and the temperatures kT by extrapolation from observations. They obtained black hole masses from the mass-velocity dispersion relation (MBH -σ relation) of Tremaine et al. (2002), except in the case of M87 for which a direct MBH measurement was available. For powerful, distant radio galaxies in poorer environments, we generally do not have either a direct measurement of Q, an MBH measurement, a galactic velocity dispersion σ, or a particularly good estimate of the density at the Bondi radius ρA, and so more indirect methods must be used.
It is well known that radio observations alone cannot be used to measure Q for a given radio galaxy without making numerous assumptions about the particle and field content of the lobes, the validity of conventional spectral ageing techniques and the fraction of the jet power that has gone into doing work on the external medium. However, there is a class of low-power objects where a model-dependent but robust jet power measurement can be made. These are the twin-jet FRI sources, where the behaviour of surface brightness and polarization in the (presumed intrinsically symmetrical) jet and counterjet can be used to model jet dynamics (Laing & Bridle 2002a;Canvin et al. 2005;Laing et al. 2006). When combined with X-ray information on the pressure gradient surrounding the jets the dynamical model allows a jet power to be derived. The only source to have a published jet power derived from this method is 3C 31 (Laing & Bridle 2002b) but since 3C 31 is one of the archetypes of its class, and has a relatively powerful jet for an FRI, we begin by considering it in more detail.
To derive black-hole masses for radio galaxies we use the relationship between MBH and K-band absolute bulge magnitude derived by Marconi & Hunt (2003) for nearby sources: log 10 MBH = 8.21 + 1.13 × (log 10 LK − 10.9) where LK is the K-band luminosity in solar units. We choose to use this particular relation because it is well calibrated against an MBH σ relation -indeed, Marconi & Hunt argue that the dispersion in the MBH -K relation is as small as that for the MBHσ relation for objects with well-determined black hole masses -and because Kband magnitudes for powerful radio sources are readily available.
Marconi & Hunt carried out a bulge-disc decomposition for their objects, which included a number of spirals, but we assume that the total K-band magnitude is the appropriate number to use for the elliptical galaxies that host radio sources. The observed scatter in the MBH -K relation is about 0.5 dex, implying (eq. 3) an uncertainty of about one order of magnitude in the available Bondi power. It should be noted that if Bondi accretion is responsible for powering radio jets, so that radio luminosity is correlated with Bondi power, then objects taken from a flux-limited radio sample are likely to be biased in the sense that their black hole masses will be above the expected value for galaxies of their observed properties, given the strong dependence of P b , and thus Q, on MBH (eq. 3). We make no attempt to correct for this, but it should be borne in mind in what follows. For 3C 31 Laing & Bridle (2002b) quote a jet power Q ≈ 10 37 W. From the K-band 2MASS observations (here and subsequently we use the total K-band magnitude provided by the NASA Extragalactic Database, NED) and the MBH -K relation we infer a black hole mass of 1.1 × 10 9 M⊙. Hardcastle et al. (2002, hereafter H02) studied the small-scale X-ray halo of the host galaxy of 3C 31 (the dominant galaxy of a rich group) and measured a central gas temperature of 0.66 keV. If we assume an efficiency of conversion of accretion power to jet power of η = 0.1 [following Allen et al.: Nemmen et al. (2006) have recently argued that efficiencies of this order are possible in various jet-formation models provided that the central black hole is rapidly spinning] then we can infer that an electron density at the Bondi accretion radius (assuming ρ = 1.13nemp as Allen et al. do) ne,A ≈ 6 × 10 5 m −3 is required to power the jet. H02 give a central density from their β-model fit of ne ≈ 2×10 5 m −3 , which is already consistent within the uncertainties on MBH: however, we know from the H02 fits to the inner 1.5 arcsec (0.5 kpc) of the 3C 31 X-ray emission that the density is higher in that region than the β-model would predict, and in fact the mean density inferred from spectral fitting is around 5 × 10 5 m −5 , while clearly if there is any density gradient in this inner region of the source the density at the Bondi accretion radius (rA = 50 pc) will be higher than the mean value. Thus there is no difficulty in producing the jets of 3C 31 by accretion of the hot phase of the IGM. We have repeated this calculation for several other FRI radio galaxies for which both X-ray estimates of the central density (e.g. 3C 296, Hardcastle et al. 2005) and jet power estimates from jet modelling (Laing, private communication) are available, and find that this conclusion is generally true: the central densities in the galaxy-scale components of these sources, even those in considerably poorer large-scale environments than 3C 31, are sufficient for Bondi accretion at a nominal 10 per cent efficiency to power the jet.
We next investigate whether there are sources that cannot be powered by Bondi accretion under these conditions. A widely used estimator of jet power is that derived by Willott et al. (1999): where L151 is the observed radio luminosity in units of 10 28 W Hz −1 sr −1 . The factor f parametrizes our ignorance of true jet powers, and is likely to depend in practice on the type of source and its environment, as discussed by Willott et al.. However, Blundell & Rawlings (2000) estimate that f may be ∼ 10 for FRII sources, while we find by normalizing this relation to jet powers of FRIs (Laing, private communication) that f lies in the range 10-20 for these objects as well. If we adopt a common f value for all sources then an object's jet power Q and available Bondi power PB for given density and temperature at the Bondi radius can be calculated simply from its redshift, radio flux density and K-band apparent magnitude (using the MBH -K relation as discussed above). In Fig. 1 we plot the observational quantities, radio luminosity and K-band luminosity, for radio galaxies from 3CRR (Laing, Riley & Longair 1983) with available K-band magnitudes, which are taken from the compilation 1 of Willott et al. (2003) for sources with z > 0.05 and from 2MASS for nearby objects. The figure also shows the conversion of the observational quantities to Bondi power and jet power, assuming values of the central density and sound speed appropriate for nearby FRI sources. Sources are divided according to their emission-line classifications in the manner discussed by H06. Quasars and BLRG are not plotted, as they have a substantial AGN-related contribution to the observed K-band magnitude, but we would expect them to behave similarly in unified models. For reference, we also plot the position of the high-excitation, powerful non-3CRR object Cygnus A (3C 405), using the black hole mass determined by Tadhunter et al. (2003).
While of course we do not claim that this plot gives the accurate position of any given source on the Q -PB plane -it is essentially just a version of the well-known magnitude/radio-power plot of Ledlow & Owen (1996) -it is instructive in several ways. Firstly, it shows that the nearby FRI radio galaxies (for which the adopted densities and temperatures are comparable to those directly measured where X-ray observations exist) almost all lie within a factor 1 Data available online at http://www-astro.physics.ox.ac.uk/∼cjw/kz/3ckz.html of a few of the line of QW = PB, as we would expect for the more detailed analysis carried out above. Secondly, it shows that the majority of low-excitation FRII radio galaxies in our sample also lie close to this line, within the uncertainties due to the scatter in the MBH -K relation. And thirdly, it shows that there is a population of FRII sources, encompassing most of the narrow-line FRII sources (and therefore, presumably, all high-excitation sources), that have jet powers exceeding the available Bondi powers (for our choice of central gas properties) in some cases by more than two orders of magnitude. For these sources to be powered by accretion of the hot IGM they would have to have a much higher central density than nearby FRIs, f factors much lower than the values appropriate for the twin-jet FRIs, or central black holes an order of magnitude more massive than the local MBH-K relation predicts. While we cannot rule out high central gas densities for distant objects, nearby FRII sources in group environments with detailed Chandra observations show that the luminosities and masses of any central hot-gas component in these objects are typically much lower than in those of nearby FRI sources (Croston et al., in prep.). Thus, for these sources at least, we feel confident in the claim that Bondi accretion of hot gas almost certainly cannot be responsible for powering the jet.
We note that several powerful low-excitation radio galaxies (with estimated jet powers Q ∼ 10 39 W) lie more than an order of magnitude away from the line of PB = QW on Fig. 1. One or two of these may not be true low-excitation objects (H06). Setting that aside, though, we already know that these powerful FRII LERGs tend to trace cluster environments, much richer than those of NLRGs, BLRGs and quasars of comparable power (Hardcastle 2004), and so it is possible that for these objects our choice of central density (comparable to those in group-centre FRIs) is too low, which would move them up on Fig. 1: at the same time, their denser environment would tend to lead to higher radio luminosity for a given jet power (Barthel & Arnaud 1996), moving them to the left. Both these effects would bring them closer to the region of parameter space where accretion from the hot phase could power the jets. While this picture needs to be tested by detailed X-ray studies of the environments of powerful LERGs, we do not feel that these objects present an insuperable problem for the model at present.
Finally, it is worth commenting on one observational point that could be used to argue against the picture we present herethe widespread detection of nuclear dust features in the host galaxies of nearby FRI sources (e.g. Martel et al. 1999). In some cases these structures have even been described as being related to the accretion disc itself (e.g. Jaffe et al. 1993), and, although they are clearly on scales much too large to be directly related either to any true accretion disc or to the larger-scale torus, it is possible that they represent a reservoir of cold gas accreting on to the central black hole. However, as we know the central cooling rates of the hot gas in these objects are high (e.g. H02) and the jet in classical twin-jet systems at least is likely to be a relatively inefficient source of heating for this central hot gas component, it is possible that small-scale cold material will naturally appear as a result of cooling, mirroring the processes seen on a larger scale in massive central cluster galaxies. Tan & Blackman (2005) argue that formation of a cold disc with a scale comparable to the Bondi radius is in fact a natural consequence of Bondi accretion in massive ellipticals. Given these possibilities, we do not consider that the observations are inconsistent with the picture presented here. Figure 1. K-band host galaxy luminosity against 151-MHz luminosity for 3CRR narrow-line and low-excitation radio galaxies with available K-band magnitudes. The top and right-hand axes show the conversion of these observational quantities into jet power derived from the Willott et al. (1999) relation (Q W ) and available Bondi power (P B ) respectively, derived from eqs 3 and 5 using a single density at the Bondi accretion radius, (ρ A = 5 × 10 5 m −3 ), a single temperature at that radius (kT = 0.7 keV), a single factor f in the Willott et al relation (f = 10) and a single Bondi efficiency (η = 0.1). Black hole masses are inferred from the K-band magnitudes as described in the text. Open stars are low-excitation radio galaxies and filled stars are narrow-line objects. A circle round a data point indicates an FRI. The filled square marks the position of the powerful NLRG Cygnus A, as discussed in the text (note that here the adopted K-band luminosity is derived from the known black hole mass rather than vice versa). The central solid line shows equality between the predicted Bondi power and the Willott et al jet power. The lines on either side are separated from the solid line by one order of magnitude, and so give an idea of the scatter expected on the Bondi luminosity from the observed dispersion in the M BM -K relation. The horizontal dash-dotted line shows the galaxy luminosity (corresponding to a black hole mass) at which the Bondi accretion rateṀ for our chosen gas parameters is equal to 0.01 times the Eddington rate (so that P B = 10 −3 L Edd ), while the dotted line shows the line at which the Willott jet power Q W equals 0.015 times the Eddington luminosity, both (for clarity) assuming the nominal M BM -K relation. See the text for discussion of these lines.
IMPLICATIONS OF THE MODEL
The analysis of the previous section has shown that it is possible that all the low-power low-excitation radio galaxies are powered by accretion from the hot phase, consistent with the fact that their nuclear spectra show no evidence for cold material close to the nucleus (i.e., no evidence for the 'torus'). On the other hand, we have seen that narrow-line radio galaxies (and therefore also broad-line radio galaxies and radio-loud quasars) which have clear evidence for accretion discs and tori, cannot be powered in this way -the large amounts of cold material in the nucleus and the radiative efficiency of accretion are naturally explained if these objects are powered by accretion of cold material via a thin disc in the standard manner.
Does accretion from the hot phase necessarily imply a radiatively inefficient accretion flow? We know that, by definition (eq. 2) the sound speed of the gas exceeds the Keplerian velocity v k at the Bondi radius. For γ = 5/3, this condition is maintained throughout the Bondi flow (Bondi 1952). Thus true spherical Bondi accretion is incompatible with the formation of a thin disc, which requires cs ≪ v k . However, the effects of a two-temperature plasma and of viscous dissipation must be considered, the assumption of spherical symmetry must break down at some point, and adequate radiatively inefficient cooling models close to the black hole should probably resemble a quasi-spherical accretion-dominated advection flow (ADAF: Narayan & Yi 1995a,b) or one of the numerous variants discussed in the literature. We know that these solutions in general are inconsistent with accretion rates of order of or greater than the Eddington rate, which we define as L Edd /c 2 , i.e.
where σT is the Thomson cross-section and mp is the mass of a proton. Therefore an important check on whether our picture is consistent with ADAF-type solutions is to ask whether it requires accretion rates of the order of the Eddington rate. Comparing eqs 3 and 6 it can be seen thatṁ =ṀB/Ṁ Edd is linear in black hole mass for given parameters of the external gas, and that the black hole mass required to giveṁ ∼ 1 is high for plausible external thermal parameters. In fact, all of the black hole masses for the low-excitation sources giveṁ ≪ 1 for our choice of Bondi parameters. The maximum value ofṁ for a low-excitation source close to the line of PB = QW in Fig. 1 is ∼ 0.02, and most lie belowṁ = 0.01. To illustrate this we have plotted the black hole mass (and therefore galaxy mass) corresponding toṁ = 0.01 for our hot-gas parameters as the dash-dotted line in Fig. 1. Thus all the low-excitation objects consistent with being powered by Bondi accretion (i.e. within the solid lines in Fig. 1) also have significantly sub-Eddington accretion rates, as required by ADAF-type solutions. In addition, we have plotted (dotted line) the line of QW = 0.015L Edd for the inferred black hole masses. This line of jet power vs. Eddington luminosity was used to divide FRI and FRII sources by Ghisellini & Celotti (2001). In the present plot we would argue that, while it does indeed separate FRIs and FRIIs reasonably well, it (or any variant on it represented by shifting the line to right or left) separates the low-excitation and high-excitation objects less well than the Bondi lines: in particular, there is a class of high-excitation low-power FRIIs with low QW/L Edd that could not lie to the right of such a line without also including a number of low-excitation FRIIs. Although the sample size is very small, this provides some weak evidence that it is genuinely the origin of the accreting material, and not simply the value of QW/L Edd , that determines the accretion mode of radio galaxies (cf. section 5.2 of Narayan & Yi 1995b).
In the rest of this section of the paper we therefore explore some of the consequences of a picture in which there is a causal connection between the origin of the accreting gas and the accretion mode, as indicated by the emission-line type of the galaxy. We know that most, though not all, low-power radio galaxies are low-excitation objects, while essentially all the high-power radio galaxies (and of course all powerful radio-loud quasars) are highexcitation objects. Thus it is not too much of a simplification to say that low-power radio galaxies (roughly in the FRI regime, but including a handful of less powerful FRIIs) are likely to be powered by 'hot-mode' accretion, while powerful FRII radio galaxies and quasars in general will trace 'cold-mode' accretion. This allows us to interpret some previous results that have been stated in terms of luminosity differences, or FRI/FRII differences, as being more naturally understood in terms of a dichotomy in accretion mode, bearing in mind that the FRI/FRII difference, in our picture, is a result of the jets' interaction with the large-scale environment rather than a direct consequence of the nature of the accretion.
Feedback
Feedback from AGN outbursts is now thought to be an important ingredient in galaxy formation models (e.g. Croton et al. 2006), enabling successful reproduction of the high-mass end of the galaxy luminosity function and solving the 'cooling flow' problem in the centre of massive clusters. An important feature of current models in which AGN heating prevents cooling of the ICM in the centre of massive clusters is that the AGN should both be able to influence, and should be influenced by, the X-ray emitting phase. Direct accretion of the hot phase provides an elegant way of ensuring that the AGN activity is regulated by the gas properties at the cluster centre, which was of course the motivation for the work of Allen et al. (2006). It is clear, though, that this is only possible for a 'hot-mode' radio source. Radio galaxies and quasars accreting in the cold mode do not have this direct connection between the hot phase and the rate of fuelling of the AGN: instead, the jet power is controlled solely by the accretion rate of cold gas, which may have nothing to do with the state of the hot phase. It is thus possible for cold-mode sources to inject catastrophic amounts of energy into the hot phase of the IGM. This is borne out by recent studies of the poor environments of nearby NLRG FRIIs Hardcastle et al. 2007) that show that the work done by the radio sources plus the internal energy in their lobes of the radio sources is comparable to the entire thermal energy of their host poor-group environments. Cold-mode systems can nevertheless play some role in feedback models. Churazov et al. (2005) suggest an evolutionary feedback model in which elliptical galaxies go through an early stage of rapid black hole growth at high accretion rates, until the black hole becomes sufficiently massive that radio-mode feedback turns on, slowing the rate of black hole growth. The radio outbursts of cold-mode systems may occur at an intermediate stage in this evolution, rather than forming part of an eventual feedback loop; the un-self-regulated energy input from this type of outburst into the ICM may instead be responsible for the "entropy excess" observed in galaxy groups and clusters (e.g. Pratt, Arnaud & Pointecouteau 2006).
Environments of active galaxies
In the model we have outlined we expect different types of active galaxies to be found in different environments. Cold-mode accretion requires a supply of cold gas: the easiest way for an elliptical galaxy to acquire this is by a merger with a gas-rich system. Samples of high-excitation radio galaxies should thus show evidence for mergers and interactions, consistent with many observations showing evidence for recent or ongoing mergers in the hosts of powerful sources (e.g. Heckman et al. 1986). Of course, since the timescale for transport of cold gas to the galactic centre may be much longer than the timescale for removal of the obvious optical signature of a merger, we do not expect a one-to-one correlation between observed elliptical-spiral mergers and AGN activity. We note, though, that this picture is consistent with observations of the few low-power, FRI, radio galaxies that we know to have heavily obscured nuclear X-ray emission, including Cen A (e.g. Evans et al. 2004) and NGC 3801 . Host galaxies of cold-mode systems do not need a rich environment, or to be at the bottom of a deep potential well, so long as galaxygalaxy mergers can take place.
By contrast, hot-mode accretion requires a supply of hot gas and a massive central black hole. Both the black hole mass and the mass of the galaxy-scale X-ray halo (e.g. Mathews & Brighenti 2003) are correlated with the mass of the host galaxy. Thus we expect hot-mode systems -which, observationally, include almost all FRI radio galaxies -to favour massive galaxies, and the most powerful radio sources to tend to be group-or cluster-dominant systems, as is observed (e.g. Longair & Seldner 1979;Prestage & Peacock 1988;Owen & White 1991). In samples that are not radio-selected, and in which the radio population (given the luminosity function) will therefore be dominated by low-luminosity radio galaxies accreting in the hot mode, we expect a strong correlation between radio activity and host galaxy/black hole mass, as found by Best et al. (2005). Best et al. (2006) have already pointed out the potential link between this observation and the fuelling of these radio sources by accretion of the hot phase, but in our picture their findings cannot be generalized to high-power, high-excitation, cold-mode radio sources.
AGN populations
The luminosity function of hot-mode radio galaxies will be determined by a combination of the black hole mass function and the distribution of properties of central hot gas. The cold-mode luminosity function, on the other hand, will be determined by the rate of accretion of cold material on to the central black hole, with no direct effect of the black hole mass until the Eddington luminosity is reached. We therefore expect the luminosity functions to be different, and the local radio luminosity function of radio-loud AGN, being the composite of two different luminosity functions, should have a break at a luminosity comparable to the transition between populations dominated by low-excitation and high-excitation radio sources, as is observed (e.g. Machalski & Godlowski 2000). The 'dual-population' unified model of Jackson & Wall (1999) proposes that the luminosity functions of FRI and FRII populations evolve differently with cosmic time. Since we know that the conditions for hot-mode and cold-mode accretion must vary with time, we would expect that this type of model would more properly be applied to the low-excitation and high-excitation sources.
Jet production and accretion history
We have so far not attempted to comment on the formation of jets in these systems. In our picture, the FRI/FRII difference is not in origin a function of accretion mode: the FRII LERG population provides the clearest evidence for this feature of the model. While the nuclear and large-scale properties are correlated in general (in 3C 31 and other twin-jet FRI sources, for example, the dense central concentration of hot gas required to fuel the active nucleus in the hot mode also provides the pressure gradient needed to collimate the jet and keep it stable to large distances: H02) they need not be in particular cases, which explains the lack of a one-to-one relationship between accretion mode and FR class. It is thus a requirement of the model that the two accretion modes must be capable of producing jets that are similar in most of their observable parsec-scale properties, as FRI and FRII jets are known to be (e.g. Pearson 1996). In models where the jet is produced from the accretion flow (Blandford & Payne 1982) it seems likely that this would require similar structure in the innermost regions of the accretion flow, but clearly we cannot distinguish between particular jet formation models.
A potentially more interesting question is whether hot-mode sources necessarily form a jet. Cold-mode sources can be divided into radio-loud and radio-quiet classes: is the same true for hotmode accretion? At least some hot-mode sources at cluster centres require a more or less continuously active jet, to reproduce the nearly universal detection of radio galaxies in high cooling-rate cluster cores (e.g. Eilek & Owen 2006) and it may be this is true for hot-mode sources in general: that is, it may be that this mode of accretion always produces a jet. Certainly it has been argued (e.g. Ho 2002;Nagar et al. 2002) that low-power AGN (with low values of L bol /L Edd ), where identified as AGN, tend to be dominated by jet emission. However, it would be hard to identify a class of AGN accreting in the hot mode in a radio-quiet manner, without powerful jets -we only know about the active nuclei in LERGs because of their jet-related radio, optical and X-ray emission. The only accretion-related radiation in this situation would be the extremely weak emission from the radiatively inefficient flow itself, but these sources would still contribute to black hole growth. Although it is generally argued that radiatively inefficient accretion has only a small effect on the evolution of the black hole mass func-tion (BHMF), direct estimates of its effect (e.g. Merloni et al. 2004;Hopkins, Narayan & Hernquist 2006) are based on samples of AGN that are detectable as such, either by radio or optical emission, and so might not include jetless hot-mode objects. Sample-independent constraints on the effects of radiatively inefficient accretion independent of the population of objects being considered come from work like that of Cao (2007), who shows that radiatively inefficient accretion cannot be very important in the evolution of the BHMF if it is not to overproduce the hard X-ray background, but this relies on theoretical assumptions about the spectral energy distribution of a radiatively inefficient accretion flow, possibly still leaving some loophole for hot-mode objects. In any case, we would expect that if radiatively inefficient accretion does have any effect on the BHMF, it will do so primarily in sources where the Bondi accretion rate is high. This would predict an environmental dependence of the BHMF, though the effect is probably too weak to detect at present.
Analogy with X-ray binaries
Recently there has been considerable discussion of the relationship between the accretion modes and jet behaviour of X-ray binaries ('microquasars'; hereafter XRB) and those of AGN (e.g. Fender, Belloni & Gallo 2004, Körding, Jester & Fender 2006. Support for a connection between XRB jet/accretion states and those of various different types of AGN comes from the so-called 'fundamental plane of black-hole activity' (e.g. Merloni, Heinz & Di Matteo 2003, Falcke, Körding & Markoff 2004, and from similarities in their variability properties (e.g. McHardy et al. 2006). Radio-quiet AGN are usually associated with the highṀ high/soft spectral state of XRB, and low-power (FRI) radio galaxies with the low/hard state (e.g. Maccarone, Gallo & Fender 2003). Powerful FRII radio galaxies and radio-loud quasars have sometimes been associated with the radio outbursts seen as XRB transition between states (e.g. Körding, Jester & Fender 2006). Churazov et al. (2005) recently suggested that the central AGN of massive elliptical galaxies evolve through an early stage of radiatively efficient, high accretion rate black hole growth (analagous to the radioquiet high/soft XRB state) to a state of stability regulated by feedback from radiatively inefficient feedback (analogous to the jetdominated low/hard state).
In the picture of radio source activity we have presented, the two different modes of accretion (radiatively inefficient accretion of the hot medium and radiatively efficient accretion of cold gas) both have to operate to produce radio jets; in this model the jet properties are essentially independent of the accretion mode. There is a strong relationship between radio morphology and accretion mode in our picture, but this is likely to be due to the need for an accretion rate higher than that available from Bondi accretion of the hot phase (in most environments) in order to produce powerful jets. By removing the direct connection between accretion mode and radiojet properties, our model causes some difficulties for a simple connection between XRB and AGN jet/disc states. In our picture, the low-excitation radio galaxies (including the FRIs) can be straightforwardly associated with the radiatively inefficient low/hard XRB state; however, it is unclear how the high-excitation sources fit into the picture, since they are clearly undergoing radiatively efficient accretion with steady jet emission. It remains possible that high-excitation sources are in some sense transitional objects (as discussed above, many such systems are associated with mergers, which plausibly cause a dramatic increase in accretion rate), but these systems can maintain steady jets for ∼ 10 7 years in a radiatively efficient accretion mode, so that it is not clear whether a direct analogy with XRB behaviour can really be made for the high-power, cold-mode systems.
SUMMARY AND FUTURE WORK
The idea that low-excitation radio galaxies are fuelled by the accretion of the hot, X-ray emitting phase of the IGM, while highexcitation radio sources are powered by accretion of cold material, can be used (qualitatively) to explain • their different optical and X-ray nuclear properties (H06, this paper) • the close relationship between the power output of low-power radio galaxies and the energy needed to solve the cooling flow problem (Allen et al. 2006) • the association of low-power radio galaxies with the most massive host galaxies (Best et al. 2006) • the observed differences in cosmic evolution of low-and highpower radio-loud AGN (this paper), and • the different environments of low-and high-excitation radio sources (this paper) In this paper we have shown quantitatively that the required difference between low-excitation and high-excitation objects in the well-studied 3CRR sample is at least plausible. Clearly, though, further testing of this picture is essential. Observational tests would include a significant expansion of the available database of sensitive, high spatial resolution X-ray spectroscopic observations of nuclei of radio galaxies -our conclusions on the nature of lowexcitation FRII sources in particular are based on small samples. Better constraints on the small-scale hot-gas environments of radio sources are vital, and forthcoming mid-infrared studies of the nuclei of radio-loud AGN will also provide important information. In future we expect studies of the evolution of the luminosity functions of the two populations, combined with cosmological simulations that give indications of the availability of fuel to the two accretion processes, to provide the most stringent tests of the model. | 2014-10-01T00:00:00.000Z | 2007-01-30T00:00:00.000 | {
"year": 2007,
"sha1": "324971f45d9b1094f1467edfc6884a8eeb0c7c72",
"oa_license": null,
"oa_url": "https://academic.oup.com/mnras/article-pdf/376/4/1849/18673593/mnras0376-1849.pdf",
"oa_status": "BRONZE",
"pdf_src": "Arxiv",
"pdf_hash": "84dfaafd087f6d95c854e7a28f762ea20cc3178c",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
34263090 | pes2o/s2orc | v3-fos-license | Claudin-3 and Claudin-5 Protein Folding and Assembly into the Tight Junction Are Controlled by Non-conserved Residues in the Transmembrane 3 (TM3) and Extracellular Loop 2 (ECL2) Segments*
Background: The transmembrane claudins assemble into polymeric tight junction strands. Results: Residues involved in differential folding and assembly of claudin-3 and claudin-5 were identified. Conclusion: Subtype-specific cis-dimerization contributes to the differing ultrastructure of tight junction strands. Significance: The molecular insights improve the understanding of the formation of paracellular barriers to molecules. The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.
The paracellular barrier in epithelia and endothelia is formed by tight junctions (TJ). 4 Freeze-fracture electron microscopy (FFEM) revealed TJ as an anastomosing network of strands composed of transmembrane particles (1). The tetraspan membrane proteins of the claudin (Cldn) family constitute the backbone of TJ (2,3). On the basis of their barrier properties, claudins can be functionally grouped in barrier-forming or channel-forming claudins (4,5). The ability of claudins to form paracellular ion channels is mainly determined by their first extracellular loop (ECL1) (4,6,7). The crystal structure of claudins is unknown, and the mechanism of TJ assembly is unclear. Assembly and disassembly of TJ is regulated by a panel of TJassociated proteins controlling, e.g., the transport of claudins or their linkage to the cytoskeleton. However, the assembly of claudins into TJ strands probably depends directly on the following claudin-claudin interactions: (I) intramolecular folding and intermolecular assembly by (IIa) co-or posttranslational cis-oligomerization (within one membrane) and (IIb) trans-interaction (between opposing plasma membranes) that mediates the formation of polymeric strands (2,8). Hereafter, (I) is mentioned as "folding" and (II) as "assembly." It is assumed that heteropolymeric TJ strands are formed by interactions between the same claudin family members (homophilic) and between different ones (heterophilic) at cell-cell contacts (3,9,10). However, the biochemical and biophysical analysis of the assembly of TJ strands is limited by their sensitivity to detergents. This, so far, prevented solubilization or in vitro reconstitution of intact TJ strands. To circumvent this limitation, we analyzed claudin assembly in a native cellular environment.
TJ strands were reconstituted in HEK293 cells by transfection with claudin constructs C-terminally tagged with YFP. In this system, strand formation can be analyzed independently of endogenous claudins and TJ-regulating proteins containing a PDZ domain (2). TJ strands were detected by FFEM or single molecule-based nanoscopy (11). Trans-interaction between claudins was detected by enrichment of claudins at contacts between claudin-expressing cells (hereafter designated "contact enrichment"). Cis-interaction was analyzed by FRET assays. This reconstitution approach and mutagenesis were used to identify the determinants for the trans-interaction of Cldn5 (2), heterophilic compatibility of claudins, and TJ-associated MARVEL proteins (9,12) together with a bioinformatics analysis to distinguish between classic and non-classic claudins and modeling of the ECL2 of Cldn3 and Cldn5 (4,13). Residues identified as relevant for claudin assembly were verified as contributing to paracellular barriers in vitro and in vivo (14,15).
In this study, we focused on two prototypes of barrier-forming claudins, Cldn3 and Cldn5, both of which are relevant to the blood-brain barrier. Cldn5 tightens the blood-brain barrier for molecules smaller than 800 Da (16). Hints of Cldn3 expression in the brain, loss of Cldn3 under pathologic conditions, and transcriptional regulation of Cldn3 in brain endothelial cells suggest that Cldn3 contributes to blood-brain barrier tightness (17,18).
Cldn3 and Cldn5 are both capable of sealing the TJ (19,20), but they form TJ strands with different ultrastructures (9): continuous-type strands associated with the protoplasmic (P) face of the membrane (Cldn3) or particle-type strands with spaced intramembranous particles associated with the exoplasmic (E) face (Cldn5). To identify claudin segments and residues involved in this difference, a panel of Cldn3/Cldn5 chimeras (ChA-ChG) was created by mixing the different predicted segments (transmembrane segments (TM1-4), intracellular loops (ICLs), and ECLs. The chimeras were expressed in HEK293 cells and screened for their capability to form TJ strands (2) with microscopic and biochemical analyses. The results provide novel insights into the molecular mechanism of paracellular barrier formation.
Live-cell Imaging-For live-cell imaging, 2 days after transfection, cells were transferred to 1 ml Hanks' balanced salt solution with Ca 2ϩ , Mg 2ϩ , glucose, and sodium bicarbonate and without phenol red (Invitrogen). The plasma membrane was visualized by addition of 20 l of trypan blue (Sigma-Aldrich, Hamburg, Germany), 0.05% in PBS. Cells were examined with a LSM 510 META system containing an Axiovert 135 microscope equipped with a PlanNeofluar ϫ100/1.3 objective (Carl Zeiss, Jena, Germany), (2).
To quantify claudin enrichment at contacts between two claudin-expressing cells, confocal images of living cells were analyzed using the LSM 510 software (Carl Zeiss). Two different methods were used.
The enrichment factor (EF) was measured using intensity profiles of confocal images. Contacts between two cells were identified by the trypan blue fluorescence peaks indicating the plasma membrane. For each cell pair, three fluorescence intensity profiles were quantified. For cultures of Cldn/chimera-YFP-expressing cells, the EF was calculated as (intensity at contact between two Cldn/chimera-expressing cells)/(sum of intensities at contact between the two expressing cells and nonexpressing cells) (2).
The EF could not be usefully determined for constructs that showed a saturation of the signal at contacts between two Cldn/ chimera-expressing cells but no clear signal between Cldn/ chimera-expressing and non-expressing cells. For these constructs, the percentage of enrichment-positive contacts was quantified as contacts between two Cldn/chimera-YFP-expressing cells with a strong YFP signal.
To quantify the ratio of claudin signals in the plasma membrane versus the nuclear membrane, images of single Cldn/chimera-expressing cells were taken. Either the plasma membrane identified by trypan blue or the nuclear membrane identified morphologically were detected close to the saturation of the signals (depending on which signal was stronger). Five fluorescence intensity profiles per cell were quantified and averaged. FRET experiments at cell-cell contacts were performed using Cldn-CFP/Cldn-YFP-cotransfected HEK293 cells, as described previously (2).
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE)-Pellets of 10 cm 2 of transiently transfected HEK293 cells were resuspended in sample buffer (1ϫ NativePAGE TM sample buffer, 1% DDM (Invitrogen), and EDTA-free protease inhibitor mixture (Roche Applied Science)). Lysates were centrifuged for 30 min at 15,000 ϫ g and 4°C. To 5 l of supernatant, 5 l of sample buffer including 0.5% NativePAGE TM G-250 sample additive (Invitrogen) was added. As a marker, we used NativeMark TM unstained protein standard (Invitrogen) and applied a correction factor of 0.6 similar to that used for claudins, as described previously (23). BN-PAGE was performed according to the instructions of the manufacturer (Invitrogen) on the basis of Ref. 24. Gels were transferred to PVDF membranes. Membranes were fixed in 8% acetic acid for 15 min, destained with 62.5 mM Tris/HCl, 2% SDS for 15 min, washed with H 2 O, and blocked with 3% milk powder for 1 h. Western blotting was performed with mouse anti-GFP (catalog no. JL-8, Clontech) and HRP-conjugated goat anti-mouse (Invitrogen).
FRET Analysis by Spectrofluorometry/FRET Ratio-A close proximity between claudin molecules was detected by FRET between CFP and YFP tags. Cldn-CFP/Cldn-YFP-cotransfected HEK293 cells were lysed in PBS containing 1% DDM (Sigma-Aldrich) and EDTA-free protease inhibitor mixture. After centrifugation (30 min, 15,000 ϫ g, 4°C), the supernatant was analyzed in a FP-6500 spectrofluorometer (Jasco, Gross-Umstadt, Germany). CFP fluorescence was detected with excitation of 425 Ϯ 5 nm and emission of 475 Ϯ 5 nm; YFP with excitation of 490 Ϯ 5 nm and emission of 525 Ϯ 5 nm; and the FRET signal with excitation of 425 Ϯ 5 nm and emission of 525 Ϯ 5 nm. Cross-talk of YFP fluorescence to the CFP and FRET signal was determined with samples containing either YFP or CFP only; for -excitation of 425 Ϯ 5 nm and -emission of 475 Ϯ 5 nm, the signal was 2% of the YFP signal (⑀1 ϭ 0.02), and for -emission of 525 Ϯ 5 nm, the signal was 5% of the YFP signal (⑀2 ϭ 0.05). As a measure of the FRET efficiency, the FRET ratio was calculated as (FRET signal Ϫ ⑀2 ϫ YFP fluorescence)/(CFP fluorescence Ϫ ⑀1 ϫ YFP fluorescence). For negative controls, a FRET ratio of about 0.4 was obtained (9).
Freeze-fracture Electron Microscopy-HEK293 cells were transfected with Cldn/chimera-YFP constructs. Three days later, they were washed with PBS with Ca 2ϩ /Mg 2ϩ , fixed with 2.5% glutaraldehyde (electron microscopy-grade, Sigma-Aldrich) in PBS Ca 2ϩ /Mg 2ϩ for 2 h, washed, and processed for freeze-fracture electron microscopy as reported previously (25). For quantification of the continuity and the P-/E-face association of the TJ strands, the particle coverage along strand axis was determined as follows. FFEM images were analyzed with ImageJ by measuring the length of the strands and counting the intramembranous particles at a given position along the strand axis in 10-nm steps. The particle coverage was defined as the number of particles ϫ 10 nm/total length of analyzed strands. This was done for the P-and E-faces. For each construct, more than seven strand network areas were analyzed.
Bioinformatics-Sequence alignments for murine Cldn3 and Cldn5 were created with the GCG program package (GCG Wisconsin package, Accelrys Inc., San Diego, CA) and visualized with Geneiouse Pro 5.4.4 (Geneious Pro 5.4.4 created by Biomatters). Transmembrane helix predictions were performed with the GCG program package and TMHMM (26).
Statistics-Unless stated otherwise, results are shown as mean Ϯ S.E. Statistical analyses were performed using Prism version 5.0 (GraphPad, San Diego, CA). First, normality tests were performed (D'Agostino and Pearson omnibus and Shapiro-Wilk and Kolmogorov-Smirnov test). Data sets exhibiting normal distribution were analyzed using unpaired, two-tailed Student's t test. Data sets not showing normal distribution were analyzed using Mann-Whitney U test. Fig. 1, A and B) with a C-terminal YFP tag were expressed by transfection in the TJ-free cell line HEK293. Similarly to Cldn3 and Cldn5, the chimeras ChA, ChF, and ChG colocalized with the plasma membrane. However, in contrast with Cldn3 and Cldn5, the chimeras ChA, ChF, and ChG were not enriched at contacts between claudin-expressing cells (Fig. 2, A, B, D, E, G, and H). This indicates inhibition of the trans-interaction. FFEM was performed to analyze whether this inhibition affects strand formation. Although extended networks of TJ strands were easily detected for Cldn3 and Cldn5 (Refs. 2, 9 and Fig. 2C), almost no strands were detected for ChA, ChF, and ChG (Figs. 2F and 10, A-C). These results show that ChA, ChF, and ChG are strongly impeded in trans-interaction and strand formation.
Subcellular Distribution of Cldn3/5 Chimeras Indicates Hampered Folding or Trans-interaction-Seven chimeras (ChA-ChG,
In contrast with Cldn3, Cldn5, ChA, ChF, and ChG, the chimeras ChB, ChC, ChD, and ChE were restricted to intracellular compartments and did not localize within the plasma membrane ( Fig. 2, I-L). This suggests that the folds of ChB, ChC, ChD, and ChE do not pass the intracellular quality control necessary for efficient plasma membrane targeting.
Although the cells showed different expression levels and, partly, a pronounced intracellular localization of the respective claudin construct, the enrichment of the construct between cells that express the construct depended on the molecular properties of the construct and not on whether it was expressed at low or high levels. In addition, stable expression of these constructs also led to similar subcellular distribution and contact enrichment, as did the transient expression (data not shown).
Taken together, all the chimeras showed an impediment to form TJ strands. This indicates that the different segments (TMs, ECLs, and ICL) of Cldn3 and Cldn5, which were mixed in the chimeras (Fig. 1), do not match structurally.
Identification of ECL2 Residues Involved in Trans-interaction by Rescue of Contact Enrichment for ChF-ChF represents
Cldn3 with the putative ECL2 of Cldn5 (Figs. 1 and 3A). The lack of contact enrichment of ChF (Fig. 2G) suggests that the exchange of ECL2 of Cldn3 by that of Cldn5 was sufficient to diminish trans-interaction. To identify residues involved in this inhibition, Cldn3-like residues were reintroduced into ECL2 of ChF. Because the transition between ECL2 and TM3 could not be predicted exactly, the ECL2 sequence of Cldn5 in ChF was also N-terminally expanded (Fig. 3A).
First, multiple substitutions of neighboring residues in the ECL2 region differing between Cldn3 and Cldn5 were analyzed. T141I/I142V/I143V and Y157R/L159M, but not N148D/ L150T or V153E/S154A, strongly increased contact enrichment (Fig. 3B). Analysis of single substitutions revealed that T141I and Y157R were responsible for increased contact enrichment (Fig. 3, B and C). Combining T141I/I142V/I143V with Y157R/L159M resulted in an additive increase in contact enrichment (Fig. 3B). A similar additive increase was obtained by combining T141I with Y157R (Fig. 3C). These results indicate that the residues differing between Cldn3 and Cldn5 at positions 141 (in Cldn3 ϭ 142 in Cldn5) and 157 (in Cldn3 ϭ 158 in Cldn5) are involved in trans-interaction.
To investigate the role of ECL2 residues in contact enrichment in more detail, Cldn3-mimicking residues were introduced into Cldn5. At transition to TM3 I142T, but not V143I or V144I, strongly inhibited contact enrichment (Fig. 3D). V154E/ S155A only led to a minor decrease. D149N/T151L double substitution inhibited contact enrichment for the Cldn5 construct, whereas neither D149N nor T151L single substitutions exhibited any inhibition (Fig. 3D). Taken together, the data obtained with ChF and Cldn5 demonstrate that ECL2 residues differing between Cldn3 and Cldn5 are involved in contact enrichment.
Identification of Incompatible Residues Responsible for the Different Subcellular Localization of Chimeras-The finding that ChA but not ChB was detected in the plasma membrane (Fig. 2, E and I) suggests that residues differing between these chimeras (TM3 and ICL) are structurally incompatible with other residues in ChB, resulting in misassembly and intracellular retention of ChB. A comparison of TM3 of Cldn3 and Cldn5 revealed the presence of two Cldn5-specific cysteines (Fig. 4A, top panel). Because the sulfhydryl group of Cys possesses special biophysical properties (e.g. electrostatic potential), its contribution to the different phenotypes of ChA and ChB was analyzed by Ser substitutions. Strikingly, C127S, but not C136S, in ChB enabled contact enrichment, and C136S inhibited contact enrichment induced by C127S in ChB (Figs. 4, A and B, and 10, I-L). This indicates that, in ChB, the sulfhydryl group of Cys-127, but not of Cys-136, in the Cldn5-like TM3, prevents contact enrichment, presumably by disturbing folding/assembly of the chimera. In addition, the corresponding C137S, but not C128S, in Cldn5 inhibited contact enrichment (Fig. 4B), indicating that the sulfhydryl group of Cys-137, but not that of Cys-128, is necessary for the correct folding and assembly of Cldn5.
Substitution of the Incompatible C127 in TM3 Is Sufficient to Rescue Strand Formation for ChB-Consistently, with contact enrichment, FFEM showed that ChB-C127S is able to form TJ strands (Fig. 4C). The strands formed by ChB-C127S showed a particle-type morphology with intramembranous strand particles on the E-face as well as on the P-face of the membrane (particle coverage along strand axis of 65.8 Ϯ 2.2% on the E-face and 37.0 Ϯ 1.5% on the P-face). Interestingly, this mixed P-/Eface association was similar to that found for Cldn3/Cldn5 copolymers (9) and was in between the strand morphology found for Cldn5 (particle coverage of 61.9 Ϯ 1.2% on the E-face and 5.3 Ϯ 1.3% on the P-face, particle type) and the one for Cldn3 (particle coverage of 4.0 Ϯ 1.0% on the E-face and 94.7 Ϯ 1.9% on the P-face, continuous type). C128S substitution in FIGURE 1. Sequence alignment and constructs used in this study. A, sequence alignment of mouse Cldn3 and Cldn5. Putative TM segments (red), ECLs, and the ICL were predicted as described under "Experimental Procedures." Note the shift in one position between Cldn3 and Cldn5. Green, hydrophobic residues; blue, basic; magenta, acidic; dark blue, hydrophilic; yellow, cysteines; cyan, tyrosines; black, proline. B, chimeric Cldn3/5 constructs used in the study to identify regions involved in claudin folding and assembly. Transitions between segments are as indicated in A. Yellow and blue segments correspond to Cldn3 and Cldn5, respectively. For clarity, the chimeras are labeled with letters and not named according to the mixed segments.
Cldn5 (corresponding to C127S in ChB) did not change E-face association nor the particle-type appearance of the strands (Fig. 4D, particle coverage of 64.0 Ϯ 4.0% on the E-face and 5.8 Ϯ 1.8% on the P-face). The very few strands found for ChA showed Cld3-like continuous (Ͼ 90% particle coverage) and P-face-associated strands (Fig. 10A). Together, these FFEM data and the sequence differences between ChA and ChB ( Fig. 1) indicate that residues in TM3/ICL of Cldn5 but not C128 alone are strongly involved in the E-face association and particle-type appearance of strands.
Coexpression of ChB-C127S with Cldn5 increased, whereas coexpression with Cldn3 decreased, E-face association of the resulting strands (Fig. 10, F and G). FRET at cell-cell contacts demonstrated a close proximity of ChB-C127S with the coex-pressed Cldn3 or Cldn5 (Fig. 10H). These data suggest that ChB-C127S is able to copolymerize with Cldn3 and Cldn5 and that this copolymerization results in a shift of P-/E-face association.
The Block of Contact Enrichment of ChA and ChF Is Rescued by Substitutions in TM3-ChB-C127S showed contact enrichment in the plasma membrane and many intracellular signals (Fig. 4A). In contrast, ChA was homogenously distributed in the plasma membrane (Fig. 2, D and E). To identify residues involved in this different subcellular localization, ChA was expanded stepwise toward ChB-C127S by introducing Cldn5like residues N-terminally of ECL2 (Fig. 5A). The substitution T141I/I142V/I143V did not lead to contact enrichment (Fig. 5B). In contrast, S136C/S138F drastically increased contact enrichment. A similar increase was obtained with S138F, but not S136C, in ChA. T141I/I142V/I143V reduced the increase induced by S136C/S138F (Fig. 5B, ChA-S136C/S138Fϩ141TII/ IVV143 (ChA CFIVV)), and T141I reduced the increase induced by S138F (Fig. 5B, ChA S138F/T141I). The data show that S138F is sufficient to rescue contact enrichment of ChA and that T141I counteracts this rescue effect.
To analyze the influence of TM4 on S138F-mediated contact enrichment of ChA, similar substitutions were introduced in ChF, which differs from ChA in TM4 (Fig. 5, C and E, pictograms). Similar as in ChA, in ChF, substitutions S136C/S138F and S138F, but not S136C, strongly increased contact enrichment (Fig. 5, B and E). In contrast to ChA, in ChF, the substitutions T141I/I142V/I143V and T141I increased contact enrichment, and T141I/I142V/I143V did not reduce the increase induced by S136C/S138F (Fig. 5B, ChF-S136C/S138Fϩ141TII/ IVV143 (ChF CFIVV)). Taken together, these data indicate that S138F in TM3 is sufficient to rescue contact enrichment independently of the differences between Cldn3 and Cldn5 in TM4, whereas the effect of T141I depends on TM4.
Substitutions in ChA and ChF Leading to Contact Enrichment Enable Strand Formation-To verify that chimeras showing contact enrichment are able to form TJ strands, FFEM was performed. ChF-S136C/S138F, ChF-S136C/S138FϩT141I/I142V/ I143V (ChF CFIVV), and ChA-S136C/S138F formed many strands (Fig. 5, H, J, and K). For these constructs, extensive networks of branched strands similar to Cldn3 and Cldn5 were found. In addition, the strongest tendency to form multiple parallel bundles was observed for ChA-S136C/S138F (Fig. 5K). The strands formed by these chimeras were rather continuous with particles associated with the P-face. In summary, continuity and P-/E-face association of the strand particles decreased in the following order: Cldn3 Ն ChF-S136C/S138F Ն ChF-S136C/S138FϩT141I/I142V/I143V Ն ChA-S136C/S138F Ͼ Ͼ ChB-C127S Ͼ Ͼ Cldn5-C128S ϭ Cldn5-T142I ϭ Cldn5. The most striking difference in P-/E-face association of the strand particles was found between ChA-S136C/S138F and ChB-C127S. This indicates that Cldn3-like P-face association mainly depends on Cldn3-specific residues N-terminally of Ser-136 but not on Cldn3-specific residues in ECL2 or TM4.
Further Analysis of Substitutions in Cldn3 and Cldn5-Because residues differing in Cldn3 and Cldn5 strongly affected strand formation of chimeric constructs, the role of differing residues was analyzed further by introducing Cldn5-like residues into Cldn3 and Cldn3-like residues in Cldn5. In Cldn3, S138F and T141I increased contact enrichment (Fig. 6A), whereas the reciprocal substitutions in Cldn5, I142T (Fig. 3D) and F139S (Fig. 6B), decreased contact enrichment. However, F139S and I142T in Cldn5 did not prevent formation or change P-/E-face association or the particle-type appearance of strands (Figs. 5I and 10, D and E). Taking wild types and chimeras together, the presences of Phe at position 138/139 and Ile at 141/142 correlate with strong contact enrichment.
Because C128 and C137 of Cldn5 were found to be relevant for contact enrichment (Fig. 4B), Cys was introduced at the corresponding position in TM3 of Cldn3 (A127C, S136C). However, contact enrichment was not changed (Fig. 6A). Similarly, C14A in TM1 of Cldn5 did not affect contact enrichment. These results underline that the effect of a sulfhydryl group depends on the protein context.
Further substitutions found to affect contact enrichment were A132T in TM3 of Cldn5 (Fig. 6B) and exchanging residues in the ICL that differ greatly between Cldn3 and Cldn5 (Fig. 6, A and B, 108QDET111/109APGP112). However, these effects were weaker than those from other substitutions in TM3 or ECL2. and Y157R/L159M substitution in ChF increased contact enrichment. Combining 141TII/IVV143 and Y157R/L159M resulted in an additive increase. C, Y157R, but not L159M, increased contact enrichment. T141I/Y157R showed a stronger increase than Y157R. D, in Cldn5, I142T and D149N/T151L inhibited contact enrichment. As a measure of contact enrichment, the contact enrichment factor was determined and normalized to ChF (B and C), or the percentage of enrichment-positive contacts was determined and normalized to Cldn5 (D) (see "Experimental Procedures." Data are mean ϩ S.E. *, p Ͻ 0.01 versus ChF (B and C) or Cldn5 (D) or as indicated; #, p Ͻ 0.05. n Ն 93 for V153E/S154A; n ϭ 64 (B); n Ն 22 (C); n Ն 10 images with an average of Ն 16 contacts analyzed (D).
Effect of the Substitutions on the Amount of Claudin Constructs in the Plasma Membrane-
The claudin constructs colocalized differentially with the plasma membrane (summarized in supplemental Table S1). Cldn3 and Cldn5 both exhibited contact enrichment but differed in the amount detected in the plasma membrane outside contacts between claudin-expressing cells. To investigate plasma membrane localization independently of trans-interaction, single claudin-expressing cells without contact to another claudin-expressing cell were analyzed (Fig. 7, A and B). The ratio of the claudin signals in the plasma membrane versus the nuclear membrane (as an intracellular reference membrane) was quantified using confocal intensity profiles (Fig. 7C, PM/NM ratio). The PM/NM ratio of Cldn3 was much higher than that of Cldn5. Removal of the Cldn5-specific sulfhydryl group by C128S and Cldn3-mimicking I142T increased the PM/NM ratio in Cldn5. ChB-C127S and ChB-C127S/I141T exhibited similar PM/NM ratios as Cldn5-I142T. For ChA, the PM/NM ratio was similar to that of Cldn3wt and was unchanged by S138F or S138F/T141I in ChA (Fig. 7C). These results indicate that the amount of claudin constructs in the plasma membrane is affected by residues in TM3.
Biochemical Analysis of Claudin Oligomerization-For biochemical analysis of claudin oligomerization, BN-PAGE of claudins solubilized with 1% DDM was used as described pre-viously (23). Cldn5 migrated consistently with being a dimer (Fig. 8A). Only a faint band consistent with a monomer was detectable. In contrast, Cldn3 was detected as a monomer, whereas a dimer was hardly detectable. Because Cldn4 has been described as migrating as a monomer on BN-PAGE (23), it was used as control. As expected, Cldn4 migrated consistently with being a monomer, similarly to Cldn3 (Fig. 8A). Furthermore, Cldn5-F147A, which has been shown previously to lack the ability for trans-interaction (2), was detected as a dimer, similarly to Cldn5. This indicates that the detected Cldn5 dimer is formed by cis-interaction.
To verify the different extents of dimerization for Cldn5 versus Cldn3, FRET assays (for detection of close proximity) were performed. HEK293 cells were cotransfected with Cldn-CFP/ Cldn-YFP, the cells were lysed as for BN-PAGE, and the FRET signals were analyzed by spectrofluorometry. A strong FRET signal was obtained for Cldn5 but not for Cldn3 (Fig. 8B). In addition, the FRET signal for Cldn5 was strongly inhibited by the addition of 0.5% SDS. Together, the BN-PAGE and FRET data strongly suggest that Cldn5, but not Cldn3, forms a stable non-covalent cishomodimer that is DDM-resistant but SDS-sensitive.
Residues in TM3 of Cldn5 Stabilize Cis-dimerization-To identify segments and residues involved in cis-dimerization, chimeras were analyzed by BN-PAGE with respect to the shows mixed P-/E-face-associated, particle-type strands (C, arrow), whereas Cldn5-C128S shows E-face-associated, particle-type strands (D, arrow). Scale bars ϭ 0.2 m. MARCH 14, 2014 • VOLUME 289 • NUMBER 11 FIGURE 5. S138F substitution is sufficient to rescue contact enrichment and strand formation of ChA and ChF. A, schematic highlighting the sequence differences between ChA, ChB, and ChF. Alignment of Cldn3 and Cldn5 sequences and substitutions that were analyzed are indicated (boxes). B, quantification of contact enrichment of ChA and ChF constructs. S138F substitution is sufficient to rescue contact enrichment in ChA and ChF. The effect of 141TII/IVV143 but not of S138F depends on TM4. CFIVV, S136C/S138Fϩ141TII/IVV143. Data are mean ϩ S.E. n Ն 39. For ChA-T141I, n ϭ 22. *, p Ͻ 0.001 versus ChA (dark blue columns) or versus ChF (light blue or red columns) or as indicated. C-F, representative images for subcellular distribution of claudin-YFP constructs (green) in living HEK293 cells. The plasma membrane was stained with trypan blue (red). Contact enrichment is indicted by arrows. Scale bars ϭ 5 m. G-K, FFEM analysis. Similar to Cldn3wt (G), ChF-S136C/S138F (H) and ChF-CFIVV (J) form huge networks of rather continuous-type strands with a strong P-face association. K, ChA-S136C/S138F forms many bundles of continuous or fragmented strands with more particles on the P-face than on the E-face. I, Similar to Cldn5wt, Cldn5-I142T forms networks of particle-type strands with a very high E-face association. Scale bar ϭ 0.2 m. dimer/monomer ratio (Fig. 8A). A low dimer/monomer ratio, similar to Cldn3, was found for ChF, ChF-S138F, ChA, ChA-S138F, and ChA-S136C/S138FϩT141I/I142V/I143V (ChA-CFIVV). In contrast, ChB-C127S showed a clearly higher dimer/monomer ratio. Because ChB-C127S and ChA-CFIVV differ in the N-terminal half of TM3 and ICL, this region appears to be strongly involved in differential dimerization. However, substitution of an ICL motif strongly differing between Cldn3 and Cldn5 (108QDET111/109APGP112) did not change the dimer/ monomer ratio (Fig. 8A). In contrast, for ChB-C127S/I141T, the dimer was even more pronounced than for ChB-C127S. In addition, Cldn5-I142T, but not Cldn5-F139S, exhibited more monomers than Cldn5wt. Together, the data indicate that position 141 (Cldn3)/142 (Cldn5), but not 138 (Cldn3)/139 (Cldn3), and also residues in the N-terminal half of TM3 are involved in dimerization.
Residues Contributing to Claudin Assembly
TJ Strands Formed by Chimeras Because of the S138F Substitution Represent a Diffusion Barrier for a 870-Da Marker-The TJ strands reconstituted by claudin expression in HEK293 cells do not form a continuous belt-like paracellular barrier in the monolayer (2). Hence, standard permeability assays using a filter insert, as performed for polarized epithelial cells with endogenous TJs (14), are inapplicable. To demonstrate that the constructs showing contact enrichment and strands in freezefracture replica constitute a functional diffusion barrier, a tracer exclusion assay was performed (9). Standard incubation of cells with trypan blue labels the whole plasma membrane (Figs. 2, 4, 5, and 7). In contrast, after a short incubation with trypan blue (873 Da) or Cellmask TM (ϳ2 kDa) in a lower concentration, the tracer also labels the plasma membrane but is excluded from cell-cell contacts with enriched claudin signals (9). This tracer exclusion was found for different chimeras showing contact enrichment and strands but not for chimeras lacking both. Representative images for the different constructs are given for ChA-S138F and ChA (Fig. 9). This indicates that ChA-S138F, but not ChA, forms a diffusion barrier at cell-cell contacts for markers of ϳ1 kDa.
DISCUSSION
In this study, a panel of Cldn3/Cldn5 chimeras was screened to identify protein segments and residues involved in folding and assembly of classic claudins such as Cldn3 and Cldn5. Experimental reconstitution of TJ strands, mutagenesis, native gel electrophoresis, and confocal and electron microscopy revealed novel insights into the molecular determinants of claudin folding and assembly and the formation of paracellular barriers.
Mislocalization of Cldn3/5 Chimeras Revealed Segments of Cldn3 and Cldn5 That Do Not Match-Analysis of subcellular localization of ChA to ChG showed that, in contrast to wild- . Trans-interaction-independent colocalization of claudin constructs with the plasma membrane. Representative images of single Cldn5expressing (A) and ChB-C127S-expressing (B) HEK293 cells are shown. C, quantification. As a measure for plasma membrane localization, the PM/NM ratio was determined as a logarithm (log) of the ratio of the claudin signal in the plasma membrane (A and B, arrowhead) to the claudin signal in the nuclear membrane (C and D, arrow) using confocal intensity profiles. The PM/NM ratio for Cldn3 was much higher than that for Cldn5. Substitutions in TM3 affected the PM/NM ratio. Scale bars ϭ 5 m. Data are mean ϩ S.E. n Ն 10. *, p Ͻ 0.01 versus Cldn3 or as indicated.
type claudins, none of the chimeras exhibited contact enrichment as an indicator for trans-interaction (Fig. 2). In addition, the lack of efficient strand formation was verified by FFEM. This indicates that, for all chimeras, the mixed segments of Cldn3 and Cldn5 do not match structurally.
Two different phenotypes were obtained for the chimeras: the "plasma membrane type," with a presence in the plasma membrane but lack of contact enrichment indicating direct or indirect inhibition of trans-interaction, and the "intracellular type," an intracellular accumulation indicating misfolding or misoligomerization that does not pass intracellular protein quality control (8,27). Chimeras with at least two TMs of Cldn5 or TM1 and ECL1 of Cldn5 belong to the intracellular type. Regarding the plasma membrane type, exchanging ECL2 of Cldn3 with that of Cldn5 (ChF) was sufficient to strongly inhibit trans-interaction and strand formation. Even the presence of ECL1 and ECL2 of Cldn5 in ChG was not sufficient to enable strong contact enrichment. These results indicate that residues in the ECL2 that differ between Cldn3 and Cldn5 and the transition between the ECLs and the TMs contribute to the ability to interact in trans. To identify mismatching residues in chimeras and, thus, residues involved in claudin folding or assembly, we subsequently tried to rescue the deficiency phenotypes of the chimeras by mutagenesis.
Identification of ECL2 Residues Involved in Trans-interaction-The block of trans-interaction by exchanging the ECL2 of Cldn3 by that of Cldn5 (Fig. 2G) enabled us to search for nonconserved residues involved in trans-interaction. Interestingly, Y157R strongly increased contact enrichment in ChF (Fig. 3C). The corresponding Y158 in Cldn5 is necessary for homophilic trans-interaction (2), but Cldn3-Cldn5 trans-interaction was found to be relatively weak (9). These findings indicate that the R157/Y158 difference in the ECL2 contributes to claudin subtype-specific trans-interaction.
Furthermore, T141I in ChF increased, whereas the inverse I142T in Cldn5 decreased contact enrichment (Fig. 3, B and D). In Cldn5, D149N/T151L diminished contact enrichment (Fig. 3D). Together, the data indicate that positions 141/2, 148/9, 150/1, and 157/8 along the ECL2 of Cldn3/Cldn5 contribute to transinteraction. Whether these positions directly or indirectly affect trans-interaction is addressed by a modeling approach. 5 Identification of Incompatible/Mismatched Residues by Comparison of Different Chimera Phenotypes-Differences between chimeras concerning their subcellular localization had to be caused by sequence differences. In particular, the different phenotypes for ChA (plasma membrane type) and ChB (intracellular type) had to be due to the different residues in TM3 or ICL (Fig. 1). Consequently, substitutions in this region were generated and analyzed by confocal and electron microscopy. Residues were identified that influence plasma membrane localization, contact enrichment, and strand formation. Two points were revealed as discussed in the following paragraphs.
1) The sulfhydryl group of Cys-128 but not that of Cys-137 in TM3 of Cldn5 prevented contact enrichment of ChB (Fig. 4). In Cldn5, Cys-128 was not necessary for contact enrichment. This indicates that, in a putative TM helix bundle, Cys-128 in TM3 of Cldn5 is not necessary for the assembly with other TMs of Cldn5 (Fig. 11, dashed box). However, in ChB, Cys-127 prevented contact enrichment, indicating that Cys-128 in TM3 of Cldn5 does not fit with TM1 and TM2 of Cldn3, which are present in ChB.
In contrast, the sulfhydryl group of Cys-136/137 in TM3 was necessary for strong contact enrichment of ChB and Cldn5 (Fig. FIGURE 8. Analysis of claudin dimerization by blue native PAGE and FRET. A, claudins from transfected HEK293 cells were solubilized with 1% DDM and analyzed by blue native PAGE. The claudin constructs differ in their dimer/monomer ratios. Bands corresponding to cis-dimers were detected for Cldn5 but hardly at all for Cldn3. I142 and other residues in TM3 of Cldn5 stabilize cis-dimers. Representative Western blot analyses are shown. ChA-CFIVV, ChA-S136C/ S138FϩT141I/I142V/I143V; Cldn3 ICL, Cldn3 Q108A/D109P/E110G/T111P; Cldn5 ICL, Cldn5 A109Q/P110D/G111E/P112T. B, FRET assays indicated non-covalent homodimerization of Cldn5. DDM lysates of HEK293 cells cotransfected with Cldn3-CFP/Cldn3-YFP (Cldn3 WT) or Cldn5-CFP/Cldn5-YFP (Cldn5 WT) were analyzed. As a measure of FRET efficiency, the FRET ratio was calculated (see "Experimental Procedures"). Addition of SDS (Cldn5 WT ϩ SDS) strongly reduced the FRET ratio. Data are mean ϩ S.E. n Ն 4. *, p Ͻ 0.001 versus Cldn3. FIGURE 9. ChA-S138F forms a diffusion barrier at cell-cell contacts. For HEK cells expressing ChA, trypan blue (red) homogenously labels the plasma membrane, including contacts between HEK cells that express ChA (A-C, green, arrows). In contrast, trypan blue is excluded at contacts between HEK cells that express ChA-S138F (D-F, green, arrows). This indicates a diffusion barrier formed by ChA-S138F. Scale bars ϭ 5 m. Cells were labeled with trypan blue and imaged within 5 min. 4B). The corresponding S136C substitution increased contact enrichment in ChA-S138F (generating ChA-S136C/S138F) and ChF-S138F but not in ChA, ChF, and Cldn3 (Figs. 5E and 6A). These data indicate that a sulfhydryl group at position 136/137 in TM3 matches Cldn5-like TM3 and TM4, e.g. to F139. Whether the sulfhydryl groups of residues 128 and 137 influence intramolecular interactions or cis-interaction between the TMs is unclear. However, as a result, trans-interaction and strand formation are affected indirectly.
2) S138F in TM3 is sufficient to rescue contact enrichment, and strand and diffusion barrier formation of ChA and T141I counteracts this (Figs. 5B and 9). S138F rescued contact enrichment in ChA (Fig. 5B) without changing the ChA amount in the plasma membrane (Fig. 7C). A similar rescue was obtained in ChF. Because ChA and ChF differ in TM4, the effect of S138F in TM3 is independent of sequence differences between Cldn3 and Cldn5 in TM4. In contrast, the effect of T141I depends on TM4.
In Cldn3, S138F and T141I increased contact enrichment (Fig. 6A), whereas the reciprocal substitutions in Cldn5, I142T (Fig. 3D) and F139S (Fig. 6B), decreased contact enrichment. I141/142T differentially affected Cldn5 and ChB-C127S, FIGURE 10. Additional analysis of chimeric mutants. A-G, freeze-fracture EM analysis of chimeric mutants. A-C, for ChA, ChF, and ChG, almost no strands were found. However, the few and rudimentary strands that were only rarely detected showed a rather continuous type and particles associated with the P-face. A and B, on the E-face, grooves with only a few particles were found. D and E, in contrast, for Cldn5-F139S, networks of particle-type strands associated with the E-face were found, similar to Cldn5wt. respectively, regarding contact enrichment (Figs. 3D and 4B), the amount in the plasma membrane (Fig. 7C), and dimerization (Fig. 8A). These results and sequence comparisons suggest that the I141/142T-mediated changes depend on differences in TM1 and TM2.
Taking wild types and chimeras together, the presences of Phe at position 138/139 and Ile at position 141/142 correlate with strong contact enrichment. The molecular mechanism by which these residues affect claudin folding and assembly is further addressed by a modeling approach. 5 BN-PAGE and FRET Reveal TM3-mediated Cis-dimerization for Cldn5 but Not for Cldn3-Several gel electrophoresis systems have been used to analyze oligomerization of solubilized claudins. We obtained the most consistent results with the DDM/BN-PAGE system (23). Cldn5 migrated consistently with being a dimer (Fig. 8A), similar to Cldn2 (23). In contrast, Cldn3 was found mainly as a monomer, similar to Cldn4 (Fig. 8A and Ref. 23). Cldn5-F147A, which is deficient for transinteraction (2), was detected as a dimer. This suggests that the Cldn5 dimer is formed by cis-interaction. FRET supported noncovalent homodimerization of Cldn5 but not of Cldn3 (Fig. 8B).
DDM-resistant claudin dimers have been suggested to be a fundamental structural unit of larger detergent-sensitive complexes that might represent the 10-nm particles of TJ strands seen in FFEM (23). Our findings support this idea and indicate that channel-forming claudins (e.g. Cldn2) as well as barrierforming claudins (e.g. Cldn5) are able to form these dimers. It has been assumed that the lack of dimerization found for Cldn4 could be due to the lack of the ability to self-organize into strands (23). Here we show that Cldn3, which forms strands, does not form stable dimers. Hence, DDM-resistant cis-dimerization is not a precondition for the ability to form TJ strands but reflects claudin subtype-specific assembly properties.
BN-PAGE analysis of Cldn3/Cldn5 chimeras showed that Cldn5-specific residues in TM3 critically contribute to cisdimerization. This is consistent with cross-linking experiments with Cldn2/Cldn4 chimeras, suggesting dimerization via TMs and proximity of TM2 (23).
TM3 Contributes to the Ultrastructure of TJ Strands-Paracellular tightness correlates with the number of strands and the extent of their cross-linking detected with FFEM after aldehyde fixation (28,29). Breaks of Ͼ20 nm in the strand network are often found under pathological conditions with increased permeability. In contrast, gaps Ͻ 20 nm define the "particle type" of strands formed by Cldn2, Cldn5, or Cldn10b (30 -32), whereas Cldn1 and Cldn3 form a continuous "strand type" without gaps (3,33). In addition, these continuous strands are found on the P-face of the membrane, whereas, for the particle-type, particles are found on the E-face (Cldn5) or E-and P-faces (Cldn2, Cldn3/5 copolymers). Importantly, mixed P-/E-face association correlates with blood-brain barrier-specific tightness (9,17,34). However, the molecular base for these ultrastructural differences remained unclear.
Here we reveal a relationship between this ultrastructural appearance and cis-dimerization of claudins. For Cldn3, Cldn5, and chimeras of these, E-face association and particle type (Figs. 2C; 4,C and D; 5, G-K; and 10, A-F) correlated with cis-dimerization (Fig. 8A). A similar correlation is found for Cldn2 and Cldn4 by comparison of literature (7,23).
The most striking difference was found between ChA-S136C/S138F and ChB-C127S (Figs. 4C and 5K). This shows that the C-terminal half of Cldn5, including TM3 but not C128, is essential for stable cis-dimerization and, thereby, for strands with particles on the E-face (Fig. 11).
In summary, we revealed subtype-specific cis-dimers as subunits of claudin polymers that contribute to the distinct ultrastructure of TJ strands. We identified non-conserved residues in TM3 and the ECL2 of classic claudins, affecting cis-and/or trans-interaction, thereby influencing the morphology and barrier function of TJ strands (Fig. 11). These mechanistic insights advance the molecular understanding of paracellular barrier formation and could facilitate the identification of key interactions for specific TJ modulation for the improvement of drug delivery or for therapeutic barrier protection. | 2018-04-03T02:37:28.055Z | 2014-01-29T00:00:00.000 | {
"year": 2014,
"sha1": "042d2214cc07618cb0ee8b71eb609c02f452be3e",
"oa_license": "CCBY",
"oa_url": "http://www.jbc.org/content/289/11/7641.full.pdf",
"oa_status": "HYBRID",
"pdf_src": "Highwire",
"pdf_hash": "5283ad293ff3e5d5cf16ab7cc64a7d060d0cd254",
"s2fieldsofstudy": [
"Biology",
"Chemistry"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
251325892 | pes2o/s2orc | v3-fos-license | Assessing the robustness of radiomics/deep learning approach in the identification of efficacy of anti–PD-1 treatment in advanced or metastatic non-small cell lung carcinoma patients
Administration of anti–PD-1 is now a standard therapy in advanced non-small cell lung carcinoma (NSCLC) patients. The clinical application of biomarkers reflecting tumor immune microenvironment is hurdled by the invasiveness of obtaining tissues despite its importance in immunotherapy. This study aimed to develop a robust and non-invasive radiomics/deep learning machine biomarker for predicting the response to immunotherapy in NSCLC patients. Radiomics/deep learning features were exacted from computed tomography (CT) images of NSCLC patients treated with Nivolumab or Pembrolizumab. The robustness of radiomics/deep learning features was assessed against various perturbations, then robust features were selected based on the Intraclass Correlation Coefficient (ICC). Radiomics/deep learning machine-learning classifiers were constructed by combining seven feature exactors, 13 feature selection methods, and 12 classifiers. The optimal model was selected using the mean area under the curve (AUC) and relative standard deviation (RSD). The consistency of image features against various perturbations was high (the range of median ICC: 0.78–0.97), but the consistency was poor in test–retest testing (the range of median ICC: 0.42–0.67). The optimal model, InceptionV3_RELF_Nearest Neighbors classifiers, had the highest prediction efficacy (AUC: 0.96 and RSD: 0.50) for anti–PD-1/PD-L1 treatment. Accuracy (ACC), sensitivity, specificity, precision, and F1 score were 95.24%, 95.00%, 95.50%, 91.67%, and 95.30%, respectively. For successful model robustification, tailoring perturbations for robustness testing to the target dataset is key. Robust radiomics/deep learning features, when paired with machine-learning methodologies, will work on the exactness and the repeatability of anticipating immunotherapy adequacy.
Introduction
The introduction of programmed death 1 receptor (PD-1)/ programmed death ligand 1 (PD-L1) blocking antibodies and targeted agents have substantially changed the therapeutic strategies for advanced lung cancer. In the setting of pre-treated patients with advanced non-small cell lung carcinoma (NSCLC), Nivolumab and Pembrolizumab monotherapy showed significantly better overall survival (OS), compared with traditional chemotherapy (1)(2)(3). Several predictive biomarkers based on cellular phenotypes, immunohistochemical, mutational tests, and expression-based approaches have been proposed to predict response to immune checkpoint inhibition. However, the predictive power of these methods was far from perfect. For example, only 44.8% of PD-L1-positive NSCLCs were responsive to Pembrolizumab in a first-line setting (4). Furthermore, it is difficult to identify the current status of immune profiles from an archival sample due to the dynamical evolution of the immune-escape mechanism during anti-cancer treatment (5,6). Therefore, non-invasive methods, understanding the dynamics of the tumors in clinical practice, and assessing the immune landscape of tumors are critical.
Radiomics/deep learning (DL) image features are becoming a promising non-invasive method to obtain quantitative measurements for tumor classification and assessment for therapy response in oncological research (7)(8)(9)). An imaging biomarker should be reproducible, robust, and accurate. However, image features are susceptible to several factors, such as imaging protocol variability, different vendors, image reconstruction processes, inter-rater tumor segmentation variability, patient motion artifact, overall image quality, and tumor phenotype (10-13). Ideally, only features that are robust to these variations would be incorporated into a predictive model for good generalizability (14).
We hypothesized that the combination of machine learning (ML) technologies and high-dimensional radiomics/DL features would facilitate the prediction of immunotherapy efficacy. Therefore, we investigated the robustness of radiomics/DL features against different perturbations and then determined the optimal model by combining feature extractors, feature selectors, and ML classifiers.
Whuh (Wuhan Union Hospital) data
The medical records of patients with advanced NSCLC who had received Nivolumab (3 mg/kg every 2 weeks) or Pembrolizumab (200 mg every 3 weeks) monotherapy between January 2019 and January 2021 were retrospectively reviewed at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Treatments were provided until disease progression, intolerable side effects, or consent to the withdrawal. The retrospective study was approved by the Ethics Committee of Union Hospital, which also waived the written informed consent, because the data were analyzed anonymously.
Patient inclusion criteria were (1) pathologically confirmed NSCLC, (2) enhanced computed tomography (CT) performed fewer than 15 days before treatment, and (3) availability of clinical data. The exclusion criteria were (1) missing or low- quality treatment CT, (2) suffering from other tumor diseases at the same time, (3) combining other treatments while using immunotherapy, (4) Patients with no measurable lesion by Immune-Modified Response Evaluation Criteria In Solid Tumors (imRECIST) or no available response evaluation (15). Tumor response to Nivolumab or Pembrolizumab monotherapy was objectively assessed by experienced radiologists (QQ. R, QN. J) using imRECIST in the third month. The details regarding the response assessment were described in the supplemental. The data pertaining to demographics, smoking history, histology type, TNM stage, and molecular testing and the number of prior lines of therapy were extracted from electronic medical records (Table 1).
Test-retest cohorts
The test-retest cohort with 31 NSCLC patients was available from the Cancer Imaging Archive (16,17). Images in the testretest cohort using the same scanner and acquisition protocol were acquired every 15 min. Informed consent was waived.
Computed tomography acquisition and segmentation
CT scans were acquired using a multi-slice spiral CT system (Philips Healthcare, General Electric Health Care, and Siemens Healthcare) with a tube voltage of 100-120 kVp, slice thickness (spacing) of 1-5mm, and in-plane resolution of 0.75 mm × 0.75 mm. All scans were acquired using the facilities' CT chest protocol and standard image reconstruction.
Pre-processing and tumor segmentation
The tumor regions of interest (ROIs), which corresponded to the biggest target lesion, were manually performed using threedimensional Slicer software, which was based on a consensus reached by two experienced radiologists (one with 5 years of experience, another with 10 years of experience). For those cases with a blurred edge around the lesion, the maximum range was drawn and regarded as the border. Large vessels, adjacent organs, and air cavities were excluded. On contrast-enhanced CT, difficult-to-identify lesions were labeled with reference to the corresponding nuclear positron emission tomography (PET) image (some patients had PET scans) or with the permission of two physicians. The two readers repeated the same procedures 2 weeks later and any disagreement was resolved through consultation.
Feature extraction
To be consistent with DL features, three consecutive slices with the maximum cross-sectional area of the tumor lesion were selected. Radiomic feature calculations were automatically done using the PyRadiomics package implemented in Python (18). Radiomics features with or without wavelet filtration included three groups: (1) first-order statistics, (2) shape features, and (3) second-order features: gray-level co-occurrence matrix (GLCM), gray-level size zone matrix (GLSZM), gray-level run-length matrix (GLRLM), neighborhood gray-tone difference matrix (NGTDM), and gray-level dependence matrix (GLDM) features (18). ImageNet, which has numerous object categories and manually annotated training photos, was used to pre-train InceptionResnetV2, InceptionV3, Resnet50, VGG16, VGG19, and Xception (19). The six pre-trained CNNs were used as an arbitrary feature extractor while executing DL feature extraction, allowing the input picture to propagate forward, halting at the penultimate layer, and using the outputs of that layer as our features. We used global max pooling to extract the feature map's maximum value before converting it to its original value.
Image normalization
An image interpolation procedure was needed to standardize the images after CT image acquisition and segmentation. The image brightness was adjusted through the adaptive window level. The histogram equalization method was applied to CT images to get better visualization. The size of the three axial slices was adjusted to 224 mm × 224 mm, consistent with the input layer size of the pre-trained CNN models. The Gaussian filter was used to remove noise in images since CT images were mainly affected by quantum noise, which would be caused by the variability of the electron density of tissue voxels, and represented by random Gaussian process statistics (20).
Robust features for test-retest imaging and image perturbations
We tested feature robustness against various perturbations in Whuh data, then feature robustness was verified in the testretest cohort.
According to the imaging guidelines (21) and the radiologist's visual inspection, we defined the expected perturbations in a multicenter setting.
(2) Rotation (R): The depicted tumor rotation would be affected by the patient's position. Therefore, we generated a set angle q [−30°, −15°, 15°30°] and rotated the image, and segmented tumor in the axial (x, y) plane.
(3) ROI variation (Seg): The depicted tumor edge might be affected by the patient's respiratory motion artifact and the variability of intra-and inter-observer ROI segmentation. Therefore, ROI enlargement and shrinking were considered (enlargement and shrinking were shown in Figure 1) (14, 22).
Robust features evaluation: ICC (2,1) for each feature was calculated and only those that reach the cutoff (ICC > 0.75) for all tested perturbations were entered following the feature selection and modeling process. Raw feature vectors were further standardized by being centered to the mean and scaled to unit variance. Features with zero median absolute deviation (MAD), regarded as nonpredictive features, were further removed.
The 12 ML classifiers included logistic regression, k-nearest neighbors, quadratic discriminant analysis (QDA), Support Vector Classifiers (SVCs) with linear and radial basis function (RBF) kernels, XGBoost, multilayer perceptrons, Gaussian processes, decision trees, naive Bayes, random forests, and AdaBoost. These classifiers were all imported from a Python (version 3.6.4) ML library named scikit-learn (version 19.0) (23). Further details about the feature selection methods were in Supplementary S2, and the parameter settings and tuning range of ML classifiers were detailed in the Supplementary Materials.
Machine learning and model performance evaluation
Seven feature extractors, 13 feature selectors, and 12 classifiers were combined, then 1,092 (7 × 13 × 12 = 1092) ML models were generated. The nomenclature of each model combined the feature exactor, the names of the feature selector, and the classification method. For example, Rnest50_ RELF _ nearest neighbors was a model trained by a k-nearest neighbors classifier with features selected by the ReliefF and extracted from Rnest50.
Each of the 1,092 models was trained during the 10-fold stratified cross-validation using the StratifiedKFold iterator in scikit-learn, which is a variation of kfold cross-validation that ensured each set contained approximately the same percentage of samples of each target class as the whole training dataset.
Synthetic minority over-sampling technique was adopted to handle the imbalanced data. The best performing model was selected based on AUC and relative standard deviation (RSD). RSD was defined as the ratio between the standard deviation and mean of the 10-fold crossvalidated AUC values: RSD = (sdAUC/mean AUC) ×100. The lower the RSD value, the higher the stability of the predicting model. The model with the highest AUC value and the lowest RSD was considered the best performing model. The performance of the best performing model was further measured by accuracy (ACC), sensitivity, specificity, F1 score, and precision.
Statistical analysis
Continuous variables were presented by using median with mean + SD and the statistic difference was compared by Wilcoxon signed-rank test. For differences in categorical variables, Fisher's exact test was adopted, and the results were shown as the number of events followed by relative frequencies (%). A two-sided p < 0.05 was used as the criterion to indicate a statistically significant difference.
Results
The study flowchart was presented in Figure 1.
Patient characteristics
Of 157 patients with advanced NSCLC (128 men, 29 women), 109 patients underwent nivolumab monotherapy and 48 underwent pembrolizumab monotherapy during the study period. The median age was 59 (range: 29-78) years. One h u n d r e d f o u r ( 6 6 % ) w e r e d i a g n o s e d a s h a v i n g adenocarcinoma, 46 (29.3%) were squamous cell carcinoma, five (3.2%) were undifferentiated large cell carcinoma, and two (1.3%) were adenosquamous carcinoma. Mutations in epidermal growth factor receptors were present in 17 patients (10.8%). Thirty-one patients (19.7%) had received one course of The study flowchart. After pre-processing and tumor segmentation, the images were artificially perturbed. Robust features were evaluated by machine learning (ML) models. chemotherapy, 51 patients (32.5%) had received two courses, and 75 patients (47.8%) had received three or more courses. The expression of PD-L1 was abundant (tumor proportion score [TPS] ≥ 50%) in 42 patients (26.8%), at low levels (1% ≤ TPS < 50%) in 29 patients (18.5%), and unknown in the remaining 86 (54.8%). According to Response Evaluation Criteria in Solid Tumors, version 1.1, after anti-PD-1 immunotherapy, 65 patients (41.4%) had a partial response, 59 patients (37.6%) had stable disease, and 33 patients (21.0%) had progressive disease (Table 1).
In DL and radiomics features, ICCs ranging from 0.80 to 0.90 demonstrated favorable feature reproducibility for S (axial slice spacing). The features from InceptionResnetV2 and InceptionV3 were robust against R(rotation) but have a lower agreement if the ROI changed. For features from Resnet50 and Xception, robustness against S and Seg (ROI variation) were comparable. The features from VGG16 and VGG19 were robust against Seg but had a lower ICC for R. Radiomics features were robust against each perturbation, especially against Seg. The percentage of robust features against all perturbations for each feature extractor was presented in Figure 3 (The performance of each feature extractor against each image perturbation was reported in Supplementary Table 1 with median and the interquartile range (IQR)). The number of robust features for different ICC threshold settings was reported in the Supplementary Material Figure 1.
Compared with the consistency test for various perturbations, the repeatability in the test-retest group was much worse. The ICC of the best radiomic features in the above robustness testing was 0.6 in the test-retest group. The performance of each feature extractor regarding the test_retesting images was reported in Supplementary Table 2 with median and IQR.
We then reduced the number of features by removing features with zero MAD across the two cohorts. With the ICC threshold set to 0.75, the numbers of features remaining after robustness testing were radiomics 233, InceptionResNetV2 25, InceptionV3 74, Resnet50 109, VGG16 30, VGG19 73, and Xception 50. These features were first screened by the 13 feature selectors mentioned, and then the best combination The heatmap of features generated from InceptionV3 for representative patients. was further screened by the wrapper feature selection method based on the recursive feature addition algorithm.
Feature selection and machine learning models
The optimal model InceptionV3_RELF_ Nearest Neighbors was selected with the AUC value 0.96 and RSD 0.50 among the 1,092 machine-learning models (list of all feature selectors were in Supplementary Table 3, and the parameter settings and tuning range of ML methods were presented in Supplementary Material). Analysis of the confusion matrixrelated classification metrics of InceptionV3_RELF_ Nearest Neighbors showed that the ACC, sensitivity, specificity, precision, and F1 score were 95.24%, 95.00%, 95.50%, 91.67%, and 95.30%, respectively. The illustration of the 10-fold crossvalidated AUC for InceptionV3 features was presented in Figure 4A. Interestingly, the radiomics models had equal performance. The AUC value of Radiomics_CIFE_Nearest Neighbors, Radiomics_CIFE_QDA, Radiomics_CMIM_Nearest Neighbors, and Radiomics_CMIM_Multilayer Perceptron) was 0.96 in each model, and the RSD was 0.61, 0.67, 0.61, and 0.67. The heatmap of the 10-fold cross-validated AUC concerning radiomics features were presented in Figure 4B
Discussion
In this study, by utilizing quantitative image analysis to extract features in conjunction with a ML classifier, we constructed accurate and reproducible models to predict immunotherapy response for advanced NSCLC. Importantly, these efficient models were obtained using cross-validation, and the inputs of the models were robust.
PD-L1 immunohistochemistry (IHC) expression, tumor mutation burden, and tumor-infiltrating lymphocytes have been suggested to predict the response to immunotherapy (24,25). However, tissue-based biomarkers rely on individual tumor samples from accessible lesions in clinic practice and may not truly reflect the complexity of inter-tumoral heterogeneity. Furthermore, it is difficult to determine the current status of immune profiles from archival samples, as immune-escape mechanisms evolve dynamically during anti-cancer treatment (5,6). The main idea of DL is to employ a deep neural network, which provides a unique set of novel tools to improve NSCLC detection (26), characterization (27), survival prediction, and treatment outcome (28). However, compared with statistical ML models, DL models typically required a much larger amount of data to train for optimal results. To overcome the limitations of small datasets, transfer learning patterns (29) facilitate DL models as powerful extractors of useful feature sets.
Radiomic features have been used to predict the benefit of adjuvant chemotherapy, disease risk in early stage lung cancer (30), treatment response to concurrent chemoradiation in locally advanced lung cancer (31), and response to immune checkpoint inhibition in advanced NSCLC (32,33). Most studies focused on the AUC of predictive models on a given dataset without considering the robustness of imaging features.
Our model is reliable and reproducible, because it uses robust features following the standardization of the model's input images and can be applied to CT data of various institutions. This model can minimize possible differences between different medical centers, inspection machines, and image reconstruction methods.
The evaluation of the robustness feature is based on the assumption that test-retest images and perturbations do not have consistent bias. We tested the robustness of features against perturbations, such as slice thickness spacing(S), rotation(R), and ROI variation (Seg). Both DL and radiomic features show excellent robustness to S perturbation and have a modest performance to Seg perturbation. The Seg perturbation captured the range of variability that occurred with human inter-observer variability and patient respiratory motion artifact. It is better to underestimate rather than overestimate the ROI when segmenting.
Several major limitations remained in the present study. First, our data were relatively small, and baseline characteristics maybe not in accordance with the population-based dataset. For example, the objective response rate was higher than in the previous study (34,35). Thirty-two patients chose immunotherapy, because they could not tolerate chemotherapy toxicity rather than disease progression, which partly explained the high efficiency. Second, three consecutive slices of the tumor were sampled for the analysis, and volumetric assessments were not performed. In a previous study, data from a single slice were found to be sufficient for this type of analysis (36). Third, whether our algorithm model for predicting immunotherapy response can be applied to cancer types other than NSCLC is another potential research question to be solved. Fourth, our model lacks external verification. Compared with the DL model, the characteristic stability of radiomics model was higher; however, the prediction capabilities of the DL and radiomics model were comparable. Which model is better requires further verification. Fifth, the factors involved with image features, such as histogram equalization approaches, noise removal methods, and image reconstruction methods, require more in-depth study. Sixth, more study is required to determine whether transfer learning may take the role of the specifically created model for NSCLC due to the heterogeneity between the source and destination databases. In addition, PD-L1 expression data were unavailable for a majority of patients in our cohort. The correlation between PD-L1 expression, which was a clinically validated biomarker of benefit from PD1/PD-L1 blockade, and the instructed model, was not involved in our study.
To the best of our knowledge, this is the first work assessing the robustness of image features in CT imaging of NSCLC patients. In addition, we perform a comparative analysis to select the best machine-learning methods with favorable predictive AUC and stability. Inception V3_RELF_Nearest Neighbors classifiers provided a robust, non-invasive way to identify NSCLC patients who may benefit from immunotherapy. We believe that combining machine-learning methods and radiomics/DL features will improve the AUC in predicting immunotherapy efficacy.
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 Committee of Union Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
Author contributions
The study was designed by NH, QJ, and QR with the help of the others. QR, PZ, FX, XC, NH, and QJ analyzed and interpreted the data. QR developed a model. QR, QJ, FX, and PZ performed the main computational works. NH, XC, and GW collected surgical data and supported transcriptome analyses. QR, FX, PZ, XC, NH, and QJ collected and analyzed the clinical data. QR, FX, PZ, and QJ performed image analysis and interpretation. GW and NH acquired funding for this study. QR, FX, PZ, NH, and QJ mainly wrote the manuscript, and all authors edited the manuscript. All authors contributed to the article and approved the submitted version.
Funding
This study was supported by the National Natural Science Foundation of China (No. 82172755) and Wuhan Knowledge Innovation Special (Item Number: 2022020801020532). | 2022-08-05T13:36:54.336Z | 2022-08-05T00:00:00.000 | {
"year": 2022,
"sha1": "e8216748515392f5f20e54fc5876471c09cb71bd",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Frontier",
"pdf_hash": "e8216748515392f5f20e54fc5876471c09cb71bd",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
52186972 | pes2o/s2orc | v3-fos-license | Pursuit of Low-Rank Models of Time-Varying Matrices Robust to Sparse and Measurement Noise
In tracking of time-varying low-rank models of time-varying matrices, we present a method robust to both uniformly-distributed measurement noise and arbitrarily-distributed “sparse” noise. In theory, we bound the tracking error. In practice, our use of randomised coordinate descent is scalable and allows for encouraging results on changedetection.net, a benchmark.
Introduction
Dimension reduction is a staple of Statistics and Machine Learning. In principal component analysis, its undergraduatetextbook version, possibly correlated observations are transformed to a combination of linearly uncorrelated variables, called principal components. Often, a low number of principal components suffice for the so-called low-rank model to represent the phenomenon observed. Notoriously, however, a small amount of noise can change the principal components considerably. A considerable effort has focussed on the development of robust approaches to principal component analysis (RPCA). Two challenges remained: robustness to both sparse and non-sparse noise and theoretical guarantees in the time-varying setting.
We present the pursuit of time-varying low-rank models of time-varying matrices, which is robust to both dense uniformly-distributed measurement noise and sparse arbitrarily-distributed noise. Consider, for example, background subtraction problem in Computer Vision, where one wishes to distinguish fast-moving foreground objects from slowly-varying background in video data (Liu et al. 2013). There, a matrix represents a constant number of frames of the video data, flattened to one row-vector per frame. At any point in time, the low-rank model is captured by a shortand-wide matrix. The time-varying low-rank model makes it possible to capture slower changes, e.g., lighting conditions slowly changing with the cloud cover. There may also be slight but rapid changes, e.g., leaves of grass moving in the wind, which could be captured by the uniformly-distributed dense noise. Finally, the moving objects are captured by the sparse noise. Clearly, low-rank modelling has wide-ranging applications beyond Computer Vision, wherever one needs Copyright c 2020, Association for the Advancement of Artificial Intelligence (www.aaai.org). All rights reserved.
to analyse high-dimensional streamed data and flag abnormal observations to operators, while adapting the model of what is normal over time.
Our contributions are as follows: • we extend the low-rank + sparse model to low-rank + dense uniformly-distributed noise + sparse, where low-rank can be time-varying • we provide an algorithm with convergence-rate guarantees for the time-invariant case • we provide an algorithm with guarantees for the timevarying case. In Theorem 2, we bound the tracking error of an algorithm for any low-rank factorisation problem for the first time. That is: we show that a sequence of approximately optimal costs eventually reaches the optimal cost trajectory. • we improve upon the statistical performance of RPCA approaches on changedetection.net of (Goyette et al. 2012), a well-known benchmark: the F1 score across 6 categories of changedetection.net improves by 28%, from 0.44643 to 0.57099. On the baseline category, it is 0.80254. • we improve upon run time per frame of the same RPCA approaches, as detailed in Table 1. Compared to TTD 3WD, to give an example of a method which is still considered efficient in the literature, our single-threaded implementation is 103 times faster.
Related Work
Traditional approaches to robustness in low-rank models (Candès and Recht 2009, to name some of the pioneering work) are based on a long history of work in robust statistics (Huber 1981). In such approaches (Candès et al. 2011;Feng et al. 2013;Guo, Qiu, and Vaswani 2014;Mardani, Mateos, and Giannakis 2013), sometimes known as "Lowrank + Sparse", one balances the number of samples of the "sparse" noise and the rank of the model, or the nuclear norm as a proxy for the rank. There are a number of excellent implementations, including some focused on the incremental update (Lin, Liu, and Su 2011;He, Balzano, and Lui 2011;Balzano and Wright 2013;Oreifej, Li, and Balzano, Chi, and Lu 2018;Yong et al. 2018, e.g.). In our comparison, we focus five of the best-known implementations and one very recent one. LRR FastLADMAP (Lin, Liu, and Su 2011), RPCA FPCP (Rodriguez and Wohlberg 2013), and MC GROUSE (Balzano and Wright 2013) use the low-rank + sparse model. ST GRASTA (He, Balzano, and Lui 2011) uses rank-1 + sparse. TTD 3WD (Oreifej, Li, and Shah 2013) uses low-rank + turbulence + sparse.
The most recent formulation we consider is OMoGMF (Yong et al. 2018), which utilises a Gaussian mixture model (GMM) structure over the low-rank model, plus sparse noise on top. We refer to (Bhojanapalli, Neyshabur, and Srebro 2016;Boumal, Voroninski, and Bandeira 2016;Jain and Kar 2017;Boumal, Absil, and Cartis 2018;Bhojanapalli et al. 2018) for the present-best theoretical analyses in the off-line, timeinvariant case, but stress that no guarantees have been known for the on-line, time-varying case. We refer to the recent handbook (Bouwmans, Aybat, and Zahzah 2016) and to the August 2018 special issue of the Proceedings of the IEEE (Vaswani, Chi, and Bouwmans 2018) for up-to-date surveys.
Problem Formulation
Consider N streams with n-dimensional measurements, coming from N sensors with uniform sampling period h from t k till t k +hT (possibly with many missing values), packaged in a (possibly partial) matrix M k ∈ R T ×nN . Every time a new observation comes in, its flattening is added at the bottom row to the matrix and the first row is discarded. In this way, the observation matrix slowly varies over time, i.e., M k+1 is different from M k , in general. It is natural to assume that any row d may resemble a linear combination of r T prototypical rows. Prior to the corruption by sparse noise, we assume that there exists where the row vector c d ∈ R 1×r weighs the rows of matrix R k , while e d ∈ R 1×nN is the noise row vector, where each entry be uniformly distributed between known, fixed −Δ and Δ. Further, this formulation (1) is extended towards the contamination model (Huber 1981), where "sparse errors" replace readings of some of the sensors. That is: Either we receive a measurement belonging to our model, or not: where index i enumerates sensors, s i ∈ R 1×n is a generic noise vector, while the Boolean vector I i,k ∈ {0, 1} n has entries that are all zeros or ones depending on whether we receive a measurement belonging to our model or not. The operation • represents element-wise multiplication.
Considering the matrix representation, we assume that the matrix M k can be decomposed into slowly varying low-rank model (C k R k ) and additive deviation (E k ) from the model comprising noise and anomalies: where T is the number of samples stacked in rows of matrix M k , r is the number of prototypes in the low-rank approxi- The missing entries in M k can represent either really absent data or outliers, such as moving objects in the case of video-processing applications. One can assume that normal behaviour exhibits certain regularity, which could be captured by a low-rank structure, and that events or anomalies are sparse across both time and space. The sparsity should be construed quite loosely, for example, comprising dense blobs of pixels moving coherently in video data, while occupying a relatively small fraction of image pixels in total. This notion of anomaly detection is widely used in monitoring streamed data, event recognition, and computer vision.
If we can identify the low-rank model, any deviation from the measurement model (1) can be interpreted as an anomaly or event. When there are few measurements for which I i,k = 1 n and those are different from standard measurements, i.e., the aggregated I k ∈ {0, 1} nN , which stacks all the individual I k for a specific time k, is sparse, and samples of s i fall outside of some range [M k,ij , M k,ij ] (defined below), it is possible to identify samples of s i perfectly. In this paper, we provide a way to detect such anomalies, i.e., measurements for which I i,k = 1 n . Hence, we are effectively proposing a principal component pursuit algorithm robust to uniform and sparse noise.
We compute matrices C k and R k by resorting to a lowrank approximation of the matrix M k with an explicit consideration of the uniformly-distributed error in the measurements. Let M k,ij be the (i, j) element of M k . Consider the interval uncertainty set [M k,ij − Δ, M k,ij + Δ] around each observation. Finding (C k , R k ) can be seen as matrix completion with element-wise lower bounds M k,ij := M k,ij − Δ and element-wise upper bounds M k,ij := M k,ij + Δ. Let C k,i: and R k,:j be the i-th row and j-th column of C k and R k , respectively. With Frobenius-norm regularisation, the completion problem we solve is: where: where : R → R is the square of the maximum of the twoelement set composed of the argument and 0, as detailed in Section "A Derivation of the Step Size" of (Akhriev, Marecek, and Simonetto 2018), and ν > 0 is a weight.
Our only further assumption is that we have the elementwise constraints on all elements of the matricial variable: This assumption is satisfied even for any missing values at ij when the measurements lie naturally in a bounded set, e.g., [0,255] in many computer-vision applications.
Proposed Algorithms
In this section, we first present the overall schema of our approach in Algorithm 1. Second, we present Algorithm 2 for on-line inequality-constrained matrix completion, a crucial sub-problem.
The Overall Schema
Overall, we interleave the updates to the low-rank model via the inequality-constrained matrix completion, detection of sparse noise, and updating of the inputs to the inequalityconstrained matrix completion, which disregards the sparse noise.
At each time step, we acquire new measurements x d and compute their projection coefficients onto the low-rank subspace as where p can be the 1, 2, ∞ norm, or the 0 pseudo-norm. Since for a very large number of sensors, even solving (6) can be challenging, we subsample x d by picking only a few sensors uniformly at random. Let i ∈Ñ be the sampled sensors, with Input: Initial matrices (C 0 , R 0 ), rank r Output: (C k , R k ) and events for each k 1: for each time t k : k = 1, 2, . . . , t k+1 − t k = h do computeṽ via the subsampled projection (7) 5: for each sensor i in parallel do 6: compute λ as a function of {r i } i as described in (Akhriev, Marecek, and Simonetto 2018) compute (C k , R k ) via Algorithm 2 with rank r 18: end for 19: return (C k , R k , y) Algorithm 1: Pursuit of low-rank models of time-varying matrices robust to both sparse and measurement noise.
where (R i k−1 ) i∈Ñ ∈ R r×nÑ is the matrix whose columns corresponds to the sensors, which are sampled uniformly at random. Solving (7) yields solutionsṽ such that the norm v −ṽ p is very small, while being considerably less demanding computationally.
Once the projection coefficients v have been computed, we can compute the discrepancy between the measurement (x d ) i coming from sensor i and our projection (7), (x d ) i − (vR k−1 ) i p , also known as the residual for sensor i. We use the residuals in a two-step thresholding procedure inspired by (Malistov 2014). In the first step, we use residuals to compute a coefficient λ > 0. In the second step, we consider the individual residuals as samples of an empirical distribution, and take the value at risk (VaR) at λ as a threshold. We provide details in (Akhriev and Marecek 2019;Akhriev, Marecek, and Simonetto 2018). The test as to whether residual at each sensor is below the threshold results in a binary map, suggesting whether the observation of each sensor is likely to have come from our model or not. For a positive value at i in the map, the measurement (x d ) i is kept in M k . Otherwise, it is discarded.
On-line Matrix Completion
Given M k , we utilise inequality-constrained matrix completion, to estimate the low-rank approximation (C k , R k ) of the input matrix considering interval uncertainty sets.
Clearly, solving the non-convex problem (4) for non-trivial dimensions of matrix M k to a non-trivial accuracy at highfrequency requires careful algorithm design. We propose an algorithm that tracks the low-rank R k over time, increasing the accuracy of the solution of (4) while new observations are brought in, and old ones are discarded. In particular, we propose the on-line alternating parallel randomised blockcoordinate descent method summarized in Algorithm 2.
For each input k, the previously-found approximate solutions (C k−1 , R k−1 ), are updated based on the new observation matrix M k , the correspondingly-derived element-wise lower and upper bounds M k,ij , M k,ij , and the desired rank r. The update is computed using the alternatig least squares (ALS) method, which is based on the observation that while f (4) is not convex jointly in (C k , R k ), it is convex in C k for fixed R k and in R k for fixed C k . The update takes the form of a sequence {(C T,τ k , R T,τ k )} of solutions, which are progressively more accurate. If we could run a large number of iterations of the ALS, we would be in an off-line mode. In the on-line mode, we keep the number of iterations small, and apply the final update based on M k at time t k+1 , when the next observation arrives.
The optimisation in each of the two alternating leastsquares problems is based on parallel block-coordinate descent, as reinterpreted by (Nesterov 2012). Notice that in Nesterov's optimal variant, one requires the the modulus of Lipschitz continuity restricted to the sampled coordinates (Nesterov 2012, Equation 2.4) to compute the step δ. Considering that the modulus is not known a priori, we maintain an estimate W T,τ ir of the modulus of Lipschitz continuity restricted to the C T,τ k,ir sampled, and estimate V T,τ rj of the modulus of Lipschitz continuity restricted to the R T,τ k,rj sampled. We refer to (Akhriev, Marecek, and Simonetto 2018) for the details of the estimate and to (Nesterov 2012) for a high-level overview.
Overall, when looking at Algorithm 2, notice that there are several nested loops. The counter for the update of the input is k. For each input, we consider factors C and R as the optimisation variable alternatingly, with counter T . For each factor, we take a number of block-coordinate descent steps, with the blocks sampled randomly; the counter for the block-coordinate steps is τ . In particular, in Steps 3-8 of the algorithm, we fix R T,τ k , choose a randomr and a random setŜ row of rows of C k , and, in parallel for i ∈Ŝ row , update C T,τ +1 k,ir to C T,τ k,ir + δ ir , where the step is: and P ir is the n×r matrix with 1 in entry (ir) and zeros elsewhere. The computation of ∇ C k f (C T,τ k , R T,τ k ; M k ), Pr j can be simplified considerably, as explained in in Section "A Derivation of the Step Size" of (Akhriev, Marecek, and Simonetto 2018).
Likewise, in Steps 9-14, we fix C T,τ +1 k , choose ar and a random setŜ column of columns of R k , and, in parallel for j ∈Ŝ column , update R T,τ +1 k,rj to R T,τ k,rj + δr j , where the step Input: updated M k , M k,ij , M k,ij , previous iterate (C k−1 , R k−1 ), rank r, limit τ Output: (C k , R k ) 1: Initialise: Algorithm 2: On-line inequality-constrained matrixcompletion via randomised coordinate descent. is: and Pr j is the r × m matrix with 1 in entry (rj) and zeros elsewhere. Again, the computation of , Pr j can be simplified.
Convergence Analysis
For the off-line inequality-constrained matrix completion problem (4), (Marecek, Richtarik, and Takac 2017) proposed an algorithm similar to Algorithm 2 and presented a convergence result, which states that the method is monotonic and, with probability 1, converges to the so-called bistable point, i.e., Here, we need to show the rate of convergence to the bistable point and a distance of the bi-stable point to an optimum f * : Theorem 2. There exists τ > 0, such that Algorithm 2 with the initialization to all-zero vector after at most T = O(log 1 ) steps has f (C T , R T ) ≤ f * + with probability 1.
Building upon this, we can prove a bound on the error in the on-line regime. In particular, we will show that Algorithm 2 generates a sequence of matrices {(C k , R k )} that in the large limit of k → ∞ guarantees a bounded tracking error, i.e., f (C k , The size of the tracking error E depends on how fast the time-varying matrices change: Assumption 3. The variation of the observation matrix M k at two subsequent instant k and k − 1 is so to guarantee that for all instants k > 0.
Now, let us bound the error in tracking, i.e., when M k changes over time and we run only a limited number of iterations τ of our algorithm per time step, before obtaining new inputs.
Theorem 4. Let Assumptions 1 and 3 hold. Then with probability 1, Algorithm 2 starting from an all-zero matrices generates a sequence of matrices
where η 0 < 1 is a constant. In the limit, In other words, as time passes, our on-line algorithm generates a sequence of approximately optimal costs that eventually reaches the optimal cost trajectory, up to an asymptotic bound. We bound from above the maximum discrepancy between the approximate optimum and the true one at instant k, as k goes to infinity. The convergence to the bound is linear and the rate is η 0 , and depends on the properties of the cost function, while the asymptotic bound depends on how fast the problem is changing over time.
This is a tracking result: we are pursuing a time-varying optimum by a finite number of iterations τ per time-step. If we could run a large number of iterations per each time step, then we would be back to a off-line case and we would not have a tracking error. This may not, however, be possible in settings, where inputs change faster than one can compute an iteration of the algorithm.
Experimental Evaluation
We have implemented Algorithms 1 and 2 in C++, and released the implementation 1 under Apache License 2.0. Based on limited experimentation, we have decided on the use of a time window of T = 35, rank r = 4, and half-width of the uniform noise Δ = 5. We have used dual simplex from IBM ILOG CPLEX 12.8 as a linear-programming solver for solving solving (7) in Algorithm 1. To initialise the C 0 and R 0 in Algorithm 1, we have used the matrix completion of Algorithms 2 with 1 epoch per frame for 3 passes on each video 1 https://github.com/jmarecek/OnlineLowRank (4,000 to 32,000 frames), starting from all-zero matrices. We note that in real-world deployments, such an initialisation may be unnecessary, as the the number of frames processed will render the initial error irrelevant.
First, let us highlight two aspects of the performance of the algorithm. In particular, on the top in Figure 1, we illustrate the effects of the subsampling on the projection (7). For projection in L 1 and L ∞ , we present the L 2 norm of the differenceṽ − v as a function of the sample period of the subsampling (7), where v is the true value obtained in (6) without subsampling andṽ is the value obtained in (7) with subsampling, and the sample period is the ratio of the dimensions of x d andx d . It is clear that L 1 is very robust to the subsampling. This corroborates the PAC bounds of ) and motivated our choice of L 1 with a sampling period of 100 pixels in the code. For completeness, we also present the performance of the Geman-McLure loss (Sawhney and Ayer 1996), where we do not consider subsampling, relative to the performance of L 1 norm without subsampling.
Next, on the bottom in Figure 1, we showcase the L 2 norm of residual C k R k − M k and the per-iteration run-time Table 2: Results of our Algorithm 2, compared to 6 other approaces on the "baseline" category of http://changedetection.net, evaluated on the 6 performance metrics of (Goyette et al. 2012 (He, Balzano, and Lui 2011) 0.45340 0.98205 0.01795 0.54660 0.44009 0.42367 TTD 3WD (Oreifej, Li, and Shah 2013) 0 We have also conducted a number of experiments on instances from changedetection.net (Goyette et al. 2012), a benchmark often used to test low-rank approaches. There, short videos (1,000 to 9,000 frames) are supplemented with ground-truth information of what is foreground and what is background. These experiments have been run on a single 4-core workstation (Intel Core i7-4800MQ CPU, 16 GB of RAM, RedHat 7.6/64) and results have been deposited 2 in FigShare. In Tables 2 and 3, we summarise the results. In particular, we present the false positive rate (FPR), false negative rate (FNR), specificity, precision, recall, and the geometric mean of the latter two (F1) of our method and 6 other lowrank approaches, which have been used as reference methods recently (Bouwmans, Aybat, and Zahzah 2016). These reference methods are implemented in LRSLibrary Bouwmans et al. 2015) and by the original authors of OMoGMF (Meng and Torre 2013;Yong et al. 2018), and have been used with their default settings. Out of these, OMoGMF (Yong et al. 2018) is the most recent and considered to be the most robust. Still, we can improve upon the results of OMoGMF by a considerable margin: the F1 score across the 6 categories is improved by 28% from 0.44643 to 0.57099, for example.
Further details and results are available in (Akhriev, Marecek, and Simonetto 2018). At http://changedetection.net/, a comparison against four dozen other methods is readily available, although one should like to discount methods tagged as "supervised", which are trained and tested on one and the same dataset. A further comparison against dozens of other methods is available in ).
Conclusions
We have presented a tracking result for time-varying low-rank models of time-varying matrices, robust to both uniformlydistributed measurement noise and arbitrarily-distributed "sparse" noise. This improves upon prior work, as summarised by the recent special issues Vaswani, Chi, and Bouwmans 2018).
Our analytical guarantees improve upon the state of the art in two ways. First, we provide a bound on the tracking error in estimation of the time-varying low-rank sub-space, rather than a result restricted to the off-line case. Second, we do not make restrictive assumptions on RIP properties, incoherence, identical covariance matrices, independence of all outlier supports, or initialisation. Broadly speaking, such analyses of time-varying non-convex optimisation (Liu et al. 2018;Tang et al. 2018;Fattahi et al. 2019;Massicot and Marecek 2019), seems to be an important direction for further research. 2 https://figshare.com/articles/AAAI2020 results zip/10316696 In practice, our use of randomised coordinate descent in alternating least-squares seems much better suited to highvolume (high-dimensional, high-frequency) data streams than spectral methods and other alternatives we are aware of. When the matrix M k does not change quickly, performing a fixed number of iterations within an inexact step (4) upon arrival of a new sample makes it possible to spread the computational load over time, while still recovering a good background model. Also, our algorithm is easy to implement and optimize. It has very few hyper-parameters, and this simplifies tuning. Our results are hence practically relevant. | 2018-09-10T19:00:34.000Z | 2018-09-10T00:00:00.000 | {
"year": 2020,
"sha1": "d9f0924419fbd7e11728fa8e24ef735d191c8974",
"oa_license": null,
"oa_url": "https://ojs.aaai.org/index.php/AAAI/article/download/5714/5570",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "d627e13897c541c37dd002dd4cc163dbd6c78a57",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Mathematics",
"Computer Science"
]
} |
256503149 | pes2o/s2orc | v3-fos-license | MIRN: A multi-interest retrieval network with sequence-to-interest EM routing
Vector-based retrieval have been widely adopted to process online users’ diverse interests for recommendations. However, most of them utilize a single vector to represent user multiple interests (UMI), inevitably impairing the accuracy and diversity of item retrieval. In addition, existing work often does not take into account the scale and speed of the model, and high-dimensional user representation vectors need high computation cost, leading to inefficient item retrieval. In this paper, we propose a novel lightweight multi-interest retrieval network (MIRN) by incorporating sequence-to-interest Expectation Maximization (EM) routing to deal with users’ multiple interests. By leveraging representation ability of the Capsule network, we design a multi-interest representation learning module that clusters multiple Capsule vectors from the user’s behavior sequence to represent each of their interests respectively. In addition, we introduce a composite capsule clustering strategy for the Capsule network framework to reduce the scale of the network model. Furthermore, a Capsule-aware module incorporating an attention mechanism has been developed to guide model training by adaptively learning multiple Capsule vectors of user representations. The experimental results demonstrate MIRN outperforms the state-of-the-art approaches for item retrieval and gains significant improvements in terms of metric evaluations.
Introduction
Recommendation systems are an effective way to alleviate information overload and suggest relevant options to end users to satisfy their individual needs and interests, especially in many user-oriented online services, such as e-commerce (Amazon, Taobao) and social media (Facebook, Instagram) sites [1]. Recommendation systems usually consist of two stages, namely the matching stage and the ranking stage. Fig 1 shows an illustrative diagram of this process. Here, the purpose of matching stage is to efficiently retrieve a subset of items from the entire corpus that are relevant to user's interests. That is, the massive multi-category rating dataset stored in the corpus is retrieved by our model to generate thousands of candidate items related to users' multiple interests during the matching stage. In the ranking stage, the retrieved items not only need to be refined by accurate recommendation algorithms and scoring algorithms but also need to be adjusted in conjunction with specific business rules to produce the final items to be recommended. As the initial phase of the entire recommendation process, the performance of the retrieval model determines the upper limit of the final recommendation. Therefore, it is critical to model user interests and find user representations for matching stage in order to retrieve items that satisfy user interests.
Recently, vectorization-based retrieval methods have been widely adopted, such as DSSM [2], Deep FM [3], YouTube DNN [4], etc. The basic idea is to embed users and items into a potential vector space and utilize the inner product of vectors to represent the preferences between users and items. Although these methods have made some progress in the matching stage, existing recommendation models still have bottlenecks in learning multiple interest embeddings from user behavior sequences. Besides, knowledge graph-based approaches, such as Deep Walk [5], EGES [6], and Graph SAGE [7], are used to investigate recommendation diversity. The fundamental idea is to construct a knowledge graph utilizing users' behavioral data and then develop a function that performs an aggregated representation of surrounding vertices to provide an embedding vector of target items. While the knowledge graph-based approach makes a small contribution to solving the UMI problem, requirements for huge computational cost and storage space make it difficult to deploy on devices with limited resources. On the one hand, the size of the model and the algorithm's inference speed are the pressing issues that we need to address in our deployment. On the other hand, the amount of user-item interactions amassed in online service platforms grows over time, and user behavior sequences keep becoming larger, so extracting merely a single embedding vector from them is no longer feasible. In fact, the matching stage should take more responsibility for diversity since it is more concerned with the coverage of user-interest items, which directly determines the upper limit of recommendation effectiveness. The diversity of recommenders needs to be ensured in the matching stage first. Otherwise, the homogeneity of candidate items generated by the matching stage will inevitably lead to a lack of diversity in the final recommendations.
In this paper, to overcome above limitations, we focus on the problem of modeling different interests of users, which is crucial in real-world recommender systems. We propose a lightweight Multi-Interest Retrieval Network (MIRN) with Sequence-to-Interest Expectation Maximization routing (EM) to reflect user representations with different interests. Specifically, we design a multi-interest representation learning module that uses Sequence-to-Interest Expectation Maximization routing (S2I-EM) to adaptively cluster users' historical behaviors into user representations. For a particular user, MIRN outputs multiple representation vectors, and An illustrative diagram shows how to use MIRN in an industrial recommendation system. MIRN focuses on the matching stage, which aims to retrieve user-interested items efficiently. Note that the matching stage is concerned with retrieving good items, rather than specific item ranks.
https://doi.org/10.1371/journal.pone.0281275.g001 each of them represents one interest tendency of the user, respectively. Ultimately these representation vectors can be used in the matching stage to retrieve user-interest related items from a large corpus. We conduct extensive experiments on two public datasets. The experimental results confirm that MIRN exhibits excellent performance compared to current state-of-theart models. To summarize, the main contributions of this work are as follows: • To obtain UMI from behavior sequences, we designed a multi-interest representation learning module incorporating S2I-EM routing, which adaptively clusters users' behavior sequences into multiple representation vectors. This work lays a good foundation for the successful application of EM routing algorithms in the matching stage.
• In order to reduce the size of our model and to be able to deploy it smoothly in real scenarios, we designed a composite capsule clustering strategy and applied multiple ways to speed up the computation of algorithm iterations. This is the first study to apply a model that integrates three characteristics (accuracy, diversity, and complexity) for item retrieval.
• Extensive experiments conducted on two real-world datasets demonstrate that MIRN achieves significant better performance consistently.
Related work
Early work on item retrieval typically used collaborative filtering (CF) to model user preferences based on their interaction history [8,9]. Model-based collaborative filtering approaches [10][11][12][13][14][15] are an increasingly common work line. These approaches project users and items into a shared vector space and estimate a user's preference for an item by measuring the similarity between the user and the items in her/his interaction history using a pre-computed item-toitem similarity matrix. In practice, however, the number of users is frequently much greater than the number of items, resulting in a significant overhead in maintaining and storing these similarity matrices. Furthermore, a user's historical data is frequently minimal, and the accuracy of finding similar users who have only made a few transactions or clicks is low.
In the last few years, tree-based approaches have been extensively studied in the item matching stage [16][17][18], especially in capturing the diversity of user interests. Tree-based retrieval approaches [19][20][21][22][23] use a tree structure as an index and attach each item in the corpus to a leaf node by clustering [23] or joint learning [22]. Specifically, a node in the tree represents an embedding vector, and the model outputs a probability value corresponding to that vector by computing it. The probability value is used to describe the user's interest in a particular node, and the whole tree represents the hierarchical information about the user's interest (diversity of user interests). In addition, although the tree-based retrieval algorithm allows the model to learn more information about the interaction between users and candidate items (improve the accuracy of item retrieval), the complexity of the structure and algorithm limits it from wide application in industry. Therefore, tree-based retrieval algorithms have greater potential for improvement both theoretically and engineering-wise.
Since then, a number of vector-based approaches for diversifying retrieval results have been proposed. Embedding approaches have had a major impact in both academia and industry. Covington et al. [4] mapped users and items into a low-dimensional dense vector in a You-Tube video recommendation system, and then performed item retrieval by an efficient retrieval method. In the deep-attention network (DAN) [24], the self-attention mechanism guides the model to appropriately filter feature vectors to reduce the interference of redundant information. Huang et al. [25] developed a vector embedding-based retrieval framework and applied it to an inverted index-based search system (Facebook). This work is a successful practice of vector embedding-based retrieval algorithms deployed to large search systems. Nie et al. [26] proposed a novel cross-domain learning network (CLN) for 2D image-based 3D shape retrieval task. Xinyang, Yi et al. [3] proposed a neural deep retrieval (NDR) algorithm based on a two-tower network architecture for retrieving personalized suggestions from a corpus of tens of millions of videos (YouTube). The core idea is to encode a wide variety of item feature vectors using a network called item tower. An advantage of these vector embedding-based retrieval methods is that quantization-based indexing [27] techniques and hierarchical graph indexing [28] techniques can be employed to speed up their retrieval efficiency.
Recently, several researchers have attempted to use graph neural networks [29][30][31][32][33] for item retrieval [34,35]. Hamilton et al. presented GraphSAGE [7], a new graph learning model that learns the embedding representation of the target node by aggregating the feature information of the node's neighbors, i.e., to improve the user-side representation and accuracy in the matching stage. Ying et al. [36] created PinSAGE, an efficient graph convolution approach that enriches item-side representation by generating item-based knowledge graphs. Besides, to improve the diversity and robustness of item retrieval, the model is trained with increasingly difficult samples. While graph-based retrieval approaches can take into consideration the diversity of user interests, the complexity of knowledge graph construction, as well as the higher storage capacity and processing capability requirements, are the most prominent challenges for deployment in real industry applications.
Problem formalization
In this article's recommendation scenario, we let U ¼ fu 1 ; u 2 ; . . . ; u jUj g be the user set and I be the complete items pool, where |u| is the total number of unique users. Each user u 2 U can be associated with an item set I u 2 I, i.e., I u stores all items interacted by user u (also called user behavior). The goal of our framework is to retrieve a subset of items from a million-scale items pool I À I u for each user u 2 U such that the subset contains only a few thousand items and each item is relevant to the user's interests. In addition, the basic information (like user gender and age) of user u can be represented by B u , and F i is used to represent the characteristics of the target item, such as item id and category id.
The core task of MIRN is to learn a function that maps raw features into user representations, which can be expressed as where the M u ¼ fm 1 u ;m 2 u ; . . .m K u g 2 R d�K denotes the representation vectors of user u. d is the dimensionality, and K denotes the number of representation vectors. In this paper, we use a dynamic strategy to replace the fixed parameter K. The details are described in Eq (16). Besides, the representation vector of target item i is obtained by an embedding function as whereṽ i 2 R d�1 denotes the representation vectors of item i. Now a similarity score can be computed based on the user representation vector and the item representation vector to filter out the top N items related to the interests of each user u from the item candidate pool. The scoring function is as follows: where N is the final number of candidates, which is set in advance based on the whole system. The main notations we used are listed in Table 1.
Embedding layer
As shown in Fig 2, the input of MIRN contains three parts, user basic information B u , user behavior sequence I u , and label item F i . The label item is split from user behaviors and belongs to a recent real purchase made by a user. Due to the large size of the dataset, the dimensionality of each feature is high, especially some categorical id features, so it is difficult to operate with one-hot encoding and the similarity between features is difficult to capture. To reduce the number of parameters in the computation process, we adopt the word2vec [37] embedding technique to embed high-dimensional features into low-dimensional dense vectors, and each feature of this dense vector can be considered to have practical value. Specifically, for users containing features such as age and gender from user basic information B u are encoded into user feature embeddingsb u , while item ids along with other categorical ids from F i are encoded to form the label item embeddingṽ i after averaging the pooling layer. And the items interacted by users from I u , corresponding item embeddings are obtained to form the user behavior embedding.
Multi-interest representation learning
The multi-interest representation learning module is developed to learn multiple representation vectors from the user's behavior sequence, and finally to form the corresponding interest Capsule of the user.
Expectation maximization routing.
We begin with a brief review of the important basics about Expectation Maximization (EM) routing, an iterative algorithm that can also be considered as a new form of neural units represented by matrices. The original EM routing was designed for image processing by computing pixel values to update background images, cannot being applied for recommendation task. However, the powerful feature capture and attribute processing capabilities exhibited by EM routing at the fine-grained level provide us an important insight that we may use it to handle category properties with strong entity representation in recommendation tasks.
Another key basis is the Gaussian Mixture Model (GMM), a clustering method utilized in EM routing. The advantages of the GMM can be summarized as follows: on the one hand, it can handle nonlinear decision boundaries and produce good clustering results. On the other hand, it can cluster data based on its distribution and interpret the clustering results more
PLOS ONE
accurately. In this paper, we suppose that the user historical behavior data can be separated into K categories and that they all follow a Gaussian distribution, the probability density function of GMM may be stated as: where j represents the category, PðjÞ denotes the probability of each category being chosen, usually written as π j . The probability distribution PðxjjÞ for each category/interest can be expressed as: N ðx; m j ; S j Þ ¼ 1 ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi where d represents the dimensionality of input vector/matrix x. μ j , S j are the mean (vector) and covariance (matrix) of the GMM distribution, respectively. Intuitively, the values of three parameters π j , μ j and S j need to be determined during the UMI modeling. There is a deduction based on the Wiener-khinchin law of large numbers, for a Gaussian distribution, the results of default maximum likelihood estimation are equal to the second-order moment estimation in a large enough amount of data. We arrive at a simplified version of the derivation process based on Bayesian definition and the formula is as follows: The mean μ can be calculated as: Similarly, the covariance matrix S and the parameter π j are calculated as follows: The above parameters vary in the S2I-EM routing algorithm inexorably, regulating the quality of user interest diversity. Obtaining their maximum likelihood estimates would be ideal, however they do not have a close form analytical solution. Therefore, we propose the S2I-EM routing algorithm for solving the above problem.
Sequence to interest expectation maximization routing.
There are two layers in our routing framework, the lower layer is the sequence of user's behaviors (behavioral Capsules) and the upper layer is the Capsules obtained by algorithmic clustering, which are user's interest Capsules. In this paper, P i ; ð1 � i � nÞ is used to denote lower layer behavioral Capsules, and n is the number of users' behavioral Capsules. We let Q j ; ð1 � j � KÞ denote the upper Capsules, where K is the number of interest Capsules/categories. The goal of S2I-EM routing is to compute the values of upper Capsules in an iterative manner once the values of lower Capsules are known. The specific details will be discussed in the following four ways.
1) Covariance matrix replacement with a diagonal matrix. Without loss of generality, the covariance matrix S j is defined as a diagonal matrix, i.e., S j ¼ diags 2 j , where s 2 j is the variance vector of category j. Therefore, the variance vector is used to describe the covariance matrix mainly for the following considerations. On the one hand, we can avoid solving the inverse of a matrix as well as the determinant, which can greatly reduce memory consumption and computation, improving efficiency of algorithm. On the other hand, σ j is a standard deviation vector, and s l j denotes the l-th component of this standard deviation vector, which makes each component of input vector/matrix x decoupled and remains independent. Based on the above analysis, the simplification of Eq (2) can be obtained as: where m l j denotes the l-th component of mean vector of interest j. Similarly, the solution expression of S j is converted to the expression of s 2 j by the following: As a result, we may construct a local representation of the S2I-EM routing, as shown in Algorithm 1.
for all behavior capsule i: R ij In this paper, p ij and R ij are two simple notations for Eqs (9) and (10), respectively. And R ij is the connection probability between behavioral and interest Capsules, which we initialize to a uniform distribution. The calculation of upper interest Capsule Q j will be discussed in Eq (15). The connection probability R ij is used to quantify the correlation between the behavior Capsules and the interest Capsules. For example, if cluster B (lower-level behavioral Capsule) and cluster A (upper-level interest Capsule) are independent of each other, then the connection probability between them is 0. The high correlation between them corresponds to a high connection probability value. Furthermore, the connection probability of a Capsule is governed by the activation probability, i.e., the Capsule with a zero activation probability value also has a zero connection probability.
2) A composite capsule clustering strategy. It is important to select K behavioral Capsules at random to represent the mean value (centroid) of each large interest cluster in the GMM clustering procedure, and then update this mean value in iterations. However, the cost of iterative updates to the GMM is enormous, which not only increases the computing overhead of our model but also severely affects the convergence speed of the algorithm. In addition, largesize models are difficult to deploy in real scenarios where storage space is limited. To ligthen the architecture of our model and retain the advantages of GMM clustering, we designed a composite capsule clustering strategy. Specifically, we first apply a lightweight clustering algorithm, namely K-means, to accomplish coarse-grained clustering of user behavioral Capsules and generate K data clusters. The GMM algorithm is then utilized to complete a finegrained partitioning of the K data clusters, with the cluster results/centroids acquired from Kmeans serving as one of the inputs to the GMM algorithm. The following are some of the benefits of this strategy: 1) Since the cluster centroids supplied by K-means are identical to the mean μ discovered by GMM, the computation time of mean μ in GMM can be considerably reduced.
2) The composite clustering technique minimizes the number of GMM iterations, reduces the size of the model, and speeds up the algorithm's convergence. Note that we also pre-set a value for the stop iteration operation, which can be used to force termination if the algorithm does not converge for a long time.
3) Information entropy describes activation value. Similar to traditional neural networks, we use a scalar a j to measure the salience of Capsule features in the information propagation process of S2I-EM routing, where we call a j the activation value of a Capsule. Then we ask: how to obtain the activation value? A promising solution is as follows: We can cluster users' interests by using GMM and obtain a probability distribution p(x|j), which can describe a user-specific interest Capsule/category. The salience of Capsules can be indirectly measured by the degree of solidarity, so we introduce information entropy in S2I-EM routing to quantify the activation value. Specifically, the salience of a Capsule can be represented by the information entropy, and the higher information entropy (close to uniform distribution), the smaller activation value. On the contrary, the lower information entropy (the closer to the Gaussian distribution), the higher activation value of the category. The information entropy S j in our S2I-EM routing is defined in the following way: We believe that the high information entropy of a category group indicates that the current category group is still chaotic. Take for example, there is still interest belonging to category B clustered in category A, making the Capsules of category A clusters hold less weight in the voting process. Conversely, when the information entropy is small, it means that the cluster is close to convergence and can contribute to the upper-level Capsule, hence the corresponding activation value should be larger.
Here we have supplemented the derivation of Eq (11). Information entropy can be used to measure the degree of confusion of a category, which in turn can describe the activation value of the category. Firstly, we give the formula for calculating information entropy as: Note that, one of the essential reasons why we use the above procedure to calculate the information entropy instead of integrating it directly is that the latter calculates the theoretical result and the former obtains the actual result for this batch of data. Next, we work on the derivation of the information entropy in S2I-EM routing. Here, it is assumed that p ij corresponds to an independent normal distribution of d elements with the following equation: This concludes the derivation of the information entropy in S2I-EM routing.
Since the value of information entropy is inversely proportional to the significance of Capsule features, it is a more reasonable choice to use À S j to measure the activation value of features. Ultimately, activation value a j is compressed (interval 0-1) by the sigmoid function to more intuitively reflect the activation level of Capsule features.
where γ a , γ b is a set of training parameters assigned to each upper-level Capsule, optimized by backpropagation.
4) Number of dynamic interests.
Let's explore more details of the upper-level interest Capsule Q j calculation. The probability that behavior Capsule i is clustered into the same interest Capsule j is influenced by the similarity between voting matrices V ij . Specifically, the voting matrix V ij is obtained by the transformation of behavioral Capsule i, i.e., behavior Capsule i and weight matrix W ij are multiplied by the product operation, where the weight matrix is learned by cost function and backpropagation.
Note that the voting matrix V ij of each behavior Capsule i is associated with the predicted value of an element in interest Capsule j. That is, the mean value m h j of the Gaussian distribution of elements in voting matrix indicates the h-th element in interest Capsule j. We essentially explain the process of S2I-EM routing execution, i.e., extracting multiple interest Capsules from the lower-level behavior Capsules to represent the UMI.
In addition, since user behaviors with implied interest diversity exhibit a distinct multipeaked distribution, where the number of peaks corresponds to the number of interest Capsules. The value of K must be adjusted for different users. We employ heuristic rules to adjust how many interest Capsules will be given in the end. Specifically, the number of dynamic interests is determined by a combination of the Gaussian distribution of user behaviors and a predetermined K. The number of dynamic interests K u for user u is calculated as: We do not propose that all users employ the same value of K, because this dynamic technique of altering the number of user interests is primarily designed to provide better assistance for those users with fewer interests, in the meaning while, saving computing and memory resources. Algorithm 2 lists the whole S2I EM routing process.
Capsule-aware module
With the multi-interest representation learning module, we obtain multiple interest Capsules from user's behavioral embeddings to represent different interests. Then we ask: How to measure the importance of these interest Capsules?
We attempt to answer this question in the following perspective: A user's final behavior will be influenced by a certain interest, i.e., there is a clear correlation between the user's actual behavior and a specific interest Capsule. Therefore, we designed a module based on a dot product attention mechanism during the training process, called Capsule-aware, for easier selection of interest Capsules to guide the model training. In Capsuleaware module, the label item is the query and the interest Capsules are both keys and values, as shown in Fig 2. Specifically, we first calculate the compatibility between each interest Capsule and the label item embedding. Second, we selected an interest Capsule corresponding to the maximum compatibility and calculated its weighted values, where the weight of the interest Capsule is determined by the corresponding compatibility. Finally, the user representation vector of the label item is represented by the weighted values of the selected interest Capsule and used to guide the training of our model. It's worth noting that one of the primary reasons we didn't utilize the weighted sum of all interest Capsules as the user representation vector for the label item throughout the training phase was to avoid confounding UMI and compromising the user's maximum expectation. The target interest Capsule is obtained by the following equation:q whereq j 2 Q denotes representation vector of user's j − th interest Capsule, j 2 f1; . . . ; K u g. f att is used to compute the similarity between labeled items and the interest Capsule. Thus, the final expressionm u for user u is computed as follows: where f concat represents the splicing function of user vector and the interest Capsule. Assign each objectṽ i to its closest centroid 9 Update the cluster means, i.e., compute the new centroid (mean) of each cluster 10 until no change; 11 return C ¼ fc 1 ; c 2 ; . . . ; c j g is a set of k cluster centroids 12 Procedure S2I-EM ROUTING 13 8i 2 I u ; j 2 K u : InitializeR ij ¼ 1=jK u j 14 for iter = 1,. . .,r do 15 8j
Multi-interest representation training objective
After the final user representationm u and the label item embeddingṽ i are learned, we calculate the likelihood score p(i|u) with respective to candidate item i as: After that, we train MIRN by minimizing the probability of user u interacting with the target item i. In this sense, we can give the model a high level of flexibility and nonlinear modeling capabilities. The complete loss function of our MIRN is as follows: The sum operator of Eq (19) is computationally expensive; thus, we use the sampled softmax technique [4] to make the objective function trackable and optimize the network by applying Adam [38], which is a variant of Stochastic Gradient Descent (SGD) to adapt the learning rate automatically. After training, the MIRN network except for the Capsule-aware module can be used as a user representation mapping function f user . And in the matching stage, the user's behavior sequence and user base information are fed into the f user function, generating multiple representation vectors for each user. These representative vectors are then used to retrieve the top N items through some approximate nearest neighbor methods, such as the Elasticsearch search engine [39]. Note that once the training is completed, we fix the model and construct approximate preference embeddings by fusing other attribute features of users to solve the cold start problem for new users.
Experiments
In experiments, we conduct extensive offline evaluations to verify that MIRN can improve both accuracy and diversity. In this section, we attempt to answer the following three research questions: • RQ1: How does the proposed MIRN perform against different types of competitive models on recommendation accuracy in the matching stage?
• RQ2: What effect do different essential parameters have on MIRN's accuracy and diversity?
• RQ3: How do the efficiency and complexity of MIRN compared to other models?
Offline evaluation
In this section, we present a comparison of the performance of MIRN and existing methods in the offline setting.
Datasets and experimental setup.
We evaluate recommendation performance on two publicly available datasets from different domains. One of them is the popular movie rating dataset MovieLens (https://grouplens.org/datasets/movielens/) provided by [40], which contains about 10 million user ratings from January 1996 to July 2014. The other is Amazon Product Data (http://jmcauley.ucsd.edu/data/amazon/) provided by [41], including user reviews (ratings, text, useful/unused votes, etc.) and product metadata (category, price, brand, etc.) on Amazon from May 1996 to July 2014. For MovieLens-Latest, due to the short user behavior sequence and simple data labels, we only consider the factors such as age, gender, occupation, and movie category. For Amazon Product Data, we deleted users whose operation records were less than 10 and items whose interaction records were less than 8. In addition, we consider the evaluation of fewer than three stars and above as positive, recorded as 1, and the evaluation of fewer than three stars as bad, recorded as 0. The statistics of the two datasets are shown in Table 2.
Compared methods.
• WALS [42] WALS, abbreviated as Weighted Alternating Least Squares, is a classical matrix decomposition algorithm. It performs the corresponding recommendation by confidence weights, giving a larger weight to items with high user preference and a smaller weight to items with no feedback.
• YouTube DNN [4]. In YouTube's recommendation process, the division of the recommendation process into two phases, retrieval and sorting, is the most successful example in the field of recommendation systems. • MaxMF [43]. It is a non-linear model. It uses multiple vectors to represent users, and then a vector to represent products. Finally, the largest score in the user vector is taken as the matching score.
• MIND [44]. The vectorized retrieval approach, represented by MIND, is one of the mainstream recommendation algorithms today. It generates multiple embeddings characterizing users' interests to enhance the matching stage and avoids the problem of causing head effects.
Evaluation metrics.
We use the idea of sequential recommendation for prediction, i.e., we predict the next interaction between a user and an item, and thus evaluate the performance of the above method accordingly. We randomly divided the user-item interaction data into training set and test set by a ratio of 17:3. For each user, we first use the last interaction data as the target item and then use the remaining interaction data for that user as the user behavior.
• Hit rate. Hit rate (HR) is a commonly used metric for measuring recall in current top N recommendation studies. It calculates the percentage of underlying true items in the top N positions of the correctly ranked list, and a larger metric indicates better recommendation performance. The HR@N score is defined as: where D test denotes the test set consisting a large number of users and target items (u, i). T n is a set of the top N items in item ranking list and f i (�) is an indicator function.
• Normalized discounted cumulative gain. NDCG is a ranking performance evaluation metric widely used in recommender systems, which indirectly evaluates the performance of model by measuring the relevance of retrieved Top N item list to user u. The higher the relevance, the better the performance. So, it is defined as: where Rank(�) represents a ranking function on input sequence or values. sim i u denotes the similarity score between the representation vector of user u and the embedding vector of candidate item i, and label is the true rating value of the item.
Implementation details.
In our experiments, we set the size of the embedding vector to 1024. We set the mini-batch size to 128 and use the Adam optimizer [38] with a 0.001 learning rate to minimize the total loss. In addition, we dynamically adjust the dropout rate in the range of {0.2, 0.4, 0.7} to prevent the overfitting of our models. And for other parameters, we use a grid search strategy to find the optimal settings. Finally, we adjust the l2 regularization term λ from 0.0001, increasing by a factor of 10 each time, and the learning rate from {0.001, 0.005, 0.01}. The implementation of the experiments is based on the server with the opensource framework Tensorflow-gpu2.4.0 [45], keras2.4.3 and python3.7, CUDA11.2. The GPU used for the experiments is NVIDIA GeForce RTX 3090 with 128GB memory.
Model complexity analysis.
In this subsection, we investigate the complexity of the proposed MIRN. It is obvious that the calculation of dynamic interest capsules and S2I-EM routing are the main operations. Given |u| users and jIj items, suppose that each user directly connects with jI u j items. In the process of calculating dynamic interest capsules, the computational complexity of procedure K-means is OðK u Þ, where K u denotes the number of interest capsules. In the process of S2I-EM routing, the time cost of the E-Step is OðrjI u jÞ, and the M-Step requires time complexity OðrjK u jÞ, where |r| represents the iteration times. Since the number of interest capsules jK u j is smaller than the number of items jI u j with which users interacted, we can regard it as a constant. The total time complexity is approximately OðrjI u jÞ. Under the same experimental settings (i.e., representation sizes and initialized clusters), MIRN has a smaller complexity than MIND. Therefore, the total time complexity is acceptable in practice.
Results analysis (RQ1).
We begin with the comparison w.r.t. HR@50 and NDCG@50. The empirical results are reported in Table 3, where %Imp. denotes the relative improvements of the best performing method (starred) over the strongest baselines (underlined). We find that: • MIRN consistently outperforms most baselines across four datasets in terms of all measures.
Specifically, it achieves significant improvements over the strongest baselines w.r.t. HR@50 by 4.80%, 2.05%, 1.55% in Amazon-Electron, Amazon Movie-TV, and MovieLens-Latest, respectively. This demonstrates the rationality and effectiveness of MIRN. We attribute these improvements to the multi-interest extraction capability of MIRN: (1) By mining users' different interests, MIRN is able to better describe the relationships between users and items, and produce more robust representations of users and items. In contrast, other baselines ignore the importance of multiple interest representations, and use a single vector to model UMI; (2) Benefiting from our S2I-EM routing algorithm, MIRN can preserve the overall semantics of interest propensity and collect more information from behavioral Capsules, than USI-based baselines (i.e., WALS, YouTube DNN).
• Combining four datasets for MIRN analysis, we find that the improvement is more significant on Amazon-Electron than on Amazon-Video-Games. This is reasonable since (1) the user-item interaction data on Amazon-Electron provides denser and richer information than the data on Amazon-Video-Games; and (2) sparse interest categories dominate on Amazon-Video-Games. This suggests that MIRN is good at exploiting the potential of multiinterest extraction.
• Our MIRN achieves the best performance on several datasets, including Amazon Electron and Amazon Movies-TV. Compared to MIND the improvement is around 1% to 4%. For Amazon datasets, the improvements introduced by mining UMI are more significant, suggesting that users in Amazon tend to have more diverse interests in filtering items than in MovieLens-Latest.
• The matrix decomposition method, WALS, is defeated by other methods, indicating that deep learning is effective in improving the retrieval performance of the model. However, MaxMF performs much better than WALS without deep learning, which can be explained by the fact that MaxMF is a nonlinear model that uses multiple user representation vectors for retrieval. And we can also see from the results that the method using multiple user representation vectors (MaxMF, MIND) shows better performance than other methods (WALS, YouTube DNN). Thus, using multiple user representation vectors can effectively model UMI and boost the accuracy of recommendations.
In addition, we analyzed the experiments in which MIRN performed poorly by evaluating HR@10 and HR@50 in Table 4. An obvious finding is that these datasets (i.e., Amazon Office-Products, Amazon Beauty, and Amazon Digit-Music) are generally small (see Table 2 for details), making it difficult for the model to fit the data completely. While MaxFM performs better than MIRN on these datasets, we conjecture that (1) MaxFM benefits from the advantages of matrix factorization and can produce appropriate recommendations even with sparse data.; (2) It alleviates the high-dimensional disaster by introducing hidden vectors to decompose the high-dimensional behavioral sequences into multiple different representation vectors. This also demonstrates the effectiveness of using multiple Capsule vectors to represent UMI.
The visualization results of the performance comparison of different models on different datasets are shown in Fig 3. From the experimental process of Amazon Electron with the Amazon Movies-TV dataset, it can be found that although MIRN requires longer iteration rounds compared to MIND, the performance achieved by MIRN is more advantageous. Besides, we found that there may be oscillations in our model-fitting curve when the data is small, which indicates that our model needs further optimization to learn enough relevant features when dealing with small datasets. However, the information collected by commercial platforms servicing millions of users is generally in the tens of thousands, so MIRN's recommendation is more in accordance with the needs in real scenarios. Finally, we fine-tune our experiments on the Amazon Home-Kitchen and Amazon Video-Games datasets directly using the model already trained on Amazon Movies-TV. The model fitting curves show that our model learns the common behavioral patterns of users with good portability.
Discussion of hyperparameters (RQ2)
In this section, we conduct experiments on the Amazon Electron and Movielens-Latest datasets to investigate the effect of hyperparameters within the multi-interest representation learning module and Capsule-aware module, respectively. 1) Parameter K sensitivity. We performed a corresponding sensitivity study on the hyperparameter K in the model architecture. K controls the overall capacity of the model. If K is small, the number of clusters is small for all the items; if K is big, the time complexity of the training and matching stage grows linearly with K. Moreover, a large K may increase the possibility of overfitting. Table 5 summarizes the performance of the model when the hyperparameter K is changed. And we can understand that the performance of our model steadily increases as K increases, peaking at K ¼ 5. For the Movielens-Latest dataset, the best performance of MIRN is obtained when K ¼ 5. For the Amazon Electron dataset, strong performance is still achieved when K ¼ 5. Fig 4 shows the iterative fitting speed and accuracy of MIRN on the Amazon Electron dataset. We notice that the performance is worst when K ¼ 1. This is because users show the same intention for all items, making the accuracy of the model suboptimal due to the lack of interest diversity. However, as K rises, user interest diversity gradually surfaces, user-side representations get richer, and the model is able to capture more accurate information to improve the ability of item retrieval. Compared to MIND, our model exhibits higher performance with different K values, which demonstrates the efficacy of our modeling strategy for extracting multiple interests.
2) Connection probability. The connection probability R ij is used to quantify the correlation between the behavior Capsules and the interest Capsules. The value of the connection probability fluctuates continually over the algorithm's iterations, and in this study, we visualize its value to show that the multi-interest extraction process is interpretable. Fig 5 illustrates the connection probabilities associated with a user, where each row represents one of the user's interest Capsules, and each column corresponds to one of the user's behaviors. The intensity of the color in heat map indicates the user's preference for the item. A dark color indicates a strong preference for the item and, conversely, a light color implies a lack of interest. From the results, we can see that the user interacts most frequently with three categories of goods (cell phones, computers, and headphones), and their respective connection probabilities are the largest in the interest Capsule, i.e., these three categories of goods form the user's corresponding interest. Regarding this result, we believe that the work of extracting multiple interests of the user has been achieved, i.e., the UMI are divided into different interest Capsules.
Model efficiency and complexity (RQ3)
We analyze the efficiency and complexity of retrieval models in various terms. In Table 6, we report the number of parameters, the number of floating-point operations (FLOPs), and the inference latency of item retrieval in the matching stage. MaxFM learns nonlinear latent factors to represent multiple user vectors, leading to a disadvantage in parametric number and inference time. YouTube DNN represents a user with a long single vector, which has fewer parameters and FLOPs than MaxFM, but its accuracy is likewise low. This demonstrates that the use of multiple vectors to represent the user can make a greater contribution to improving models' accuracy. In addition, some of the more advanced models, such as MIND and MIRN, take the form of multi-vector representations, as indicated in Table 6. It can be observed that MIRN with a composite clustering strategy not only outperforms its much larger counterpart, MIND, in terms of performance; its reduced model size also significantly reduces inference costs and greatly facilitates the use of smaller hardware in downstream tasks. Second, in our analysis, the number of initialized interest categories K and the length of user behavior sequences are the key factors affecting FLOPs. Increasing the value of K increases the number of parameters in our model marginally while also improving its performance. In conclusion, MIRN outperforms other methods in terms of model size and multi-interest extraction.
Conclusion and future works
In this paper, we propose a new model named MIRN, which can generate multiple Capsule vectors of users' different interest representations for item retrieval in the matching stage. Specifically, we design an S2I EM routing algorithm to dynamically extract the different interests of users, and then these interests are trained with the Capsule-aware module. Besides, a composite clustering strategy is applied in this paper to make our model more lightweight. To investigate the performance of MIRN, we conducted extensive offline evaluations, results analysis. Experiments have shown that our model achieves significant improvements compared to the baseline approaches on two large public datasets, Movielens-Latest and Amazon Product.
In the future, we will work in two directions. The first is to build a multi-channel network to capture the temporary interests generated by users in a short time. The second direction is to investigate the embedding of user preference with the GNN and metric-based learning techniques in order to enhance the performance of cold-start recommendation. | 2023-02-03T06:16:44.566Z | 2023-02-02T00:00:00.000 | {
"year": 2023,
"sha1": "55510b3d36e7eb7e16a839b8914ad04c3c7f612f",
"oa_license": "CCBY",
"oa_url": null,
"oa_status": null,
"pdf_src": "PubMedCentral",
"pdf_hash": "6aed9146577c7412d4a7f62c1fc72956ca95e4f2",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
258003432 | pes2o/s2orc | v3-fos-license | Astrovirology: how viruses enhance our understanding of life in the Universe
Viruses are the most numerically abundant biological entities on Earth. As ubiquitous replicators of molecular information and agents of community change, viruses have potent effects on the life on Earth, and may play a critical role in human spaceflight, for life-detection missions to other planetary bodies and planetary protection. However, major knowledge gaps constrain our understanding of the Earth’s virosphere: (1) the role viruses play in biogeochemical cycles, (2) the origin(s) of viruses and (3) the involvement of viruses in the evolution, distribution and persistence of life. As viruses are the only replicators that span all known types of nucleic acids, an expanded experimental and theoretical toolbox built for Earth’s viruses will be pivotal for detecting and understanding life on Earth and beyond. Only by filling in these knowledge and technical gaps we will obtain an inclusive assessment of how to distinguish and detect life on other planetary surfaces. Meanwhile, space exploration requires life-support systems for the needs of humans, plants and their microbial inhabitants. Viral effects on microbes and plants are essential for Earth’s biosphere and human health, but virus–host interactions in spaceflight are poorly understood. Viral relationships with their hosts respond to environmental changes in complex ways which are difficult to predict by extrapolating from Earth-based proxies. These relationships should be studied in space to fully understand how spaceflight will modulate viral impacts on human health and life-support systems, including microbiomes. In this review, we address key questions that must be examined to incorporate viruses into Earth system models, life-support systems and life detection. Tackling these questions will benefit our efforts to develop planetary protection protocols and further our understanding of viruses in astrobiology.
Introduction
Viruses are numerically abundant and an integral part of life on Earth.However, there is very little known about viruses across many of Earth's environments, including the many extreme habitats that serve as astrobiology analogues, when compared with the understanding of viruses relevant to human health.Even with respect to human health, relatively little is known about viral responses to the space environment beyond Earth's atmosphere.Additionally, almost no research has been conducted to identify the impact of these viruses on the spacecraft systems used to sustain humans.Understanding viruses and virus-host interactions in space-relevant contexts will inform our grasp of their role(s) in both human spaceflight missions, including environmental control and life support systems (ECLSS), and in the search for life elsewhere.Here, we review viruses in diverse Earth environments, the very limited studies conducted on viruses in space and lay out a roadmap for future research.For a more extensive overview of environmental viruses and introduction to viruses, see Berliner et al. (2018); for a specific review on the roles of viruses in spaceflight affecting human health, see Pavletic et al. (2022); for a complementary review on the need to incorporate viruses more in astrobiology, see De La Higuera and Lázaro (2022).
Viruses are sometimes defined as 'very small obligate intracellular parasites' (Acheson, 2011), and more broadly as 'entities whose genomes are elements of nucleic acids that replicate inside living cells using the cellular synthetic machinery and causing the synthesis of specialized elements that can transfer the viral genome to other cells' (Luria et al., 1978).These 'specialized elements' or virions are crucial to the definition of viruses.Viruses contain genetic information that can be transferred on massive scales due to the very large numbers of virions on Earth.
The sheer ubiquity of virions on Earth, with up to 10 31 in the Earth's oceans alone (Suttle, 2005;Mushegian, 2020), makes them an attractive target for the search for life on other worlds if one only knew what to search for!Viruses that infect bacteria and archaea typically range in size from tens to a few hundred nanometres, while viruses that infect eukaryotic organisms (e.g.amoebae or humans) can even range from tens to thousands of nanometres (Fig. 1).These so-called giant viruses can be larger than some bacteria or archaea and can even be infected by viruses themselves (Sommers et al., 2021).For example, virophages have been observed to infect giant viruses that are themselves infecting cellular organisms (e.g.amoebae).In these cases, the virophage infection co-opts the giant virus and may improve the condition of the host organism (Roux et al., 2017;Backstrom et al., 2019;Schulz et al., 2020).Viruses can also alter the genetic architecture, phenotypic characteristics, reproduction strategy, infection dynamics and evolution of nearby viruses, which in turn influences how viruses interact with their hosts and ultimately, ecosystems.This spectrum of virus behaviours across all domains of life is also reflected in the productivity of their infected host cells (i.e.modulation of host-cell metabolism and tens to thousands of progeny virus particles per cell) and timing of single-cycle infection (e.g.tens to thousands of minutes).Notably, the timing for one cycle of virus growth correlates with the doubling time of healthy host cells (Jin and Yin 2021).
The diverse morphology of viruses which extends beyond that typically found in cellular life (Fig. 1), and the presence of unique genes for the virus coat or capsid, have already been used as a model for other life: 'capsid-encoding organisms' (Forterre and Prangishvili, 2009).Virus genomes can be comprised of DNA or RNA, double-stranded or singlestranded, in multiple variations of each.Unlike cellular life, there are no universal viral genes to serve as a backbone upon which to build consistent evolutionary trees (Roux et al., 2019;Tisza et al., 2020).The diversity of both virus morphology and viral nucleic acids makes them difficult to search for comprehensively, but also drives expanded toolkits that could be employed in the search for life on other worlds and therefore deserves further investigation.
Virus-like entities may even be detectable on other worlds where life-like processes are only just evolving.Although humans have become explicitly aware of viruses and their effects only in the past few centuries (Loeffler and Frosch, 1897;Beijerinck, 1898), they were likely present as soon as -or even before -the first cell arose on Earth (Moelling and Broecker, 2019).Viral infections have played a key role in the evolution of life on Earth via the exchange of genes between viruses and their hosts, providing genetic diversity, killing dominant hosts thus maintaining balanced populations and possibly even inventing DNA itself (Forterre and Prangishvili, 2009;Enard et al., 2016).Viral genome remnants are present in most, if not all, cellular genomes, including in humans, where viruses play key roles in human physiology (Bannert and Kurth, 2004;Arneth, 2021).Further studies of the history of Earth's viruses may highlight opportunities for detecting emergent life on other worlds.
On Earth, viruses have such a substantial impact that they drive major global biogeochemical cycles, and the same may be true on other worlds.The vast majority of marine viruses infect microbes (Suttle, 2005;Parikka et al., 2017), thus affecting many biological processes, including encoding photosynthesis genes that substantially contribute to atmospheric oxygen (O 2 ), and help regulate global nutrient cycling (Greene and Reid, 2013).Further understanding of the persistence of virus particles and viral impacts on microbial cellular chemistry may therefore help illuminate biosignatures on distant planets, or signatures of past life in the geologic records of other worlds.
Targeting the search for biosignatures should focus on locations that are currently or once were within the environmental ranges compatible with the persistence and replication of information molecules.The more that research is conducted on viral persistence and replication in extreme environments, the more that remarkable feats of persistence and replication may be discovered (e.g.viruses thawed from permafrost could infect a modernday version of their host; Legendre et al., 2014).
Viral persistence in and response to extreme conditions also has important implications for humans voyaging into space, as recently reviewed by Pavletić et al. (2022).However, in addition to viral impacts on human physiology via direct infection and impacts on the microbiome, viral infection of plants or microbes could impact critical life support systems on deep space missions, including algal bioreactors (Matula and Nabity, 2019).Conditions such as microgravity affect fluid dynamics with implications for bacterial biology (as reviewed in Diaz et al., 2019;Acres et al., 2021;Sharma and Curtis, 2022), and presumably also virus-host interactions, but this is still an underdeveloped field.
The diversity of unique viral qualities and their integral role in life on Earth compels us to better understand the potential roles of viruses and virus-like entities in both astrobiology and space biology.There are multiple critical knowledge gaps in our understanding of viruses in the space environment and their potential role in other hypothetical biospheres in our Solar System and beyond.
In this review, we outline what new virus research here on Earth should investigate to address key scientific questions in planetary science, space biology and astrobiology.These include:
1.
What role(s) did viruses play in the origin and evolution of life on Earth?
2.
What are the environmental limits to the preservation and propagation of virions?
3.
What role(s) could viruses play in potential biospheres elsewhere, and how might those impacts be detectable as biosignatures?
4.
Can the detection of viruses beyond Earth be interpreted as a sign of life?
5.
How do artificial environments affect the human virome?
6.
Can virions be fossilized or otherwise preserved in a recognizable state?
Potential role(s) of viruses in the early stages of life
Increasing knowledge of the roles of viruses in the origins of life on Earth will benefit the search for life elsewhere.Viruses have and will continue to play a critical role in the evolution of life on Earth.Precursors of virus-like entities may even mark the beginning of life itself, making the virosphere potentially as old and significant as the cellular biosphere (Fig. 2; Janjic, 2018;Moelling and Broecker, 2019).Viral signatures may be pivotal in the search for life elsewhere and in understanding evolutionary mechanics of other biospheres.Theoretical models predict that (depending on the information-transfer properties of the system) parasitic replicators will emerge anywhere in the Universe that life-like processes evolve (Eigen, 1971;Bresch et al., 1980;Vignuzzi and López, 2019;Vlok et al., 2019).Indeed, even viruses themselves fall prey to parasitic replicators in the form of defective virus genomes and virophages that multiply at the expense of fully intact virus genomes (Perrault, 1981;Vignuzzi and López, 2019;Roux et al., 2023).The 'RNA-world' hypothesis characterizes the origin of life with self-replicating RNA, followed by ribonucleoproteins that later evolved into DNA and larger proteins (Müller et al., 2022).Viruses and virus-like replicators are the only known extant biological entities that contain all types of nucleic acid genomes (Table 1), including single-stranded and double-stranded RNA and DNA or mixtures thereof.RNAviruses serve as models for how RNA and ribonucleoproteins could have propagated via simple self-replicating RNA structures and ribozyme activity (Landweber et al., 1998;Koonin et al., 2006;Tyler, 2008;Durzyńska and Goździcka-Józefiak, 2015;Matsumura et al., 2016;Weinberg et al., 2019).Viruses may have helped support the transition from the early RNAworld to the current DNAworld (Forterre, 2006;Diemer and Stedman, 2012).Further, the recent acceptance of network-based clustering for virus taxonomy (Jang et al., 2019) in addition to incorporation of three-dimensional (3D) structures (and other viral characteristics) have allowed deep evolutionary inferences that have identified newly proposed phyla of RNA viruses which may be a previously missing link in the transition from the RNA-peptide world to the DNA-RNA-peptide world (Zayed et al., 2022).Further, beyond the origins of life, viruses played a role in the origins of the mitochondrion (Shutt and Gray, 2006) and of the placenta (Mi et al., 2000).Viruses may even have been critical in the evolution of eukaryotes and multicellularity (Forterre and Gaïa, 2016;Lee et al., 2018;Guglielmini et al., 2019).Overall, viruses appear to have played many roles in the origin and evolution of early life.
While uncovering the history of viruses is key to understanding the origin and evolution of life on Earth, an additional benefit is the development of new technologies that are potentially valuable for characterizing the diversity and history of non-terran life.For instance, comparative analysis of 3D atomic structures is a non-genomic method being used to interrogate virus origins that could also be applied to non-terran samples that may lack nucleic acids (Bamford et al., 2005).This method is employed to detect and classify viruses because there are no genes shared by all extant viruses (Roux et al., 2019;Tisza et al., 2020).
In comparison, all organismal cells conserve some genomic features such as ribosomal RNA (Table 1).Thus, the search for life elsewhere can tap into such techniques.In particular, we propose the following future research directions.
a.
Investigate evolutionary relationships between RNA viruses and ribozymes (ribonucleotide enzymes) to evaluate the potential role of viruses in an RNA world, and the transition to a DNA world.
b.
Create tools and methods to enable inferences about early life through comparative analysis of extant viral life.
c.
Reconstruct ancient events to illuminate the origin(s) of viruses on Earth, and their possible roles in the emergence of all life.
d.
Develop phylogenies that consider all biological entities to attempt to connect and better constrain the shared and divergent evolutionary histories of viruses and cellular organisms.
Habitability, persistence and process limits of viruses and cellular life
Viruses have been known to remain active in a variety of harsh conditions, sometimes influencing the functioning and preservation of their hosts and sometimes reinfecting hosts after being preserved themselves yet still viable.Cellular life can also persist and operate under a variety of extreme conditions on Earth.However, the monitoring of virusvirus and virus-host activity together in extreme environments has been limited, despite these interactions being inherently different than those in milder environments.Therefore, fundamental concepts are not well understood about the types of environmental conditions and processes that are most conducive to the preservation and propagation of information molecules or molecular signatures of life.For example, understanding the upper and lower limits of viruses' persistence and activity under a range of conditions would inform where we might find viruses and other life on both Earth, in space and on other worlds.Additionally, long-term monitoring of viruses in extreme environments could inform their ability to persist under similar conditions on another planetary body.Multiple analytical techniques could be used to better determine viral activity in both dormant and active host environments and to better characterize viruses and low-level host activity as well.While viral process limitations are currently unknown for a range of conditions, viruses persist and interact with their hosts under a wide range of extreme conditions on Earth (Gil et al., 2021), including extremes in temperature, pH, pressure and salinity.These conditions manifest in both natural and built environments, and viruses have been observed in a variety of settings (Table 2).Understanding the extreme ranges of conditions in which viruses and their hosts can survive informs the search for life on other worlds, the operational considerations for planetary protection and viral monitoring during human spaceflight.
In a variety of environmental settings, viruses can persist, maintain their integrity and even help preserve their hosts for extended periods of time.For example, viruses that adsorb to clays are protected from inactivation, thus enabling them to persist in soils for longer periods of time in the absence of a host (Bitton, 1975;Lance and Gerba, 1984;Lipson and Stotzky, 1985;Syngouna and Chrysikopoulos, 2010).Virus particles can also be preserved via silicification and can become reactivated (i.e.infectious once again) when the silica is removed (Laidler and Stedman 2010;Laidler et al., 2013).Further, upon thawing, viruses preserved in frozen permafrost for ~30 000 years were able to infect modern day versions of their hosts (Legendre et al., 2014).Viruses can also influence the preservation of their biological hosts.For example, viruses that infect microbial mats can cause or expedite the microbial mats' fossilization into a stromatolite by (1) acting as a nucleation site for organomineralization, (2) altering microbial host metabolism to promote carbonate precipitation or (3) increasing the production of microbial extracellular substances (Pacton et al., 2014;White et al., 2021).In summary, viruses can function as key structures for preservation and propagation of biological information, especially in extreme environments.
In this context, a better understanding of which environmental and molecular factors drive this long-term persistence will enhance future searches for virus-like elements outside of Earth.
Throughout extreme environmental conditions, viruses can also extend protection to their hosts.For instance, viruses can confer host heat tolerance (Márquez et al., 2007), and carry genes for sporulation that may drive their host to form an inactive spore, that is robust to unfavourable conditions (Trubl et al., 2018;Van Goethem et al., 2019;Pelusi et al., 2021;Travers et al., 2022).In another instance, viruses allow photosynthesis in cyanobacteria under desiccating conditions (Azua-Bustos et al., 2012).Harsh environments, such as polar regions or hydrothermal vents, have a high incidence of temperate viruses, which can reside within their microbial hosts (lysogeny) until conditions are favourable for viral replication (Anderson et al., 2014;Brum et al., 2016).While in this lysogenic state, viruses may express genes that alter their microbial host's physiology and metabolism, thus increasing the Table 2. Example locations where viruses have been observed through isolation or genomic studies host cell's ability to survive conditions in which resources are limited, including within sea ice, dry soil or acidic environments (Chen et al., 2005;Yu et al., 2015;Howard-Varona et al., 2017;Lee et al., 2021;Wu et al., 2021).
By integrating into hosts that are dormancy capable, viruses may be able to persist through conditions that are incompatible with activity then reactivate when conditions improve.Extreme examples of organisms regaining function after dormancy include: (1) cyanobacteria reviving after a 672 day exposure to space outside of the International Space Station (ISS; Laranjeiro et al., 2021), (2) cyanobacteria in Antarctica metabolizing within a week after rewetting following 20-30 years without stream flow (McKnight et al., 2007), (3) nematodes reviving from 30 000-year-old permafrost in Siberia (Shatilovich et al., 2018) and (4) rotifers in northeastern Siberia reactivating from 24 000-year-old permafrost (Shmakova et al., 2021).Microbes living under low-energy conditions in the South Pacific Gyre are claimed to have even retained their metabolic response after 101.5 million years (Morono et al., 2020).The diverse evidence of microbial and eukaryote dormancy noted above shows the potential for organisms to survive and even grow in space-like environments and planetary analogues.However, not much is known about any hitchhiking viruses, that have accompanied these long-persisting hosts, nor how they may have contributed to long host dormancies.Evaluating the persistence and reactivation of viruses in dormant hosts would hint at the plausibility of their presence in extreme non-terran environments.We posit that similar virus-host interactions are likely on any planetary body where life exists, thus it is critical to expand our understanding of these interactions on Earth to enable the detection and identification of such phenomena during space missions.For example, it is now feasible to computationally integrate how functions encoded in virus genomes interact with the material and energy resources of their hosts, thereby predicting the timing and levels of virus growth (Yin and Redovich, 2018).Successfully predicting viral activity in human-support space environments would help assure astronaut health.
Viral activity in low-energy Earth environments can be explored through development of high sensitivity tracer approaches.Bulk tracer methods, such as nucleic acid stable isotope probing (SIP; Radajewski et al., 2000;Manefield et al., 2002), can provide detailed information about the level of stable isotope incorporation by microbes and have been applied to investigate virus activity in complex environments (Pasulka et al., 2018;Lee et al., 2021Lee et al., , 2022;;Starr et al., 2021;Trubl et al., 2021).Bulk methods such as SIP integrate over relatively large sample mass (Nuccio et al., 2022), thus requiring more enriched substrate in complex systems.However, investigators have recently developed imaging mass spectrometry approaches that can detect viral replication at the single-particle level based on incorporation of rare stable carbon and nitrogen isotopes (Gates et al., 2018;Pasulka et al., 2018;Mayali et al., 2019).Because virion enrichment has been detected in individual particles using nanometre-scale secondary ion mass spectrometry (NanoSIMS), the approach is clearly equally viable for other very small samples.These studies have demonstrated sensitivity down to 100 nm-diameter virions.Further research is necessary to extend these methods to bacteriophage in the 50 nm-diameter range and experimentally apply them to extreme environmental and space-like conditions.Such powerful techniques could be used to evaluate the resilience of viruses to astrobiologically relevant conditions, thus we propose the following experimental directions.
a.
Increase of environmental surveys and long-term monitoring of viral persistence and activity in extreme environments that can be used as analogues of planetary bodies and early life on Earth.
b.
Experimental work on the lower and upper limits to viral persistence and activity under a variety of environmental parameters.
c.
Evaluation of the persistence and activity of viruses in dormant and active hosts, combining techniques (e.g.SIP with metagenomics) to better characterize viruses and detect low-level host activity.
Biogeochemical cycling and biosignature detection
Viruses play critical roles in biogeochemical cycles on Earth.Our understanding of viruses as major players in Earth's biogeochemical cycles has been reshaped by the advent of metagenomic approaches that have enabled the study of uncultivated viruses (Kristensen et al., 2010;Roux et al., 2016Roux et al., , 2019)).Marine virology has been particularly intensely studied, highlighting the potential for worlds with liquid oceans, such as Enceladus or Europa, to contain informational molecules such as virus genomes and proteins amongst their organic compounds (Postberg et al., 2018).On Earth, viruses have a huge impact on O 2 concentrations by infecting the marine cyanobacteria that are responsible for ~25% of the O 2 in Earth's atmosphere.At any moment, about half of marine cyanobacteria are infected, which can lead to either a decrease in O 2 production (i.e. cells lysed by viruses) or an increase in the efficiency of their O 2 production (Sieradzki et al., 2019).
We need an improved understanding of what controls that balance, and how can that be used in biogeochemical modelling.Other work has demonstrated the activity of viruses in cold to sub-freezing temperature soils (Trubl et al., 2021;Wu et al., 2022) and illustrates the potential for viruses in biogeochemical processes of cold terrestrial worlds, such as Mars.Terrestrial worlds, or at least partly rocky bodies, such as comets, may host ice-lidded cryoconite holes in their polar ice caps that could result in the presence of liquid water to support active life (Zawierucha et al., 2017).Could terrestrial worlds with thick mid-deck atmospheres, such as Venus, harbour life and virus-like entities?On Earth, we know that viruses exist within droplets in the atmosphere and can augment iron and sulphur metabolisms (Anantharaman et al., 2014;Bonnain et al., 2016;Roux et al., 2016;Dalcin Martins et al., 2018).Since Venus's clouds contain sulphuric acid, viruses could be influencing biogeochemical processes in the planet's atmosphere as well.More intimately studying the role of viruses in biogeochemical processes in a variety of Earth analogue environments could inform any potential role viruses might play in biogeochemical processes on other worlds.
The widespread and frequent detection of genes used by viruses to hijack the metabolism of their host cell(s) and manipulate them to produce viral progeny strengthens the need for a conceptual shift towards calling virus-infected cells 'virocells', as this will help emphasize their differences from uninfected cells (Forterre, 2011(Forterre, , 2013)).Viral infections can change a microbe's metabolic outputs, impacting the composition and quantity of their biosignatures in ways as profound as the organism's own genome.Combined experimental and in silico studies of virus growth on bacterial hosts have shown how the physiological state of the host cell can be reflected in the timing and level of virus production, whereas virus production is intimately linked to the availability of resources for protein synthesis (Mahmoudabadi et al., 2017).Since nutrient availability in the host's environment impacts its ability to produce viruses, the productivity of virus infection can provide a readout of the metabolic demands of the living host (You et al., 2002).Further, computational modelling suggests that viruses evolve to optimally use the finite metabolic energy resources of their host cells (Kim and Yin, 2004).Virus growth may also be linked to host physiology in ways not previously appreciated; studies of viruses that infect bacteria, eukarya and archaea revealed delay times for virus production that correlate with the doubling times of their host cells (Jin and Yin, 2021).Similar virus-host interactions could be at play in other systems, and we must be prepared to identify them.
Virus-host interactions and viral effects on microbial biosignatures can be evaluated through tracking stable isotopic signatures.Autotrophic microbial life preferentially incorporates the lighter isotope available for each biogenic element, a feature often used to identify ancient microbial life and sources of input materials in extant life.Yet the exact signatures of this process depend on which organisms are performing a given metabolic process, how many enzymatic reactions take place in relevant metabolic pathways and how the enzyme(s) work.For example, fractionation factors are different for denitrification via fungi versus bacteria (Ostrom and Ostrom, 2017), or for methanogenesis depending on the initial substrate and the organisms involved (Whiticar, 1999;Hornibrook et al., 2000;Penning et al., 2006).When a virus infects a microbial host, it redirects its metabolism, thus possibly impacting these isotopic values.Such impact could be more dramatic if a virus were to encode an auxiliary metabolic gene that has a different fractionation factor than the host version of that gene.For example, kinetic measurements of virus and host versions of the same enzyme have revealed that the virus enzyme had a significantly lower k cat /K M value than the host enzyme (Thompson et al., 2011).Such viral influences can lead to large variations in isotopic signatures, leading to uncertainty when distinguishing between abiotic and biological processes and in the utility of a particular biosignature (Schwieterman et al., 2018).While complex and potentially difficult for distinguishing biotic from abiotic processes, isotopic fractionation has the potential to reveal fundamental characteristics of biosphere metabolism, including the impact and contribution of viruses.
Isotopic fractionation measurements have long been applied to the geologic record of Earth to infer characteristics of the metabolisms of past biospheres and changes over time (Johnson et al., 2021).The isotope history of marine ecosystems throughout Earth's history is well-studied (Zerkle and Mikhail, 2017;Krissansen-Totton et al., 2015).However, the isotopic composition of icy environments through time on Earth is less well-characterized but may provide useful analogue environments applicable to other planetary bodies such as Mars (Havig and Hamilton, 2019).Distinguishing viral influences on both present isotopic signatures and on their variation over multiple timescales may therefore provide a reference for interpreting isotopic biosignatures of past life on other worlds.To better understand the role of viruses in biogeochemical cycling on Earth in order to interpret and model geochemical cycles elsewhere, we propose the following experimental directions.
a.
Improve understanding of how different viruses hijack their host's cellular machinery.
b.
Further explore the broad range of virus-host interactions and dynamics in various ecosystems.
c.
Quantitatively estimate the role and impact(s) of virocell metabolism on Earth's different biogeochemical cycles.
d.
Improve understanding of the extent and distribution of viral impacts on biosignatures for majorEarth biogeochemical cycles, including the potential magnitude of such effects.
e.
Search for the existence of any generalizable biosignatures associated with viral metabolic reprogramming to reduce uncertainty associated with biosignatures for life detection.
Impact of viruses on human space exploration
Human spaceflight continues to evolve, and many new technologies and tests are being developed for the safety of crew members and planetary protection for future human-landed missions.However, many studies exclude viruses, despite their threats (and potential benefits) to many types of cellular life.Thus, questions remain as to how viruses interact with other viruses and their hosts in space environments.For example, how do natural versus artificial environments drive changes in the virome and how are viruses impacted and shaped by their environment?As human spaceflight expands to sustained missions beyond low Earth orbit (LEO), nearly every aspect of the mission (including ECLSS, human health and performance, lander operations and extravehicular activities (EVA)) must consider the impact of virus-host interactions.Otherwise, the impacts may prove to be analogous to the current concern about volatile contamination on the ISS where current instrument capabilities and measurements are less sensitive or erroneous due to environmental conditions untested prior to flight (Regberg et al., 2022).
Viral response to spaceflight environments-Viral studies in microgravity have been very limited, with most microbiology investigations focused on bacteria.Those have shown a variety of species-specific and even strain-specific morphological and physiological responses to the low fluid shear force and lack of liquid media convection in microgravity (as reviewed in Diaz et al., 2019;Acres et al., 2021;Sharma and Curtis, 2022).Without externally applied forces, nearly everything in a microgravity environment can remain suspended or quiescent.Brownian motion dominates, thus reducing the ability for host cells to gather nutrients (Zea et al., 2016), and thereby influencing the size, structure and organization of these host cells.Additionally, the physical limitations of microgravity could potentially modify the infiltration capabilities of viruses.Further, the lack of buoyancy effects may also influence viral dispersal and host encounter rates in both the spacecraft cabin atmosphere and quiescent fluid systems.Another response to microgravity is aggregation (Zea et al., 2018;Domnin et al., 2022), which might limit the ability of viruses outside an aggregate to encounter a host surface or increase host encounter rate once one host in an aggregate is lysed.Even with external forces (fans, pumps, etc.) moving liquid and gas loops, it is still unknown how well viruses can adhere to encountered surfaces.Simulated microgravity has been associated with a thicker cell membrane envelope in Escherichia coli (Zea et al., 2018), which could inhibit the ability of viruses to infect hosts.In contrast, lower membrane integrity was observed in Vibrio fischeri (now known as Aliivibrio fischeri; Vroom et al., 2021), which could make hosts more susceptible to membrane-disrupting events.
The effects of microgravity on virus dynamics can be measured through environmental sequencing of surfaces aboard space stations and crewmember microbiomes, yet there is a dismaying lack of virus-centric metagenomic analyses (according to Mora et al., 2019, there has been a single study).Although there may be limitations in sampling size, frequency and depth of coverage due to the technical and logistical challenges of obtaining spacecraft samples, several data sets from the ISS are already available for reanalysis (Be et al., 2017;Singh et al., 2018;Urbaniak et al., 2018Urbaniak et al., , 2022;;Checinska Sielaff et al., 2019;Avila-Herrera et al., 2020).In future studies, the virus-to-host ratio for known virus-host pairs is one statistic that can be estimated, and its distribution explored spatially, temporally and in response to natural or experimental perturbations.Targeted and untargeted metagenomic sequencing of microgravity samples would have complementary strengths allowing for comprehensive analyses that include data from broad surveys at the species and genus taxonomic levels and to narrowly focused studies concerning specific strain and variant population dynamics.Viral dispersal dynamics, host adsorption, infection and progeny productivity in spaceflight environments are extremely understudied and deserve significant further work.
Environmental control and life support systems-ECLSS are imperative for supporting human spaceflight.These flight-proven technologies on the ISS control air composition and temperature, food and water and waste remediation.However, longduration missions beyond LEO will require robust alternatives that do not rely on frequent resupply missions (Anderson et al., 2019).Closing the carbon loop through bioregenerative technologies is one approach for providing ECLSS.Algal photobioreactors can remove carbon dioxide (CO 2 ), liberate O 2 , remove or alter waste and produce edible biomass (Matula and Nabity, 2019;Fahrion et al., 2021).These photobioreactors can withstand the dynamic temperature environment experienced within the ISS thermal control loops (Matula and Nabity, 2021;Matula et al., 2021).Preliminary spaceflight studies using algae for ECLSS observed thriving cultures (Helisch et al., 2020;Poughon et al., 2020).Likewise, extremophilic algae, lichen, cyanobacteria and fungi included in experiments mounted on the outside of the ISS and Space Shuttle survived weeks to months-long missions (de Vera et al., 2019;Malavasi et al., 2020).However, these studies did not characterize the full microbiome within the non-axenic cultures.This current lack is extremely important.For example, virophages can have profound implications for microbial nutrient cycling, often referred to as the microbial loop.Predator-prey simulation models indicate that the presence of virophages regulates helper virus and algal host dynamics altering carbon flux through the microbial loop in aquatic ecosystems (Yau et al., 2011).To survive unfavourable conditions (e.g.microgravity, viral infection and antimicrobials), some microbes can form protective barriers such as biofilms.Bacteria exude a variety of organic matter dubbed extracellular polymeric substances, which forms the biofilm and acts like glue securing and protecting the bacteria allowing them to remain hydrated, control local pH and other services.Biofilms can benefit certain systems such as the rhizosphere (plants root zones) aiding in food production.However, biofilms can also be detrimental by corroding or clogging machinery that supports ECLSS, or preventing wound healing in skin abrasions, to name just a couple of examples (Landry et al., 2020).Recently, highlighted in Justiniano et al. (2023) are the knowledge gaps in our understanding of microbiomes (including viruses) and biofilm formation in space, although we do know that viruses can adapt new strategies to prevent bacterial growth or kill organisms within biofilms.Understanding potential biome or virome shifts within these systems over long durations may elucidate the need for specific algal species selection, causes of viral or bacterial-based system failures and operational considerations (Fig. 3; Matula and Nabity, 2019).
Bioregenerative ECLSS-Efforts on the ISS have focused on plants for CO 2 removal and O 2 production, as a source of multivitamins, for urine reuse and to promote psychological well-being (Dzhos et al., 2021).On Earth, plant viruses can be a threat to agriculture, and disease management relies on rapid and accurate viral identification (Rubio et al., 2020).Many plants have been grown under microgravity, including flower-producing species, herbs and vegetables.However, due to the stress of spaceflight, viral infection of these plants can be hugely influential on functionality.A recent study found that spaceflight factors significantly affect tomato plants (Dzhos et al., 2021), increasing the productivity of the plants and the concentration of some vitamins such as carotenoids, which are important for crew members on long-term space missions.Importantly, plants grown from seeds that were in space for half a year prior to germination were resistant to viral infection.Moreover, seed resilience of many plant species (amongst other organisms) was tested by keeping them outside the ISS for 558 or 682 days, thus exposing them to high levels of radiation (Tepfer and Leach, 2017).All seeds were able to germinate after the shorter flight, but only seeds with a stronger coat could survive the longer radiation exposure.While these studies show promise for cultivating plants on long-term voyages, our understanding of plant-virus interactions on short flights is extremely limited and we have no information for long-term flights.
Human physiology and the viral ecology of crewed spacecraft-In space-bound crewmembers, latent viruses are shown to reactivate more than in matched controls.Sometimes this reactivation occurs before leaving Earth and is presumably stress related.However, there are several plausible reactivation triggers associated with spaceflight itself, such as ultraviolet or ionizing radiation (Pavletíc et al., 2022), but precisely which mechanisms might be responsible for increased virus reactivation in crewmembers have not been determined.Reactivation has included herpes simplex virus, Epstein-Barr virus and varicella zoster virus (Stowe et al., 2000;Mehta et al., 2004;Pierson et al., 2005;Rooney et al., 2019).After 6-12 months in space, crewmembers experienced significant changes in their microbiome (both internal and external) that led to rashes and hypersensitivity episodes (Voorhies et al., 2019).These symptoms warrant further investigation into the effects of long-term space environment exposure on humans combined with their accompanying microbes and viruses.
The community composition of viruses and other organisms on the ISS is mostly comprised of viruses that only infect non-human cells, such as bacteriophages.This composition differs from that of terrestrial space analogues (e.g.MDRS and HI-SEAS), thus pointing towards the space environment as the likely trigger (Mora et al., 2019).Although these viruses do not infect humans, they can be transported back to Earth and released into Earth environments via plants, humans and other entities that can harbour them.Continued efforts are needed for quarantining people within space stations for long-term space travel and increased screening for crewmembers returning to Earth.A viable way to test viral transmission and educate humans, especially those preparing for space travel is through simulations that consider epidemiology parameters (Patel et al., 2021).Although it might be tempting to contemplate elimination of viruses from spacecraft wherever possible, we should be sensitive to the unanticipated positive roles viruses may play and the unreasonable resources that viral elimination would entail.As well, a virus's presence can be beneficial by sometimes warding off more pathogenic relatives by cross-protection or superinfection exclusion (Folimonova et al., 2020).Immunologic 'eustress' can promote a healthy organism, as is seen by comparison to the physiological deficits of germ-free animals (Round and Mazmanian, 2009).Indeed, in some cases, viruses can be symbiotic with their host organisms (Roosinck, 2011).As such, viruses are key players in crewmember health and safety and in the ecology of crewed environments.
Spaceflight operational impacts-Given the differences in viral dispersal patterns observed in low-gravity environments and the extreme conditions under which viruses persist and interact with hosts (see Section 'Viral response to spaceflight environments'), even surfaces often considered sterile, or at least clean, must be investigated.Some bacterial species -for example, Bacillus pumilis strain SAFR-032 (Tirumalai et al., 2013) -are refractory to pre-flight sterilization, and some could be tolerant as well given the extreme diversity of viral morphotypes (including lipid-free viruses, see Fig. 1).Even with perfect sterilization of equipment (which is essentially unobtainable), it is not currently possible to achieve a virus-free environment on a manned space mission because of post-launch viral shedding by crewmembers (vide supra).The frequency of sampling, maintaining and cleaning life support systems will impact day-to-day spaceflight operations, during both cruise phase and orbital or surface sustained missions.EVA and associated planetary protection protocols must also be accommodated: for example, protecting areas with highscientific value from contamination (e.g.Rummel et al., 2014).Modifications of operations may entail landing further from a planetary surface sample site, designing new vehicles, requiring more stringent cleaning protocols or considering entirely new sample sites (e.g.Meyer et al., 2019).Additionally, contamination restrictions during EVAwill narrow the design of space suit systems (Willson et al., 2014).Attempts to dictate planetary protection protocols without fully understanding viral dispersal and survivability will potentially result in overly restrictive constraints that ultimately hinder scientific discovery or alternatively inadequate protocols that do not achieve proper containment.
To understand the impact of viruses on human space exploration, we suggest that the following experiments be prioritized.
Future directions
a.
Characterize differences in dispersal, aggregation and adsorption processes of viruses in both fluid and air microgravity environments.
b.
Compare the ability of viruses to adsorb to and infect hosts during and after microgravity-induced cellular changes.
c.
Investigate virus-host interactions, especially in response to perturbation effects on virus-to-host ratios.
d.
Develop enhanced capabilities for onboard biosurveillance.
e.
Investigate viromes in built environments relevant to space, including the ISS.
f.
Explore virus-host dynamics for both human-and plant-associated microbiomes in space.
g.
Assess space-associated triggers that cause latent viral infections to become virulent.
h.
Quantitatively estimate the role and impact(s) of virocell metabolism in spaceflight environments.
i.
Identify viral loading impacts significant to both planetary protection requirements, and preservation of human health.Explore desirable modifications to spaceflight operations (travel, landing and EVA) that could ameliorate such loading impacts.
j.
Characterize viral influence on microbial biofilm composition and growth dynamics in human, algal and plant systems under various space-related conditions.
Detection of viruses on Earth and elsewhere
A variety of instrumentation exists that can detect both microbial biomarkers and viruses.These types of techniques have often been used on Earth to study viruses (Sommers et al., 2021;Vincent and Vardi, 2023).However, these same techniques can be difficult to conduct with automated systems in space and have yet to be designed specifically for space missions.Therefore, it is currently unclear if and how informational molecules can be reliably detected in environmental samples beyond Earth.Engineering investigations must be paired with science goals to further develop innovative, flight-ready technologies to detect and characterize viruses, virus-like particles and virus genomes efficiently and accurately.Once developed, such technologies could then be applied in Earth analogue systems to test performance and limitations for detecting viruses and virus-like particles.
Additionally, these instruments and protocols can be incorporated into standard operating procedures for planetary sample missions.
While autonomous virus-detection technology is not yet ready, the future is promising.For example, solid-state nanopore-based biosensors are currently being designed for spaceflight and have proven capability for evaluating different biomarkers (i.e.DNA/RNA, proteins and whole viral particles -spanning from a few nanometres to over 100 nanometres in diameter).Solid-state nanopore sensors would be particularly useful in space missions because they also can measure particle-size distributions within virus populations and discriminate between viral particles by monitoring the change in electrical current as particles pass through an electrically biased pore or by measuring the mass density of viruses (Zhou et al., 2011;Arjmandi et al., 2014;McMullen et al., 2014;Akpinar and Yin, 2015;Wu et al., 2016).Researchers have adapted flow cytometry to the scale of virus particles; specifically, flow virometry employs more powerful lasers, reduced diameter fluid flows, wider-angle sampling of scattered light and fluorescent labelling of particles (Bhat et al., 2022).Additionally, microscopy has been used to evaluate microbial and viral populations on Earth.Microscopy technologies paired with spectroscopic techniques, can inform the physical shapes and boundaries belonging to certain chemical or mineralogic compositions (Zhang et al., 2013).While an atomic force microscope was used during the Phoenix Mars Lander to investigate Martian soils (Pike et al., 2011) and recently, a scanning electron microscope was added to the ISS (Own et al., 2018) higher performance complex space-qualified microscopes have yet to be developed that can reliably detect virus-like particles.Another technique that could be applied to space virology is NanoSIMS, which performs in situ quantitative trace sample analysis with exceptional sensitivity and spatial resolution (Mayali, 2020;Pett-Ridge and Weber, 2022).NanoSIMS has been used to detect viruses (Gates et al., 2018) and map their elemental and isotopic distributions in complex communities and in mineralized samples (Pacton et al., 2014).Also, the Network for Life Detection is currently using NanoSIMS to discern between abiotic and biological signatures based on the differences in elemental and isotopic patterns of cellular organisms versus minerals and other precipitates.These technologies complement spectroscopic methods by providing orthogonal evidence for viral and non-viral life (Zhang et al., 2013).Overall, there are multiple instrument designs either waiting to be developed or currently being developed for detecting viruses and life beyond Earth.
Environmental virology techniques could also complement life detection instrumentation.In conjunction with other previously described instruments, high fidelity sequencing methods and high-resolution structure analysis could also confirm the presence of viruses on other worlds (Bamford et al., 2005).Moreover, to reduce signal-to-noise problems, relic or environmental DNA can be removed from a system using propidium monoazide to enhance detectability of intact viruses and microbes (Wagner et al., 2008;Weinmaier et al., 2015).Thus, methods of environmental virology could also be necessary for life detection and returned samples (Janjic, 2018;Ricciardi et al., 2022; Table 3).Much work remains to integrate both engineering and science perspectives.While some instrumentation, such as light, electron and atomic force microscopy, has been used on Mars and in space on the ISS (Pike et al., 2011;Own et al., 2018), instrumentation in development must also be tested in extreme analogue environments to explore and overcome limitations.Moreover, for planetary protection purposes, utilizing instrumentation for detecting viruses should be incorporated into standard operating procedures for flight and sample return missions.To fully understand the presence of viruses in space environments, new technologies need to be developed and include the following.
a.
Develop innovative autonomous flight-ready technologies for efficient and accurate detection and characterization of virus genomes.
b.
Apply such technologies to Earth analogue systems to explore prospects and limitations for detecting viruses and virus-like particles.
c.
Incorporate measurements of viruses in standard operating procedures for sample return missions and subsequent examinations.
Conclusion
For effective pursuit of astrobiology questions, it is critical to understand how life on Earth functions, as it is currently the only place where life is confirmed to exist.Viruses are key contributors to Earth's ecosystems; however, much remains unknown regarding their influence on cellular life, role in evolutionary history and their fundamental physical interactions with the Earth system.Basic ecological factors, such as persistence and decay Review of spaceflight ECLSS potentially impacted by viruses.A simplified overview of environmentally controlled life-support systems for spacecraft and surface habitats that could be positively or negatively impacted by viruses.
Comparison of cellular and non-cellular entities-cells possess each of these four significant genetic and structural materials In the case of viruses and subviral elements, only reduced suites of these key molecules are present.Since non-cellular entities can possess either DNA or non-DNA genomes and may lack lipids, the search for extraterrestrial DNA, RNA or lipid alone would fail to detect some of these entities.
Figure 1 .
Figure 1.Morphological comparison of viruses, and a common bacterium at the same scale.Viral morphology and size are highly diverse, ranging from a few to thousands of nanometres.Here, an example bacterium (Escherichia coli, red text) is contrasted with example viruses that infect humans (black text), amoebae (purple text), archaea (orange text) and bacteria (blue text).Virus images adapted from ViralZone, Swiss Institute of Bioinformatics.
Figure 2 .
Figure 2.Uncertainty in the origin(s) and evolution of life on Earth.Viruses play a critical role in the evolution of life as we currently know them, and viruses or precursors of virus-like entities must be considered in origin experiments.Adapted fromHarris and Hill (2021).
(Dávila-Ramos et al., 2019)xtremophiles involve virus-host interactions that are fundamentally different relative to ideal environments(Dávila-Ramos et al., 2019).The monitoring of viral persistence and activity in a wide variety of extreme environment types may indicate how viruses survived during early Earth, and what is applicable to alien biospheres.We need expanded experimentation to determine end member limits of viral persistence and activity under conditions like aridity, humidity, low/high O 2 content, trapping in amber or soil/sediments, etc. Scoping out the broad viral environmental envelope would point to where virus-like entities might be found on both Earth and other worlds. | 2023-04-07T15:27:22.753Z | 2023-04-05T00:00:00.000 | {
"year": 2023,
"sha1": "b2ebd5f25c537da4f24c57c2ccd271ce9e8c9dbb",
"oa_license": "CCBY",
"oa_url": "https://www.cambridge.org/core/services/aop-cambridge-core/content/view/D85B7E5AA045A366E2E87652144FA533/S1473550423000058a.pdf/div-class-title-astrovirology-how-viruses-enhance-our-understanding-of-life-in-the-universe-div.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "c2c89231dd25839072e0ba9cbcfccfaf92c056b5",
"s2fieldsofstudy": [
"Environmental Science",
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
266224295 | pes2o/s2orc | v3-fos-license | Deciphering Immune Landscape Remodeling Unravels the Underlying Mechanism for Synchronized Muscle and Bone Aging
Abstract Evidence from numerous studies has revealed the synchronous progression of aging in bone and muscle; however, little is known about the underlying mechanisms. To this end, human muscles and bones are harvested and the aging‐associated transcriptional dynamics of two tissues in parallel using single‐cell RNA sequencing are surveyed. A subset of lipid‐associated macrophages (triggering receptor expressed on myeloid cells 2, TREM2 + Macs) is identified in both aged muscle and bone. Genes responsible for muscle dystrophy and bone loss, such as secreted phosphoprotein 1 (SPP1), are also highly expressed in TREM2+ Macs, suggesting its conserved role in aging‐related features. A common transition toward pro‐inflammatory phenotypes in aged CD4+ T cells across tissues is also observed, activated by the nuclear factor kappa B subunit 1 (NFKB1). CD4+ T cells in aged muscle experience Th1‐like differentiation, whereas, in bone, a skewing toward Th17 cells is observed. Furthermore, these results highlight that degenerated myocytes produce BAG6‐containing exosomes that can communicate with Th17 cells in the bone through its receptor natural cytotoxicity triggering receptor 3 (NCR3). This communication upregulates CD6 expression in Th17 cells, which then interact with TREM2 + Macs through CD6‐ALCAM signaling, ultimately stimulating the transcription of SPP1 in TREM2 + Macs. The negative correlation between serum exosomal BCL2‐associated athanogene 6 (BAG6) levels and bone mineral density further supports its role in mediating muscle and bone synchronization with aging.
Introduction
Aging-related changes in skeletal muscle and bone begin early in life and manifest as a reduction in both the mass and function of these tissues. [1]Disorders resulting from such aging-related changes in either or both tissues have been termed Sarcopenia, Osteoporosis, or osteoSarcopenia (SOS), which significantly constrain the daily activities of elderly people.Patients with SOS are prone to falls and fractures, leading to increased disability, frailty, hospitalization, and mortality, and, along with an aging population, the disease burden of SOS is surging. [2,3]Currently, no pharmaceutical intervention is available for sarcopenia. [3]herapeutic agents have long been clinically applied and shown effectiveness for osteoporosis, whereas side effects such as necrosis of the jaw, atypical fractures, etc., are presented. [4]Therefore, it remains critical to obtain an in-depth understanding of the mechanisms driving muscle and bone aging and to identify potential therapeutic targets to prevent the loss of muscle and bone in elderly individuals.
Numerous studies have revealed a synchronization of muscle and bone mass for decades, and a close functional and developmental relationship exists between the two tissues; [5] thus, the concept of a muscle-bone (M&B) unit has emerged. [6]Nevertheless, the mechanism underlying the coordination of muscle and bone mass has been previously simplified and viewed as mechanical in nature, where the muscle is recognized as the primary source of anabolic mechanical stimuli for bone tissue. [7]Recent findings have identified the endocrine functions of both bone and muscle. [8]Therefore, more complex interactions between muscle and bone, in terms of biochemical crosstalk, metabolic communications, and extracellular vesicle-mediated coupling, are gaining increasing attention. [8]Moreover, both tissues consist of diverse cell types at different stages, among which immune cells play an essential role in regulating their homeostasis. [9,10]ecent studies have emphasized the impact of conserved immune responses on aging physiology across various organs and shared immune responses may contribute to various agingrelated diseases. [11]However, a comprehensive analysis of the cellular composition of human bone and muscle and how these cells adapt and interact during aging is lacking.In this study, we established a single-cell landscape of human muscle and bone in parallel, depicting the aging features of human M&B units, which could advance the understanding of synchronized musculoskeletal aging and lay the molecular foundation for further translational studies.
Aging Increases the Diversity of Musculoskeletal Executive Cells and Myeloid Cells
To explore the cellular diversity and transcriptional adaption of human musculoskeletal tissue during aging, four old (aged over 70 years) and two young (29 and 34 years) individuals were enrolled in this study (Figure 1a).Tissue samples from the gluteus maximus and femoral neck were harvested during hip replacement surgery from each individual.Histologically, we observed that the muscle fiber size was reduced in the elderly group compared to that in the young group (Figure 1b).In addition, we found an increase in extracellular collagen deposition in the interstitial tissues of all older muscles compared to young subjects (Figure 1b).Regarding the bone, micro-CT reconstruction images showed a decline in trabecular bone number and thickness in older adults compared to that in young adults (Figure 1c).Thereafter, cells in the harvested muscle and bone were isolated, purified, and subjected to droplet-based single-cell RNA sequencing (scRNA-seq) to investigate cell type-specific remodeling during aging (Figure 1d).After we removed low-quality or contaminated cells, a total of 40238 cells were obtained, comprising endothelial cells (ECs), fibroblasts (FBs), lymphatic ECs, mesenchymal stem cells (MSCs), pericytes, smooth muscle cells (SMCs), myocytes, satellite cells, myeloid cells, lymphoid cells (mainly T cells and natural killer (NK) cells), plasma B cells, mast cells, and a population of cycling cells (Figure 1e).A UMAP plot showing individual donors' contributions to the identified cell subsets indicated consistency between donors within the same group (Figure S1a, Supporting Information).By comparing tissue distributions, most cells in the bone were immune cells, while only a few ECs and MSCs were identified.Consistent with the current knowledge , [12] satellite cells were dominant in young individuals, and an increased lymphoid cell composition was observed in old individuals (Figure 1e; Figure S1b, Supporting Information).The signature expression of each cell type indicated robust filtering and clustering (Figure 1f).Fibrogenic signatures in FBs were minimally expressed in both old and young individuals, suggesting the absence of muscle injury in the enrolled individuals; therefore, we could assume any experimental differences would reflect aging-driven alterations (Figure S1c, Supporting Information).Furthermore, somatic mutations accumulate during aging; consequently, more noise or transcriptional instability should be observed.Hence, the concept of transcriptional instability was introduced by Enge et al. [13] and considered as a feature of mammalian aging , [13] we calculated the transcriptional noise of the 12 identified cell clusters, six clusters displayed increased noise namely myocytes, satellite cells, lymphatic EC, MSC, myeloid cells, and EC, while the rest 6 clusters were either unchanged or decreased in the old samples (Figure 1g).To understand the underlying mechanisms of differential transcriptional regulation in aging, we subsequently subclustered the major cell types, with a focus on the immune compartment, and delineated their interactions with tissue executive cells in the following sections.Additionally, a landscape of ECs, pericytes, MSCs, and SMCs is also displayed in Figures S1d and S2 (Supporting Information).
A Unique TREM2 + Macrophage Enriched in Aged Musculoskeletal Tissues Highlights a Potential Culprit of Aging-Related Pathologies
As a central player in innate immunity, we first investigated agerelated changes in the myeloid cell compartments. [14]Myeloid cells, including macrophages, monocytes, dendritic cells, and neutrophils, were also identified (Figure 2a).We observed an increased proportion of macrophages among the myeloid cell population in old individuals compared to young individuals (Figure S3a, Supporting Information).Moreover, increased transcriptional noise was also observed in aged macrophages, indicating that aging caused higher variability and potential fate drift in macrophages (Figure 2b).Re-clustering of macrophages identified three subsets (Figure 2a; Figure S3a,b, Supporting Information): 1) a subset that highly expressed TREM2, named TREM2 + Macs; 2) a subset that was distinguished by the high expression of the inflammatory marker, IL-1B, named inflammatory (Inf) Macs; and 3) tissue-resident (Trm) Macs expressing tissue residence markers including LYVE1 and MRC1 (Table S1, Supporting Information).By assessing tissue distribution, we found that Trm Macs were exclusively located in skeletal muscle, whereas Inf Macs and TREM2 + Macs were found in both tissues (Figure 2a; Figure S3a, Supporting Information).Stratified by age, Trm Macs and Inf Macs were found in both young and aged individuals, whereas TREM2 + Macs emerged uniquely in old muscles and bones (Figure 2a; Figure S3a,c, Supporting Information).The transcriptional signature genes of Trm Macs were analyzed using gene ontology (GO) analysis to gain a deeper understanding of their function.As shown in Figure S3d (Supporting Information), genes enriched in Trm Macs were associated with three major function categories: 1) cellular response to multiple niche signals including tumor necrosis factor, insulin stimulus, and interferon-gamma, and transforming growth factor beta, etc.; 2) muscle homeostasis maintaining, namely angiogenesis, endothelial cell proliferation, aging, skeletal muscle cell differentiation; 3) immune cell-intrinsic function, namely regulation of apoptotic process and receptor-mediated endocytosis, positive regulation of T cell activation, antigen processing and presentation of exogenous peptide antigen via MHC class II, and inflammatory response.[17] Regarding Inf Macs, GO analysis revealed an enrichment of signature genes associated with inflammatory processes, including antigen processing and presentation, positive regulation of T cell activation, neutrophil chemotaxis, inflammatory response, and leukocyte cell-cell adhesion (Figure S3e, Supporting Information).To explore the effects of age-related changes, we further explored the enriched functions of differentially expressed genes in each macrophage cluster with aging.Being the most prevalent macrophage population in the muscle, Trm Macs in old individuals exhibited enhanced gene expression in regulating leukocyte differentiation, cell chemotaxis, regulation of the ERK1/2 cascade, and exerted a pro-angiogenic effect (Figure S3f, Supporting Information).Aged Inf Macs displayed features associated with an apoptotic signaling pathway, oxidative stress, positive regulation of NF-B transcriptional factor activity, and positive regulation of leukocyte activation (Figure S3g, Supporting Information).
Since TREM2 + Macs emerged exclusively in older individuals, we postulated that this subset might show distinguishing features in relation to the aging phenotype of both muscle and bone.We first confirmed the presence of TREM2 + Macs in aged muscles and bones by immunofluorescence staining (Figure 2c,d).It has been known that TREM2 is a key sensor of metabolic pathologies and tissue damage .18]Prior rodent study has also found that a subset of macrophages with high expression of Trem2 was chronically activated in the dystrophic muscle of X-linked muscular dystrophy (MDX) mice, where constant damage occurs .19]Therefore, the emergence of TREM2 + Macs in aged individuals indicates a loss of tissue-level homeostasis in both muscles and bones.To further understand the functional phenotypes of TREM2 + Macs, we explored the functional enrichment of the transcriptional signature genes according to Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways and GO terms.Results showed that transcriptional signatures of TREM2 + macs in muscle and bone were highly associated with lipid metabolism, lysosome, phagocytosis, and tissue homeostasis (Figure 2e).Furthermore, compared to other macrophage subsets, TREM2 + Macs also exhibited less inflammatory activation, as indicated by the decrease in the levels of inflammatory response and the upregulation of peroxisome proliferator-activated receptor (PPAR) signaling and oxidative phosphorylation pathways (Figure S3j, Supporting Information).Such features of TREM2 + Macs closely resembled that of adipose tissue tumor-associated macrophages (TAMs) or brain diseaseassociated macrophages (DAMs), which were recently identified in mouse fibrotic lung and fatty liver tissues, as well as in mouse and human adipose and brain tissues, indicating that this cluster may be their musculoskeletal counterpart .18]Both DAMs and TAMs have been reported to contribute to the pathogenesis of certain tissues by inducing fibrosis (LGALS3 and SPP1) or cellular senescence (CTSB and CD36).Indeed, genes involved in these biological processes are also highly expressed in TREM2 + Macs found in our study (Figure 2f; Figure S3h, Supporting Information).Muscle and bone undergo significant tissue remodeling, including fibrosis, loss of mass, and damage to microarchitecture with aging. [2,20]Therefore, the emergence of TREM2 + Macs highlights a potential culprit of these aging-related pathologies.Previous research has also indicated that SPP1 promotes fibrosis in muscular dystrophy [21] and degradation of the bone matrix responsible for bone loss ,22] both of which are recognized as aging phenotypes.Nonetheless, the source of SPP1 in muscle remains unknown; however, SPP1 accumulated in bone was recently believed to originate from macrophages in epididymal adipose tissue through the circulatory system. [22]The finding that TREM2 + Macs highly expressed SPP1 in the muscle and bone tissue, which was validated by immunostaining (Figure S3i, Supporting Information), suggests that there is a major source of SPP1 in the local musculoskeletal niche.Accordingly, the identification of TREM2 + Macs in aged muscle and bone indicates a loss of tissue homeostasis, and this subset of macrophages may contribute to the pathogenesis of aging-induced musculoskeletal phenotypes.
To better understand the developmental connection between TREM2 + Macs and other macrophages, we constructed pseudotime trajectories for the macrophages. [23]The in silico trajectory indicated that TREM2 + Macs showed distinctive features, as they were distributed at the terminus of the bifurcated branch and showed a minimal connection with Trm Macs, monocytes, and Inf Macs (Figure 2g).Prior rodent experiments have evidenced that the TREM2 + Macs counterparts emerged in fatty liver, acne lesions, and tumors originating from recruited circulating monocytes. [24,25]Our trajectory also suggested a differentiation potential from monocyte to TREM2 + Macs.Along this trajectory, we also revealed that nuclear receptor subfamily 1 group H member 3 (NR1H3) was specifically expressed in TREM2 + Macs (Figure 2g).Furthermore, gene regulation network analysis using SCENIC identified several transcription factors (TFs), including MAFF, REL, and CEBPB, that modulate macrophagespecific gene regulatory networks (Figure 2h). [26]NR1H3 was exclusively identified in TREM2 + Macs, which agrees with the trajectory inference (Figure 2i).The protein encoded by NR1H3 belongs to the NR1 subfamily of the nuclear receptor superfamily.NR1 family members are key regulators of macrophage function, controlling the transcriptional programs involved in lipid homeostasis and inflammation. [27]Combined with our study results, this suggests a potential role for NR1H3 in the establishment of a specific transcription program in TREM2 + cells.
T Cells Programing Toward Tissue-Specific Inflammatory Lineages Provides a Mechanistic Basis for Aging-Associated Chronic Inflammation
A hallmark of aging is chronic system-wide inflammation, termed inflammaging.Recent evidence has highlighted intrinsic alterations in T cells actively contribute to inflammaging, and infiltration of these dysfunctional T cells could accelerate tissue-specific aging phenotypes. [28]However, the diversity and age-associated alterations in T cells infiltrating musculoskeletal tissues are not yet fully understood.Detailed clustering analysis further categorized T and NK cells into 14 subsets (Figure 3a; Figure S4a, Supporting Information), and the percentage of each cell type in both young and old individuals is shown in Figure S4b (Supporting Information).CD4 + T cells resided in both muscles and bones, while CD8 + T cells were mainly detected in the bone (Figure 3b; Figure S4c, Supporting Information).A bias toward enrichment in memory CD4 + T cells and a decrease in the naïve pool was seen both in the muscle and bone during aging (Figure 3b; Figure S4c, Supporting Information), which echoes the notion that aging causes a systemic naïve memory CD4 + T cell imbalance. [28]In particular, the fraction of effector memory CD4 + T (T EM ) cells was elevated in both the muscle and bone, whereas Th17 was almost exclusively detected in bone tissues among old individuals (Figure 3b; Figure S4c, Supporting Information).To gain insight into lineage relationships among these CD4 + T cell subsets, we performed a pseudo-trajectory analysis using a monocle.Naïve T (T N ) cells were diffused at the left end, whereas T EM cells were distributed to the right end.A bifurcated distribution was observed in the CD4 + T EM cells, indicating the existence of two different T EM cell phenotypes (Figure 3c).Therefore, we compared the differentially expressed genes between bifurcated T EM cells.Prior research has suggested that T EM cells maintain effector functionality; [29] here, we found that the upper branch T EM cell cluster presented a Th1-lineage transcription pattern (Th1-like) with high IFNG expression, whereas the lower branch mimicked a Th2-like phenotype (Th2-like) with AREG (Figure S4d, Supporting Information).To confirm our findings, we implemented another trajectory algorithm, URD, [30] to delineate the relationships between these T cell clusters.The results showed that Th1-and Th2like T EM cells were both rooted in T N cells and distributed separately in independent branches other than Treg or Th17 clusters.In addition, the master regulator genes known for the Th1 (RUNX3) and Th2 (GATA3) lineages were uniquely highlighted in the corresponding developmental branch (Figure 3d).Therefore, these data indicate that T EM cells not only maintain effector functionality but could also be programmed to various effector lineage-like phenotypes corresponding to the master regulator genes.
Tissue-specific CD4 + T cell programming was observed in aged individuals, as Th1-and Th2-like T EM cells were mainly found in aged muscle (Figure S4e, Supporting Information), while Th17 was dominant in aged bone (Figure S4b,c, Supporting Information).We focused on age-dependent alterations in the function of terminally differentiated CD4 + T cells.In agreement with previous studies, Th17 cells were enriched in genes involved in the pro-inflammatory interleukin-17 (IL-17) and tumor necrosis factor (TNF) signaling pathways, both of which are essential for Th17-mediated tissue inflammation and bone destruction (Figure 3e). [31]Th1-and Th2-like T EM cells also exhibited pro-inflammatory features and strong cytotoxicity (Figure 3f; Figure S4f, Supporting Information) ,32] which provided a mechanistic explanation for the inflammation-induced muscle loss observed in aged muscle. [33]Accordingly, these data indicate that a common transition toward pro-inflammatory phenotypes occurs in aged T cells across tissues.To unravel gene expression regulation beyond such a transition in musculoskeletal aging, we performed gene network analysis by SCENIC for CD4 + T cell subsets and discovered that the NFKB1 regulon was actively transcribed in both aged T EM and Th17 cells (Figure S4g, Supporting Information).Furthermore, as the core component of the NF-B signaling pathway, REL was a direct downstream target in both Th1-like and Th17 cells (Figure 3g); therefore, we speculated that targeting the NF-B signaling pathway may slow down aging phenotypes by attenuating tissue-level inflammation in both muscle and bone.
Moreover, prior research has identified an accumulation of a small subset of CD8 + T cells, termed "virtual memory" CD8 + T (T VM ) cells, with aging. [34]In mice, T VM cells are phenotypically characterized by high expression of ll2rb and low expression of Itga4.These two markers reflect the semi-differentiated state of the T VM cells.In our sample, a putative T VM cell was also identified and showed striking differences from T EM cells (Figure 3h).Consistent with a previous study, these cells are naïve-like and lack the expression of CCR7 and CD27 compared to central memory T cells (Figure 3i). [35]In our study, T VM cells emerged within the bone tissue and increased in proportion with age (Figure S4b,c, Supporting Information).Notably, given that putative T VM cells lose the expression of CCR7, they may have been included in classical gating strategies for T EM cells.Using single-cell analysis, distinct transcription features were observed between these two subtypes of memory cells.Genes that regulate memory T cell trafficking were less abundant, such as ITGAE (Figure 3i), [36] while genes that might predict increased responsiveness to the inflammatory milieu (e.g., STAT4 and CCR2) were downregulated in human T VM cells (Figure 3i). [34]In addition, aged human T VM cells exhibited a lower score of exhaustion but a relatively higher score of senescence compared to other memory cells (Figure 3j). [37]T cell exhaustion is a state of T cell dysfunction that arises during many chronic conditions and cancer.It is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. [38]Thus, such results indicate that dysfunctional and senescent T VM cells are present in older individuals.www.advancedscience.com
Elevated Blood Interleukin-18 Levels and Intramuscular LDHA/LDHB Ratios Indicate a Decline in Muscle Function
Re-clustering of muscle cells identified six subsets of myocytes and one group of muscle stem cells (MuSCs) (Figure 4a).Among these myocytes, three subsets exhibited slow-twitched fiber features (highly expressed MYH7), including Slow C1, Slow C2, and MYL12A hi Slow; two subsets, namely Fast C1 and SAA1 hi Fast displayed fast-twitched fiber features (highly expressed MYH1); and one cluster showed Bag fiber features (highly expressed MYH15). [39]We calculated scores for aging-related phenotypes, including muscle atrophy, denervation, ubiquitylation, and reactive oxygen species (ROS) stress.In old individuals, myocytes with fast-twitch or slow-twitch phenotypes displayed increased activities regarding these features, suggesting a higher degree of aging (Figure 4b).As hallmark genes for sarcopenia (aging-related muscle atrophy), FBXO32 and TRIM63 were also increased in aged myocytes (Figure S5a,b, Supporting Information). [40]We applied trajectory analysis to delineate the relationships between these muscle cells, and subsequently, five functional states were identified (Figure 4c).MuSCs and the majority of cells from young individuals were enriched in State 1, whereas States 2-5 were dominantly occupied by aged myocytes (Figure S5c, Supporting Information).Intriguingly, State 5 was exclusively distributed on one branch of the trajectory while States 2-4 were diffused on the other, which suggested that myocytes of State 5 may present a unique transcript feature.We further examined the functional enrichment score of aging-related phenotypes and expression levels of sarcopenia markers across each state.State 5 exhibited the highest score for aging phenotypes and expression levels of sarcopenia markers, indicating that myocytes in this State displayed the highest degree of aging (Figure 4d; Figure S5d, Supporting Information).Consistent with this finding, biological processes associated with aging were also enriched in State 5, including DNA damage response, ubiquitination, and autophagy (Figure 4e; Figure S5e, Supporting Information).Differential expression analysis identified that IL18 was highly expressed in State 5, which encodes a secreted protein, interleukin-18 (IL-18) (Figure S5f, Supporting Information).To test whether the expression level of IL-18 correlated with muscle function, we measured IL-18 levels in the blood serum of 90 subjects for whom grip strength was evaluated.IL-18 was negatively correlated with grip strength (R = −0.01731,P = 0.035), suggesting that IL-18 could be a potential biomarker for the evaluation of muscle function (Figure 4f).
Compared to fast fibers, slow fibers display higher resistance to aging-associated mass loss according to histological analysis, but the molecular alterations remain unknown at the transcriptome level. [41]As a result, we focused on Slow C1, Slow C2, and MYL12A hi Slow, which exhibited slow fiber features.Among these, Slow C1 was present in both young and aged individuals, whereas the other two were exclusively present in aged individuals (Figure S5g, Supporting Information).Thus, we hypothesized that the Slow C2 and MYL12A hi Slow might degenerate from normal slow fibers to various extents.Details of signature genes for Slow C2 and MYL12A hi Slow are listed in Tables S2 and S3 (Supporting Information).To confirm our hypothesis, we first compared the proportion of trajectory State 5 in the two subsets, where State 5 was preferentially distributed in Slow C2 rather than MYL12A hi Slow, indicating that Slow C2 may exhibit a more deteriorated function (Figure 4g).Decreased mitochondrial function and content are indicators of aging in slow fibers. [33]Compared to MYL12A hi Slow, Slow C2 had a decreased overall mitochondrial function (Figure 4h).Moreover, aging-related phenotypes were more prominent in Slow C2 than in MYL12A hi Slow (Figure S5h, Supporting Information).These data are in accordance with our deductions based on the proportion of State 5. Additionally, we characterized the genes involved in fatty acid (FA) -oxidation and glucose metabolism in Slow C2 and MYL12A hi Slow.The results indicated that MYL12A hi Slow preserved higher expression of genes mediating FA uptake (CD36, FABP3, SLC27A1, and SLC27A4), transport to the mitochondria (CPT1B), and -oxidation, which suggested active FA metabolism (Figure 4i).Notably, prior research using bulk sequencing and proteomics has suggested that FABP3 is upregulated in aged skeletal muscles, causing endoplasmic reticulum (ER) stress, and disrupting muscle homeostasis. [42]e found that MYL12A hi Slow may be capable of adapting to this change by increasing the transport of FAs to the mitochondria and -oxidation.In contrast, Slow C2 showed aging-prone LDHA expression, whereas LDHB was more prominent in MYL12A hi Slow (Figure 4i).This was further confirmed by in situ hybridization of LDHA and LDHB mRNA (Figure 4j,k; Figure S5i, Supporting Information).Prior research in the brain has shown that a high lactate level is a hallmark of aging and is caused by an increase in the LDHA/LDHB ratio. [43]Given our evidence of Slow C2 as more aging myocytes, an increase in the LDHA/LDHB ratio might be a predictive value for muscle aging phenotypes.
Exosome-Driven Long-Distance Cell-Cell Communication Facilitates Muscle-Bone-Synchronized Aging
To rewire cell-cell interactions during aging, CellChat [44] was used to construct a comprehensive signaling interaction map between all identified subsets in young and old individuals separately.We then calculated the differential interaction weight between young and old individuals and found that old-enriched cell subsets had extensively increased crosstalk (Figure S6a, Supporting Information).To further dissect the cellular crosstalk that emerged in old individuals, we investigated communication signals for old-enriched cell subsets in the aged group (Figure 5a).We focused on the cell subsets with significantly altered communication.Interestingly, we found that Slow C2 communicated with Th17 in the bone in the aged group (Figure 5b).To further examine the exact ligand-receptor molecules that participate in the aging network, we delineated the communication signaling for these aging-related cells.Surprisingly, we identified Slow C2-secreted BAG6 signaling molecules exclusively in old individuals (Figure 5c).BAG6 signaling functioned in a paracrine manner and was secreted by Slow C2 and monocytes in the muscle (Figure 5d; Figure S6b, Supporting Information).We focused on the BAG6 from Slow C2 and hypothesized that Slow C2 may interacted with Th17 via the BAG6-NCR3 axis (Figure S6b,c, Supporting Information).Prior research has shown that BAG6-encoded protein, also known as BAG6, is released extracellularly via exosomes. [45]As Slow C2 in muscle and Th17 in bone were not physically juxtaposed, we assumed that BAG6 might be secreted by aged and malfunctioned muscle via exosomes and thereafter delivered to Th17 through blood vessels.As mentioned above, Th17 cells are known for age-related bone loss; therefore, to understand the impact of BAG6 on Th17 cells, we further dissected the increased crosstalk between Th17 and other cell clusters in aged bones.Consequently, the CD6 signaling pathway was of particular interest, as it was enriched in Th17 cells (Figure 5c).Furthermore, ALCAM was the only expressed receptor for CD6, which was detected in TREM2 + Macs in the bone (Figure 5e; Figure S6d,e, Supporting Information).In addition, we applied another algorithm, CellPhoneDB, to compare cell interactions during aging and thereafter validated the increased BAG6 and CD6 signaling in silico (Tables S4 and S5, Supporting Information).The aforementioned results indicated that TREM2 + Macs were the major macrophage population in aged bone and highly expressed genes that promote osteoclastogenesis and bone destruction, such as SPP1. [46]To this end, we applied NicheNet [47] to predict the downstream targets of TREM2 + Macs upon CD6 stimulation.We found that the transcription factor STAT3 was significantly upregulated in TREM2 + Macs, which could bind to the promoter region of SPP1 and enhance its transcription (Figure S6f, Supporting Information). [48,49]o confirm the in silico findings, we performed validation experiments.First, we assessed BAG6 expression in human muscle and blood exosomes from both aged and young subjects.As expected, the expression of BAG6 was higher in aged skeletal muscles than in young skeletal muscles (Figure S6g, Supporting Information).Blood plasma exosomes were isolated from the peripheral blood of the included subjects using size-exclusion chromatography (SEC) and characterized using Transmission electric microscope (TEM), nanoparticle tracking analysis (NTA), and western blotting (Figure 5f; Figure S6h,i, Supporting Information), in accordance with the guidelines recommended by the International Society for Extracellular Vesicles (ISEV).Thereafter, higher expression of BAG6 was found in exosomes derived from aged and osteoporotic participants (Figure 5g; Figure S6j, Supporting Information).To test the effects of BAG6 on Th17 cells, we stimulated human Th17 cells using blood exosomes with higher expression of BAG6 (BAG6 hi Exos) from aged individuals and those with lower expression in young individuals (BAG6 low Exos) (Figure 5h).In line with the bioinformatic analysis, increased expression of CD6 was detected in Th17 cells from the BAG6 hi Exos group after treatment compared to those from the BAG6 low Exos group (Figure 5i).The sorted Th17 cells from old individuals also showed an increased CD6 ex-pression, as compared to those from young ones (Figure S6k, Supporting Information).Additionally, to determine whether such interactions could ultimately promote the production of SPP1 in macrophages, we co-cultured human macrophages with Th17 cells in a conditioned medium with either BAG6 hi Exos or BAG6 low Exos.Higher expression of the SPP1-encoded protein osteopontin was detected by ELISA in the BAG6 hi Exostreated group (Figure 5j).We further treated Th17 cells and macrophages with BAG6 hi Exo, respectively.Results showed that BAG6 hi Exos did not directly stimulate the SPP1 expression in either macrophages or Th17 cells separately (Figure S6l, Supporting Information).Accordingly, these results support our findings from the bioinformatic analysis, where BAG6 is secreted by aged and malfunctioning muscle via exosomes and thereafter delivered to Th17 cells.This communication upregulates CD6 expression in Th17 cells and ultimately stimulates the production of SPP1 in macrophages, which is responsible for osteoclastogenesis and bone loss.Such long-distance cell-cell interactions facilitate synchronized aging within the M&B unit (Figure 5k).
Discussion
In this study, we established a single-cell landscape showing heterogeneous aging features of diverse cells within the M&B unit.Aging increased the diversity of myeloid cells, which was indicated by higher levels of transcriptional noise.A distinct subset of macrophages, TREM2 + Macs, was identified in aged muscles and bones.TREM2, a transmembrane receptor, interacts with a wide array of ligands that are hallmarks of tissue damage resulting from the loss of metabolic homeostasis and a chronic low-grade inflammatory state. [18]Macrophages highly expressing TREM2 are found in pathological sites across multiple organs, including the fatty liver, [24] obese adipose tissue, [50] and Alzheimer's brain. [51]TREM2 + Macs identified in aged muscle and bone shared similar characteristics with these macrophages, implying similar stresses induced by aging within the muscle and bone compartments.Indeed, an increase in fat infiltration has been recognized as a shared metabolic aberration seen in both muscle and bone during aging. [52,53]In addition, a systemic low-grade inflammatory state has been observed as a common feature of aging. [54]The findings of this study have broadened our understanding that the emergence of TREM2 + Macs may represent a conserved macrophage alteration within the bone and muscle compartments in response to aging-induced homeostasis aberrations.M for _Muscle, _B for _Bone.g) BAG6 expression in exosomes from human participants in either young or aged groups was determined by enzymelinked immunosorbent assay (ELISA)(n = 24).h) Representative immunofluorescent images showing the uptake of exosomes by Th cells.Exosomes were labeled by PKH26 and incubated with Th cells for 12 h.i) Intracellular CD6 expression of T cells after incubation with BAG6-shuttled exosomes compared to PBS controls, as tested by qPCR (n = 3 independent experiments).j) Expression level of SPP1 in the conditioned medium of macrophages and Th17 cells co-culture system, determined by ELSIA.The conditioned medium contained the same quantity of either BAG6 hi exosomes (BAG6 hi Exos) or BAG6 low exosomes (BAG6 low Exos), or the same volume of PBS.k) Schematic representation of predicted cell-cell interactions across aged muscle and bone.Degenerated muscle secretes exosomes containing BAG6, the latter of which travel through blood vessels and promote the interaction between Th17 cells and TREM2 + Macs in bone.The TREM2 + Macs potentially facilitate the differentiation of osteoclasts and bone resorption.The significance (p-value) was calculated using the Mann-Whitney t-test (g and i) and the one-way ANOVA test (j); * p < 0.05; ** p < 0.01; *** p < 0.001.
Regarding the functions of TREM2 + Macs in aged individuals, we found that SPP1, which promotes fibrosis in muscular dystrophy and bone destruction, was highly expressed in TREM2 + Macs.Similarly, in mice with metabolic-associated fatty liver disease, Trem2 + Macs were found in the fibrotic zone and expressed genes involved in fibrosis, such as Spp1 and Des, suggesting a role in the mechanism of steatohepatitis. [24]Consistent with our findings, blockade of TREM2 inhibited bone resorption, and TREM2 stimulation enhanced the formation of mature osteoclasts that were generated from bone marrow macrophages or RAW264.7 cells treated with receptor activator for nuclear factor B Ligand (RANKL) and macrophage colony-stimulating factor 1 (M-CSF1).Furthermore, loss-of-function mutations of TREM2 are seen in the context of Nasu-Hakola disease (NHD), a human disease characterized by osteoporotic features leading to recurrent bone fractures associated with pre-senile dementia. [55]Patients with NHD present osseous symptoms typically at a young age (between 20 and 30 years), where impairment in TREM2 + signaling may disrupt the coupling of bone resorption and formation, leading to low turnover osteoporosis.A lack of data comparing aged subjects with and without NHD impedes the understanding of the role of TREM2 signaling in bone homeostasis.However, the high expression of SPP1 in TREM2 + Macs suggests that this subset of macrophages is a potential culprit in aging-related pathologies.Given the importance of TREM2 + Macs in various diseases, including obesity, Alzheimer's disease, and cancer, numerous efforts are underway to pharmacologically target TREM2 signaling. [18]Our study highlights a novel possibility that harnessing the TREM2 function might have beneficial effects on the aging phenotypes of M&B units beyond the aforementioned contexts.
Subsequently, we found that T cells residing in aged muscles and bones exhibit pro-inflammatory features.Most older individuals develop inflammaging, a condition characterized by elevated levels of inflammatory blood markers, which increases the risk of high chronic morbidity, disability, frailty, and premature death. [54]Although inflammaging was initially attributed to the accumulation of non-immune senescent cells, recent evidence has highlighted T cells as major drivers of this age-associated inflammation. [56]However, the effects of aging on T lymphocytes in humans are largely limited to the peripheral blood, which has been more extensively studied than tissue-residing T cells . [28]n our study, biased differentiation of CD4 + T cells was seen in bone and muscle during aging, where Th1-like cells were mainly enriched in muscle while Th17 was enriched in bone.Th1 has been previously described to contribute to aging-related obesity and insulin resistance, [57] while Th17 contributes to bone loss. [10]uch biased differentiation echoes the aging phenotypes of host tissues.Attempts have been made to modulate certain inflammatory markers to prevent aging-associated conditions; however, clinical trials have shown controversial results.According to our findings, immune cells may adapt to various phenotypes shaped by the tissue-specific niche; therefore, a deeper understanding of the immune cell diversity and its role in tissue-level inflammation is needed to facilitate a more precise modulation.Furthermore, we identified that the NFKB1 regulon is actively transcribed in Th1-like and Th17 cells, which are the core components of the NF-B signaling pathway.NF-B is a pleiotropic transcription factor that has been reported to be closely associ-ated with aging-related dysfunction in various contexts. [58,59]In the context of muscle and bone, a new perspective on the role of NF-B as a master regulator-coordinating the aging features of M&B units-has been provided here in our study.Nonetheless, the downstream regulons of NF-B in Th1 and Th17 were quite different, which further confirmed that T cells were shaped by the local niche and, therefore, operate in a variety of specialized functions.
Muscle has long been recognized as the driving force for bone adaptation through mechanical loading. [60,61]Recent evidence has identified muscle as an endocrine organ. [62]Therefore, beyond the conserved hallmarks of aging, we wondered if there are certain factors released from aged muscle to facilitate the synchronized aging of the two tissues.Prior research has suggested that fibers with a slow-twitch phenotype are more resistant to aging-induced mass loss. [63]By analyzing these cells at a singlecell resolution, we found that the function of slow fibers may also be damaged to various extents, as indicated by the metabolism switch, and secreted factors that are closely correlated with grip strength.In addition, we identified that exosome-shuttled BAG6 might play a crucial role in mediating muscle and bone coupling, as BAG6 is highly expressed in muscles with a higher degree of aging and interacts with Th17 in bone through the BAG6-NCR3 axis.Moreover, increased expression of exosomal BAG6 was observed in patients with osteoporosis compared with that in normal subjects.Therefore, beyond soluble factors, including irisin [64] and myostatin , [65] our findings may provide a new perspective that muscle and bone could interact through exosomemediated signaling.
Experimental Section
Human Participants: This study was approved by the Ethics Committee of the Chinese PLA General Hospital, and written informed consent was obtained from all patients.All experiments were performed in accordance with approved protocols.Samples, including muscle, bone, and blood plasma, were harvested from patients recruited from the Chinese PLA General Hospital hip fracture cohort. [66]Ninety human serum samples were used to examine the relationship between serum IL-18 levels and grip strength.The samples were randomly selected from the China Hainan Centenarian Cohort Study (CHCCS). [67]ematoxylin and Eosin (H&E) Staining of Muscle Tissue: Human muscle tissues were fixed with 4% paraformaldehyde for 24 h and dehydrated and embedded in paraffin.Five micrometers thick muscle slides were obtained and stained with HE according to the manufacturer's instructions.In brief, tissue sections were dewaxed and dehydrated, and then incubated for 10 min with hematoxylin solution and washed with water.Eosin was added for 1 min and sections were dehydrated with ethanol.Sections were captured using bright field microscopy (Leica DM IL LED).
Microcomputed Tomography (CT) Analyses: The micro-CT scanning of bone specimens was performed using the Inveon MM system. [68]The exposure time was set to 1500 ms in each of the 360 rotational steps with an effective pixel size of 8.89 μm, a voltage of 60 kV, and a current of 220 μA.The images have 1536 slices, and the voxel size was 8.89 μm in all three axes.The 2D images were then reconstructed with Inveon Research Workplace to create 3D visualization images.A circle with a diameter of 3.71 mm on consecutive trans-axial sections (with a height of 4.4 mm) was used to create a cylindrical volume of interest.
Tissue Dissociation and Cell Isolation: Bone and skeletal muscle were obtained from patients who underwent hip replacement surgery.For skeletal muscle, tissues were dissociated by enzymatic digestion with a skeletal muscle dissociation kit according to the manufacturer's instructions.The bone tissue was cut into small pieces and digested with trypsin for 10 min at 37 °C and followed by type II collagenase overnight at 37 °C.Enzymatic digestion was stopped with DMEM supplemented with 10% fetal bovine serum (FBS).The cell pellet was collected by centrifugation at 300 × g for 5 min, resuspended in red blood cell (RBC) lysis buffer, and incubated on ice for 2 min.To remove cell debris and dead cells, RBC-lysed cells were further purified using the Dead Cell Removal Kit.Finally, the isolated cells were passed through a 70 mm filter and subjected to a single-cell experiment.
10x Genomics 3 Single-Cell RNA-Seq: The single-cell suspension was processed through the Chromium Single-Cell platform using the Chromium Single-Cell 3′ Library and Gel Bead Kit v3.Briefly, ≈15 000 individual cells were loaded onto the Chromium Single-Cell A Chip Kit and partitioned into gel beads in emulsion in the chromium instrument, where the cells and barcoded reverse transcription occurred, followed by amplification, fragmentation, and 5′ adaptor and sample index attachment.Libraries were sequenced using an Illumina Nova-seq system.
Computational Analysis of scRNA-Seq Data: Sequencing reads were aligned to the GRCh38 human genome using the CellRanger toolkit with default parameters.Low-quality cells were discarded according to the following criteria: 1) cells that had fewer than 400 genes, 2) cells that had fewer than 600 UMI or over 10000 UMI, and 3) cells that had more than 15% mitochondrial UMI counts.After quality control, SCTranform wrapped in the Seurat package was applied to integrate the expression matrices of each sample and remove the batch effects of donors.The integrated matrix was analyzed (dimension reduction, graph-based clustering, marker gene detection, and visualization) using Seurat software.Genes presented in fewer than ten cells were filtered out (UMI > 0).Briefly, highly variable genes (HVGs) were calculated with the "FindVariableFeatures," and the top 3000 HVGs were selected for downstream analysis.Data were scaled using the "ScaleData" function, setting the parameters "vars.to.regress" to "percent.mito" and "nUMI."Principal component analysis (PCA) was performed using the "RunPCA" function with the top 3000 HVGs.The number of principal components (PCs) was selected using a visualization plot with the "ElbowPlot" function.A shared nearest neighbor (SNN) graph was constructed using the "FindNeighbors" function with the top 40 PCs, and the cells were clustered by the "FindClusters" function with the "resolution" parameter set to 0.5.The "RunUMAP" function was used for the visualization plot.Marker genes for each cluster were detected using the "FindAllMarkers" function, setting the parameter "min.pct" to 0.2 and "logfc.threshold" to 0.4.
For myeloid, TNK, myocytes, smooth muscle cells/pericytes, and endothelial cell populations, the number of PCs used in the "FindNeighbors" function was 30, 30, 15, 30, and 30, respectively, while the "resolution" parameter of the "FindClusters" function was set to 0.5 for all five populations.Subsequently, cell clusters were manually annotated to the major cell types according to known markers, and any cluster with multiple markers for the two cell types was manually discarded as a doublet.Functional enrichment for certain cell subsets was performed using the clusterPro-fileR package with differentially expressed genes.
Pseudo-Trajectory Analysis of Myeloid Cells, T Cells, and Myocytes: The monocle package (version 2.12.0) was used to infer the potential pseudotimes for myeloid cells, CD8 + T cells, and myocytes.After size factor calculation and dispersions estimation, differentially expressed genes among clusters along the trajectory were identified by the "differentialGeneTest" function.The q values were set to 10E-15, 10E-35, 10E-10, and 10E-25 to determine the significance of macrophages, neutrophils, CD4 + T cells, and myocytes, respectively.For CD8 + T cells, the top 1000 genes ordered by q value were selected.Dimension reduction was performed using the "re-duceDimension" function with the "DDRTree" method.After cell ordering, the "plot_cell_trajectory" function was used for visualization.
To better understand the developmental connections among CD4 + T cells, another algorithm, URD (version 1.0.1), [30]was implemented to reconstruct the trajectory.Naïve CD4 + T cells were set as the root cells, and Th17, Treg, and monocle-defined Th1/Th2 cells were included.The following parameters were adopted in the study: variable genes were defined as diffCV.cutoff= 0.7, knn = 200, sigma = 8, the k-divergence method, and 100 for cells.per.pseudotime.bin.
Gene Expression Regulatory Analysis: To infer the gene regulatory network, the single-cell regulatory network inference and clustering (SCENIC) [26] algorithm was used to identify regulons specifically involved in different cell subsets.The raw expression matrix was extracted, and the transcription factor (TF) activities (AUCell) for each cell were calculated using motif collections version mc9nr.The significantly upregulated regulon was defined by a log fold change of more than 0.1 and an adjusted p-value < 10E-5.The transcriptional network of TF and predicted target genes were visualized using Cytoscape and the igraph package.
Cell-Cell Interaction Analysis: To investigate cellular communication in multiple dimensions, three algorithms were adopted.First, the SCtranformed data were used as input for CellphoneDB, [73] and the parameter "iterations" was set to 1000, "threshold" to 0.1, and "p-value" to 0.05.The outgoing and incoming signals were inferred by the CellChat package. [44]ellular communications found in more than ten cells for downstream analysis were maintained.After selecting candidate ligand-receptor pairs, NicheNet [47] was applied to further predict the target genes in the receiver cells for a specific ligand.Upon ligand CD6 stimulation, the predicted downstream target genes in TREM2 + Macs were visualized using Cytoscape.
Quantification of Serum Interleukin 18 in the Blood: A simple Plex assay was performed using the Ella System to determine the protein concentration of IL-18 in serum, according to the manufacturer's instructions.Briefly, 50 μL of diluted sample was loaded into separate wells of the cartridge, and 2 mL of washing buffer was loaded into the respective wells.The assay was then run using Simple Plex Runner Software and analyzed using Simple Plex Explorer.
RNAscope Assay for LDHA and LDHB mRNA Detection: RNAscope 2.0 Assays were performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 according to the manufacturer's instructions.Hs-LDHA, Hs-LDHB-C2 targeting label probes, and control probes were ordered from the ACD.After counterstaining and mounting the slides, all images were captured using a confocal laser scanning microscope.Staining data were recorded according to the semiquantitative guidelines provided by ACD: 0 for no staining or < 1 dot/10 cells, 1 for 1-3 dots/cell, 2 for 4-9 dots/cell, and none or very few dot clusters, 3 for 10-15 dots/cell and < 10% of the dots in clusters, and 4 for > 15 dots/cell and > 10% of the dots in clusters.
Immunostaining for TREM2 + Macs in Muscle and Bone: The muscle samples were fixed in 4% paraformaldehyde (PFA) for 24 h, dehydrated and embedded in paraffin, and finally sectioned to obtain 4 μmthick paraffin-embedded muscle sections.Following deparaffinization and dehydration, the slides were immersed in citrate buffer and boiled for 10 min for antigen retrieval.After blocking with QuickBlock blocking buffer at room temperature for 30 min, the muscle sections were incubated overnight at 4 °C with primary antibodies against CD68 (1:500) and TREM2 (1:250).After washing three times, the sections were incubated for 1 h at room temperature with the secondary antibodies Alexa Fluor 488 (1:500) and Alexa Fluor 568 (1:500).After counterstaining and mounting the slides, all images were captured using a confocal laser scanning microscope (Leica TCS SP5, Germany).For bone samples, cryosection was used for immunostaining following the established protocols.
Exosome Isolation and Characterization: Exosomes were isolated from the plasma of both young and aged individuals using size-exclusion chromatography (qEV single 35 nm iZON columns) according to the man-ufacturer's instructions.The exosomes collected from 6 to 11 fractions were characterized by nanoparticle tracking analysis using the NanoSight NS300.The morphology of the isolated exosomes was characterized using transmission electron microscopy (TEM).Exosomes were then characterized for ALIX, CD9, TSG101, CD63, and CD81 markers using western blotting, according to a previously described protocol.
Enzyme-Linked Immunosorbent Assay (ELISA) for BAG6 Detection: BAG6 levels were measured using the Human HLA-B-associated transcript 3 (BAT3) ELISA kit according to the manufacturer's protocol and based on the double antibody sandwich technique.
Exosome Uptake and Confocal Microscopy: The exosomes extracted by the size exclusion method were incubated with PKH26 at a ratio of 1:200 at 4 °C overnight.The exosomes were washed three times with PBS using 30 kDa ultrafiltration tubes to remove the free dye.The sorted T helper cells were incubated with 1 × 10 8 PKH26-labeled exosomes for 24 h.The cells were then washed three times with PBS.The DiO dye and PBS were prepared as working solutions at a ratio of 1:100, after which 200 μL of the working solution was added to each well, incubated for 1 h at room temperature, and washed three times with PBS.Finally, 200 μL of DAPIcontaining mounting medium was added to each well of the Petri dish and imaged using a Nikon confocal microscope.
RT-qPCR Analysis: Total mRNA was extracted from cultured cells and muscle tissues using an RNA isolator and reverse transcribed into cDNA using the HiScript III All-in-one RT SuperMix Perfect for qPCR.Real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix on a CFX96 Real-Time System.ACTB was used to normalize the RNA content of the samples, and the 2 −ΔΔCt method was used to calculate relative expression.The primer sequences used were listed in the Key resources table.
Co-Culture of Macrophages and Th17 Cells: Fresh venous blood was drawn from healthy donors and anticoagulated with sodium citrate.The PBMCs were isolated from human blood by density gradient centrifugation using Ficoll-Paque.For macrophage differentiation, PBMCs were cultured in RPMI-1640 supplemented with 10% FBS, 1% penicillin-streptomycin, and 100 ng mL −1 macrophage colony-stimulating factor (M-CSF) for 6 days.Then human Th17 cells (1 00 000 cells per well) were co-cultured with macrophages for 3 days supplemented with or without human plasma exosomes (high BAG6 and low BAG6).The cell suspensions were pelleted, and the cell supernatants were harvested and stored at −80 °C for quantification of cytokines.SPP1 in cell supernatants was measured using ELISA according to the manufacturer's instructions.
Figure 1 .
Figure 1.Study design and the cellular landscape of the aging human musculoskeletal system.a) The baseline information of enrolled patients is shown.b) Representative H&E staining images showing a cross-section view of myofibers from young and older individuals.c) Representative micro-CT reconstruction images showing the trabecular bones from young and older individuals.d) Schematic representation of our workflow; for enrolled patients, both bone and muscle were dissected.Cells from bone and muscle tissues were isolated, purified, and generated as a single-cell library separately.e) Cell clustering projected by UMAP plots showing major cell types in musculoskeletal tissues detected by scRNA-seq; colored by tissue (top) and age distribution (bottom).EC: endothelial cells, FB: fibroblasts.SMC: smooth muscle cells, MSC: mesenchymal stem cells.f) Dot plot showing signature gene expression in each cell type.Circle size indicates the cell fraction expressing the signature gene, and color indicates the gene expression level.g) Transcriptional noise comparison of major cell types between young and old individuals.Blue: young individuals.Red: old individuals.
Figure 2 .
Figure 2. Cellular diversity of myeloid cells in human musculoskeletal tissues.a) UMAP plots of myeloid cell subsets, colored by tissue (bottom) and age distribution (up).b) Transcriptional noise comparison of myeloid cell subsets between young and old individuals.Blue: young individuals.Red: old individuals.M_ = Muscle_; B_ = Bone_.c,d) Representative images of immunofluorescent staining for TREM2 + macrophages (Macs) in both muscle and bone from young and aged individuals.e) KEGG enrichment of highly expressed genes in TREM2 + Macs.f) Violin plots of expression levels of representative genes enriched in the GO terms, namely tissue homeostasis in (e).g) Pseudo-time analysis by Monocle estimating macrophage development in musculoskeletal tissues and pinpointing that NR1H3 may play a distinct role in the development of TREM2 + Macs.h) SCENIC analysis indicated the regulon of NR1H3 was switched on in TREM2 + Macs.Black bars suggested that TF regulon was active in the corresponding cell subsets.i) Transcription factor (TF) in myeloid subsets.
Figure 3 .
Figure 3. Lymphoid subset reprogramming in aged human musculoskeletal tissues.a) UMAP plots of T and NK cells identified in bone and muscle.b) The proportion of CD4 + T cell subsets across age groups, according to tissue type.c) Trajectory inference of CD4 + T cells assessed by Monocle.d) Differentiation trajectories of CD4 + T cells by URD showing naïve CD4 + T cells gave rise to Th1, Th2, Th17, and Treg.The right panel displays the expression of classic transcription factors on each developmental tree.e) GO enrichment for highly expressed genes in Th17.f) The functional enrichment of highly expressed genes in Th1, Th2, Th17, and Treg, visualized by a Radar plot.g) Gene regulatory network inferring NFKB1 targets in Th1 and Th17, respectively.h) Trajectory analysis of CD8 + T subsets highlighted that virtual memory CD8 + T cells were significantly distinct from effector CD8 + T cells.i) Dot plot showing differential gene expression across CD8 + T subsets.Circle size indicates the cell fraction expressing the signature gene, and color indicates the gene expression level.j) Exhaustion score and senescence score comparison among CD8 + T subsets.
Figure 4 .
Figure 4. Sub-clustering analysis of myocytes in aging human muscle.a) UMAP plots showing the diversity of myocyte subsets.b) The functional score of aging-related terms in fast and slow myocytes between young and old individuals._O is for _Old; _Y is for _Young.Fast_ is for Fast myocytes; Slow_ is for Slow myocytes.c) The pseudo-trajectory analysis identified five distinct States in myocytes.d) Violin plot for the expression of muscle-specific aging hallmark genes (TRIM63 and FBXO32) across the five States.e) GO enrichment of highly expressed genes in State 4 and State 5. f) Pearson's correlation analysis between interleukin-18 (IL-18) levels in human serum and grip strength (n = 90).g) Comparison of State composition in each subset.h) Overall expression comparison of five mitochondrial respiratory chain complexes in Slow C2 and MYL12A hi Slow in old individuals.i) Detailed illustration of energy metabolism difference in Slow C2 and MYL12A hi Slow.The red arrow indicates genes upregulated in the MYL12A hi cluster, and the grey arrow infers genes upregulated in Slow C2. j,k) Representative Fluorescence in situ hybridization (FISH) images by RNAscope and quantification for LDHA (green) and LDHB (red) expression in young and aged human muscle (n = 3).The significance (p-value) was calculated using Pearson's correlation analysis (f) and the Mann-Whitney t-test (k); * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 5 .
Figure 5. Cellular communication among different cell subsets in human bone and muscle tissues.a) Incoming and outgoing interaction strength of different cell subsets in old individuals.b) Autocrine and paracrine counts of selected cell subsets in old (top) and young (bottom) individuals.c) Cellular communication signaling variation in different cell subsets between old and young individuals.d,e) Circos plot showing the expression of ligandreceptor pairs BAG3-NCR3 and CD6-ALCAM.f) Characterization of exosomes by TEM harvested from the blood plasma of young and old individuals.M for _Muscle, _B for _Bone.g) BAG6 expression in exosomes from human participants in either young or aged groups was determined by enzymelinked immunosorbent assay (ELISA)(n = 24).h) Representative immunofluorescent images showing the uptake of exosomes by Th cells.Exosomes were labeled by PKH26 and incubated with Th cells for 12 h.i) Intracellular CD6 expression of T cells after incubation with BAG6-shuttled exosomes compared to PBS controls, as tested by qPCR (n = 3 independent experiments).j) Expression level of SPP1 in the conditioned medium of macrophages and Th17 cells co-culture system, determined by ELSIA.The conditioned medium contained the same quantity of either BAG6 hi exosomes (BAG6 hi Exos) or BAG6 low exosomes (BAG6 low Exos), or the same volume of PBS.k) Schematic representation of predicted cell-cell interactions across aged muscle and bone.Degenerated muscle secretes exosomes containing BAG6, the latter of which travel through blood vessels and promote the interaction between Th17 cells and TREM2 + Macs in bone.The TREM2 + Macs potentially facilitate the differentiation of osteoclasts and bone resorption.The significance (p-value) was calculated using the Mann-Whitney t-test (g and i) and the one-way ANOVA test (j); * p < 0.05; ** p < 0.01; *** p < 0.001. | 2023-12-16T12:39:20.103Z | 2023-12-13T00:00:00.000 | {
"year": 2023,
"sha1": "b5555bda525cd41f8da70171453511e348e5f51a",
"oa_license": "CCBY",
"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/advs.202304084",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "394c8d975b6e3bfdb351fe328828d93b8b457257",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
251748487 | pes2o/s2orc | v3-fos-license | Self-Assembly of Porcine Parvovirus Virus-like Particles and Their Application in Serological Assay
Porcine parvovirus (PPV) is widely prevalent in pig farms. PPV is closely related to porcine respiratory disease complex (PRDC) and porcine circovirus disease (PCVD), which seriously threatens the healthy development of the pig industry. Although commercial antibody detection kits are available, they are expensive and unsuitable for large-scale clinical practice. Here, a soluble VP2 protein of PPV is efficiently expressed in the E. coli expression system. The VP2 protein can be self-assembled into virus-like particles (VLPs) in vitro. After multiple steps of chromatography purification, PPV-VLPs with a purity of about 95% were obtained. An indirect, enzyme-linked immunosorbent assay (I-ELISA), comparable to a commercial PPV kit, was developed based on the purified PPV-VLPs and was used to detect 487 clinical pig serum samples. The results showed that the I-ELISA is a simple, cost-effective, and efficient method for the diagnosis of clinical pig serum and plasma samples. In summary, high-purity, tag-free PPV-VLPs were prepared, and the established VLP-based I-ELISA is of great significance for the sero-monitoring of antibodies against PPV.
Introduction
Porcine parvovirus (PPV), the common causative agent of reproductive failure associated with swine, belongs to the genus Parvovirus within the family Parvoviridae. The genotypes of PPV have been characterized to date, including seven strains from classical PPV type 1 (PPV1) to six novel strains (PPV2-PPV7). Among the seven viruses, PPV1, first discovered in cell culture contaminants in Germany in 1965 [1], is the most prevalent and is considered to be one of the main pathogens causing infertility and abortion in sows [2]. PPV2-PPV7 were discovered successively through detection techniques, such as metagenomic sequencing [3][4][5][6][7][8]. A PPV capsid is a small, non-enveloped, icosahedral, and spherical shell, with a diameter size of about 20~25 nm [9], that contains a single-stranded, linear DNA with a genome size of about 5~6 kilobases (kb) [10,11]. There are two main open reading frames (ORF) in the genome of PPV: ORF1, located at the 5 end, encodes three nonstructural proteins (NS1, NS2, and NS3); and ORF2, located at the 3 end, encodes three structural proteins (VP1, VP2, and VP3), among which VP2 is the main capsid component and the protective antigen [12].
PPV disease is widespread and mainly affects fetal pigs. PPV can cross the placental barrier to the fetus, which causes porcine reproductive disorder. In pig farms, sows often have clinical manifestations, such as mummification, reduced litter size, sow dystocia, and repeated mating, suggesting that the disease may break out in pig herds [13]. The disease mostly occurs in the spring, summer, sow parturition, and mating season, and once infected, it will quickly spread to the whole herd, making it challenging to eradicate. When
Gene Amplification and Optimization
The VP2 sequence of PPV obtained from Genbank (Accession No. MF447833) was used as a reference. Subsequently, the VP2 gene was codon-optimized and synthesized by the Genscript Corporation and ligated into the expression vector pET28a.
Construction and Expression of Recombinant VP2 Protein in E. coli
The recombinant vector pET28a-PPV-VP2 was transformed into BL21(DE3)-competent cells containing chaperone pTf-16. Monoclonal bacteria were selected on the plate containing kanamycin and chloramphenicol, then activated for 12 h at 37 • C and 220 rpm/min in 5 mL of TB medium, which contained 160 ug/mL of chloramphenicol and 50 ug/mL kanamycin. Subsequently, 4 mL of bacterial solution was transferred to 200 mL of TB culture medium at the ratio of 1:50 at the same temperature and shaking speed for 2 h 30 min, after which the temperature was reduced to 16 • C, and IPTG and L-Arabinose with the final concentration of 0.1 mmol/L and 2 mg/ mL were added to induce VP2 expression for 20 h. Upon completion of protein induction, the bacteria culture was centrifuged at 6000 g/min for 10 min. The cell pellets were weighed (g) and resuspended in a disruption buffer (200 mM NaCl, 20 mM Tris-HCl, 10% glycerol, pH 8.0) at a ratio of 1 (wet bacteria weight (g)): 10 (disruption buffer (mL)). After mixing well, sonication was performed with an ultrasonic cell disruptor (Cole Parmer, Vernon Hills, IL, USA). SDS-PAGE and Western blot were used to check the expression and solubility of the recombinant VP2 protein. After protein electrophoresis, one part was stained with Coomassie brilliant blue, and the other part was transferred to a PVDF membrane in PBST for 2 h. The PVDF membrane was washed three times with PBST for 15 min and probed with the PPV-specific positive serum (1:5000 dilution) for 1 h at RT. The PVDF membrane was washed three times again and detected with fluorescently-labeled anti-pig secondary antibody (Biodragon, Beijing China, 1:10,000) for 40 min at RT. The PVDF membrane was then washed three times again in the dark and then scanned on a near-infrared fluorescence scanning imaging system (Odyssey CLX, USA).
Purification of PPV-VLPs
The sonicated solution was clarified at 12,000 g/min for 30 min to remove bacterial debris and inclusion bodies. The supernatant was precipitated with PEG6000, and the protein precipitate was centrifuged at 12,000 g/min for 30 min. The pellet was resuspended in resuspension buffer (20 mM Tris, pH 8.0). The resuspended solution was loaded on a DEAE Bestarose Fast Flow column (Bestchrom, Shanghai, China) in an automated FPLC system (AKTA, GE-Healthcare Life Sciences, USA), and the fractions containing effluent from the bulk VLPs were collected. The effluent fractions were subjected to a Sepharose 6FF 16/96 column (Bestchrom, China) equilibrated with equilibration buffer (20 mM Tris-HCl, pH 8.0) at the flow rate of 1.5 mL/min, and the second half of the first peak was collected. The collected protein fractions were further loaded onto a heparin-agarose column (Bestchrom, China) and eluted with elution buffer (500 mM NaCl, 20 mM Tris-HCl). Finally, the eluted fraction was passed through a Sepharose 6FF 16/96 column (Bestchrom, China), which was equilibrated with equilibration buffer (20 mM Tris-HCl, pH 8.0) at a flow rate of 1.5 mL/min. The first peak was collected in separate 15 mL centrifuge tubes, with 10 mL per tube. The protein elutions were analyzed with SDS-PAGE and TEM.
TEM Procedure of PPV-VLPs
The sample was incubated for 10 min at RT. Subsequently, the PPV-VLPs were fully adsorbed onto the copper mesh and dried at RT. The copper mesh was negatively stained with 2.5% phosphotungstic acid for 1 min after being dried, and the excess stain was Viruses 2022, 14, 1828 4 of 12 blotted off using filter paper and carefully placed on the TEM (HITACHI, Tokyo, Japan) for observation.
Determination of Hemagglutination of PPV-VLPs
The hemagglutination activity of PPV-VLPs was determined by a hemagglutination assay. In a 96-well V-plate, 25 uL PBS was added to each well, then 25 uL PPV-VLPs were added to the first column of wells and mixed to generate 2-fold dilution. Then, 25 uL of the 2-fold-diluted PPV-VLPs were added to the second column of wells and mixed, repeating the dilution pattern across the plate to complete the 2-fold serial dilutions of antigen PPV-VLPs. A total of 25 uL 1% chicken erythrocyte suspension was added to the plate and shaken for 2 min to mix well. The control group used only 25 uL of PBS and 25 uL of 1% chicken red blood cell suspension. The results were determined 1 h later, and the highest dilution that allowed 100% agglutination of red blood cells was the hemagglutination titer of PPV-VLPs.
Optimization of the PPV-VLP-ELISA Procedure
Purified PPV-VLPs were used as antigens to develop I-ELISA. The checkerboard method was applied to determine the optimal serum dilution and antigen-coating concentration. The concentration of PPV-VLPs was determined by a BCA kit (Thermo, Waltham, MA, USA). PPV-VLPs were diluted to 0.5 ug/mL, 1 ug/mL, 2.5 ug/mL, 5 ug/mL, 7.5 ug/mL, and 10 ug/mL in carbonate coating buffer (pH 9.6) and then plated on ELISA plates (Biofil, Guangzhou, China) to determine the optimal antigen-coating concentration. Positive and negative sera were diluted at 1:50, 1:100, and 1:150 (v/v) with PBST to determine the optimal serum dilution. Additionally, the reaction time, temperature, and other conditions were optimized.
Standardization of PPV-VLP-ELISA Procedure
PPV-VLPs were coated with 100 µL/well, at a concentration of 1 µg/mL, in a 96-well ELISA microplate (Biofil, China) overnight at 4 • C. The coated plates were washed three times with PBST and then blocked with an enzyme plate stabilizer (InnoReagents, China) for 2 h in a 37 • C incubator (Yiheng, Shanghai, China). The blocked plates were washed three times, and 100 µL of the diluted serum samples were added and incubated at 37 • C for 1 h. Then, the plates were washed three times, and 100 µL HRP-SPA dilution (Bosterbio, Wuhan, China, 1:10,000) was added and incubated for 1 h at 37 • C. After the three-time washes, 100 µL of tetramethylbenzidine (TMB, Solarbio, Beijing, China) was added to each well and incubated for 15 min at 37 • C in the dark. Finally, 50 µL of stop solution (1M HCl) was added to stop the reaction, and the OD450nm value was measured with an ELISA plate reader (PE, Corona, CA, USA).
Determination of Cut-Off Value
Fifty-five negative sera were employed to determine the cut-off value. All sera were analyzed by PPV-VLP-ELISA three times independently to reduce the deviation. The mean OD450nm value (X) and standard deviation (SD) were calculated, and the cut-off value was defined as X + 3SD.
Reproducibility and Cross-Reactivity Assay
Twelve serum samples were selected to evaluate the reproducibility of the PPV-VLP-ELISA. For each sample, the coefficient of variation (CV) values between plates (inter-assay variation) and within plates (intra-assay variation) were calculated. The results showed that both the inter-assay and intra-assay variation were less than 10%. Four positive sera and one negative serum diluted at 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400 were applied to evaluate the sensitivity of this method. The specificity of the method was evaluated by comparing the OD450nm values of the standard positive serum with those of the selected, potentially interfering viruses (African swine fever virus (ASFV), Japanese encephalitis virus (JEV), PRRSV, foot-and-mouth disease virus (FMDV), PCV2, simian immunodeficiency virus (SIV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV)).
Cultivation and Proliferation of PPV
Pig kidney (PK-15) cells were obtained and preserved by the Harbin Veterinary Research Institute (Harbin, China). The PK-15 cells were revived in the cell culture flask, and when the bottom of the flask was confluent with PK-15 cells, the cells were digested and transferred to a new flask. PPV was then inoculated into the flask and cultured in a cell incubator (Thermo, USA) at 37 • C and 5% carbon dioxide. When about 80% of the inoculated cells became cytopathic, they were repeatedly incubated. The viruses were fully released by three freeze-thaw cycles and were harvested at 2000 r/min for 10 min to remove cell debris and frozen at −80 • C.
Comparison of the PPV-VLP-ELISA with the Commercial PPV ELISA Kit for Detection of Anti-PPV Antibodies
Sixty-four samples were randomly selected from 487 serum samples for comparison between commercial ELISA kits (Ingenasa, Spain) and PPV-VLP-ELISA. Sixty-four samples were tested using a commercial kit, according to the instructions, and positive and negative samples were placed and labeled separately. These samples were subsequently tested using PPV-VLP-ELISA. Each serum was analyzed three times independently, using commercial kits and PPV-VLP-ELISA to minimize bias.
Indirect Immunofluorescence Assay Verification of Five Positive Samples Determined by PPV-VLP-ELISA
The five positive serum samples to be tested were named A, B, C, D, and E, respectively, and then an indirect immunofluorescence assay (IFA) was performed. First, PK-15 cells were infected with PPV for 48 h, after which the cells were fixed with 4% paraformaldehyde for 30 min at RT and then washed with PBS three times. Permeabilization fluid (0.25% Triton-X100) was added at 100 uL/well and was placed on ice for 15 min. After washing three times with PBS, a blocking solution (5% BSA) was added for 1 h. After another three-time wash, the serum samples and SPF serum were diluted at 1:100, added to the wells, and incubated at 37 • C for 2 h. After another three-time wash, a FITC-labeled goat anti-pig secondary antibody (Sigma, Burlington, MA, USA) was added at a dilution of 1:200 and incubated at 37 • C for 1 h. After the last three-time washes, results were measured with an inverted fluorescence microscope (AMG, Denver, CO, USA).
Expression and Purification of PPV-VLPs from E. coli
The recombinant plasmid pET28a-PPV-VP2 was constructed and transformed in E. coli BL21 (DE3) competent cells, and cells containing the plasmid were inoculated in TB medium. The recombinant VP2 protein (molecular weight~64 kDa) was IPTG-and L-Arabinose-induced with or without the co-expression of the chaperone plasmid pTf-16. When expressed in the absence of chaperone pTf-16, the VP2 protein was poorly expressed and less soluble ( Figure 1A). When co-expressed with chaperone pTf-16, both the expression and solubility of the VP2 protein significantly increased ( Figure 1B). Western blot analysis also confirmed the folding of the VP2 protein obtained from chaperone pTf-16 co-expression, as it reacted with specific positive sera ( Figure 2A). As described above, expressed VP2 proteins were purified by three columns: ion-exchange column (IEC), size-exclusion column (SEC), and heparin-agarose column (HP). While self-assembled PPV-VLPs were observed after HP ( Figure 2B), a few heterogeneous protein bands and oddshaped impurity particles around 25 nm could also be clearly observed under SDS-PAGE ( Figure 2C) and TEM ( Figure 2D). PPV-VP2 proteins were further purified via another SEC ( Figure 3A-C). Homogeneous and intact particles without any impurities were observed under TEM ( Figure 3D). VLPs were observed after HP ( Figure 2B), a few heterogeneous prot shaped impurity particles around 25 nm could also be clearly observe ( Figure 2C) and TEM ( Figure 2D). PPV-VP2 proteins were further p SEC ( Figure 3A-C). Homogeneous and intact particles without any served under TEM ( Figure 3D). medium. The recombinant VP2 protein (molecular weight ~64 kDa) was IPTG-and L-Arabinose-induced with or without the co-expression of the chaperone plasmid pTf-16. When expressed in the absence of chaperone pTf-16, the VP2 protein was poorly expressed and less soluble ( Figure 1A). When co-expressed with chaperone pTf-16, both the expression and solubility of the VP2 protein significantly increased ( Figure 1B). Western blot analysis also confirmed the folding of the VP2 protein obtained from chaperone pTf-16 co-expression, as it reacted with specific positive sera ( Figure 2A). As described above, expressed VP2 proteins were purified by three columns: ion-exchange column (IEC), sizeexclusion column (SEC), and heparin-agarose column (HP). While self-assembled PPV-VLPs were observed after HP ( Figure 2B), a few heterogeneous protein bands and oddshaped impurity particles around 25 nm could also be clearly observed under SDS-PAGE ( Figure 2C) and TEM ( Figure 2D). PPV-VP2 proteins were further purified via another SEC ( Figure 3A-C). Homogeneous and intact particles without any impurities were observed under TEM ( Figure 3D).
Hemagglutination Activity of PPV-VLPs
The hemagglutination activity of PPV-VLPs was confirmed to be 2 8 (1:256) (Figure 4), indicating that the PPV-VLPs correctly displayed the epitope of PPV required for hemagglutination activity, and the structure was similar to that of the native PPV.
Standardization of the PPV-VLP-ELISA Procedure
The optimization of the VLP-based I-ELISA was guided by OD450nm and P/N values (ratio of the OD value of a test sample to the average OD value of negative control). A total of 50 mM of carbonate-bicarbonate buffer (pH 9.6) and plate stabilizer were selected as the final coating and blocking buffers, respectively. The concentration of purified PPV-VLPs was determined to be 0.1 mg/mL using the BCA protein concentration measurement kit (Thermo, USA). Checkerboard titration experiments showed that when coating concentration for PPV-VLPs was 1 μg/mL and the serum dilution was 1:50, the P/N ratio can be ultimately optimized. All experiments were performed in triplicate (Table 1). Fifty-five negative serum samples were used to determine cut-off values, with the mean OD and SD values of 0.149 and 0.059, respectively ( Figure 5A). Therefore, the cut-off value for the PPV-VLP-ELISA was 0.326 (X + 3SD). Serum with an OD450nm value greater than or equal to this threshold was considered positive. Otherwise, it was determined to be negative for PPV antibodies. The experiments showed that this optimized method has good sensitivity and specificity ( Figure 5B,C).
Hemagglutination Activity of PPV-VLPs
The hemagglutination activity of PPV-VLPs was confirmed to be 2 8 (1:256) (Figure 4), indicating that the PPV-VLPs correctly displayed the epitope of PPV required for hemagglutination activity, and the structure was similar to that of the native PPV.
Hemagglutination Activity of PPV-VLPs
The hemagglutination activity of PPV-VLPs was confirmed to be 2 8 (1:256) (Figure 4), indicating that the PPV-VLPs correctly displayed the epitope of PPV required for hemagglutination activity, and the structure was similar to that of the native PPV.
Standardization of the PPV-VLP-ELISA Procedure
The optimization of the VLP-based I-ELISA was guided by OD450nm and P/N values (ratio of the OD value of a test sample to the average OD value of negative control). A total of 50 mM of carbonate-bicarbonate buffer (pH 9.6) and plate stabilizer were selected as the final coating and blocking buffers, respectively. The concentration of purified PPV-VLPs was determined to be 0.1 mg/mL using the BCA protein concentration measurement kit (Thermo, USA). Checkerboard titration experiments showed that when coating concentration for PPV-VLPs was 1 μg/mL and the serum dilution was 1:50, the P/N ratio can be ultimately optimized. All experiments were performed in triplicate (Table 1). Fifty-five negative serum samples were used to determine cut-off values, with the mean OD and SD values of 0.149 and 0.059, respectively ( Figure 5A). Therefore, the cut-off value for the PPV-VLP-ELISA was 0.326 (X + 3SD). Serum with an OD450nm value greater than or equal to this threshold was considered positive. Otherwise, it was determined to be negative for PPV antibodies. The experiments showed that this optimized method has good sensitivity and specificity ( Figure 5B,C).
Standardization of the PPV-VLP-ELISA Procedure
The optimization of the VLP-based I-ELISA was guided by OD450nm and P/N values (ratio of the OD value of a test sample to the average OD value of negative control). A total of 50 mM of carbonate-bicarbonate buffer (pH 9.6) and plate stabilizer were selected as the final coating and blocking buffers, respectively. The concentration of purified PPV-VLPs was determined to be 0.1 mg/mL using the BCA protein concentration measurement kit (Thermo, USA). Checkerboard titration experiments showed that when coating concentration for PPV-VLPs was 1 µg/mL and the serum dilution was 1:50, the P/N ratio can be ultimately optimized. All experiments were performed in triplicate (Table 1). Fifty-five negative serum samples were used to determine cut-off values, with the mean OD and SD values of 0.149 and 0.059, respectively ( Figure 5A). Therefore, the cut-off value for the PPV-VLP-ELISA was 0.326 (X + 3SD). Serum with an OD450nm value greater than or equal to this threshold was considered positive. Otherwise, it was determined to be negative for PPV antibodies. The experiments showed that this optimized method has good sensitivity and specificity ( Figure 5B,C).
Coincidence Rate with the Ingezim PPV ELISA Kit for Detection of Anti-PPV Antibodies
Clinical pig serum samples (n = 64) were used to determine the reliability of the PPV-VLP-ELISA compared to the commercial PPV-ELISA kit. These sera were randomly selected from 487 serum samples and tested using both commercial ELISA kits and PPV-VLP-ELISA developed in-house. When tested using the commercial ELISA kit, 36 (56.25%) of 64 serum samples were positive and 28 (43.75%) of 64 serum samples were negative. When tested using the PPV-VLP-ELISA method, 41 (64.1%) of 64 serum samples were positive and 23 (35.9%) of 64 serum samples were negative ( Table 2). The results were similar to the commercial ELISA kit, as the overall concordance rate was 92.2% (59/64) between PPV-VLP-ELISA and the commercially available PPV-Ingenasa-ELISA kit. Among 41 samples tested positive by PPV-VLP-ELISA, 36 samples also tested positive in the commercial kit and the other 5 samples tested negative in the commercial kit. The results suggested that the sensitivity and specificity of PPV-VLP-ELISA were at least comparable to those of the commercial kits. Table 2. Comparison of the PPV-VLP-ELISA with the commercial PPV ELISA kit for detection of anti-PPV antibodies. Positive Negative Total Positive 36 5 41 Negative 0 23 23 Total 36 28 64
Confirmation of PPV-VLP-ELISA Tested Positive Samples with Indirect Immunofluorescence Assay
The IFA results agreed with the PPV-VLP-ELISA results. Five positive sera determined by PPV-VLP-ELISA were also confirmed positive by IFA. SPF serum as a negative control was proved to be negative by IFA (Figure 6).
Coincidence Rate with the Ingezim PPV ELISA Kit for Detection of Anti-PPV Antibodies
Clinical pig serum samples (n = 64) were used to determine the reliability of the PPV-VLP-ELISA compared to the commercial PPV-ELISA kit. These sera were randomly selected from 487 serum samples and tested using both commercial ELISA kits and PPV-VLP-ELISA developed in-house. When tested using the commercial ELISA kit, 36 (56.25%) of 64 serum samples were positive and 28 (43.75%) of 64 serum samples were negative. When tested using the PPV-VLP-ELISA method, 41 (64.1%) of 64 serum samples were positive and 23 (35.9%) of 64 serum samples were negative ( Table 2). The results were similar to the commercial ELISA kit, as the overall concordance rate was 92.2% (59/64) between PPV-VLP-ELISA and the commercially available PPV-Ingenasa-ELISA kit. Among 41 samples tested positive by PPV-VLP-ELISA, 36 samples also tested positive in the commercial kit and the other 5 samples tested negative in the commercial kit. The results suggested that the sensitivity and specificity of PPV-VLP-ELISA were at least comparable to those of the commercial kits.
Confirmation of PPV-VLP-ELISA Tested Positive Samples with Indirect Immunofluorescence Assay
The IFA results agreed with the PPV-VLP-ELISA results. Five positive sera determined by PPV-VLP-ELISA were also confirmed positive by IFA. SPF serum as a negative control was proved to be negative by IFA (Figure 6).
Application of PPV-VLP-ELISA to Screen Clinical Pig Serum Samples
Swine serum samples collected in northeast China in 2020 and 2021 were tested with PPV-VLP-ELISA. A total of 432 samples (88.7%) tested positive, and 55 samples (11.3%) tested negative. The result showed a high positive rate of PPV antibodies in the tested herd (Figure 7), indicating that PPV is widespread in pig farms and poses a serious threat to the local pig industry. In conclusion, PPV-VLP-ELISA has high application value in the monitoring and eradication of PPV in the future.
Discussion
Currently, different expression host systems can express VLPs, including bacteria, insect, yeast, mammalian cell, cell-free, and plant expression systems [24]. Recombivax HB, the world's first genetically engineered vaccine for the prevention of hepatitis B, is a VLP vaccine expressed by the saccharomyces cerevisiae expression system [25,26]. From then on, multiple VLP vaccines were approved, including human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis E virus (HEV), and H1N1 vaccine, etc [27]. About 30% of approved VLP vaccines are produced by bacterial expression systems, mainly in E. coli. Other VLP bacterial expression systems besides E. coli, Lactobacillus, and Pseudomonas aeruginosa are responsible for vaccines against HPV and cowpea chlorosis mottle virus (CCMV) [28,29]. While bacterial expression systems may have many advantages in expressing VLPs, such as high growth rate, being easy-to-scale up, and low cost, their appli-
Application of PPV-VLP-ELISA to Screen Clinical Pig Serum Samples
Swine serum samples collected in northeast China in 2020 and 2021 were tested with PPV-VLP-ELISA. A total of 432 samples (88.7%) tested positive, and 55 samples (11.3%) tested negative. The result showed a high positive rate of PPV antibodies in the tested herd (Figure 7), indicating that PPV is widespread in pig farms and poses a serious threat to the local pig industry. In conclusion, PPV-VLP-ELISA has high application value in the monitoring and eradication of PPV in the future.
Discussion
Currently, different expression host systems can express VLPs, including bacteria, insect, yeast, mammalian cell, cell-free, and plant expression systems [24]. Recombivax HB, the world's first genetically engineered vaccine for the prevention of hepatitis B, is a VLP vaccine expressed by the saccharomyces cerevisiae expression system [25,26]. From then on, multiple VLP vaccines were approved, including human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis E virus (HEV), and H1N1 vaccine, etc [27]. About 30% of approved VLP vaccines are produced by bacterial expression systems, mainly in E. coli. Other VLP bacterial expression systems besides E. coli, Lactobacillus, and Pseudomonas aeruginosa are responsible for vaccines against HPV and cowpea chlorosis mottle virus (CCMV) [28,29]. While bacterial expression systems may have many advantages in expressing VLPs, such as high growth rate, being easy-to-scale up, and low cost, their applications should be evaluated cautiously due to the lack of post-translational modification
Discussion
Currently, different expression host systems can express VLPs, including bacteria, insect, yeast, mammalian cell, cell-free, and plant expression systems [24]. Recombivax HB, the world's first genetically engineered vaccine for the prevention of hepatitis B, is a VLP vaccine expressed by the saccharomyces cerevisiae expression system [25,26]. From then on, multiple VLP vaccines were approved, including human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis E virus (HEV), and H1N1 vaccine, etc. [27]. About 30% of approved VLP vaccines are produced by bacterial expression systems, mainly in E. coli. Other VLP bacterial expression systems besides E. coli, Lactobacillus, and Pseudomonas aeruginosa are responsible for vaccines against HPV and cowpea chlorosis mottle virus (CCMV) [28,29]. While bacterial expression systems may have many advantages in expressing VLPs, such as high growth rate, being easy-to-scale up, and low cost, their applications should be evaluated cautiously due to the lack of post-translational modification of proteins. Concerns about endotoxin contamination may further reduce the scope of its application [30]. The yeast expression system is widely used to produce VLPs for HPV and HBV. Insect and mammalian cell expression systems have been used to produce VLPs since the 1980s. The plant cell expression system and cell-free expression system have recently been found capable of producing VLPs.
VLPs only contain viral capsid proteins but no viral genetic material. Their conformational epitopes and morphological structure are similar to those of natural viruses and have good immunogenicity [31,32]. VLPs can induce strong humoral and cellular immune responses and are considered a highly efficient vaccine platform [33]. In recent years, the technologies of expressing PPV VP2 proteins and self-assembly into VLPs have been greatly advanced. These new technologies have been advancing the production and future development of VLP-based vaccines [18]. PPV VP2 is a non-enveloped protein that can self-assemble into VLPs under suitable conditions, without additional modifications. Therefore, compared with other eukaryotic expression systems, the E. coli expression system, without protein modifications, is proper and desirable for producing VP2 proteins. Assembled PPV-VLPs are highly likely to form conformation similar to that of natural viral particles. The suitability of the E. coli expression system to produce the VP2 protein was verified by both the results from PPV-VLP-ELISA and previous studies regarding VLP-based vaccines [18,34].
In this study, the E. coli expression system has been employed successfully to express and purify tag-free PPV-VLPs. PPV-VLPs have many advantages, including costeffectiveness, high-yield expression, and high-density cultivation. Thus, it is significant for diagnosis and vaccine development.
The VLP has only viral structural proteins without viral genetic material, and its morphological structure is similar to that of natural virus particles [35,36]. It is capable of mimicking external epitopes and conformational epitopes of viruses [37,38]. Therefore, the VLP is more suitable for establishing the ELISA diagnostic method. Recent studies have shown that the PPV is closely related to porcine respiratory diseases, so monitoring and vaccination against the PPV plays an important role in the prevention and treatment of PRDC and PCVD [39,40]. Therefore, establishing a diagnostic method with high sensitivity, high specificity, and suitability for large-scale screening is of great significance for the control of PPV. This paper successfully showed a high-sensitivity, high-specificity, and low-cost PPV-VLP-ELISA method using high-purity PPV-VLPs. Subsequently, the VLP-ELISA method was successfully used for PPV antibody detection in 487 clinical serum samples collected in northeast China in 2020 and 2021. It was found that PPV was widely prevalent in Chinese pig farms, indicating that this method can be widely used in PPV epidemiological studies.
Conclusions
The E. coli expression system has been employed for the expression and purification of tag-free PPV-VLPs, which could be used for the development of a VLP-based PPV vaccine. In addition, this is the first report of the I-ELISA method based on PPV-VLPs for testing the PPV-specific antibodies in clinical pig serum. The PPV-VLP-ELISA is highly specific, sensitive, and reproducible. It is a valuable tool for monitoring the prevalence of PPV.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. | 2022-08-24T15:16:45.943Z | 2022-08-01T00:00:00.000 | {
"year": 2022,
"sha1": "bff3ae9ba5b7b337a92f0663523edf8225d21d5a",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1999-4915/14/8/1828/pdf?version=1660991453",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "818449edce7da6912b361cddd2f9b272a0ae1d79",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
258430783 | pes2o/s2orc | v3-fos-license | Determination Analysis of Leading Commodity in the Melonguane National Border Strategic Area (NBSA)
Border areas are the gateway for a country; generally, it is an underdeveloped area. This study aims to develop the economy based on only commodities in the National Strategic Activity Center (NSA) Melongguane, Kepulauan Talaud Regency, North Sulawesi Province. This study uses qualitative and quantitative approaches. The qualitative approach is based on the policy formulation of the North Sulawesi Provincial government and the Kepulauan Talaud Regency, namely the Regional Spatial Planning (RTRW) document. While the quantitative method with the Location Quotient (LQ) analysis. Based on the North Sulawesi Provincial Spatial Planning (RTRW) document shows that the leading commodities at the Melongguane National Strategic Activity Center (NSA) are Nutmeg, Coconut, Clove, and Tuna-Skipjack-cob. The Location Quotient (LQ) analysis shows that the sub-districts with potential nutmeg commodities are Kabaruan and Damau sub-districts. At the same time, the potential locations for developing Tuna-Skipjack-cob are Nanusa and Miangas Districts.
INTRODUCTION
Border area development is an integral part of national border area development.The border areas have considerable natural resource potential, especially fisheries, agriculture, and plantations.Based on a socio-economic perspective, people living in border areas tend to lag behind development in other areas compared to the socio-economic conditions of neighboring countries.Generally, people living in border areas have a much lower level of welfare.A higher gap will cause various social vulnerabilities (Partnership for Governance Reform, 2011).The development and management of national border areas have been one of the main agendas of President Jokowi's government, namely building from the periphery (Priyarsono, 2017).They explain that development is carried out more than in urban areas (Centralization).It must be evenly distributed throughout Indonesia (Decentralization).
The Social-economic conditions in national border areas are not only related to the territorial or geographical areas adjacent to the borders of neighboring countries but also a concern to improving the quality of human resources who have been marginalized and live at a low level of welfare.The indicators are low public access to productive infrastructures, such as education, health, and other infrastructures.These are a result of development that has only focused on urban areas, considered the center of growth (Setiawan, 2019).Another area for improvement in border areas is the disparity in the development of border areas within the country as abroad (for example, Malaysia).The facts show that the potential in border areas is excellent and diverse, including natural resources such as fisheries, agriculture, and plantations.Likewise, the quality of human resources living at the border needs to catch up and be more developed than in other areas.The limitations of infrastructure, facility, institutional capacity, and human resources inhibit regions' growth and development in border areas.There is strategic value in maintaining the state of sovereignty regarding geopolitical, social, economic, defense, and security aspects (Marif, 2016).
One of the Strategic Directions for the Management of Internal State Borders as contained in the Master Plan for the Management of State Boundaries and Border Areas for 2020-2024 is to realize increased economic activities, development of the facility, and the infrastructure as well as improve the quality of human resources.The directive aims to create the national border area as a center of growth through increasing economic activities based on Leading potential and improving the quality of Human Resources (Bappenas, 2020).The rise of border areas based on Leading sectors/commodities has been one of the strategic steps to achieve accelerated development in border areas.Therefore, developing border areas based on Leading sectors/commodities is one of the strategic steps to achieve accelerated development in border areas.
The development of the economic sector regarding the Leading sector and impacting accelerating economic growth will also affect fundamental changes in the economic structure.The Leading sector is a sector whose existence at this time has played a significant role in the region's economic development because it has advantages.The Leading sector has high toughness and capability so that it can be used as the foundation, backbone, and Determination Analysis of Leading Commodity in the Melonguane National Border Strategic Area (NBSA) (Hanim et al.,) 57 driving force of the economy, so it can also be referred to as a critical sector, or sector leading the economy of the region (Hajeri et al., 2015).Building a regional economy that utilizes the potential of local resources will create quality economic growth, namely economic growth accompanied by reducing income inequality between residents and regions and reducing poverty (Hanim, 2021).In general, people who live on state borders are economically dependent on the economy of neighboring countries (Marwasta, 2016).Maximizing available local resources will improve the standard of living of the people living within the borders of the country and reduce the lag behind residents of neighboring countries.
The theory of economic basis is one of the methods commonly used to identify the leading sectors of a region.The theory states that the determining factor for the economic growth of a region is the demand for imports and exports of goods and services.In other words, a region with a primary sector means that the region is competitively superior compared to other regions within the same sector (Hutapea et al., 2020;Maspaitella & Parinussa, 2021;Suryani, 2013;Tutupoho, 2019).In the theory of the economic basis, the economic sector consists of two sectors, namely the base sector and the non-base sector.The base sectors are the main driver of economic growth.Any change in the base sector will have a multiplier effect on the regional economy.An increase in the base sector will increase the flow of income to the region concerned.It will improve the performance of the non-base sector (Atmayanti et al., 2021).An important activity to identify base economic sectors is fundamental for determining priority economic sectors in development programs.
Melongguane is a sub-district located in the Kepulauan Talaud Regency, North Sulawesi Province.Apart from being the capital of the Kepulauan Talaud Regency, Melongguane is also the Melonguane National Strategic Activity Center (NSA) with 4 (four) supporting sub-districts, namely Kabaruan District, Damau District, Nanusa District, and Miangas District which are referred to as priority location districts.NSA Melongguane borders the Philippines country, namely Davao City.The location of the sub-district which is directly adjacent to the Philippines is The Miangas District on the island of Miangas.
As with the condition of the border area in general, the border area of North Sulawesi has the same problem.It has abundant natural resource potential, especially for fisheries and plantation commodities, but has yet to develop optimally.Based on the reason necessary to explore local potential as a source of regional economic growth.Formulating the Leading commodities as sources of economic growth that have the potential and accelerate the level of community welfare in the Melonguane National Strategic Activity Center (NSA) area, Kepulauan Talaud Regency, North Sulawesi Province.The development of the economic sector concerning the Leading sectors in addition to impacting and accelerating economic growth, will also affect fundamental changes in the economic structure, reduce backwardness with other regions and reduce poverty.
Regional economic development is a process in which local governments and their communities manage resources to increase regional economic growth.The resources available in an area are economic potential that could be developed to become a source of regional income and livelihood for the local community's economy.Identifying potential and only commodities can be a reference for economic planners to focus on and support unique activities that will improve the regional economy (Alhowaish, 2015).The Kepulauan Talaud Regency is the gateway to the border between Indonesia and the Philippines and needs to explore the local economic potential as an engine of growth.
Several empirical studies related to potential and leading sectors have long attracted the interest of economists and policymakers because they involve empirical results and use various approaches.Adhitama (2012) uses the Location Quotient (LQ) Technique, Shift share, and Klasen typology to identify leading sectors at the sub-district level in Magelang Regency.Arafah & Matheos (2017) used static LQ and dynamic location quotient analysis to identify leading sectors in Bantaeng Regency, South Sulawesi Province.Atmayanti et al. (2021) and Maspaitella et al. (2021) use the LQ and Shift share models to identify potential sectors in Teluk Bintuni Regency.Kharisma and Ferry combined the Location Quotient, SWOT, and ANP models to analyze potential sectors and subsectors in Maluku Province.Other researchers, Niyimbanira et al. (2019), identified the main sub-industry between metropolitan cities on the coast of South Africa by applying the location quotient technique, while Alhowaish (2015) used the percentage share, multiplier effect, and location quotient models to identify critical sectors in Tabuk City, Saudi Arabia.
This potential has been developed optimally by identifying the potential of the leading sector owned by a region.This sector would grow and become the base sector in the region.The increase in economic activity in the base sector and regional potential would increase GRDP.Sectoral specialization or superior sub-sectors in each region can increase the effectiveness and efficiency of the community in carrying out economic activities.Thus, local governments might know precisely the base and non-base sectors and the potential sectors for new base sectors in the region (Kharisma & Hadiyanto, 2019).
In general, researchers identify the potential of a region in terms of sectoral aspects, where the analysis is macro and the conclusions formulated are the identification of leading regional sectors.When the regional government puts it into a policy, it still has to conduct further analysis, namely an analysis of the superior regional commodities concerned.Therefore, this study identified superior commodities in the sea border area of Kepulauan Talaud Regency and synchronized the identification of only commodities contained in local government policies.It, namely the Regional Spatial Plan (RTRW), ratified in the regional regulations of kepulauan Talaud Regency.It is hoped that the identification results will be more implementable and make it easier for local governments to determine policies related to their development.Literature Review Regional economic development Regional economic development is a process in which local governments and communities work together to manage available resources into an economic activity capable of creating employment opportunities for local communities to improve the economic welfare of the people in the area concerned.The community must jointly take regional development initiatives (Siwi, 2017).
The concept of regional development as a new policy approach to regional development is increasingly being used in various countries.Both developed and developing countries, including in the Talaud Islands Regency, North Sulawesi Province, especially regarding the readiness of a region to increase its competitiveness in the face of regionalization and globalization (Kharisma & Hadiyanto, 2019).
Economic growth has been an indicator of the economic progress of a region and is the main requirement for the continuity of economic development and increased welfare.The goals of economic development in the regions are similar to those of national development.However, the regional development process is much more specific (Raqib, 2018).Where the implementation is adjusted to the conditions and potential of each region (Gafur et al., 2016).One of the efforts must be made to identify an area's various potentials.If this potential is developed optimally, it will benefit the area.Optimizing economic activities in potential sectors will create a base sector in the area concerned (Kharisma & Hadiyanto, 2019).Economic Base Theory Economic Base Theory (Economic Base Theory) is related to the main determinants of economic growth in a region.In the economic basis theory, the economic sector has been divided into 2 (two) classifications, namely the economic base sector and the non-economic basis sector.The base sector is a sector that has great potential in determining regional development as a whole, while the non-base sector is supporting regional development (Hutapea et al., 2020).
The economic base theory states that the magnitude of the increase in exports from that region to other regions determines a region's economic growth level.The base sector is the primary driver of economic growth in a region.Any changes that occur in the base sector will cause a multiplier effect on the regional economy.Increasing the main activities in an area will increase the flow of income to the area concerned.It will improve the performance of the non-base sector (Atmayanti et al., 2021).
The base sector is a source of regional economic growth.Increased economic activity in the base sector means sectoral/subsectoral specialization or superior regional commodities.So that economic activity becomes more effective and efficient.Thus, local governments might know precisely the base and non-base sectors.By analyzing the sector basis, it means that the potential of the leading regional sectors/commodities will be identified so that it would make it easier for local governments to formulate economic development policy priorities in their regions (Kharisma & Hadiyanto, 2019).
METHOD Data
This research is descriptive, namely qualitative descriptive and quantitative descriptive.The data used in this study is secondary data.The data collected consists of Gross Regional Domestic Gross (GDRB) Kepulauan Talaud Regency and North Sulawesi Province 2016-2020 and commodities data of sub-district at kepulauan Talaud Regency in 2020.Government Regulation that is the Kepulauan Talaud Regency Regulation Number 1 concerning the Regional Spatial Planning (RTRW) of the Kepulauan Talaud Regency for 2014-2034.
Analysis Method
The Regional Spatial Plan (RTRW) of the Kepulauan Talaud Regency is one of the policies that the Melonguane National Strategic Activity Center (NSAC) employs to formulate leading commodities.On the other hand, the Location Quotient (LQ) analysis has been used in the second method.The determination of the Leading commodity resulting from the calculation of the Location Quotient (LQ) must be under the determination of the Leading commodities in the Regional Spatial Planning (RTRW) policy of North Sulawesi Province and Kepulauan Talaud Regency.a.The policy of the Regional Spatial Plan (RSP) of North Sulawesi Province 2014-2034.
The policy formulation of North Sulawesi Province Government in the 2014-2034 Regency Spatial Plan (RTRW) is related to Leading Commdities commodities.b.Analisis Location Quotient (LQ) The Economic Base Theory emphasizes the role of exports as a source of growth (Gomez & Stair, 2017), along with the increase in exports, which implies a flow of income from other regions that would encourage one region's economy (Rahadiantino & Fathurrohman, 2021).One way to determine which sector is the basis of the local economy is to use Location Quotients.Location Quotient (LQ) measures the concentration of a region's economic activity by comparing its role in the regional economy with the contribution of similar economic activities in a higher regional economy, for example, provincial or national (Fikri & Fafurida, 2018;Sandra Yulia Setiawati, 2014).The Location Quotient (LQ) technique, namely, by reviewing production and productivity documents (Yos Wahyu Harinta, Joko Setyo Basuki, 2016).The location quotient (LQ) is a very important technique, as it denotes areas where there is a concentration of workers with transferable skills, interconnected businesses, suppliers, and related industries, or areas of a growing industry where employment in the sector is weak but growing (Niyimbanira et al., 2020).The formulation of the Location Quotient (LQ) is as follows: Criteria: • LQ > 1, Leading commodities, apart from meeting their own needs, also have the potential to be exported to other regions.The region specializes in that commodity (base).• LQ = 1, the commodity produced can only meet the needs in its area.
• LQ < 1, not Leading, the area is not specialized in that commodity.
The Location Quotient (LQ) calculation is carried out twice, namely at the sectoral level at the district level, meaning to find out the base sector based on the Location Quotient (LQ) value of the economic sectors in the Kepulauan Talaud Regency.Then calculate the Location Quotient (LQ) to determine the location of the Leading commodity base at the Melonguane National Strategic Activity Center (NSAC).
FINDING AND DISCUSSION
Based on the Spatial Planning Document for North Sulawesi Province, it is explained that the leading commodities in Kepulauan Talaud Regency are Nutmeg and Capture Fisheries, namely tuna-skipjack-cob.Furthermore, in the 2014-2034 Regional Spatial Plan (RTRW) for Kepulauan Talaud Regency, nutmeg production centers are spread across 19 (nineteen) sub-districts, while tuna-skipjack-cob production centers are spread over 11 sub-districts.The distribution of the primary commodities of nutmeg and tuna-skipjack-cob is as follows: The production centers for tuna -skipjack -cob has been spread over 11 sub-districts.It is the sub-districts of Lilinabu, East Melonguane, Pulutan, Beo, North Beo, South Beo, Tampan'Amma, Rainis, Nanusa, Kalongan, and Miangas District.
Based on the details regarding the identification of only commodities as described in the RTRW of Kepulauan Talaud Regency, it is necessary to analyze using the Location Quotient (LQ) method.The Location Quotient (LQ) size is one of the determinants of the strategic value of each sector as the main driver of economic growth.The sector has a Location Quotient (LQ) >1 is called the base sector, which is a sector that has a broad economic impact in an area.The sector can serve the domestic market as well as the export market.Economic activities generated by the base sector will provide a high income and Tampaan Amma 14 Damau employment multiplier.The value of LQ> 1 indicates the sector's ability as a driver of economic growth and will encourage the competitiveness of a region's economic sector.
The impact multiplier has been one of the best approaches to determining the welfare change potential of a new economic activity.The basic assumption is that changes in the production sector will increase people's income.The impact of the labor indicator determines the magnitude of the opportunities for new job creation by economic activities developed in an area.Increasing investment by empowering the potential of the base sector would create economic growth and be accompanied by an increase in employment and people's income.The other impact would reduce income inequality between regions and decrease poverty.The creation of investment opportunities can be done by empowering the potential of the leading sector owned by the region concerned.
The results of the calculation, Location Quotient (LQ) of the economic sectors of the Kepulauan Talaud Regency are as follows: Based calculation shows that several economic sectors that get The Location Quotient (LQ) > 1 are called the base sectors.The sectors that play a vital role in the economy of the Kepulauan Talaud Regency are Agriculture, Forestry, Fisheries, Wholesale, Retail, Car, and Motorcycle Repair Sectors.The Kepulauan Talaud Regency's economy is characterized by agriculture, forestry, and fisheries.The sector contributed 41.23%, while the Wholesale, Retail, and Repair sector contributed 14.75% (BPS, 2021a).
In addition, the infrastructure sector consists of the Electricity, Gas, and Clean Water sectors in the base sector category.In other words, the development of the agricultural, forestry, and fisheries sectors will be able to run smoothly because of infrastructure is ready to support the development of the base sector.
The Kepulauan Talaud Regency's economy is characterized by agriculture, forestry, and fisheries.Based on calculation shows that several economic sectors that get The Location Quotient (LQ) > 1 are called the base sectors.The sectors that play a vital role in the economy of the Kepulauan Talaud Regency are Agriculture, Forestry, Fisheries, Wholesale, Retail, Car, and Motorcycle Repair Sectors.The sector contributed 41.23%, while the Wholesale, Retail, and Repair sectors contributed 14.75% (BPS, 2021a).
In addition, the infrastructure sector comprises the Electricity, Gas, and Clean Water sectors in the base sector category.In other words, the development of the agricultural, forestry, and fisheries sectors will run smoothly because the infrastructure is ready to support the development of the base sector.
The results of calculating the Location Quotient (LQ) of the economic sector are in line with the policies of the Government of North Sulawesi Province.The Spatial Planning document for North Sulawesi Province states that the leading commodities are capture fisheries commodities (Article 52 paragraph 2) and nutmeg (Article 51 paragraph 11).The main commodity of capture fisheries in the Kepulauan Talaud Regency is tuna-skipjackcob.Kepulauan Talaud Regency is one of the centers of producers of tuna-skipjack-cob because tuna-skipjack-cob have a Location Quotient (LQ) is 1,78 or more than 1.
Determining the location for the development of the commodity at the National Strategic Activity Center (NSA) of Melonguane is based on the calculation of Location Quantity (LQ) commodity in all sub-districts of the Kepulauan Talaud Regency.The calculation results of Location Quantity (LQ) for Nutmeg, clove and coconut are as in the Table 3.
Based on the Location Quotient (LQ) calculation, a location was found which was the leading producer of nutmeg, clove and coconut.The locations for the development of the nutmeg commodity are spread over 10 (ten) sub-districts in the Melongguane national strategic area (PKSN), namely Melonguane, Kabaruan, Damau, Nanusa, and Miangas subdistricts of the 10 (ten) sub-districts, Damau and Moronge are the sub-districts with the highest productivity of nutmeg compared to other sub-districts in the Kepulauan Talaud Regency.Coconuts are spread over 10 (ten) sub-districts, namely Kabaruan, Lirung, Melongguane, North Beo, Rainis, Tampan Amma, South Essang, Gemeh, Nanusa, and Miangas.At the same time, Clove Production Centers are spread across 8 (eight) districts, namely Kalongan, Tampan Amma, Pulutan Essang, South Essang, Beo, North Beo, and South Beo.Based on the location quotient (LQ) analysis of the National Strategic Activity Center (PKSN) Melongguane is a commodity base area for cloves, coconuts, nutmeg, and tuna-skipjack-cob.The high and increasing value and volume of tuna-skipjack-cob exports provide hope and optimism in developing the tuna-skipjack-cob commodity as a leading commodity at the National Strategic Activity Center (PKSN).
Meanwhile, the development of Indonesian nutmeg exports on the world market from 1991 -2019 fluctuated but showed an increasing trend.The increase in the volume and value of nutmeg exports in the world market illustrates the potential of demand for nutmeg.Indonesia, China, and Vietnam are the largest importers of nutmeg from Indonesia.The large area will encourage an exporter country that ranks first in the export of nutmeg in the world market (Sekretariat Jenderal Kementerian Pertanian, 2020).
Based on data on the average production of nutmeg in Indonesia for 2014-2020, there are 5 (five) central provinces of nutmeg production in Indonesia, namely Aceh, North Maluku, North Sulawesi, West Papua, and Maluku.In 2014, Aceh Province was ranked first in nutmeg production.Then in 2020, this position will be shifted to North Sulawesi.Kepulauan Talaud Regency is in the first rank for nutmeg production, which is 3,958 tons or 34.90% of the nutmeg production in North Sulawesi Province.The Kepulauan Talaud Regency is not only the center of the largest nutmeg producer in North Sulawesi Province but also the producer of nutmeg with the highest productivity.Besides that, it has the most significant number of farmers compared to other districts in North Sulawesi Province (BPS, 2021a).
Likewise, clove is one of Indonesia's export commodities.Even in 2020, Indonesia has been named the largest clove-producing country in the world.In 2020, national clove production will reach 133,604 tons, according to data from the Food and Agriculture Organization (FAO).Not only to meet domestic needs but national clove production is also aimed at meeting export needs and become one of Indonesia's leading commodities.Export destination countries for Indonesian cloves are India, the United Arab Emirates, China, Bangladesh, and Singapore.The potential for coconut resources is enormous.It allows for developing a solid agribusiness with an agro-industrial structure from upstream to downstream.Exports of Indonesian processed coconut products include the Netherlands, USA, China, Malaysia, South Korea, and others (Subekti et al., 2018).
The potential for the high demand for the three leading commodities is a massive opportunity for the development of the leading commodity processing industry, namely cloves, coconut, nutmeg, and tuna-skipjack-cob.The development of these commodities will directly impact the regional economy.The LQ analysis clarifies the location of the production centers of each leading commodity in the Melongguane Sea Border Area.It could be used as a direction for the local government of the Kepulauan Talaud Regency in formulating policies for developing superior commodities from both the upstream and downstream sides.
CONCLUSION
Based on the 2014-2034 Kepulauan Talaud Regional Spatial Plan (RTRW) document, it has been stated that the leading commodities at the Melongguane National Strategic Activity Center (NSA) are commodities from the plantation sector, namely nutmeg, coconut, cloves, and abaka, with all sub-districts in Kepulauan Talaud as a production center.Meanwhile, from the fisheries sector, capture fisheries for tuna-skipjack-cob commodities with production centers spread over 11 (eleven) sub-districts, namely Salibabu, East Melonguane, Pulutan, Beo, North Beo, South Beo, Tampan'amma, Rainis, Nanusa, Kalongan, and Miangas.
The results of identifying only commodities and the locations of their production centers were obtained.Furthermore, by calculating the location quotient (LQ) in the subdistricts in Kepulauan Talaud Regency, potential locations for nutmeg development were obtained in the National Strategic Activity Center (NSA) Melongguane, namely Kabaruan and Damau Districts.At the same time, the potential locations for the development of tunaskipjack -cob are Nanusa and Miangas sub-districts.
Viewed from the demand aspect, tuna-skipjack-cob and nutmeg are Indonesia's mainstay export commodities.The Kepulauan Talaud Regency is one of the production centers for nutmeg and tuna-skipjack-cob, so the designation of Nutmeg, Coconut, clove and tuna-skipjack-cob as leading commodities at the Melongguane National Strategic Activity Center (NSA) shows the integration between policy and economic facts.Melongguane, as the capital of the Kepulauan Talaud Regency and Melongguane NSA with Leading commodities tuna-skipjack-cob, Nutmeg, Coconut and clove, has further implications.Namely the need to develop facilities and infrastructure and increase human resources to support regional development in the Melongguane National Strategic Activity Center (NSA).Economic development at the Commodity-based Melongguane National Strategic Activity Center (NSA) will improve the welfare of the people in Melongguane and catch up with other regions, even the border areas of neighboring countries.
value of a sector of each sub-district in the Melonguane National Strategic Activity Center (NSA) Vts : Commodity value of a sector of all sub-districts in the Melonguane National Strategic Activity Center (NSA) Vik : Commodity value of a sector of each sub-district in the Melonguane National Strategic Activity Center (NSA) Vtk : Commodity value of a sector of all sub-districts in the Melonguane National Strategic Activity Center (NSA)
Table 1 .
Main Commodity Location Districts in Kepulauan Talaud Regency | 2023-05-02T15:02:28.389Z | 2023-04-30T00:00:00.000 | {
"year": 2023,
"sha1": "63fe7045e46e8bc404be29524c1fe0898f3a8183",
"oa_license": "CCBY",
"oa_url": "https://journal.uny.ac.id/index.php/economia/article/download/51388/pdf",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "f35c1426d329f840c996ccad2470906943cd0437",
"s2fieldsofstudy": [
"Economics"
],
"extfieldsofstudy": []
} |
119085733 | pes2o/s2orc | v3-fos-license | Antiferromagnetic ordering in the Kondo lattice system Yb$_2$Fe$_3$Si$_5$
Compounds belonging to the R$_2$Fe$_3$Si$_5$ series exhibit unusual superconducting and magnetic properties. Although a number of studies have been made on the first reentrant antiferromagnet superconductor Tm$_2$Fe$_3$Si$_5$, the physical properties of Yb$_2$Fe$_3$Si$_5$ are largely unexplored. In this work, we attempt to provide a comprehensive study of bulk properties such as, resistivity, susceptibility and heat-capacity of a well characterized polycrystalline Yb$_2$Fe$_3$Si$_5$. Our measurements indicate that Yb$^{3+}$ moments order antiferromagnetically below 1.7 K. Moreover, the system behaves as a Kondo lattice with large Sommerfeld coefficient ($\gamma$) of 0.5~J/Yb mol K$^{2}$ at 0.3 K, which is well below T$_N$. The absence of superconductivity in Yb$_2$Fe$_3$Si$_5$ down to 0.3 K at ambient pressure is attributed to the presence of the Kondo effect.
I. INTRODUCTION
Ternary rare earth transition metal silicides, which form in a variety of crystal structures have been widely investigated due to their remarkable physical properties [1,2]. A few of them also undergo superconducting transition at low temperatures [3,4]. In the early 80's, several investigations have been carried out to understand the superconductivity and magnetism exhibited by compounds belonging to the R 2 Fe 3 Si 5 system [5][6][7][8]. In this family the Fe atoms do not carry any magnetic moment but help in building a large density of states at the Fermi level [9]. It is now well established that a member of this series, namely, Tm 2 Fe 3 Si 5 [10] is the first reentrant antiferromagnetic superconductor (perhaps the only one down to 50 mK). It is worthwhile to point out that we still do not know why the antiferromagnetic order among Tm 3+ ions is a deterrent to the superconductivity in Tm 2 Fe 3 Si 5 given that the neutron scattering studies [10] could not find any ferromagnetic component in the magnetic unit cell in this compound. A recent report has suggested that an antiferromagnet Er 2 Fe 3 Si 5 [11] (below 2.5 K) also shows superconductivity below 1 K whereas an earlier heat-capacity study [8] indicated quadruple magnetic transitions without any superconductivity down to 1.5 K. A previous study [8] on an arc melted Yb 2 Fe 3 Si 5 sample reported the observation of antiferromagnetic ordering of Yb moments around 1.7 K. However, that study did neither establish the Kondo lattice behaviour nor the high value of the Sommerfeld coefficient (γ) as we will see later. In this context, the physical properties of Yb 2 Fe 3 Si 5 are largely unexplored as compared to other compounds of the R 2 Fe 3 Si 5 series. Moreover, compounds with magnetic trivalent or nearly trivalent Yb are interesting since valence instability and Kondo interaction can lead to unusual properties. Thus, we need to understand the absence of superconductivity in Yb compound given that both Tm and Lu of the same series exhibit superconductivity. In this work, we provide a comprehensive study of bulk properties such as, resistivity, susceptibility, and heat capacity of a well characterized polycrystalline sample of Yb 2 Fe 3 Si 5 .
II. EXPERIMENTAL DETAILS
Samples of Yb 2 Fe 3 Si 5 were made by melting the individual constituents (with 2% Yb excess and other elements taken in stoichiometric proportions) in an Alumina crucible which is placed inside a Tantalum container and sealed in an arc furnace under high purity argon atmosphere. The whole assembly is heated in a vertical furnace. The purity of the rareearth element Yb and the transition element Fe were 99.99% whereas the purity of Si was 99.999%. The X-ray powder diffraction pattern of the sample did not show the presence of any parasitic impurity phases. The quality of the sample was checked with EDAX analysis confirming that the sample is essentially single phase with 2-3-5 composition. The nearest rare-earth distances in Yb 2 Fe 3 Si 5 is 3.8Å.
The temperature dependence of the magnetic susceptibility (χ) was measured using the commercial Squid magnetometer (MPMS5, Quantum Design, USA) in a field of 1 kOe in the temperature range from 1.9 to 300 K. The ac susceptibility was measured using a home built susceptometer [13] from 1.5 to 20 K. The resistivity was measured using a four-probe dc technique with contacts made using silver paint on a bar shaped sample of 1 mm thick, 10 mm length and 2 mm width. The temperature was measured using a calibrated Si diode (Lake Shore Inc., USA) sensor. The sample voltage was measured with a nanovoltmeter (model 182, Keithley, USA) with a current of 10 mA using a 20 ppm stable (Hewlett Packard, USA) current source. All the data were collected using an IBM compatible PC/AT via IEEE-488 interface. The heat-capacity in zero field between 0.3 to 30 K was measured using an automated adiabatic heat pulse method. A calibrated germanium resistance thermometer (Lake Shore Inc, USA) was used as the temperature sensor in this range.
A. Magnetic susceptibility studies
The temperature dependence of the inverse dc magnetic susceptibility (1/χ dc ) of a polycrystalline Yb 2 Fe 3 Si 5 in a field of 1 kOe from 1.9 to 300 K is shown in the Fig. 1. The high temperature susceptibility (100 K<T<300 K) is fitted to a modified Curie-Weiss expression which is given by, Here, χ 0 is the temperature independent susceptibility (sum of Pauli, Landau and core susceptibilities), C is the Curie constant and θ p is the Curie-Weiss temperature. The Curie constant C can be written in terms of the effective moment as, where x is the concentration of Yb 3+ ions (x = 2 for Yb 2 Fe 3 Si 5 ). The value of χ(0) is extremely small (7×10 −4 emu/mol) while the values of the effective magnetic moment (µ ef f ) and θ p are found to be 4.5 µ B and −14.4 K, respectively. The estimated effective moment is found to be nearly equal to the free ion moment of the magnetic Yb 3+ ion which rules out the possibility of mixed valent nature of Yb as conjectured in an earlier study [8]. One can also observe that below 100 K, the χ data show deviation from Curie-Weiss plot which could be due to the combined effect of crystal field and Kondo lattice contributions. The inset shows the ac-susceptibility behavior of the sample at low temperature. This inset clearly indicates antiferromagnetic ordering of Yb 3+ moments at 1.7 K. It must be emphasized here that we do not see the influence of any impurity contribution to χ(T) and especially the absence of Yb 2 O 3 (T N =2.3 K) which is generally present in many Yb based compounds.
The presence of a single distinct anomaly in χ at 1.7 K clearly suggests the high quality of the sample.
Earlier study [8] showed that the antiferromagetic ordering temperature of the heavy rareearth R 2 Fe 3 Si 5 compounds do follow the deGennes scaling [14] [∼(g J -1) 2 J(J+1)] indicating that the dominant magnetic interaction is the RKKY interaction. Surprisingly, the same study . But the same study revealed that the antiferromagnetic ordering temperature of Yb 2 Fe 3 Si 5 lies far above the theoretical curve [8]. In our opinion, there is no reason to invoke something else other than the RKKY interaction for explaining the relatively large value of T N . An ordering temperature of 1.7 K is a very common value for Yb-compounds. A slightly enhanced 4f-hybridization, besides inducing some Kondo interaction, leads at first to an increase of the magnetic ordering temperature.
B. Resistivity studies
The temperature dependence of the resistivity (ρ) data of Yb 2 Fe 3 Si 5 is shown in Fig. 2.
The two insets (a) and (b) display the ρ behaviour from 1.4 to 5 K and 1.4 to 50 K, respectively. The large value of the ρ(T) at room temperature and a low value of the residual resistivity ratio down to 5 K could be due to either the structural disorder (inter site change between Fe and Si) or to the strong hybridization effects. It must be recalled here that the properties of the 2-3-5 silicides are highly anisotropic [15] and the over all shape of the temperature dependence of ρ(T) of a polycrystal can easily be influenced by the anisotropy. The important aspect is the the low temperature ρ data on an expanded scale as shown by the inset (b). From this inset one can clearly see the non-monotonic dependence of ρ(T) with a log T dependence from 9 to 25 K. The low temperature ρ data below 30 K display a typical Kondo lattice response with a minimum at 25 K followed by a maximum of ρ(T) at 6 K. Subsequently, ρ(T) falls sharply below 4.5 K due to coherence.
A small kink in the resistivity data at 1.7 K [shown in the panel(a)] indicates a possible magnetic transition at 1.7 K, which is in agreement with the susceptibility data. at the T N clearly shows bulk magnetic ordering of Yb 3+ moments. The antiferromagnetic ordering temperature obtained from the heat capacity measurements is in accordance with those obtained from the χ and ρ measurements. The specific heat data of Lu 2 Fe 3 Si 5 in the normal state is also shown in the same panel.
In the second panel of Fig. 3, the temperature dependence of C/T data are shown. Well below, T N , these data can be nicely fitted with a linear and a cubic term, C = γ T + βT 3 , as shown in the inset. The cubic term in this case corresponds to the contribution of antiferromagnetic magnons, since the phonons are completely negligible in this temperature range. The linear term obtained from this extrapolation, γ = 0.4J/Yb-mol K 2 , can be considered as a lower limit for the Sommerfeld coefficient, since the experimental data show an upward curvature at the lowest limit of the measurement. This would indicate freezing out of the magnons due to an anisotropy gap. In this case, C/T = 0.5 J/Yb-mol K 2 at the lowest measured temperature gives the upper limit for this Sommerfeld coefficient. The magnetic contribution to the heat capacity of Yb 2 Fe 3 Si 5 (which is obtained after subtracting the C p data of Lu 2 Fe 3 Si 5 ) is shown in the third panel (c) of the Fig. 3. The estimated entropy from the experiment is also shown in the same panel. The entropy is nearly temperature independent from 7 to 12 K and reaches a value of 0.9 R ln 2 (R is a gas constant) at 20 K, which establishes that the ground state is a well separated doublet. The heat capacity also shows a small negative curvature above 1.8 K to about 3 K. There are two possibilities for the tail in C p above T N : short range fluctuations or Kondo effect. We would argue that the sharpness of the transition and the weak T-dependence of C/T above T N are in disagreement with short range correlations, whereas the Kondo scenario is supported by the resistivity maximum at 6 K and the large γ value. Moreover, the entropy at T N [S mag = S(T N )]is found to be 0.39 R ln 2, which strongly indicates that the ordered moment is heavily compensated by Kondo interactions. In the absence of short range order, the reduction in S mag at T N is due to the partial lifting of the two fold degeneracy of the ground state above T N by Kondo effect. In such cases, one can show that S mag = S K (T N /T K ), where S K (t) is the entropy of a single ion Kondo impurity at the normalized Kondo temperature, t = T N /T K [16].
The entropy has been calculated by Desgranges and Schotte using the Bethe ansatz for a spin-1/2 Kondo model [17]. Sharp heat capacity anomaly at T N and the possible absence of short range order facilitate the use of the theory [17] and with the experimental value of S mag , we estimate a value of 5 K for the Kondo temperature of Yb 2 Fe 3 Si 5 . However, accurate estimation requires inelastic scattering data. The value of the entropy at 20 K is only 0.7 R which is much smaller than the theoretical value of 2.08 R [R ln(2J+1) with J=7/2 for the free Yb 3+ ion]. This indicates that the CEF levels are well splitted by the tetragonal symmetry.
IV. CONCLUSION
To conclude, we have observed antiferromagnetic ordering and heavy electron behaviour | 2019-04-14T02:02:14.450Z | 2002-07-01T00:00:00.000 | {
"year": 2003,
"sha1": "f1a4126ebd5977fc8e0e8d98ced09c163d3c0a82",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/cond-mat/0304110",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "c713d8af613f54dacddd4d7073948a61b31c832e",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
225697122 | pes2o/s2orc | v3-fos-license | Implementation of STEM-Robotics as High School Intra-curricular
Keyword: STEM Robotic Intra-curricular The study aim to describe STEM-Robotic implementation as an intra-curricular in regular curriculum. STEM-Robotics is usually implemented as an extracurricular program in many schools. In the 2019/2020 school year, Edu Global Senior High School Bandung implemented STEM-Robotics as an intra-curricular for the ten-grade science program. STEM-Robotic implementation as an intra-curricular based on the challenges of 21st-century learning that lead to innovative creative learning and skills development. The method in this study is descriptive qualitative research method through the observations, questionnaires, and interview with teacher teams. The results of this study show that, 1) The implementation of STEM-Robotic as an intra-curricular has a curriculum that focuses about robots NXT and Arduino to provide the basics of engineering and technological skills in robotics and coding, while the concepts of science and mathematics have not been directly linked in STEM-Robotic, 2) The implementation of STEM-Robotic dominated by hands-on and mind-on activities in the learning process and its assessment. 3) Generally, students responded positively to the implementation of STEM-Robotics as an intra-curricular that makes learning more interesting, useful, fun, challenging, and develop engineering and technology skills. This result indicates that STEM-Robotic is an alternative subject or learning strategy in regular curriculum to accommodate science learning with educational robotics. To cite this article: Latip, A.,Hardinata, A. (2020). Implementation of STEM-Robotics as High School Intracurricular. Thabiea: Journal of Natural Science Teaching, 3(1), 11-19.
Introduction
Industrial revolution 4.0 exerted influence on various fields, including the learning process in classroom. In the era of the rapid development of science and technology, the learning process must be more creative and innovative by paying attention to the needs of the era. Learning in the classroom is expected to equip students with a variety of 21st-century skills that were oriented to the development of problem-solving, cognitive, creative, critical, collaborative, and communicative skills. These skills are skills demands that must be mastered by students living in the 21st-century (Greenstein, 2012).
The provision of 21st-century skills aims to prepare students to face the increasingly fierce and tangible globalization competition. Turiman, et al (2012) states that the 21stcentury demands and offers the challenges of life without borders, globalization, and internationalization. Referring to these demands, learning expected to accommodate hands-on and mind-on through various learning activities that are not just transferring knowledge from the teacher to students. More than that, learning is expected to involve technology and digitalization as part of adjusting to the era and collaboration between individuals, groups, and machines (McCoog, 2008). In line with that, Jiea, et al (2018) stated that digitalization in education can lead learning to collaboration between individuals and groups.
The development of various skills, technology engagement, and digitalization in learning has been carried out by many stakeholders in education, including teachers. The teachers were package learning through the selection of media, models, approaches, and learning methods. STEM is one approach that accommodates the development of various skills and involved technology in classroom learning. In STEM implementation, learning is packaged by integrated science, technology, engineering, and mathematics. Lantz (2009) states that STEM is a subject related to the development of students' skills to succeed in global work. Moreover, (Permanasari, 2016) states that STEM is an alternative learning approach that can build a generation to face the challenges of the 21st-century through training students to apply their knowledge to solve environmental problems using technology.
The implementation of STEM in the classroom learning was also integrated with other fields, such as the field of art or STEAM (science, technology, engineering, art, and mathematics). In other implementations, STEM also is integrated with robotics as part of the development of engineering and technological designs. Withehead (2010) states that robotics is a technology that can be used by teachers to develop STEM curricula that more specific to technology and engineering. In line with this, integration of robotic in STEM subject can create an environment learning that encourages hands-on activity, creativity and problem solving (Jiea, et al, 2018), creates more meaningful learning and equips technology application experience to students (Carik and Guven, 2019), and improving basic STEM skills such as mathematical skills, scientific methods, and skills in engineering design (Eguchi, 2013;Karaahmetoglu and Korkmaz, 2019).
Integration of robotic in STEM can also increase students' interest in STEM, both in STEM learning and in future career selection related to STEM (Ebelt, 2012;Khanlari, 2013;Mosley, Ardito, and Scollins, 2016;Chen and Chang, 2018). However, although these robotics are popular in the field of education, the facts in the field show that robotics is still used as an extracurricular program (Gura, 2011;Eguchi, 2013). In the 2019/2020 school year, Edu Global Senior High School made STEM-Robotics as an intra-curricular for ten-grade students of Sciences program. The implementation of robotics in STEM learning aims to develop students' various skills and answer the challenges of the rapid development of science and technology. Based on the description, this article will be discussed and described regarding the results of study on the implementation of STEM Robotics as an intra-curricular in Edu Global Senior High School Bandung.This study focused on the discussion of the development of STEM-Robotic curriculum, the implementation of STEM-Robotic learning as an intra-curricular/regular curriculum, and the students' responses of participating in STEM-Robotic learning.
Method
The research method of this study is a descriptive qualitative method. Qualitative research methods lead to a variety of interpretation techniques and assessment of participant perspectives with multiple strategies, namely interactive strategies such as direct observation, questionnaires, and reviewing documents (Merriam, 2009). In this research, researchers conducted observations on STEM-Robotic learning in ten-grade students of Edu Global Senior High Bandung during 10 meetings of learning. The researchers observed the implementation of curriculum Lego NXT in STEM-Robotic learning for the first up to fourth meetings of learning. In the fifth up to end meetings of learning, the researchers observed the implementation of curriculum Arduino device to support STEM-Robotic learning. Besides, the researchers also reviewed the documents of the STEM-Robotic curriculum. This document developed by the STEM-Robotic teacher team. The researcher also gave the questionnaires to students in STEM-Robotic learning.
The data obtained from each stage of the research process were analyzed, processed, and described as research findings and results. The direct observation in classroom learning using the instrument of observation and obtained the descriptive data of implementation STEM with robotic as a tool to support the learning process. The reviewed document curriculum STEM-Robotic obtain the descriptive data of curriculum Lego NXT and Arduino device in STEM learning. The student responses about STEM-Robotics collected by questionnaire with a Likert scale. The result of the questionnaires displayed in percentage of students' responses about implementation of STEM-Robotics.
Results and Discussion
The results and discussion in this article based on an analysis of research findings of the implementation of STEM-Robotic as an intra-curricular in Edu Global Senior High School Bandung. Results and discussion focused on 3 main studies, namely 1). Development of STEM-Robotic curriculum, 2). Implementation of STEM-Robotic as an intra-curricular, and 3). The students' responses to STEM-Robotic learning. The results and discussion of the three main studies are as follows:
Development of STEM-Robotic Curriculum as intra-curricular
The development of STEM-Robotic curriculum conducted by STEM-Robotic teacher team, the development taking into the minimum achievement of skills that must be mastered by students, the minimum achievement of skills must strengthen STEM concepts, namely science, technology, engineering, and mathematics. Besides, the development of the STEM-Robotic curriculum must also pay attention to supporting learning facilities in schools, such as the availability of tools and robotics equipment.
Development of STEM-Robotic curriculum does not refer to the national curriculum, because the national curriculum has not STEM-Robotic as a subject in the regular curriculum. The development of STEM-Robotic curriculum based on various skills that will support when learning STEM concepts. Jiea, et al, (2018) stated development of Robotic curriculum in STEM dose not to replace the existence of science and mathematics curriculum, but it is an alternative way and media that can be used by teachers to prepare students to be literate of STEM and get to know the world of work in the STEM field.The description of STEM-Robotic curriculum about main subject and sub-topics is as follows: Arduino basic theory (understanding, types, basic syntax), Microcontroller, Led and Button, and Sevensegment Table 1 shows the main and sub-topics in the STEM-Robotic curriculum for one semester. The main STEM-Robotic curriculum for the first semester focused on material about Lego NXT and Arduino. Lego NXT is an educational robotics device that can be programmed as desired. Programming can be run in lego NXT include NXT-G and Next Byte Codes. The selection of lego NXT and Arduino based on many results of research, it showed that lego NXT and Arduino are educational robotic devices can develop many skills, such as science process skills, problem-solving skills, and cooperative skills (Eguchi, 2013). Besides, lego NXT and Arduino can also provide more opportunities for effective learning when learning physics, biology, mathematics, geography, science, electronics, and mechanical engineerin (Eguchi, 2013). Irigoyen, et al (2013) stated Lego NXT and Arduino have various elements that connected with various devices and linked with variables studied in science, such as pressure, light, sound, and temperature. More specifically, Carik and Guven (2019) state that Arduino and coding can help students to visualize abstract scientific concepts and make learning science more enjoyable. According to that, lego NXT and Arduino will help students to develop and strengthening knowledge and skills in the STEM field.The selection of lego NXT and Arduino also based on the ease of getting this platform and having an affordable price for the School. Irigoyen, et al (2013) states that Lego NXT and Arduino are educational robotic platforms that have low prices. Moreover, Lego NXT and Arduino are also basic of robotics platforms that can be developed by educated participants on other educational robotic platforms.
Developing of STEM-Robotic curriculum can also review from the expected achievements by students after studied the STEM-Robotic. The basic competence that must be mastered by students after studied STEM-Robotic during the odd semester on lego NXT material were: 1) assemble the basic form NXT robotic, 2) programming the NXT to move the wheels, 3) moving the robot using sound sensors, ultrasonic, touch and light, and 4) assemble the Lego NXT in the form of a steering wheel and run it along the line until the finish. The basic competence of students' skill for Arduino material are: 1) understanding the basic theory of Arduino (microcontroller, type, and syntax), 2) understanding the concepts of LEDs and resistors (making circuits and counting resistors), 3) practicing how to turn on LEDs, 4) understanding the LED concept and button (understanding polarity and making a circuit), 5) practicing how to light an LED through a button, 6) understanding the concept of seven-segment (understanding polarity, and circuit), and 7) practicing how to turn on sevensegment until it forms zero to nine number (0-9).
Based on the elaboration of basic competence in lego NXT and Arduino, it can summarize that the STEM-Robotic curriculum for the first semester has more focus on handson and mind-on activities during the learning process. Besides, the STEM-Robotic curriculum in the first semester also dominated by concepts that lead to strengthening engineering and technology skills, while the concepts of science and mathematics still get a smaller portion.
Implementation of STEM-Robotic Learning as Intra-curricular
The implementation of STEM-Robotics learning as an intra-curricular is a description of the implementation of STEM-Robotics learning during 10 learning meetings. Description of STEM-Robotic implementation explains the learning process and assessment in STEM-Robotic learning. Explanation of the implementation of STEM-Robotics based on the result of the analysis of observations in class. STEM-Robotic or educational robotic learning is one of the tools in an extracurricular activities program. Although educational robotics is very popular in the education field, robotics does not yet include in the regular curriculum schedule (Gura, 2011;Eguchi, 2013). In the 2019/2020 school year, Edu Global Senior High School Bandung made the integration of STEM-Robotic as an intra-curricular for the ten-grade science program.
Implementation of STEM-Robotic subjects gots 2 hours of learning (2 x 45 minutes) in one week. In the odd semester of the 2019/2020 school year, STEM-Robotic learning consists of twelve for learning meetings, one meeting for midterm assessment, one meeting for final semester assessment, and four meetings for the daily skills exam. Every STEM-Robotic lesson, students make grouped with members of each group consisting of 4-5 people. Each group was given one lego NXT and Arduino. Every group also required to bring a laptop to make a simple program of coding that supports lego NXT and Arduino. The program of coding used to run the lego NXT and Arduino robots that studied at the lesson.
The teacher team of STEM-Robotics has the role of main and accompanying teacher. The main teacher delivered the main subject material to all students. The accompanying teacher assisted each group during the learning activities, especially in completing project STEM-Robotic learning. The implementation of STEM-Robotic learning does not just a transfer of information from the teacher to students. More than that, the hands-on activities of students are very dominating during the learning process. The result same with the opinions and research findings (McKay, 2015;Hinton, 2017;Jiea, et al. 2018;Blackley and Howell, 2019) states that integration of STEM learning with robotics can increase the hands-on activities of students.
The hands-on activity in STEM-Robotic learning can show from the activities of students in designing a basic form of lego NXT robotics, steering wheels, and designing Arduino devices with all components. STEM-Robotic learning accompanied by minds-on activities in the making programs and coding to run lego NXT and Arduino robots. According to these activities, students in his group must take a strategy to complete each learning material and project during STEM-Robotic learning. The setting of STEM-Robotic learning leads to the development of various skills, including collaborative skill, problem-solving, critical, and creative skills. This statement same with several studies which state that integrated STEM with robotics can improve students' collaborative skills (Eguchi, 2013;Mosley, 2016;Hinton, 2017;Jiea, et al, 2018;Cakir and Guven, 2019;Anwar, et al, 2019;Chen and Chang, 2019), improve problem-solving skills (McKay, 2015;Menekse, 2017;Jiea, et al, 2018;Chen and Chang, 2019) and improve critical and creative thinking skills (Menekse, et al, 2017;Chen and Chang, 2019).
Besides the learning process, the assessment of STEM-Robotics also focuses on many skills. The assessment consists of daily skills tests, midterm tests, and final semester tests. All assessments lead to skills tests that involve hands-on and mind-on activities in designing and programming a STEM-Robotic project. The results of the assessment presented in a special report card STEM-Robotic which contains the scores obtained for each project, the average score of project, predicate, and description of the skills that have been achieved by students.
Based on the analysis of STEM-Robotic students' report cards during one semester, the final score of the student in STEM-Robotic subjects as follows: Table 2 shows that most students have been able to complete each STEM-Robotics project well, starting from the assembly process, design to making the program/coding to run the lego NXT and Arduino robots. Even so, there are still students who have difficulties in the main subject of STEM-Robotic, such as assembly Arduino devices and making programs/coding to run the lego NXT or Arduino robots.
Students' Responses to STEM-Robotic Learning
In this study, data of students' responses collected through questionnaires at the end of STEM-Robotic learning in the odd semester of the 2019/2020 school year. The questionnaire consists of two main contents, namely the existence of STEM-Robotics as a subject in regular curriculum and the relationship of STEM-Robotic learning with fields in STEM. The questionnaire prepared using a Likert scale with a choice of responses, namely strongly agree (SA), agree (A), doubtful/neutral (N), disagree (D), and strongly disagree (SD). The results of this questionnaire were processed and presented as a percentage. Table 3 showed students' responses to the existence of STEM-Robotic subjects as a regular curriculum. Generally, students gave positive responses about the existence of STEM-Robotics as an intra-curricular. The students' responses showed that STEM-Robotics is a useful, interesting, challenging, and fun. The result was the same with several research findings that state educational robotics can increase students' interest in STEM learning, make learning more interesting and enjoyable (Ebelt, 2012;Mosley, et al, 2016;Chen and Chang, 2018;Carik and Guven, 2019). The questionnaire also collected students' responses about the relationship of STEM-Robotics with science, technology, machinery, and mathematics. The results of the questionnaire are as follows: Table 4 showed that students gave positive responses about relationship STEM-Robotic with engineering skills and technology literacy. Engineering and technology are the main fields that dominate STEM-Robotic learning, especially in the process of designing, assembling, and making a coding. Other responses, students gave a neutral response to the relation of the STEM-Robotics with science and mathematics. The lesson of STEM-Robotics has not received more tangible benefits to be applied in the fields of science and mathematics. The majority of neutral responses based on the packaging of STEM Robotic content that has not been directly linked with science and mathematics. Besides that, the portion of content science and mathematics in STEM-Robotic learning has not been given as much as the portion of engineering and technology content.
Conclusion
STEM as a part of the learning approach has been conducted by many teachers in the learning process. In its development, STEM has also been integrated with many other fields. In this research, STEM was integrated with robotics as an effort to develop 21st-century skills, especially technology and engineering skills. In its implementation, STEM-Robotic is usually an extracurricular activity that is not included in the regular curriculum. In the 2019/2020 school year, Edu Global Senior High School Bandung made STEM-Robotic as a subject (intra-curricular) in regular curriculum. The results of study about STEM-Robotic Implementation as an intra-curricular show that: 1) STEM-Robotic as an intra-curricular has a curriculum that focuses on material about lego NXT and Arduino robots. The curriculum aimed to provide students with the basics of engineering and technology skills in the field of robotics and coding. The STEM-Robotic curriculum has not been directly linked with science and mathematics concepts. 2) The implementation of STEM-Robotic as an intra-curricular dominated by hands-on and minds-on activities in the learning process and its assessment. 3) Generally, students gave a positive response to the implementation of STEM-Robotic as an intra-curricular. STEM-Robotic makes learning more interesting, useful, fun, and challenging. Besides, the students also responded that STEM-Robotic provides opportunities to improve engineering and technology skills. | 2020-07-09T09:15:19.727Z | 2020-06-24T00:00:00.000 | {
"year": 2020,
"sha1": "503f9e0cc453cfff7d420365ad299ce5a213017b",
"oa_license": "CCBYNC",
"oa_url": "https://journal.iainkudus.ac.id/index.php/Thabiea/article/download/6770/4418",
"oa_status": "HYBRID",
"pdf_src": "Anansi",
"pdf_hash": "a2b8e3c6b35bcc0c0a034afcad3d618938bedd3f",
"s2fieldsofstudy": [
"Engineering",
"Education"
],
"extfieldsofstudy": []
} |
21348693 | pes2o/s2orc | v3-fos-license | xenoGI: reconstructing the history of genomic island insertions in clades of closely related bacteria
Background Genomic islands play an important role in microbial genome evolution, providing a mechanism for strains to adapt to new ecological conditions. A variety of computational methods, both genome-composition based and comparative, have been developed to identify them. Some of these methods are explicitly designed to work in single strains, while others make use of multiple strains. In general, existing methods do not identify islands in the context of the phylogeny in which they evolved. Even multiple strain approaches are best suited to identifying genomic islands that are present in one strain but absent in others. They do not automatically recognize islands which are shared between some strains in the clade or determine the branch on which these islands inserted within the phylogenetic tree. Results We have developed a software package, xenoGI, that identifies genomic islands and maps their origin within a clade of closely related bacteria, determining which branch they inserted on. It takes as input a set of sequenced genomes and a tree specifying their phylogenetic relationships. Making heavy use of synteny information, the package builds gene families in a species-tree-aware way, and then attempts to combine into islands those families whose members are adjacent and whose most recent common ancestor is shared. The package provides a variety of text-based analysis functions, as well as the ability to export genomic islands into formats suitable for viewing in a genome browser. We demonstrate the capabilities of the package with several examples from enteric bacteria, including an examination of the evolution of the acid fitness island in the genus Escherichia. In addition we use output from simulations and a set of known genomic islands from the literature to show that xenoGI can accurately identify genomic islands and place them on a phylogenetic tree. Conclusions xenoGI is an effective tool for studying the history of genomic island insertions in a clade of microbes. It identifies genomic islands, and determines which branch they inserted on within the phylogenetic tree for the clade. Such information is valuable because it helps us understand the adaptive path that has produced living species. Electronic supplementary material The online version of this article (10.1186/s12859-018-2038-0) contains supplementary material, which is available to authorized users.
GI-finding methods can be broadly divided into those that operate on a genome from a single species, and comparative genomics methods that operate on genomes from several species [5,6].
Many single genome methods are compositional, making use of various attributes of sequence composition such as GC content, oligonucleotide frequency or codon bias. Because genomes differ in these compositional characteristics, when a foreign piece of DNA arrives into a genome, it may differ in some of these characteristics from the genome it is entering. For insertion events that are sufficiently recent, this can be a mechanism to identify foreign DNA. Such methods have been developed to try to take advantage of many compositional features, including GC content [7], oligonucleotide frequencies [8][9][10][11][12][13][14][15][16], and codon bias [17,18]. Single genome methods also sometimes target specific sequence features that are associated with GI insertion such as tRNA genes [19]. And a number of such methods use combinations of multiple attributes including composition and/or specific sequence features [20][21][22][23][24][25][26][27][28].
The basic idea of comparative genomics methods is to compare related genomes to identify regions that are unique to certain genomes and likely result from horizontal transfer. These methods are closely related to methods for identifying the core and pan genomes of a set of species or reconstructing ancestral gene order [29][30][31][32][33][34]. Comparative methods typically involve whole genome or protein alignments and then some methods built on top of this to identify orthologs and recognize events such as horizontal transfer, deletion, and so on.
Several automated comparative genomics methods for finding GIs have been developed to date. The tRNAcc package combines comparative genomics with a feature specific search [35]. It identifies islands that have inserted near tRNA genes by creating alignments between closely related species using MAUVE [36], and then looking for regions of DNA that are unique to one species near tRNA genes. This approach is good at finding those GIs that insert near tRNA genes, but will miss others. It is included in the web-based MobilomeFINDER service [23].
Another widely used method is IslandPick [37] which has been incorporated into the web service IslandViewer [38][39][40]. IslandPick is provided with a single input genome where the user desires to find genomic islands. It first identifies a set of comparison genomes, then creates pairwise whole genome alignments using MAUVE, and finally analyzes the alignments to identify regions that are unique in the input genome. This comparative approach allows accurate identification of GIs that are unique to the input genome, and is widely used as a part of the IslandViewer website.
Given continuing reductions in the cost of sequencing, in the future comparative genomics methods are likely to be increasingly important for finding GIs. However existing methods have one important limitation: they are not able to automatically place GIs in the context of a phylogenetic tree. Existing methods such as tRNAcc and Island-Pick make use of multiple genomes, but they are best at identifying regions which are unique to one genome compared with others. If we want to study the history of genomic island insertions in a clade of microbes, these methods allow us to find islands that are unique to various strains of the clade. But they do not automatically identify genomic islands which are shared between some strains in the clade, and they do not determine the branch on which those islands inserted within the phylogenetic tree.
Here we describe xenoGI, a system that identifies genomic islands, and maps their origin within a clade of closely related bacteria. Every gene present in a clade has one of two possible origins. Either it originated in the most recent common ancestor of the clade, or it originated in a subsequent horizontal transfer event. The goal of xenoGI is to group genes by origin, identifying islands of genes that entered via horizontal transfer events, and mapping those events onto the phylogenetic tree. Such information is often of interest because it helps us understand the adaptive path that has produced living species. Implementation xenoGI is a command line program implemented in Python. It can be downloaded from http://www.cs.hmc. edu/xgiWeb/ or via GitHub (https://github.com/ecbush/ xenoGI).
Input, output, basic structure
Input consists of a set of sequenced genomes in GenBank format, and a tree specifying their phylogenetic relationships. The GenBank files provide protein sequences and their genomic order in each strain. Because the algorithm makes use of synteny information, the genomes need to come from a clade of bacteria that are closely related enough to preserve gene order. For the same reason, the genome assemblies should be at the scaffold level or better. The algorithm is not suitable for analyzing plasmid sequences because of the rapid rate of change of gene content and order on plasmids. Typically, the set of input genomes would include a focal clade that we wish to study, and one or two outgroups (Fig. 1). These outgroups help us to better recognize core genes given the possibility of deletion in some lineages. For cases where the phylogenetic tree is unknown, xenoGI includes optional tools to help users determine it. A provided script allows users to obtain multiple alignments from the set of conserved core genes (discussed below). These alignments can then be used with existing methods to reconstruct a phylogenetic tree. Every gene in the input genomes must have one of two origins. Either it is a core gene present in the most recent common ancestor of the strains, or it arrived via a horizontal transfer event. The goal of the algorithm is to determine this origin for each gene, grouping genes that arrived together in the same horizontal transfer events as islands. The output is a text file specifying these islands. The output can be visualized further with several textbased visualization functions included in the package, and can also be exported for visualization in a genome browser.
There are three basic steps the algorithm takes. It first calculates a set of scores between genes in the input genomes based on their protein sequences. This includes scores based on sequence similarity and scores based on synteny. It next groups genes into families in a tree-aware way. Finally, it groups these families into islands where the families in an island are interpreted to have a common origin (they either arrived in the same horizontal transfer event or are core genes).
An important aspect of our approach is the use of species tree and synteny information directly in the process of making gene families. The logic is that because we ultimately want to know about the history of GI insertions within a specific species tree, it helps to have gene families that reflect homology due to common decent within that tree, ignoring deeper homology. Every gene family thus formed has a most recent common ancestor that falls on some node in the input tree.
Calculating scores between genes
The first step is to calculate a set of similarity and synteny scores between genes in the input data set. We initially run protein BLAST between every gene and genes in all the other strains. For those pairs of genes above a certain E-value threshold, we calculate a number of other types of scores.
A raw score is a similarity score between a pair of proteins. We take the global alignment score and scale it to be between 0 and 1, using the following formula: where g is the global alignment score between two proteins. a is a floor value for the alignment score between these two proteins based on what we would get if they were aligned with all gaps (among the pairs we look at, which have significant BLAST hits, there will be nothing lower than this). b is a ceiling value for the alignment score (the score of the shorter sequence aligned against itself ). The global alignment is calculated using Parasail [41]. The use of global alignment here reflects the fact that we are operating in a clade of closely related strains and the gene families we build consist of closely related genes. Because of this, we expect alignments between homologs within families to span entire proteins making global alignment preferable to local.
The calculation of raw scores can be run in parallel on multiple processors.
We also calculate a normalized similarity score, which normalizes for the average level of protein distance between a pair of species. Such scores make it easier to set thresholds based on similarity in family formation.
To begin, we identify sets of orthologs where there is exactly one copy in each strain. We do this with the all around best reciprocal hit method, identifying sets of orthologs where every gene is a best reciprocal hit with every other gene, and has one copy in each strain. These sets of orthologs are very conservative and high confidence. We'll refer to them below as conservative core genes.
Then for each pair of strains we calculate the mean and standard deviation of raw scores between all pairs of orthologs in these sets of conservative core genes. Using this, we take a raw score comparing proteins in two strains and normalize it as follows: where m is the mean and s is the standard deviation of raw scores from the conservative core genes for that pair of strains. We calculate two kinds of synteny scores based on similarity in the neighborhood of genes.
Our core synteny score makes use of the conservative core genes just discussed. To calculate this score, we define a neighborhood for each gene consisting of a user specified window of conservative core genes in either direction. For example, the neighborhood might be a region encompassing 20 conservative core genes, the first 10 in either direction. To compare a gene A from one strain with a gene B from another, we determine how many conservative core genes from the neighborhood of A are also found in the neighborhood of B, and divide this by the number of conservative core genes in the neighborhood (twenty in the example above). This core-gene synteny score is then: core synteny score = number of shared genes number of core genes in neighborhood and is thus a value between 0 and 1.
Because the conservative core gene neighborhoods tend to be large, this measure of synteny looks at a comparatively large region around a gene.
To calculate synteny in a more local region, we also calculate a synteny score based on all genes, including non-core genes.
To compare a gene A from one strain with a gene B from another, we obtain lists of neighboring genes for each. Let N a be the set of neighboring genes for gene A, taken from within a window of size W measured in number of genes, and including both core and non-core genes. Let N b be the same for gene B. Our local synteny score is calculated as follows. We find the pair of genes with the highest norm score between N a and N b and keep it. We remove those genes from N a and N b , find the next highest pair between them and so on. The local synteny score for gene A and gene B is the average of the T highest scoring pairs. local synteny score = T highest norm scores from N a , N b T The calculation of synteny scores can be run in parallel on multiple processors.
Forming gene families in a tree-aware way
We wish to build gene families that fit into the known species tree.
For our purposes, a gene family is a set of genes that have descended from a single ancestor gene that existed within that species tree. The most recent common ancestor (MRCA) in such a gene family will fall on the species tree, and its location there will reflect the origin of the family. Gene families whose MRCA falls outside the focal clade are core genes relative to that clade. Gene families whose MRCA falls within the focal clade have arrived via horizontal transfer. This implies that genes that share a deeper homology predating the root of the species tree, will be placed in different families. In cases where the same gene has entered our clade multiple times as a part of distinct transfer events, we want each insertion to correspond to a different gene family.
Our approach is to use a version of the PHiGs algorithm [42] which we have modified to consider synteny information. The PhIGs algorithm operates on the species tree beginning at the root node and moving successively to descendant nodes. At each node, we group all genes descending from species on the left branch (call this group 1), and also group all genes descending from species on the right branch (call this group 2). For nodes other than the root, there are also genes in outgroup species, which we ignore. For every gene in one group, we find the most similar gene in the other, e.g. for all genes in group 1 we find the closest gene in group 2. A pair identified in this way constitutes a seed upon which we build a larger family via single linkage clustering. Because order matters for this approach, the seeds are processed in order of similarity, so that the most similar seeds are worked on first. To build a family from a seed, we identify all previously unclustered genes in either group 1 or group 2 that are closer to a member of the growing family then the original seeds in 1 and 2 were to each other. In our implementation, similarity is measured by the raw score.
We have modified the original algorithm in several ways. In order to add a gene to a family, the basic algorithm requires that it be more similar to some family member than the similarity level of the seed. We have added an absolute threshold for similarity measured using the norm score. This is typically set low, and is a sanity check to make sure we're not clustering very distantly related proteins together into a family. We also incorporate synteny in a number of ways. We have synteny thresholds for both core and local synteny. Below these thresholds, we do not add a gene to a family. We also use high synteny on the other end, to increase the raw score between a pair of genes we're considering, potentially helping them get over the bar of seed similarity set by the basic algorithm.
This step runs on a single processor.
Grouping families into islands
Our goal is to group gene families that arrived together as a part of the same horizontal transfer event. We refer to such groups as islands.
We first sort families according to their MRCA because families that belong in the same island will have the same MRCA. We then build islands using a greedy approach that progressively adds families that are inferred to be adjacent to each other in the MRCA.
We identify genes to add to an island using a parsimonybased metric. Our metric uses a very simple notion of evolutionary changes in gene order. If a pair of genes were adjacent, but due to rearrangements move apart, we assess a cost of one. Similarly if two genes were non-adjacent, but due to rearrangements move to be adjacent, we also assess a cost of one. Consider two gene families with the same MRCA, for example i0 in Fig. 1. We have adjacency information for those families in species A and species B. Our approach is to calculate a rearrangement cost for these families under two cases: either assuming they were adjacent at their MRCA, or assuming they were not. We define the rearrangement score for the families as follows: rearrangement score = cost starting non-adjacent − cost starting adjacent This score can range between -2 and 2 with more positive values indicating families that are more likely to have been adjacent in the common ancestor.
We begin by creating a set of one-family islands for all families with a particular MRCA. We then calculate the rearrangement score for every pair of islands and identify the pair with the highest score, arbitrarily breaking ties. Then we merge this pair into a two-family island and recalculate its rearrangement score with other islands. Note that because multi-family islands have a gene order (the inferred order in the MRCA), to calculate rearrangement scores for them, we consider adjacency on each of their two ends. The algorithm continues merging islands until all the rearrangement scores are below a certain threshold. It then repeats the procedure relaxing the criterion for adjacency (e.g. we can count as adjacent genes that are one gene away from each other). When all the rearrangement scores are below threshold, the algorithm terminates.
The island-making step runs on multiple processors.
Analysis tools
Included in the package are command line tools for identifying and visualizing islands at particular nodes, for finding islands associated with particular genes and for examining gene families. Also included are scripts for exporting xenoGI island output to bed or gff format for visualization in a genome browser such as IGB [43].
Validation via simulation
We assessed the effectiveness of xenoGI in two ways: using simulations, and detecting known genomic islands. We used simulations to produce test data sets where the location of GI insertions within a phylogenetic tree was known. To do this we implemented a custom simulator that evolved sequences over a user provided phylogenetic tree, allowing for horizontal transfer of novel genes (from outside the clade) as well as for genomic scale deletions, duplications and inversions, and amino acid level sequence change. The latter was done using the pyvolve module [44]. Figure 2 shows the results of a simulation on a tree with 11 species. The simulation was run on a tree matching the branch lengths and topology of a tree from the enteric bacteria, discussed below. Species A-I form the focal clade, with J and K as outgroups. The simulation contained a total of 495 horizontal transfer events which mapped onto the various branches. These ranged in size from 2 to 51 genes. There were also 1009 deletions (from 1 to 50 genes in size), 494 duplications (from 1 to 48 genes in size), and 125 inversions (from 5 to 147 genes in size).
xenoGI achieved a base-wise true positive rate of 0.85 and a positive predictive value of 0.51 over the whole tree. What we mean by base-wise, taking the example of the true positive rate, is that among all nucleotides that were truly in genomic islands, xenoGI correctly identified (and placed on the correct branch) 0.85 of them. The majority of islands (0.83) had just a single xenoGI predicted island overlapping them. As can be seen from the figure, xenoGI's accuracy is best for GIs inserting on tips, and declines as we move to internal branches.
Validation via GIs from the literature
Simulations have the advantage of providing a situation where ground truth is known. However they are necessarily simplified, and may not adequately capture the features Fig. 2 Phylogenetic tree used in genome simulations. We ran xenoGI on simulated genomes that were generated on the tree shown. On each branch we show the true positive rate (red) and the positive predictive value (blue) for xenoGI on that branch of real genome evolution. For this reason, we also examined how well xenoGI identified genomic islands that have been reported in the literature.
Wei et al. compiled a list of known genomic islands from 11 bacterial strains for the purposes of validation [28]. We used six of these strains for testing as we built the system, and for developing a set of default parameters. We held the five remaining strains as a validation data set, the results of which we report here. For each of the five strains, we identified five or six closely related species with genomes in GenBank. We reconstructed the history of genomic island insertions in the resulting clade using xenoGI with default parameters. A listing of the strains and trees used here can be found in Additional file 1. We note that there were not many cases where these known islands were shared among multiple already sequenced genomes. Thus most of the cases we look at here, the islands are on the tips of the phylogenies we created. In the section below we give several examples of cases where islands can be identified on internal branches of a tree.
The islands in this validation set were reported in genome papers for their respective strains [45][46][47][48][49]. They are chiefly based on comparative work involving human curation, and in some cases on experimental evidence as well. They are likely to represent true islands. However we cannot be sure that these represent exhaustive lists of all genomic islands in a strain. For this reason, it does not make sense to calculate a true positive rate or positive predictive value here. The validation ranges are given in nucleotide positions, whereas xenoGI identifies genes that are part of an island. For the purposes of comparison, we consider the nucleotides of a xenoGI island to be those between the beginning of the first gene and the end of the last gene. Table 1 shows our results for this analysis. As can be seen from the table, the base coverage, that is the proportion of validation island bases that are covered by a xenoGI island, is in the upper 90 percent range for 4/5 strains, and 88% for Cronobacter. In addition, in the majority of cases, xenoGI identified a single island corresponding to each validation island. And in most cases (6/11) where a validation island was broken into more than one xenoGI island, this was actually correct, due to the fact that the validation island had genes with a most recent common ancestor at different times on the tree, and likely resulted from multiple horizontal transfer events.
Additional file 1 contains a more detailed description of our comparison between xenoGI islands and validation islands. One of the measures included is extra coverage, which refers to cases where a xenoGI island extends beyond the range of the validation island. There were 21 cases where the extra coverage was more than 10% of the length of the island. In the majority of these (16/21) further examination suggested that in fact xenoGI was correct to extend the range of the island. Additional file 2 contains comparison results with several widely used tools from the IslandViewer web site [18,19,22,37,40]. These tools have been run on the same five species used in Table 1, and using the same two metrics. The file shows that xenoGI's performance compares favorably with these tools. For both base-wise coverage and the proportion validation ranges covered by a single predicted island, xenoGI has values greater than (or in one case equal to) those for the other methods.
Reconstructing the timing of GI insertions: two examples from enteric bacteria
We look at two examples of genomic islands from the enteric bacteria with the goal of demonstrating the sort of analysis one can do with xenoGI. We do this on a tree of eleven enteric species (Fig. 3a).
The first example is Salmonella pathogenicity island 2 (SPI-2), from Salmonella enterica [50,51]. This island is essential for virulence in S. enterica and contains a type-III secretion system that is expressed while the bacterial cell is inside host cells [52]. The island was originally defined as the region between but not including the genes ydhE and pykF [53]. It is known to consist of several components with different evolutionary origins [54].
xenoGI's results are consistent with what we would expect from the literature. The largest part of SPI-2 is colored dark green in Fig. 3b. It includes the type III secretion system and is shared between Salmonella enterica strains (LT2 and Arizoniae in our sample), but is not Each row corresponds to a strain. Num. islands represents the number of validation islands and total bases represents the total number of nucleotides in those islands. Base coverage is the proportion of all bases in the validation islands that xenoGI correctly recognized as an island. In single island indicates the proportion of validation islands that xenoGI captured as a single island a b c d Fig. 3 Examples from enteric bacteria. a Phylogenetic tree of 11 enteric species. Symbols indicate the branches of insertion of GIs in b-d. The images in b-d were made by outputting xenoGI islands and then displaying in the IGB genome browser. Note that the scale for the three is not exactly the same. In the figures, different islands are given different colors. All islands with an mrca at or before the point where C. rodentium diverges are colored black. b Salmonella pathogenicity island 2 shown in three Salmonella species. c The acid fitness island as reconstructed by xenoGI in two E. coli species and E. albertii. d The island around gadB in our four Escherichia species present in Salmonella bongori. xenoGI puts this region in one island, reflecting the common origin of the genes. All the genes in the region are included, with the exception of one gene specific to S. enterica LT2 which is shown in red. xenoGI identifies another island (shown in pink) corresponding to a second part of SPI-2. This region contains the tetrathionate reductase gene cluster. Consistent with the literature, xenoGI identifies this region as being shared between S. enterica and S. bongori [54]. The final part of SPI-2, shown in brown in Fig. 3b, contains genes that are present in S. enterica LT2, but not S. enterica arizoniae.
Our second example is the acid fitness island (AFI) in E. coli [55]. This island runs from slp to gadA inclusive in E. coli K-12 (Fig. 3c) and is about 15 kb long [56].
It encodes a number of genes involved in resistance to acid stress including a glutamate decarboxylase enzyme (coded for by gadA) and its regulators. xenoGI identified the island from slp and gadA, and placed it on the branch after the divergence of E. fergusonii, but before the divergence of E. albertii. One internal gene which is part of the island, yhiD, was left out because it is not present in E. albertii.
Further exploration reveals some additional features of the evolution of acid fitness genes in Escherichia. The acid fitness island was originally described in E. coli K-12 [55], and contains 12 genes in that strain. However the island in both E. coli O157:H7 and E. albertii contains an additional 8 genes. As it turns out, these additional genes, which are involved in the scavenging of iron from hosts, have been identified and studied previously. They were identified first in Shigella dysenteriae, but were found to also be present in a number of E. coli strains [57]. (It's worth noting that Shigella strains nest within the E. coli clade.) This so-called heme transport locus falls in the middle of the AFI, but because the AFI has mostly been studied in E. coli K-12, and that strain lacks the heme transport locus, it was never recognized that the two are co-localized. xenoGI places them in the same island because they have the same most recent common ancestor, being present in some E. coli strains and E. albertii. It is possible that these two sets of genes with seemingly distinct functions arrived as a part of a single event.
A second question relates to the time of arrival of the AFI. xenoGI places it on the branch before E. albertii diverged (Fig. 3a). However, an analysis of AFI homologs in other strains using several functions included in the package reveals that E. fergusonii contains a nearly complete copy of the AFI (missing only the heme transport genes and gadA), but in a non-syntenic location. The question then is whether this island in E. fergusonii represents an independent insertion. Alternatively, it may have resulted from the same insertion as in the other Escherichia strains, but have been moved to a different location via some rearrangement or translocational process. It has been observed that E. fergusonii has undergone a large number of genome rearrangements since the time of its divergence from E. coli [29]. That notwithstanding, the complete lack of synteny here favors an independent insertion, and is the reason xenoGI placed E. fergusonii's AFI in a separate island.
Another issue is the phylogenetic distribution of the heme transport genes. The original paper on these genes observed a puzzling phylogenetic distribution in E. coli [57]. We observe something similar in our enteric species. The heme transport locus is not present in E. coli K-12 and E. fergusonii, but is present in E. coli O157:H7 and E. albertii. This distribution would require an insertion into and then a clean deletion from the AFI, or else some process involving horizontal transfer and perhaps gene conversion.
The status of another set of genes can also shed light on the evolution of acid tolerance in Escherichia. Escherichia genomes contain a second glutamate decarboxylase enzyme, gadB. gadB has typically been seen as the result of a gene duplication, the idea being that the AFI was inserted, and then gadB arose by duplication [58]. However xenoGI finds that the gadB gene lies in an island consisting of 8 genes that is shared by the entire Escherichia clade (Fig. 3d), including E. fergusonii, but is not found outside that group. That is, it appears to have arisen on the branch leading to Escherichia before the divergence of E. fergusonii. This fact raises the question as to whether gadB is really a duplication or the result of an independent insertion event (albeit one that may have been followed by gene conversion events between gadA and gadB [58]). The other genes in the island surrounding gadB do not have close paralogs in other parts of the genome, a fact which may favor the idea of an independent insertion via horizontal transfer.
Acid tolerance has often been seen as a feature unique to E. coli [58], however our results show that this is in fact a characteristic of the whole Escherichia clade. This fact does have some practical significance, as AFI genes have been the basis for assays attempting to identify E. coli in samples [59,60]. Beyond this our data suggest that the evolution of acid fitness in this group was more complicated than previously appreciated, likely involving multiple insertion events.
Resource usage
We have run xenoGI on up to 115 strains. There is a tradeoff between time and RAM usage, because using more processors requires more RAM. Additional file 3 shows plots of RAM usage, user time and wall clock time for up to 40 strains running on 50 processors. On our machine, 40 strains required approximately 500 GB of ram and around 20 h from start to finish.
Discussion
The results presented above show that xenoGI performs well on both simulated and real data. In the validation using simulations, xenoGI's true positive rate and positive predictive value were high, and most insertions were recognized as a single island. We did observe that xenoGI's effectiveness declined as we moved to more internal branches. This trend is not surprising. Internal branches are older, and in the extra time that has passed, events may have occurred that obscure the evidence for a horizontal transfer event. xenoGI also correctly identified most of the previously identified validation islands in real genomes (Table 1). Often in cases where it seemingly made a mistake, e.g. split a single validation range into several islands, upon closer examination we found that its result was correct (Additional file 1). Furthermore, xenoGI performs favorably compared with several widely used previous methods [18,19,22,37,40]. It has higher values for both base coverage and the proportion of a validation range represented by a single island (Additional file 2). We note that in assessing the effectiveness of xenoGI and other packages, we have chosen not to use metrics such as the true positive rate or the positive predictive value. This is because the validation ranges we are using are unlikely to represent an exhaustive list of all GI's in those species. Metrics like the true positive rate and the positive predictive value require an exhaustive list. Finally, our examples from enteric bacteria illustrate the novel aspect of xenoGI's functionality, the ability to identify genomic island insertions that have occurred on internal branches of the tree for a set of strains. Taken as a whole, these results show that xenoGI can effectively reconstruct genomic island insertions in clades of closely related bacteria.
One topic it is worth addressing briefly is the relationship between the number of strains used and the effectiveness of xenoGI. In general, we have found that as we increase the number of strains, the system becomes better at accurately placing genes in families. However, when the number of strains becomes too large (on the order of dozens) it has a tendency to inaccurately break islands on deep internal branches into multiple parts.
As a comparative genomics approach, xenoGI is relatively resource intensive compared with methods that examine composition in a single genome. However, in return it provides a comprehensive assessment of the history of GI insertions into a clade. The fact that it operates in the context of a clade makes it distinctive compared with previous automated comparative genomics methods. Other distinctive features include the fact that it is gene based, doesn't depend on MAUVE, and integrates species tree and synteny information from an early stage of its analysis. In the past, reconstructing the history of GI insertions into a clade typically required heavy human involvement. Our hope is that xenoGI will make this sort of thorough comparative approach accessible to more users.
It is also worth reflecting briefly on some of the assumptions and limitations of our approach. To create gene families, xenoGI makes extensive use of synteny information. In situations where synteny is poor due to changes in gene order and composition, then family formation will be inaccurate. If the reduction in synteny is moderate, it is possible to adjust the parameters to compensate for this to some extent, e.g. by shrinking the synteny window sizes. However, in sequences such as plasmids which have undergone rapid changes in gene order and composition, the system does not perform well.
Another caveat has to do with genomic island insertion hotspots. It has been observed that certain regions are more likely than others to receive insertions [29]. When there are multiple insertions of the same or very similar GIs, xenoGI distinguishes these insertions using synteny. However, if similar islands insert multiple times in the same region, it will not be able to recognize those events as distinct.
Future work might attempt to use additional information such as that available in gene trees to supplement decisions we are currently making based on synteny. This could potentially enable us to better recognize cases where related islands inserted in the same location multiple times.
More generally, the problem of making tree-aware families is one that might be aided with machine learning approaches. The creation of such families involves integrating multiple pieces of information. However it is challenging to create a single set of rules that captures what we want to do. It may be easier for a human to annotate a clade of bacterial genomes, creating a set of gene families, and then let a machine learning algorithm learn from that. The algorithm would be using similar information to our current algorithm, but would have learned the rules based on a training set.
Finally, genomic islands which have inserted multiple times in different strains seem to be relatively common. The AFI discussed above is a potential example. It would be helpful to have a more systematic way to identify such cases. To do this, we could potentially add an additional step to xenoGI which involves comparing all the islands found in a clade against each other.
Conclusions
As more and more microbial genomes are sequenced, it becomes desirable to analyze genomic adaptation in the context of phylogenetic trees. Here we have presented xenoGI, a software package that takes a clade of closely related microbes and identifies islands of genes that entered via common horizontal transfer events, placing those events on the phylogenetic tree for the clade.
Additional files
Additional file 1: A more detailed description of the correspondence between xenoGI islands and validation islands. A tab-delimited text file containing each validation range used in the five species we looked at. Includes the assemblies used to compare with each strain and the tree. For each validation range gives the base-wise coverage, the amount of extra coverage, the number of overlapping xenoGI islands and any comments. (TSV 6 kb) Additional file 2: Comparisons with commonly used GI finding software. A tab-delimited text file containing comparisons between xenoGI and four other commonly used GI finding programs: SIGI-HMM, IslandPick, IslandPath-DIMOB, and Islander. These were done using validation ranges from the same five species used for in Table 1 and Additional file 1. | 2017-09-23T21:08:08.251Z | 2017-09-14T00:00:00.000 | {
"year": 2018,
"sha1": "212578afd652848d75c99918d24841e37a630634",
"oa_license": "CCBY",
"oa_url": "https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-018-2038-0",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "5f444accac1513578d7687b85e4c4c4a34094efd",
"s2fieldsofstudy": [
"Biology",
"Computer Science",
"Environmental Science"
],
"extfieldsofstudy": [
"Medicine",
"Biology",
"Computer Science"
]
} |
4530480 | pes2o/s2orc | v3-fos-license | The influence of cytokine gene polymorphisms on the risk of developing gastric cancer in patients with Helicobacter pylori infection
Background Helicobacter pylori infection is the main cause of gastric cancer. The disease progression is influenced by the host inflammatory responses, and cytokine single nucleotide polymorphisms (SNPs) may have a role in the course of the disease. The aim of our study was to investigate proinflammatory cytokine polymorphisms, previously associated with the development of gastric cancer, in a Slovenian population. Patients and methods. In total 318 patients and controls were selected for the study and divided into three groups: (i) patients with gastric cancer (n = 58), (ii) patients with chronic gastritis (n = 60) and (iii) healthy control group (n = 200). H. pylori infection in patient groups was determined by serology, histology and culture. Four proinflammatory gene polymorphisms were determined (IL-1β, IL-1ra, TNF-α, TLR-4) in all subjects. Results We found a statistically significant difference between males and females for the groups (p = 0.025). Odds ratio (OR) for gastric cancer risk for females was 0.557 (95% confidence interval [CI]: 0.233–1.329) and for chronic gastritis 2.073 (95% CI: 1.005–4.277). IL-1B-511*T/T homozygous allele for cancer group had OR = 2.349 (95% CI: 0.583–9.462), heterozygous IL-1B-511*T had OR = 1.470 (95% CI: 0.583–3.709) and heterozygotes in TNF-A-308 genotype for chronic gastritis had OR = 1.402 (95% CI: 0.626–3.139). Other alleles had OR less than 1. Conclusions We could not prove association between gastric cancer and chronic gastritis due to H. pylori in any cytokine SNPs studied in Slovenian population. Other SNPs might be responsible besides infection with H. pylori for the progression from atrophy to neoplastic transformation.
Introduction
Gastric cancer is the fourth most commonly diagnosed cancer and the second most common cause of cancer-related death worldwide. 1 The incidence of gastric cancer in Slovenia is among the highest in Europe with the crude incidence rate in male population of 28.6/100 000. [2][3][4] Gastric cancer is multifactorial disease. Environmental and host genetic factors influence the development of gastric cancer. The most important is Helicobacter pylori (H. pylori) infection. It is belived that roughly 65-80% of gastric cancers are associated with H. pylori infection. However, only a minority (1-2%) of infected individuals will develop gastric cancer during their lifetime. 5,6 Gastric carcinogenesis is a multistep process that starts with chronic active gastritis and continues through the development of gastric atrophy, and metaplasia, to reach gastric cancer stage at the end of that process, lasting tipically between 30-50 years. [7][8][9][10] In addition to H. pylori infection, several host genetic factors are important for the development of gastric cancer, especially several single nucleotide polymorphisms (SNPs) and/or point mutations in genes that affect gastric acid secretion and innate immune response to infection. [11][12][13][14] Polymorphisms in cytokine genes may influence the level of the cytokine production by the host, and consequently influence the disease outcome. 15 The immune response to H. pylori is important for the development of gastric cancer due to the recognition of pathogenic elements and induced synthesis and secretion of inflammatory cytokines, resulting inflammation, what can lead to severe gastric immunopathology and cancer. 16 Interleukin 1b (IL-1b) is the main cytokine secreted in response to H. pylori infection. It has a strong pro-inflamatory activity and inhibits gastric acid secretion. IL-1b is 100-times more potent inhibitor of acid secretion than proton pump inhibitors. 17 Inhibition of acid secretion may lead to the spread of bacteria from the antrum to the corpus, and consequently the development of corpus predominant gastritis which further leads to the development of gastric cancer. 18,19 Three polimorphisms were described in the IL-1B gene at positions -31, -511 and +3954 from the transcription start site. 18,20 IL-1B-31*C and IL-1B-511*T alleles are associated with decreased acidity in the stomach (hypochlohydria) in response to the infection with H. pylori. 18 IL-1b receptor antagonist (IL-1ra) polymorphisms have also been associated with the level of IL-1b secretion. Genotype IL-1RN*2 is associated with higher secretion of IL-1b, most probably through the reduction of its receptor antagonist IL-1ra. 20,21 Tumor necrosis factor-a (TNF-α) is a central mediator of the immune response. Several polymorphisms are known in the promoter region of TNF-A gene of which -308*G>A was associated with increased production of TNF-α in response to the infection, and increased gastric cancer risk. [22][23][24] El-Omar et al. 25 and Machado et al. 26 found that subject with this polymorphism have almost two-fold increased risk of gastric cancer.
Recently, a functional polymorphism at the position +896, in exon 4 of the Toll-like receptor-4 (TLR-4) gene, has been described. This A>G transition results in an alteration of the extracellular domain of TLR-4, that causes hyporesponsiveness to LPS, reduced epithelial TLR-4 density and exaggerated inflammatory cytokine response. 27 A recent study has reported an association of TLR-4 gene polymorphisms with gastroduodenal diseases such as gastric atrophy and hypochlorhydria. 28,29 TLR-4 substitution was associated with noncardia gastric cancer. 30,31 The aim of our study was to determine the prevalence of the selected pro-inflammatory cytokine polymorphisms in the Slovenian population of patients with gastric cancer and chronic gastritis, and compare its prevalence with the prevalence in the normal healthy population, to see if high incidence of gastric cancer in Slovenian population could be, at least partially, attributed to the higher prevalence of those proinflammatory polymorphisms in the genes for IL-1β, IL-1ra, TNF-α and TLR-4.
Patients
In total 318 patients and controls were included in the study devided into three groups: (i) consecutive patients with gastric cancer (n = 58), (ii) consecutive patients with chronic gastritis due to H. pylori (n = 60) and (iii) healthy control group (n = 200). Study was conducted as a case-control study, where the cancer patients represented one group and the gastritis patients represented the other group. Subjects for the healthy control group were randomly selected from the pool of representative blood samples of Slovenian healthy adults, to be matched for age and sex. All subjects were informed about the inclusion in the study and agreed to it in writing form. National medical ethics committee reviewed and cleared the protocol of the study.
Histopathology, serology and culture
Patients in the gastric cancer group had the histological type of cancer determined using the Lauren's classification that differentiates among intestinal, diffuse and mixed or indetermined type adenocarcinoma. In the group of patients with chronic gastritis two biopsies were obtained from corpus and antrum, and the histological diagnosis was determined in accordance with the Huston modification of Sydney classification for gastritis. 7,32 Serological confirmation of H. pylori infection in the gastric cancer group was confirmed by the quantitative IgG ELISA test GAP®-IgG (Biomerica, USA) from human serum. Test was performed in accordance to instructions by the manufacturer. 33 H. pylori culture was performed in the gastritis group from two biopsy samples of antrum and corpus, respectively. Biopsy samples were transported to the laboratory in Portagerm pylori transport medium (Biomerieux, France). In the laboratory, samples were homogenized in 1 mL of phosphate buffer (PBS) and 0.5 mL of the homogenate was inoculated onto two selective agar plates: Pylori agar (Biomerieux, France) and Brucella agar supplemented with human blood and antibiotic mixture (BBL, USA). Culture media were incubated at 37 °C for 72 hours in microaerophilic conditions. The identification of typical colonies was confirmed using Gram stain and the proof of enzymes: urease, catalase and oxidase.
Genotyping
Genomic DNA was extracted from the whole blood samples with EDTA using automated system for DNA isolation Magna Pure Compact Nucleic Acid Isolation Kit I (Roche Applied Science, Germany) on fully automated platform MagNa Pure Compact System in accordance to the instructions by the manufacturer. 34 Complete nucleotide sequences of individual genes for inflammatory cytokine IL-1β (rs16944), TNF-α (rs1800629) and TLR-4 (rs4986790) were looked into online databases National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov) and ENSEMBLE (www.ensembl.org). The sequences were examined with the help of the software package Vector NTI Advance 11 (Invitrogen, Carlsbad, CA, USA). 21,25,26 Polymorphisms genotyping was performed using the KASP technology (KBioscience competitive Allele-Specific PCR) using primers and reagents Kasp On Demand (KOD) (KBioscience, UK). 120 bp long reference sequences were sent to the manufacturer, upon which the appropriate primers and probes were designed ( Table 1).
The amplification of genomic DNA and the detection of polymorphisms were performed using the real-time polymerase chain reaction (PCR) apparatus LightCycler 480II (Roche Diagnostics GmbH, Germany). A touchdown protocol provided by the manufacturer was used: 94 °C for 15 min; 10 cycles of 94 °C for 10 s, 61 °C for 60 s (the anniling temperature dropped 0.6 °C per cycle to reach the annealing temperature of 55 °C) then; 26 cycles of 94 °C for 10 s, 55 °C for 60 s. IL-1RN gene contains a variable number of 86 base pair long tandem repeats (VNTR). 19 Genomic DNA was amplified and PCR products were separated by the 1.5% agarose gel electrophoresis. Primers to detect IL-1RN*2/2 (TIB Molbiol, Germany) were used. We have used forward primer: There are 5 versions of alleles. Allele 1, 2, 3, 4 and 5 carries 4, 2, 5, 3 and 6 repeats, respectively. 20,35 Due to easier statistical analysis the allele polymorphisms were divided into short and long, the short allele being allele 2 and the long allele being those with 3 repeats or more (alleles 1, 3, 4, and 5). 26
Statistical analysis
The SPSS Statistics 21 (IBM, USA) software package was used for the statistical analysis. The Hardy-Weinberg equilibrium (HWE) of alleles in each individual locus was assessed. The degrees of freedom for HWE were calculated as the number of genotypes subtracted with the number of alleles. If the value of the c 2 was less than 3.84, the frequencies of the population were in HWE. For all genotypes, the homozygote of the common allele was used as the reference. The IL-1B, IL-1RN, TNF-A and TLR-4 genotype frequencies for each polymorphism were compared by 2-sided Pearson c 2 test, to evaluate the genotype distributions of categorical variables between each group of cases and controls, and to see if there was any association between the tested variables. The odds ratios (ORs) and the 95% confidence interval (95% CI) were assessed using logistic regression analysis with the reference category being healthy controls. ORs for different groups were adjusted for sex only. Statistical differences were considered to be significant at a P value < 0.05.
Results
Patients with diagnosed chronic gastritis due to H. pylori and gastric cancer were investigated compared to healthy controls. The average age of individuals and gender ratio were comparable in all groups (Table 2). We included 198 subjects and controls in the study meeting the necessary initial criteria: 108 healthy control subjects with no underlying conditions, 32 patients with intestinal type of gastric adenocarcinoma and 58 patients with chronic gastritis and positive H. pylori infection were included and processed for statistical analysis. The genotype frequencies distribution among cytokine polymorphisms are presented in Table 3. Comparison of genotype frequencies between intestinal adenocarcinoma group and atrophic gastritis group and healthy controls showed no significant difference (p > 0.05). P-value of 0.084 for IL-1β showed closest statistical difference between the diagnosis severe progression and influence of genetic polymorphisms. However, there was a statistically significant difference between males and females compared between the groups (p = 0.025) ( Table 3). The sex-adjusted OR of gastric cancer among H. pylori positive subjects was 0.557 (95% CI: 0.233-1.329; p = 0.187) and of chronic gastritis 2.073 (95% CI: 1.005-4.277; p = 0.048). Males were taken as reference.
Pearson correlation model for all IL-1B-511, IL-1RN VNTR, TNF-A-308 and TLR-4+896 genotypes was performed and showed no statistical significance between them (p > 0.01). However correlation between IL-1B and IL-1RN was found (Pearson's R = 0.300; p < 0.001). Furthermore, there was no evidence of the association between the 55 (28.9%) carriers of IL-1B-511*T and IL-1RN*2 alleles (OR = 1.489; 95% CI: 0.660-3.361) for the risk of gastric cancer (data not shown). There was also no association for chronic gastritis. Moreover, combined T and 2 allele carriers had even lesser risk associated with developing gastric cancer than each allele separately.
Discussion
This is the first study on Slovenian population that checked variants or polymorphisms in genes responsible for cytokine secretion that may contribute to the different outcomes of infection and the development of gastric lesions. Our results showed that there was a statistical difference between genders on the outcome of infection with H. pylori (p = 0.025). Males were more predominant to develop gastric cancer than females (female OR = 0.557). Meanwhile females had 2-fold greater probability to develop chronic gastritis (OR = 2.073; 95% CI: 1.005-4.277). Our results were consistent with reported results in studies stated by Chandanos and Lagergren 4 , and Dixon et al. 32 All investigated polymorphism unfortunately showed no associations with disease prediction.
IL-1B polymorphisms were not statistically associated with the prediction of each diagnose as according, however p-value to determine association between polymorphism and outcome of infection (diagnose severity: gastritis or cancer) was 0.084. Frequency distribution in our population showed that IL-1B-511*C homozygote allele was most frequent in chronic gastritis group (58.8%). According to our knowledge such results were not found in any other study. Genotype frequencies for cancer group were coincided with control group. Studies in Caucasian and Asian populations have shown that polymorphisms in the genes IL-1B and IL-1RN were in conjunction with an increased risk for hypochlohydria and gastric carcinoma. 13 According to our findings, individuals carrying the IL-1B-511*T/T allele compared to control group showed an increased OR for gastric cancer. Heterozygotes for IL-1B gene (IL-1B-511*T carriers) and both homozygotes and heterozygotes for T allele, also showed increased OR for developing gastric cancer. Although the OR values were evaluated it would be exaggerated to affirm that these polymorphisms could indicate on the risk for developing intestinal adenocarcinoma, because the power of our statistical anysis was really poor with p-values less then 0.05 and wider 95% CI. However allele combination (T/T and C/T) showed statistically significant association with diagnose prediction (p = 0.021). Percent of IL-1B-511*T carriers in cancer group has reached almost 69% of tested individuals. El-Omar et al. 24 have identified the inflammatory profile of genetic polymorphisms in the genes for IL-1β (IL-1B -511*T) and IL-1ra (IL-1RN*2/2) to increase the risk of developing gastric cancer. 17 In our population there was 40.6% of short allele carriers diagnosed with cancer but no statistical difference to predict the disease was observed ( Table 4). The correlated association between IL-1B and IL-1RN proinflammatory genotypes (IL-1B-511*T carriers and IL-1RN*2 homozygotes) and risk for gastric cancer was also determined (p < 0.001 and Pearson's R = 0.300). These results indicated that IL-1RN*2/2 gene is recessive in combination with T carriers in IL-1B. 36 The genotype frequencies for individuals with gastric cancer or even chronic gastritis were even smaller than in control group. Results should be taken caustiously because in our population only 2% of cancer patients or patients with gastritis and 12% of controls had IL-1RN*2/2.
The present study has showed that TLR-4 polymorphism is not associated with the development of the premalignant gastric abnormalities of hypochlorhydria and atrophy, or with increased risk of gastric adenocarcinoma. No association was seen with cancer although this polymorphism has been associated with risk of other inflammatory conditions. The polymorphism was associated with hyporesponsiveness to bacterial LPS. 37 The association of the TLR-4+896A>G polymorphism identifies subjects who have an increased risk of severe inflammation and subsequently, development of hypochlorhydria and gastric atrophy, which are regarded as the most important precancerous ab-normalities. 27 However, our results were comparable to those by Garza-Gonzales 28 that the TLR-4 polymorphism did not play a role in the development of gastric premalignancies.
H. pylori infection also enhances the mucosal production of TNF-α. TNF-α is not as potent inhibitor of gastric acid secretion as IL-1β. 38 Although El-Omar et al. 20 and Machado et al. 26 found an almost two-fold increase in risk for gastric cancer, several studies have not found an association between TNF-A-308*A and gastric cancer risk. [39][40][41] The TNF-A-308*A allele has been found in association with an increased risk of cagA positive infections and gastric cancer by Zambon et al. 23 and Yea et al. 42 also found no significant association between the TNF-A-308 polymorphism and the severity of gastric disease (carcinoma, gastritis, gastric ulcers, duodenal ulcers). However our results have not confirmed that and were coincided with results of Tseng et al. 43 , who investigated polymorphisms in Jamaican children. Meanwhile the G allele has been found to be associated with peptic ulcer, which commonly accompanies gastritis 32 , and concomitant H. pylori infection, compared to those without ulcerations. 44 Mucosal expression levels of TNF-α was lower in H. pylori-infected individuals with duodenal ulcers. Heterozygous G carriers in our population were slightly drawn near with development of chronic gastritis (OR = 1.402; 95% CI: 0,626-3,139), but again the p-value was 0.411 and the association was not confirmed.
The reduced number of samples available for statistical analysis may have harmed our results. We have found no indications that the infection with H. pylori in a given inflammatory genotype of could result in an inflammatory response, and then gastritis or cancer. We have also showed that the presence of IL-1B-511 genotype for the inflammatory cytokine was inclined to the difference between intestinal type of gastric cancer, chronic gastritis and healthy controls. However statistically it was not associated entirely and could not be used to identify people at increased risk. On the other hand, cytokine gene polymorphisms represent just one component of complex interactions among host, pathogen, and environmental factors involved in gastric carcinogenesis, what was definitly confirmed with statistical difference between genders. Only combination of H. pylori and host-associated risk factors do not always allow evaluation of gastric carcinoma risk. The progression from atrophy to neoplastic transformation depends on other factors, including diet and different pathogenesis of H. pylori strains. 5,7 Ando et al. 45 have found that patients who develop duodenal ulcer disease are protected from gastric cancer. Both conditions are associated with H. pylori, but duodenal ulcers are associated with an antrum predominant gastritis, low prevalence of gastric atrophy, and very high acid secretion. On the contrary, gastric cancer patients develop corpus predominant gastritis, multifocal atrophic gastritis, and hypochlorhydria. Proinflammatory genotypes of the IL-1B gene, through its induction of gastric atrophy and gastric acid inhibition, increase the risk of gastric atrophy.
The number of cases in our study was small. In the study, in cancer group, we only included patients with intestinal type of gastric cancer, however in gastritis group we included all gastritis types, not only those with accompanied atrophy. Individuals with extensive corpus gastritis develop hypochlorhydria and gastric atrophy, which are presumptive precursors of gastric cancer. 20 Another drawback is that we have not determined bacterial strain (vac A, cag A) as it was done by Figueiredo et al. 46 and Zambon et al. 23 Anyway, now we have learnt that the assessment of patients with H. pylori infection and its strain is very important and concluded that eradication of bacteria has essential meaning. We recommend that not only screening for H. pylori also the strain determination should have some diagnostic value, especially in the patients who already developed gastritis. Furthermore, for such patients assessment of disease progression (atrophic or metaplastic gastritis) could be followed by polymorphism determination. The statistical power of our pilot study was very poor and we could not evaluate it to the whole Slovenian population, but for further polymorphism investigations it is necessary to include more patients with different disease progression. Our study design was considered good, because our study population was not heterogenic. Untill now we cannot predict the disease based only on single polymorphism.
Conclusions
Altogether, our findings indicated that host genotype as well as H. pylori infection could be important for greater risk for developing gastric cancer. However, those parameters alone could not predict the incidence of the disease. For more accurate analysis of the impact of genetic polymorphisms and identification of people with an increased risk for developing the disease, it would be necessary to expand the study and include a larger number of subjects, especially patients with gastric cancer. | 2016-05-12T22:15:10.714Z | 2015-08-21T00:00:00.000 | {
"year": 2015,
"sha1": "65644c2bde9cef83dcd889e302ba613dc0c214c5",
"oa_license": "CCBY",
"oa_url": "https://content.sciendo.com/downloadpdf/journals/raon/49/3/article-p256.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "65644c2bde9cef83dcd889e302ba613dc0c214c5",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
30060141 | pes2o/s2orc | v3-fos-license | Features of the formation and nanostructure of the film with the basic hexagonal phase TiN 0 . 3 by arc evaporation
Forming and nanostructuring processes of TiN film by electric arc evaporation under the conditions of the reactive nitrogen gas deficit in the gas mixture (30%) have been investigated. The results of a technological experiment, electron microscopic examination, X-ray diffraction phase analysis and mechanical testing of the film revealed that a significant increase in ion density and mobility leads to deterioration of the formation temperature conditions, structural and phase changes in TiN film and change of the main cubic phase (111)TiN on a hexagonal (101)TiN0.3. In the end repeated decrease of the the film microhardness with (101)TiN0.3 was caused not only by erosion of the film, but also because of change in the processes of its formation and nanostructuring in comparison with similar processes of the film with (111)TiN.
INTRODUCTION
It is known that films' exploitation conditions depend on raw materials, methods and technological parameters of the application, providing the necessary energy mass transfer of precipitable atoms, ions, molecules, nano and ionized particles.The main technological parameters are: the pressure of the gas mixture, the bias voltage on the substrate, the contents of the reaction gas in the gas mixture, the cathode-substrate distance, arc current (for electric arc evaporation), the temperature of the substrate and film, the maximum rate of deposition.The aim of this paper is to study the effect of the nitrogen minimum concentration in the gas mixture at the temperature conditions, the direction of preferential crystallographic orientation, phase composition, mechanical conditions, formation and nanostructuring of TiN film during electric-arc evaporation.
Methods of the Preparation of Substrates and Formation of TiN Film
TiN film was formed on an industrial installation NNV-6.6-I4 with a single arc evaporation with a titanium cathode (s BT1-00) with 30% nitrogen content in the gas mixture.To increase the adhesive strength of TiN film on the surface of the test samples from Article 2 [2], Ti underlayer was applied after its ion cleaning-heating.
The Method of Substrate and TiN Film Temperature Control
Temperature of test pieces surface as well as the film temperature after each 10 minutes of the film deposition have been measured by infrared contactless pyrometer after both ionic cleaning and sublayer applying, a whole duration of the precipitation process was 30 minutes.
Methods of Studying the Structure, Phase Composition and Conditions of TiN Film
Morphological traits of the formed films have been investigated by bitmapped electron microscope BS 300 with prefix for microanalysis EDAX Genesis 2000.X-ray diffraction phase analysis of TiN film was carried out using X-ray diffractometer DRON-4 in Cu Kα radiation.Microhardness of the composition has been measured by microhardness tester PМТ-3 with indenter load of 0.5 N after the film precipitation process.
SIMULATION RESULTS AND DISCUSSION
Technological parameters of processes for preparing the substrate surface before precipitation process of the film: ion cleaning-heating and deposition of Ti underlayer are shown in Table 1, the technological parameters of electric arc evaporation process and microhardness of the composition: the TiN film -substrate (hereinafter the microhardness of the composition) are listed in Table 2.
The results of X-ray diffraction phase analysis of TiN film plots, formed by arc evaporation at a nitrogen content in the gas mixture of 30% do not correspond with the previous similar studies of TiN film, formed at different contents of bias potential on the substrate (Figure 1, Tables 3,4).It was found previously [2] that regardless of the bias voltage at a maximum temperature of the film > 700 K and the rate of heating on the substrate > 14.2 K/min, the film is formed, consisting of the main cubic phase of TiN with the direction of preferred crystallographic orientation (111) and morehexagonal phase TiN 0.3 .
It was established for the first time that the nitrogen content decrease in the gas mixture to 30%, a significant increase in ion density and its mobility, as well as deterioration of the formation temperature conditions (the original film temperature was 613 K, the rate of its heating -12.0 K/min) lead to a change in the basic cubic phase 111(TiN) to the hexagonal (101)TiN 0.3 phase, the emergence of new additional phases with volume fractions: tetragonal Ti 2 N -3.7% and hexagonal Ti -2.1% and more than twofold decrease of the composition microhardness.It ought to be noted that minimal deviation of the interplanar distance from the table value, a maximum width of the peaks of the hexagonal phase (101)TiN 0.3 and homogeneous internal stresses in the film happened due to unidirectional displacement of the peaks (111)Ti 2 N and (101)TiN 0.3 , (111)Ti.
To determine the cause of changes in the mechanical conditions of TiN film, based on the structural characteristics and phase composition, which differs from all previously received TiN film, the morphological features of its surface were studied.
Through a small increase it was possible to find out only the rough surface of TiN film (Figure 2).
Based on the results of electron microscopic studies of the film surface at higher magnification it was revealed that the formation and nanostructure processes of the film with the basic hexagonal TiN 0.3 , or cubic TiN phase are different.In constructed thermal conditions two processes consistently occur: the formation of coarsegrained film and when the temperature of the film gets close to 700 K (650 K) -nanostructuring polycrystalline part of the film.
When the initial temperature conditions of formation is 613 K and heating rate is 12.0 K/min coarse-grain film of laminose structure is formed (Figure 3).Erosion of the film surface and its maximum surface roughness, as a result of intense bombardment and sputtering lead to It was discovered for the first time that film temperature increase up to 650 K leads to change of processe from coarse-grain film formation to nanostructuring of the polycrystalline film constituent, connected with the following indispensable stage consequence.
Integration of the globules (Figure 5(b)) followed by surface texturing at the end (Figure 5(c)).
Nucleation of polycrystalline constituent of the film (Figure 5(f)).
Nanostructuring of the film polycrystalline constituent (Figure 5(g)) followed by seed integration to microsystems with incoherent boundaries (Figure 5
The whole process of nanostructuring polycrystalline constituent of the film is shown in Figure 6.
CONCLUSION
Phase-structural state of TiN film, formed by electric arc evaporation with a minimum content of nitrogen in the gas mixture (30%) was investigated by X-ray dif- fraction phase analysis method.For the first time ever was established that the maximum possible content of argon in the gas mixture leads to a change in the main phase of the film from a cubic (111)TiN to the hexagonal (101)TiN 0.3 .Results of electron-microscopic studies of the films surface morphology revealed that at the film temperature less than 650 K coarse-grained film with various defects is formed on the substrate surface.Nanostructuring process of polycrystalline constituent of the film with the main hexagonal TiN 0.3 phase differs from the same film process with the main cubic (111)TiN phase.
Based on previous experimental studies of the structure and conditions of films formed by nitrogen content in the gas mixture of 90% and a variable bias to the substrate 80 ... 250 V was found that the nucleation of a polycrystalline film with the main component of the cubic phase (111)TiN occurs only at temperatures of films > 700 K and the rate of heating > 14.2 K/min [2].
The obtained results allow to suggest that the main reason for changing the phase ratio in the film, the original course of the process of coarse-grained film formation, and then the process of nanostructuring of poly crystalline constituent of the film with the main component of the hexagonal phase (101)TiN 0.3 , under conditions of low nitrogen content and intensity ion bombardment of the surface forming a film by argon is in sufficient temperature conditions, characterized by the temperature of the film and its rate of heating, nucleation,formation and nanostructure of the film with the main cubic phase (111)TiN.
We can assume that one of the main reasons for double reduction in the microhardness composition is different structural states of formed continuous film-grain lamellar structure and formed on its surface as a result of nanostructuring 3D structures with a lamellar substructure.
Figure 1 .
Figure 1.Comparative band of diffractogram fragments for TiN-based film pieces produced by arc spraying with 30% nitrogen content in the gas mixture.defects in the form of discontinuity at the interface of grains (Figure 4(a)), cracking (Figure 4(b)), grain pitting (Figure 4(c)), and grain boundary fracture of the film Figure 4(d)).It was discovered for the first time that film temperature increase up to 650 K leads to change of processe from coarse-grain film formation to nanostructuring of the polycrystalline film constituent, connected with the following indispensable stage consequence.Forming of the globules (Figure5(a)).Integration of the globules (Figure5(b)) followed by surface texturing at the end (Figure5(c)).Forming of 3D-formations with grain substructure (Figure5(d)).Surface coarsening of the 3D-formations with grain substructure (Figure5(e)).Nucleation of polycrystalline constituent of the film (Figure5(f)).Nanostructuring of the film polycrystalline constituent (Figure5(g)) followed by seed integration to microsystems with incoherent boundaries (Figure5(h)).Forming of cone textures <111> on the surface of the 3D-formations with laminose structure (Figure5(i)).
Figure 2 .
Figure 2. Photomicrographs of TiN films formed by arc spraying at 30% nitrogen concentration in mixed gas.
Figure 5 .
Figure 5. Photomicrograhs of the film having main hexagonal TiN 0.3 phase on various stages: (a) globular with globule dimension up to Ø9.0 μm; (b) integration of the globules with maximum dimensions of Ø7.5 μm and Ø6.5 μm into structures with length L = 14.0 μm and/or (c) integration of the globules into islands dimensioned up to Ø17.0 μm having globular structure; forming of 3D formations with grain substructure (d) and further their surface coarsening (e) (respectively Ø12.0 μm and Ø9.0 μm); (f) forming of seeds of polycrystalline constituent of the film having form of frustums with upper base dimension of 120x250 μm; (g) forming of laminar 3D formations (Ø4.0 μm) sustaining border coherence, (h) their integration into laminar microsystems (Ø9.0 μm) having non-coherent borders and discontinuity, (i) forming of conic surface structures <111>.
Table 1 .
Process conditions of ion cleaning-heating and deposition of Ti underlayer.
Table 2 .
Process conditions of the TiN film applying.
Table 3 .
Structural characteristics of the TiN-based films produced by arc spraying.V is phase inclusion volume fraction, dTi 2 N/dTi 2 N table is cleavage spacing, I Ti2N /I TiN0,3 is intensity ratio of all reflexes of tetragonal Ti 2 N phase and hexagonal TiN 0,3 the one, maxI 111Ti2N /I Σ and maxI 101TiN0.3/I Σ are ratios of maximum reflex intensities (111) or (101) to total intensity of all TiN phase reflexes, and β 0 is breadth of X-ray diffraction line.
Table 4 .
Positions of the diffraction peaks. | 2018-02-20T10:18:31.053Z | 2011-02-22T00:00:00.000 | {
"year": 2011,
"sha1": "9c9f73241109a9e27deb28f1dd4684db18080ae3",
"oa_license": "CCBY",
"oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=3862",
"oa_status": "HYBRID",
"pdf_src": "ScienceParseMerged",
"pdf_hash": "9c9f73241109a9e27deb28f1dd4684db18080ae3",
"s2fieldsofstudy": [
"Materials Science"
],
"extfieldsofstudy": [
"Materials Science"
]
} |
207869763 | pes2o/s2orc | v3-fos-license | Needlet thresholding methods in component separation
Foreground components in the Cosmic Microwave Background (CMB) are sparse in a needlet representation, due to their specific morphological features (anisotropy, non-Gaussianity). This leads to the possibility of applying needlet thresholding procedures as a component separation tool. In this work, we develop algorithms based on different needlet-thresholding schemes and use them as extensions of existing, well-known component separation techniques, namely ILC and template-fitting. We test soft- and hard-thresholding schemes, using different procedures to set the optimal threshold level. We find that thresholding can be useful as a denoising tool for internal templates in experiments with few frequency channels, in conditions of low signal-to-noise. We also compare our method with other denoising techniques, showing that thresholding achieves the best performance in terms of reconstruction accuracy and data compression while preserving the map resolution. The best results in our tests are in particular obtained when considering template-fitting in an LSPE like experiment, especially for B-mode spectra.
Introduction
The search for primordial polarization B-modes is the main and most exciting challenge for both the current and coming generation of Cosmic Microwave Background (CMB) experiments. A detection of B-mode CMB polarization would carry huge implications for our understanding of the Early Universe, essentially allowing for a smoking-gun confirmation of Inflation, as well as for the measurement of its energy scale [1][2][3][4]. At the same time, the quest for B-modes presents formidable challenges. The polarization signal from the inflationary stochastic Gravitational Wave (GW) background is expected to be very faint. Its detection, if achievable, will require an exquisite and unprecedented level of accuracy in controlling systematic biases in the data. One of the major sources of systematic contamination, besides instrumental effects, is the astrophysical foreground [5][6][7][8][9][10][11][12]. A variety of methods have been so far designed, with the purpose to separate the CMB signal from foreground components. Of course, state-of-the-art component separation methods work very efficiently on the best available datasets, such as Planck [5,13]. The target sensitivity for future B-mode surveys is, however, orders of magnitude below that of current experiments [14,15]. This -together with the morphological complexity and incomplete understanding of polarized foregrounds -makes further study and advancements in this area still crucial.
Different component separation algorithms exploit characteristic features of foreground emission to disentangle them from the background radiation, (mostly, but not only, their non-blackbody spectrum) [16][17][18][19][20][21][22]. In this work, we present an investigation on a technique relying on the assumption that the foreground signal is "sparse" in a proper representation, i.e. the majority of the signal is concentrated in few expansion coefficients. To this purpose, we will rely on a needlet expansion of the CMB map. Needlets are a special kind of spherical wavelets, directly defined in harmonic space and not relying on any tangent plane approximation (see next section for details). They were developed as a functional analysis tool by [23] and applied to CMB analysis in various works [17,[24][25][26][27]. Like all wavelets, needlets display the property to be localized both in space (or time) and frequency, this is a key property to induce sparsity in the representation of coherent signals, such as foreground emission. In our analysis, we will exploit sparsity to either reconstruct foreground templates via thresholding procedures or for template denoising purposes. The idea is that these approaches can be combined with -and be used as preliminary steps in -different standard component separation methods: for example, initial template denoising in needlet space can be followed by standard template fitting; alternatively, needlet thresholding can be used for pre-cleaning single frequency channels, by exploiting morphological information (non-Gaussianity and anisotropy of foregrounds); the channels can then be combined with a common approach such as Internal Linear Combination (ILC). The idea is therefore not that of developing a component separation method per se, but rather to explore a range of applications in a general context. We will specify case by case what are the main novelties introduced in the different techniques which we are going to explore, and in which regime they find their best application. Our general finding is that the methods we explore here can be useful in situations characterized by limited frequency coverage, as it can be the case for current and forthcoming ground-based and balloon experiments. The paper is structured as follows: in section 2 we will review the main characteristic of the spherical needlet and we will introduce the notions of sparsity and thresholding. In section 3 we will show how these properties can be exploited for component separation and we will describe the techniques developed in this work. In section 4 we will check the performance of our thresholding methods on simulations of different CMB surveys, showing a comparison with alternative techniques for template denoising, as well as possible applications in which thresholding is used in synergy with other methods. Finally, section 5 is dedicated to the conclusions.
Needlet Regression
Spherical wavelets are usually constructed by relying on a local flat sky approximation. This means that the basis function is defined on a flat tangent plane and then implemented on the sphere. The needlet basis, instead, is defined directly in harmonic space, in terms of spherical harmonics. As we will show in the following, this is a great advantage for the exact computation of the needlet coefficients.
More specifically, needlets are defined starting from the window function b( , j) that sets the harmonic support for each needlet layer j. This function must satisfy three properties: 1. compact support: b( , j) > 0 if min,j ≤ ≤ max,j and b( , j) = 0 otherwise. This ensure that each needlet layers represents a fixed range of scales. Moreover, each layer j will have equal support in log( ).
Given a window function with these properties (see [28] for a complete derivation), the spherical needlet basis function can be defined as: where j represents the needlets scale, λ jk and ξ jk are respectively the weights and the cubature points at the level j, and B is a parameter fixing the coverage of the needlets layers. As shown in [29], the support of b( B −j ) extends over ∈ (B j−1 , B j+1 ), this is the multipoles window spanned by the needlets layer j. From Eq. 2.1 we can define the needlets coefficients β jk of a square integrable function on the sphere f (x) as: where we use: 2) is the direct needlet transform, and β jk are the needlet coefficients at scale j in the position defined by the cubature point ξ jk . The harmonic coefficients a m can be computed numerically with a high level of precision [30], this makes the numerical implementation of needlets very convenient. The inverse of equation (2.2) is: With these preliminary definitions in hand, let us now review the main properties which make needlets a particularly suitable choice for CMB analysis. Needlets owe their name to their localization properties. The basis functions are localized quasi-exponentially around their centers, represented by the cubature points ξ jk . In their seminal paper [23], Narcowich and collaborators proved this statement showing that for any point x on the sphere surface, there exists a constant c M , such that: (2.5) Note that the function arccos in the above formula represents the distance on the sphere; this states exactly that the function ψ jk decreases faster than any power law. This property is of significant importance in CMB analysis, where the presence of missing observations poses a problem for the computation of harmonic coefficients due to the onset of spurious correlations between harmonic coefficients a m . These correlations represent a limitation for the evaluation of the power spectrum and the other cumulants and must be corrected for, generally with high computational costs. As proven in [29], needlet coefficients are instead much less sensitive to gaps in the map, therefore working in needlet space allows avoiding to correct for missing observation. Needlets are also particularly well suited for the representation of random fields on the sphere, due to their uncorrelation properties. The fact that the window function b( B −j ) has compact support in (B j−1 , B j+1 ) indeed ensures that theoretical correlations between β jk cancel if the difference in levels is greater than 2, so that if j − j > 2 we have: as the simple consequence of the fact that the supports of the two basis functions do not overlap. If we consider instead fixed scale correlations, it was proven in [24] that the needlet representation of a Gaussian random field with smooth power spectrum satisfies: for any positive integer M and c M > 0. This implies that, for growing scale j, needlets coefficients are asymptotically uncorrelated. Therefore, under Gaussianity, the small scales coefficients behave approximately as a sample of i.i.d. random variables.
Sparsity and Thresholding
A signal is said to be sparse if -in a given basis or frame -it can be reconstructed by using only a small amount of basis elements (see e.g. [31] for a complete review on the subject). Wavelets and needlets present several crucial features which allow for sparse representation of signals. First, they are not an orthonormal basis but instead a tight frame. This implies that the basis contains redundant elements. Furthermore, their tight space-frequency localization properties make them suitable to identify discontinuities in the signal and represent them with just a small number of modes. In such conditions, sparsity can always be achieved if the signal under study is smooth (although this is not a necessary condition). This makes sparsity a key element in wavelet regression methods [32], since it allows developing efficient techniques to separate a coherent signal (which is sparse, for the reasons just mentioned above) from a stochastic "noise" component. Note that in our setting, CMB itself can be viewed as part of this "noise" (for the purpose of foreground estimation). In general, stochastic fields do not admit a sparse representation, due to their lack of smoothness and, in the case of CMB, to its isotropy. Such separation can be essentially achieved by setting to zero all the coefficients under a certain threshold and eventually rescaling the remaining ones. This clearly filters the few large signal coefficients from the noise background. Such a procedure is referred to as thresholding and it is, in spirit, similar to principal component analysis, since it aims to reduce the complexity of a multidimensional data-set, by identifying the most significant modes. The simplest thresholding scheme is called hard thresholding (HT); the effects of the hard thresholding operator on the needlet coefficients are simply: where λ is a given threshold. In the case of a coherent signal, only few significant coefficients survive this operation, providing an optimal representation as well as efficient data compression. A second option is soft thresholding (ST). In this case, the significant coefficients are rescaled, proportionally to the chosen threshold: where the operator ( * ) + stands for the positive part of the argument. It is known that the soft thresholding solution can be interpreted -from a Bayesian perspective -as a maximum posterior estimator from a Gaussian Likelihood with a leptokurtic Laplace prior on the parameters, which in our case are the needlet coefficients. Let us briefly show this. Consider a dataset x = θ + n, where θ is a signal that is sparse in some basis and n is a Gaussian noise with known variance σ 2 . Assume also that the scale parameter 1/λ of the Laplace prior on θ is known and its mean is 0, so that we can write: where we use N ( * ; µ, σ) and L( * ; µ, λ) to define respectively the Normal and the Laplace distributions, that is: The maximum posterior estimator (MPE) is obtained by minimizing equation (2.11). We start by taking the derivative with respect θ (that we denote with ∂ θ ): since the absolute value is not differentiable around zero (and equivalently the L1 norm ||θ||, if dealing with multidimensional data), we should take the subgradient, so that we have: note that for θ = 0 the subgradient is actually an interval of values. We can understand the soft thresholding solution applying the conditions (2.15) at equation (2.14). First notice that the term σ 2 λ∂ θ |θ| can only take values in the interval [−σ 2 λ, σ 2 λ]. Therefore,considering the case |x| > σ 2 λ we must have θ = 0 to satisfy condition (2.14). From the same equation we see that it must be sgn(θ) = sgn(x). In this case, the solution is given by θ = x − sgn(x)σ 2 λ. For |x| = λσ 2 instead, P (θ|x) is maximum in θ = 0 since lim x→σ 2 λ (x − sgn(x)σ 2 λ) = 0 (remind that P (θ|x) is continuous). At last, if we have |x| < σ 2 λ , the only admissible solution of (2.14) is θ = 0 otherwise, due to condition (2.15), it would give θ < 0 for θ > 0 and vice versa. After these considerations it is clear that the solution coincides with the soft thresholding operator, that in this case is:
Methodology
The general idea behind this investigation is that foreground signals and the CMB fluctuations can be disentangled when the data are represented in a proper basis, frame or dictionary, and that needlets represent an ideal choice to this purpose. Foreground emission comes, in the j=1 j=3 j=5 j=6 larger part, from coherent sources concentrated around the galactic plane and in few large structures that extend at higher galactic latitudes. As we saw in the previous sections, a space-frequency representation of coherent signals naturally tends to be sparse. We will thus expect that the contribution from the galactic foreground will be concentrated in few large coefficients that can be identified and fitted with a needlet thresholding technique. On the other hand, CMB has very different features, since it is a random isotropic field and not a coherent signal. Thus, unlike foregrounds, a needlet-space representation of the CMB signal will not be sparse. The reason is that the CMB mostly does not form coherent structures, but it is instead a homogeneous fluctuations field at all scales.
The different behaviour of the two components can be immediately appreciated by looking at the needlet decomposition of a CMB realization and a foreground template. We show in figure 1 the needlet decomposition of a random CMB realization: the signal is spread over all the coefficients, as expected, given its stochastic nature. Furthermore, it is easy to notice that only adjacent layers show some level of correlation. Needlets split a continuous field in several independent realizations, the layers, each one covering a limited range of frequencies. Figure 2 shows instead the corresponding decomposition of a thermal dust template. We see how the information is actually concentrated only in few coefficients located near the galactic plane. The lowest layers trace the diffuse emission while the higher frequency levels contain only few small scale corrections. Moreover, all scales are highly correlated at any distance in frequency level, and this is expected since the signal is coherent.
To get further insight into how the thresholding procedure operates in a component separation context, we now illustrate and justify it within a general Bayesian framework. As shown in e.g., [33], a Bayesian approach provides a way to describe different component separation techniques within a unified, general formalism. This approach shows in particular how different common component separation methods amount to different choices of priors and marginalized parameters. We start as usual by assuming to have observations from K channels, with a mixture of N components and M elements (pixels) in each channel (by "pixel" we mean real space pixels, a m , needlet coefficients or the elements of whichever basis is adopted to represent the signal). We recall then the linear mixture model: where d i is a vector of K elements representing the observations in a given pixel i, s i is a vector of N elements representing the contribution of the components in the same pixel, A is the mixing matrix of dimension N × K, which weighs the contributions of different components at different frequencies; finally, n i is the noise in the pixel i. The Bayesian formulation of the component separation problem aims to solve: as shown e.g., in [33]. With specific assumptions on the priors and eventually variables to marginalize over, the formulation above can be used to define typically adopted component separation techniques, such as ILC and SMICA [16,34]. In our case, we want to introduce the sparsity assumption on the foreground templates. A similar hypothesis is also at the basis of the development of other algorithms as, for example, GMCA [20]. We will soon clarify the differences between the approach discussed here and GMCA.
As noticed in the previous section, the usual way to enforce sparsity in a Bayesian context is to assume leptokurtic priors, with the Laplace distribution as a common choice. We thus want to study the implementation of this kind of prior in a Bayesian component separation framework.
First, we must rewrite the linear mixture model in a form more suited to our scope, which is ultimately that of recovering the CMB signal. We assume that the foreground signal is sparse in the needlet domain, but the CMB is not, this is the main difference with the GMCA algorithm, which instead implicitly assumes sparsity also for the CMB component. In more detail, GMCA aims at the reconstruction of the full mixing matrix A and the signal s by exploiting the morphological diversity of the components in a given basis [35,36]. In this work, instead, our purpose is to isolate the cosmological stochastic signal from the other emissions, avoiding the full reconstruction of the components. We thus rewrite the linear mixture model assuming that components with a "Gaussian" and a "non-Gaussian" prior probability coexist in the data. Besides noise, we assume that the only "Gaussian" component is the CMB; thus we have, in the single pixel: where we explicitly separate the CMB from the other components, by denoting it with c i , times the vector of ones e of length K (since the CMB signal is constant between channels). Furthermore f i = A s i , where A is the mixing matrix with the row corresponding to the CMB set to zero. We rewrite the model in this way because at this stage we are not interested in the mixing matrix A. Therefore instead of explicitly estimating the underlying N templates, we consider the linear combination, f i , of all of them, in each of the K channels. We assume that a Laplace distribution is a proper prior also for this combination.
We now split the problem as follows: We can then solve the problem of recovering the CMB component in two steps. We first find the valuef which maximize 3.4, followed by replacing such value in 3.5 and maximizing again.
Since the noise component is uncorrelated between channels and since in our approach we are not interested in recovering explicitly the full mixing matrix, we can assume we will repeat our thresholding procedure independently at each frequency. Therefore, in our present derivation, we treat d, f and c as single-channel maps. The data likelihood takes the usual form (Gaussian noise): where C n and C c are respectively the noise and CMB covariance matrices. Therefore we can write: Marginalization over c can be carried out via standard Gaussian integration. We remind: to obtain: We then implement the Laplacian prior, again assuming uncorrelation between the channels. Thus we have P (f ) ∝ exp −λ||f || where || * || is the L1 norm. We add this to (3.10) define R ≡ C −1 n + C −1 c for simplicity of notation. Finally, we differentiate posterior with respect to the foreground component, to find: As derived in section 2.1, the solution to this problem is the soft thresholding operator, with threshold C −1 n − C −1 n R −1 C −1 n −1 λ. Following the above derivation, the central idea of our study is therefore that of using thresholding as a preliminary tool for foreground cleaning or foreground template reconstruction, in single channels. This captures morphological information in the foreground spatial distribution, at any fixed frequency. Channels can then be combined and the overall cleaning procedure further refined by applying standard algorithms that exploit CMB and foreground spectral properties. To this purpose, in the following, we combine thresholding with template fitting and Internal Linear Combination. Before concluding this section, let us stress here that our thresholding operator is applied to reconstruct foregrounds and not the CMB component. Reconstructing the CMB via thresholding in specific representations could break isotropy and Gaussianity, and we explicitly avoid this potential issue with our approach [37,38].
Template Fitting
Template fitting provides estimates of the amplitudes of each component from the fit of known templates to the data of interest. The results are then subtracted from the data to remove the spurious signal.
In the linear mixture model (3.1), the distribution of the components over the data is stored in the matrix s. The templates used should then reproduce the elements of s, other than the CMB, or a linear combination thereof. Assuming to have the exact templates, the linear fit would provide the entries of the mixing matrix A corresponding to the given source and channel. The result for a collection of N temp known templates T fitted to a map y, is the standard linear regression solution. Calling α the vectors of estimated amplitudes we have: where C is the N pix × N pix covariance matrix of the map, which depends on the noise and the CMB, and T is the N pix × N temp matrix representing the template. The estimation and the inversion of C is a major limitation in template fitting since, given the high number of data points collected by modern surveys, it is very large and computationally expensive. The choice of templates is, obviously, the crucial part of any template fitting technique. A possibility is that of resorting to external templates from previous experiments or theoretical modeling. This approach requires a lot of a-priori information on the emissions of interest, which can be unavailable or unreliable. Relying on external data-sets also runs into the issue of having to deal with additional systematic effects, cross-calibration problems and so on. For this reason, most of the modern CMB surveys have been designed with several channels at foreground dominated frequencies, allowing us to track spurious contaminant components without having to rely on external information. These templates obtained directly from the data are called internal templates. The most straightforward approach would be just to use these foreground dominated maps as templates, for example, a high-frequency channel as a dust template and a low frequency as synchrotron. However, these maps contain also the CMB contribution, which would be removed together with the contaminants, so that a correction factor must be introduced in formula (3.13). Another widely implemented solution is thus to build linear combinations of different channels so that the constant component (the CMB, providing that the observations are calibrated to its black body spectrum) vanishes [21,39]. These combinations usually are computed as the subtraction of adjacent frequency channels. The major drawback of this approach is that the noise in the internal templates is enhanced compared with the single channel. A natural application of the thresholding techniques just described is thus the denoising of the internal difference templates.
In other words, our goal is to clean a "target" map with a given number of noisy foreground templates; a needlet thresholding algorithm is then used to obtain optimal templates in which the noise component is minimized while preserving the resolution of the starting template. In the next section, we will discuss in more detail the "optimality" of the thresholding solution for this purpose, and we will show a comparison with other viable estimators. In our procedure, these templates are then fitted to the target channel, and the fitting coefficients are then used to combine the original templates. The map obtained is an estimate of the foreground contribution in the target channel, which is then cleaned by subtracting it. We start by decomposing the data (the target channel and the internal templates) in needlet coefficients. For each needlet scale j, the templates are then thresholded with threshold λ i and fitted to the data to obtain the amplitude coefficients α i (where i runs over the different templates). The optimal thresholds are selected in recursive way to maximize the goodness of fit of the templates with the target channel; namely, we iteratively threshold and fit the internal templates so that: is minimized. Here, β map jk represents the target channel at the needlet scale j (With k running over pixels), β T i jk (λ i ) are the templates thresholded with threshold λ i and the coefficients α i are obtained with the standard template fitting solution. We will discuss other threshold selection method in the next section. The templates are thus linearly combined with weights α i and subtracted to the target map as a standard template fitting procedure. We stress the fact that the cleaned template are used only to obtain the fitting coefficient, while the full templates are used to clean the map. This allows us to preserve the linearity of the template fitting procedure. In section 4.3.1, we will show the results of the application of this algorithm to different simulated data-sets.
ILC
As a further case study and an example of the versatility of our approach, we merge our thresholding algorithm with an Internal Linear Combination cleaning procedure. The general idea is as follows: a thresholding algorithm is "inverted" to remove from the map the most contaminated coefficients. This is followed by combining the channels, following the usual ILC prescription. Since we work in needlet space, such method can be straightforwardly implemented in needlet-based ILC pipelines, such as NILC. However, this is by no means mandatory, and any other signal representation domain can be chosen in the ILC step.
The overall rationale of the approach is as usual that thresholding exploits complementary foreground information, compared to ILC, since it allows us to minimize the foreground contribution in single channels, on the basis of spatial -rather than spectral -features. Merging the two methods could therefore in principle lead to useful improvements, especially in experiments where a limited number of frequency channels are available. In the section 4.3.2 we will show the results of the application of our ILC with thresholding procedure, using simulated data-set and comparing it with the standard approach.
The algorithm developed in these tests is structured as follows. First of all, each channel map is decomposed in needlet coefficients, and we treat different scales separately. In other words, we exploit the flexibility of the needlet representation to identify and remove large, spurious coefficients scale by scale. Note that, to avoid distortions in the spectral energy distribution of foregrounds, which would compromise the ILC step, in this analysis the masked coefficients after thresholding must be the same in each channel. In practice, we construct an initial linear combination of the channels (for example with a preliminary ILC) to identify the foreground dominated regions as the isotropic residuals in this co-added map. We then go back to single-channel maps and remove these regions at each frequency. The criterion to choose the threshold is again recursive and based on the minimization of the anisotropy of the residual coefficients. More specifically, in the thresholding step, we minimize: where, as usual, j set the scale, β jk represents the map in needlet space and β T λ jk is the map thresholded with threshold λ, k is the position index (the HEALPix pixels, in our algorithm), N j is the number of coefficients at the given layer j and the variance σ j can be estimated from the coefficients themself. After this "pre-cleaning" via thresholding, the different channels are then combined again with an ILC algorithm to produce a final CMB map. The overall method can be essentially interpreted as a needlet space masking, where the masked area varies with the scale, followed by Internal Linear Combination. At the end of this procedure, we extract the power spectrum from the cleaned maps, correcting for the missing coefficients with the standard MASTER technique [40]. The final power spectrum is used as a figure of merit to assess the performance of both our template fitting and ILC algorithms. The performance of our algorithms is discussed in the next section.
Results
The initial goal of our analysis is that of verifying how well we can reconstruct noisy foreground templates at high resolution, using needlet thresholding. We then focus on the issue of combining our thresholding procedure with standard component separation methodsnamely ILC and template fitting -with the aim of improving their performance. However, before moving to these points, we start by quantitatively checking the foreground sparsity assumption, on which the entire procedure is based.
Measuring sparsity
Our main goal is to separate the CMB and foreground components, by exploiting the sparsity of the latter. It is, therefore, useful to start our analysis by defining a metric to quantify sparsity and verify in detail whether and to which extent this assumption holds in our context of interest.
It has been proven that a good measure of sparsity is the so-called Gini index [41,42], defined as: where P (x) is a positive valued probability distribution, with cumulative distribution C(x).
Originally, the Gini index was introduced in social economics to measure the degree of inequality of the income distribution of a population. It runs from 0 (perfect equality, every person has the same wealth) to 1 (perfect inequality, one person owns all the goods) [43]. It is immediate to notice that the notion of inequality as defined above corresponds to the notion of sparsity in signal processing. To check the sparsity assumption, we thus measure the Gini index of different foreground templates and we compare it to a Gaussian realization. Since the Gini index is defined for positive valued distributions, we use the square of the needlet coefficients β jk . Table 1 shows the results for intensity templates of different components, at different scales j, setting the needlet scaling parameter B = 2 (we remind that each scale j cover the multipole interval [B j−1 , B j+1 ], and the corresponding window function peaks at B j ). We report the values computed both on the full sky and outside the galactic plane, with a galactic mask covering the 20% of the sky. The first row refers to a random CMB realization. The following rows refer instead the principal sources of foreground emission. Since they roughly follow the shape of the Galaxy, the results are similar between components and are less sparse outside the galactic plane, where the diffuse emission dominates. We generate the templates with the PySM software, based on the Planck sky model [44,45]. If we start by looking at the Gini index of the square of the CMB coefficients, we notice that it remains substantially constant between scales and it converges to 0.637 for high j. This is the expected value for the χ 2 distribution, i.e. the distribution of the squares of a Gaussian random variable. This behaviour reflects what stated in section 2, namely that the needlet coefficients are asymptotically uncorrelated/independent at higher frequencies. If we now focus on the foreground components, the first thing we see is that lower frequencies display lower values of the Gini index. This is due to the fact that lower frequencies represent diffuse emission, which is not sparse. Layers corresponding to higher multipoles are instead significantly more sparse, with a high degree of sparsity achieved at j ≥ 3 or 10 in the corresponding harmonic representation. This reflects the fact that, at smaller scales, we track single structures rather than a homogeneous fluctuations field. The small scale portion of the signal can thus be recovered keeping only the coefficients corresponding exactly to the size and positions of these structures, which are few compared to the total number of cubature points. In light of these observations, we, therefore, expect our thresholding-based methods to achieve significantly better performance at 10. Let us point out that the fact that very large scales are not sparse does not refute in any way the overall sparsity assumption. This is of course due to the fact that the cardinality of the needlet coefficients at a given frequency scales as B 2j , thus the number of low j β jk is negligible compared to the total. Summarizing, we have just verified that foreground templates can be faithfully represented taking almost all the (few) large scale needlet coefficients and a minimal portion of the high-frequency ones, selected with a thresholding algorithm. This highlights how thresholding can also achieve a remarkable level of data compression.
Template reconstruction
After this preliminary investigation, we now move on to show the actual performance of thresholding on the recovery of a signal template from noisy data. In this analysis, we use as benchmark a map obtained from a foreground template at 200 GHz and an isotropic Gaussian noise realization, with HEALPix nside 512. In order to highlight the denoising properties of thresholding, we set a very high noise level, so that a large portion of the available scales are where N is the dimension of the map x, i runs over the pixels indices and, in the specific case of soft thresholding, the last summation corresponds simply to minus the number of thresholded coefficients. As expected, thresholding is very effective in recovering the power of the input signal, deep into the noise dominated region. This is shown in the left panel of figure 3, where the angular power spectrum of the recovered template (in green) follows faithfully the one of the input signal (in red) at all scales and well below the noise level (in black). The excellent performance of thresholding is directly related to what discussed in the previous section. At high multipoles, corresponding to the noise dominated region in this example, the signal power is concentrated in a tiny number of very large β jk , whereas the noise power is spread among all the coefficients. In the right panel, we show the real space reconstruction of signal structures, compared to the noisy map and the input signal. We also show the map smoothed at the signal-dominated scale. The comparison with the thresholding results shows clearly how the latter removes a large part of the noise while also preserving the smallest structures.
In summary, this test shows that thresholding out small coefficients produces an almost complete suppression of the noise with only marginal loss of the signal power while maintaining the full resolution of the starting template. Furthermore, as said before, the dimension of the data set is greatly reduced: the reconstruction presented here use only 7% of the total
number of coefficients.
Moreover, the denoising of a foreground template is not the only applications of these methods. Needlet properties also allow us to separate the coherent (foreground) and stochastic (CMB, noise) components. Since we have just reconstructed a foreground template, using only 7% of the map coefficients, it is natural to assume that the residual coefficients provide a reconstruction of the stochastic component, being it the noise (as in our previous example) or the CMB. To check for this, we build a map from a foreground template and a CMB realization. We are interested in the recovery of specific features of the CMB power spectrum, e.g. the acoustic peaks, thus, for the purpose of this test, we do not need to include noise in the map. We show the results of this analysis in figure 4, considering two realizations at 143 and 217 GHz respectively. In this case, we used a hard thresholding algorithm implemented with a threshold selection criterion based on the minimization of the difference between the Gini index of the residuals with the expected value for a Gaussian field. In both cases we remove a small part of the galactic plane (f sky = 90%). Note that the size of the mask does not affect the results of thresholding. We see that the power spectrum of the residuals (black cross) remarkably follows the CMB one (cyan line) for 20 irrespective of the relative amplitude of the foreground component (red line). On the other hand, at low multipoles the reconstruction completely fails. As we commented before, these multipoles represent the diffuse signal, that is not sparse and cannot be separated from the stochastic background with this technique.
As a final interesting result we show that, in consequence of the sparsity of foregrounds, thresholding (and in particular soft thresholding) dominates all linear estimators in terms of mean square error, when we attempt to reconstruct a foreground template from a noisy realization. The problem we have dealt with so far is that of estimating a foreground template f from a single noisy observation d = f + n, where the noise is a Gaussian realization n = N (0, Iσ), so that d = N (f, Iσ). The usual least square solution would be simplyf = d.
In contexts of this type, it however proven that the asymptotically optimal estimator is not least square but it is the so-called James-Stein (JS) estimator. More specifically, if we focus method
James-Stein
Thresholding smoothing real space needlets harmonic hard soft rms (σ n = 27.10mK) 0.020σ n 0.026σ n 0.018σ n 0.023σ n 0.020σ n 0.016σ n rms (σ n = 0.75mK) 0.295σ n 0.735σ n 0.259σ n 0.259σ n 0.189σ n 0.170σ n rms (σ n = 0.28mK) 0.493σ n 0.925σ n 0.429σ n 0.426σ n 0.315σ n 0.286σ n Table 2. Root mean square errors of the real space template in unit of the noise standard deviation per pixel. The noise levels are set so that the power spectrum match the signal one at = 20, 250, 600 (top-down).
on linear estimators, and the dimension of the data-set is greater than 3, the JS estimator is known to provide be the lowest mean square error, always dominating the least square solution [47,48]. We will therefore focus here on this class of estimators to set our performance benchmark, which we will use to assess thresholding results later on. Following the notation just defined, the JS estimator of the template f is defined as: where i runs over the mode indices in the chosen representation, N is the dimension of the data-set and the apex + stand for the positive part. We develop a simple implementation of this estimator and test it in needlet, harmonic and real space. In real space, the application is straightforward: d is the map, σ is the noise standard deviation, and N is the number of pixels. For needlet (harmonic) space we apply the estimator adaptively, scale by scale and multipole by multipole, so that -for each scale j (multipole )d in formula 4.4 corresponds to β j k (a m ), while σ 2 corresponds to the needlet noise variance σ 2 j (noise power spectrum C ). A general implementation would require the computation of the full noise covariance. However, in our idealized situation, where the noise is white and uncorrelated both in space and frequency, the solution we provide here is exact. Note that, under the assumption that signal and noise are independent Gaussian random fields, the harmonic James-Stein estimator just described would be formally analogous to the Wiener filter solution. Basically, in its scale/multipole dependent implementation, this estimator suppresses the noise dominated scales.
As mentioned above, after applying the JS estimator to our case of interest, we compare its performance to thresholding. In particular, we compare two thresholding algorithms: a soft thresholding where the threshold is selected minimizing SURE, and a hard thresholding based on the so-called universal threshold defined as: where σ j is the noise variance at the scale j and N in the number of needlet coefficients. We find that, in the case of sparse signals, thresholding dominates (i.e. provides lower mean square error) even this estimator (and therefore any other linear estimator). We show this in Table 2, where we list the root mean square errors of the real space template with respect to the input signal, in units of σ noise for different estimators and noise levels for a 200GHz foreground template. The central row corresponds to the configuration used in the analysis shown in figure 3. As said earlier, we compare the results obtained with thresholding with the JS estimates in real, harmonic and needlet space, and with a simple smoothing at signal dominated scales (respectively = 20, 250, 600 for the three configurations top-down).
Channel 140 220 240 Beam (arcmin) 110 110 110 σ (µK CMB arcmin) 30 40 80 Table 3. SWIPE specifications Besides optimality issues, another advantage of needlet methods is that they are completely blind (under the assumption of white noise), whereas the other approaches assume knowledge of either the noise Power Spectrum or σ pixel . For the smoothing case, also some knowledge about the signal Power Spectrum is necessary, in order to set the scale of the low-pass filter.
On the contrary, in the needlet methods, the noise standard deviation (that appear both in SURE and in the JS shrinking factor) is computed directly from the data using the median absolute deviation (MAD) 1 of the highest frequency layer, knowing that this is noise dominated. As expected, thresholding always provides the lowest rms in these tests. In particular, soft thresholding with SURE based threshold selection achieves the best results in all cases. The hard thresholding estimator, based on the universal, threshold also provides good results, being slightly outperformed by the needlet JS estimator only in the noisiest case. The improvement is always higher for higher level of noise for all, while the differences between them are more pronounced for lower noise. Summarizing, our analyses so far have shown that thresholding achieves the best results in the extraction of foreground templates from noisy realizations. In the next section, we discuss some examples of applications of these techniques, in synergy with other component separation methods.
Synergy with other methods
In this section, we present the results obtained by applying the techniques described in the previous sections to different simulated data-sets. We generate two mock datasets using the PySM software [45], which mimic respectively observations of the Planck mission and of the SWIPE instrument of the forthcoming LSPE mission [49][50][51]. LSPE (Large Scale Polarization Explorer) is a forthcoming ASI mission aimed at the measurement of large scale CMB polarization fluctuations. It will scan a large region of the sky (20%), with two instruments, SWIPE and STRIP. In this work, we concentrate on the former, SWIPE (Short Wavelength Instrument for the Polarization Explorer). SWIPE will measure CMB polarization in three frequency channels from a stratospheric balloon flying long-duration in the northern polar region during the winter night. Due to its configurations, SWIPE represents a good benchmark for our methods: since it observes in just three different frequency channels, needlet thresholding can in principle significantly improve the results of blind component separation techniques as internal templates fitting and ILC. Planck simulations, instead, are useful to test the performance of the algorithms on the current state of the art CMB maps and, more in general, on full-sky data-sets with significant frequency coverage.
Template Fitting
In this section, we test the performance of our internal template fitting pipeline, equipped with the needlet thresholding method described before. Our aim here is not that of present- ing a full component separation pipeline, but rather that of quantifying the impact of the application of thresholding techniques on simple template fitting procedures. As a first test, we produce a set of 100 simulations of the three SWIPE channels, assuming the Planck cosmology and tensor-scalar ratio r = 0.1, while the foregrounds are generated following the Planck sky model (see [45] for reference). Given the coarse angular resolution of the experiment under exam, simulations with HEALPix nside=128 are sufficient. The noise is assumed to be white, Gaussian and isotropic. With just three frequency bands, only one internal template can be used. This regime provides therefore an ideal benchmark to verify the improvement from the pre-processing of the templates with our denoising algorithm. The internal template is obtained from the difference between the 240 GHz and the 220 GHz channels, and it is used to clean the 140 GHz cosmological channel. As a probe of the flexibility of this technique, we implement two different pipelines. The former strictly follows the steps described in section 3. The latter still uses the same procedure to obtain the thresholded template, but the final fit is now performed in real space. The overall approach to assess the performance of our method is quite straightforward: we decompose each set of polarization maps in E and B modes, followed by applying our template fitting algorithms with and without thresholding. We then measure EE and BB power spectra in all cases and use them as figure-of-merit, by comparing cleaned spectra with the input ones. We show the results of this analysis in figure 5 and 6. We find that, for both techniques (needlet and real space fit), pre-cleaning the templates with the needlet thresholding provide noticeable improvements. Especially in the case of B-mode reconstruction, where the noise dominate the templates, we found that preliminary denoising allows us to successfully recover full template denoised template Figure 6. Power spectrum reconstruction using a template fitting algorithm in real space -with and without thresholding -for SWIPE simulations, corrected for the average noise contribution. The black line represents the average of the maps' power spectra and the shaded blue area is its standard error. Green and the red lines represent the mean of the power spectra of the cleaned maps with and without thresholding respectively; the error bars are the respective standard errors. the input power spectrum in a large portion of the multipole space under examination. We repeat the same kind of analysis on simulations of Planck data. Planck, with 9 frequency channels, has a much larger frequency coverage than SWIPE, therefore we expect the effects of template thresholding to be less relevant in this case. Given the preliminary level of this investigation, we do not produce full resolution simulations but we limit the maps to nside=256 and we restrict the analysis to lmax=500. Since we are looking at diffuse foregrounds on large scales, this does not alter significantly our final performance assessment. For consistency, all the template are smoothed to match the Planck channel with lowest resolution, i.e. the 30 GHz channel with a 30 arcmin beam. Our internal templates are built following the SEVEM pipeline described in [13]. In figure 7 we show results for the cleaning of the 147 GHz channel. Following the Planck team, we use as internal templates the difference between the channels: (30-44)GHz, (217-100)GHz, (353-217)GHz. The first traces the synchrotron, while the last two will trace the dust emission. Our results are obtained on a set of 50 simulation; as expected in this case, the impact of thresholding is much less relevant than in the LSPE case. However, some improvement can still be noticed in the B-mode spectrum reconstruction, where the signal to noise is very low. Let us note here that, in principle, other denoising techniques can be applied to this problem, with a good performance in terms of the mean square error. For example, if we focus on the B-mode maps, a simple smoothing of the template at very low resolution can still produce an accurate fit. This is because -using the foreground model of our simulations (based on PySM)-the result is mostly driven by the first few multipoles, where the foreground amplitude is well above the template noise. However, even in cases where the error improvement by using thresholding is only marginal, we stress the fact that soft thresholding does maintain a number of important advantages. The main advantage is that thresholding preserves the original resolution of the maps after denoising, thus allowing us to reconstruct and study the actual structure of the template on smaller scales in real observations. All this can be achieved at the same time with a huge amount of data compression, as discussed and tested in detail in the previous section. This last property is of particular interest in the context of template fitting, because a smaller data-set requires to invert a smaller covariance matrix, with a remarkable computational gain. No other technique among those we analyzed allows us to achieve this combined result, i.e., optimality, no loss of resolution and data compression. If we focus for example on the denoising method described in our previous section, we already commented how smoothing the maps has the obvious drawback of removing potentially relevant information at small scales. The JS estimator, on the other hand, preserve resolution, but does not allow for data compression.
ILC
Here we present a similar analysis as in the previous section but we focus on ILC techniques rather than template fitting. We work on the same set of simulations described before. We consider a needlet space ILC approach, implemented via the algorithm described in section 3, where the map needlet coefficients are thresholded before combining the channels. We compare this technique with a needlet space ILC where the weights are left free to vary between scales. Before applying this "standard" needlet ILC with no thresholding, we mask the galactic plane with the Planck component separation common mask in polarization. On the other hand, our method does not need any masking since the thresholding automatically remove the most contaminated regions. We want to verify how our blind threshold selection criterion based on the minimization of anisotropy performs with respect to a standard, "a priori" masking procedure. Figures 8 and 9 show the results for LSPE and Planck respectively. We find that the results are comparable, proving that our technique performs well in selecting the foreground contaminated regions. We point out here that our currently implemented ILC technique can be improved in several ways, e.g. changing the weight in different areas of the sky. However, we did not introduce these additional refinements since -as for the previous template-fitting analysis -they are not required for what we are strictly interested about in this work, namely the specific impact of thresholding on the cleaning procedure. These results show how this technique can act as an automatic, blind masking method, with similar performance as the standard real space mask. It is relevant to notice that this approach allows recovering the input power spectrum without external assumptions on the contaminated areas of the sky (i.e., the masked part of the sky is recovered internally via thresholding, and no externally generated galactic mask is required). We also stress again that the role of thresholding, in this case, is conceptually completely different from the denoising performed in the template fitting algorithm. This is an additional probe of the large flexibility of this method.
Conclusions
In this paper, we showed several applications of needlet thresholding techniques to the problem of CMB component separation. The unifying idea behind our study is that of exploiting sparsity of foreground components in the needlet representation, as a tool to separate foregrounds from the stochastic background by exploiting their peculiar morphological features (anisotropy, non-Gaussianity).
In the first part of our analysis, we tested explicitly on simulations that our needlet expansion of foreground templates is sparse, by using Gini coefficients as a measure of sparsity. We then showed how thresholding allows reconstructing a noisy template with high accuracy, up to small scales, well below the noise level. We also made a comparison between different denoising techniques, showing that for our purposes, needlet thresholding has the best performance in terms of reconstruction accuracy, while preserving the full resolution of the templates and at the same time achieving strong data compression. After this investigation, in the second part of our study we implemented specific needlet thresholding procedures as extensions of existing component separation techniques. We then verified whether and in which situations this could improve the final CMB reconstruction. To this purpose, we focused on two well-known component separation procedures, namely ILC and template-fitting, considering simulated data sets and using as figure of merit the reconstruction of the input CMB polarization power spectrum. We compared bot soft-and hard-thresholding schemes and developed different procedures to set the optimal threshold level.
In the case of ILC, the role of thresholding is that of "pre-cleaning" single channels, before combining all the frequencies. This captures information on the foreground spatial distribution, which complements spectral frequency information and can in principle lead to a more accurate foreground cleaning procedure. In the case of template fitting, we have instead already discussed how thresholding is a powerful denoising method for internal templates.
After applying our algorithms to realistic simulations of different experimental setups, we found in practice that thresholding can be useful in experiments with few frequency channels, in conditions of low signal-to-noise. This is logical, since in these cases the original internal foreground templates are very noisy and the small frequency coverage reduces the accuracy of the standard approaches.
The best performance of thresholding in our tests are in particular found when considering a template fitting technique in an LSPE like experiment, especially for B-mode. Our ILC-thresholded algorithm, where we set the threshold level to maximize isotropy, gives instead similar results to standard ILC. Similarly, as anticipated, only marginal improvements are obtained in both cases for a Planck-like experiment with many frequency bands.
After the preliminary exploration discussed in this paper, we will therefore focus in a future work on developing in detail a full thresholding-based, needlet template-fitting pipeline. We will also explore the performance of this approach in a different context, namely for foreground cleaning and template reconstruction in intensity mapping experiments. | 2019-11-04T15:58:28.000Z | 2019-11-04T00:00:00.000 | {
"year": 2019,
"sha1": "70752cb006a27e71b22e143140ea5b7b4213575b",
"oa_license": null,
"oa_url": "https://www.duo.uio.no/bitstream/10852/79349/1/1911.01298.pdf",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "70752cb006a27e71b22e143140ea5b7b4213575b",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
20564618 | pes2o/s2orc | v3-fos-license | A job analysis of selected health workers in a district health system in Kw aZu lu-Natal Part one : Jo b analysis of nurses in hospital settings
The aim of this descriptive survey was to do a job analy sis of different categories of nurses in a District Health System in order to clarify job expectations, describe current practice of nurses in hospitals and clinics and to make recommendations about skills mix in district services. A mail questionnaire requested the sampled nurses to rate the frequency and importance of the tasks they perform. Only 19% of the nurses (41 nurses of all cat egories) returned the questionnaire, and an index tak ing into account frequency and importance, was calcu lated. The self-report data was compared with data from non-participant observation done over 19 days in 14 units in all three hospitals. A total of 39 tasks were done more than six times per week, of which most (16) were in the category of clinical assessment and recording. Counselling and teaching ( 8 tasks), were the second most frequent type of task. Only two tasks were rated as very important (giving injections and assessing respiratory status). When fre quency and importance were combined into a Task In dex, a large number of tasks scored in the middle range, with very few very high or low. Respondents identified 33 tasks that did not appear on the questionnaire. The observations showed that all categories of nurses shared many tasks in hospital settings. However, Reg istered Nurses were involved in specialized treatment and care, as well as administration of the unit. The spe cialists type tasks of Registered Nurses were also clear in Operating Theatre settings. The implications of the study are discussed and recom mendations are made. Introduction Problem statem ent In the light of the fact that about 60-80% of operating costs are from staffing, cost containment programmes demand that the right skills are employed at the right place. The term skills mix has been coined to describe a range of hu man resource options to contain cost while delivering opti mal care. It may involve the mix of posts in the establish ment, the mix of employees in the post, the combination of skills available at a specific time and/or the combination of activities that comprise each role (Buchan, Ball and O ’May, 2000: 18). These authors summarized the eight main ap proaches to determining skill mix, which includes task analy sis and job analysis, interview s/role reviews. The ap proaches vary in terms of the level of staff involvement (and therefore acceptance of results), quality of data, cost and time demands. In South Africa the nursing resources consists o f three categories of nurses: • registered nurses, with at least four years of educa tion after 1 2 years of school, • enrolled nurses, with at least two years of educa tion after ten years of school, and • nursing auxiliaries, with at least one year of educa tion after eight years o f school. The Scope of Practice regulations of the South African Nursing Council makes inadequate distinction between the registered and enrolled categories, with almost all func tions listed exactly the same (SANC, 1984). A recent gov ernment task team has also requested that these regula tions be revised, since they do not allow for the shortage of 32 Curationis November 2003 professionals to be addressed creatively (Department of Health, 2000). To ensure the most rational human resource planning with regard to skills mix, job analyses can be done. However, Buchan, Ball and O ’May (2000) point out that most skills mix studies have been done in the USA, most have meth odological flaws and most did not provide appropriate evaluation of outcomes in terms of quality or cost. In accordance with the South African Qualifications Au thority Act of 1995 (South Africa, 1995) a Nursing Standard Generating Body was constituted in South Africa to estab lish the standards for all nursing qualifications. One of the methods used in establishing the standards for an occupa tion is to do a job analysis of the current practice. This has never been done in South Africa. A study was therefore done to describe the jobs of all cat egories of nurses and selected other health workers at Pri mary Health and Secondary Care level in one health district in order to allow for human resource planning and training decisions to be based on empirical data. Although the study involved nurses in hospital and community settings, this article deals only with hospital settings. The study will be described in three sections. Section one will deal mainly with data about nurses work ing in hospital settings. Section two will deal with nurses working in PHC settings, as well as contextual factors and a comparative analysis of the burnout risk of nurses in both settings. Section three will deal with other health workers in both settings, and conclude with the recommendations of the total study. Literature survey Job analysis: In a discussion of job analysis, Landau and Rohmert (1989: 4) highlight that a job analysis process should adhere to the following principles: 1. It should be based on a theoretical model that al lows a practical interpretation of the results obtained. 2. Offer complete coverage of all demands that are present on a worker. 3. Offer maximum cost-effectiveness with regard to application, data processing and data evaluation. 4. Go beyond merely verbal work description and al low quantitative statements at least at the ordinal scale level. They also identify a number of issues that should be taken into account or form part of the job analysis (Landau and Rohmert, 1989:10). These include: • Preparation for the job (setting up the environment for the job). • The possibility of the worker influencing the dura tion or the tasks which makes up the job (full, lim ited, no). • Type of utilization of other workers and equipment (full, limited, no). • Work order (interrupted, uninterrupted). • The types of demands made by the job (information reception, information processing, information out put or activity). In Diagram 1 the thick arrow refers to the tasks the worker performs. Each task comprises of a specific performance, which is either mainly mental or physical or a combination. Figure 1 : Jo b analysis fram ew ork (adapted from La n d au and Rohm e rt, 1 9 8 9 :1 7 )
Introduction
Problem statement In the light of the fact that about 60-80% o f operating costs are from staffing, cost containm ent program m es demand that the right skills are em ployed at the right place.The term skills mix has been coined to describe a range o f hu man resource options to contain cost while delivering opti mal care.It may involve the mix o f posts in the establish ment, the mix o f em ployees in the post, the com bination of skills available at a specific time and/or the com bination of activities that comprise each role (Buchan, Ball and O 'May, 2000: 18).These authors summarized the eight main ap proaches to determining skill mix, which includes task analy sis and jo b analysis, interview s/role review s.The ap proaches vary in terms o f the level of staff involvement (and therefore acceptance o f results), quality o f data, cost and tim e demands.
In South A frica the nursing resources consists o f three categories of nurses: • registered nurses, with at least four years of educa tion after 1 2 years o f school, • enrolled nurses, with at least two years o f educa tion after ten years o f school, and • nursing auxiliaries, with at least one year of educa tion after eight years o f school.The Scope o f Practice regulations o f the South African Nursing Council makes inadequate distinction between the registered and enrolled categories, with almost all func tions listed exactly the same (SANC, 1984).A recent gov ernment task team has also requested that these regula tions be revised, since they do not allow for the shortage of professionals to be addressed creatively (Department of Health, 2000).
To ensure the m ost rational hum an resource planning with regard to skills mix, job analyses can be done.However, Buchan, Ball and O 'May (2000) point out that most skills mix studies have been done in the USA, m ost have meth odological flaws and m ost did not provide appropriate evaluation of outcomes in terms of quality or cost.
In accordance with the South African Qualifications Au thority Act of 1995(South Africa, 1995) a Nursing Standard Generating Body was constituted in South Africa to estab lish the standards for all nursing qualifications.One of the methods used in establishing the standards for an occupa tion is to do a job analysis of the current practice.This has never been done in South Africa.
A study was therefore done to describe the jobs of all cat egories o f nurses and selected other health workers at Pri mary Health and Secondary Care level in one health district in order to allow for hum an resource planning and training decisions to be based on empirical data.Although the study involved nurses in hospital and com munity settings, this article deals only with hospital settings.The study will be described in three sections.
Section one will deal m ainly with data about nurses w ork ing in hospital settings.Section two will deal with nurses working in PHC settings, as well as contextual factors and a com parative analysis of the burnout risk of nurses in both settings.Section three will deal with other health workers in both settings, and conclude with the recom m endations o f the total study.
Literature survey
Job analysis: In a discussion o f job analysis, Landau and Rohmert (1989: 4) highlight that a job analysis process should adhere to the following principles: 1.
It should be based on a theoretical model that al lows a practical interpretation of the results obtained.
2.
Offer complete coverage of all demands that are present on a worker.
3.
Offer maximum cost-effectiveness with regard to application, data processing and data evaluation.4.
Go beyond merely verbal work description and al low quantitative statements at least at the ordinal scale level.They also identify a num ber of issues that should be taken into account or form part of the job analysis (Landau and Rohmert, 1989:10).These include:
•
Preparation for the job (setting up the environment for the job).
•
The possibility o f the worker influencing the dura tion or the tasks which makes up the job (full, lim ited, no).
• Type o f utilization o f other workers and equipment (full, limited, no).
•
The types of demands made by the job (information reception, information processing, information out put or activity).
In Diagram 1 the thick arrow refers to the tasks the worker performs.Each task comprises o f a specific performance, which is either mainly mental or physical or a combination.
►
Each task also comprises of an object, which can also be mental or physical, and may require means such as material or equipment.It is done somewhere and at a specific time for a specific time.M any o f the job analysis approaches described in the literature (Fisher, Schoenfeldt and Shaw, 1999:28-42) have the aim of comparing the weight of differ ent jobs.Since that is not the purpose of this study, many of these methodologies, which aim to classify all tasks into a few general categories, are not appropriate for this study.
For educational purposes: T anner(2000:141-2) criticized as being biased the curriculum development approach in which the individual or group devises programmes, which decide on the content based on their opinions and experi ences rather than reality o f nursing practice.She compared the content of the current theatre nursing course devised by individual schools against the content determ ined through research observed skill undertaken by nurses and knowledge required to perform those activities. In
•
The participants are also asked to do frequency rat ing and critical rating for each o f 2 2 2 activity state ments.Frequency rating and critical rating are then com bined to provide an "importance" rating.The importance weight for each of the activities is deter mined.D ata is collected by means of m ailed ques tionnaires, and factor analysis perform ed to group activities that cluster together.D ata is given to the examination committee for interpretation and reflect the current practice o f nurses (National Council of State Boards of Nursing, NCSBN, 1991).According to Burgel, Wallace, Kem eer and Garbin (1997: 581), educational programmes need to be based on current practice to maintain validity.In a job analysis that was performed by the American Board for Occupational Health Nurses, four approaches were used, that is local analysis, direct observation, critical incident technique and task in ventory.Job analysis reflected com prehensive description of the diverse knowledge skills needed by occupational health nurses.
In South Africa Troskie (1998:3) evaluated the competency of newly qualified nurses by looking at com m unication skills, managem ent and clerical skills.The instrument the researcher used for the study was constructed based on a number of scales from the literature.These instruments need to be re-evaluated and expanded, so as to be used for job analysis purposes.
Objectives
The objectives o f the study were to: 1.
Clarify the job expectations o f the identified catego ries based on all documentation related to job de scriptions, including the core package and service description.
2.
Describe the current practice of the nurses in hospi tals in terms of frequency and importance o f tasks performed, environmental factors impinging on the job, task demands and immediate outcomes: 3.
Identify the skills and knowledge gaps in current practice o f these workers in relation to job expecta tions.4.
Make recommendations about skills mix in district services, by also referring to cost.This article deals only with objectives one and two and only related to nurses in hospital settings.Definition of terms A task: is a meaningful unit o f work activity generally performed on the jo b by one worker within some limited time period.It is a discrete unit, which represents a com posite o f methods, procedures and techniques.A job: is a group o f positions that are identical with respect to their m ajor or significant tasks and sufficiently alike to justify them being covered by a single analysis.Environm ental factors: This refers to the availability of necessary resources for the performance o f the jo b and any other physical or social factors influencing the level demands or strain o f the job.
Research design
This was a descriptive study.A mail survey was done, asking nurses to rate the frequency and importance o f listed tasks.The data from this survey was complemented by non-participant observation by an expert practitioner of the functioning o f all targeted workers.This focused spe cifically on tasks performed, job demands, and environ mental factors.
Sam pling for the mail survey
Two stratified random samples of nurses were drawn from a sample frame of the district for the task analysis (see table 1.1).The planned sample in hospital settings (216) was big enough to compare district hospitals and regional hospital, and also to compare the different categories o f workers, except for supervisors.It was small enough to allow for two samples that do not overlap to be drawn from the popula tion o f all nurses in district and regional hospitals in the district (720).
Mail survey instrument
The job analysis questionnaire was developed based on the core Prim ary Health Care Package (D epartm ent of Health, 2001:21-35).The list of tasks developed in this way was checked against the list of activities used in the job analysis of entry level registered nurses in the USA (Kane, Kingsburg, Colton & Estes, 1986).The list was then final ized and the 141 tasks were listed in a format which required respondents to rate how often they performed each task (less than 1 per week, 1 -5 per week, 6 -10 per week and over 1 0 times per week) and how im portant they thought the task was (whether it could sometimes be om itted or could not be omitted).The questionnaire also included a dem o graphic section that included gender, age, professional rank and the area where the respondent works.A third section dealt with activities that the respondent perform but were not included in the list provided.
Three experts checked the instrum ent for clarity.The as sessment o f the stability of the instrument was done dur ing a pilot study using test-retest reliability.Five nurses were selected for the pilot study and com pleted the ques tionnaire on two separate occasions at an interval of two weeks and the scores obtained were compared.A 100% correlation was obtained for 139 tasks, 80% for ten tasks, and 60% for three tasks.No changes were made to the instruments.
Developing an instrument based on the Primary Health Care (PHC) Package and services provided ensured content va lidity.Criterion-related validity was ensured by checking that all items covered by Kane et al (1986) in the American instrument, was also covered in this instrument: Preventive and prom otive services (45 items) Curative Service (39 items) Maternal and Child Health Service ( 8 items) Mental Health Service (16 items) Rehabilitative Service (10 items) Planning and M anagem ent (33 items) A total o f 21 items address more than one category.Since all categories of the PHC package were well represented in the instrument, and provision was made for respondents to add items, it can be argued that the instrument was valid.
The instrument was mailed to the first sample with a cover ing letter explaining the research and asking for participa tion.Respondents were supplied with a stamped envelope to return the com pleted questionnaire to the University.The m ailing was followed up with a rem inder letter four weeks later.Three months later the same questionnaire was sent to the second sample.This was done to allow for enough respondents, without nurses in the same setting working on the questionnaire at the same time and there fore influencing each other.
O b se rva tio n schedule
The observation schedule was developed to focus on the contextual factors of tasks performed, such as environmen tal factors (physical and social environment), interruptions, and control over speed o f task performance and task de mands.The observations were done over one-hour peri ods, with each category being observed at least on two different days and at least once in the m orning and once in the afternoon.Sampled units were approached by mail to explain the research and ask for their participation.They were requested to answer on the answ er sheet provided.
On receipt o f a positive answer, arrangements for the ob servation visit were made with the person in charge of the service, who arranged with individual units.The field worker was trained to use all the data collection m ethodologies in the research plan.This com ponent of the research could be seen as intrusive, and the presence o f the observer was therefore explained to patients/clients, and their permis sion was obtained.The intrusiveness was limited by meas ures such as sitting outside o f the nurse-patient circle, and using a registered nurse as observer.
The project was approved by the University o f Natal Eth ics Committee and by the provincial and district health au thorities.Individual institutions and sampled individuals were then approached and their informed, voluntary par ticipation sought.
Data analysis
The frequency and importance rating of each task was cal culated, and an index of frequency x importance was calcu lated for each item.
Frequency was calculated, based on the following classifi cation:
•
Very frequent: all tasks performed 6 times or more per week as indicated by 70% or more o f respond ents.
• Frequent: all tasks performed 6 times or more as in dicated by 50% of respondents • Rarely performed tasks performed less than once per week by 50% or more o f respondents.
• Very rarely performed: all tasks performed less than once per week by 80% or more of respondents.
The m ethodology used to calculate the frequency-importance index was based on that highlighted in the article "Certified Occupational Health Nursing -Job A nalysis in the United States o f America" (Burgel et al, 1997:45).This m ethodology enables one to com pare the both the fre quency and the importance of a task, giving more weight to the latter.As highlighted by the abovementioned article, this methodology is common in job analysis studies for the health professions, as the less frequent tasks are often the most important tasks, e.g.administering cardio-pulmonary resuscitation (CPR).
The calculation is done as follows: 1.
2.
Importance was rated on a 3 point scale: 1 = does not apply, 2 = can sometimes be omitted, 3 = can never be omitted.
3.
The ratings for the frequency index and importance index was summed, with importance given twice the weight o f frequency.The sum o f the tw o indices yields an index score per task.
4.
The highest possible index score is 10 (frequency 4 + importance 3x2).A mean index was calculated by dividing the sum ratings from all respondents for each task by the num ber o f respondents.To compare the roles of different categories o f nurses, cross tabulations of frequency o f task perform ance was done based on category, and the Chi Square correlation was cal culated to identify significance o f observed differences.
Sample realization and description
The response rate was poor, even though services were telephoned, and nurses rem inded once by mail.Only 19% (42) of the randomly selected nurses in hospitals responded, and this represents only 6 % o f the total population in the hospitals o f the District (see table 1.1).N evertheless, the distribution across the different strata was m aintained as planned, and all the relevant groups are represented.The sam ple was accepted because the data was also being checked against direct observation data.
Not a single respondent from one district hospital returned the questionnaire.This hospital had no m ember on the re search team, and was also going through significant tur moil during the research period.The response rate from enrolled and enrolled auxiliary nurses was poorer than that from registered nurses.These groups are not used to com pleting questionnaires, and a mail survey might not be the best way of involving them in a survey.
O f the 38 respondents who gave their ages, there was a relatively equal spread of about 60% ( 1 2 ) o f the regional hospital nurses between the ages o f 25 and 49, and the rest (9 or 43%) were over 50.In the dis trict hospitals the majority o f re spondents (13 or 76%) were under the age o f 39.Older nurses seem to dominate in the regional hospital, while younger nurses dominate the district hospitals.O f the 41 respondents, only five were males, and three worked in the regional hospital.
Sample Description for observations
A
Task frequency
According to the respondents only three tasks were very frequently performed, that is 6 or more times per week by 70% or more o f respondents, and these were assessing the patient's health status, taking a blood pressure, and at taching m onitoring equipm ent to the client.How ever if one looks at the items perform ed frequently, or more than six times per week by more than 50% of respond ents, the num ber increases to 39.These 39 tasks can be classified as follows: Clinical assessm ent and recording (16 items) Planning (3 items) Giving treatment (5 items) Doing counseling and teaching ( 8 items) Collaborating (2 items).There were no very rarely perform ed tasks or rarely per form ed tasks, but the tasks most infrequently done were preparing a patient for investigating procedures, assess ing m aternal and fetal status during labour.The last item indicates that few respondents worked in labour units.
Task importance
Only two tasks (assess respiratory status and giving an intramuscular injection) was rated as very important, or can never be omitted, by more than 50% o f respondents.
Task frequency and importance (task index)
In contrast to the task index o f the PHC settings, only 6 tasks (4%) were given a 10, but only 11 got indexes lower than 5 (8 %).The tasks were categorized into six roles, and the average task index for each role calculated.These cat egories and the tasks data o f each are reflected in table 1.3.Nurses rated their counselling and teaching tasks most highly (8,67) and their management o f the unit the lowest (5,56).Tasks infrequently done are highest in the role of preventive and promotive health care.
Additional tasks
A t the end o f the questionnaire respondents were asked to add any task, which they perform and could not find in the questionnaire.Respondents identified 128 such tasks.On analysis it was found that 56 of them (44%) actually did appear in the questionnaire, but in a more general form.For instance, "weighing pregnant women" was already included in task 19.One respondent indicated that paediatric proce dures such as "Inserting an IV infusion to a paediatric pa- tient" should be listed separately.This is a valid point, but will depend on whether the research has a specific focus on paediatric care.Another respondent listed separately the activities included in task 4 and 92 (hygiene and activi ties of daily living).
Two items were not clear ("creativity clinics" and "sew ing").However, a total of 33 tasks could be added from the list provided by respondents, and 4 tasks could be changed to make them more inclusive.The categories in which the additional tasks were added, are indicated in table 1.3.
Observation of tasks
In the Hospital settings, the most frequent tasks were tak ing blood pressure (total 174 of which 142 by ENA's); inter preting for doctor (total 166 o f which 78 were by ENs, and 59 by RNs); recording (133 of which 54 by ENAs and 52 by RNs); taking vital signs (total 94 o f which 71 is by ENAs); directing patients and fam ilies (total 81 o f which 30 by ENAs); bedmaking (total 79 of which 32 by ENs, and 26 by RNs) and history taking (total 62 of which 28 were by RNs).
The variety of tasks is bigger in the hospital setting than that found in the PHC settings, but they are done less sel dom.Nevertheless the RN has a num ber o f therapeutic or specialised tasks (e.g.Applying Plaster of Paris and splints 10 times; Putting up or discontinuing IV 28 times; Checking patients, sorting them out 32 times), The administrative tasks o f the RNs seems to be higher, especially arranging movements of patients (13 times), carry out stock checks (7 times), com pleting forms ( 8 times).Two tasks that seem to take a lot o f time o f all categories of nurses are interpreting for doctors (166 times) and directing patients and families (81 times).Although both of these tasks were also seen in the PHC settings, it is on a much smaller scale.
Operating theatre nursing
The tasks observed in this setting differs greatly from those in the more general settings, and the observation data is therefore listed in a table.This data is based on 24 hours of observation, 12 o f RNs, five of ENs, and seven o f ENAs in one regional and one district hospital (table 1.4).
Twenty-three tasks were identified, with only the role of the scrub nurse (seven tasks from f to m) exclusive to the registered nurse.In one hospital the Central Sterilization Department is attached to the Operating Theatre (OT), which explains tasks v and w.
The more general tasks also found in the OT, such as re cording, supervising and teaching was discussed with the general data above.
Environment
Observations of the context within which nurses work, were recorded.The district has very hot sum mers and very cold winters.
Regional hospital:
Security is poor during the night, and some patients come in drunk or drugged.Poor patients stay long after they have been discharged.
Surgical ward: Not enough toilets, and only one bath, which is used to soak linen from clean and septic cases.Walls need painting, and the roof leaks.Double-adapters are needed.Patients steal from each other.
M edical ward: Psychiatric patients cause a problem, and the ward is not equipped to deal with them.There is a short age o f bed linen.Elderly and terminal patients put a strain on nurses.
Casualty: Spacious and well-equipped, but toilets next to nurses station is not a good idea.Security is a problem, especially at night, and with psychiatric patients.It was very hot, since the air conditioning was out o f order.Staff who take unpaid leave without notice, and poor supervi sion is a problem.
Operating Theatre: Staff feel over-worked.They often can take no tea -time, have to work over-time, and have to help out in other areas within the theatre.Off-duty roster is un pleasant because o f the shortage, and orientation to the unit is poorly done.The ceiling is in poor condition, light ing poor and air conditioning not very good.
Outpatient Departm ent(O PD):
Inadequate space for pa tients to wait for treatment increases pressure on staff.All communities use the service.There are not enough doc tors, and this causes delays, which are stressful.Also, there is no social worker to attend to Social grants.Air conditioner not working, and it gets very hot.There is no isolation area.
District hospital one:
OPD: There is no toilet for staff, and male and female pa tients share a toilet.Doctors come late, and this causes stress for nurses.The waiting room is small, and so is the dressing room.The community has many problems, such as unem ploym ent and poverty.
Theatre: There is no toilet in CSSD section, and they have to use theatre toilet.There is no porter in theatre, so that two nurses from theatre has to take patients to ward.The theatre also acts as CSSD for the whole hospital.The staff has no resting place, and no lockers to keep bags.
District hospital tw o:
There is only one kit -room for the whole hospital, and it is too small.
Casualty:
The building is old.The place is over-crowded, especially the corridor.All kinds of patients are mixed and casualty and OPD are not separate.There is no privacy to attend to confidential matters.There is no change room, and no duty-room.The dressing room is small.The bed in the POP (Plaster of Paris) room cannot be adjusted.
M edical ward:
The building and ventilation is good, but there are partitions in the ward that are good for privacy, but bad for view.The kitchen is next to the toilets.There is a relative staff shortage, with the categories o f nurses not well balanced, so that there is a skills shortage.Staff also change very often.Staff is often away for meetings and courses.Doctors do rounds late, so that medication to take • home is late and patients cannot go home, and blood sam ples are too late for the laboratory.They nurse many term i nal patients, who need home based AIDS care.
Surgical ward:
The toilets are out o f order and the rough floor looks dirty.They also have many cases that need AIDS home care.They also need a social worker to deal with abuse cases.There is no wall suctioning and oxygen, making it difficult to handle emergencies.Shortage o f staff and over-crowding leads to poor patient care.There are good relationships betw een staff.
Discussion
The Hospital nurses are involved in a far wider range of tasks than the PHC nurses, but perform s each less fre quently.Thus only 42 tasks rate as frequently or very fre quently done, in com parison with the 50 tasks rated as such by PHC nurses.Nurses in hospital settings also rated their tasks as less important than the PHC nurses did.This might be because there are other team members who can take up the slack in a hospital setting, when a nurse omits a task, while that is not the case in PHC settings.The task index o f different tasks indicates the wide range o f tasks done by hospital nurses.This finding is supported by the observations, in which Hospital nurses were observed do ing 67 tasks com pared to the 56 o f PHC nurses.
The role com ponents according to the task indexes, reflect counselling and teaching as first priority, with diagnosis and planning, assessm ent and recording and then preven tion and prom otion next.O bservations put assessment and recording at the top, with treatm ent and care and m anage m ent next.The difference might be based on the fact that tasks such as planning are less visible (observable), but this cannot explain the discrepancy totally.It might be that nurse's under-estim ate the time they spend on the routine tasks o f assessm ent (such as taking vital signs and blood pressure) recording of treatment and care and management.
A num ber o f tasks that were observed very frequently or frequently deserve further discussion.
•
Bed making was observed 79 times and 33% of this was by RNs.It is granted that beds have to be made, but it seems a very low skilled job for RNs to engage in.However, it may be that they do this while wait ing for the doctor to arrive, since this is a task from which they can easily withdraw.
•
The task of directing patients and families, observed 81 times, is interesting.Although this also happens in the clinic, it is m uch less.In the old hospitals in the region, full o f nooks and crannies, and a confus ing layout, getting people from one place to another, seems a m ajor problem.A number o f workers re ferred to the fact that there is no easy way (such as a line on the floor), which can assist people with finding their way.This simple solution needs to be explored, and the time spent on these tasks m oni tored.
The high rate o f interpretation for doctors (166 ob servations) needs to be pointed out, especially since 36% of these observations involved RNs.RNs might be interpreting for doctors during doctors' rounds.This practice should be explored more fully.If the ward round is in effect a multi-disciplinary meeting to discuss the treatment o f care o f patients, even though only two people take part, that might be a good use of time.However, if the RN is in effect mainly there as interpreter, this needs work rede sign.It might then be better to supply an ENA to accompany the doctor, and for him to read nursing notes for a patient report.In any case, it would seem that the task of interpretation has always just fallen on the nearest person who speaks the language.Perhaps it is time to plan and implement different models o f supplying an interpretation service for doctors, and evaluate their effectiveness and cost.
The rate o f taking o f vital signs and blood pressure is also high.Perhaps it would be useful to investi gate whether all these observations are really nec essary, or whether much of it is done because of routine.
RNs seem to have an important task o f generally checking on patients, prioritising their care and monitoring their condition (A 8 ,32 times).This is an important task, since the RN does not do routine observations, and therefore might not be in contact with patients on a continuous basis.The continual checking keeps her in touch with what is happening to patients.This was particularly mentioned in rela tion to new patients, and patients in waiting rooms.Another task that takes much o f the RNs time is arranging for transfer of patients (A 1,13 tim es).Al though ENs assist with this task, it is mostly the duty o f the RN.
The roles of nurses in this setting seem to be less differen tiated, with ENs and even ENAs sharing much o f the tasks with RNs.ENAs have an especially wide range o f tasks in this setting.RNs however, remain responsible for some specialized tasks and for managem ent tasks.
The University o f Natal has launched a one-year "Unit management" course that aims to address the continuing education needs of mainly this group of nurses.It includes one semester o f advanced clinical skills, and one semester o f unit m anagement skills.The clinical skills include an in troduction to intensive care nursing, resuscitation, selected mental health and community health competencies.If one compares this programme with the tasks of nurses in the Hospital setting, it seems that there is a basic fit.However, it might be necessary to include a module on splinting tech niques.
With regard to Operating Theatre Nursing the role o f scrub nurse was exclusive to the RN, but all other tasks were done interchangeably by RNs, ENs, and ENAs.The use of the ENAs for duties such as assisting the scrub nurse seems a particularly useful way o f using a person with limited training.It allows for direct supervision in a useful and essential job.
Recommendations
In terms of educational needs, it would seem that hospital nurses in a D istrict Health System need unit management and advanced clinical skills for RNs to enhance current roles.
Planning for and designing an efficient translation service should be considered in these settings, where it is cur rently not a planned activity, and might therefore not be done in the m ost cost-effective manner.
To decrease directing time, hospitals should be equipped with clear markers, understandable to the whole popula tion, which will save time nurses spend on directing pa tients and families.
Table 1 .
1 Sam ple realization (planned sample numbers in brackets)
Table 1 .
3 Task index for hospital nurses with regard to six task categories
Table 1 .
4 Task frequency per category in Operating Theatre | 2017-04-30T06:37:43.390Z | 2003-09-28T00:00:00.000 | {
"year": 2003,
"sha1": "a812fcbe64687af3d5820a3c88b9f46870e6a68f",
"oa_license": "CCBY",
"oa_url": "https://curationis.org.za/index.php/curationis/article/download/828/765",
"oa_status": "GOLD",
"pdf_src": "ScienceParseMerged",
"pdf_hash": "a812fcbe64687af3d5820a3c88b9f46870e6a68f",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
216642920 | pes2o/s2orc | v3-fos-license | Exosomal MicroRNA-221-3p Confers Adriamycin Resistance in Breast Cancer Cells by Targeting PIK3R1
Drug resistance in breast cancer (BC) cells continues to be a stern obstacle hindering BC treatment. Adriamycin (ADR) is a frequently employed chemotherapy agent used to treat BC. The exosomal transfer of microRNAs (miRNAs) has been reported to enhance the drug-resistance of BC cells. Herein, we first sought to elucidate the possible role of the exosomal transfer of miR-221-3p in the drug resistance of MCF-7 cells to ADR. Differentially expressed genes (DEGs) were initially screened through microarray analysis in BC drug resistance-related datasets. Next, the expression of miR-221-3p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was quantified in ADR-resistant MCF-7 (MCF-7/ADR) and ADR-sensitive MCF-7 (MCF-7/S) cell lines, after which exosomes were separated and identified in each cell line. Target relationship between miR-221-3p and PIK3R1 was validated by a dual-luciferase reporter assay. Next, the expression of miR-221-3p and PIK3R1 was altered to clarify their effects on the resistance of MCF-7 cells to ADR in vitro and in vivo. PIK3R1 was identified as a BC drug resistance-related DEG, with the regulatory miR-221-3p subsequently obtained. Moreover, the MCF-7/ADR cells exhibited a low expression of PIK3R1 and a high expression of miR-221-3p. Notably, PIK3R1 was identified as a target gene of miR-221-3p. The overexpression of miR-221-3p in MCF-7/ADR cell-derived exosomes promoted ADR resistance in MCF-7/S cells via the PI3K/AKT signaling pathway. The in vitro results were reproducible in in vivo assays. Taken together, drug-resistant BC cell-derived exosomal miR-221-3p can promote the resistance of BC cells to ADR by targeting PIK3R1 via the PI3K/AKT signaling pathway in vitro and in vivo. These findings provide encouraging insights and provide perspectives for further investigation into the BC drug resistance mechanism.
INTRODUCTION
Breast cancer (BC) remains one of the most common and deadly malignancies afflicting the female population (1). The incidence of the young population with BC is particularly higher in China in comparison to the western world (2). Several treatment agents exist for the treatment of BC, all with varying efficacies. Adriamycin (ADR) has been well-documented to play a crucial role in treating BC, owing to its ability to kill cancer cells in humans (3). However, the emergence of drug resistance remains a complex issue that can accelerate drug efflux or even alter an individual's immune system, highlighting a significant obstacle regarding the treatment of patients with cancer (4). A previous report has defined BC drug resistance as a clinical condition implicated by molecular variations (5).
Investigations into the underlying molecular mechanisms associated with drug resistance continue to be conducted. Exosome transfer has been highlighted as a potent medium capable of elucidating the mechanisms associated with drug resistance (6). Exosomes can be found in human body fluids, and their anti-cancer effects, by transferring drugs outside of BC cells, have been proven in a previous study, resulting in drug resistance (7). Accumulating evidence has emphasized the significance of exosomal microRNAs (miRNAs) in drug resistance. At present, 22 exosomal miRNAs have been identified in BC cell resistance, whose altered expression has been suggested to be indicative of prognostic or diagnostic markers for drug resistance in BC (8).
Although the role of exosomal miRNAs in BC drug resistance has been noted, the specific mechanisms by which exosomal miRNAs contribute to drug resistance remain largely unknown and thus require further investigation. The upregulation of miR-221-3p has been implicated in cervical cancer, whereby cancer-derived exosomal miR-221-3p was found to significantly promote tumor growth (9). Moreover, oncogene-miR-221 has also been reported to play a role in the mechanism of drug resistance in non-small cell lung cancer (10). Although miR-221-3p has been extensively investigated in BC (11)(12)(13)(14), the specific function of BC-secreted exosomal miR-221-3p in BC drug resistance still remains unclear. A recent study explored the role of miR-221 targeting phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) in cholangiocarcinoma cells (15). The forced expression of PIK3R1 possesses the ability to significantly enhance paclitaxel resistance in BC cells and xenograft tumors and enhanced PIK3R1 expression also leads to activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) (16). Based on the aforementioned exploration of the literature, we hypothesized that an exosome-mediated transfer of miR-221-3p in ADR-resistant BC cells may be involved in BC drug resistance, by regulating PIK3R1 and the PI3K/AKT signaling pathway. The central objective of the study was, therefore, to determine if the aforementioned hypothesis was valid and to further explore the mechanisms by which exosomal miR-221-3p promoted the resistance of BC cells to ADR through the regulation of the PIK3R1-dependent PI3K/AKT signaling pathway.
Ethics Statement
The study was approved by the Animal Ethics Committee of Huadu Hospital, Southern Medical University. All animal experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Extensive efforts were made to minimize the number and suffering of the included animals.
Cell Culture and Treatment
Human normal breast epithelial cell line MCF-10A, and human BC epithelial cell line MCF-7/S and MCF-7/ADR purchased from the Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Science (Shanghai, China) were cultured with Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS) and 10% penicillinstreptomycin (Gibco Life Technologies, Grand Island, NY, USA) in a 5% CO 2 incubator at 37 • C. The cells were then detached using 0.25% trypsin, and subsequently sub-cultured at a ratio of 1:3. Next, the cells were seeded into a six-well-plate at a density of 3 × 10 5 cells/well. When reaching 70 -80% confluence, the cells at logarithmic growth phase were selected for further experiments. MCF-7/S and MCF-7/ADR cell lines were seeded into sixwell-plates at a density of 4 × 10 5 cells/well and then transfected in accordance with the protocol provided by lipofectamine 2000 (11668-019; Invitrogen, Carlsbad, CA, USA). A total of 10 µg diluted plasmids (a final concentration of 50 nM) by 250 µL serum-free Opti-MEM and 5 µL diluted lipofectamine 2000 by 250 µL serum-free Opti-MEM were mixed, allowed to stand for 20 min, and added into the six-well-plate, followed by 48 h culturing at 37 • C in 5% CO 2 and saturated humidity. Next, the cells were then further cultured for 24-48 h with RPMI-1640 medium containing 10% FBS for subsequent analyses. The MCF-7/S cells were transfected with plasmids of overexpression negative control (NC) (oe-NC), oe-PIK3R1, short hairpin RNA (shRNA) against NC (sh-NC), and sh-PIK3R1. MCF-7/ADR cells were transfected with plasmids of mimic NC, miR-221-3p mimic, inhibitor NC, miR-221-3p inhibitor, oe-NC, oe-PIK3R1, sh-NC, and sh-PIK3R1. The aforementioned sequences and plasmids were all purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China).
Isolation and Identification of Exosomes
The MCF-7/S and MCF-7/ADR cells were cultured overnight in serum-free RPMI-1640 medium, followed by the collection of supernatant after 24 h. Cell debris were removed by centrifugation at 2,000 g and 4 • C for 20 min. The supernatant was harvested and subsequently centrifuged at 10,000 g and 4 • C for 1 h. The cells were next suspended and precipitated using the RPMI-1640 medium [treated as the way described in a previous study (17)] with the supernatant, then discarded in accordance with the same procedure stated above. The precipitated cells were stored at −80 • C for subsequent use.
The exosomes were identified using a transmission electron microscope (TEM). Briefly, 30 µL exosomes were added onto the grid, supplemented with 30 µL phosphotungstic acid solution (pH = 6.8), counterstained at room temperature for 5 min, dried and photographed under a TEM.
The content of CD63, an exosome surface marker, was measured by flow cytometry. The exosomes were first extracted, resuspended in 1 mL phosphate buffer saline (PBS) with 1% bovine serum albumin (BSA) and cultured at room temperature for 30 min to block non-specific antigen. Exosomes were then resuspended in a 200 µL eppendorf (EP) tube and supplemented with the CD63-PE antibody, respectively. The tubes free of antibody addition were regarded as blanks, while tubes with PE conjugated anti-human immunoglobulin G (IgG) were regarded as the isotype control. The cells were centrifuged followed by the removal of the supernatant. The exosomes were resuspended again, loaded, and determined using the Guava easyCyte TM flow cytometry based on the provided instructions. The expression of the exosome surface markers CD63 and HSP70 was evaluated by western blot analysis, while the level of miR-221-3p contained in the exosome was detected by reverse transcription quantitationpolymerase chain reaction (RT-qPCR).
The diameter of exosomes was evaluated using dynamic light scattering. The evaluation was performed at an excitation wavelength of 532 nm based on the instructions provided by Zetasizer Nano-ZS90 (Malvern Corporate, Malvern, UK). Th exosome sample was diluted by 0.15 M NaCl (1:50) and detected by optical signal.
Endocytosis of MCF-7/ADR Cell-Derived Exosomes by MCF-7/S Cells
The extracted MCF-7/ADR exosomes were labeled by a PKH67 (Green) staining solution (MINI67-1KT, Sigma-Aldrich, St. Louis, MO, USA) according to the provided instructions. The fluorescence-labeled exosomes were then co-cultured with confluent BC cells that had been seeded into 24-well-plates for 48 h of culturing and observed under an inverted fluorescence microscope. The expression of miR-221-3p and PIK3R1 was then detected by RT-qPCR, with the protein expression of PIK3R1 determined by western blot analysis.
RNA Isolation and Quantification
Total RNA was extracted by TRIzol reagent (15596-018; Beijing Science & Technology Co., Ltd., Beijing, China). All primers in this study were synthesized by Takara (Dalian, China; Table 1). Reverse transcription was then conducted following the instructions of the complementary DNA reverse transcription kit (K1622; Beijing Reanta Biotech Co., Ltd., Beijing, China). The relative transcription level of target genes was calculated
based on the 2 − Ct method, with U6 and β-actin serving as the loading controls. Cell quantification was performed using the same method.
3-(4,5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) Assay
A total of 200 µL cell suspension was added into a 96-well-plate at a density of 5 × 10 5 cells/well, followed by the addition of 500 ng/mL ADR (20 µL ADR/well) with four repeated wells for each concentration, and zero-setting and blank control wells set. The well without the addition of ADR was regarded as blank. The cells were then incubated on days 1-5 in a 5% CO 2 incubator at 37 • C and reacted with ADR at different concentrations. After the removal of the medium, 90 µL serum-free RPMI-1640 medium and 20 µL MTT solution (5 mg/mL; 0.5% MTT) was added to each well and incubated for 4 h in a 5% CO 2 incubator at 37 • C. After the incubation was terminated, 200 µL dimethyl sulfoxide was added to each well and shaken for 10 min to repeatedly dissolve the crystals. The cell survival curve was drawn after measuring the optical density at 570 nm (OD 570 ) value using an automatic microplate reader with the cell survival rate (%) calculated (OD 570 in the experimental group/OD 570 in the control group × 100%).
Flow Cytometry
Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (
Dual-Luciferase Reporter Assay
The target relationship between PIK3R1 and miR-221-3p was verified by dual-luciferase reporter assay. The target and mutant sequences were designed based on the binding sequence of PIK3R1 3 ′ untranslated region (UTR) and miR-221-3p. The luciferase activity of PIK3R1 3 ′ UTR in cells transfected with miR-221-3p was determined in accordance with the instructions provided by the Genecopoeia's dual-luciferase reporter assay kit (D0010; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Luciferase intensity was then evaluated by Promega's GLomax20/20 Luminometer.
Tumor Xenografts in Nude Mice
A total of 30 BALB/c nude mice (aged 3-5 weeks; weighing 14-18 g) were housed in a specific pathogen-free barrier facility with regular ultraviolet radiation, sterilized cages, bedding materials, food, and water under controlled temperature and humidity conditions (24-26 • C; 40-60%). The cultured MCF-7 cells were selected, and its suspension concentration was adjusted to 1 × 10 6 cells/mL using PBS. Next, 50 µL cell suspension was extracted and subcutaneously inoculated into the right flank of each mouse. One week later, the mice were randomly treated (six mice in each group) with (1) PBS, (2) ADR (25 mg/kg), (3) MCF-7/ADR exosomes added ADR, (4) MCF-7/ADR exosomes with inhibitor NC, and ADR and (5) MCF-7/ADR exosomes with miR-221-3p inhibitor and ADR. After 30 days had elapsed, the mice were euthanized after anesthesia by injections of pentobarbital sodium (100 mg/kg; P3761; Sigma-Aldrich, St. Louis, MO, USA). Their tumors were then isolated and dissected. Tumor volume was then calculated using the formula: volume = π (short diameter 2 × long diameter)/6. The weight of tumors was finally measured using a balance.
Statistical Analysis
All data were processed with the SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. Unpaired data in compliance with normal distribution and homogeneity of variance between two groups were compared using an unpaired t-test. Comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test. Data at different time points and different concentrations were compared by repeated measures ANOVA with Bonferroni post-hoc test. A value of p < 0.05 indicated significant difference.
PIK3R1 Was Poorly Expressed in Drug-Resistant BC Cells
The BC drug resistance-related microarray GSE76540 was obtained from the GEO database, including the cell lines MCF-7/S and MCF-7/ADR. A total of 2745 DEGs were obtained through differential analyses on gene expression in the two cell lines ( Figure 1A). The relationship between the DEGs was analyzed by PPI ( Figures 1B,C), the results of which revealed five genes (UBB, GNGT1, PIK3R1, GNB2, and ESR1) located at the center of PPI network. Differential expression analysis of these five genes was subsequently conducted in order to identify their expression in normal breast epithelial cell MCF-10A and ADRsensitive BC cell line MCF-7/S, which displayed that PIK3R1 was the gene with the most variation ( Figure 1D). Next, to further determine the expression of PIK3R1 in drug-resistant BC cells, PIK3R1 expression in normal MCF-10A, ADR-sensitive MCF-7/S and ADR-resistant MCF-7/ADR cell lines was evaluated by RT-qPCR and western blot analysis. The results obtained demonstrated that PIK3R1 was downregulated in MCF-7/ADR cells in contrast to that in MCF-10A and MCF-7/S cells (p < 0.05; Figures 1E,F). Altogether, the results obtained indicated that PIK3R1 was involved in BC drug resistance.
PIK3R1 Regulated Cell Apoptosis and Drug-Resistance of BC Cells
Following the confirmation of PIK3R1 contribution to BC drug resistance, we set out to further investigate the role of PIK3R1 in drug-resistance BC cells. After PIK3R1 was overexpressed or knocked down, the expression of PIK3R1 in the MCF-7/S and MCF-7/ADR cells was determined by RT-qPCR and western blot analysis. As depicted in Figures 2A-D evaluated by flow cytometry. The results showed that PIK3R1 overexpression led to significantly augmented value of IC 50 (Figures 2E,F), decreased cell viability (Figures 2G,H) and enhanced cell apoptosis (Figures 2I,J). However, the value of IC 50 was notably diminished, while cell viability was elevated and cell apoptosis was declined in MCF-7/S and MCF-7/ADR cells when PIK3R1 was knocked down when compared with sh-NC treatment (all p < 0.05). The aforementioned results provided evidence suggesting that PIK3R1 could affect drug resistance, cell viability, and apoptosis in BC cells.
Isolation, Identification, and Endocytosis of MCF-7 Cell-Derived Exosomes
As previously reported, BC-secreted exosomes participate in the drug resistance transfer (6,18,19). Hence, we set out to elucidate the impact of exosomes contained in BC on cell drug resistance. Exosomes from MCF-7/S and MCF-7/ADR cells were isolated using high-speed centrifugation. TEM revealed that exosomes isolated from different cell lines had identical form (circular or oval membranous vesicle; Figure 3A). In addition, the diameter of the exosomal particles ranged from 30 to 120 nm in the determination of dynamic light scattering ( Figure 3B). The expression of CD63 and HSP70 was then detected by western blot analysis, the results of which ( Figure 3C) demonstrated that CD63 and HSP70 were highly expressed in exosomes. The exosome surface marker CD63 content was subsequently identified by flow cytometry, which indicated that the CD63 content was markedly elevated in exosomes isolated from cell lines (p < 0.05; Figure 3D). The aforementioned results suggested that cellular exosomes were successfully isolated. In order to verify whether ADR-sensitive cells could endocytose MCF-7/ADR cell-derived exosomes, ADR exosomes traced by PKH67 (Green) were co-cultured with MCF-7/S cells for 48 h. MCF-7/S endocytosing ADR exosomes were analyzed under a confocal microscopy. The results indicated notable MCF-7/S endocytosing ADR exosomes following 48 h of coculture ( Figure 3E). The results clearly demonstrated that drugresistant exosomes could be endocytosed by ADR-sensitive cell line MCF-7/S.
MCF-7/ADR Cell-Derived Exosomes Could Transfer Drug Resistance
Next, to further investigate the role of exosomes in BC drug resistance transfer, exosomes secreted from different cells were processed with MCF-7/S cells and then treated with 500 ng/mL ADR for 48 h. The value of IC 50 , cell viability and apoptosis in MCF-7/S cells were detected by MTT assay and flow cytometry, respectively. The results (Figures 4A-C) exhibited that MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes exhibited an elevated IC 50 -value and cell viability along with reduced cell apoptosis in contrast to MCF-7/S cells (all p < 0.05); however, the MCF-7/S cells co-cultured with MCF-7/S cellderived exosomes displayed no significant difference (p > 0.05). These results demonstrated that exosomes in ADR-resistant cells could transfer drug resistance to ADR-sensitive cells, while ADR-sensitive cell-derived exosomes did not exert any influence on cell activity. Next, the expression of PIK3R1 was analyzed by RT-qPCR and western blot analysis. As illustrated in Figures 4D-E, protein level of PIK3R1 was decreased in MCF-7/ADR cell-derived exosomes (p < 0.05), but showed no significant change in MCF-7/S cell-derived exosomes (p > 0.05), when compared to the MCF-7/S cells. Taken together, MCF-7/ADR cell-derived exosomes could inhibit PIK3R1 to regulate cell viability, apoptosis and drug resistance.
PIK3R1 Was a Target Gene of miR-221-3p
In an attempt to clarify the upstream regulatory mechanism of drug-resistant BC cell-derived exosomes affecting BC drug resistance, upregulated miRNAs differentially expressed in BC exosomes were obtained from the EVmiRNA database. Next, based on the mirDIP and TargetScan databases, the upstream regulatory miRNA of PIK3R1 was predicted, which was jointly analyzed via the EVmiRNA database ( Figure 5A). There were 5 miRNAs emerged from the above analyses (hsa-miR-221-3p, hsa-miR-320a, hsa-miR-93-5p, hsa-miR-21-5p, and hsa-miR-222-3p), among which miR-221-3p displayed the most distinct altered expression (Figure 5B). A specific binding region was detected between the sequence of PIK3R1 and miR-221-3p based on the analysis conducted by the online software ( Figure 5C).
The expression of miR-221-3p was detected by RT-qPCR in exosomes of drug-sensitive cells and drug-resistant cells. Furthermore, miR-221-3p exhibited higher expression in the exosomes of ADR-resistant cells than that in exosomes of drugsensitive cells (p < 0.05) (Figure 5D). The targeting relationship between miR-221-3p and PIK3R1 was subsequently verified using dual-luciferase reporter assay. The results shown in Figure 5E presented that the luciferase activity of wild type (WT)-PIK3R1 was reduced in cells upon miR-221-3p mimic treatment in contrast to mimic NC treatment (p < 0.05) while the luciferase activity of mutant (Mut)-PIK3R1 showed no significant change (p > 0.05). These results indicated that miR-221-3p could specifically bind to PIK3R1. The altered expression of miR-221-3p in MCF-7/ADR cells was further identified, and meanwhile, PIK3R1 expression in the MCF-7/ADR cells was analyzed by RT-qPCR and western blot analysis (Figures 5F,G). The measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test. Each experiment was repeated three times independently.
Compared with cells transfected with mimic NC, PIK3R1 mRNA and protein level were notably decreased in cells transfected with miR-221-3p mimic, while they were obviously elevated in cells transfected with miR-221-3p inhibitor in contrast to inhibitor NC-transfected cells (all p < 0.05). The above results indicated that PIK3R1 was regulated by miR-221-3p. Upregulated miR-221-3p could inhibit PIK3R1 expression, while downregulated miR-221-3p promoted PIK3R1 expression.
Exosomal miR-221-3p Promoted BC Cell Drug Resistance by Targeting PIK3R1 and Inhibiting the PI3K/AKT Signaling Pathway We next aimed to further investigate whether drug-resistant exosomes promoted drug resistance via miR-221-3p or not, MCF-7/ADR cells were transfected with miR-221-3p mimic or inhibitor. Then, MCF-7/S cells were processed with isolated exosomes. miR-221-3p expression and PIK3R1 mRNA expression were detected by RT-qPCR, while PIK3R1 protein level was determined by western blot analysis. As illustrated in Figures 6A,B, when MCF-7/ADR cell-derived exosomes were co-cultured with miR-221-3p mimic, miR-221-3p was upregulated but PIK3R1 was downregulated, while a contrasting trend was identified in the expression of miR-221-3p and PIK3R1 in MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p inhibitor. After ADR treatment, the value of IC 50 was measured in MCF-7/S cells by MTT assay. The IC 50 -value was increased in MCF-7/ADR cell-derived exosomes treated with miR-221-3p mimic, but it was decreased in MCF-7/ADR cell-derived exosomes treated with miR-221-3p inhibitor ( Figure 6C). MTT assay and flow cytometry (Figures 6D,E) revealed that cell viability was increased but cell apoptosis was decreased in MCF-7/ADR cell-derived exosomes treated with miR-221-3p mimic, all of which was reversed in MCF-7/ADR cell-derived exosomes treated with miR-221-3p inhibitor (all p < 0.05). These results revealed that miR-221-3p contained in exosomes promoted drug resistance of BC cells. As previously suggested in literature, PIK3R1 regulated drug resistance through the PI3K/AKT signaling pathway (20). In order to validate this, western blot analysis was performed to detect the extent of PI3K and AKT phosphorylation as well as protein expression of PI3K and AKT. The results shown in Figure 6F displayed an upward trend in the protein expression of PI3K and AKT as well as the extent of PI3K and AKT phosphorylation in MCF-7/ADR cell-derived exosomes treated with miR-221-3p mimic, which was abrogated in MCF-7/ADR cell-derived exosomes treated with miR-221-3p inhibitor (all p < 0.05). All the results indicated that exosomes containing miR-221-3p targeted PIK3R1 and affected drug resistance via the PI3K/AKT signaling pathway.
Inhibited Exosomal miR-221-3p Derived From MCF-7/ADR Cells Suppressed Tumor Formation in vivo
Lastly, we attempted to study the impact of MCF-7/ADR exosomal miR-221-3p on BC tumor formation in nude mice. Xenograft tumor model was first induced in nude mice. After tumor formation, the mice were then subjected to different treatments. Tumor growth curve, volume, and weight were then analyzed. The results shown in Figures 7A-D presented that tumor growth speed, volume, and weight of mice were significantly reduced in MCF-7/ADR cell-derived exosomes treated with miR-221-3p inhibitor and ADR, when compared with those in MCF-7/ADR cell-derived exosomes treated with inhibitor NC and ADR (p < 0.05).
Cell apoptosis was determined using flow cytometry, and protein expression of PI3K and AKT as well as the extent of PI3K and AKT phosphorylation was evaluated by western blot The measurement data were expressed as mean ± standard deviation. Data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test while data comparison at different time points was analyzed by repeated measures ANOVA, followed by Bonferroni post-hoc test. *p < 0.05, compared with MCF-7/S cells; #p < 0.05, compared with MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes. Each experiment was repeated three times independently.
analysis. Results exhibited that cell apoptosis was decreased in MCF-7/ADR cells following ADR treatment in contrast to that following PBS treatment. Cell apoptosis, along with protein expression of PI3K and AKT as well as the extent of PI3K and AKT phosphorylation was reduced in MCF-7/ADR cell-derived exosomes treated with both miR-221-3p inhibitor and ADR in contrast to those in MCF-7/ADR cell-derived exosomes treated with inhibitor NC and ADR (all p < 0.05; Figures 7E,F). These findings further proved that inhibition of exosomal miR-221-3p derived from MCF-7/ADR cells and ADR could retard tumor formation in nude mice.
DISCUSSION
BC, the second major cause of cancer deaths in women, affects ∼1.7 million individuals every year worldwide and the drug resistance remains an obstacle of developing more effective and efficient therapeutics (5). Exosomal miRNAs possess the ability to shuttle between cells to communicate and exchange genetic material valuable, highlighting them as candidate biomarkers for tumorigenesis and drug resistance (21), particularly in BC (22). The gathered findings from the present study demonstrated that exosomal miR-221-3p could enhance the resistance of The measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test, while data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, compared with mimic NC-transfected cells; #p < 0.05, compared with inhibitor NC-transfected cells. Each experiment was repeated three times independently.
BC cells to ADR by targeting PIK3R1 via the PI3K/AKT signaling pathway.
During the current study, the MCF-7/ADR cell line exhibited high levels of miR-221-3p expression. In a recent study, miR-221-3p was reported to be up-regulated in both resistant BC tissues and cells (23). More recently, the exosome-mediated transfer of miRNAs has been delineated to alter BC cell resistance to drugs, unearthing potential biomarkers for BC progression Western blot analysis of PIK3R1 protein in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor. (C) IC 50 -value in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor measured by MTT assay. (D) Viability of MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor measured by MTT assay. (E) Apoptosis of MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor measured by MTT assay. (F) Western blot analysis of PI3K, and AKT as well as the extent of AKT and PI3K phosphorylation in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor. The measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test, while data comparison at different time points was conducted by repeated measures ANOVA, followed by Bonferroni post-hoc test. *p < 0.05, compared with the cells treated with MCF-7/ADR cell-derived exosomes co-cultured with mimic-NC; #p < 0.05, compared with the cells treated with MCF-7/ADR cell-derived exosomes co-cultured with inhibitor-NC. Each experiment was repeated three times independently. and treatment (24). Exosomal miR-221 exerts promoting effects on the resistance of glioma cells to temozolomide, thus inducing tumor progression (25). In pancreatic cancer, miR-221-3p has also been reposted to be responsible for the drug resistance (26). These findings are partially in agreement with ours, whereby exosomal miR-221-3p could promote the resistance of BC cells to ADR. Furthermore, the expression of hsa-miR-221-3p may be associated with the diagnosis and prognosis of BC patients (27).
The downstream regulatory mechanism of miR-221-3p was subsequently analyzed. PIK3R1 was proven to be a target gene of miR-221-3p. Through analysis of BC drug resistance-related microarray data, PIK3R1 was identified. PIK3R1 expression was decreased both at mRNA level and protein level in MCF-7/ADR cells. PIK3R1 is defined as a differentially expressed gene in invasive BC (28), which was consistent with our research. In addition, PIK3R1 is also found to be expressed at a poor level in BC cells (29). In a previous study, PIK3R1 is linked to insulin resistance in vivo (30). Moreover, PIK3R1 is regarded as one of the biomarkers predicting drug resistance in human epidermal growth factor receptor-2 + cancer cells (31). Also, CapG-mediated hyperactivation of PIK3R1 results in increased sensitivity to chemotherapy in BC cells (16). The role of PIK3R1 in BC was further analyzed, which revealed the role of PIK3R1 in the invasion and migration of MCF-7 cells (32), confirming that PIK3R1 is associated , and AKT as well as the extent of AKT and PI3K phosphorylation in tumor tissues of nude mice. The measurement data were described as mean ± standard deviation. Data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test, while data comparison at different time points was conducted by repeated measures ANOVA, followed by Bonferroni post-hoc test. N = 6. *p < 0.05, compared with the mice following PBS treatment; #p < 0.05, compared with ADR treatment. &p < 0.05, compared with the mice inoculated with MCF-7/ADR cell-derived exosomes treated with inhibitor-NC. Each experiment was repeated three times independently.
with human BC epithelial cell line. However, there are few studies investigating the altered expression of PIK3R1 in BC and the relationship between PIK3R1 and BC drug resistance, while the current study conducted an in-depth investigation into this topic. The influence of PIK3R1 in the event of BC drug resistance was determined using ectopic expression experiments and the results in our study proved that the overexpressed PIK3R1 regulated drug resistance and resulted in an increase in cell apoptosis and diminished cell viability in BC. PIK3R1 has been reported to be negatively regulated by miR-21 in BC (20). Besides, the binding between PIK3R1 and miR-221 had been previously reported, with miR-221 FIGURE 8 | The graph of the molecular mechanism of miR-221-3p in ADR-resistant BC cells. In ADR-resistant BC cells, miR-221-3p promotes drug resistance by negatively regulating PIK3R1, thereby promoting BC cell viability and inhibiting apoptosis. In addition, miR-221-3p can be packaged into the exosomes derived from ADR-resistant BC cells, and transferred to ADR-sensitive BC cells.
found to target PIK3R1 in endothelial progenitor cells (33). During the current study, bioinformatics analysis and dualluciferase reporter assay confirmed that exosomal miR-221-3p could target PIK3R1 and inhibit its expression in BCresistant cells.
Based on the known regulatory mechanism underlying miR-221-3p/PIK3R1, our data further verified the mechanism by which exosomal miR-221-3p/PIK3R1 influences BC drug resistance. Our results indicated that exosomal inhibited the PI3K/AKT signaling pathway by targeting PIK3R1. Activation of the PI3K/AKT signaling pathway is customarily regarded as an emblem of BC development (33). Furthermore, the mutant PI3K/AKT/mTOR pathway is frequently found in BC drug resistance (34). Consistent with the findings of the current study, inhibition of the PI3K/AKT signaling pathway has been reported to induce drug resistance in gastric cancer cells (35). As highlighted in our study, exosomal miR-221-3p-mediated PIK3R1 downregulation promoted BC drug resistance by suppressing the PI3K/AKT signaling pathway. The in vivo experimental results further confirmed that the regulatory network contributed to tumor formation.
In conclusion, drug-resistant BC cells-derived exosomal miR-221-3p could promote drug resistance of BC cells to ADR by targeting PIK3R1 via PI3K/AKT signaling pathway in vitro and in vivo (Figure 8). These findings may provide further perspective and insight for future investigations into the mechanism of BC drug resistance. However, this research is still at a preliminary stage, further investigation is still required regarding the action of the mechanism.
DATA AVAILABILITY STATEMENT
Publicly available datasets were analyzed in this study, these can be found in the NCBI Gene Expression Omnibus (GSE76540).
ETHICS STATEMENT
This study was approved by the Animal Ethics Committee of Huadu Hospital, Southern Medical University. Nude mice were employed in this study according to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health.
AUTHOR CONTRIBUTIONS
XP, XH, and RW wrote the paper and conceived and designed the experiments. JL, NH, and LC analyzed the data. YC and MY collected and provided the sample for this study. All authors have read and approved the final submitted manuscript.
ACKNOWLEDGMENTS
We would like to express our sincere appreciation to the reviewers for critical comments on this article. | 2020-04-30T09:11:22.716Z | 2020-04-30T00:00:00.000 | {
"year": 2020,
"sha1": "3b0761ded779d476677931093e984cd93190f074",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/articles/10.3389/fonc.2020.00441/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "883d106144b2af9c823ef361530f4b7b45f76a0c",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Biology"
]
} |
119189109 | pes2o/s2orc | v3-fos-license | Calculations of $K^-$ nuclear quasi-bound states based on chiral meson-baryon amplitudes
In-medium ${\bar K}N$ scattering amplitudes developed within a new chirally motivated coupled-channel model due to Cieply and Smejkal that fits the recent SIDDHARTA kaonic hydrogen 1s level shift and width are used to construct $K^-$ nuclear potentials for calculations of $K^-$ nuclear quasi-bound states. The strong energy and density dependence of scattering amplitudes at and near threshold leads to $K^-$ potential depths $-Re V_K \approx 80 -120$ MeV. Self-consistent calculations of all $K^-$ nuclear quasi-bound states, including excited states, are reported. Model dependence, polarization effects, the role of p-wave interactions, and two-nucleon $K^-NN\rightarrow YN$ absorption modes are discussed. The $K^-$ absorption widths $\Gamma_K$ are comparable or even larger than the corresponding binding energies $B_K$ for all $K^-$ nuclear quasi-bound states, exceeding considerably the level spacing. This discourages search for $K^-$ nuclear quasi-bound states in any but lightest nuclear systems.
Introduction
The study of the interaction of antikaons with baryonic systems, such as kaonic atoms, K − nuclear clusters or dense strange kaonic matter, is an interesting issue with far-reaching consequences, e.g. for heavy-ion collisions and astrophysics. The closely related problem of K − nuclear quasi-bound states is far from being settled, despite much theoretical and experimental effort in the last decade [1,2].
Our first studies of K − quasi-bound states in nuclear many-body systems based on an extended relativistic mean field (RMF) model were focused on the widths expected for K − quasi-bound states [3]. The subject of multi-K − (hyper)nuclei was studied in Refs. [4,5,6] with the aim to explore whether kaon condensation could occur in strong-interaction self-bound baryonic matter.
In the RMF formulation, the energy independent real K − nuclear potential was supplemented by a phenomenological imaginary potential fitted to kaonic atom data, with energy dependence that accounted for the reduced phase space available for in-medium K − absorption, including 2-nucleon absorption modes.
Weise and Härtle performed calculations of K − nuclear states in 16 O and 208 Pb using chiral-modelKN amplitudes within a local density approximation [7]. This paper reports on our latest calculations of K − nuclear quasi-bound states within a chirally motivated meson-baryon coupled-channel separable interaction model [8]. We apply a self-consistent scheme for constructing K − nuclear potentials from subthreshold in-mediumKN scattering amplitudes which was introduced in Refs. [9,10]. This time, theKN amplitudes are constructed using a recent in-medium coupled channel model NLO30 [11] that reproduces all available low energyKN observables, including the latest 1s level shift and width in the K − hydrogen atom from the SIDDHARTA experiment [12]. We demonstrate the crucial role of the strong energy and density dependencies of the K − N scattering amplitudes, leading to deep K − nuclear potentials for various considered versions of in-medium modifications of the scattering amplitudes. Using several versions of the chirally motivated coupled-channel model, we demonstrate the model dependence of our calculations. Moreover, we discuss the effects of p-wave interactions and the K − NN → Y N absorption modes. Finally, we present binding energies and widths of all K − quasi-bound states -including excited states -in selected nuclei.
The paper is organized as follows. In Section 2, we briefly describe the model and underlying self-consistent scheme for constructing K − nucleus potentials from in-medium subthresholdKN scattering amplitudes. In Section 3, we present results of our calculations of K − quasi-bound states in various nuclei across the periodic table. Conclusions are summarized in section 4.
Model
In this section, we briefly outline the methodology which forms the framework of our calculations of K − nuclear quasi-bound states. We concentrate only on basic ingrediences of the model since the details can be found in Refs. [9,10,11].
The K − nuclear quasi-bound states are determined by self-consistent solution of the in-medium Klein-Gordon equation: whereω K is complex energy of antikaon containing the Coulomb interaction V C introduced by minimal substitution: with Γ K being the width of K − nuclear state of energy ω K = m K − B K , where B K is the binding energy of antikaon. The self-energy operator Π K = 2(Re ω K )V K is constructed in a "tρ" form with the amplitude calculated in a chirally motivated coupled-channel approach: where F K − p(n) is the K − -proton (neutron) in-medium scattering amplitude in a separable form, p is the relative K − N momentum, and √ s is the K − N total energy. The realistic proton ρ p and neutron ρ n density distributions in the core nuclei are taken from relativistic mean-field nuclear-structure calculations. The K − N scattering amplitudes F K − N are constructed within a chirally motivated coupled-channel separable interaction model [8]. In this work, we applied the latest version NLO30 [11] which reproduces the recent K − hydrogen 1s level shift and width from the SIDDHARTA experiment [12]. For the sake of comparison, we applied as well the TW1 model [10,11] fitted also to the SIDDHARTA data, and the older CS30 model [8]. It is to be noted that in the TW1 (NLO30, CS30) model, the effective separable meson-baryon potentials are constructed to match the equivalent amplitudes derived from the chiral effective Lagrangian at leading (next-to-leading) order, respectively.
When the elementary K − N system is embedded in the nuclear medium of density ρ, one has to consider in-medium modifications of the scattering amplitude, in particular Pauli blocking in the intermediate states (this in-medium version is marked 'no SE') [13]. The other version (marked '+SE') adds selfconsistently meson and baryon self-energies generated by the interaction of hadrons with the nuclear medium [14,15]. Figure 1 illustrates a typical resonance-shape energy dependence of the inmedium reduced 1 scattering amplitudes for nuclear matter density ρ 0 = 0.17 fm −3 , corresponding to the interaction of the K − meson with symmetric nuclear matter. The amplitudes were calculated within the TW1 and NLO30 models and for each of the models, two in-medium versions, 'no SE' and '+SE', are shown for comparison. The pronounced energy dependence of the scattering amplitude appears crucial in the self-consistent calculations of kaonic nuclear states. In particular, the real part of the '+SE' amplitudes in both models changes from weak attraction at and above threshold to strong attraction at ∼ 30 MeV below threshold. As a result, the '+SE' and 'no SE' amplitudes become close to each other at energies relevant for self-consistent calculations of kaonic nuclei. While both models give similar imaginary parts Imf K − N for each in-medium version, they differ considerably (≈ 20%) in real parts of the scattering amplitudes below threshold. This indicates the extent of the model dependence of the calculations of K − nuclear quasi-bound states.
The scattering amplitude and relative momentum p. For nuclear bound-state applications it is necessary to transform the two-body K − N arguments into theK-nuclear c.m. frame. For the relative momentum p we have which upon averaging over angles yields p 2 of the form Similarly, expanding near threshold energy where B N stands for the binding energy of nucleon, and the last two terms represent corrections due to the kinetic energies of nucleon and antikaon. The nucleon kinetic energy is approximated in the Fermi gas model p 2 N /(2m N ) = 23(ρ/ρ 0 ) 2/3 MeV and the kinetic energy of antikaon is obtained by means of local density approximation p 2 This finally leads to We note that the K − potential V K and the K − binding energy B K appear as arguments in the expression for √ s , which in turn serves as an argument for the self-energy Π K , and thus for V K . This suggest a self-consistency scheme in terms of both V K and B K for solving the Klein-Gordon equation (1).
It is to be stressed that the present chiral model of K − -nucleus interaction does not account for the absorption of K − mesons in the nuclear medium through nonpionic conversion modes on two nucleons To estimate the contribution of two-nucleon absorption processes to the decay widths of K − nuclear states we introduced phenomenological term into the K − self-energy: where W 0 was fixed by kaonic atom data analysis and f Y N (B K ) is kinematical suppression factor taking into account reduced phase space available for decay products of K − nuclear bound states [3].
The present chiral model also does not address the p-wave part of the K −nucleus interaction. Since p-waves may play an important role for tightly bound K − nuclear systems [16] we studied their contribution by adding the self-energy term: with C K − N p-wave amplitude constrained by the Σ(1385) resonance phenomenology and parametrized following Ref. [7].
Results and discussion
We adopted the above methodology to calculations of quasi-bound K − states in selected nuclei across the periodic table. In most cases, we solved KG equa-tion self-consistently in a static approximation. For the sake of comparison, we performed also fully dynamical calculations upon taking into account the polarization of the nuclear core by the strongly bound antikaon. Figure 2 shows the K − nuclear potentials for 1s state in Ca at threshold ('E th ') and for √ s (Eq. (8) in-medium modification, in the 'no SE' case, the depths of ReV K evaluated at threshold and at √ s are close to each other. This weak energy dependence of ReV K has its origin in rather weak energy dependence of the 'no SE' NLO30 scattering amplitude below threshold, as shown in Fig. 1. The NLO30 model yields ∼ 20 MeV deeper ReV K than the TW1 model, as could be anticipated from Fig. 1. The imaginary parts of V K and consequently the widths which represent only K − N → Y N decays, are considerably reduced in the selfconsistent calculations of the subthreshold amplitudes owing to the proximity of the πΣ threshold (see both right panels of Fig. 2).
The peculiar role of the energy dependence of the K − N scattering amplitudes is illustrated for the TW1 model in Fig. 3. Binding energies B K of 1s K − nuclear quasi-bound states obtained by solving Eq. (1) self-consistently (denoted 's 1/2 ') for several nuclei are compared with B K calculated using threshold amplitudes (E th ). It is worth noting that the self-consistent calculations of B K using 'no SE' and '+SE' in-medium amplitudes give very similar results. These B K values are remarkably close to those calculated within a static RMF approach, when the K − -nucleus interaction is mediated exclusively by vector mesons with purely vector SU(3) F-type couplings (denoted 'RMF') [4]. For comparison, we present also binding energies B K calculated self-consistently using the NLO30 '+SE' amplitudes which are more than 10 MeV larger than the corresponding binding energies calculated using the TW1 amplitudes. Figure 4 shows the effect of particular hadron self-energies in the intermediate states on the K − binding energies B K and widths Γ K , calculated selfconsistently within the NLO30 model. The widths Γ K are plotted as function of B K for various options of implementation of the self-energies, as indicated in the legend of Fig. 4. The binding energies B K in all nuclei under consideration differ in all cases by less than 5 MeV and the effect of self-energies seems to be A independent. Implementation of pion self-energies leads to a sizable reduction of the widths Γ K in lighter nuclei (C, O). On the other hand, the role of hyperon self-energies seems to be marginal.
It is to be noted that the calculated widths shown in the figure represent only K − N → πY decays, accounted for by the coupled-channel chiral model. When phenomenological energy dependent imaginary ρ 2 terms are added selfconsistently to simulate two-nucleon K − NN → Y N absorption modes and their available phase space [3], the resulting widths of order Γ K ≈ 50 MeV become comparable in light nuclei to the binding energies B K . Figure 5 illustrates the model dependence of the K − nuclear state calculations by presenting B K and Γ K of 1s K − states in selected nuclei calculated selfconsistently using various chiral-model scattering amplitudes -TW1, CS30, and NLO30. The CS30 and NLO30 models yield larger binding energies and lower widths of the nuclear K − quasi-bound states than the TW1 model. is thus more pronounced than the effects of self-energies in the self-consistent calculations of kaonic nuclei. considered. This leads to considerable overlap of the K − quasi-bound states even in the lightest nuclei (except Li, where only the 1s K − quasi-bound state exists). It is to be stressed that the 2N absorption which was not considered here, adds additional sizable contribution to the widths, particularly of low lying states. Such large widths thus inevitably obscure experimental study of the K − nuclear quasi-bound states in heavier nuclei. Table 1 shows, as representative examples, binding energies B K and widths Γ K of K − nuclear quasi-bound states in Ca, calculated self-consistently using the NLO30 '+SE' scattering amplitudes. The results of fully dynamical RMF calculations which take into account the polarization of the nuclear core by the strongly bound K − meson, are compared with the static RMF scheme in the first 2 blocks. The dynamical calculations give, in general, higher binding energies B K and smaller widths Γ K . As could be anticipated, the polarization effect is A dependent: while it increases B K by ∼ 6 MeV in Li, it is less than 2 MeV in Ca (shown in Table 1), and in Pb the difference between static and dynamical calculations is less than 0.5 MeV. Effects of adding a p-wave K − N interaction assigned to the Σ(1385) resonance are demonstrated within the static RMF scheme in the third block of the table. The p-wave interaction increases the K − binding energy only by few MeV, being more pronounced in light nuclei where surface effects are relatively more important. Nevertheless, even in Pb the p-wave interaction increases B K by ∼ 2 MeV. Finally, phenomenological energy dependent imaginary ρ 2 terms are included to simulate 2N absorption processes K − NN → Y N. Whereas the K − binding energies B K decrease only slightly, the absorption widths Γ K > 50 MeV become comparable to the binding energies B K or even much larger in the case of higher K − nuclear quasi-bound states. Table 1 Binding energies B K and widths Γ K (in MeV) of the K − nuclear quasi-bound states in Ca, calculated self-consistently using NLO30 '+SE' amplitudes. Dynamical and static RMF schemes are compared in the first two blocks. Results of a static RMF scheme including p-wave amplitudes is shown in the third block, and K − N N → Y N decay modes are included in the last block ('+2N abs.').
Conclusions
We performed extensive study of the K − nuclear quasi-bound states within a chirally motivated meson-baryon coupled-channel separable interaction model. We considered two in-medium versions of the K − N scattering amplitudes: the 'no SE' version which takes into account only Pauli blocking in the intermediate states, and the '+SE' version which adds self-consistently hadron selfenergies. In addition, we used several versions of the model to explore model dependence of our calculations. In this contribution, we demonstrate on few selected examples main results of the calculations with the aim to assess the role of various ingredients of the approach that influence binding energies and widths of the K − nuclear states. Energy dependence of the in-medium scattering amplitudes, particularly in the K − N subthreshold region, is the decisive mechanism that controls the self-consistent evaluation of corresponding K − optical potentials. While the two in-medium versions of the K − N scattering amplitudes yield considerably different potential depths ReV K at threshold, they give similar depths in the self-consistent calculations with the subthreshold extrapolation of √ s. The role of hadron self-energies in the self-consistent calculations of the K − binding energies B K is less pronounced than the model dependence of predicted B K which amounts to ∆B K ≈ 15 MeV. As for the calculated widths Γ K , the model dependence and the effects due to the hadron self energies are comparable. The p-wave interaction generated by the Σ(1385) subthreshold resonance was found to play only a marginal role.
The widths of low-lying K − states due to K − N → πY conversions are substantially reduced in the self-consistent calculations, thus reflecting the proximity of the πΣ threshold. On the contrary, the widths of higher excited K − states are quite large even if only the pion conversion modes on a single nucleon are considered. After including 2 body K − NN → Y N absorption modes, the total decay widths Γ K are comparable or even larger than the corresponding binding energies B K for all K − nuclear quasi-bound states, exceeding considerably the level spacing.
The above conclusions should discourage attempts to search for isolated peaks corresponding to K − nuclear quasi-bound states in any but very light nuclear systems. | 2012-06-01T15:23:22.000Z | 2012-05-01T00:00:00.000 | {
"year": 2012,
"sha1": "db490ead6e8a15a1d30fb3f6336d24528d4b7200",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1206.0223",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "db490ead6e8a15a1d30fb3f6336d24528d4b7200",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
220963397 | pes2o/s2orc | v3-fos-license | PrescrAIP: A Pan-European Study on Current Treatment Regimens of Auto-Immune Pancreatitis
Introduction: Treatment of autoimmune pancreatitis (AIP) is based solely on consensus and has yet to become standardized. Consequently, therapeutic regimens vary greatly between countries and centers, and largely depend on the experience of the physician. At this moment, the optimal regimen for inducing disease remission and preventing relapse is unknown. Objectives: The primary objective of this study is to describe current treatment regimens used in Europe, and to compare their effectiveness in inducing remission and preventing and treating relapse. The secondary objectives are: to identify risk factors for relapse; to assess the diagnostic accuracy of the Unified-AIP criteria; to assess the performance of the M-ANNHEIM score for predicting relapse; and to assess long-term outcomes including pancreatic exocrine insufficiency and pancreatic cancer. Methods: This is an international, retrospective, observational cohort study, performed in over 40 centers from 16 European countries. Eligible are all patients diagnosed with AIP from 2005 onwards, regardless of the used diagnostic criteria. Data on study subjects will be retrieved from the hospital's electronic medical records and registered with a standardized, web-based, electronic case report form (eCRF). To compare the effectiveness of treatment regimens in inducing remission, preventing relapse, and treating relapse, subjects will be stratified in groups based on: type of therapy; initial therapy dose; cumulative therapy dose; therapy tapering speed and duration; and having received maintenance therapy or not. Ethics and Dissemination: Ethical and/or institutional review board approvals are obtained by all participating centers according to local regulations. The study complies with the General Data Protection Regulation (GDPR). All manuscripts resulting from the study will be submitted to peer-reviewed journals. Conclusion: This is the first pan-European retrospective registry for AIP. It will produce the first large-scale data on treatment of European patients with AIP, providing answers on the use and effectiveness of treatment regimens. In the future, this collaboration may provide a network for continuation into a prospective European registry.
Introduction: Treatment of autoimmune pancreatitis (AIP) is based solely on consensus and has yet to become standardized. Consequently, therapeutic regimens vary greatly between countries and centers, and largely depend on the experience of the physician. At this moment, the optimal regimen for inducing disease remission and preventing relapse is unknown.
Objectives: The primary objective of this study is to describe current treatment regimens used in Europe, and to compare their effectiveness in inducing remission and preventing and treating relapse. The secondary objectives are: to identify risk factors for relapse; to assess the diagnostic accuracy of the Unified-AIP criteria; to assess the performance of the M-ANNHEIM score for predicting relapse; and to assess long-term outcomes including pancreatic exocrine insufficiency and pancreatic cancer.
Methods: This is an international, retrospective, observational cohort study, performed in over 40 centers from 16 European countries. Eligible are all patients diagnosed with AIP from 2005 onwards, regardless of the used diagnostic criteria. Data on study subjects will be retrieved from the hospital's electronic medical records and registered with a standardized, web-based, electronic case report form (eCRF). To compare the effectiveness of treatment regimens in inducing remission, preventing relapse, and treating relapse, subjects will be stratified in groups based on: type of therapy; initial therapy dose; cumulative therapy dose; therapy tapering speed and duration; and having received maintenance therapy or not.
Ethics and Dissemination: Ethical and/or institutional review board approvals are obtained by all participating centers according to local regulations. The study complies with the General Data Protection Regulation (GDPR). All manuscripts resulting from the study will be submitted to peer-reviewed journals.
INTRODUCTION
Autoimmune pancreatitis (AIP) has recently been recognized as an immune-mediated disease of the pancreas with distinct features (1). It is a rare disease with an annual incidence of ∼3.1/100,000, which varies substantially between geographical regions (2). To date, two types of autoimmune pancreatitis have been described (1). Type 1 AIP is the pancreatic manifestation of IgG4-related disease (IgG4-RD). It shares clinical and histological hallmarks with IgG4-RD, namely increased serum Immunoglobulin G4 (IgG4) levels, dense storiform fibrosis, and IgG4-positive plasma cell infiltration of the affected organ (3). Type 2 AIP is known as idiopathic duct-centric pancreatitis and is characterized by neutrophil-mediated duct destruction, in the form of granulocytic epithelial lesions (GEL) (4). Both types can cause abdominal pain and jaundice and can ultimately lead to chronic pancreatitis (1). This in turn, might even increase the risk of developing pancreatic cancer (5). Yet, the incidence of these complications has not been clearly established.
In the last two decades, several efforts have been made to establish diagnostic criteria (1,(6)(7)(8), which are all based on the combination of clinical, serological, and pathological features. Nevertheless, AIP cases are still missed or even mistaken for pancreatic ductal adenocarcinoma (9, 10). As such, sequelae of chronic pancreatitis with endocrine and exocrine insufficiency can develop or the diagnosis of type 1 and type 2 AIP is made after unnecessary surgery (11). In addition, a discrete percentage of AIP cases does not fulfill diagnostic criteria for type 1 or type 2 AIP and thus are referred to as Not-Otherwise-Specified (NOS) AIP (1).
All AIP subtypes respond dramatically to steroid treatment (up to 99% in the different cohorts) (11)(12)(13)(14), but the optimal dose to induce remission remains controversial. Reported induction doses have ranges between 30 and 60 mg daily (11)(12)(13)(14) Recently, other therapeutic options, that induce B-cell depletion, have also been employed in inducing AIP remission, with promising results (15,16). In a cohort from France, the reported efficacy was 94% with two infusions given in most patients (17). However, in spite of the dramatic response to initial treatment, the risk of relapse within 1 year from disease remission ranges between 30 and 50% (12), being higher in type 1 than in type 2 AIP patients (12). Several risk factors for AIP relapse have been proposed, but have not been validated prospectively (11,12,18,19). Due to the high frequency of relapse after induction, some authors recommend maintenance treatment with low-dose steroids or immunomodulators, but the patient group that would benefit from maintenance is currently unknown (20)(21)(22)(23)(24)(25)(26).
Thus far, data on the epidemiology and natural history of AIP are still scant due to its rarity and its relatively recent appraisal. Most of the data comes from Asian or North-American cohorts, while data on large European cohorts are lacking. As a consequence, treatment options applied in Europe are largely based on retrospective studies from Asian and North-American patients.
We established the PrescrAIP (A Pan-European Study on Current Treatment Regimens of Auto-immune Pancreatitis) study network to retrospectively describe the current status of AIP treatment in Europe on a large scale. In addition, our effort will create the opportunity for a subsequent international prospective registry that will be able to provide definite answers in the future. In particular, in this retrospective multicentre study, we aim to define and compare the AIP treatment regimens used throughout different European centers, highlighting their differential impact on disease remission and long-term outcomes. These results will foster our knowledge of this rare disease, yielding to better patient care.
OBJECTIVES
The primary objective of this study is to describe AIP treatment regimens across Europe, and to compare the effectiveness of treatment regimens in inducing remission, preventing and treating relapse. The secondary objectives include identifying risk factors for relapse, assessing the diagnostic accuracy of the U-AIP criteria (6), assessing the performance of the M-ANNHEIM score for predicting relapse (19), and assessing longterm outcomes including pancreatic exocrine insufficiency and pancreatic cancer. Thirdly, we are aiming to assess whether standard treatment was altered due to pre-existing diabetes mellitus, determine the prevalence of diabetes mellitus before and after steroid treatment, assess glycemic control in patients with diabetes mellitus and to describe the clinical, radiological and pathological characteristics of a cohort of patients diagnosed with AIP through pancreatic resection.
Study Design
This is an international, retrospective, observational cohort study including all AIP patients. We used the Pancreas2000 framework (www.pancreas2000.org) to create a study network starting with the six centers in the PrescrAIP core group. Additional European centers with expertise in the treatment of AIP patients have been recruited, accumulating to a total of 44 collaborating centers.
Study Population
Every patient with an AIP diagnosis (type 1, type 2 or NOS-AIP) will be included, regardless of diagnostic criteria used (U-AIP, HISORt, ICDC). Patients with AIP diagnosed prior to 2005 will be excluded due to lack of uniformity in diagnostic standards.
Setting
The collaboration involves a large number of European centers, most of which are academic hospitals (Figure 1). Starting with the eight centers of the PrescrAIP core group, the study now encompasses centers in Germany (12), the United Kingdom (5), the Netherlands (3)
Data Collection
Patient data will be collected from the hospitals' medical records. Variables were selected to answer the research questions and meet the objectives of the study. Variables will be recorded in a REDCap-based (https://www.project-redcap.org) electronic datasheet (electronic case record form, eCRF), hosted by The North Denmark Region. The principal investigator of each site will ensure that the data in the eCRF are accurate, complete and legible. REDCap is a secure, online application designed to support data acquisition and storage by providing a shapeable interface for validated data entry.
Assessment Variables
At the time of inclusion, all relevant variables will be recorded in the eCRF. These will include variables on demography and epidemiology, disease characteristics (radiological, laboratory and clinical), the set of diagnostic criteria employed, treatment (type, dose, duration), and short and long-term clinical outcomes. The complete variable list and definitions are reported in Appendix 1. AIP subtypes will be defined following the analysis of the above-mentioned variables. Given that patients with elevated serum IgG4 but without other organ involvement or biopsy sample can potentially be misdiagnosed, a subgroup with these characteristics will be created. Then a sensitivity analysis with and without excluding this subgroup will be performed to evaluate potential differences in the outcomes.
Study Endpoints
Our primary endpoints consist of remission of disease (defined as the absence of clinical symptoms and the resolution of pancreatic abnormalities on imaging), relapse of disease, relapse rates compared between patients with a low or a high dose regimen, cumulative maintenance therapy dose and relapse-free survival time. Our secondary study endpoints are gold standard diagnostic tools of AIP compared to U-AIP, the prevalence of pancreatic exocrine insufficiency and the cumulative incidence of pancreatic ductal adenocarcinoma. The diagnosis of diabetes mellitus [as defined by the American Diabetes Association (27)] either before or after steroid treatment is our tertiary study endpoint.
Statistical Analysis
Continuous, non-normally distributed values will be presented as median and interquartile range (IQR), unless otherwise specified. Discrete variables will be presented as frequency (percentage). Normal distribution of continuous variables will be assessed with the Kolmogorov-Smirnov algorithm. Normally distributed variables among the different groups will be compared using the Student's t-test. Non-normally distributed variables will be compared using the Mann-Whitney U-test. Categorical variables will be analyzed by the Fisher's exact test or chi-square test.
To compare remission rates, relapse rates, relapse-free survival and long-term outcomes between participants, we will stratify participants according to the date of diagnosis, meeting the different diagnostic criteria, the type of treatment, the glucocorticoids starting dose, the cumulative dose, tapering speed, and treatment with maintenance treatment.
Moreover, to assess the impact of the treatment effect being part of the diagnostic criteria, we will perform sensitivity analyses comparing the study outcomes between those classified as AIP regardless of the steroid trial, and those in whom the diagnosis was dependent on the steroid trial. Sensitivity analyses will also be performed to compare the study outcomes including or excluding individuals that do not meet any available diagnostic criteria, and those classified as NOS-AIP.
Kaplan-Meier curves will be used to assess time-to-relapse. Time-to-relapse will be compared between subgroups using the log-rank test. Univariable and multivariable analyses with a backward selection procedure will be performed to identify possible predictors for relapse, based on a Cox-proportional hazards regression model. A significance threshold of P < 0.05 will be used.
Data from national pancreatic cancer registries will be used to determine age-and sex-specific incidence rates for pancreatic ductal adenocarcinoma. Standardized incidence ratios (SIRs) will be then calculated by obtaining the ratio of the observed to the expected number of cases, and 95% confidence intervals (95% CIs).
ETHICS AND DISSEMINATIONS
This study was reviewed and approved by the ethics committees of the centers in the PrescrAIP core group, namely those at the San Raffaele Scientific Institute (Milan, Italy), the South East Regional Committee for Medical and Health Research Ethics (Oslo, Norway), the Erasmus University Medical Center (Rotterdam, The Netherlands), the Aalborg University Hospital (Aalborg, Denmark), the Marmara University School of Medicine (Istanbul, Turkey), the Institute for Clinical and Experimental Medicine (Prague, Czech Republic), the Karolinska University Hospital (Stockholm, Sweden), and the Martin Luther University Halle-Wittenberg (Halle, Germany). In addition, participating centers had the protocol reviewed and approved wherever required by local regulations.
Patients' data will be collected retrospectively from preexisting electronic patient records. Study data will then be collected and managed using a REDCap database hosted at Aalborg University Hospital, North Denmark Region, Denmark. All data will be coded (pseudonymized). The key to the coded data will be stored locally in the participating site, in a password-protected file controlled by the Principal Investigator, and separately from any research data. For the German centers data will be anonymized immediately. When data is exported from the REDCap system for analysis, the data will be made completely unidentifiable and potential identifier variables will be removed. Additional processing of already collected data for the purpose of scientific research is exempt from specific consent according to Articles 5(1)(b) and 89(1) of the General Data Protection Regulation (GDPR). The study adheres to the Declaration of Helsinki. No patient will be exposed to any inconvenience in relation to the present study because all data are obtained retrospectively. The findings of the study will be published in a peer-reviewed journal and disseminated at national and international conferences.
DISCUSSION
AIP has only been acknowledged as a discrete entity in the last 20 years (1), even though the first reports date back to the 1960's (28). In the last decade, international efforts led to the creation of several sets of diagnostic criteria (1,(6)(7)(8), definitely raising awareness on AIP and providing guidelines for its treatment. Despite significant progress in the field, key questions related to the pathophysiology, diagnosis, treatment, and treatment-related complications of AIP remain unanswered. Therefore, AIP still poses a clinical challenge and the diagnosis is still overlooked. In particular, the role and efficacy of glucocorticoids in the induction of remission has been widely accepted and reported, but the optimal starting dose as well as tapering speed is far from being elucidated. In addition, several risk factors for relapse have emerged recently, but only few derive from large cohorts and none have been validated prospectively (11,12,18,19). Finally, long term outcomes in terms of pancreatic exocrine and endocrine dysfunction, as well as incidence rates of malignancies, have rarely been addressed, ultimately impacting on patients' nutritional status and survival (29)(30)(31).
To a certain extent, the scarcity of data available can be linked to the low incidence of AIP. Together with its relatively recent appraisal, this complicates the implementation of adequately powered randomized controlled trials. Through an international European-based, multicenter effort, we plan to shed light on this multifaceted condition in order to develop evidence-based treatment strategies in the future. As such, our aim is to collect all available AIP cases, reporting clinical, laboratory, and treatment-related features in order to create an accurate picture of the current European AIP management and to obtain novel data on the natural history of AIP in Europe. Stemming from these, new questions may be formulated and evaluated in a prospective continuation of the present study. In addition, as mentioned above, due to the relatively young age of AIP long-term sequelae have often been overlooked by seminal studies in this field. Yet, a better knowledge in the development of pancreatic exocrine and endocrine (type 3c diabetes or "pancreatogenic" diabetes) insufficiency or treatment related diabetes is warranted in order to deliver better quality of life.
This project has several strengths. Based on a multicentre cohort, this pan-European AIP study will provide a large dataset, dealt by field experts. Indeed, the vast majority of AIP reports derive from North-American or Asian cohorts, with most likely distinct genetic or environmental features that might not be shared by European AIP patients. Therefore, our study will plausibly provide a more homogenous AIP population thus far not included in recent trials. Due to the rarity of AIP, multicentric collaboration offers the unique possibility of obtaining more reliable results since larger patients' cohort can be analyzed. Moreover, the electronic datasheet will guarantee data quality and safety and will also stimulate data homogeneity across the various very heterogeneous countries and practices.
The study does not come without limitations, it being a descriptive retrospective study. There is a risk of the clinical and diagnostic guidelines being applied differently throughout the recruitment period. To mitigate this, we have collected clinical symptoms, laboratory and radiological findings rather than using the derivative AIP subtyping of the clinician. For example, as in most institutions pancreatic biopsies are not performed, seronegative type 1 AIP confined to the pancreas could be misdiagnosed as type 2. Conversely, atypical type 2 AIP with elevation of serum IgG4 might be classified as type 1 AIP. By subgrouping patients according to clinical, histopathological (where available) and radiological findings we aim to better judge the AIP subtype of the patient we aim to better judge the AIP subtype of the patient and increase transparency and uniformity of the AIP diagnoses made. As the response to a steroid trial might in some cases establish the diagnosis of AIP incorrectly, we will perform a subgroup analysis in patients in whom the AIP diagnosis was based on the successful steroid trial. Hereby, we will be able to estimate the effect of this group on the overall results of our analysis. In addition, we will be enabled to perform sensitivity analyses in that distinct subgroups, as described above. Lastly, many patients have a long follow-up, which should strengthen the true AIP diagnosis.
In conclusion, our work will provide detailed description on the natural history and management of AIP in Europe, representing a unique framework for future prospective studies that may be able to provide definite answers to the questions that remain after the current retrospective evaluation.
AUTHOR CONTRIBUTIONS
All named authors contributed to planning, conduct, and reporting of the work. | 2020-08-05T13:09:06.015Z | 2020-08-05T00:00:00.000 | {
"year": 2020,
"sha1": "c8a343c41741eeb982a4280112bb4b80caf89c62",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/articles/10.3389/fmed.2020.00408/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "c8a343c41741eeb982a4280112bb4b80caf89c62",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
252355476 | pes2o/s2orc | v3-fos-license | Perspectives of Participation in Daily Life From Cancer Survivors: A Qualitative Analysis
Objective To characterize how survivors of cancer define participation. Design Cross-sectional qualitative study. Setting Participants were enrolled from a large academic medical center in the Midwestern United States. Interviews were conducted over Zoom or phone. Participants Survivors of cancer (N=40) with brain, breast, colorectal, or lung cancer (n=10 per group). Participants were purposively sampled to maximize variation in the study sample. Participant ages ranged from 26-83 years, with a mean age of 55 years. Seventy percent of participants were receiving active cancer treatment at the time of the interview. Interventions Not applicable. Main Outcome Measures Participant perspectives gathered from 1-on-1 semistructured interviews. Qualitative description and thematic analysis were used to analyze interview transcripts and develop themes from the data. Results Survivors described participation as doing valued activities and highlighted 4 common aspects: (1) control; (2) social connection; (3) engaging in various contexts; and (4) cultivation of joy and purpose. Fully participating in life involved being able to do what they want to do without restrictions or limitations. Survivors’ perspectives of control outlined how competence, choice, adaptations, and locus of control influence broader feelings of control and participation. Interviews highlighted that participation remains central to daily life among survivors of cancer. Conclusions Rehabilitation researchers and clinicians need to establish a standard and comprehensive definition of participation. Rehabilitation providers need to consistently evaluate how participation is affected among survivors of cancer and use measures that include core aspects of participation identified in this study and previous research. Comprehensively defining participation will improve the design and selection of measurement tools and support comprehensive assessment of survivor experiences.
Conclusions: Rehabilitation researchers and clinicians need to establish a standard and comprehensive definition of participation. Rehabilitation providers need to consistently evaluate how participation is affected among survivors of cancer and use measures that include core aspects of participation identified in this study and previous research. Comprehensively defining participation will improve the design and selection of measurement tools and support comprehensive assessment of survivor experiences. © 2022 The Authors. Published by Elsevier Inc. on behalf of American Congress of Rehabilitation Medicine. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Participation is recognized as the ultimate goal of rehabilitation, 1 yet there remains no universally accepted definition or standard approach to its measurement. [1][2][3][4][5] Participation was defined as "involvement in a life situation" in the International Classification of Functioning, Disability, and Health, 6(p10) but this broad definition does not fully encompass the construct. 7 Continued challenges defining and measuring participation may contribute to its limited uptake beyond the field of rehabilitation.
Survivors of cancer, identified as survivors at diagnosis, 8 are a rapidly growing population 9 and a key group of individuals among whom we need to better understand participation and rehabilitation needs. 10 While researchers, clinicians, and survivors recognize the need to support survivors in returning to work, school, and life activities, 11 they do not identify these as domains of participation. Cancer care does prioritize quality of life, 12 a related, but separate construct from participation. 13,14 Ultimately, how rehabilitation scientists explain participation to other disciplines may support or further hinder its clinical integration 15 and opportunities to refer survivors of cancer to rehabilitation.
As we work toward integrating participation into cancer care, we first need to understand what participation means to survivors of cancer. To further elucidate this complex construct we aimed to characterize how survivors of cancer with brain, breast, colorectal, and lung cancer define participation. Secondarily, we aimed to describe how cancer affects survivors' perspectives of participation.
Methods
The institutional review board and protocol review and monitoring committee at Washington University in St. Louis School of Medicine approved this cross-sectional study.
Study sample
This study was conducted with patients of a large academic medical center located in the Midwestern United States. Participants included adults (older than 18 years) with any stage/grade brain, breast, colorectal, or lung cancer. Eligible individuals were in any phase of treatment or post treatment. We included multiple common cancer types 16 to evaluate variations and commonalities across diagnoses. We aimed for 10 participants per stratum (N=40), which is typically a sufficient number to reach thematic saturation, 17 the point when new themes are no longer emerging from the data. 18 Individuals were excluded who were not English-speaking or had aphasia because their ability to engage in a conversation would be limited. Purposive sampling was used to maximize variation, identifying individuals on and off treatment and with varying participation challenges. Potential participants were informally screened by the medical and research team to develop a basic understanding of their participation restrictions. A purposive sampling approach was used to ensure we were not gathering a sample of extremes or overrepresenting the perspectives of 1 group of individuals, for example, those with severe impairments.
The oncology treatment team approached patients about the study during clinic visits. Contact information of interested patients was shared with the study lead. One individual was recruited via snowball sampling. Verbal informed consent was completed over the phone. Most participants selected a preferred date and time of interview at the time of consent.
Data collection
Individual semistructured interviews were conducted over Zoom with video (n=16), without participant video (n=4), or over the phone (n=20) between September and November 2020. All participants were in a private location, typically the home, during the interview. A researcher not part of the treatment team conducted all interviews (mean, 57 minutes; range, 30-81 minutes). An interview guide (online supplement appendix) was developed based on previous research 7 and clinical knowledge to understand the effect of cancer on survivors' daily lives. Interviews were audio recorded, transcribed verbatim, and deidentified. Participants self-reported demographic characteristics and treatment history.
The data presented in the current report are part of a broader mixed-methods research study focused on the participation experiences of survivors of cancer. The interview guide is divided into 2 sections in which participants first discussed their participation experiences and then provided feedback on 3 measures of participation. Please refer to the online supplement for additional details on the quantitative participation measures completed before the interviews. The entirety of each transcript was coded and included in this analysis, but only data specific to our current research question are presented.
Data analysis
Data were analyzed through an iterative team-based process by 5 researchers. Guided by qualitative description 19 and thematic analysis, 20 a primarily inductive approach to coding was used. 20 Thematic analysis is a process through which researchers synthesize participant experiences to identify collective meaning within the data. 20,21 Codes and definitions were documented in a codebook that was piloted by the team (A.L., A.M., A.T., N.H.) on 4 transcripts. The codebook was modified to improve clarity and tested on 1 additional transcript. Two coders independently coded each transcript; 1 team member reviewed coded text and coding discrepancies. When indicated, team discussion was used to resolve coding discrepancies; the single code reconciler (A.L.) made the final decision on text coding. Themes were developed (A.L., N.S.) by continuously analyzing and reviewing coded text to identify data patterns. 20 Data were coded using NVivo 12 software. a For additional methodology, see the online supplement.
Results
Sixty-one survivors of cancer were contacted to participate after expressing interest in the study during an oncology clinic visit. Ten could not be reached, and 6 were no longer interested (reasons: request of spouse, too busy, did not feel their life was affected by cancer). Forty-five consented; 3 were lost to follow-up, 1 withdrew because of time limitations, and 1 wanted an inperson interview, which was not possible because of COVID-19 research restrictions. Throughout the data collection process, ongoing data analysis was conducted to identify emerging themes and assess saturation. After 35 interviews, we determined no new themes were emerging; however, only 6 individuals with colorectal cancer had participated. To ensure themes unique to the colorectal group were not missed, additional interviews were completed for an equal sample of 10 per group. Forty survivors aged 26-83 years participated (table 1).
African American or Black 6 (15) 1 (10) 4 (40) 1). The final theme highlighted that participation remains central to daily life among survivors of cancer. Themes were assessed for differences across diagnosis groups, but no major differences were observed.
Control
Participants emphasized their desire to do what they want to do without restrictions. They wanted control and choice over what, how, and when they participate: "To physically be able to do whatever it is I want to do. Um, whatever that may be. Not to have any limitations." (50 years, male, colorectal.) Embedded within control were the concepts of competence, or the skills or abilities needed to participate, locus of control, adaptation, and choice.
Competence (skills and abilities)
Cancer dominated the lives of many participants. Thus, their cancer experiences were often embedded within how they defined participation: I did a lot of cooking and baking. Um I loved doing that for my family [voice cracks and gets high-pitched] and I can't really do that now because-and all of this is difficult because my left side is affected. The motor function has been um affected. . .so a lot of things have been lost." (62 years, female, brain.) Survivors' definitions emphasized treatment and disease adverse effects (eg, fatigue, colostomy issues, etc) and cancer-related limitations. Cancer and its treatment resulted in a change in the underlying skills of many individuals. With a change in skills, such as inability to raise an arm or stand for 5 minutes, survivors experienced a loss of control over their ability to do what they valued in life.
Participants felt they needed certain skills (eg, physical, cognitive) to be competent and able to fully participate: Perceptions of what skills are needed to participate can be highly individual and yield different perspectives of participation. These differences are related in part to an individual's locus of control.
Locus of control
Two perspectives of control, internal and external loci, were apparent within survivors' definitions of participation. With an external locus of control, cancer was often in control of the individual's life and survivors felt unable to change their abilities: "I'd want to get rid of my physical restraints and go back to doing things." (65 years, male, brain.) Many felt that if they had specific abilities they would be able to participate: "More and more, I come to think up of, well, if I could breathe, I used to like to work in the yard . . . I used to plant bulbs and flowers . . . I just absolutely can't do that anymore." (75 years, female, lung.) A loss of skill was often associated with the cancer experience and seen as outside of an individual's control.
With an internal locus, some survivors had a "don't let cancer stop me" mentality. These individuals focused on pushing past challenges to participate in life. An internal locus was also more dominant among survivors who experienced fewer adverse effects and participation restrictions. Overall, the complexity of survivors' experiences often highlighted both loci of control: Most of it feels outside of my control. Yeah, I guess there's always more you could do. You know you could be a lot more self-motivated . . . I could take more chances, you know with the getting out of the house than I do. But I mean even yesterday, like I ran to [store], which is probably 5 minutes from our house and I had an accident, you know, while I was out so it kind of discourages you from wanting to get out." (59 years, female, colorectal.) Some survivors were still processing what control they had over their participation. Negative experiences often reinforced the control that cancer had over their lives.
Adaptation
Adapting, putting forth effort, or making specific plans to participate were strategies used by some to support participation. Adapting was a way to take action, control a situation, and control their participation. Some struggled to problem solve through how, when, or what to adapt to participate. After thinking through multiple ways to modify a leisure activity, a 39-year-old female participant with a brain tumor explained: "There's no way I could do that [activity] right now." Despite wanting to adapt to participate many survivors expressed they were restricted in what or how they could participate because of a lack of control over their abilities and circumstances.
Choice
There was also an emphasis on having opportunities to choose to do what they want to do, another way of expressing they want control over their participation. However, many felt limited control over their lives because of their cancer experience. When an external locus of control was dominant, participants expressed they had fewer choices in what and how they participated.
Participants felt personal control when in a more stable situation with their disease and when they had the skills and resources needed to participate. Resources provided choices and helped survivors participate in what they desired.
Social connection
Participation often involved doing activities for or with others, typically family and friends. Examples included cooking, going out with friends, or having a game night. Participation was often discussed in the context of life roles, such as being a spouse, parent, grandparent, or friend.
There was a desire among participants to be present and visit or spend time with others. Family members (spouse, children) were frequently identified as survivors' primary motivation and support to participate. Even when a few individuals discussed more solitary activities (eg, writing), some still connected these activities to other individuals (eg, motivated by other people enjoying her stories). For many, their cancer experience highlighted the importance of relationships and some made it a priority to participate with others: "I just spent more time trying to do things with other people . . . I had a kayak for a year by myself and I was like, 'why am I doing this by myself?' So I got my wife a kayak." (26 years, male, brain.) Communicating and socializing were central to participation and were important throughout the entire cancer continuum: "To be able to interact with others and um being able to talk with them back and forth." (67 years, male, brain, considering hospice.) Survivors expressed their desire to form connections with others through participation, but these opportunities sometimes became limited after a cancer diagnosis: I used to be able to get up and do stuff, I was interacting with people, um you know, seeing them at the university, seeing them at church, you know what I mean? I was always interacting with people. Um, now I don't at all, and it's been [short pause, sounds emotional] something I really, I'm sorry about." (75 years, female, lung.)
Engaging in various contexts
Survivors discussed the importance of doing valued activities in various contexts such as the home, neighborhood, city, and work. For a subset of the sample, their concept of full participation included doing valued activities outside the home. Examples included golfing, shopping, driving, walking outdoors, traveling, and going to restaurants and appointments. Survivors wanted to "go out" to do things with others and to enjoy life: "Be able to travel and see things and experience the things in nature that I would like to enjoy." (49 years, male, colorectal.) Some survivors acknowledged that their cancer experiences limited their participation outside the home because of physical challenges, body image or infection concerns, bathroom urgency, and shifts in what they found important in life. For some, their home environment became their place of safety, and they became hesitant to participate outside the home. When engaging less outside the home, social networks also became smaller, as discussed in the previous theme.
Survivors rarely focused on engaging with the broader community. While some discussed volunteering, valued activities outside the home were often done independently or with close family and friends, with little emphasis on broader community-focused activities: [Being sick from chemotherapy was] when I realized how much I'd like to participate. Not necessarily in like group or community activities but just the basics, going to the store, uh you know, going shopping, taking the dog for a walk again, golfing with my kids, hanging out outside with my kids." (50 years, male, colorectal.) participation. Participants explained they wanted to continue to live life and do what they enjoyed even while dealing with cancer. There were phases when participating was very limited for some, but it was still something they highly valued and fought to continue to do. Participating in life was identified as a priority among survivors. When participation restrictions were experienced, distress was common.
Cultivation of joy and purpose in life
Cancer altered many survivors' perspectives of what was important in life and their participation priorities: "My interest and desire for things that used to be there, it just kind of dropped off . . . I guess that the analogy of before it was a floodlight and now it's a focused pin light on what I find enjoyable." (49 years, male, colorectal.) While some developed a narrower focus, others broadened their horizons and cancer was a catalyst to participate more fully: "I think I've found my purpose . . . before I was just alive. And now I think I am living. I am doing more exciting things . . . and seeing different things that I've always wanted to see. And I love it." (54 years, female, breast.) Survivors repeatedly emphasized their focus on not taking life for granted and appreciating life, which was central to how they viewed and defined participation.
Discussion
Research aiming to broaden the conceptualization of participation has historically focused on the perspectives of individuals who receive intensive rehabilitation after an injury or illness (eg, spinal cord injury, stroke). This study is the first to investigate how survivors of cancer conceptualize participation. Survivors' perspectives confirmed previous findings about core aspects of participation including control, choice, competence, social connection, community engagement, and meaning generation. 1,2,7,22 Findings also highlight how perspectives of participation may vary among individuals with different types of disabilities or medical conditions. The study by Hammel et al examining what participation means to individuals with disabilities primarily included individuals with spinal cord injury, stroke, or traumatic brain injury. 7 Societal perceptions were emphasized in the participation values outlined by Hammel, including societal inclusion, rights to resources, and the meta theme of respect and dignity. 7 The current study did not confirm these findings among survivors of cancer. Participants in Hammel's work likely had more permanent and static disabilities than survivors of cancer. Cancer is a dynamic process 23 and can result in a disability experience that is constantly evolving. Instead of focusing on how society views them and can adapt to their needs, many survivors are occupied by their disease, treatment, and associated changes within their body. Survivors identify the source of their cancer-related impairments (eg, fatigue) as coming from within; consequently, environmental adaptations and societal influences are underrecognized. Disability 7,24 and survivor of cancer identitites 23,25 can further shape perspectives of participation and rehabilitation. 26 Cancer-related disability is common, 16 but it is unknown whether survivors see themselves as people living with disabilities. Many of the disabling conditions experienced by survivors (eg, cognitive impairments) result in hidden disabilities. The hidden nature of these disabilities may explain why participants' perspectives of participation did not reflect the respect and dignity meta theme. 7 Survivors may not see themselves as having disabilities, and neither does society, so survivors may not face the stigmatization experienced by other groups living with disabilities.
Survivors also frequently anticipate resolution of cancerrelated symptoms once they have completed treatment. They expect, and are often told by medical providers, it may take time to get back to their "old self" and return to normalcy. 27 However, the complexities of cancer-related disability make it difficult to assess when and if patients will return to baseline and resume participating. Participation is a critical outcome of rehabilitation, 28 and we have demonstrated it is important to survivors of cancer.
Future research needs to define participation for a broad group of individuals with varying disabilities. Facets of participation identified in this study and previous research 7,22,29 need to be systematically tested to develop an accepted comprehensive definition of participation. Measurement tools can then be refined and tested to support the clinical integration of participation measurement. Evaluating variability in participation experiences can help providers identify individuals who may benefit from rehabilitation. 10
Study limitations
Environmental influences were rarely discussed by survivors. This may be because of the interview guide design; we were seeking to understand participation broadly and did not probe on environmental factors. However, survivors did not discuss environmental factors when asked about barriers or supports to participation, suggesting survivors may be less aware of environmental influences on participation. Prior to the interviews, survivors completed 3 quantitative participation measures because a portion of the interviews focused on engaging survivors in providing feedback on these measures. The content of the participation measures may have primed survivors about what the construct of participation means, as defined by assessment developers. It is unclear how this conceptualization of participation by survivors translates to all survivors of cancer and other populations. Our sample was intentionally broad to capture a wide range of experiences. It is encouraging that the core aspects of participation identified in this study are consistent with previous research.
Conclusions
Rehabilitation researchers and clinicians need to establish a standard and comprehensive definition of participation, which will support the design and selection of measurement tools. Rehabilitation providers need to consistently evaluate how participation is affected among survivors of cancer and use measures that include the central aspects of participation identified in this study and previous research. | 2022-09-19T05:05:51.901Z | 2022-07-02T00:00:00.000 | {
"year": 2022,
"sha1": "fbae9555924e5984c3a7f27c076950c03c558b84",
"oa_license": "CCBYNCND",
"oa_url": "https://doi.org/10.1016/j.arrct.2022.100212",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "fbae9555924e5984c3a7f27c076950c03c558b84",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
116624032 | pes2o/s2orc | v3-fos-license | Experimental and Numerical Simulation of the Heat Transfer Enhancement on the Twin Impingement Jet Mechanism
: This article presents a study which sought to enhance heat transfer by employing a twin jet impingement mechanism (TJIM) and investigating the impact of the distance between the nozzles and plate on the Nusselt number (Nu) and heat transfer coefficient. This investigation was additionally based on the measurements of the heat flux temperature micro foil sensor and IR thermal imaging. A computational study of the cooling heated plate, through simulating the electronic components by the TJIM, was investigated using the RNG k- ε turbulence model. The jet-plate position was changed at the different jet-to-plate distances S = 1, 6, and 11 cm, Reynolds number = 17,000. The main flow structure, the static pressure, local and average Nus and heat transfer coefficient, were also examined. The findings have yielded new information about TJIM, and represent a new contribution about the flow and heat transfer characteristics of TJIM, and means of improving the rate of heat transfer in the passive heat transfer technique. The results of the various positions of the TJIM determined that the first model is, in fact, the best model for the heat transfer coefficient and the highest Nu, when S = 1 cm and H = 1 cm. Furthermore, the irregular distribution of the local Nu and the local heat transfer coefficient (h) on the impinged surface are due to the increase or decrease in the turbulence of flow on the measured surface.
Introduction
Impingement jets are widely used in many engineering applications, especially in last few decades, to enhance heat transfer rates [1][2][3]. In this study, experimental and numerical methods are employed to investigate twin impingement jet flow and impingement heat transfer. The results from the experimental work and simulations are then analysed and discussed, along with the impacts relating to the heat transfer that occurred due to the impingement of the twin jets on the hot plate surface. The study further investigates the twin impingement jet flow, by examining the influence and effects of applying different parameters, such as Nozzle-Nozzle spacing and Nozzle-plate distance [4,5]. The flow characteristics of twin jets incorporate the velocity of the air jet in the external hole when the Reynolds number is equivalent to 17,000. The issues associated with heat transfer are correlated using five factors that affect heat transfer, and where further investigation of the heat transfer enhancement associated with the twin jets impingement is conducted. Importantly, the experiments in this study were performed following a complete verification of the experimental setup [6].
In this article, a computational study is undertaken on the cooling of industrial and engineering components, in an attempt to enhance the heat transfer coefficient [7]. Jet impingement has a significant impact on the cooling process of a flat surface, as well as on electronic applications [8,9]. Many researchers over the years have been motivated by the characteristics associated with heat transfer in the cooling process [9,10]. The flow and heat transfer characteristics of a single jet impingement mechanism on the protrusion surface have been investigated using numerical simulation and particle image velocimetry (PIV) [11]. A ground fast cooling simulation device was employed to investigate the impact of arrangement and nozzle geometry on the impinging jet's heat transfer [12]. Large eddy simulation was carried out to study the flow dynamics and heat transfer characteristic of multiple impinging jets at high Reynolds numbers, and in a hexagonal configuration [13]. A numerical investigation and design optimization were carried out for impingement cooling system with an array of air jets [14].
However, there has been limited research to investigate the twin impingement jets mechanism numerically and experimentally. Accordingly, one of the key objectives of the present study is to simulate the characteristics of heat transfer in twin jets on a heated plate surface which is cooled by TJIM at different jet positions. This provides information on the most suitable model through the best heat transfer rate from different arrangements of jets on the surface measured, and is of considerable significance in the performance of industrial applications, and in efforts to improve the rate of heat transfer in passive heat transfer techniques using TJIM. The current study is conducted both experimentally and numerically, based on ANSYS FLUENT software (version 18, Ansys, Inc., Canonsburg, PA, USA). SOLIDWORKS software (Dassault Systèmes SolidWorks Corporation, Waltham, MA, USA) will be used to create and grid the system geometry, and to simulate the cases for the geometry model with two jets.
Turbulence Model
Computational Fluid Dynamics (CFD) provides a qualitative (and sometimes even quantitative) prediction of fluid flows for solving problems, which consists of four subject domains: geometry and grid generation, establishing a physical model, solving it, and post-processing the computer data. Using this approach, many of the CFD problems may be solved along with the acquired data that is computed using several tools to assist in creating the correct geometrical designs and grid patterns. However, no single model suits all conditions or requirements-especially for industrial and engineering applications-for establishing a physical model for turbulence flows, given that all models differ in some way. The importance of understanding the influencing factors for selecting the right model is paramount and often requires researching previous models used by other researchers that touch upon heat transfer and turbulence models, including their limitations and advantages.
Therefore, in selecting an appropriate turbulence model, many factors may influence the final decision, including: the physics associated with the overall flow model, whether the model is based on a proven method and approach, resolving of specific problems, the level of accuracy, assumptions, resource requirements, and computing power required to process large volumes of data quickly and precisely. Often overlooked is the allocated time needed for the simulation exercise, as this can be considerable, and may require additional processing power and effort.
One model that has been commonly used by many researchers is referred to as the common K-epsilon (k-ε) model, due to its abilities to provide accurate results, its affordability, the fact that it is relatively straightforward to implement and provides reasonable predictions for many flows, and that it has been widely used to simulate mean flow characteristics for turbulent flow conditions [15]. Notably, this model has been used to investigate turbulent flow problems through various simulation exercises and experiments in many countries.
Furthermore, what makes the K-epsilon (k-ε) model stand out, is that it is a two-equation model which gives a general description of turbulence using two transport equations, partial differential equation (PDEs): turbulent kinetic energy (k) and turbulent dissipation rate (ε), which are solved, and the eddy viscosity (µ t ) is computed as a function of k and ε. The Boussinesq hypothesis is also part of this model, given its low computational cost, and is applied to model the Reynolds stress term. Therefore, in applying this model, several assumptions are made. The working fluid is air, and the flow characteristics are: steady flow, Newtonian fluid, an incompressible fluid, and turbulent flow. The Boussinesq approach also utilizes the Boussinesq hypothesis to reduce the Reynolds stresses to the mean velocity gradients as given below in Equation (1): where the eddy viscosity (µ t ) at each grid point is related to the values of the (k) and (ε), [16]: Moreover, where C µ is an empirical constant; and k is the turbulent energy is defined by the fluctuating velocities as: The assumption here is that the flow is entirely turbulent, and the impact of the molecular viscosity is negligible.
Experimental Procedures and Methodology
Two types of impingement plates form part of the experimental setup according to the application of jet impingement heat transfer. First, an electrically heated plate is used for the jet impingement cooling application, and second, the thin film relatively cold plate is used in the hot jet impingement application. Accordingly, in both plates, a heat flux was generated due to the temperature difference between the air jet and the plate surface. Furthermore, the impingement plate surface used for the cooling application was designed and fabricated to be used as a heat source [17]. The plate was made of aluminum, chosen for its high thermal conductivity (204 W/m·K) and light weight (2707 kg/m 3 ) [18]. The dimensions of the plate were 4 mm × 300 mm × 300 mm; an electric plate heater of equivalent surface dimensions were fixed onto the back of the plate, and a steel plate supported the back of the heater with the same surface dimensions. All three components were then bolted together and fixed to an aluminum frame holder. It was possible to adjust the height of the plate. The impingement plate of the cooling application is shown in Figure 1. The experimental procedures consisted of five steps:
1.
For each jet in the steady state, the airflow was initially set to acquire a Reynolds number of 17,000 by measuring the twin jet middle (center) point velocity at the nozzle exit using a pitot tube.
2.
To measure the flow rate and steady jet flow, a flow meter anemometer was installed in a constant temperature mode of 100 • C by Dantec Dynamics, who specialize in the development, manufacture and application support of measurement systems. Next, the flow meter was positioned between the refrigerated air dryer and the TJIM pipes, which pass through to the twin jets. It is important in this step to verify the speed of the flowmeter from the pitot tube through to the operation of the twin impingement jets. By capturing the heat transfer per unit (q) via the data logger, the highest Reynolds number is determined by calculating the convective heat transfer coefficient (h) by the units (W/(m 2 ·K)). From the measured surface, the average Nu is then obtained for the points at the radial distance on the surface from the stagnation point.
3.
In this step, differential pressure produces a pressure difference as an analog that is then inputted to the data acquisition Ni 6008 device, which converts it to a signal. Using the Scilab code (developed beforehand), the signal is converted to a value. Notably, the differential pressure occurs between the pitot tube and the Ni 6008 data acquisition device.
4.
Three models are next constructed for the experimental tests using aluminum foil at a measured distance of 1, 6 and 11 cm from the nozzle exit and surface, with spacing between the nozzles of 1 cm. The purpose of this step is to allow measurements of the heat flux and surface temperature to be taken on the impingement surface.
5.
To capture the thermal images and temperature distribution at the surface, an infrared thermal imager (Fluke Ti25) is used to simultaneously capture images at the surface region until heat transfer to the steady state is achieved. When the heat inlet to the aluminum foil by the jets equals the heat loss by the effect of natural convection, heat transfer is achieved. During this step, to minimize errors in the heat flux temperature, several samples were recorded, to determine the average values. (see Figures 2 and 3). Before conducting the tests, various constant parameters were maintained related to the infrared thermal imager and TJIM as shown in Table 1. Next, the fluid mechanics and heat-transfer are considered in addressing the problems of jet impingement heat transfer, and consequently, the dimensionless numbers that are related must also be specified. The computation of the Reynolds number (the velocity) of the twin air jet-which correlates to the inertial forces because of viscous effects-is given as follows [19]: where µ represents the fluid's dynamic viscosity (Pa·s or kg/(s) or N·s/m 2 ), ν signifies the fluid velocity x (m/s), and ρ denotes the fluid density (kg/m 3 ). Forced convection dominated during the jet impingement heat transfer. The Newton's law equation can be employed to determine the heat-transfer coefficient (h), i.e., [20] where T s represents the surface temperature, T j signifies the air jet temperature and q denotes the amount of heat flux (W/m 2 ). The Nu equation is used to calculate the ratio of convective to conductive heat transfer, as follows [19]: where h indicates the convective heat-transfer coefficient, d represents the pipe diameter, and k signifies the fluid's thermal conductivity.
Governing Equations
A series of governing equations are established as follows: • The governing equations to be solved include the momentum, continuity and energy equations [21,22].
•
The mass of fluid is conserved.
•
According to (Newton's second law), the rate of change in momentum is equal to the sum of the forces on a fluid particle.
According to the first law of thermodynamics, the rate of change of energy is equal to the sum of the rate of heat added, and the rate of work performed on a fluid particle.
Geometrical and Flow Description
The schematic diagram of a twin jet impingement mechanism (TJIM) of the three models that depend on the spacing between the nozzle and distance between the nozzle and plate and the cross-flow between the twinjet and the impinging plate is illustrated in
FLUENT Software Package
The flow equations are solved using two modules: 1.
The first module is a pre-processor module, a program structure that creates the geometry and grid using ANSYS FLUENT for: (a) Modeling of the geometry; (b) Mesh generation; and (c) Boundary condition.
2.
The second module is the 'Solution' module, for solving the Navier-Stokes equations (which include momentum, continuity and energy equations), as well as the turbulent flow model.
Mesh Generation
There are two kinds of approaches used in volume meshing: structured and unstructured meshing. FLUENT can use grids comprising of tetrahedron or hexahedron cells in three dimensions. However, the type of mesh selection depends on the application.
In a structured mesh, the governing equations are transformed into the curvilinear coordinate system aligned with the flat surface. While it is trivial for simple shapes [23], it becomes incredibly inactive and time consuming for complex geometries, and therefore is excluded in this study. In the unstructured process, the integral form of the governing equations is discretized, and either a finite-volume or a finite-element scheme is used. The information regarding the grid is directly inserted into the discretization. Unstructured grids are generally successful for complex geometries, rather than applications such as that described above. Notably, unstructured tetrahedron grids were used in the present work. A higher order element type is used for mesh generation to approximate the precise the boundaries of high curvature, including the following: • For a three-dimensional mesh, a higher order tetrahedral element with hanging nodes (i.e., nodes on faces that are not vertices of all the cells sharing those faces) is used.
Three Dimension Mesh Generation
Although many mesh generation codes (e.g., FLUENT and ANSYS) support mesh generation of solid geometry and three-dimensional models from a single phase with minimum input from the user, it is more durable to divide this process into subsequent steps, including the following two key issues to further control the mesh [24].
Surface Mesh Generation
The surface mesh is created for the flat aluminum surface, the TJIM, and other boundaries as follows: (a) The edges are meshed by assigning an interval size for each boundary comprising a closed loop of the area; (b) The meshed edges are controlled by specifying a grading scheme for each edge; (c) On the edges of the mesh, a triangular element is used to generate a three-dimensional pave unstructured surface mesh.
Volume Mesh Generation
For the surfaces meshed for each region, the volume mesh can next be created for each area comprising of a closed loop of the area using the T-Grid, ANSYS scheme. Notably, building the mesh requires fine cells in the zones near the surfaces and edges in a TJIM. Furthermore, the mesh should be manipulated and controlled manually to maintain a smooth mesh transition, and to retain accurate mesh for a three-dimensional model. Moreover, with a minimal computational expense, this is accomplished by applying the size function. The features of the cases : the relevance center used was medium, the normal curvature angle of 12-degrees, the minimum size = 3 × 10 −4 , max face size 4 × 10 −3 , and the max Tet size 4 × 10 −3 (see Figure 5).
Mesh Quality and Mesh Dependency
The generation of the mesh and mesh control for the three-dimensional model has been previously discussed in this article. Therefore, it is important to assess the mesh for both quality and dependency before running to guarantee that accurate results are successfully achieved. Furthermore, there are some general guidelines to produce good mesh, known as the rules of QRST standing for Quality, Resolution, Smoothness, and Total cell count [25].
Quality of Mesh
Investigating the mesh quality and to check the orientation of the elements is important. The face alignment represents the importance of the quality parameter; it is the parameter that calculates skewness of the cells. Also, the elements with high skewness should be averted. The face skewness can be calculated as given in Equation below as: Accordingly, this value is about (0.2-0.5) according to [24].
Mesh Resolution
The grid-dependence analysis is held by creating a grid with more cells and to compare the solutions of the two models. The grid refinement tests for the average static temperature on a hot surface indicated that a grid size of approximately (one million cells) could produce sufficient accuracy and resolution to adopt as the standard for the models. Furthermore, the number of cells should be more near viscosity, influencing areas like the walls and the smaller at non-critical zones.
Total Cell Count
The last point in producing a good mesh is through the total number of cells produced. Accordingly, it is necessary to acquire a sufficient number of cells to achieve a good resolution, but memory requirements will increase as the number of cells increase. For the current case, an average of one million cells was used, and the number of iterations is the maximum number of iterations performed before the solver terminates, and in the present case, 3000 iterations were carried out.
Boundary Conditions
The boundary conditions are prescribed at all boundary surfaces of the computational domain. The mainstream conditions were maintained in all cases as follows:
Model Validation
The cooling process adopted for engineering and industrial applications within the manufacturing industry has employed many models. One of the most popular models is the RANS turbulence model given the advantages associated with this approach for predicting the heat transfer procedure and the model's minimal computational time. In the present chapter, the RNG k-ε turbulence model simulates all the cases of the twin impingement jets mechanism on the aluminum plate surface. Validation of the present model was undertaken using the experimental results of the average heat transfer coefficient presented by [6] for the same operating conditions and geometry. Indeed, the experimental results will determine the heat transfer coefficient numerically on the plate surface. Also, it was observed that the distribution of the average heat transfer coefficient had a minimal deviation from the experimental data and reported a good agreement with the experimental data, as illustrated in Figure 6.
The distribution of the average heat transfer coefficient (h) on the aluminum surface for all models at Reynolds number = 17,000 of the twin impingement jet mechanism is shown in Figure 6. The temperature distribution over a flat surface for the different models is illustrated in the above table to validate the simulation model. Also, it is observed that the distribution of the average heat transfer coefficient had a slight variation from the experimental data and reported a good agreement with the experimental data. The percentage of thermal enhancement factor resulting from the difference in the arrangement of the nozzles in the three models was approximately 10% to 20% as indicated in Table 2.
The concave profile on the front face is also in good agreement with the experimental data. The results show that the higher values of the heat transfer coefficient were obtained on the first model when S = 1 and H = 1.
Experimental Results on Twin Impingement Jet Mechanism
The experimental setup of TJIM was verified through performing several tests and through comparing the data obtained with other work. The analyses of the air leakage, flow characteristics, heat transfer performance, and the thermal behavior at the impingement surface were conducted to verify the overall setup and the twin jets performance. The work of [26] was selected for comparative purposes in this study, due to the similarities between their experimental setup and the present work. However, there are several differences between both practices that were considered in the comparison of the results. The "Test Results in The Interference Zone of Twin Jets Impingement" as shown in Figure 7, illustrates the intersection of the Nu and the Reynolds number at a nozzle-nozzle spacing equal to 1 cm, and the nozzle-plate distance equal to 1, 6 and 11 cm subsequently. Obviously, the Nu at the near nozzle-plate distance level is higher than that at the high nozzle-plate distance level. Furthermore, at all nozzle-plate spacing distances, the Nu increases as the Reynolds number increases with a similar slope. Indeed, this slope similarity denotes a similar effect on the Nu which is performed as the nozzle-plate distance varies at all considered Reynolds number levels. Also, there is a noticeable enhancement in Nu compared with the other results [27]. Figure 7 displays the intersection of the Nu at the nozzle-nozzle spacing equal to 1 cm, and the nozzle-plate distance equal to 1, 6 and 11 cm subsequently. Obviously, the Nu at the near nozzle-plate distance level is higher than that at the high nozzle-plate distance level. Moreover, at all nozzle-plate spacing distances, the Nu increases as the Reynolds number increases with a similar slope. Indeed, this slope similarity denotes the similar effect on the Nu which is performed as the nozzle-plate distance varies at all considered Reynolds number levels. There is a noticeable enhancement in the Nu comparing with another result. Figure 8 presents the images captured by a thermal imager and illustrate how the twin jets impingement mechanism (TJIM) is affected and distributed on the plate surface that measures the impinged target. The images are labeled with the center of the temperature values. The illustrated steady jet cases below are based on three models. The images were captured for all models, with one image captured for each model at Re = 17,000. Furthermore, in these images, some observations can be made. First, the hottest spots can be observed due to the impact of the twin jets impingement. Secondly, the midlevel point of temperature, an elliptical temperature distribution can be observed, and higher temperature rates were achieved through the steady jet case because of the high flow rates for the jets. In contrast, in TJIM, due to its duty principles, the lower flow rate was supplied. Figure 8 also displays the higher center temperature of 82.6 • C when H = 1 and S = 1 (i.e., the first model). Notwithstanding, the twin jets had the highest temperature result to the high sensitivity of the thermal imager that can detect even the smallest temperature changes between both jets.
Convective Heat Transfer
Numerical simulations are performed using the RNG k-ε model to investigate the cooling process on the flat plate heated by an electrical heater using the TJIM for the three models according to jet-jet spacing (S) = (1 cm), jet-plate distance (H) = (1, 6 and 11 cm) and the Reynolds number at 17,000. The impact of the jet position on the average Nus and heat transfer coefficient are examined. The values of the average Nu and heat transfer coefficient (h) are chosen based on the jet-jet spacing and jet-plate distance.
The distribution of the heat transfer coefficient (h) on the aluminum surface for all the models at the Reynolds number =17,000 of the twin impingement jet mechanism is shown in Figure 9. The temperature distribution over a flat surface for the different models are: (a) S = 1 and H = 1 (b) S = 1 and H = 6 (c) S = 1 and H = 11. It can be observed that the distribution of the average heat transfer coefficient has a convex profile on the front face that is in good agreement with the experimental data. The results show that the higher values of the heat transfer coefficient are obtained on the first model when S = 1 and H = 1. The remaining models, as seen from the images, are not dissimilar from each other regarding thermal distribution. The jet arrangement will help to determinate the turbulence intensity in the air flow that limits the distribution of the heat transfer coefficient and reflecting the impacts of the twin jets on the plate surface. For Model 1, when the spacing between the nozzles is equivalent to 1 cm, and the distance between the nozzle and plate equals 1 cm, the contour of the predicted static temperature for the k-ε model for model 1; displays contours of the total temperature, as presented in Figures below. A tiny part for the thermal distribution of the hot plate as shown in Figure 10 below, displays the heat transfer coefficient being high in the middle of the plate surface and then decreases gradually when moving away from the plate center. Furthermore, the high-temperature region is located on the plate surface where the presence of strong recirculating air flow exists resulting from the twin jet under high velocity. The remaining models, as shown in the figures below, are not dissimilar from each other. The surface Heat Transfer Coefficient in this model (h) = 51.76 (W/m 2 ·K). The distribution of the fluctuating heat transfer coefficient (h) is presented in Figure 11. Notably, these distributions display many characteristics which are like the mean heat transfer coefficient distributions. The peak in the magnitude of the fluctuations occurs at the stagnation points of the twin jets, which gradually decreases moving from the center of the stagnation points. The velocity vectors and temperature contours in the below-mentioned models, (Figures 12 and 13) illustrate the combined plot of the velocity vectors colored by the velocity magnitude and the temperature contours for all cases of the models described above. From these figures, it is evident that an air flow on the heated flat plate surface and the apparent turbulence on the aluminum plate occurs mainly when the jets become too close to the plate surface. Furthermore, it is also observed that the flow directions have changed and that swirls are generated, in-turn increasing the disturbance that leads to the increased heat transfer coefficient and thus increases the Nu. The temperature attained on the plate surface was 100 • C with the air velocity Reynolds number was equal to 17,000. Figure 12. Velocity vectors colored by the static temperature of the twin impingement jet mechanism at the Nozzle-Nozzle spacing equal to 1 cm and the Nozzle-Plate distance equal to 1 cm. Figure 13. Velocity vectors colored by the velocity magnitude for the twin impingement jet mechanism at the Nozzle-Nozzle spacing equal to 1 cm and the Nozzle-Plate distance equal to 1 cm. 3.6.2. Model (2) Nozzle-Nozzle Spacing = 1 cm and the Nozzle-Plate Distance = 6 cm Figures 14-17 present the contours of the static temperature of the cooling process for the plate surface for Model 2 when the nozzle-nozzle spacing is equivalent to 1 cm, and the nozzle-plate distance equals 6 cm. The contour of the predicted static temperature for the k-ε model for model 1; the contours of the total temperature is presented in Figure 15. The high-temperature region is located on the plate surface where the presence of strong re-circulating air flow results from the twin jet under high velocity. The temperature is 100 • C, and the Surface Heat Transfer Coefficient in this model (h) = 44.55 (W/m 2 ·K). The shape of the heat transfer coefficient distributions shown in Figure 14 indicates that the heat transfer decreases dramatically when moving away from the direction of the stagnation point. Once again, the difference is more pronounced at different parameters such as an increase or reduction in the S and H values.
The velocity vectors and temperature contours in the below-mentioned model, (Figures 16 and 17) show the combined plot of the velocity vectors which are colored by the velocity magnitude and the temperature contour for the second model. From these figures, it is evident that an air flow on the heated flat plate surface and the noticeable turbulence on the aluminum plate occurs mainly when the Jets are near to the plate surface. The flow directions are also observed to have changed, and swirls are generated, thereby increasing the disturbance leading to the increased heat transfer coefficient and in-turn, increases the Nu.
Model (3) Nozzle-Nozzle Spacing = 1 cm and the Nozzle-Plate Distance = 11 cm
For Model 3 when the spacing between the nozzles is equivalent to 1 cm, and the distance between the nozzle and plate is equal to 11 cm, the contour of the predicted static temperature for the k-ε model for model 2; contours of the total temperature are presented below. Also, a tiny part representing the thermal distribution of the hot plate is shown in the figures below, heat transfer coefficient is high in the middle of plate surface then decreases gradually when moving away from the plate center. The high-temperature region is located on the plate surface where there is the presence of a strong recirculating flow of air that is originating from the twin jet under high velocity. The Surface Heat Transfer Coefficient in this model = 41.59 (W/m 2 ·K).
By observing the heat transfer coefficient distributions at different points with the vortex development, a better understanding of the influence of the vortices can be achieved. Figure 18 presents the heat transfer distributions at the low nozzle to the impingement surface spacings. The axial heat transfer coefficient distributions are consistent with the flow behavior and the heat transfer coefficient. Figures 19-21 below present the contours of the static temperature of cooling process for the plate surface for model 3 when the nozzle-nozzle spacing is equivalent 1 cm and the nozzle-plate distance is equal to 11 cm. The contour of the predicted static temperature for the k-ε model for model 3; contours of the total temperature is also presented in the figures. The high-temperature region is located on the plate surface where the presence of strong re-circulating air flow air is observed coming from the twin jet under high velocity.
The values of the Nu exhibited a reasonable change with an increase or decrease in the distance values [28]. These results appear more logical when comparing to other research works [29]. Moreover, when the steady or where the impinging air twin jets on a hot plate surface occur near the interference zone's center line passing through all the twin jets' holes towards the end of the surface plate [30]. Accordingly, this section presents the results from the experimental data. Also, the figures above demonstrate the effect of TJIM on the surface temperature as measured through a heat flux-temperature sensor placed on the front of the flat plate surface and the thermal image on the surface. Indeed, at the first 4 or 5 points, there was an observed increase in the surface temperature on the plate surface, which then begins to gradually decrease after the 5-point measured distance on the aluminum surface plate [6]. It is important also to demonstrate how TJIM can impact the Nu in the midpoint or center between the air twin jets passing to the end of the interference zone near the terminal aluminum plate surface. The figures demonstrate how the Nu numbers are influenced based on the measurements by the micro-foil sensors. The recorded Nu number was found to decrease with increases in the distance from the center of the aluminum plate surface towards the terminal of the surface (low rates at distant points away from the flat plate surface center). Accordingly, this result was logical, as well as the simulation and experiment, where the increase in the heat-transfer rate occurred until the twin jets were near to the flat plate surface. Especially, when under the direct impact experienced by the heat flux sensor under the twin jets air flow on the surface. There was a gradual decrease in the distance from the center of the interference zone. However, this was an increase when compared to other research studies. Eventually, as presented in the figures of the heat transfer coefficient distribution, this indicated that the heat transfer gradually decreases when moving away from the direction of the stagnation point. Likewise, the difference is more evident at different parameters such as an increase or reduction in the spacing between the nozzles and the distance between the nozzle and plate. Furthermore, there is an apparent mixing of air in the intersection area between the nozzles, as well as the presence of the measured area under the direct effect of the air coming from the nozzles, which decreases when the spacing increases between the nozzles and in-turn, increases the distance between the nozzle and plate.
Conclusions
Numerical simulation and experimental work were carried out based on the RNG k-ε turbulence model and TJIM to investigate the cooling process of a heated aluminum plate surface using TJIM in three different models. The impact of the jet position (i.e., the 'Jet-Plate' distance) and the high Reynolds number on the convective heat transfer to obtain the average heat transfer coefficient and average Nu. The main conclusions drawn from the study are that the temperature distribution over a flat surface for the different models is demonstrated to validate the simulation model. Secondly, it was also observed that the distribution of the average heat transfer coefficient had a slight variation from the experimental data and was reported to be in good agreement with the experimental data. Therefore, in summary, the findings have given more information about TJIM that has not been done before to provide the new contribution about the flow and heat transfer characteristics of TJIM. The main conclusions from work performed in this study are: (a) Simulation of three models at the Reynolds number = 17,000 was used to compare and validate the results comparing with the experimental tests. (b) The jet position was investigated (as the jet-plate distance), influencing the heat transfer coefficient, the average Nu and distribution of static pressure. (c) The contour of the predicted static temperature, the velocity vector colored by the static temperature and the velocity vectors' colored velocity magnitude for the k-ε model were captured and discussed. the Nu is the third model when the S/D = 1 cm and H/D = 11 cm. (i) As the twin nozzles are close to each other, the nearest distance between the nozzle and impinged surface plate shows significant variation especially at interference zone more than that as nozzle-nozzle spacing increases. | 2019-04-16T13:28:03.733Z | 2018-04-13T00:00:00.000 | {
"year": 2018,
"sha1": "9b2bb5e20639274022cc7c6156280ee74d09b858",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1996-1073/11/4/927/pdf",
"oa_status": "GOLD",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "7471f756038dfe9ad187d4ba6a808486f30ee012",
"s2fieldsofstudy": [
"Engineering",
"Physics"
],
"extfieldsofstudy": [
"Materials Science"
]
} |
132256292 | pes2o/s2orc | v3-fos-license | Arrival timing diagnostics at a soft X-ray free-electron laser beamline of SACLA BL11
Arrival timing diagnostics between a soft X-ray free-electron laser and synchronized optical laser pulses were performed at SACLA BL1.
Introduction
The soft X-ray free-electron laser (XFEL) beamline BL1 of SACLA (Ishikawa et al., 2012), which employs a dedicated 800 MeV LINAC, started user operation in July 2016. In this beamline, SASE-FEL in the photon energy range 40-150 eV is available with an average pulse energy of $80 mJ at 100 eV (Owada et al., 2018a,b). BL1 has been providing research opportunities for various fields of science, such as materials science (Yamamoto et al., 2018) and atomic, molecular and optical (AMO) science (Minemoto et al., 2018).
The recent progress in synchronization techniques has enabled the reduction of the relative arrival timing jitter between FELs and optical lasers to less than 100 fs (Schulz et al., 2015;Kang et al., 2017). However, arrival timing diagnostics are still necessary to compensate for the jitter to perform ultrafast pump-probe experiments. In hard XFEL beamlines, transient optical reflectivity or transmissivity changes of semiconductors induced with XFEL irradiation have been probed using a spatial or spectral encoding scheme. A spectral encoding method combining white light with a transmissive target such as a thin film of Si 3 N 4 has been adopted at hard XFEL endstations of the LCLS (Bionta et al., 2011;Harmand et al., 2013). At SACLA BL3, a beam branching method using transmissive grating combined with one-dimensional focusing enabled the performance of pumpprobe experiments simultaneously with the arrival timing diagnostics (Sato et al., 2015;Katayama et al., 2016). In the extreme ultraviolet (EUV) and soft XFELs, the THz field streaking method with gas targets has been applied (Grguras et al., 2012;Juranic et al., 2014;Ivanov et al., 2018) because it is non-destructive in this photon energy region.
As a beamline instrument, stable and simple operation of the timing monitor is very important. For this purpose, transient transmissivity probed by the spatial encoding manner would be promising. One of the advantages of this scheme is ISSN 1600-5775 requiring only a fundamental beam of a Ti:sapphire laser without white light or THz generation. While the beam branching method using transmissive optics cannot be simply applied to the EUV and soft X-ray regions, a wavefront splitting scheme is applicable even in those photon energy regions. Recently, we performed a proof-of-principle experiment on the arrival timing diagnostics. Here, a beamline slit and elliptical mirror have been used for extracting a small portion of the incident soft X-ray beam and focusing the beam in one dimension (Owada et al., 2018a). Based on the result, we developed an arrival timing monitor for SACLA BL1 that employs the wavefront splitting method. In this paper, we report the design of the timing diagnostics system and the commissioning results including the arrival-timing correlation between the branched beam and the main beam.
Design
A key point of our arrival timing monitor is the combination of the wavefront splitting for beam branching with onedimensional focusing for efficient pumping of a semiconductor. For this purpose, we designed a new elliptic cylindrical mirror which has a sharp edge of silicon substrate coated with carbon. A schematic view of our arrival timing monitor is shown in Fig. 1. The upper portion of the incident beam is reflected at a glancing angle of $3 and one-dimensionally focused onto the surface of a mirror-polished GaAs wafer, while the remaining lower part is introduced to the endstation EH4a for the experiments. Fig. 2 shows the soft X-ray beam profile at 100 eV before and after inserting the branching mirror into the incident beam axis. We note that beam branching does not affect the focus size of the KB mirrors installed in the endstation as shown in Fig. 3.
As a probe pulse, the synchronized Ti:sapphire laser pulses with a 1.57 eV photon energy and a pulse duration of $35 fs were one-dimensionally focused using a cylindrical lens. The polarization of the optical laser is horizontal because the reflectivity change ratio of the p-polarized optical beam is a few times larger than that of the s-polarized beam (Owada et al., 2018a). The transient change of reflectivity on the surface of GaAs is detected with a visible CCD camera (OPAL-2000D) combined with a micro-imaging lens (ULWZ-200-IR). The spatial resolution of the camera is 2.1 mm per pixel, which corresponds to a temporal resolution of 5.1 fs per pixel considering the optical geometry of the spatial encoding method.
Since the probe beam irradiated the GaAs wafer at an incident angle of 45 , the focal depth of the imaging lens limits the effective temporal window. Under the current magnification conditions, the effective temporal window is $2.5 ps. Schematic of the arrival timing monitor at SACLA BL1. The incident soft XFEL beam is introduced to the beam branching mirror. A small portion of the XFEL beam is reflected and focused to a GaAs wafer, while the major part of the beam is introduced to the KB mirror system and focused to the sample position. The optical laser pulses were reflected on the GaAs wafer and imaged onto the visible CCD camera with an imaging lens. (a) Beam profile of the incident soft X-ray pulse at 100 eV. (b) Beam profile after inserting the beam branching mirror. The distance between the centre of the incident beam and the mirror edge is $5 mm, which corresponds to an $15% beam splitting ratio.
Figure 3
Spatial profiles of the focused beam without beam branching in the (a) horizontal and (b) vertical directions, measured at h = 100 eV with the knife-edge scan method. The red circles are measured results, while the dashed lines are Gaussian fits. Panels (c) and (d) represents the focused beam profiles with beam branching in the horizontal and vertical directions, respectively.
Single-shot arrival timing diagnostics
At first, we analysed single-shot CCD images to evaluate the arrival timing in the same manner as our previous paper (Owada et al., 2018a). At 100 eV with $80 mJ of incident pulse energy, $15% of the incident energy was branched and focused onto the GaAs wafer. The soft X-ray fluence on the wafer was $3 mJ cm À2 , which was sufficiently high to induce the transient optical reflectivity change and low enough to avoid permanent damage of the GaAs wafer after 12 h irradiation. Under this condition, the edge of reflectivity reduction was detected, as shown in Fig. 4. While the higher XFEL intensity easily causes permanent damage to the surface of GaAs, the lower XFEL intensity cannot induce sufficient transient reflectivity change, which makes the accurate arrival timing analysis difficult. In order to optimize the XFEL intensity, one can change the branching ratio by varying the vertical position of the branching mirror.
Temporal resolution
The temporal resolution was evaluated by changing the relative delay time between the soft XFEL and the Ti:sapphire laser pulses. Fig. 5 shows the dependence of the 1000-shotaveraged edge position with respect to the relative delay time. The conversion coefficient from pixel location to relative arrival timing was determined to be 4.9 (2) fs per pixel, which is consistent with that derived from the optical geometry. Although the higher magnification of the imaging lens improves the temporal resolution up to <2 fs per pixel, which was achieved at the timing monitor of SACLA BL3 (Katayama et al., 2016), the effective time window becomes narrower due to the limitation of the focus depth. The temporal resolution of $5 fs per pixel with an effective temporal window width of $2.5 ps is an optimized condition considering the relative arrival timing jitter of $500 fs (FWHM; Owada et al., 2018a), which mainly originated from the synchronization system of the oscillator, and the pulse duration of the soft XFEL and the optical laser pulses, which are estimated to be <100 fs (FWHM) and $35 fs (FWHM), respectively.
Correlation measurement
In order to evaluate the performance of this system, we calculated the correlation by independently measuring the arrival timing at the timing monitor and at the sample position located $5 m away from this system. We analyzed 36 000 shots of images and plotted the correlation in Fig. 6(a). The histogram in Fig. 6(b) represents the arrival timing jitter. The residual error of linear fitting, which corresponds to the accuracy of the arrival timing measurement and its histogram, are plotted in Figs. 6(c) and 6(d), respectively. The error width includes the fitting accuracy and systematic errors such as mechanical vibration of the optics and sample. The histogram in Fig. 6(d) shows the FWHM width of 22 fs (i.e. RMS width of 13 fs). This means one can achieve $20 fs temporal resolution The dependence of 1000-shot-averaged edge position on the CCD images with respect to the delay time between the soft X-ray and the optical pulses. The horizontal bars represent the 2 width of the edge positions to be evaluated. using the arrival timing monitor, which is sufficiently accurate considering the temporal duration of the FEL and the optical pulses.
Summary
We have developed the arrival timing monitor for the soft XFEL beamline of SACLA BL1. We adopted the wavefront splitting method for beam branching, which enabled simultaneous operation of the arrival timing diagnostics and ultrafast pump-probe experiments. By measuring the arrival timing correlation between the branched beam and the main beam, an $22 fs (FWHM) error is obtained. The arrival timing monitor enables the improvement of the experimental temporal resolution down to a few tens of femtoseconds. | 2019-04-26T14:16:02.753Z | 2019-04-01T00:00:00.000 | {
"year": 2019,
"sha1": "75488f85dcd9b62aee5a30c27a1301f197b39390",
"oa_license": "CCBY",
"oa_url": "https://journals.iucr.org/s/issues/2019/03/00/xq5001/xq5001.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "75488f85dcd9b62aee5a30c27a1301f197b39390",
"s2fieldsofstudy": [
"Physics",
"Engineering"
],
"extfieldsofstudy": [
"Physics",
"Medicine"
]
} |
49642385 | pes2o/s2orc | v3-fos-license | De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures
Next-generation sequencing has been invaluable in the elucidation of the genetic etiology of many subtypes of intellectual disability in recent years. Here, using exome sequencing and whole-genome sequencing, we identified three de novo truncating mutations in WAS protein family member 1 (WASF1) in five unrelated individuals with moderate to profound intellectual disability with autistic features and seizures. WASF1, also known as WAVE1, is part of the WAVE complex and acts as a mediator between Rac-GTPase and actin to induce actin polymerization. The three mutations connected by Matchmaker Exchange were c.1516C>T (p.Arg506Ter), which occurs in three unrelated individuals, c.1558C>T (p.Gln520Ter), and c.1482delinsGCCAGG (p.Ile494MetfsTer23). All three variants are predicted to partially or fully disrupt the C-terminal actin-binding WCA domain. Functional studies using fibroblast cells from two affected individuals with the c.1516C>T mutation showed a truncated WASF1 and a defect in actin remodeling. This study provides evidence that de novo heterozygous mutations in WASF1 cause a rare form of intellectual disability.
Neurodevelopmental disorders (NDDs), which include intellectual disability (ID), epilepsy, and autism spectrum disorder, are a heterogeneous group of disorders caused by abnormal development of the central nervous system (CNS). The complexity of CNS development is reflected in the fact that over 700 genes to date have been associated with ID, and very few occur at high prevalence. 1,2 Because of the extreme genetic heterogeneity of ID, the utilization of next-generation sequencing (NGS) technology provides an efficient method of determining the genetic cause of ID in individuals and discovering ID-associated genes. In addition, NGS of trios enables detection of de novo mutations, 3 including single-nucleotide variants (SNVs) and small indels, which are a major contributing factor to the genetic etiology of moderate to severe ID and NDDs. [4][5][6][7] In this study, we used NGS approaches to identify three de novo variants in WAS protein family member 1 (WASF1 [MIM: 605035]), which encodes WASF1 (also known as WAVE1), in five unrelated individuals with overlapping neurodevelopmental abnormalities, including severe ID with autistic features and seizures. We used Matchmaker Exchange (MME) 8 to connect the four international centers, which had each independently identified WASF1 as a candidate gene. All three de novo variants, including a recurrent truncating variant, cluster within the C-terminal actin-binding WCA domain of WASF1 and are predicted to result in a truncated protein.
The five affected individuals described in this report are from non-consanguineous families and are unrelated. All participants and parents gave informed consent, and the studies were approved by the appropriate institutional research ethics boards (Children's Hospital of Eastern Ontario, Ottawa, Canada; IWK Health Centre, Halifax, Canada; Groupe Hospitalier Pitié-Salpêtrière, Paris, France; East of England Cambridge South, Cambridge, UK; Santa Maria Hospital, Lisbon, Portugal; and Radboud University Medical Center, Nijmegen, the Netherlands ). The five affected individuals (P1-P5) have moderate to profound ID with autistic features, seizures, severe impairments in speech, gross motor delay, and a paucity of significant congenital abnormalities. A detailed clinical overview is provided in Table 1. The affected individuals have midfacial hypoplasia but lack a recognizable dysmorphic facial phenotype (Figures S1A-S1D). P5 started walking at 25 months, P1 and P2 began walking at age 3-4 years, and P4 did not walk until age 10 years. P1 requires wheelchair assistance when traveling out of his home. P3 has never achieved independent ambulation. All affected individuals either are non-verbal or have limited speech with a few or single words. All affected individuals except P5 have seizures, although these include a range of seizure types, including generalized and focal seizures; all require antiepileptic therapy. Four of the affected individuals (P1, P2, P4 and P5) had significant hypotonia in infancy, and two (P1 and P4) were described as having a wide-based gait, poor balance, and hyperactivity of movements. Musculoskeletal findings included joint hyperflexibility, ankle valgus, and pes planus in the more severely affected individuals. P5 presented with upperlimb dystonia in the first year of life. A high pain tolerance was observed in P1 and P3, whereas P4 and P5 exhibited automutilation, which is observed in those with an abnormal response to pain. Computed tomography of P1 showed mild atrophy near the Sylvian fissures, magnetic resonance imaging (MRI) of P2 and P3 was normal, and MRI of P4 revealed abnormalities of the periventricular white matter, although this individual also suffered a traumatic birth. MRI of P5 showed enlarged ventricles. Toe abnormalities (short third and fourth toes) were noted in three of the four affected individuals (Figures S1E-S1G).
Testing for a range of other genetic conditions was undertaken in the affected individuals but resulted in no alternate diagnoses. Specific gene testing included MECP2, ATRX, UBE3A, CDKL5, MEF2C, FOXG1, TCF4, and NRXN1, reflecting the differential diagnosis and developmental severity of the condition. All had a normal result on diagnostic microarray testing. Metabolic testing was normal, as was a muscle biopsy of P3. Because the initial genetic tests were negative, all affected individuals had either exome sequencing or whole-genome sequencing (WGS) performed at their respective centers. Details of the methods used for each affected individual are provided in Table S1. Genomic coordinates throughout this report refer to GRCh37, and coding sequence and protein coordinates refer to the canonical transcript (Ensembl: ENST00000392589; GenBank: NM_003931.2).
Trio exome sequencing was performed on individual P1 and his parents as part of the Care4Rare Canada research program according to our standard approach as previously described. 9 After filtering for rare variants (with a frequency less than 0.1% in gnomAD and present fewer than six times in our in-house controls), all variants in known disease-related genes were assessed, but no variants that could explain this individual's phenotype were identified. In the search for potential novel genes, possible biallelic or X-linked recessive variants were examined, but there were no rare homozygous or hemizygous variants. Compound-heterozygous variants were identified in CROCC (MIM: 615776), but this gene was ruled out as a likely candidate because it has many loss-of-function variants in control databases (Table S2). Finally, de novo variants in WASF1, ATP5J (MIM: 603152), SLC38A4 (MIM: 608065), and ZNF175 (MIM: 601139) were identified (Table S2). Assessment of protein localization patterns and function and in silico mutation predictions determined that ATP5J, SLC38A4, and ZNF175 were unlikely to be responsible for this condition (refer to Table S2 for further details). Given the role of WASF1 in actin polymerization and the importance of actin regulation in achieving synaptic plasticity, the de novo heterozygous variant in WASF1 (c.1516C>T [p.Arg506Ter]) was judged to be the strongest candidate for causing this individual's condition and was entered into MME.
Individuals P2 and P5 underwent trio exome sequencing as part of routine diagnostic testing at the Département de Génétique of Hôpital Pitié-Salpêtrière (Paris, France). After filtering for rare variants (with a frequency less than 0.1% in the ExAC Browser), no pathogenic variants, likely pathogenic variants, or variants of unknown significance (VUSs) were identified in known developmental-diseaseassociated genes. Next, rare variants in genes not previously known to be associated with disease were considered. A heterozygous de novo stop-gain variant in WASF1 (c.1516C>T [p.Arg506Ter]), the same variant identified in P1, was identified in both P2 and P5. A de novo missense variant in CDCA7L (MIM: 609685) was also identified in P2 but was not considered likely to be pathogenic (Table S2). No additional variants that required consideration of pathogenicity were identified in P5. Individual P3 and his mother underwent WGS as part of the National Institute for Health Research (NIHR) BioResource study (UK) as previously described. 10 No pathogenic or likely pathogenic variants were found in known developmental-disease-associated genes, but a heterozygous stop-gain variant in WASF1 (c.1558C>T [p.Gln520Ter]), which was not present in the unaffected mother, was identified. Sanger sequencing of P3 and his parents confirmed that the variant occurs de novo in the affected individual ( Figure S2B). A hemizygous missense variant in X-linked ACSL4 (MIM: 300157), in which variants can cause X-linked ID (MIM: 300387), was also identified in P3 and was heterozygous in the mother. This was classified as a VUS because the variant was not previously associated with disease (Table S2).
Individual P4 underwent trio exome sequencing as part of routine diagnostic testing (Groningen, the Netherlands). No pathogenic variants, likely pathogenic variants, or VUSs in known developmental-disease-associated genes were identified. Next, de novo variants in genes not previously known to be associated with disease were considered. A heterozygous de novo frameshift variant in WASF1 (c.1482delinsGCCAGG [p.Ile494MetfsTer23]) was identified. No other coding variants that occurred de novo were identified.
Initially, the four groups independently identified WASF1 as a strong candidate because of features consistent with those of developmental-disorder-associated genes. This gene is constrained for loss-of-function variation in the ExAC Browser (pLi ¼ 0.91) 11 and is highly and specifically expressed in the adult human brain. 12 All three WASF1 variants are absent from 1000 Genomes, the ExAC Browser, and gnomAD. 11,13 The variants in individuals P1 and P3-P5 were confirmed to be de novo by Sanger sequencing of the trio ( Figure S2B). The read depths for P2 and his mother and father were 127 (with 69 read counts for the alternate allele), 143, and 124, respectively. MME connected three of the groups, and the fourth was connected by personal correspondence with the UK group.
Interestingly, the three de novo variants appear to cluster around the WASP-homology 2 (WH2) domain of WASF1 ( Figure 1A). A previously published method was used to determine that the clustering is statistically significant (p ¼ 1.31 3 10 À6 ). 15 The C-terminal actin-binding WCA region, which includes the WH2 domain, is highly conserved throughout evolution ( Figure 1B). The WCA region plays an important role in regulating WASF1 16,17 so that actin and the Arp2/3 complex can bind to the WCA domain to promote actin polymerization. 18 All three variants identified in the affected individuals fall either within the last 50 bp of the penultimate exon or within the last exon ( Figure 1C) and are therefore predicted to result in the generation of a truncated protein that partially or fully eliminates this WCA domain. 19 Next, the potential effect of the identified WASF1 variants on protein function was determined. Primary fibroblasts were obtained from individuals P1 and P2, who carry the same c.1516C>T variant (predicted to introduce a premature stop codon at amino acid 506). Amounts of WASF1 mRNA and WASF1 were examined. Real-time PCR showed variable levels of mRNA between the two affected individuals and control individuals (Figure 2A). For western blot analysis of WASF1, total protein extracts were probed with either a C-terminal antibody (epitope located after amino acid 506; Abcam, ab50356) or an N-terminal antibody (Sigma-Aldrich, W0267) against WASF1. Comparison of control and affected individuals revealed that the cells from affected individuals had both the full-length WASF1 (75 kDa) and a truncated $70 kDa protein that was not observed in control cells (Figures 2B and 2C). Densitometry quantification of these bands showed that the fulllength protein was present at approximately 50% of the control levels, reflecting the presence of one wild-type allele, whereas the truncated protein was present at 14%-25% of control levels ( Figures 2B and 2C). This suggests that although a truncated isoform is produced, it is unstable at either the mRNA or protein level such that the amount of protein is reduced. Therefore, the WASF1 c.1516C>T variant causes the production of a shorter mutant protein rather than the absence of a protein due to complete nonsense-mediated decay of the primary transcript.
WASF1 plays a critical role in binding actin to initiate actin polymerization. Examination of the reorganization of the actin cytoskeleton during lamellipodia formation in fibroblasts was used for testing this role. [20][21][22] Serumstarved fibroblasts were trypsinized, re-plated onto poly-L-lysine-coated coverslips, and stimulated with platelet-derived growth factor (PDGF; Sigma-Aldrich, P3201) for inducing the formation of lamellipodia, as previously described. 20 Then cells were fixed, filamentous actin was visualized by labeling with phalloidin (Thermo Fisher Scientific, A12349), and the actin phenotype was quantified in each genotype. In the majority of control cells (77%), actin at the cell periphery formed well-organized, sheet-like lamellipodia structures ( Figures 3A and 3B, white arrowhead; Figure S3). This was interspersed with cells in which the actin sheets were interjected by filopodia, which are finger-like actin projections ( Figures 3A and 3B, red asterisk; Figure S3). We next assessed fibroblasts from affected individuals and found that although a sheet-like lamellipodia structure was observed along the periphery of 34% and 24% of P1 and P2 cells, respectively, the actin bundles were thinner and less organized than in the control cells ( Figures 3A and 3B). We also noted that a portion of cells from P1 and P2 had severe disruptions in actin organization such that no lamellipodia delineated the cell periphery and only filopodial projections were present (12% and 11% for P1 and P2, respectively; Figures 3A and 3B). This phenotype was not seen in control cells. Therefore, cells from affected individuals have an alteration in actin organization, suggesting that the presence of a truncated WASF1 results in defective actin remodeling during the formation of lamellipodia.
Finally, WASF1-dependent actin polymerization has been shown to mediate mitochondrial trafficking into dendritic spines in primary neurons; 23 therefore, we assessed mitochondrial morphology in fibroblasts with the c.1516C>T variant. Mitochondria were visualized and the average length was quantified as previously described. 24 As expected, a dense and complex network of mitochondria was present in both control and affected fibroblasts. Quantification revealed that mitochondria in the cells from affected individuals were significantly longer than those in control fibroblasts ( Figure 3C). This result suggests that the presence of the c.1516C>T variant in WASF1 disrupts the regulation of mitochondrial dynamics and alters the normal balance between fission and fusion in affected fibroblasts.
This report provides evidence that de novo truncating variants in WASF1 in five unrelated individuals cause a NDD comprising severe ID with autistic features, seizures, and developmental delay. Interestingly, three of the five individuals in this study have the same de novo variant Figure 2. Amounts of WASF1 mRNA and WASF1 in Fibroblasts Derived from Affected Individuals with the c.1516C>T Variant (A) RT-qPCR shows variable amounts of WASF1 mRNA between primary fibroblasts derived from individuals P1 and P2 and healthy control fibroblasts. (B) Western blot analysis using an antibody with an epitope downstream of Arg506 showed that the amount of full-length WASF1 was approximately 50% lower in affected fibroblasts than in control fibroblasts. (C) Western blot analysis using an antibody with an epitope in the N-terminal region of WASF1 showed the presence of the full-length and truncated WASF1 in affected fibroblasts. The truncated WASF1 was not present in control fibroblasts. All experiments were performed with fibroblasts derived from three healthy control individuals. Western blots were performed in triplicate, and band intensity was quantified with Image Lab Software (Bio-Rad).
Error bars indicate the range of measurement of triplicate samples.
(c.1516C>T [Ensembl: ENST00000392589]). Three of the four individuals have VUSs in other genes in addition to the WASF1 variants. Population-level sequencing initiatives have enabled increased recognition of the prevalence of recurrent benign de novo mutations. 25 Although it is unlikely, the possibility that they contribute to the respective individuals' phenotypes cannot be excluded.
The variants described as associated with this NDD are all stop-gain or frameshift variants and significantly cluster around the C-terminal WH2 domain in the WCA region of WASF1. The truncated protein observed for c.1516C>T (p.Arg506Ter) suggests that all three variants are likely to lead to altered function of the mutant protein rather than complete protein loss or haploinsufficiency from degradation through nonsense-mediated decay. In a disease context, recurrent de novo events are known to be associated with specific dominant-negative or gain-of-function effects, such as FGFR3 (MIM: 134934) variants causing achondroplasia (MIM: 100800), and are usually missense variants. 26 Clustering and recurrence of de novo protein-truncating mutations also do occur, albeit less frequently because the genic localization of a pathogenic mutation resulting in haploinsufficiency is generally not critical. 15,27,28 Additional individuals with rare WASF1 variants are required for determining whether any pathogenic variants lie outside of this WCA region and/or whether a spectrum of phenotypes is perhaps associated with different variants in this gene.
WASF1 is an essential component of the actin pathway where RAC1 activation triggers a conformational change in WASF1 to allow binding of actin and ARP2/3 to the WCA domain to initiate actin polymerization. 20,21,29,30 The presence of a truncated protein that lacks the WCA region, as observed here, most likely disrupts the WASF1 complex itself, its interactions with CYFIP1, its proteasomal degradation, and the binding of actin ( Figure 2C). 16,17,31 Like mutations in WASF1, mutations in RAC1 similarly disrupt the formation of lamellipodia in fibroblasts, 32 indicating that the organization and stabilization of actin bundles during the formation of lamellipodia is likely to be compromised by truncated WASF1.
In summary, de novo heterozygous truncating variants in WASF1 cause a NDD in individuals with ID associated with autistic features, seizures, and developmental delay. The three de novo variants, identified in five unrelated affected individuals, are all predicted to affect the actin-binding C-terminal WCA region of WASF1. The clustering of truncating pathogenic variants reported here and the presence of a truncated protein in cells from affected individuals imply either a gain-of-function or dominant-negative mechanism of disease. Because WASF1 functions within a large protein complex with ABI2, CYFIP1 or CYFIP2, BRK1, and NCKAP1, the hypothesis that these variants have a most likely dominant-negative effect remains to be tested. This study further expands the list of actin-regulatory-pathway genes associated with NDD and demonstrates the value of sharing genomic data through MME to identify the consequence of extremely rare mutational events.
Supplemental Data
Supplemental Data include three figures and two tables and can be found with this article online at https://doi.org/10.1016/j.ajhg. 2018.06.001. | 2018-07-12T07:59:37.309Z | 2018-06-28T00:00:00.000 | {
"year": 2018,
"sha1": "97fcef3603ccdeb8b954eb883290f0b428a9c4d7",
"oa_license": "CCBYNCND",
"oa_url": "http://www.cell.com/article/S0002929718301940/pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "eb687827a427a281017740699665b0a2c981aa2a",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
1682570 | pes2o/s2orc | v3-fos-license | Hodgkin's disease and infectious mononucleosis: Is there a causal association?
that persons at relatively high risk for the disease during young adulthood are those who as children belonged to small fami lies, lived in single-family homes, had rel atively few neighborhood playmates, and had relatively well-educated parents. They were more likely to be Jewish and to have a history of infectious mononucleosis. On the other side of the world, Cohen and Hamilton2 studied the epidemiologic and histologic patterns of Hodgkin's dis ease among the black and white popula tions of Johannesburg, South Africa, com paring 88 black patients and 54 white patients diagnosed over the four-year pe riod from 1971 to 1974. They did not em phasize any potential viral etiology, but they did note that with respect to histology and age and sex distribution, their data strengthened “¿ the existing general hypoth esis that host immune capacity, influenced by socioeconomic factors, may have a strong and measurable effect on the patho genesis of Hodgkin's disease.― No refer ence was made to infectious mononucleo sis. Cohen and Hamilton did place much emphasis on differences in histologic pat terns of Hodgkin's disease (lymphocyte predominant, nodular sclerosis, mixed cel lularity, and lymphocyte depleted types) as well as differences in age-specific in cidence curves. They noted, for instance, the similarity between white Johannesburg patients, and black patients in the United Introduction
Introduction
Until more facts are known, theories about the etiology of Hodgkin's disease will con tinue to flourish. One of the most prevalent of these postulates an etiological link be tween Hodgkin's disease and a virus, spe cifically the Epstein-Barr virus (EBV). The pursuit of proofs for this theory has produced considerable conjecture, argu ment, disagreement, and even dispute about the way data are used to support individual points of view.
Infectious Mononucleosis and Hodgkin's Disease
Gutensohn and Cole' attempted to estab lish a model that would mimic the polio myelitis model. They recently published data concerning a case control study of 225 patients in whom Hodgkin's disease was diagnosed between July 1, 1973 opment of â€oe¿ an unexpected lead―which became available when Elaine Hutkin, a young laboratory technician whose blood plasma had been initially tested and found free from antibodies to the EB virus, de veloped fever, sore throat, and enlarged lymph nodes. Mononucleosis was diag nosed on the basis of blood counts and a positive heterophile antibody test. As soon as she returned to the laboratory her blood plasma was again tested and found to be positive for the presence of antibodies to the EB virus. Subsequent to this, various studies linked the development of infec tious mononucleosis to a rise in the level of the EB virus titer. â€oe¿ Thus, infectious mononucleosis was unexpectedly found to be immunologically related to the EB vi rus,―Dr. Gross concluded, â€oe¿ and, in all probability, caused by that virus or by a closely related viral agent.―4Hence, Dr. Gross cautiously pointed out that the re lationship between infectious mononucleo sis and a rise in the EB virus titer seems to be a distinct phenomenon.
Note The authors did express concern, how ever, that the sex ratio markedly skewed towards the male bias was extraordinary and that this exceededthenumber expected to be found with Hodgkin's disease in this cohort. Yet the number of cases of leu kemia that developed in the same popu lation of patients with prior infectious mononucleosis was no greater than that expected to be found by their mathematical incidence predictions.
Therefore, their findings did support the suggestion that infectious mononucleosis and Hodgkin's disease may be associated.
More RecentStudies
In the more recent study, by Drs. Kvâle, Høiby, and Pedersen'2 (1979), it was dis covered that among 5,840 patients with a positive Paul Bunnell test, a total of six developed malignant lymphoma, three of these more than one year after the positive '4 (1977) also concluded that prior infectious mononucleosis was not associated with a significant increase in the subsequent development of Hodg kin's disease.
Of special interest are the case reports of patients who already have an established diagnosis of Hodgkin's disease and who then subsequently develop infectious mononucleosis. Langenhuysen'5 reported the occurrence of infectious mononucleo sis in a 20-year-old male who had initially been diagnosed and treated for Hodgkin's disease at the age of 17. At the time of the development of the infectious mononu cleosis he was in complete remission, and it was noted that he developed a high heterophile titer after guinea pig kidney absorption in addition to the clinical syndrome of infectious mononucleosis. Furthermore, he developed sero-conver sion of anti-EBV-capsid antigen (VCA) antibodies and EBV-associated nuclear an tigen (EBNA) antibodies. It was, there fore, concluded that this patient contracted a primary infection associated with a risk in the anti-EB virus titer more than three years after curative treatment for Hodg kin's disease. Langenhuysen considered this to be clear evidence of a lack of causal relationship between EBV and Hodgkin's disease in this patient. Furthermore, the finding of EBV sero negative patients with Hodgkin's disease may be interpreted as additional evidence against any etiologic role of EBV, at least in these cases of Hodgkin's disease.
Davidson and Lessels'6 treated a 26year-old female in 1968 whom they di agnosed as having Hodgkin's disease of the lymphocyte-depleted type. While the patient was being treated with nitrogen mustard and steroid therapy in March 1969, they diagnosed infectious mononu cleosis and did obtain a positive hetero phile test as well as an elevated EBV an tibody titer. On serologic examination, the patient exhibited a rising EBV titer and a falling infectious mononucleosis hetero phile antibody titer; this was accompanied by a gradual improvement in her clinical condition. The development of infectious mononucleosis was considered to be an intercurrent illness during the course of her Hodgkin's disease. Her Hodgkin's disease progressed, and she died in April, 1970, while receiving cytotoxic chemotherapy. Davidson and Lessels were convinced, however, that the patient's typical periph eral blood picture and pattern of infectious mononucleosis, heterophile, and EBV an tibody titers established beyond doubt the diagnosis of infectious mononucleosis in their patient. Obviously, cases such as this further complicate investigation of the re lationship between infectious mononucleo sis, the EB virus, and Hodgkin's disease.
Summary
There is a very strong desire to discover an oncogenic virus in humans. The Ep stein-Barr herpes virus is certainly the most provocative agent stimulating such re search investigation over the past decade. Failure to establish a simple and clear re lationship between the Epstein-Barr herpes virus and Hodgkin's disease has led to ad ditional research that has attempted to link the potential relationship with the possible genetic differences among patients with Hodgkin's disease. Hodgkin's disease has been characterized by ethnic variations in incidence, as well as a high frequency of certain HL-A antigens. In several ethnic groups, Hodgkin's disease was found to correlate significantly with the mean gene frequency of HL-Al and HL-A8. Vianna andHodgkin's disease. Because ofthefre quency of incidence of infectious mono nucleosis and the rarity of Hodgkin's dis References ease, it is important to maintain an emotionally balanced point of view, to avoid causing unnecessary anxiety among those who may develop infectious mono nucleosis. On the basis of current data, it is incorrect to imply that those who de velop infectious mononucleosis may be significantly more susceptible to Hodg kin's disease.
In the absence of firm data, it is better to conclude, for the time being, that the true association between infectious mono nucleosis, Hodgkin's disease, and the EB virus is at best worthy of continuing sci entific investigation by a variety of disci plines. | 2018-04-03T03:18:21.434Z | 1981-11-01T00:00:00.000 | {
"year": 1981,
"sha1": "9fb36d66ccbe9479ba7180ae85853ac142cd6d1f",
"oa_license": null,
"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.3322/canjclin.31.6.359",
"oa_status": "GOLD",
"pdf_src": "Wiley",
"pdf_hash": "843d47df3bece815d3b6d2a94c1f6dc1ac3020d6",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
248186232 | pes2o/s2orc | v3-fos-license | Innovative Technology–Based Interventions to Reduce Stigma Toward People With Mental Illness: Systematic Review and Meta-analysis
Background Stigma toward people with mental illness presents serious consequences for the impacted individuals, such as social exclusion and increased difficulties in the recovery process. Recently, several interventions have been developed to mitigate public stigma, based on the use of innovative technologies, such as virtual reality and video games. Objective This review aims to systematically review, synthesize, measure, and critically discuss experimental studies that measure the effect of technological interventions on stigmatization levels. Methods This systematic review and meta-analysis was based on PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines and included studies in English and Spanish published between 2016 and 2021. Searches were run in 5 different databases (ie, PubMed, PsycInfo, Scopus, Cochrane Library, and ScienceDirect). Only randomized controlled trials were included. Two independent reviewers determined the eligibility, extracted data, and rated methodological quality of the studies. Meta-analyses were performed using the Comprehensive Meta-Analysis software. Results Based on the 1158 articles screened, 72 articles were evaluated as full text, of which 9 were included in the qualitative and quantitative syntheses. A diversity of interventions was observed, including video games, audiovisual simulation of hallucinations, virtual reality, and electronic contact with mental health services users. The meta-analysis (n=1832 participants) demonstrated that these interventions had a consistent medium effect on reducing the level of public stigma (d=–0.64; 95% CI 0.31-0.96; P<.001). Conclusions Innovative interventions involving the use of technologies are an effective tool in stigma reduction, therefore new challenges are proposed and discussed for the demonstration of their adaptability to different contexts and countries, thus leading to their massification. Trial Registration PROSPERO International Prospective Register of Systematic Reviews CRD42021261935; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021261935
Stigma Toward People With Mental Illnesses
Globally, stigma toward people with mental illness represents a serious public health problem and is considered the main barrier to social inclusion and participation of those impacted. It has a negative effect on their quality of life, worse therapeutic results, and even an increased risk of suicide and mortality [1][2][3].
Thus, discrimination, prejudice, and stereotypes present in society not only severely impact the recovery process, quality of life, and well-being of people with mental illnesses and their families, but also represent the main gap in accessing specialized mental health services by the general population [4,5]. The aforesaid has great relevance in the global context, where research has shown the presence of high levels of discrimination, stigma, and prejudice toward those impacted by mental health problems, especially schizophrenia and bipolar disorder [6,7].
Innovative Interventions Carried Out at International and National Levels
Faced with the high levels of stigmatization present in society, several initiatives and studies have been conducted that focused on its reduction. It has been demonstrated that direct contact with people with mental illnesses and educational initiatives are essential and effective interventions to reduce stigma [8,9]. Although direct face-to-face contact with people with mental illness has been shown to be a key component of successful stigma reduction programs, their implementation in virtual learning and innovative spaces is recent [10]. In the last years, several authors have shown that innovative technology-based interventions have had a great impact on the reduction of stigma toward people with mental illness, mainly due to its adaptability to different contexts and age ranges [11]. Among this type of interventions, the use of video games has been highlighted as an effective tool to reduce anxious and depressive symptomatology in patients, and has been useful to reduce misconceptions and stigmatization about severe mental illnesses, such as schizophrenia or bipolar disorder [12,13].
In addition, because of the growth of technologies, the application of virtual and immersive reality in mental health has become increasingly common [14]. For example, it has demonstrated its utility in the treatment of mental health problems, such as phobias and anxiety symptoms, among others [15], along with a reduction of the negative perceptions and attitudes toward people with mental illnesses [16]. This usefulness and effectiveness can be explained by the degree of immersion in a strongly educational environment, which promotes the change of attitudes and beliefs [17]. By contrast, the use of simulation platforms has showed controversial results, with some studies showing an increase of stigmatization when used through the simulation of hallucinatory symptomatology, which can promote negative beliefs and attitudes toward people with mental illness [18,19].
Finally, as an innovative intervention, electronic contact (e-contact)-defined as "computer-mediated real-time interactions where members of different groups interact online" through virtual media with mental health services users [20]-has been used as a strategy for promoting awareness and reducing prejudice among ideologically different groups [21]. However, e-contact implementation in the field of stigma reduction is new and innovative, and it has been demonstrated that e-contact using chat and synchronous videoconferencing can reduce anxiety, stigma, and stereotypes toward the population impacted by mental disorders, and promote an inclusive attitude [11,22].
Despite the considerable progress in this field, further research is needed on innovative technologies and their application in mental health care, such as advances in detection, treatment, and promotion of inclusion and well-being of people with mental health problems [23,24]. Regarding this growing field of application of new technologies and the need to synthesize, measure, and critically discuss the effects of the studies performed for the reduction of stigma, the objectives of this systematic review and meta-analysis are to assess the effectiveness of technology-based interventions to reduce stigma associated with people with mental health problems and to describe the experimental studies that use these types of interventions.
Data Sources and Search Strategy
The systematic review and meta-analysis protocol were registered in the Prospective Register of Systematic Reviews (PROSPERO) international database (registration ID: CRD42021261935) and was conducted according to the guidelines and recommendations of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) [25].
From January to July 2021, searches were conducted from the following 5 databases, including texts dated from March 5, 2016, to March 5, 2021: PubMed, PsycInfo, Scopus, Cochrane Library, and ScienceDirect, using the following string of search terms: [technology or technologies or simulation or virtual or digital or Internet or web or games or computer or app or online or electronic or social media] AND [stigma or discrimination or prejudice or negative attitude or stereotypes] AND [mental health or mental disorder or mental illness or schizophrenia or psychosis or bipolar disorder or depression].
In addition, a final manual trace back literature search was conducted in August 2021 to identify any recently published sources/literature.
Inclusion and Exclusion Criteria
Published articles that met the following criteria were included: (1) randomized controlled trials using innovative technologies (defined as software apps used with smartphone, videogames, e-contact, or virtual reality); (2) interventions aimed at reducing stigma toward people with mental illness (eg, schizophrenia, psychosis, or bipolar disorder), which included at least one relevant quantitative measure of public stigma (eg, attitudes, stereotypes, and social distance); (3) interventions relevant for any populations (eg, students and general population); (4) all age groups; and (5) articles written in English and Spanish, published in peer-reviewed journals.
Exclusion criteria were (1) reduction of stigma not related to mental health problems; (2) technology using only video or education (eg, films or presentations) not combined with any other innovative technology (eg, virtual reality, videogames, or e-contact); (3) reduction focused on self-stigma only; (4) interventions focused on stigma toward psychiatry or addictions; (5) research protocols; (6) measurement of stigma; and (7) studies that did not include a randomized control group as a comparison.
Study Selection and Data Extraction
After excluding duplicates using Endnote reference manager software, 2 researchers (MER-R and AJC) independently selected articles for inclusion. A third investigator (LAC) examined all the included articles to review this selection and resolve discrepancies. In addition, to check and ensure consistency and clarity at the screening and coding stages across studies, we calculated the interrater reliability using the Cohen κ coefficient [26]. We obtained a Cohen κ of 0.75 (SE 0.11), demonstrating a medium to high degree of agreement among coders. Following this calculation, coders (MER-R and AJC) reviewed the articles in which they found disagreements, and any discrepancies were resolved by a consensus discussion with a third investigator (LAC) who was not involved in the searches.
The eligibility of search results was examined in 2 stages: first by title and abstract, and then by full text. Reasons for exclusion were recorded for each document excluded.
Data were extracted using a standardized table format, which was then reviewed by a third author (LAC). Characteristics of each article included the study design, sample size, setting, type of new technology applied, control or comparison intervention, as well as the main outcomes and effect sizes of the interventions. In the cases where the data for the calculation of the effect size were incomplete, we contacted the principal investigator to request the necessary additional information.
Quality and Risk-of-Bias Assessment
To assess the quality of the selected articles, 2 researchers (MER-R and AJC) independently assessed the risk of bias (ROB) of each study using the Cochrane ROB-2 assessment tool, which assesses 7 study design quality criteria (ie, random sequence generation, allocation concealment, blinding of participant and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias), graded as high, medium, or low risk [27]. Discrepancies were resolved by further discussions and consensus among the authors. Figure 2 summarizes the assessment of the ROB, which was performed using the robvis visualization tool.
The use of the funnel plot for the evaluation of publication bias was not incorporated, because it has been demonstrated that its use is not reliable when the number of studies pooled in direct comparison is less than 10 [27].
Statistical Analysis
The meta-analysis was conducted by researchers (SV and MER-R) using the Comprehensive Meta-analysis software (version 2) [28]. Standardized mean difference and the inverse variance method with a 95% CI were used for continuous and normally distributed data, respectively. The I 2 and Q-statistic were used to explore heterogeneity of effect sizes [29]. Random effects models were used due to the heterogeneity in the type of intervention in the studies [30].
As 3 of the studies included more than 1 scale that assessed levels of stigma [11,22,31], we conducted an analysis that combined them into a single effect size. In those cases, we followed the methodology suggestion for complex data structures [30]. As a consequence, we computed a summary effect including the multiple measures; this synthetic effect size was then included in the meta-analysis.
In addition, as 2 of the studies [32,33] reported their results through standardized regression, the β coefficients were entered into the comprehensive meta-analysis software (CMA) as correlation coefficient, according to the recommendations of Peterson and Brown [34].
In addition, we used statistical procedures to quantify the effect of publication bias, by Duval and Tweedie's trim-and-fill analysis and Rosenthal's fail-safe N test [35].
Output of Searches
Of the 2876 studies retrieved from the selected databases, 1718 duplicates were removed and 1158 were screened. Upon the screening of the titles and abstracts, 1086 studies were removed. For the remaining 72 studies, their full texts were checked, among which an additional 63 were removed due to specific reasons ( Figure 1). Only 9 articles presented enough statistical data for meta-analysis.
Characteristic of Included Studies
The characteristics of the included studies are presented in Table 1. A total of 9 randomized control trial studies were included, which utilized a variety of technology-based interventions to reduce stigma, including interventions using video games (n=4), audiovisual simulation of hallucinations (n=1), virtual reality (n=2), and the use of e-contact with mental health services users through videoconferencing and online chats (n=2). Most of these studies were conducted with undergraduate students (8/9, 89%). As many as 4 studies (44%) were conducted in Europe, 2 (22%) in North America, and only 1 each in Asia (11%), Australia (11%), and Latin America (11%). Most of the participants were female. The proportion of female participants ranged from 50.3% to 81.1%, and the mean age of the participants ranged from 15.7 to 24 years. Public stigma in the studies was measured through different scales, with the most commonly used being the Attribution Questionnaire and the Questionnaire on Student Attitudes Toward Schizophrenia.
Risk of Bias of the Included Studies
As shown in Figure 2, most of the studies included in this review were considered as having low ROB in terms of their methodological quality. However, 3 studies [31,32,37] showed a high ROB in the blinding of the participants and blinding of outcome assessment. Besides, 2 other studies [11,32] showed a high ROB related to the allocation concealment. Regarding data, 2 studies [22,37] presented incomplete data. Finally, only 1 study [31] had a high ROB in selective reporting, and no study showed a high risk of other bias or random sequence generation.
Study and Quantitative Synthesis Outcomes: Public Stigma
A total of 9 articles were included in the meta-analysis, with a total sample of 1832 participants. As shown in Figure 3, the technology-based interventions had medium effects on reducing the level of public stigma (d=-0.64; 95% CI 0.31-0.96; P<.001) compared with the control group. Only 1 study [31] that used audiovisual simulation symptoms showed an increase in the level of stigma (P=.036; d=0. 32), and another that used videogame with avatars [32] did not show any statistical effect in the level of stigma (P=.14; d=-0.18). High heterogeneity was observed among the included studies (P<.001; I 2 =87.6%; Q=64.96), which was expected due to the variety of interventions.
Publication Bias Analysis
We ran statistical analyses for publication bias [35], where Duval and Tweedie's trim-and-fill analysis identified the differences in effect sizes that could potentially be attributed to bias; the technique imputes effect sizes until the error distribution gets close to normality. In this way, the test offers the best estimate of the unbiased effect [30]. Results suggest that there were no differences in effect sizes attributable to bias.
Under a random effect model, the point estimate for the combined studies did not differ when comparing the original and the adjusted estimate (in both cases, standardized mean difference 0.63; 95% CI 0.94-0.30). Based on the parameter of Duval and Tweedie's trim-and-fill analysis, it seems that no studies are missing.
Additionally, Rosenthal's fail-safe N test is a technique for computing the number of missing studies that would be necessary to nullify the found effect [38]. Small numbers of missing studies would reveal the likelihood of biased effects. Test was equal to 180, suggesting that it would be necessary to allocate and include 180 missing studies with no effects for every observed study to achieve the combined 2-tailed P value exceeding .05. Therefore, it is highly unlikely that missing studies could alter the substantive conclusion.
Principal Findings
The results of this meta-analysis support the use of new and innovative technology-based interventions to reduce stigma toward people with mental illness.
This study shows a medium effect on stigma reduction, demonstrating a positive impact and effectiveness of these interventions. Its findings are better compared with a previous meta-analysis [39], which reported only a small effect of contact interventions for people with mental illness, and for the educative intervention, both classical and common-type interventions. This is probably due to the increasing technological development, where realism, immersion, and technological interaction are greater, becoming an increasingly natural means of communication and daily application [40].
In this regard, a recent meta-analysis showed that antistigma interventions involving contact had an immediate small-to-medium effect, and it was equivalent with diverse types of contact mediums, such as videos and presentation [41].
Another finding of our study is that the intervention that used simulation of symptoms of hallucination [31] had an increase in the level of stigma, which is concordant with a previous meta-analysis that demonstrated that it can increase social distance and negative attitudes [42]. These negative results may be explained by the focus on symptoms rather than on the recovery process, which may increase stereotypes and prejudice, especially toward people with schizophrenia. Thus, it has been suggested that it should be used with caution and ideally in combination with educational or contact interventions [43]. In this sense, it is important to consider that several research studies show that the information provided is essential to reduce stigma, where, for example, it has been helpful to refer to biographical aspects (eg, related to difficulties, personal adversities), because it allows people to empathize, understand, and generate a change in their attitudes, knowledge, and stigmatizing behaviors [44]. Therefore, the aim is not only to show "symptoms," but also to promote an understanding of these experiences and the social consequences for the people who experience them [37].
As an additional result, one of the included studies, which used videogames through avatar identification [32], despite showing no significant effect on stigma in the combined effect analysis, reported a significance effect (B=-0.21; P<.05) for the reduction of social distance, making it a tool that can be considered for future research.
Our study demonstrated the usefulness of innovative interventions in stigma reduction and summarized its latest advances, in accordance with the growing interest and need for the application of new technologies in the field of mental health in the contemporary world. These types of interventions have a variety of advantages and offer innovative solutions to everyday problems, due to their adaptability to different contexts and lower associated cost, along with the possibility of privacy in a protected environment, which allows strategies to be focused and adapted to different key population.
Furthermore, it is important to mention that while stigma is currently one of the most important problems in mental health, there are also a number of socioeconomic, cultural, and structural barriers present in society that limit access to treatment and should be considered as an integral part of reducing discrimination and inequalities in mental health.
Limitations
This study has some limitations. First, we did not include gray literature, which can increase the risk of publication bias [45]. Despite this, our statistical analysis of publication bias seems to show no missing studies, and that it is also very unlikely that a possible missing study could alter our results. Second, the small number of studies and high heterogeneity prevented us from estimating which technology-based intervention was the most effective, a limitation that may be considered in future studies as the number of publications in this emerging area increases, allowing future subgroup analyses of each type of intervention and addressing the high heterogeneity. Third, all the studies found and included focused on the population of high-school and university students, which may limit the generalizability of the results to the general population. In addition, most of the included studies did not include follow-up studies, which prevented the analysis of long-term stigma reduction.
Future Research
As stigma is a complex social phenomenon transversally present in the society [1][2][3][4][5][6], innovative interventions involving use of technologies can be an effective tool for its reduction. However, one challenge in the implementation of this type of intervention is its adaptability to different contexts and countries, and so future interventions should include cross-cultural comparison. In addition, all experimental studies involving technology-based interventions in stigma reduction focus on the young population, which represents a major challenge for future studies focused on the development, implementation, and evaluation of these types of interventions for different ages, such as adults and the elderly [46,47].
Because of the extensive evidence supporting key strategies in the development of antistigma programs, such as the educative and contact components, future research should consider and adapt them to different innovation-based interventions. Its adaptation had demonstrated a great utility, for example, in e-contact with people with mental illness, as they offer the possibility of wider dissemination and even the possibility of reaching remote areas [11,22]. New lines of work should generate greater access and development of low-cost tools with the use of new technologies that allow their use and integration in workplaces, health systems, and educational communities as a daily support tool in mental health, for example, through the development and use of free apps for smartphones and freeware for computers.
Finally, it is essential to generate integrated technological systems not only to reduce stigma, but also to consider preventive interventions in mental health, together with timely referrals to specialized health services and access to treatment. In this regard, we highlight the recently developed app Help Club [48], which provides the possibility of access to communities of mutual support in mental health in virtual spaces, through the use of virtual reality, demonstrating the potential and growing impact of metaverse as a space for social interaction and an increasingly used tool.
Conclusions
Our meta-analysis showed that innovative interventions involving the use of virtual reality and communication technologies are effective tools for stigma reduction toward people with mental illness and can be an alternative and a complement for the traditional methods on stigma reduction. As this field is growing and emerging, future studies present several challenges in their adaptation and dissemination in different populations and countries. | 2022-04-16T15:04:09.943Z | 2021-11-24T00:00:00.000 | {
"year": 2022,
"sha1": "3552b158705016d874ae9c3bf411c3f4e4faf4b6",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.2196/35099",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "843bcc87136ac4480d18508f5a78ff571d35c2a9",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
11984319 | pes2o/s2orc | v3-fos-license | HIV-associated Burkitt lymphoma in a Japanese patient with early submandibular swelling
Background Patients infected with the human immunodeficiency virus (HIV) are at risk of developing malignancies and have an increased susceptibility to infection. HIV-associated Burkitt lymphoma (BL) is relatively rare in developed countries, but remains prevalent in developing counties and is sometimes compounded by the fact that patients may be unaware that they are HIV-positive. Case presentation A 37-year-old Japanese man was referred to our department for diagnosis and management of submandibular swelling. He was unaware that he was HIV-positive at the initial visit. Here, we describe our diagnostic approach, in which we used hematological and immunological investigations, biopsy, fluorescence-activated cell sorting and fluorescence in situ hybridization to confirm the diagnosis of HIV-associated BL. The patient has no risk factors for HIV infection, and the source of infection remains unclear. Conclusions In this case, submandibular swelling was the first clinical sign of pathology and the patient’s HIV-positive status only became evident later. It is highly likely that BL was triggered by HIV infection.
Background
Human immunodeficiency virus (HIV) was first reported in 1981, in a cohort of homosexual men with Pneumocystis carinii pneumonia [1]. By 2011, the Joint United Nations Program on HIV/AIDS estimated that the number of adults and children living with HIV was between 31.4 million and 35.9 million). In developing countries, especially sub-Saharan Africa, widespread HIV infection has caused severe economic and social problems as a result of decreased life expectancy and increased childhood mortality [2]. HIV-positive patients are more likely to develop malignant disease than healthy individuals because of the immunosuppressive effects of the virus. Moreover, it is not uncommon for patients to be unaware that they are infected with HIV.
Burkitt lymphoma (BL) was first reported in 1958 as a sarcoma of the jaw in a Ugandan patient [3]. Spina et al. [4] and Straus [5] have reported that BL accounts for only 1-3% of lymphomas in HIV-seronegative adults, but this figure rises to 15-40% for AIDS-related lymphomas. We report an uncommon case of HIV-associated BL with clinical signs of submandibular swelling in a Japanese man.
Case presentation
A 37-year-old man was referred to the Department of Oral-Maxillofacial Surgery, Dentistry and Orthodontics at the University of Tokyo Hospital for diagnosis and management of a swelling in the right submandibular region, which had been present for approximately 1 month and was associated with sublingual discomfort on eating ( Figure 1A). On clinical examination, the swelling was localized to the right side of the floor of the mouth ( Figure 1B), and the amount of saliva produced from the right orifice of the right Wharton's (submandibular) duct was reduced. Computed tomography (CT) views revealed a 50 × 36 mm submandibular adenopathy with a clear border ( Figure 1C) but no regional lymph node enlargement. The provisional diagnosis was of submandibular gland inflammation, with a differential diagnosis of malignant lymphoma (ML). Panoramic radiography showed no radiolucent areas that would be characteristic of bony erosion, invasion or destruction, nor were there any radiopaque areas that may be indicative of a sialolith. The medical history was unremarkable. The results of cytology investigations in the right lateral region of the neck indicated a Class II lesion. Exploration of the right orifice of the right Wharton's duct restored the volume of saliva to normal. Taken together, these findings suggested that the right submandibular swelling was caused by inflammation of the submandibular gland secondary to obstruction of the salivary duct. However, after exploration, the swelling enlarged rapidly and the patient was admitted to hospital for biopsy and further investigations without delay. Fluoro-D-glucose (FDG) positron emission tomography (PET) CT imaging showed maximum intensity signals in the submandibular area and lymphadenopathy in the axillary, inguinal and deep cervical nodes (Figure 2A), findings that are highly suspicious of ML. Surprisingly, immunological and hematological investigations revealed the presence of anti-HIV-1/2 antibodies, HIV-1 ribonucleic acid (3200 copies/ml) and increased LDH levels; however, the levels of sIL-2R and other parameters were normal ( Figure 2B). More detailed immunological examination found that anti-VCA-IgG and EBNA antibodies were increased, although anti-VCA-IgM antibodies were at normal levels ( Figure 2B). Taken together, these findings indicated a likely diagnosis of HIVassociated malignant lymphoma with no involvement of Epstein Barr virus (EBV). The patient was referred to the Department of Hematology and Oncology at our institution, and, as he was unaware of his HIV-positive status, he was also referred to the Department of Infectious Diseases. At the same time, we also undertook a biopsy of the right submandibular area under local anesthesia. The swelling had a clear border covered with a membrane and exhibited no adhesions to the surrounding tissue. Hematoxylin and eosin staining revealed numerous pathognomonic apoptotic cells with a so-called 'starrysky' appearance ( Figure 3A). The c-myc translocation was identified by fluorescence in situ hybridization, which identified that the rearrangement was positive for IgH/MYC, but negative for IgH/BCL-2 ( Figure 3B). Furthermore, representative fluorescence-activated cell sorting analysis showed negative staining for T-cell associated markers (CD3, CD4 and CD8) and positive staining for B cell-associated antigens (CD19, CD20 and the germinal center-associated marker, CD10; Figure 4). The final diagnosis of HIV-associated BL based on the revised World Health Organization (WHO) classification was confirmed from the clinical, hematological, radiological, histopathological, immunohistochemical and cytochemical analyses. According to a classification based on the Ann Arbor scheme in conjunction with FDG-PET, the disease was diagnosed as being at Stage III.
HIV infection was treated with highly active antiretroviral therapy at a specialist center, and BL was treated with four cycles of R-HyperCVAD/R-MA chemotherapy administered without radiotherapy. Two years after diagnosis, he continues to receive treatment at the same hospital with no evidence of recurrence. The patient has no apparent risk factors for HIV infection.
Conclusions
Burkitt lymphoma is a highly aggressive non-Hodgkin lymphoma (NHL) [6]. According to the WHO classification, there are three clinical variants of BL: endemic, sporadic, and immunodeficiency-associated. Endemic BL develops in parts of Africa and New Guinea, in most cases at 4-7 years of age, with boys affected twice as frequently as girls [7]. It can involve the jaw and other facial bones, kidneys, gastrointestinal tract, ovaries, breast, and other extranodal sites. Sporadic BL is a worldwide phenomenon with no specific geographic or climatic association [6], accounting for 1-2% of adult lymphomas and up to 40% of child lymphomas in the United States and Western Europe [7]. Sporadic BL most commonly presents in the abdomen, ovaries, kidneys, omentum,
MYC B
A MYC/IgH and Waldeyer's tonsillar rings. Endemic BL is strongly associated with EBV infection, but the etiology of sporadic BL has yet to be defined [8]. Immunodeficiency-associated BL occurs in HIV-infected patients and allograft recipients [9]. This case is highly likely to be an immunodeficiencyassociated BL.
HIV-infected patients have a two-fold increased risk of developing malignant disease; in the head and neck the majority of cancers are Kaposi's sarcoma or oral Kaposi's sarcoma (68%), with squamous cell carcinoma and NHL accounting for 17% and 13%, respectively, and only 2% diagnosed as BL [10]. Burkitt lymphoma is strongly associated with HIV infection, and HIV-associated BL accounts for approximately 5-40% of cases of HIVassociated NHL [6,8,11]. HIV-associated BL is rare in developed countries, despite being common in developing countries. However, the number of HIV carriers in Japan has recently increased, in part because of internationalization, which may be expected to lead to an increase in HIV-associated BL.
Generally, patients with submandibular swelling visit a dental clinic or are referred to a specialist center for investigation and treatment. In such cases, differential diagnoses that must be considered include inflammation of the submandibular gland, submental ranine, submental lymphadenitis, a tumor of the submandibular gland, a metastatic tumor, and ML. Thus, screening for infectious disease, notably for circulating antibodies against HIV and EBV, is important in cases where suspected BL cannot be differentiated from HIV-associated BL by clinical, radiological, histopathological and immunohistochemical analyses alone. Furthermore, those practicing in oral and maxillofacial medicine should take a history that includes questions regarding sexual history (e.g. sexual intercourse with different partners), travel history (especially to/from countries with many HIV carriers) and history of transfusion of blood products to facilitate differential diagnosis. In this case, the patient is neither homosexual nor has any history of travel to developing countries where HIV is prevalent.
In conclusion, this case emphasizes the importance of an integrated diagnostic approach for the early diagnosis and appropriate treatment of HIV-associated BL in the submandibular region.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images.
Ethics approval
This study was performed in conformity with the Declaration of Helsinki, and was approved by our institutional ethical committee. | 2017-08-03T02:29:32.021Z | 2013-12-01T00:00:00.000 | {
"year": 2013,
"sha1": "80659e546a4b6c995b6f29f75199efbfdf249e24",
"oa_license": "CCBY",
"oa_url": "https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/1756-0500-6-557",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "125a8cab8c201c015894f98b4aa304c26bcd38fc",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
237292823 | pes2o/s2orc | v3-fos-license | Modeling a deep transfer learning framework for the classification of COVID-19 radiology dataset
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Coronavirus-2 or SARS-CoV-2), which came into existence in 2019, is a viral pandemic that caused coronavirus disease 2019 (COVID-19) illnesses and death. Research showed that relentless efforts had been made to improve key performance indicators for detection, isolation, and early treatment. This paper used Deep Transfer Learning Model (DTL) for the classification of a real-life COVID-19 dataset of chest X-ray images in both binary (COVID-19 or Normal) and three-class (COVID-19, Viral-Pneumonia or Normal) classification scenarios. Four experiments were performed where fine-tuned VGG-16 and VGG-19 Convolutional Neural Networks (CNNs) with DTL were trained on both binary and three-class datasets that contain X-ray images. The system was trained with an X-ray image dataset for the detection of COVID-19. The fine-tuned VGG-16 and VGG-19 DTL were modelled by employing a batch size of 10 in 40 epochs, Adam optimizer for weight updates, and categorical cross-entropy loss function. The results showed that the fine-tuned VGG-16 and VGG-19 models produced an accuracy of 99.23% and 98.00%, respectively, in the binary task. In contrast, in the multiclass (three-class) task, the fine-tuned VGG-16 and VGG-19 DTL models produced an accuracy of 93.85% and 92.92%, respectively. Moreover, the fine-tuned VGG-16 and VGG-19 models have MCC of 0.98 and 0.96 respectively in the binary classification, and 0.91 and 0.89 for multiclass classification. These results showed strong positive correlations between the models’ predictions and the true labels. In the two classification tasks (binary and three-class), it was observed that the fine-tuned VGG-16 DTL model had stronger positive correlations in the MCC metric than the fine-tuned VGG-19 DTL model. The VGG-16 DTL model has a Kappa value of 0.98 as against 0.96 for the VGG-19 DTL model in the binary classification task, while in the three-class classification problem, the VGG-16 DTL model has a Kappa value of 0.91 as against 0.89 for the VGG-19 DTL model. This result is in agreement with the trend observed in the MCC metric. Hence, it was discovered that the VGG-16 based DTL model classified COVID-19 better than the VGG-19 based DTL model. Using the best performing fine-tuned VGG-16 DTL model, tests were carried out on 470 unlabeled image dataset, which was not used in the model training and validation processes. The test accuracy obtained for the model was 98%. The proposed models provided accurate diagnostics for both the binary and multiclass classifications, outperforming other existing models in the literature in terms of accuracy, as shown in this work.
INTRODUCTION
Viral pandemics are usually a serious threat to the world, and coronavirus disease 2019 is not an exception. According to a COVID-19 report of the World Health Organization (WHO), coronaviruses are from a large family of viruses that cause illness in animals or humans (WHO, 2020). Numerous coronaviruses have been reported as the cause of respiratory disease in humans, ranging from the common cold to more serious illnesses like Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). This newly discovered coronavirus has caused the 2019-novel coronavirus disease. COVID-19 was initially observed in the Wuhan province of China and has spread to all parts of the world (Nadeem, 2020). COVID-19 was recognized as a contributory virus by Chinese authorities on January 7, 2020. The Director-General of WHO, on the January 30, 2020 reported that the epidemic constitutes a Public Health Emergency of International Concern (PHEIC), based on the recommendations made by the Emergency Committee. WHO activated the R&D Blueprint in reaction to the occurrence to speed up diagnostics, vaccines, and therapeutics for this new coronavirus (WHO, 2020). The International Committee on Taxonomy of Viruses named the novel coronavirus "severe acute respiratory syndrome coronavirus 2 (SARS-Coronavirus-2 or SARS-CoV-2)". Globally, as of March 10, 2021, there have been 117,332,262 confirmed cases of COVID-19, including 2,605,356 deaths, reported to WHO. Also, as of March 9, 2021, a total of 268,205,245 vaccine doses have been administered (WHO, 2021). Specifically, in Nigeria, the Nigeria Centre for Disease Control (NCDC) report on March 11, 2021, showed 394 new confirmed cases recorded. To date, 159,646 cases have been confirmed, 139,983 patients have recovered and discharged, and 1,993 deaths have been recorded in 36 states and the Federal Capital Territory (Nigeria Centre for Disease Control, 2021).
Numerous researchers globally are putting their efforts together on collecting data and developing solutions. The persistent focus has been on advancing key performance indicators, for example, continually enhancing the speed of case detection, segregation, and early cure. The execution of these containment procedures has been sustained and enabled by the pioneering and aggressive use of cutting-edge technologies. Measures such as immediate case detection and isolation, rigorous close contact tracing and monitoring/quarantine, and direct population/community engagement have been considered in lessening COVID-19 illness and death. This work, therefore, is aimed at using Artificial Intelligence (AI), specifically machine learning, to identify those who are at risk of contracting COVID-19 to aid early diagnosis. According to British Broadcasting Corporation (BBC) (2020), a superhuman attempt is required to ease the deaths due to the global epidemic. AI may have been overestimated-but in the case of medicine, it already has established evidence. According to Arora et al. (2020), the role of AI is going to be crucial for predicting the outcome based on symptoms, CT-Scan, X-ray reports, etc.
Laboratory checking of suspected cases is characterized by extended periods of testing and an exponential rise in test requests (Kobia & Gitaka, 2020). Quick diagnostic tests with shorter turnaround times of between 10 and 30 minutes have been developed to ease the problem. However, many are presently going through clinical validation and is not in regular use (ECDC, 2020). While awaiting results, there is a need to continue to self-isolate. Once results are received, there is a need to remain on self-isolation until the symptoms resolve after being in seclusion for at least 14 days. If the symptoms worsen during the seclusion time or continued after 14 days, the patient has to contact the accredited healthcare providers. Rapid Test Kits can even deliver results after hours.
Pneumonia has been described as the most severe and frequent manifestation of COVID-19 infection ; therefore, chest imaging including readily available and affordable chest radiograph (X-rays) remains an essential factor in the diagnosis and evaluation of COVID-19 patients (Rubin et al., 2020). However, the availability of radiologists to report the chest images is another obstacle. Therefore, there is a need to develop computer algorithms and methods to optimize screening and early detection; that is the primary purpose of this research in which deep learning, most especially Convolutional Neural Network (CNN), is deployed. Deep learning provides the chance to increase the accuracy of the early discovery by automating the primary diagnosis of medical scans (Madan, Panchal & Chavan, 2019). The CNN belongs to a category of Deep Neural Networks (DNN), which comprise several layers that are hidden like convolutional layers. The convolutional layers come with the non-linear activation function, a rectified linear unit (ReLU layer), a pooling layer, and a fully connected normalized layer. CNN divides weights in the convolutional layer, thereby decreasing the memory footprint and increasing the performance of the network (Sasikala, Bharathi & Sowmiya, 2018). The objective of this paper is to classify a real-life COVID-19 dataset consisting of X-ray images using a novel Deep Learning Convolutional Neural Network Model. Data from chest X-rays were used because most hospitals have X-ray machines, and the COVID-19 X-ray dataset is now available on the web. The remaining parts of this paper are organized as follows: literature review, materials and methods, experimentation and results, evaluation of results, conclusion and future works.
LITERATURE REVIEW
COVID-19 is predominantly a respiratory illness, and pulmonary appearances constitute the main presentation of the disease. SARS-CoV-2 infects the respiratory system but may also affect other organs, as reported in some studies. Renal dysfunction (Chu et al., 2005;, gastrointestinal complications (Pan et al., 2020), liver dysfunction , cardiac manifestations , mediastinal findings (Valette, du Cheyron & Goursaud, 2020), neurological abnormalities, and haematological manifestations (Song & Shin, 2020) are among the reported extrapulmonary features. Some of the clinical symptoms of COVID-19 are cough, expectoration, asthenia, dyspnoea, muscle soreness, dry throat, pharyngeal dryness and pharyngalgia, fever, poor appetite, shortness of breath, nausea, vomiting, nasal obstruction and rhinorrhoea. A study on COVID-19 credited to WHO, stated that the disease does not exhibit distinct symptoms, and patients' symptoms can vary from fully asymptomatic to extreme pneumonia and death (WHO, 2020). Nevertheless, certain symptoms, such as dry cough, fever, dyspnea and fatigue, were confirmed to be more prevalent in COVID-19 patients. Sore throat, nasal inflammation, fever, arthralgia, chills, diarrhoea, hemoptysis, nausea and conjunctival congestion are some of the other clinical symptoms (Behzad et al., 2020). Other non-specific symptoms include loss of smell and taste, dermatologic eruptions, delirium, and a general decline in health (Recalcati, 2020). Because of the wide range of clinical indications and the growing global burden of COVID-19, it is critical to promptly scale up diagnostic ability to diagnose the virus and its risks.
Reverse transcription-polymerase chain reaction (RT-PCR) is the primary clinical instrument currently in use to detect COVID-19. It uses respiratory specimens for testing (Wang et al., 2020a). RT-PCR is used as a reference method for detecting COVID-19 patients; however, the technique is expensive, manual, complicated, time-consuming, and requires specialized medical personnel. Alternatively, X-ray imaging is an easily accessible tool that can be excellent in the COVID-19 diagnosis. Chest imaging is an essential part of evaluating respiratory complications, which remain one of the most familiar presentations ranging from acute respiratory distress syndrome to respiratory failure . Chest imaging has been defined as an efficient screening method for detecting pneumonia, with a sensitivity of 97.5 percent for COVID-19 (Nabila et al., 2020; National Health Commission of People's Republic of China, 2020). Provided COVID-19's preference for the respiratory system, chest radiography (X-ray), CT of the thorax, and/or Ultrasound have been verified not only as case management and screening methods for COVID-19, but also as a way of reducing infection transmission through early detection in initially False-negative RT-PCR tests (Rubin et al., 2020). Imaging tests are useful for generating clinically actionable outcomes, which can be used to determine a diagnosis or to guide management, triage, or treatment. Costs such as the risk of exposure to radiation to the patient, the risk of COVID-19 transmission to uninfected health care staff and other patients, the use of personal protective equipment (PPE), and the need for sanitation and interruption of radiology rooms in resource-constrained settings reduce the benefit (Kooraki et al., 2020).
The role of imaging includes the detection of early parenchymal lung disease, disease progression, complications, and alternative diagnoses, including acute heart failure from COVID-19 myocardial injury and pulmonary thromboembolism (Driggin et al., 2020). Although CT is more sensitive than chest radiography, chest radiography remains the firstline imaging modality in COVID-19 patients because of its availability, affordability, reduced radiation risk, and ease of decontamination (Fatima et al., 2020). Hence, this research work utilized chest radiographs (X-ray) for identifying COVID-19 in patients. Bilateral, peripheral, lower zone prevalent ground-glass opacities are common chest radiograph observations in COVID-19 patients (Vancheri et al., 2020). Other possible findings include normal, unilateral, or bilateral reticular alterations, consolidations, ground-glass opacities and pleural effusion (Hamid, Mir & Rohela, 2020).
COVID-19 is transmitted through droplets from coughing or sneezing and on close contacts with infected persons. Propagation of based on infected surfaces are considered as the major means of transmitting SARS-CoV-2. However, patients going through screening are protected and scanned through the use of treated tools (Kooraki et al., 2020). The incubation period of COVID-19 is usually about 14 days, during which it attacks the lung. Different countries recommend personal protection equipment (PPE). According to the Centers for Disease Control and Prevention, radiology personnel should use a face mask, glasses or face shield, sleeves, and an isolation garment. A surgical cap and foot covers are required in countries with more rigorous PPE guidelines, whereas a surgical mask, goggles, or face shield are recommended in countries with less strict PPE guidelines (Centres for Disease Control & Prevention, 2020). COVID-19 is highly contagious, and the symptoms begin to appear 5-6 days after contracting it either from the droplet or close contact with an infected person. According to Wang, Tang & Wei (2020), the period between the manifestation of symptom and demise ranges from 6-41 days, depending on the age and immune system of the patient. The period is shorter among older people Hamid, Mir & Rohela, 2020).
COVID-19 shows some unique clinical symptoms like targeting the lower airway that manifests in sore throat, sneezing, and rhinorrhoea. In addition, the chest radiographs' result in some cases possess "infiltrate in the upper lobe of the lung" related to growing dyspnea with hypoxemia (Hamid, Mir & Rohela, 2020). COVID-19 has become endemic and rapid diagnosis is imperative to identify patients and carriers for possible isolation and treatment to curb the spread of the disease. Attempts have been made to diagnose the disease, but many are slow and not accurate in that they often give false-negative and false-positive results. This section examines some of the work done in this area.
Over comprehensive detection of each aggressive spread chain, Lokuge et al. (2020) proposed an accurate, fast, and flexible tracking approach for spotting all residual COVID-19 group transmission. Using surveillance evaluation methods, they considered efficiency and sensitivity in the classification of population transmission chains by testing primary care fever and cough patients, hospital cases, or asymptomatic community members. They also varied the number of duplications, monitoring capacities, and the prevalence of COVID-19 and non-COVID-19 fever and cough. The findings of the study revealed that testing both syndromic fever and cough primary care presentations, as well as precise and diligent case and touch monitoring and assessment, allows for proper primary direct detection and elimination of COVID-19 population transmission. Even with optimized test sensitivity, measures such as combining these approaches could allow for increased case discovery if testing capacity is minimal. The impact analysis of movement restriction as a result of emergence of COVID-19 was carried out by Hyafil & Moriña (2020). The study looked at the impact of the steps put in place in Spain to combat the epidemic. The instances and the influence of the imposed restriction to movement on the multiplicative quantity of hospitalization reports were estimated.
The projected figure of instances displayed a rapid rise towards total movement restriction as imposed. The primary replication rate reduced meaningfully from 5.89 (95% CI [5.46-7.09]) before the lockdown to 0.48 (95% CI [0.15-1.17]) after the lockdown. The study found that managing a pandemic in the magnitude of COVID-19 was very intricate and required timely decisions. The significant modifications found in the infestation rate displayed that employing inclusive participation in the first phase was vital in reducing the effect of a possible transferrable threat. The paper likewise stressed the significance of dependable up-to-date epidemiological facts to precisely measure the influence of Public Health guidelines on the virus-related outburst. Yu et al. (2020) observed the mounting evidence that suggested that there remained a hidden collection of COVID-19 asymptomatic but transferable cases and that approximating the count of disease instances without symptoms was essential in knowing the virus and curtailing its transmission; though, it was reported that it was difficult to precisely calculate the spread of the infection. A machine learning-based fine-grained simulator (MLSim) was proposed to combine many practical reasons, such as disease development during the maturation phase, cross-region population movement, unobserved patients without symptoms, preventative measures and confinement resilience, to estimate the number of asymptomatic infection, which is critical in understanding the virus and accurately containing its spread. Digital transmission mechanisms with many unspecified variables were used to simulate the relationships between the variables, which were calculated from epidemic data using machine learning approach. When MLSim learned to closely compare and contrast real-world data, it was able to simulate the instances of patients without symptoms as well. The accessible Chinese global epidemic data helped the MLSim to train better. The analysis indicated that fine-grained machine learning simulators could improve the modelling of dynamic realworld infection transmission mechanism, which can aid in the development of balanced mitigation steps. The simulator equally showed the possibility of a great amount of undiscovered disease risk, posing a major threat to containing the virus. COVID-19 was modelled using a composite stochastic and deterministic concept that allowed for timevarying transmission capacity and discovery chances (Romero-Severson et al., 2020). Iterative particle sorting was used to adapt the model to a historical data study of occurrence and casualty figures from fifty-one countries. The report confirmed the fact that the spread rate is decreasing in forty-two of the fifty-one countries surveyed. Out of the forty-two countries, 34 showed a big significant proof for subcritical transmission rates, though the turndown in novel cases was moderately slow in comparison to early development rates. The study concluded that attempts to reduce the occurrence of COVID-19 by social distancing were successful. They could, however, be improved and retained in various regions to prevent the disease from resurfacing. The study also proposed other approaches to manage the virus before the relaxation of social distancing efforts.
The challenges associated with the storage and security of COVID-19 patients' data were highlighted by ElDahshan, AlHabshy & Abutaleb (2020). The authors pointed out that the variety, volume, and variability of COVID-19 patients data required storage in NoSQL database management systems (NoSQL DBMS). It was noted that available NoSQL DBMSs were fraught with security challenges that rendered them unsuitable for storing confidential patient data. Academic institutions, research centres, and enthusiasts find it difficult to select the most suitable NoSQL DBMS because there are myriads of them without standard ways of determining the best. Thus, the study presented an inventive approach to selecting and securing NoSQL DBMS for medical information. The authors outlined the five most common NoSQL database groups, as well as the most common NoSQL DBMS forms affiliated with every one of them. In addition, their research included a comparison of the various types of NoSQL DBMS. The paper provided an efficient solution to myriads of security challenges, ranging from authorization, authentication, encryption, and auditing in storing and securing medical information utilizing a collection of web service-based functions.
Guerrero, Brito & Cornejo (2020) used a mathematical model to depict a sneezing individual in an urban setting with a meteorological wind of medium strength. The proliferation of airborne route was demonstrated using a Lagrangian method and a wall-modeled Large Eddy Numerical simulation. The results showed that the dimensions of two kinds of droplets differ in size: larger droplets (400-900 µm) scatter between 2-5 m in 2.3 s, whereas smaller (100-200 µm) droplets are transported in a larger, more impressive array between 8-11 m by the windy conditions in 14.1 s on average. Knowing the ambiguity of possible infection in this way aids in the development of solutions for the possibility of adopting tougher self-care and distance policies.
To distinguish communicable acute abdomen patients suspected of COVID-19, Zhao et al. (2020) proposed a forecasting model identified as a monogram and scale. The analytical framework was built on the basis of a retrospective case study. In a training cohort, the model was formulated using LASSO regression and multivariable logistic regression method. In the training and testing cohorts, standard curve evaluated the efficiency of the monogram, receiver operating characteristic (ROC) curves, decision curve analysis (DCA), and clinical effect curves. According to the monogram, a simpler testing scale and management algorithm was developed. In the testing cohort, the CIAAD monogram demonstrated strong differentiation and standardization, which was approved. The CIAAD monogram was clinically useful, according to decision curve research. The monogram was condensed even further into the CIAAD standard. The estimated Bayesian Computation method was utilized by Vasilarou et al. (2020) to determine the parameters of a demographic scenario engaging an exponential growth of the size of the COVID-19 populations and revealed that rapid exponential development in population size could sustain the monitored polymorphism patterns in COVID-19 genomes.
Amrane et al. (2020) adopted a genetic approach using a rapid virological diagnosis on sputum and nasopharyngeal samples from suspect patients. Two real-time RT-PCR systems employing a "hydrolysis probe and the LightCycler Multiplex RNA Virus Master Kit" were used. The primary technique probes the envelope protein (E)-encoding gene and used a synthetic RNA positive control. The subsequent system targeted the spike proteinencoding gene (forward primer, reverse primer, and probe) and used synthetic RNA positive control methods. Bai et al. (2020) proposed the use of medical technology through the internet of things (IoT) to develop an intelligent analysis and treatment assistance programme (nCapp). The conceptual cloud-based IoT platform includes the basic IoT functions as well as a graphics processing unit (GPU). To aid in deep mining and intelligent analysis, cloud computing systems were linked to existing electronic health records, image cataloguing, picture cataloguing, and interaction. Li et al. (2020) examined chest images for the analysis of COVID-19. High-resolution Computed tomography (HRCT) was implemented for analysis of the virus infection. CT scans were taken with the following parameters: 120 KV; 100-250 mAs; collimation of 5 mm; the pitch of 1-1.5; and 512 × 512 matrix. The images were reconstructed by high resolution and conventional algorithms. The experiments were repeated several times, running into days for each patient. The High-resolution CT objectively evaluated lung lesions giving a better understanding of the pathogenesis of the disease. Long et al. (2020) evaluated the suitability of Computed Tomography (CT) and realtime reverse-transcriptase-polymerase Chain Reaction (rRT-PCR). A clinical experiment with life data was executed, and the results presented showed that CT examination outperformed that of rRT-PCR at 97.2% and 84.6%, respectively. In Vaishya et al. (2020), seven critical AI applications for the novel COVID-19 were recognized to perform vital roles in screening, analyzing, tracking, and predicting patients. Application areas identified comprised early detection and diagnosis of the infection, treatment monitoring, individuals contact tracing, projection of cases and mortality, drugs and vaccines development, lessening healthcare workers' assignment, and deterrence of the disease by providing updated supportive information. Zhang et al. (2020) conducted a survey that presented some proofs of mental distress and associated predictors amongst adults in the current COVID-19 pandemic in Brazil. The data was composed of 638 adults from March 25 to 28, 2020, about one month after the index case was confirmed in São Paulo. Female adults, who were young, more trained, and practiced less, recorded higher levels of distress, with 52 percent experiencing mild-to-moderate distress and 18.8 percent suffering extreme distress. The study's findings also revealed that a person's distance from Sao Paulo, the epicenter, had a direct connection with the psychological distress they were experiencing. For the older population who worked the least, the "typhoon eye effect" was more potent. Adults who lived far off the worst hit geographic area and did not go to work in the week preceding the survey were the most vulnerable. The paper concluded that recognizing the predictors of suffering would allow mental health services to improve target finding and assist the more mentally defenseless adults in the crisis. Ghafari et al. (2020) assessed the challenges and indications of concern of the COVID-19 in Iran. The heterogeneous COVID-19 casualty levels around the country in fourteen university hospitals in Tehran were investigated, and it was revealed that the recorded cases on 13/03/2020 indicated just under 10% of symptomatic patients in the population in adolescents. The finding indicated that there was a major inaccurate reporting of cases in Iran. The study suggested that strict measures be implemented throughout a period of widespread underreporting in order to prevent the healthcare system from being exhausted within a month. Further studies on efficient diagnosis, detection, and vaccine of the virus are continuing for two main reasons: partly because the disease is new, and secondly because available research efforts have not been able to address the concerns effectively. Therefore, in this paper, a deep learning modeling framework for efficient identification, classification, and provision of new insights for the diagnosis of COVID-19 was presented. Also, the prediction of probable patients of the novel COVID-19 using radiology scanned images of suspected patients were shown. Zivkovic et al. (2021) proposed a hybrid comprising machine learning Adaptive Neuro-Fuzzy Inference System (ANFIS) and enhanced Bio-inspired Beetle Antennae Search (BAS) Algorithm referred to as CESBAS-ANFIS. The hybrid study was carried out with a view to improving the existing time-series prediction algorithms for forecasting COVID-19 new cases. An improved BAS algorithm was adopted to update the parameters of ANFIS. A prediction model for the virus outbreak was formulated using ANFIS trained by the improved BAS algorithm to enhance the prediction accuracy of new cases of COVID-19. CESBAS was introduced to update ANFIS parameters, thereby solving parameters' optimization problem of machine learning techniques for prediction. Cauchy mutation operator was incorporated into the original BAS called CESBAS (Cauchy Exploration Strategy BAS) to improve exploration ability and solution diversity deficiencies observed. The proposed method consists of five layers; one input layer, two hidden layers, one layer for conclusion parameters and the output layer that presented the forecasted value. The proposed model with other hybrid models was tested under the same conditions on two datasets; one dataset from WHO and the second from the "our world in data" website, and their results were compared. CESBAS-ANFIS showed superior performance compared to other hybrid techniques such as ABC-ANFIS, BAS-ANFIS and FPA-ANFIS. Irfan et al. (2021) explored the contributions of hybrid deep neural networks (HDNNs), chest X-rays and computed tomography (CT) in the detection of COVID-19. The work employed X-ray imaging and CT to develop the HDNNs for predicting the early infection of COVID-19. The HDNNs were trained and tested on five thousand (5,000) images collected from five different sources (public and open), comprising 57% males and 32% females, and 3,500 infected and 1,500 healthy controls within an age group of 38-55 years. The proportion of the test dataset to the training dataset was 20:80, and classification accuracy of 99% was achieved with the HDNNs on the test dataset. The results of the performed experiments showed that the new multi-model and multi-data approach achieved improved performance over the traditional machine learning models. Elzeki et al. (2021) presented a new deep learning computer-aided scheme for rapid and seamless classification of COVID-19. Consisting of three separate COVID-19 X-ray datasets, the study presented the COVID Network (CXRVN) model for assessing grayscale chest X-ray images. The scheme was implemented on three different datasets using MatLab 2019b. A comparison was made with the pre-trained models of AlexNet, GoogleNet and ResNet using the mini-batch gradient descent and Adam optimizer to aid the learning process. Performance evaluation results of the model using F1 score, recall, sensitivity, accuracy, and precision, with generative adversarial network (GAN) data augmentation revealed that the accuracy for the two-class classification was 96.7%.
In comparison, the three-class classification model reached 93.07%. However, the authors pointed out that increased availability of datasets could improve the performance of future methodologies. In addition, it was stated that the model could be enhanced by employing computed tomography (CT-images) and studying different updated cases of the COVID-19 X-ray images.
From the reviews presented in this section, it was observed that some of the existing works reported low accuracies, used imbalanced datasets, and there was no evidence of the use of some standard evaluation metrics such as Matthew's Correlation Coefficient and Cohen's Kappa Statistics in their approaches. The aforementioned issues were addressed in this paper. In addition, the performance of the VGG-16 and VGG-19 networks, the forms of the VGGNet Architecture, in predicting COVID-19 X-ray datatsets were compared in this study.
The VGGNet architecture
The VGGNet, proposed by Simonyan & Zisserman (2015), is a convolutional neural network that performed very well in the ImageNet Large Scale Visual Recognition Challenge (ILSVRC) in 2014. The VGG-16 and VGG-19 networks are forms of the VGGNet Architecture. The networks accept color images with size 224 × 224 and three channels (Red, Green and Blue) as their input data. The images pass through convolutional layers that are stacked on top of each other, in which there is a limited reactive field of 3 × 3 and stride of 1 in the convolutional filter. The convolutional kernel employs row and column padding such that the resolution before the convolution is retained after the processing of the images. Max-pooling is then done over a max pool window of size 2 × 2 with a stride of 2 (Simonyan & Zisserman, 2015).
The network of the VGG-16 CNN has 13 convolutional layers (that is, 3 × 3 convolutional layers in blocks that are stacked on top of one another with growing depth). Two blocks house two 3 × 3 convolutional layers of the same setup in a sequential arrangement, while three blocks have three 3 × 3 convolutional layers of the same configuration in a sequential arrangement. As such, the VGG-16 has two contiguous blocks of two convolutional layers, with each block accompanied with a max-pooling. Also, it has three continuous blocks of three convolutional layers, with each block accompanied with a max-pooling. In all, there are five max-pooling layers in the architecture. Max pooling layer handles the reduction of the volume size after each block that contains two convolutional layers and after each block that contains three convolutional layers. The informative features are obtained by these max-pooling layers that are applied at the earlier specified stages in the network. The VGG-16 further has two fully-connected layers, each with 4,096 nodes and one fully-connected layer with 1,000 nodes, one node each for each of the 1,000 categories of images in the ImageNet database on which the network was pre-trained, and is followed by the SoftMax classifier (Simonyan & Zisserman, 2015), as presented in the VGG Architecture, which can be found in Frossard (2016).
The network of the VGG-19 CNN has 16 convolutional layers (that is, 3 × 3 convolutional layers in blocks that are stacked on top of one another with growing depth).
Two blocks house two 3 × 3 convolutional layers of the same setup in a sequential arrangement, while three blocks have four 3 × 3 convolutional layers of the same configuration in a sequential arrangement. In other words, the VGG-19 has two continuous blocks of two convolutional layers, with each block accompanied by a maxpooling. It also has three contiguous blocks of four convolutional layers, with each block accompanied with a max-pooling. In all, there are five max-pooling layers in the architecture. The Max-pooling layer handles the reduction of the volume size after each block that contains two convolutional layers and after each block that contains four convolutional layers. The informative features are obtained by these max-pooling layers that are applied at the earlier specified stages in the network. The VGG-19 further has two fully-connected layers, each with 4,096 nodes and one fully-connected layer with 1,000 nodes, one node each for each of the 1,000 categories of images in the ImageNet database on which the network was pre-trained, and is followed by the SoftMax classifier (Simonyan & Zisserman, 2015).
MATERIALS & METHODS
This study focused on diagnosing the COVID-19 chest X-ray dataset using a deep learning convolutional neural network (CNN). The CNN comprises one or more convolution layers and then followed by one or more fully connected layers as obtained in a standard multilayer neural network. COVID-19 Radiology Dataset (chest X-ray) for Annotation and Collaboration was collected from the Kaggle website (a database collated by researchers from Qatar University and the University of Dhaka) with collaborators from Pakistan and Malaysia, and some medical doctors, and Mendeley dataset repository. The data was preprocessed, and the median filter was employed to restore the image undergoing evaluation by mitigating the severity of collection degradations. Manikandarajan & Sasikala (2013) mentioned some preprocessing and segmentation strategies that were used. Each data point was replaced by the average value of its neighbors, and itself, in the median filter. As a result, data points that differed significantly to their neighbors were removed. Following the preprocessing of the image dataset, the images were sectioned by using a simulated annealing algorithm. Feature extraction and classification were done using CNN. The neural network-based convolutional segmentation was implemented in Jupyter Notebook using Python programming language and the model was built using sample datasets for the system to recognize and classify the COVID-19. The model generated can be used to develop a simple web-based application that medical personnel handling COVID-19 tests could use to input new cases and quickly predict the presence of the COVID-19, with a very high level of accuracy.
Dataset description and preprocessing
Chest X-ray images were selected from a repository of COVID-19 positive cases' chest X-ray images, as well as regular and viral pneumonia images, which were collated by researchers from Qatar University and the University of Dhaka, along with collaborators from Pakistan and Malaysia, and some medical doctors. There are 219 COVID-19 positive images in their current release, 1,341 normal images, and 1,345 viral pneumonia images (Chowdhury et al., 2020). For multiple representations, the dataset of chest X-ray images for both COVID-19 and normal cases were also selected from the Mendeley dataset repository (El-Shafai & Abd El-Samie, 2020), which contains 5,500 Non-COVID X-ray images and 4,044 COVID-19 X-ray images. This study, therefore, adopted these multisource datasets. Due to limited computing resources, in this study, 1,300 images were selected from each category for model building and validation. In other words, 1,300 images of COVID-19 positive cases, 1,300 normal images, and 1,300 images of viral pneumonia cases, totalling 3,900 images in all. Also, a different set of 470 images (containing 70 COVID-19 images, 200 ViralPneumonia and 200 Normal) were selected and used for testing to obtain an impartial evaluation of a final model. The dataset used in this study can be found at Fayemiwo et al. (2021a). It should be noted here that further descriptions of the datasets were not provided by the authors of the dataset's sources.
OpenCV (Bradski & Kaehler, 2008) was used for loading and preprocessing images in the dataset. Each image was loaded and preprocessed by performing a conversion to RGB channel and changing the size of the images to 224 × 224 pixels to be ready for the Convolutional Neural Network. Pixel intensities were then scaled to the range [0, 1] and converted the data and labels to NumPy array format. Labels were then encoded using a one-hot encoder while training/testing splits were created. To ensure that the model generalizes well, data augmentation was performed by setting the random image rotation to 15 degrees, random range zooming to 0.15, random shift of width and height to 0.2, random shear range to 0.15, randomly flipping half of the images horizontally by setting horizontal_flip = True and fill_mode to nearest. One thousand, nine hundred and fifty (1,950) images were allocated initially to train the model in the binary classification task, resulting in 78,000 training images after augmentation. Two thousand, nine hundred and twenty-five (2,925) images were allocated initially for training the model in the multiclass classification task, which resulted in 117,000 training images after augmentation.
The deep-transfer learning model
This study employed the VGG-16 and VGG-19 Convolutional Neural Networks (CNN) with Deep Transfer Learning (DTL) approach for COVID-19 detection. The DTL approach focused on the storage of weights that have been grown while unraveling some image classification tasks and then engaging them on a related task. Several DTL networks have been proposed, some of which include VGGNet (Simonyan & Zisserman, 2015), GoogleNet (Szegedy et al., 2015), ResNet (He et al., 2015), DenseNet (Huang et al., 2017) and Xception (Chollet, 2017). In this paper, VGG-16 CNN and VGG-19 CNN, forms of the VGGNet, were trained on the popular ImageNet images dataset. The VGG-16 and VGG-19 CNN were pre-trained deep neural networks on the ImageNet for computer vision (image recognition) problems, having 16 weight layers and 19 weight layers, respectively. They were used as pre-trained models to help learn the distinguishing features in COVID-19 X-ray images with the aid of a transfer learning approach, thus, trained DTL models for the identification of COVID-19 from X-ray images.
As shown in the workflow in Figs. 1 and 2, respectively, to train the VGG-16 based DTL model and VGG-19 based DTL model for the detection of COVID-19, the VGG-16 CNN and VGG-19 CNN was used as pre-trained models and were fine-tuned for COVID-19 detection based on the principles of transfer learning. The weights of the lower layers of the network, which train very common characteristics from the pre-trained model, were used as feature extractors for the implementation of transfer learning with fine-tuning. Therefore, the pre-trained model's lower layers weights were frozen and therefore not updated through the training process, thus not participating in the transfer-learning process. The higher layers of the pre-trained model were used for learning task-specific features from the COVID-19 images dataset. In this case, the higher layers of the pretrained model were unfrozen, made trainable or fine-tuned in which the weights of the layers were updated. Consequently, the layers were allowed to participate in the transferlearning process. Each of these models ends with the SoftMax layer, which produces the outputs. The weights for the VGG-16 and VGG-19 networks were pre-trained on ImageNet, and the Fully Connected (FC) layer head was removed. From there, new fullyconnected layer heads were built comprising POOL => FC = SOFTMAX layers and attached on top of VGG-16 and VGG-19. The Convolutional weights of VGG-16 and VGG19 were then frozen in such a way that only the FC layer head was trained. Both binary class scenario and three-class classification scenario were considered in the workflow, in which the DTL model determines the class of the chest X-ray images as either "COVID-19" category or "Normal" category in the binary class scenario or as either "COVID-19" category, "Viral-Pneumonia" category, or "Normal" category in the threeclass classification scenario.
The flowchart of the experimental algorithm for the deep transfer learning models based on the VGG-16 and VGG-19 networks proposed in this paper is presented in Fig. 3.
EXPERIMENTATION AND RESULTS
Four different experiments were performed to classify radiological X-ray images using Deep Transfer Learning approaches. Two of the experiments (Experiments A and B) include each of the trained two Deep Transfer Learning models (VGG-16 and VGG-19 based) on binary class X-ray image dataset (with COVID-19 and Normal classes). The other two experiments (Experiments C and D) include each of the trained two Deep Transfer Learning models (VGG-16 and VGG-19 based) on a multiclass X-ray image dataset (with COVID-19, ViralPneumonia, and Normal classes). For these experiments, out of a total of 3,900 images used, 2,925 images (75%) were used for training the models, while 975 images (25%) were used for validation and to perform hyper-parameter tuning. A different set of 470 images 200 ViralPneumonia,and 200 Normal) were used for testing to obtain an impartial evaluation of the final model. The code for this experiment can be found at Fayemiwo et al. (2021b).
The performances of the proposed models were obtained from the models' generated confusion matrices, using standard metrics such as accuracy, specificity, precision, recall (sensitivity), F1-Score, Matthews Correlation Coefficient and Cohen's Kappa statistics. Equations 1, 2 and 3 show the formulas for computing the three key metrics used in this article, namely Accuracy, Matthews Correlation Coefficient and Cohen's Kappa statistics, respectively: employing a batch size of 10 in 40 epochs, using Adam optimizer specifically for updates of weights, cross-entropy loss function with a learning rate of 1e −2 . Performance of the proposed fine-tuned VGG-16 based DTL model was evaluated on 25% of the X-ray images.
The output of the confusion matrix for the binary classification as obtained from the VGG-16 based DTL model is shown in Table 2. Figure 4 illustrates the training loss and accuracy, the validation loss and accuracy graphs of the proposed fine-tuned VGG-16 based DTL model. The validation accuracy, recall, specificity, precision, F1-Score, Matthews Correlation Coefficient and Cohen's Kappa statistics of the proposed fine-tuned VGG-16 based DTL model were also obtained.
It was observed from Fig. 4 that the validation and training losses were slightly high in the earlier epochs and then almost flattened as the training occurs in subsequent epochs. The decrease in the loss values at around the 40th epoch was attributed to the fact that the fine-tuned VGG-16 based DTL model was exposed to all the available X-ray images time and again during each of the epochs considered during training. The validation accuracy obtained for the fine-tuned VGG-16 based DTL model was 99.23%, its recall was 100%, while its specificity stands at 98.48%. The obtained values for the precision, recall, F1-Score, Matthews Correlation Coefficient and Cohen's Kappa statistics metrics for the binary classification task using the VGG-16 based DTL model are given in Table 3.
Experiment B
The second experiment was performed on the binary class dataset. A DTL model based on a pre-trained VGG-19 model was trained to classify the X-ray images into the two classes of COVID-19 or Normal; and also, to detect if X-ray images are simply of the class COVID-19 or Normal. Also, the VGG-19 based DTL model summary detailing the layers and the parameters in each layer of the model is shown in Table 4. The fine-tuned VGG-19 based DTL model consists of 20,057,346 total parameters, with 32,962 of them made trainable while 20,024,384 were non-trainable. The VGG-19 DTL model was modelled by employing a batch size of 10 in 40 epochs, using Adam optimizer for the updates of weights, categorical cross-entropy loss function with a learning rate of 1e −1 . Performance of the proposed fine-tuned VGG-19 based DTL model was evaluated on 25% of the X-ray images.
The output of the confusion matrix for the binary classification as obtained from the fine-tuned VGG-19 based DTL model are shown in Table 5. Figure 5 illustrates the training loss and accuracy along with the validation loss and accuracy graphs of the proposed fine-tuned VGG-19 based DTL model. The validation accuracy, recall, specificity, precision, and F1-Score of the proposed fine-tuned VGG-19 based DTL model were also obtained.
It was observed from Fig. 5 that the validation and training losses were slightly moderate in the earlier epochs and then decreases as the training occurs in more subsequent epochs (with a sharp increase at about epochs 12 and 20). The decrease in the loss values at around the 40th epoch were attributed to the fact that the fine-tuned VGG-19 based DTL Table 4 The precision, recall and F1-score obtained for the classification task using the fine-tuned VGG-19 based DTL model (binary classification).
Layers
Layer's type Shape of output Num of trainable param model was exposed to all the available X-ray images repeatedly on all the epochs considered during training. The validation accuracy obtained for the fine-tuned VGG-19 based DTL model was 98.00%, its recall was 95.95%, while its specificity stands at 100%. The values obtained for the precision, recall, F1-Score, Matthews Correlation Coefficient and Cohen's Kappa statistics metrics for the binary classification task using the VGG-19 based DTL model are given in Table 6.
Experiment C
The third experiment was performed on the multiclass (three-class) dataset, in which a DTL model based on a pre-trained VGG-16 model was trained to classify the X-ray images into the three classes of COVID-19, Viral Pneumonia or Normal; and to detect if X-ray images were simply of the class COVID-19 or Viral Pneumonia or Normal. The VGG-16 based DTL model summary, detailing the layers and parameters in each layer of the model, is shown in Table 7. The fine-tuned VGG-16 based DTL model consists of 14,747,715 total parameters, with 33,027 of them made trainable while 14,714,688 were non-trainable. The VGG-16 DTL model was modelled by employing a batch size of 10 in 40 epochs, using Adam optimizer specifically for updates of weights, cross-entropy loss Table 6 The precision, recall and F1-Score obtained for the classification task using the fine-tuned VGG-16 based DTL model (three-class classification). function with a learning rate of 1e −2 , the performance of the proposed fine-tuned VGG-16 based DTL model was evaluated on 25% of the X-ray images. The output of the confusion matrix for the binary classification as obtained from the VGG-16 based DTL model are shown in Table 8. Figure 6 illustrates the training loss and accuracy and the validation loss and accuracy graphs of the proposed fine-tuned VGG-16 based DTL model. The validation accuracy, recall, specificity, precision, and F1-Score of the proposed fine-tuned VGG-16 based DTL model were also obtained.
It was observed from Fig. 6 that the validation and training losses were significantly high in the earlier epochs and then slowly decrease as the training occurs in more subsequent epochs. The decrease in the loss values at around the 40th epoch was attributed to the fact that the fine-tuned VGG-16 based DTL model was exposed to all the available X-ray images repeatedly during each of the epochs considered during training. The validation accuracy obtained for the fine-tuned VGG-16 based DTL model was 93.85%, its recall was 97.98%, while its specificity stands at 94.69%. The obtained values for the precision, recall, F1-Score, Matthews Correlation Coefficient and Cohen's Kappa statistics metrics for the three-class classification task using the VGG-16 based DTL model are given in Table 9.
Experiment D
The fourth experiment performed on the multiclass (three-class) dataset, in which a DTL model based on a pre-trained VGG-19 model was trained to classify the X-ray images into the three classes of COVID-19, ViralPneumonia or Normal; and also, to detect if X-ray images are simply of the class COVID-19 or ViralPneumonia or Normal. The VGG-19 based DTL model summary detailing the layers and parameters in each layer of the model is shown in Table 10. The fine-tuned VGG-19 based DTL model consists of 20,057,411 total parameters, with 33,027 of them made trainable while 20,024,384 were non-trainable. The VGG-19 DTL model was modelled by employing a batch size of 10 in 40 epochs, using Adam optimizer specifically for updates of weights, cross-entropy loss function with a learning rate of 1e −1 , the performance of the proposed fine-tuned VGG-19 based DTL model was evaluated on the 25% of the X-ray images. The output of the confusion matrix for the binary classification as obtained from the VGG-19 based DTL model are shown in Table 11. Figure 7 illustrates the training loss and accuracy and the validation loss and accuracy graphs of the proposed fine-tuned VGG-16 based DTL model. The validation accuracy, recall, specificity, precision, and F1-Score of the proposed fine-tuned VGG-16 based DTL model were also obtained.
It was observed from Fig. 7 that the validation and training losses were significantly high in the earlier epochs and then gradually decrease as the training occurs in more subsequent epochs. This decrease in the loss values at around the 40th epoch was attributed to the fact that the fine-tuned VGG-19 based DTL model was exposed to all the available X-ray images time and again during each of the epochs considered during training. The validation accuracy obtained for the fine-tuned VGG-19 based DTL model was 92.92%, its recall was 95.95%, while its specificity stands at 89.68%. The obtained values for the precision, recall, F1-Score, Matthews Correlation Coefficient and Cohen's Kappa statistics metrics for the three-class classification task using the VGG-19 based DTL model are given in Table 12.
It was noted from the obtained confusion matrices and the computed performance evaluation metrics of the binary and three-class classification tasks that the fine-tuned VGG-16 based deep transfer learning model outperformed the fine-tuned VGG-19 based deep transfer learning model in the detection of COVID-19. Based on this, some tests were carried out on unlabeled images using the developed fine-tuned VGG-16 multiclassification model. The test was carried out to obtained an impartial evaluation of the final model. Some results of the tests are shown in Figs. 8 to 10.
Being the best performing model in this study, fine-tuned VGG-16 DTL model was tested on the test dataset of 470 images. The test accuracy obtained for the model was 98%. The results of the tests as shown in Figs. 8 to 10 show how the fine-tuned VGG-16 DTL model classified and detected each of the images as either "COVID-19", "Viral Pneumonia", or "Normal." The level of confidence in the model classification is also shown. Figure 8 shows the sample images that were detected as "COVID-19" along with the model's classification confidence accuracy values. Figure 9 shows the sample images that were detected as "Viral Pneumonia" and the model's classification confidence accuracy values. In contrast, Fig. 10 shows the sample images that were detected as "Normal" along with the model's classification confidence accuracy values.
Out of the six sample images shown in Fig. 8, only one showed a lower confidence level of 76.37%, while others were above 94%. Similar results could be seen in Fig. 10 for Normal classification, where the lowest confidence level is 78.92%. However, the lowest output for Viral Pneumonia is 96.21%, as shown in Fig. 9. These test results showed that the developed models could generalize and adapt to new data outside the training and validation dataset. These test results are necessary to show the adaptability of the developed models when related data is considered.
EVALUATION OF RESULTS
The results obtained in this work were compared with thirteen other existing approaches in the literature. Few studies conducted before this study had used twenty-five and fifty images in each class (National Health Commission of People's Republic of China, 2020; , while nine out of the twelve approaches benchmarked used imbalanced data (Table 9). Generally, the problem in modeling imbalanced data is that it could lead to the inability of the model to generalize, or the model can be biased towards a class with a high number of data points. Hence, in this study, an equal value of data (1,300 images) was used for each category, and this is believed to have contributed to increasing accuracies of the proposed models. At the moment, creating an automated diagnostic tool for the detection of COVID-19 suffers from the drawback of limited number of cases. To ensure the generalization of the models developed in this work, data augmentation was performed by setting the random image rotation setting to 15 degrees clockwise. The proposed new models were based on finetuning VGG-16 and VGG-19 methods by constructing a new fully-connected layer head consisting of POOL => FC = SOFTMAX layers and append it on top of VGG-16 and VGG-19; the Convolution weights of VGG-16 and VGG-19 were then frozen, such that only the FC layer head was trained. The fine-tuned models gave better results than other models that used ordinary pre-trained VGG-16 and VGG-19 (Apostolopoulos & Mpesiana, 2020;El Asnaoui & Chawki, 2020). Complete results of the comparison with thirteen other existing results from the literature are presented in Table 13, with the proposed model recording the best performance accuracy. The closest performing model to the proposed model is that of the DarkCovidNet model (Ozturk et al., 2020) with 98.08% accuracy, while the proposed DTL-based VGG-16 model has 99.23% accuracy, both in the binary classification task.
CONCLUSIONS AND FUTURE WORK
Several researchers around the world are combining their efforts to collect data and develop solutions for the COVID-19 pandemic problem. Laboratory testing of suspected cases characterized by long waiting periods and an exponential increase in demand for tests has hitherto constituted a significant bottleneck globally. Hence, rapid diagnostic test kits are being developed, most of which are currently undergoing clinical validation and have yet to be adopted for routine use. This paper proposed a solution using the Deep Learning Convolutional Neural Network Model to classify a real-life COVID-19 dataset of chest X-ray images into three-classes: COVID-19, Viral-Pneumonia and Normal categories. Two experiments were performed where the VGG-16 and VGG-19 CNN with DTL was implemented in Jupyter Notebook using Python programming language. Experimental results showed that the pre-trained VGG-16 DTL model classified COVID-19 data better than the VGG-19 based DTL model. The fine-tuned VGG-16 and VGG-19 models produced classification accuracies of 99.23% and 98.00%, respectively, for binary classification and 93.85% and 92.92% for multiclass classification. The proposed model, therefore, outperformed existing methods in terms of accuracy. Moreover, the fine-tuned VGG-16 and VGG-19 models have MCC of 0.98 and 0.96 respectively in the binary classification, and 0.91 and 0.89 for multiclass classification. These results showed that there are strong positive correlations between the models' predictions and the true labels. In the two classification tasks (binary and three-class), it was observed that the fine-tuned VGG-16 DTL model had stronger positive correlations in the MCC metric than the fine-tuned VGG-19 DTL model. The VGG-16 DTL model has a Kappa value of 0.98 as against 0.96 for the VGG-19 DTL model in the binary classification task, while in the three-class classification problem, the VGG-16 DTL model has a Kappa value of 0.91 as against 0.89 for the VGG-19 DTL model. This result is in agreement with the trend observed in the MCC metric. The findings of this study have a high potential of increasing the prediction accuracy for COVID-19 disease, which would be of immense benefit to the medical field and the entire human populace as it could help save many lives from untimely death. The researchers suspect that the better performance of the VGG-16 DTL model might be attributed to the volume of data used in the experiments; that is, the depth of layers in the VGG-19 architecture may not have any significant effect on the performance when the dataset is small. This suspicion would be investigated in future work when more COVID-19 data is available. Finally, the COVID-19 images from Chest CT scans are not readily available, unlike X-ray images, because of their high cost. Therefore, other future works would consider using Chest CT images to develop a more sensitive diagnostic tool for detecting viral pneumonia and COVID-19 variants. Further hyper-parameter tweaking would also be done to get more accurate results. | 2021-08-10T13:30:28.335Z | 2021-08-03T00:00:00.000 | {
"year": 2021,
"sha1": "ebe1c409049261fb6440f7c9fe6a488276786464",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.7717/peerj-cs.614",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "d6537f8c4db886488c62986df5c19cef92d59bed",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
214475453 | pes2o/s2orc | v3-fos-license | Micro-PTV technique application to velocity field measurements in immiscible liquid-liquid plug flow in microchannels
The present paper presents the results of micro-PTV technique application to velocity fields measurements in plug flow of immiscible liquids in T-shaped straight and curved microchannels. The data processing algorithms are described; the procedure for velocity circulation assessment was proposed and applied to measure velocity circulation in aqueous plugs at different flow rates. The comparison with micro-PIV technique application results is performed.
Introduction
Microfluidic devices are of great interest in the last years due to multiple advantages such as highintensity heat and mass transfer, small volumes of reagents, the possibility to control local parameters and safety of the processes carried out [1]. Immiscible liquid-liquid flows in microchannels are widely used in chemical and biomedical applications and allow to enhance the outcome of extraction processes and other chemical reactions [2,3]. Plug flow when a series of plugs of one liquid surrounded by liquid film and separated by slugs of another liquid is essential for microfluidic devices because of its unique properties. The circulations inside plugs enhance mixing and increase mass transfer through the dimeric interface. There is a need to measure velocity fields inside plugs and assess velocity circulation in order to develop and optimize microfluidic devices. Although Microresolution Particle Image Velocimetry (micro-PIV) has become an established technique for velocity field measurements in microflows since 1998 [4], it has some drawbacks in application to immiscible liquid-liquid plug flow. Due to confinement, the velocity distribution inside plugs has the areas with high velocity gradients which impose constraints on the maximum interrogation area. On the other hand, typically flow seeding density is quite low in microflows which leads to large size interrogation areas or velocity fields averaging. Latter has the meaning only for quasi-stationary flows which is not the case for plug flow. The alternative technique to micro-PIV is Microresolution Particle Tracking Velocimetry (micro-PTV). The technique is based on tracking of individual particles on the image frame rather than the correlation of interrogation areas containing a group of particles; thus it can be applied to the high gradient velocity fields. In the present work, the micro-PTV technique was applied to velocity field measurements in plug flow of immiscible liquids in T-shaped microchannels. The procedure was developed to assess velocity circulation inside plugs.
Experimental setup
Velocity field measurements were carried out in plug flow regime of immiscible liquids in T-shaped straight and curved microchannels. The top view of the microchannels is presented in Figure 1. The width of the inlet channels was 200 μm; the width of the outlet channel was 400 μm while the depth of the channels was equal to 200 μm. The curvature radius of the curved channel was 600 μm. Castor oil was a carrier liquid; distilled water played a role of the dispersed phase. Flow rates of dispersed and carried liquids were varied in the range 0.695μl/min <Q d < 4.17 μl/min and 0.695 μl/min <Q c < 8.34 μl/min by means of KD Scientific double syringe pump. The dispersed phase was seeded by 2 μm fluorescent particles with 0.07% concentration by volume. The scheme of the experimental setup is shown in figure 1. The microchannel was mounted on the Zeiss microscope stage. The velocity measurements were performed at a distance of 8.5 mm from a T-junction and the curved part. The focal plane was set to the central plane of the channel. The flow was illuminated through the microscope lens by double Nd:YAG laser with 532 nm wavelength. The light emitted by particles was directed to a dichroic mirror and captured by a double exposure CCD camera with 4 Mpix resolution. The spatial resolution achieved was 0.8 μm/pixel.
Velocity field calculation
All calculations were performed using our in-house ActualFlow software, where PTV algorithm was realized. For PTV calculations a pair of tracer images with a short time delay between them is required similarly to PIV. The algorithm detects individual particles at both the first and the second frame and then matches them to obtain the most probable displacement of each particle. The algorithm includes three main stages: particle identification, relaxation algorithm, and result correction. At the first stage particle identificationa raw image of tracer particles (Figure 2 (a)) is filtered using the Ricker wavelet convolution with a Fourier transform of an initial image. This operation allows to single out characteristic structures and to decrease noise. Further, local maxima at the image are found. A particular pixel is defined as local maxima if it has maximum intensity in the area of 3×3 pixels around The threshold parameter P should be chosen for particular experimental data because images of tracer particles can differ significantly. Too high values of P can lead to a small amount of identified particles, whereas too small values lead to misidentification and spurious particles. The example of particle identification is presented in Figure 2 (b). In this work, P was varied from 0.9995 to 0.9998.
Figure 2.
Stages of velocity field calculation using the PTV algorithm: a) a raw image of tracer particles; b) particles found at the image by the PTV algorithm; c) a raw PTV vector field; d) an irregular PTV field after range validation and mask imposition operations; e) a regular velocity field interpolated from PTV data.
The second stage is the relaxation algorithm which estimates first frame particle probabilities of displacing in corresponding particles in the second frame. This algorithm is based on two main principles: maximum particle velocity (or displacement) is defined, and continuity of velocity field is satisfied (local groups of particles moves in the approximately same direction). When the most probable displacement is determined, the corresponding velocity vector is calculated for each pair of particles. A more detailed description of the relaxation algorithm is presented in [5]. At the final stage, the correction of results is performed using the correlation function maximum [6]. Each particle from the second frame is shifted according to the previously calculated displacement and correlation between particle from the first frame, and the shifted particle are calculated. Since a maximum of the correlation function is known, corrections to the initial displacement are made. An example of the resulting irregular velocity field is presented in Figure 2 (c). If spurious or irrelevant vectors appear and it is impossible to eliminate them just by varying threshold value P, additional procedures such as image masking or velocity vector range validation should be applied. Irregular field after mask imposition and range validation is shown in Figure 2 (d). Eventually, irregular fields were interpolated into a regular grid using second order local polynomial algorithm for further calculation of derivatives (Figure 2 (e)). Amount of nodes in the grid was approximately equal to the number of vectors in the irregular field. For experiments in this paper typical grid resolution was 8×8 µm 2 . .
Velocity circulation assessment
To assess circulations inside plugs for liquid-liquid plug flow it is convenient to use vorticity fields calculated from velocity derivatives. Often, the PIV algorithm provides unsatisfactory results and resolution of velocity field insufficient to calculate the vorticity field. For instance, this case takes place for serpentine microchannels, where velocities inside plugs have different directions. In Figure 3 the comparison between vorticity fields obtained from PIV and PTV data is presented. PIV velocity field was calculated using 64×64 pixel grid without overlapping, further refinement of the grid was impossible due to initial image quality and insufficient flow seeding. As can be seen in Figure 3, the vorticity field obtained from PIV data, even the main vorticies in the front part of the plug are resolved poorly. Besides, small values of velocity vectors near the plug boundary introduce significant error into the resulting vorticity field. By comparison, using interpolated PTV results, adequate vorticity field can be calculated even for a curved channel. For the assessment of velocity circulation inside plugs, the following procedure has been developed. From calculated velocity derivatives, the vorticity component, directed orthogonally to the measurement plane, were obtained. The total circulation in the plugs was calculated from vorticity distributions using the following equation: We approximated the area integral with the sum of the z-th component of vorticity ω z multiplied by the cell area S for each cell in the circulation areas. Circulation areas were defined as areas with vorticity values greater than 25% of the maximum for a given plug. It allowed us to eliminate areas with low vorticity from consideration. To take into account the circulation regions with both positive and negative vorticity values, the values of ω z were taken in absolute value.
Using the described approach to the total circulation assessment experiments with castor oil -water plug flow in the straight microchannel were conducted. The plot of the total circulation versus Ca bulk Q d /Q c parameter, where Ca is the capillary number, is shown in figure 4. Total circulation depends linearly on Ca bulk Q d /Q c . This result is in agreement with the work [7], where Ca bulk Q d /Q c was shown to describe plug shape and velocity fields inside aqueous plugs in castor oil.
Conclusion
In the present study, micro-PTV technique was applied to velocity field measurements in plug flow of immiscible liquids in microchannels. Two types of microchannels were used: straight T-shaped microchannel and curved T-shaped microchannel. Velocity field and velocity circulation calculation procedures were described. It was demonstrated that the micro-PIV technique does not take satisfactory results for velocity circulation assessment in case of curved plug due to the restriction of seeding density on the minimum size of the interrogation area. Velocity circulation at different flow rates was calculated in the T-shaped microchannel, its linear dependence on Q d /Q c ·Ca bulk was shown. | 2020-01-02T21:50:02.026Z | 2019-12-01T00:00:00.000 | {
"year": 2019,
"sha1": "c4a94217825e3180ba59b3ca29d270c7b70bf387",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1742-6596/1421/1/012026",
"oa_status": "GOLD",
"pdf_src": "IOP",
"pdf_hash": "6359dae558a05b582391e4ce61cefc349f952bf7",
"s2fieldsofstudy": [
"Engineering"
],
"extfieldsofstudy": [
"Materials Science"
]
} |
15406805 | pes2o/s2orc | v3-fos-license | Dilaton-driven brane inflation in type IIB string theory
We consider the cosmological evolution of the three-brane in the background of type IIB string theory. For two different backgrounds which give nontrivial dilaton profile we have derived the Friedman-like equations. These give the cosmological evolution which is similar to the one by matter density on the universe brane. The effective density blows up as we move towards the singularity showing the initial singularity problem. The analysis shows that when there is axion field in the ambient space the recollapsing of the universe occurs faster compared with the case without axion field.
I. INTRODUCTION
The idea that our universe might be a domain wall embedded in a higher dimensional space [1] has attracted much interest recently. It is possible that the fundamental scale of gravity can be lowered to the electroweak scale by introducing large extra dimensions [2]. Randall and Sundrum [3] proposed scenarios that our observed universe is embedded in a five-dimensional bulk, in which the background metric is curved along the extra dimension due to the negative bulk cosmological constant. These models have been studied extensively because they might provide the solution to the gauge hierarchy problem and cosmological constant problem [4]. The cosmology can be different from the conventional four dimensional one.
One can naturally attempt to justify the scenario within a well defined framework for higher dimensional quantum theory of gravity such as string theory. Many attempts have been made to apply this idea to string theory in the context with D-branes [5], where the standard model gauge bosons as well as charged matter arise as fluctuations of the D-branes. An early example of this is the Horava-Witten picture for the nonperturbative heterotic E 8 × E 8 string [6]. The spacetime includes a compact dimension with an orbifold structure. Matter is confined to the hypersurface which forms the boundaries of the spacetime. Within the string theory context, it is natural that our observable four-dimensional world is a three-brane embedded in ten dimensional string. In such theories, one of the important issue is the cosmological evolution of our universe. Many cosmological models associated to brane universe have been suggested. The models can be classified into two categories. The first is that the domain walls (branes) are static solution of the underlying theory and the cosmological evolution of our universe is due the time evolution of energy density on the domain wall (brane) [7]. The second is that the cosmological evolution of our universe is due to the motion of our brane-world in the background of gravitational field of the bulk [8,9]. We will focus on the second approach in this paper.
It is shown that the motion of the brane in ambient space induces cosmological expansion (or contraction) on our universe simulating various kinds of matter or a cosmological constant. In other words the cosmological expansion is not due to energy density on our universe but somewhere else. This is the idea by mirage cosmology [9]. Friedman-like equations were derived for various bulk background field solutions. In [10], the motion of a three-brane, in a background of type 0 theory was examined.
In this paper, employing the formalism of [9], we will study how the presence of matter field on the background geometry affects the cosmological evolution of the brane universe. More specifically we will consider cosmological evolution of type IIB theory with two different background geometries, one without axion and the other with axion. We will compare the two and see the difference.
The organization of the paper is as follows. In Sec. II we will briefly review the formalism of mirage cosmology in ref. [9] and set up some preliminaries for our calculation. In Sec. III we consider the type IIB theory and its background solution with and without axion field. In Sec. IV, using the background solutions of Sec. III, we find the cosmological evolution of the three-brane under the background. Finally section V is devoted to conclusions and discussion.
II. FORMALISM
In this section, we consider a probe brane moving in a generic static spherically symmetric background. We ignore its back reaction to the ambient space. As the brane moves in a geodesic, the induced world-volume mertic becomes a function of time. The cosmological evolution is possible from the brane resident point of view. We will focus on a D3-brane case. For this purpose we parametrize the metric of a D3-brane as and there are dilaton field φ as well as RR (Ramond-Ramond) background C(r) = C 0...3 (r).
The probe brane will in general move in this background and its dynamics is governed by the Dirac-Born-Infeld (DBI) action. In maximally supersymmetric case, ignoring the fermions, it is given by where the induced metric on the brane iŝ with similar expressions for other fields. Generally the motion of a probe D3-brane have a nonzero angular momentum in the transverse directions. We can write the relevant part of the Lagrangian, in the static gauge x α = ξ α (α = 0, 1, 2, 3), as where h ij ϕ i ϕ j is the line element on the unit five-sphere (i, j = 5, · · · , 9), A(r) = g 3 (r)|g 00 (r)|e −2φ , B(r) = g 3 (r)g rr (r)e −2φ , D(r) = g 3 (r)g S (r)e −2φ , and C(r) is the RR background. The momenta of the system are given by Calculating the Hamiltonian and demanding the conservation of energy, we have where E is the total energy of the brane. Also from the conservation of the total angular momentum h ij p i p j = ℓ 2 , we have Substituting equation (8) into (7) and solving with respect toṙ 2 , we have the equation for the radial variable asṙ Plugging equation (9) back into (8), we have the equation for the angular variable The induced four-dimensional metric on the three-brane universe is Using equation (9) and (10), this reduces to where we defined, for the standard form of a flat expanding universe, the cosmic time η as If we define the scale factor as a 2 = g, we can calculate, from the analogue of the fourdimensional Friedman equation, the Hubble constant H =ȧ/a ȧ a where the dot denotes the derivative with respect to cosmic time and the prime denotes the derivative with respect to r. The right hand side of (14) can be interpreted as the effective matter density on the probe brane We have alsoä Equating the above to −(4π/3)(ρ eff + 3p eff ), we can find the effective pressure The apparent scalar curvature of the four-dimensional universe is We have given the formalism for simple D3-brane case. The geodesic motion of Dp-brane in the background of the Dp ′ -brane with p ′ > p can be generalized easily. In the case p = p ′ , there exists the additional Wess-Zumino term T p Ĉ p+1 in the DBI action which modifies the equation of the probe brane as well as the induced metric. This modification turn out to be the shift E → E + C where C = C 0...p [9].
III. THE TYPE IIB BACKGROUND SOLUTION
Here we will consider the background geometry of type IIB theory with five-form flux through an S 5 . We will also assume, for the metric, (3+1)-dimensional Poincaré invariance ISO(1, 3) since we need the theory defined on the Minkowski space-time. In addition we will preserve the SO(6) symmetry of the AdS 5 ×S 5 . As a result, the ISO(1, 3)×SO(6) invariant ten-dimensional metric with N units of five-form flux through an S 5 , in the Einstein frame, can be written as where χ, σ, and also the dilaton φ and the axion η are allowed to depend only on the radial coordinate r.
The equations of motion in type IIB supergravity, truncated to the fields of our interests, are given by [11] where hat means that the operators are expressed in ten-dimensional terms and M, N, · · · = 0, · · · , 9. The equation of motion for the five-form field iŝ which is satisfied with the self-duality condition (20). The Einstein equation in S 5 direction isR which is automatically satisfied if The remaining equations can be expressed in purely five-dimensional terms where the new metric should be used to compute R µν and to contract indices. The equations in (25) can be derived from the five-dimensional action [12] S = 1 2κ 2 where the gravitational couplings κ 5 and κ in five and ten dimensions are related by Note the minus sign in front of the axion kinetic term, which is the result of the Hodgeduality rotation of the type-IIB nine-form [13].
A. Solution without axion
In general one can reduce the equations of motion (25) to a set of coupled non-linear second order ordinary differential equations in φ, η, χ, and σ. These equations are too complicated to solve in general, but there is an obvious simplification with χ = η = 0 [14]. Then the equations in (25) become much simpler Equations (30) and (31) are obtained from (tt) and (rr) components of the Einstein equation. Because χ = 0 there is no distinction between the five-dimensional Einstein metric and tendimensional metric restricted to the five-dimensional noncompact subspace. The equation (29) can be integrated to give where φ ∞ is the value of the dilaton at the boundary of the asymptotically AdS 5 geometry and B is an integration constant. Substituting (32) into (30) and (31), defining new variable u ≡ r/L, we obtain The second equation follows from differentiating the first, so we see that (32), (33) and (34) are consistent system of equations despite being overdetermined. One can understand (33) as a mechanical analog of a classical particle with unit mass moving in the potential with zero enegry. If B = 0, the solution is pure AdS 5 with constant dilaton. To have a solution with nonconstant dilaton, we take B > 0. However, the B = 0 geometry is geodesically incomplete and singular at some point u = u 0 . To find u 0 explicitly, we integrate equation where F (α, β; γ; z) is the usual hypergeometric function. The second term vanishes as σ → −∞, so we find Also we find the dilaton in terms of σ by solving the equation (32) We can write the ten-dimensional Einstein metric explicitly if we use σ as the radial variable
B. Solution with axion
In the previous subsection we described the simplest case in which one can have a solution with nontrivial dilaton. Here we will consider the case with only χ = 0 to find the solution with nontrivial axion field [15]. The equations in (25) can be written Integral of the axion in equation (41) is where the prime denotes derivative with respect to u = r/L and η 0 is an integration constant. Inserting this expression for η into equation (40) we obtain the differential equation for dilaton and integrating once we have whereB is another arbitrary constant. Equations (43) and (45) are sufficient to proceed and solve for the function σ(u) that appears in the metric (42). The Einstein equation (42) becomes Inserting (43) and (45) into (46) and (47), we obtain The above equations are exactly the same as those without axion (see (33) and (34) ( 1 + (24/B 2 )e 8σ + 1) (51)
IV. BRANE COSMOLOGY
In this section we will consider the cosmology probe D3-brane when it is moving along a geodesic in the background type IIB solutions of the previous section.
A. Without axion field
The metric of D3-brane (1) using the background solution (39) is |g 00 (r)| = e 2σ , g(r) = e 2σ , g rr (r) To apply the formalism of Sec. II we also need to express RR field in terms of σ. From the ansatz for the RR field equation (22) becomes where Q is a constant. Using the solution of the metric in (52), the RR field can be integrated with appropriate normalization, Now we can calculate the effective density on the brane using equations (38), (52) and (55) If we rescale with B 2 = 24 and using a = e σ , we get where the range of a is 0 < a < ∞, while the range of σ is −∞ < σ < ∞. When the universe brane is moving towards the singularity (σ → −∞, a → 0) the universe is contracting while it is moving outward (σ → ∞, a → ∞) it is expanding. We also can calculate the scalar curvature of the four-dimensional universe from (18). Far from the black brane, one can see that ρ eff ∼ a −4 . The cosmological expansion due to the brane motion is indistinguishable from the one by radiation on the brane. This is the idea of the mirage cosmology. If we use the effective density (57) it blows up ρ eff ∼ a −8(2+ √
3/2)
as a → 0. Also if we move a → 0, the ten-dimensional metric becomes which is an AdS 5 × S 5 space. Thus the brane develops an initial singularity as it reaches a = 0 where the description of our formalism breaks down.
B. With axion field
In this case, the only difference on the effective density comes from the form of the dilaton. Still withB 2 = 24, e σ = a, we have Near the brane the effective density blows up ρ eff ∼ a −8(2+ √ 3/2) as a → 0 which has the same functional dependence as in the case without axion. However, far from the brane (a → ∞), it gives ρ eff ∼ −1/L 2 . This negative cosmological constant means that the expansion of the universe stops at somewhere and eventually recollapses. Comparing equation (59) with (57), we see that the effective density becomes negative faster if there is axion field. The presence of the axion field do not play any important role in the early stage of the evolution but its coupling to other field gives different evolution at late stage.
V. DISCUSSION
We considered the motion of a brane universe moving in a background bulk space of type IIB string theory. For two different backgrounds which give nontrivial dilaton profile, one without axion field and the other with axion, we have derived the Friedman-like equations. These give the cosmological evolution which is similar to the one by matter density on the universe brane. As the brane moves towards the singularity (smaller values of radial coordinate) it contracts and while if it moves away from the black brane it expands. So an observer on the three-brane will see that the universe is expanding. The presence of axion field in the background changes the dilaton profile but it does not change the induced metric. Since dilaton, as well as the induced metric, plays an important role in the effective density, the cosmological evolutions are different for two different backgrounds. For both cases, the effective density blows up as we move toward the singularity showing the initial singularity problem and becomes negative due to the angular momenta ℓ 2 on the brane meaning the recollapse of the universe. The functional dependence on the radial coordinate shows that when there is axion field in the ambient space the recollapsing of the universe occurs faster compared with the case without axion field. It seems that this phenomenon is true if we do the same calculation with field other than axion.
ACKNOWLEDGEMENT I would like to thank N. Kaloper, S. P. Kim and E. Kiritsis for discussions and suggestions. | 2014-10-01T00:00:00.000Z | 2000-04-24T00:00:00.000 | {
"year": 2000,
"sha1": "6e3c354b0f9665cad6203d0b8a3e7ee6cab8bd76",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/hep-th/0004155",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "6e3c354b0f9665cad6203d0b8a3e7ee6cab8bd76",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
230674586 | pes2o/s2orc | v3-fos-license | THE INTERNATIONAL JOURNAL OF BUSINESS & MANAGEMENT Integration of Students in Publicity Strategies: Opportunities, Challenges, and the Way Forward for Private University Colleges in Ghana (Part I)
a research conducted by Bowen, Gogo and Maswili in 2012 on the subject, ‘Marketing strategies that attract and increase student enrolment in institutions of higher learning: Case of private universities in Kenya’ concluded that, advertising using an institution website, advertising using the various media stations, use of social networks such as Abstract: This paper is derived from a three-part qualitative study that focuses on integration of strategic stakeholders in publicity activities of Private University Colleges (PUCs) in Ghana. Existing discussion on publicity strategies is narrow and leaves out the role of students as stakeholders. The purpose of the study was to assess how students could be effectively integrated into publicity activities of PUCs in Ghana. The objectives of the study were1) To find out the opportunities for integration of students’ as stakeholders in publicity activities of PUCs; 2) to explore the challenges of the integration process; and 3) to examine the way forward for addressing identified challenges. A qualitative approach was used for the study with in-depth interviews conducted in obtaining data. The study concludes that even though the management of the selected PUCs admit that students are key stakeholders and have a role to play in publicity activities, they have not been properly integrated and this is evident from the lack of existing policy framework that consciously addresses the issue. It is recommended that publicity theorists must develop a comprehensive framework that informs the integration of students’ in their publicity activities in a coordinated manner. The proposed framework must address issues like conceptualization, management and evaluation of integrated student publicity. It is evident from this study that a proper integration of students in publicity activities will inure tremendously to the benefit of the PUCs particularly in an era of increasing competition, lack of government support and sustainability concerns.
Facebook, encouraging word-of-mouth, career fair involvement, open day on campus and Alumni support are marketing strategies that can offer opportunity to attract and increase enrolment of students. These findings help us to appreciate the publicity options available to institutions in the context of integrated marketing communications.
In addition, Bylon and Bede in May 2013 researched on the subject, 'The role of marketing communications in student enrolment in private universities in Ghana' and found that advertising was the most important tool in attracting students to enrol in public universities in Ghana. However, a recommendation was made for the consideration of the other elements of marketing communications which are equally important. Now the question is, how has researchers explored the role of students in a coordinated and well-planned publicity activities aimed at increasing numbers and resources for management purposes? The issue of effective integration of stakeholders particularly students in publicity initiatives have not been fully explored. Most of the extant literature tends to scratch the surface of the matter leaving a significant gap in literature to guide research and practice. The purpose of this current study was to assess how students, who constitute one of the key stakeholders of tertiary education could be effectively integrated into publicity activities of PUCs in Ghana. The objectives of the study were1) To find out the opportunities for integration of students' as stakeholders in publicity activities of PUCs; 2) to explore the challenges of the integration process; and 3) to examine the way forward for addressing identified challenges.
Publicity in Perspective
The concept of 'publicity' is the deliberate attempt to manage the public's perception on a subject with the ultimate goal of promoting client's product or service (Eisa, 2003). This means that publicity is used as a communication tool for making a product known to the public. Egan (2007) describes publicity as 'a series of positive messages about an organization or its employees designed to improve the image of the organization'. These reamplifiedby Jobber (2010) who argues that publicity can be defined as communication about a product or an organization by the placing of news about it in the media without paying for the time or space directly.
Publicity offers a number of benefits to organizations irrespective of size, mission and financial muscles. Cleary (2014) identifies seven benefits in organization's marketing tools including costs. Publicity is virtually free and Cleary rightly notes that 'Apart from the costs accruable from media distribution, liaison, preparation and distribution, the cost for publicity is virtually free'. This view is very critical because there is evidence of PUCs struggling to meet operational costs due to factors such as 'unhealthy' competition from the traditional public universities, lack of government support, and dwindling number of fresh applicants. Some of the benefits of publicity include the flexibility of being noticed, forming strategic alliances, enhancing organisation identity, building your credibility and boosting your effective competitiveness.
Integration of Students: The Stakeholder Argument
Stakeholder theory was propounded by Edward R. Freeman in 1984. The theory suggests that the purpose of a business is to create as much value as possible for stakeholders. In order to succeed and be sustained over time, executives must keep the interests of stakeholders like customers, suppliers, employees, communities and shareholders like the employers to be aligned and going in the same direction. Innovation to keep these interests aligned is more important than the easy strategy of trading off the interests of stakeholders against each other. Hence, by managing for stakeholders, executives will also create as much value as possible for shareholders and other financiers. Evan and Freeman (1988) in their work, 'Stakeholder Theory of the Modern Corporation' indicate that, stakeholders have their jobs and usually their livelihood at stake; they often have specialized skills for which there is usually no perfectly elastic market. In return for their labour, they expect security, wages, benefits and meaningful work. In return for their loyalty, the corporation is expected to provide for them and carry them through difficult times.
In the context of tertiary education, students are regarded as one of the key stakeholders. Their existence and contributions cannot be underestimated. As the largest stakeholder group of PUCs, students are expected to follow the instructions of management most of the time, to speak favourably about the university, and to be responsible citizens in the local communities in which their institutions operate. Where they are used as means to an end, they must participate in decisions affecting such use.
Controlling and Empowering Students for Publicity Strategies
According to Palmer (2008), there are two basic approaches to managing people for task. On the one hand, management of the School can supervise students closely and make corrections where they fail to perform to standard. On the other hand, management can make students responsible for controlling their own actions and this is often referred to as 'empowering students'. For an organisation like the university to effectively recover from service failures and closely tailor services to individual targets' needs, there is the need for students' empowerment. Palmer argues that'one of the underlying assumptions of those advocating empowerment is that student's values will be in line with those of the university. The school must be prepared to allow students the freedom to act and to make decisions based on their own judgment' (Palmer, 2008:381).
The Role of Motivation in Student's Integration
According to Palmer (2008), 'strategies to empower [students] to make effective service encounter are less likely to be successful if [students] do not feel engaged in their job. Motivation, consent, participation and communication form essential focal points for an organization's strategy for bringing about the sense of engagement that underlies empowerment' (p. 384). Students are key primary stakeholders of an organization and when motivated, consented and allowed to form an essential participant in the publicity strategy will bring about the sense of empowerment to enhance the PUCs aim of exploiting the benefits of publicity including increasing enrolment.
Weimer (2012) states that university authorities must ensure that institutional cultures are welcoming to students from diverse backgrounds. This means that students must feel they are accepted and affirmed and feel they belong to the institution. Weimer again says that students must be enabled to build up their social and cultural capital. This kind of capital derives from a sense of belonging, from active relationships with others, participation and knowing how things work around the institution not only in the classroom work but beyond it as well to making the school known to potential students. Weimer further urges that school authorities must invest in a variety of support services for students. This support services could be reduction of fees or scholarship for students who actively participate in publicity for the school in various ways to enhance enrolment. According to Zepke and Leach (2010),'What is needed in a democraticcritical conception of engagement that goes beyond strategies, techniques, and behaviours, is a conception in which engagement is participatory, dialogic and leads not only to academic achievement but to success as an active citizen.' (p. 173). Being good and active citizens enable students to become good ambassadors of the school to the potential publics.
Training and Development of Students in Publicity Integration
According to Cole (1993), training is any learning activity which is directed toward the acquisition of specific knowledge and skills for the purpose of an occupation or task. The students being trained here will serve the task of making their PUCs known to the public. Thomas (1988) also defines training as 'a process through which experience are deliberately offered to trainees to enable them to absorb some new perspective, understanding, values, attitude, technique or skills. This requires the organization to plan activities to increase task knowledge and skills or amend the attitude and social behaviour for its participants in a way that are consistent with organizational goals and requirement of the task. Palmer (2008) adds that: 'if an organization wishes to make its stakeholders involved in publicity, it must include such an objective within its overall corporate plan and identify the required training-and development needs (p. 394). It is therefore essential to make stakeholders aware of the competitive market challenges and pressures, and how the school intends to overcome them by making use of any available channels like the students to reach the public among whom they reside. This, according to Palmer will encourage students to be morally involved in the process of change in their school. However, if the publicity skills and knowledge of the students are not developed, the PUCswill lose opportunities.
Methodology
This study was a qualitative one aimed at exploring how students could be integrated into publicity activities of PUCs. Daymon and Holloway (2002) deduce that most studies embarked upon in the field of public relations use qualitative research approach as it presents an interpretive and rational worldview. The qualitative approach therefore enables researchers in the collection of appropriate data to deal with the research questions. Exploratory design was used in this study. This design, according to Lynn and Lynn (2014) is used for a research topic which has few or no previous studies to refer to. It focuses on gaining familiarity and insight for subsequent investigation, or could be used when primary stage of investigation. The exploratory design was used because the study was in a relatively new area and literature was difficult to come by. The study selected three PUCs in Ashanti Region purposively namely Christian Service university College (CSUC), Garden City University College (GCUC) and Ghana Baptist University College (GBUC). The three PUCs were purposively but carefully selected based on factors such as student population size, depth of programmes offered, length of years of operation and their established history of publicity activities. Qualitative data in the form of structure interview were collected and analysed from Management members of the three PUCs who were familiar with the subject under study and labelled as 'management sources'. The identity of the management sources is not disclosed. The data was manually transcribed and analysed using inductive and deductive approaches. Key emerging themes were clustered and presented in a form of report per the objectives of the study.
Opportunities for Integration of Students in Publicity Activities
To find out the opportunities for integration of students in publicity activities, the views of the selected management members of the PUCs were sought and presented as follows: The management sources indicated that three main traditional communication channels have been used for publicity activities namely the radio, television and newspaper adverts. They gave the indication that these three media platforms have enabled them to engage their prospective fresh applicants although other strategies have been used. For instance, one management source explained that sometimes, they resort to senior high school visits and other times, they undertake admission outreach to churches and identifiable groups (private and public) for publicity purposes. This finding gives an indication that several publicity opportunities are available to PUCs. This finding is consistent with existing findings in the area of publicity channels available for use by PUCs (Omboi & Mutali, 2011;Sahid & Imram, 2010;Bowen, et al, 2012). Now the substantive matter is, how has students, who are considered as a key stakeholder been integrated in the publicity process? A direct question was put to the management and interestingly, they all agree that student's involvement in the publicity activities of University colleges was very crucial. For instance, one management source had this to say: 'the school is building its name so students must do their part through their testimonies or personal experience'. Another management source observed: 'students are everywhere . . . they go to their friend's houses, work places and even attend events of their friends together where they can always talk about their University Colleges'. It was a generally held view among the selected management that involving students in publicity of PUCs could be the most important move towards improving enrolment.
The opportunity for integration of students in publicity activities was further supported by a management source which argued that: 'students are students, and they will remain same' there is no need to restrict them, 'when they are treated well, they will publicize the University College wherever they may be'. Students as a group and a key stakeholder present a powerful 'asset' to enhance institutional advancement particularly in the area of admission promotion in an increasing competitive environment. We argue that PUCs should begin to utilize this 'asset' in a well-coordinated manner to achieve phenomenal results. But the irony of the matter was that PUCs didn't have established policy framework that consciously integrate students in their publicity activities. Rather, responses from management of all the three PUCs indicated to have some arrangement that involves using selected students especially for outreach programmes on adhoc basis.
The understanding is that some of the students are co-opted into publicity activities depending on what the institution seeks to achieved. It is strongly contested that such a strategy may not be effective and that a properly planned strategy that clearly understands the unique role of students in publicity, particularly for admissions purposes is the way to go. Even though some of the management sources hinted that they use students for publicity purposes, it became clear that there was no documented policy that drives this engagement. One of the possible explanations to this phenomenon is the fact that most of the publicity activities are not handled by probably professional communication units or departments within some of these PUCs. For instance, the study found that all the three management sources of the PUCs mentioned different outfits as being in charge of their publicity activities. GCUC's activities are managed by the administration headed by the Vice Registrar (Administration). CSUC is handled by the Corporate Affairs Unit (Formerly Partnership Development Office) whilst publicity activities of GCUC are managed by the marketing department of the institution. This finding was further corroborated by the management sources which reported that the mandate of the respective units/offices that manage publicity was not clearly spelt out. One management source noted: 'They plan and decide which radio station to even work with. . .'
Challenges of the Students' Integration Process
There is no doubt that redirecting students' psychology, attitudes and behaviour towards publicity activities may present challenges. But the question is, is it worth trying? And the answer in our opinion is yes. In response to the question of possible challenges in the integration process, two of the management sources were of the view that there shall always be a challenge in issues like this; the fear was that if not properly handled, it may create a deviation considering that PUCs have an academic mandate that may not support the whole idea of students' integration in the publicity process. There were concerns about capacity building, time management, ethical issues, antagonism from the student leadership, apathy among other concerns. One of the management sources made a fundament observation: 'finding the appropriate reward for students will be a challenge'. The concern expressed was how to balance academic goals with a possible 'commercialization' attitude that may derail students from their core goals for enrolling in PUCs. One of the management sources had this to say, 'there may be challenges initially, but when it is well planned, if will be attractive and students would buy into it much easily'.
However, one of the management sources was of the view that there could be no challenge and noted: 'I personally do not foresee a challenge coming from students' referrals because there is a benefit and whoever does it gets it. Those who don't do it, go their way'. This argument is valid when publicity engagement is limited to only student's referrals. But if the issue is viewed from a holistic perspective, then obviously one may conclude that there will be formidable challenges. How then do we address the challenges considering the identified opportunities? The next section addresses this concern. Zepke and Leach (2010), states; 'what is needed is a democratic-critical conception of engagement that goes beyond strategies, techniques, behaviours, and a conception in which engagement is participatory, dialogic and leads not only to academic achievement but to success as an active citizen.' (p. 173).
The Way Forward to Addressing Identified Challenges
The respondents were asked to suggest ways to address the identified challenges. As a matter of principle, all the management sources unanimously pointed out that those challenges could be dealt with when the students were given very good orientation on the need to help grow the student members in the PUCs. Such education would have to point out to students, the immediate-to-long term benefits of actively participating in publicizing the University Colleges. In as much as all the management sources agreed that there should be some sort of training for the students, none of them recommended any complex training programme. One of them said: 'I do not think they need any special training, just something small to equip them to know what to say and how to say them for maximum results.' The management sources were of the opinion that the training should be directed at how students could tell their friends about their schools without any form of influence from authorities of their schools.
Weimer (2012) also states that University authorities must ensure that institutional cultures are welcoming to students from diverse backgrounds which mean that students must feel they are accepted and affirmed and feel they belongs to the institution. They argued that when the student feels good about the treatment he receives from the school, there is the likelihood that he would gladly talk to friends about it. In essence, they said that the management of PUCs must develop student-centred policies with every aspect of the students' life -from tuition through lecture schedules, flexible fee payment plan and even how conducive the campus environment is made for teaching and learning. This expressed opinion is very critical. The recognition of students as key stakeholders must as a matter of course, illicit some responsibility on the part of management of PUCs, how well do you manage the academic environment has an effect on the conduct of students to a larger extent. Effective training sessions especially in communication skills, persuasion. Attractive reward system must be put in place to motivate and encourage entire students' participation.
Besides, capacity building in the form of training, motivation featured prominently in the way forward in dealing with the identified challenges. Two of the management sources agreed that the idea of establishing a reward system is an attractive one to serve as a source of motivation for the student who would successfully refer their friends or colleagues to enrol in the PUCs. All of them however were of the opinion that it is more prudent to consciously make the students understand the 'other' benefits of convincing more people to join their PUCs. As one of them rightly put it, 'Well, right from matriculation we tell them that the school is trying to build a name for itself and for its students and that they should be part and generally, they welcome such admonition'. The scope and nature of the motivation was not outlined by the management sources (a focus of another research yet to be published). According to Palmer (2008), 'strategies to empower employees to make effective service encounter are less likely to be successful if employees do not feel engaged in their job. Motivation, consent, participation and communication form essential focal points for an organization's strategy for bringing about the sense of engagement that underlies empowerment' (p.384).
Conclusion
The study concludes that even though the management of the selected PUCs admit that students as key stakeholders have a role to play in publicity activities, they have not been properly integrated and this is evident from the lack of existing policy framework that consciously addresses the issue.
Recommendations
Beyond the suggestions on the way forward for addressing the challenges of integration identified and discussed above, we are of the opinion that publicity theorists and practitioners must faction out a comprehensive theoretical framework that informs Management's decisions on the opportunity for the integration of students in publicity activities in a coordinated manner. It is evident from this study that a proper integration of student will inure tremendously to the benefits of the PUCs particularly in an era of increasing competition and uncertainty. The proposed framework must address the academic and extra-curricular commitments on the part of students as viable agents of change and growth in the context of publicity. | 2020-12-17T09:11:26.037Z | 2020-07-31T00:00:00.000 | {
"year": 2020,
"sha1": "72acca78f7d8e2974f6d42ae627905d0c34feb38",
"oa_license": null,
"oa_url": "http://www.internationaljournalcorner.com/index.php/theijbm/article/download/154197/106992",
"oa_status": "GOLD",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "2a864d71e0cc9a8b56867abd1b58af4e25a95ed0",
"s2fieldsofstudy": [
"Education"
],
"extfieldsofstudy": [
"Business"
]
} |
29864312 | pes2o/s2orc | v3-fos-license | Effects of Irradiation on Marrow Stromal Cells with Respect to Committed Granulocyte-Macrophage Progenitor Cells
Marrow stromal cells/Radiation/G ran ulo-macrophage progenitor cells/Colony stimulating factor The effects of irradiation on the growth regulatory function of marrow stromal cells (MSC), and on the committed granulocyte-macrophage progenitor cells (GM-CFC), were investigated using a liquid culture system. CSF activity in the supernatant of irradiated MSC (0-900 rads) increased markedly with the increase of MSC irradiation dose. CSF-inhibitory activity in the supernatant of irradiated MSC (0-900 rads) also increased with the increase of MSC irradiation dose. Furthermore, the activity of exogenous CSF added to the supernatant of 900 rad-irradiated MSC was lost more slowly than that of non-irradiated MSC. These data suggest that irradiation affected CSF production, inhibitor production and consumption of CSF by MSC.
proposed that inhibitory cells in marrow stromal cells inactivate CSF. Heard et al. 13) also showed that CSF was consumed by marrow stromal cells in a bi-layer agar culture system. Previously, we14,") reported that marrow stromal cells have three different functions with respect to the effects on the GM-CFC in vitro culture system. Thus, it is apparent that marrow stromal cells have both a positive function, such as CSF production, and negative functions, such as inhibitor production and CSF consumption (as their own growth factors). In the present study, we investigated the effects of irradiation on these functions of marrow stromal cells.
Mice
Eight-to-12 week old male DDY strain mice were purchased from Japan Laboratory Animals Co., Ltd. and were maintained in cages containing 8 to 10 mice each; the mice were provided with standard mouse food and acidified water.
Bone marrow cells
Bone marrow cells were drawn through a #23 gauge needle from the femora and tibiae into alpha medium (Flow Labs.). Single cell suspensions were then prepared in alpha medium by repeated pipetting, and the suspensions were adjusted to the desired concentration using alpha medium.
Culture of marrow stromal cells (MSC)
Two-ml samples of pooled bone marrow cell suspension in alpha medium, supplemented with 20% fetal calf serum (FCS), containing 2 x 106 nucleated cells per ml, were placed onto 35-mm plastic dishes (Falcon #3001). The dishes were incubated at 37°C in fully humidified air containing 5% CO2. On day 10, the medium and the non adherent cells were removed, and MSC adhering to the dishes were washed three times with alpha medium. These dishes were employed for preparating supernatant.
In vitro irradiation of MSC.
The MSC layers were exposed to 0, 150, 500 or 900 rads of X-rays (MBR-1505R, Hitachi Co. Ltd. Japan) operated at 140 KVP 40 mA, filtered by 0.5mm Cu plus 1.0 mm of Al, at a target distance of 40 cm.
Preparation of the supernatant of irradiated MSC
After the establishment of MSC, the medium was replaced with fresh medium supplemented with 20% FCS, either with the addition of 15% abdominal wall conditioned medium (AWCM)16> which was used as a source of CSF to examine CSF consumption by MSC, or without the addition of AWCM to examine the production of CSF or inhibitor by MSC. The MSC were then exposed to 0, 150, 500 or 900 rads at room temperature. The irradiated MSC were cultured for another 7 days. The MSC supernatant was harvested every day for 7 days, passed through a 0.45-mm millipore filter, and then kept at -20°C until use.
Coculture of the supernatant of irradiated MSC and GM-CFC
GM-CFC was assayed by employing a modified double layer agar culture, based on the method of Pike and Robinsonl7). One and a half ml of alpha medium, containing 0.5% agar, 12.5% FCS, and 50% of the final concentration of MSC-conditioned medium as an underlayer, was set in 35-mm plastic dishes. Then 0.5 ml of 0.3% agar medium containing 12.5% FCS and 5 x 104 bone marrow cells, which were supplied with or without 7.5% (final concentration) of AWCM depending on whether the inhibitory activity of GM-CFC or CSF production was to be examined, was carefully overlaid on the agar base. In the study of inhibitor production and CSF consumption by MSC, the supernatants incubated without MSC were used as controls. The supernatants were maintained under the same condition as mentioned above for 7 days. Colonies consisting of 50 or more cells were counted using an inverted microscope.
Statistical analysis of the data Student's t-test was used to determine the significance of the differences between values.
RESULTS
Colony stimulating activity in the supernatant of irradiated MSC CSF from irradiated MSC were measured for 7 consecutive days beginning at the time of irradiation (Fig. 1). The time course of CSF production by non-irradiated MSC revealed maximum activity at day 2, which then decreased gradually. After exposure to 150 rad, CSF production by MSC was significantly enhanced with a peak at day 3 which was followed by gradual decline as in the case of non irradiated MSC. However, the time course of CSF production by MSC exposed to 500 or 900 rads was quite different from that observed for 0 or 150-rad exposure groups, and the maximum activity was observed at day 7.
Inhibitory activity in the supernatant of irradiated MSC Inhibitory activity was detected in the supernatant of 900-rad irradiated MSC at day 1 (Fig. 2). It was dose-dependent and significant, although the magnitude of the inhibition was at best 30%. The production of inhibitory factors in the supernatant of irradiated MSC was measured for 7 consecutive days, beginning the first day after irradiation (Fig. 3). The inhibitory activity of the supernatant of irradiated MSC at day 1 increased with the increase of MSC irradiation dose, compared with that of non irradiated MSC. The time course of inhibitory activity in the supernatant of irradiated MSC revealed maximum activity at day 1, which then declined gradually.
Consumption of CSF by irradiated MSC Consumption of CSF by irradiated MSC was investigated. Two ml of fresh medium, composed of alpha medium was supplemented with 20% FCS and 15% CSF with 0 or 900-rad irradiated MSC for 3 days. The supernatant was collected every day and used as a source of CSF for GM-CFC assay. CSF activity in the supernatant of non irradiated MSC declined linearly with the increase in incubating time (Fig. 4). The residual CSF activity in the supernatant of irradiated MSC was different from that in the supernatant of non-irradiated MSC. CSF levels in the supernatant of 900-rad-irradiated MSC at days 2 and 3 were significantly higher than that of non irradiated MSC. 15) also reported the same phenomenon in a liquid culture system. In that experiment, we could not detect the inhibitory activity produced by MSC, 2 to 7 days after the addition of exogenous CSF15). Our preliminary study showed that the exogenous CSF, which was added to the cultured medium of MSC, stimulated the proliferation of MSC and increased the number of MSC one and half-fold in 7 days. Wang et al.20 reported that GM-CSF stimulated proliferation of MSC, especially macrophages and endothelial cells. Yan et al.27) reported that MSC-conditioned medium contained an autocrine factor, possibly IL-1, for bone marrow fibroblasts, and a paracrine factor (CSF-1) for macrophages and/or endothelial cells. These data suggested that CSF stimulated the proliferation of MSC and, in the process, consumed itself. Therefore, in our third experiment, the effect of irradiation on CSF consumption by irradiated MSC was investigated. When fresh medium containing exogenous CSF was cultured with 900-rad-irradiated MSC, the CSF in the supernatants at days 2 and 3 was higher than the CSF in the supernatants of non-irradiated MSC. These data suggest that irradiation disturbs CSF consumption by MSC. Zuckerman et al. 28) reported that total RNA, total protein and collagen synthesis in MSC were reduced by 35-60010 within two days after 900-rad irradiation. In our preliminary study, the numbers of MSC cocultured with exogenous CSF for 7 days after exposure to 0 and 900-rad irradiation were 4.55 x 105 and 1.86 x 105 per dish, respectively. Our result might be due to the fact that irradiation suppressed the MSC proliferation stimulated by CSF. Further investigation is necessary to clarify the reason for this. | 2018-04-03T01:33:19.611Z | 1991-12-01T00:00:00.000 | {
"year": 1991,
"sha1": "84c35da456d8b90a974b77f51cfccd07dbcbaef1",
"oa_license": "CCBYNC",
"oa_url": "https://academic.oup.com/jrr/article-pdf/32/4/395/9646724/jrr-32-395.pdf",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "f766da9ceaef87c27b417a1f51e958a650de7808",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Chemistry",
"Medicine"
]
} |
1856328 | pes2o/s2orc | v3-fos-license | Catalytic Function of PLA2G6 Is Impaired by Mutations Associated with Infantile Neuroaxonal Dystrophy but Not Dystonia-Parkinsonism
Background Mutations in the PLA2G6 gene have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystonia-parkinsonism. These clinical syndromes display two significantly different disease phenotypes. NBIA and INAD are very similar, involving widespread neurodegeneration that begins within the first 1–2 years of life. In contrast, patients with dystonia-parkinsonism present with a parkinsonian movement disorder beginning at 15 to 30 years of age. The PLA2G6 gene encodes the PLA2G6 enzyme, also known as group VIA calcium-independent phospholipase A2, which has previously been shown to hydrolyze the sn-2 acyl chain of phospholipids, generating free fatty acids and lysophospholipids. Methodology/Principal Findings We produced purified recombinant wildtype (WT) and mutant human PLA2G6 proteins and examined their catalytic function using in vitro assays with radiolabeled lipid substrates. We find that human PLA2G6 enzyme hydrolyzes both phospholipids and lysophospholipids, releasing free fatty acids. Mutations associated with different disease phenotypes have different effects on catalytic activity. Mutations associated with INAD/NBIA cause loss of enzyme activity, with mutant proteins exhibiting less than 20% of the specific activity of WT protein in both lysophospholipase and phospholipase assays. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity, and two mutations produce a significant increase in specific activity for phospholipid but not lysophospholipid substrates. Conclusions/Significance These results indicate that different alterations in PLA2G6 function produce the different disease phenotypes of NBIA/INAD and dystonia-parkinsonism. INAD/NBIA is caused by loss of the ability of PLA2G6 to catalyze fatty acid release from phospholipids, which predicts accumulation of PLA2G6 phospholipid substrates and provides a mechanistic explanation for the accumulation of membranes in neuroaxonal spheroids previously observed in histopathological studies of INAD/NBIA. In contrast, dystonia-parkinsonism mutations do not appear to directly impair catalytic function, but may modify substrate preferences or regulatory mechanisms for PLA2G6.
Introduction
Mutations in the PLA2G6 gene (Entrez GeneID:8398) have been identified in autosomal recessive neurodegenerative diseases classified as infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accumulation (NBIA), and dystoniaparkinsonism [1][2][3]. Although there is significant overlap between the NBIA and INAD phenotypic spectrum, the clinical features of dystonia-parkinsonism are distinct in many ways from those reported for NBIA and INAD. INAD and NBIA caused by PLA2G6 mutations typically begin in the first two years of life and involve progressive impairment of movement, speech and cognition, secondary to widespread degeneration in the peripheral and central nervous system [4][5][6]. Additional clinical features specific for the INAD/NBIA phenotypic spectrum include cerebellar atrophy and iron accumulation in the globus pallidus, both of which can be observed on magnetic resonance imaging of the brain. In contrast, dystonia-parkinsonism begins primarily as a movement disorder in the age range of 15-30 years old, and is further distinguished from NBIA/INAD by the absence of cerebellar atrophy and iron accumulation [2,3]. A combination of dystonia and parkinsonism are the common presenting features, and similar to idiopathic PD, the parkinsonism is responsive to levodopa or a dopamine receptor agonist. Cognitive impairment is observed with disease progression.
The PLA2G6 gene encodes group VIA calcium-independent phospholipase A2 (PLA2G6) also known as calcium-independent phospholipase A2 beta (iPLA 2 b). The enzyme was originally identified in Chinese hamster ovary cells based on its ability to hydrolyze the sn-2 acyl groups of phospholipids, producing free fatty acids and lysophospholipids [7,8]. Morgan et al originally mapped a gene locus containing PLA2G6 in multiple families with autosomal recessive inheritance of INAD or NBIA [1]. Sequencing of the PLA2G6 gene in INAD and NBIA revealed a total of 44 unique mutations associated with disease. In all but one case in which PLA2G6 mutations were detected, mutations were present in both alleles, indicating that disease is caused by loss of function rather than a dominant gain of function. In some INAD/NBIA cases, both alleles were affected by early frame shift and stop codon mutations, suggesting a complete loss of protein function [1,6]. However the majority of disease-associated mutations cause missense single amino acid substitutions.
Subsequent studies identified PLA2G6 mutations in patients with dystonia-parkinsonism. Paisan-Ruiz et al identified regions of homozygosity on chromosome 22 in two families with dystoniaparkinsonism [2]. Sequencing of genes in this region revealed missense mutations in PLA2G6, causing amino acid substitutions R741Q in one family and R747W in the other. In each case, affected patients were homozygous for the missense mutation in PLA2G6. A third missense mutation in PLA2G6, causing amino acid substitution R632W has been identified in association with dystonia-parkinsonism in 3 siblings [3]. The three affected siblings in this family were homozygous for the missense mutation, while 3 unaffected siblings and parents were heterozygotes. Interestingly, the R632W mutation has been identified on one allele in an INAD patient with compound heterozygous mutations in PLA2G6 [1].
Distinct phenotypes associated with mutations in the same gene may result from the influence of additional genetic and environmental factors. Alternatively, individual PLA2G6 mutations may primarily determine phenotype through distinct effects on protein function, causing either different degrees of impairment in a single function, or perhaps affecting different functions of the same protein. To examine the hypothesis that disparate phenotypes are determined primarily by distinct effects of mutations on PLA2G6 enzyme function, we developed assays to assess the catalytic activity of wildtype (WT) and mutant PLA2G6 proteins. We find that the human PLA2G6 enzyme functions as an A2 phospholipase, hydrolyzing the sn-2 acyl chain of phosphatidylcholine (PC), and as a lysophospholipase, hydrolyzing the sn-1 acyl chain of lysophosphatidylcholine (LPC), the product of its A2 phospholipase reaction. We find that mutations associated with INAD and NBIA profoundly impair enzyme function in both phospholipase and lysophospholipase assays. In contrast, mutations associated with dystonia-parkinsonism mutations do not impair catalytic function.
Results
Recombinant human PLA2G6 catalyzes the release of free fatty acids from multiple lipid substrates We produced purified recombinant wildtype human PLA2G6 protein and used in vitro assays with radiolabeled lipid substrates to examine its catalytic function. Recombinant protein was produced by transient transfection of the 293FT cell line and purified using nickel affinity chromatography to capture a six histidine tag added to the C-terminus of PLA2G6. We produced recombinant protein for the longest PLA2G6 isoform, encoded by transcript variant 1, and examined its catalytic lipase activity using two lipid substrates ( Figure 1). Recombinant PLA2G6 catalyzed the release of oleic acid from the phospholipid substrate 1-palmitoyl-2-oleyl-phosphatidylcholine (PC). Recombinant PLA2G6 also catalyzed the release of palmitic acid from the 2-lysophospholipid, 1-palmitoyl lysophosphatidylcholine (LPC). Lysophospholipids can be generated from phospholipids by the A2 phospholipase activity of PLA2G6 as well as other A2 phospholipase enzymes. Mutation of the catalytic serine residue within the GXSXG lipase consensus sequence abolished the
Mutations associated with INAD and NBIA cause loss of enzyme activity
We used site directed mutagenesis to introduce missense mutations previously identified in patients diagnosed with either NBIA or INAD. These mutations included Y790X, the most frequent mutation found in association with INAD/NBIA that results in a premature stop codon, truncating the last 15 amino acids of the WT protein. We selected several other mutations based on their location within proposed functional regions of PLA2G6, including the ankyrin repeat region that may be responsible for protein-protein interactions (A341T), the first glycine residue in the GXSXG lipase domain (G517C), and a Cterminal region that includes a calmodulin binding domain (G638R). The location of disease-associated mutations relative to functional domains in the PLA2G6 protein is illustrated in Figure 2. We produced recombinant proteins containing each of the disease-associated mutations and compared catalytic activity to the WT PLA2G6 protein. To compare specific activities of WT and mutant proteins, quantitative western blot analysis was used to determine the relative PLA2G6 protein concentration for each recombinant protein preparation and add equal amounts of WT and mutant proteins to the catalytic assays.
We found that the above mutations associated with INAD/ NBIA significantly reduced PLA2G6 phospholipase activity compared to WT protein. The A341T, G517C, G638R, and Y790X mutant proteins had less than 10% of the activity of WT protein ( Figure 3A). We also examined the catalytic activity of mutant proteins using the substrate LPC. The lysophospholipase assay yielded results that were similar to those in the phospholipase assay, with INAD-associated mutations causing impaired enzyme activity for the LPC substrate ( Figure 3B).
To further investigate the potential significance of genotype differences between INAD/NBIA and dystonia-parkinsonism, we examined the catalytic activity of two other mutations associated with NBIA/INAD. Mutations causing a missense amino acid substitution at position 741 are associated with both INAD/NBIA (R741W) and dystonia-parkinsonism phenotypes (R741Q). We examined the effect of the INAD/NBIA-associated tryptophan substitution at amino acid 741 (R741W) and found that this amino acid change reduced PLA2G6 phospholipase activity to approximately 25% of the level of WT activity and lysophospholipase activity to approximately 10% of WT activity ( Figure 4A-B). We also examined the catalytic activity of the V691del mutation because it has been identified in combination with an R632W mutation in an INAD patient with compound heterozygous mutations, which is in contrast to homozygous R632W mutations associated with dystonia-parkinsonism. To investigate the potential contribution of the V691del mutant allele observed in combina-
Mutations that cause dystonia-parkinsonism do not impair PLA2G6 catalytic activity
We also produced recombinant PLA2G6 proteins containing the three mutations associated with dystonia-parkinsonism (R632W, R741Q, and R747W). In contrast to the effects of INAD/NBIA-associated mutations, the three mutations associated with dystonia-parkinsonism did not impair phospholipase catalytic activity ( Figure 5). In fact, PLA2G6 enzyme with the R747W and R632W mutations displayed an increased rate of PC-hydrolysis relative to WT enzyme in the phospholipase assay. In the lysophospholipase assay, the catalytic rates of the three mutant proteins were not significantly different from WT. Since PLA2G6 has also been observed to hydrolyze palmitoyl coenzyme A (CoA) in vitro [9], we examined PLA2G6 catalytic activity using radiolabeled palmitoyl CoA, and observed that the dystoniaparkinsonism mutations also do not impair PLA2G6 thioesterase activity ( Figure S1).
Discussion
Our results provide insight into pathogenic mechanisms underlying the spectrum of neurodegenerative diseases caused by PLA2G6 mutations. We find that mutations associated with NBIA/ INAD impair the catalytic activity of the PLA2G6 protein. In contrast, mutations associated with dystonia-parkinsonism do not impair catalytic activity. These results clarify the mechanisms underlying the phenotypic expression associated with PLA2G6 mutations; selective effects on protein function, rather than other genetic or environmental factors, produce the two different disease spectrums, NBIA/INAD and dystonia-parkinsonism. Comparison of the effects of a glutamine (Q) versus a tryptophan (W) substitution at the 741 position further illustrates the correlation between enzymatic activity and disease phenotype. A tryptophan substitution for arginine at position 741 produces an 80% reduction in activity, associated with INAD in one patient and NBIA in another patient, while glutamine substitution results in no apparent reduction in catalytic activity and is associated with a dystonia-parkinsonism phenotype.
Although our studies do not detect a loss of catalytic function resulting from dystonia-parkinsonism mutations, the recessive pattern of inheritance suggests that they are more likely to cause a loss of function rather than a dominant gain of function. The R632W mutation has been observed in one patient with INAD in addition to three siblings with dystonia-parkinsonism. In the patient with INAD, a heterozygous R632W mutation was found in combination with a heterozygous V691del mutation, which is distinct from the situation in dystonia-parkinsonism patients who have all been homozygous for the R632W mutation. The complete loss of enzyme function caused by the V691del mutation suggests that a genotype-phenotype correlation may exist based on the degree of impairment in PLA2G6 function, and that impairment below a certain level may cause the early onset INAD/NBIA disease phenotype and more widespread effects in the nervous system. Although our assays did not detect impairment of catalytic activity, they do not exclude the possibility of impaired enzyme function in vivo. An alternative explanation is that an additional PLA2G6 mutation was present in this patient but was not detected by sequencing of PLA2G6 exons.
Our results suggest that the R632W and R747W mutations might alter PLA2G6 function by increasing the catalytic rate for PC. We did not observe an increase in the catalytic rate for LPC. Further experiments are needed to determine whether these mutations alter the relative catalytic rates for PC and LPC. Such a change in substrate preference could significantly alter enzyme function in vivo. By promoting phospholipid hydrolysis over lysophospholipid hydrolysis, the mutations may alter the relative levels of phospholipids, lysophospholipids, and fatty acids normally regulated by the PLA2G6 enzyme. It is also possible that these mutations interfere with other mechanisms regulating PLA2G6 function, such as interactions with calmodulin, calcium/calmodulin-dependent protein kinase IIb or other proteins [10,11], which may not be detected in our in vitro assays.
The capacity for human PLA2G6 to hydrolyze both phospholipid and lysophospholipid substrates supports a role for the enzyme in phospholipid homeostasis, which is also supported by histopathological changes in INAD and experimental results in cultured cells. In cultured cell lines, PC levels remain constant in the face of increased PC synthesis produced by over-expression of a rate-limiting PC synthesis enzyme. Cells compensate for increased PC production by the conversion of PC to free fatty acids and glycerophosphocholine (GPC), a process that is associated with increased expression of PLA2G6 and blocked by an inhibitor of PLA2G6 [12,13]. Our results for human PLA2G6, consistent with previous studies of Chinese hamster PLA2G6 [8,14], demonstrate the capacity of PLA2G6 to catalyze both enzymatic steps in the conversion of PC to fatty acids and GPC.
The proposed role for PLA2G6 in phospholipid turnover does not exclude additional roles in membrane remodeling or arachidonic acid signaling pathways, but a role in phospholipid homeostasis may explain the axonal accumulation of membranes in neuroaxonal spheroids, the hallmark feature of NBIA/INAD observed throughout the peripheral and central nervous system [15][16][17][18][19][20][21] The core component of neuroaxonal spheroids is tubulovesicular membrane accumulation, and this feature is reproduced in mouse models of INAD produced by mutations in the PLA2G6 gene [22][23][24], including a missense mutation that disrupts PLA2G6 phospholipase activity [23]. Interestingly mice with mutations in the PLA2G6 gene develop neurological impairment later within the mouse lifespan than observed in human INAD [22,23]. Later onset disease in the mouse may result from differences between species in neuronal vulnerability to loss of PLA2G6. Species differences in axon length, metabolic requirements or compensatory metabolic pathways could explain differences in age of onset and degree of neurological impairment.
Our results predict that PLA2G6 mutations may cause not only accumulation of phospholipid substrates but also decreased production of fatty acids. Fatty acid release by PLA2G6 may be important in the synthesis of new phospholipids, triglycerides and other lipids, or alternatively catabolic pathways such as fatty acid beta-oxidation. The INAD/NBIA phenotype is also associated with accumulation of alpha-synuclein in Lewy bodies and Lewy Figure 5. PLA2G6 mutations associated with dystonia-parkinsonism do not impair phospholipase and lysophospholipase activity. Purified recombinant protein was produced for WT PLA2G6 protein and for PLA2G6 proteins containing missense mutations associated with dystonia-parkinsonism, Equal amounts of WT or mutant proteins were added to enzymatic assays utilizing (A) PC or (B) LPC as substrate. Relative rates of fatty acid release are shown for WT and each mutant protein (mean percent WT + standard deviation, n = 3 independently prepared protein preparations for each of WT and 3 mutants). Asterisks indicate mean activities that were significantly different from WT (p,0.05, unpaired t-test). Although an initial experiment with n = 1 recombinant protein preparations indicated decreased activity of all three mutant proteins relative to WT, results similar to those shown in panels A and B were observed in all subsequent experiments, which included at least 3 additional independent protein preparations. doi:10.1371/journal.pone.0012897.g005 neurites [4]. Since alpha-synuclein binds fatty acids and regulates brain fatty acid metabolism [25][26][27][28][29][30], altered fatty acid metabolism may be a mechanism underlying alpha-synuclein accumulation. Additional studies in neuronal culture and mouse models may further define mutation-induced changes in PLA2G6 function, and mechanisms linking changes in PLA2G6 catalytic activities to these histopathological features of neurodegeneration.
Materials
Chemicals were obtained from Sigma unless otherwise indicated.
Plasmids
The full length human PLA2G6 coding sequence for transcript variant 1 was amplified by PCR from the human cDNA clone MGC:45156 (IMAGE:5166749, Genbank:BC036742), using forward primer aagaattcaccatgcagttctttggccgcctg and reverse primer aatctagagggtgagagcagcagctggat. The forward primer contains an added EcoRI restriction site upstream of the ATG start codon and the reverse primer contains an XbaI site downstream of the last codon. The PCR product was subcloned into pcDNA3.1 myc-his. Mutations were generated in the PLA2G6 transcript variant 1 pcDNA 3.1 expression vector using the Quickchange Site-Directed Mutagenesis protocol (Stratagene, La Jolla, CA). Mutations were confirmed by sequencing.
Production and purification of recombinant PLA2G6 protein
293FT cells (Invitrogen) were cultured in DMEM containing 10% fetal bovine serum (FBS), penicillin and streptomycin. Cells were transfected in 10 cm culture dishes using Lipofectamine 2000, by combining 36 ml of Lipofectamine 2000 reagent and 12 mg plasmid DNA in a total volume of 3 ml Optimem medium. After incubating the mixture for 20 min at room temperature, it was added dropwise to 5 ml DMEM with 10% FBS in a 10 cm tissue culture dish. To this mixture were added 9610 6 293 FT cells suspended in 5 ml DMEM with 10% FBS, which were prepared by trypsin digestion and trituration. The DNA liposomecontaining medium was replaced with growth medium the next morning and cells were cultured for an additional day before extraction of protein at approximately 42 hours after transfection (longer incubation times after transfection led to significant cell death that was specific to PLA2G6 plasmids). After washing in 5 ml phosphate buffered saline, cells were extracted in 1.25 ml Triton Wash Buffer (0.1% Triton X-100, 50 mM TrisHCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 2 mM 2-mercaptoethanol, 5 mg/ml aprotinin and 5 mg/ml leupeptin. The extract was frozen at 280uC, thawed, sonicated with five pulses of 1 sec, and centrifuged at 15,0006 g for 10 min. The supernatant was combined with 0.1 ml Ni-NTA Agarose beads (Invitrogen) equilibrated in Triton wash buffer, and incubated with gentle rocking for 30 min at 4uC. Beads were collected from the suspension using a 0.8 ml Handee spin column (Pierce) with centrifugation at 5006 g. Beads were washed two times in Triton Wash buffer, then washed an additional two times in glycerol wash buffer (same composition as Triton wash buffer except 20% glycerol was substituted for 0.1% Triton X-100). Bound protein was eluted in glycerol elution buffer (20% glycerol, 50 mM TrisHCl pH 8.0, 500 mM NaCl, 250 mM imidazole, 2 mM 2mercaptoethanol, 5 mg/ml aprotinin and 5 mg/ml leupeptin) using three separate 0.1 ml additions of elution buffer and collection of eluate by centrifugation.
Quantitation of PLA2G6 protein levels in purified fractions PLA2G6 protein concentrations in catalytic assays for WT and mutant PLA2G6 were normalized based on quantitative Western blot analysis of purified fractions. Typically 1.2 ml of each purified fraction was loaded on a 26-well Criterion 10% SDS-PAGE gel (Bio-Rad), with 2-3 replicate lanes per protein sample. Electrophoresed proteins were transferred to nitrocellulose membrane, incubated with mouse anti-myc monoclonal antibody 9E10, followed by horseradish peroxidase conjugated anti-mouse secondary antibody (Jackson Immunoresearch). Bound secondary antibody was detected by enhanced chemiluminescence (ECL) using Immobilon Western ECL Substrate (Millipore). Blots were imaged with a Kodak Image Station 4400 and the intensity of individual bands compared using Kodak 1D analysis software. Standard curves containing a range of WT protein amounts were included in the analysis of each set of recombinant proteins, in order to verify that ECL signal for the amount of protein loaded was in the linear range for quantitation. A graph demonstrating the linear relationship between PLA2G6 protein concentration and ECL signal intensity is shown in Figure S2. Average ECL signal intensity was used to determine the relative PLA2G6 protein concentrations in each preparation, which was used to add equal amounts of WT and mutant proteins to the catalytic assays.
Assays for PLA2G6 catalytic activity
Catalytic activities of purified recombinant enzymes were measured using in vitro reactions containing 25 mM Tris HCl pH 7.5, 1 mM EGTA, and 4.5 mM lipid substrate, using methods similar to previously reported catalytic assays [7][8][9]31]. Substrates were L-a-1-palmitoyl-2-oleyl phosphatidylcholine [oleyl-1- 14 Prior to experiments, the substrate was evaporated under a stream of nitrogen and redissolved in 100% ethanol at a concentration of 100 mM. The aqueous portion of the reaction, minus the enzyme volume, was rapidly mixed with the ethanol-dissolved substrate, and the reaction mixture was then sonicated in either a cup horn or bath sonicator for 20 minutes at 25uC prior to addition of enzyme. Immediately after addition of enzyme in a 5 ml volume, reactions (50 ml final volumes) were incubated at 37uC for 2 min for phospholipase assays or 4 min for lysophospholipase assays. Fatty acid products were extracted by addition of butanol (25 ml) immediately after the incubation period, followed by vortexing and centrifugation at 20006 g for 4 minutes. Butanol-extracted lipids were separated by thin layer chromatography on silica gel plates (Partisil LK6F, Whatmann) using a 80:20:1 petroleum ether:diethyl ether:acetic acid solvent system. TLC plates were exposed to image phosphor plates, which were analyzed using a FLA-7000 phosphorimager and Multigauge software (Fujifilm). Control reactions were included in each experiment and contained equivalent volumes of purified protein from vector-transfected cells. The average amount of product obtained in control reactions (typically less than 5% of product produced by WT PLA2G6 preparations) was subtracted to obtain the final value for each enzyme preparation. Less than 5% of substrate was converted to product under the reaction conditions used in experiments, and for the amount of enzymes added to the reactions, product formation was linear with respect to enzyme concentration ( Figure S3) and was also linear with respect to incubation time (not shown). Figure S1 PLA2G6 mutations associated with dystonia-parkinsonism do not impair PLA2G6 thioesterase activity. WT or mutant proteins were added at equal enzyme concentrations to catalytic assays utilizing radiolabeled palmitoyl CoA as substrate. Relative rates of fatty acid release are shown for WT and each mutant protein (mean percent WT + standard deviation, n = 3 independently prepared protein preparations for each of WT and 3 mutants). The asterisk indicates that the mean activity of R632W was significantly different from WT (p,0.05, unpaired t-test). Similar to the results observed in phospholipase and lysophospholipase assays, mutations associated with dystonia-parkinsonism do not impair the thioesterase catalytic activity of PLA2G6. Found at: doi:10.1371/journal.pone.0012897.s001 (0.17 MB TIF) Figure S2 Western blot analysis of purified recombinant protein to normalize WT and mutant protein concentrations in catalytic assays. The graph shows the western blot ECL signal measured for different amounts of purified WT PLA2G6 protein (diluted 15, n = 3 lanes for each volume). The graph also shows a standard curve (y = 16400x-1184) obtained by linear regression (R2 = 0.92) for the relationship between luminescence (after subtracting average luminescence of control lanes from vector transfected cells) and volume of WT PLA2G6 protein. Western blot analysis was used to determine the relative PLA2G6 protein concentration in each recombinant protein preparation and to normalize the amount of protein added to the catalytic assay as outlined in Material and Methods. Found at: doi:10.1371/journal.pone.0012897.s002 (0.26 MB TIF) Figure S3 Linear relationship between enzyme concentration and free fatty acid production in catalytic assays used to compare specific activities of WT and mutant PLA2G6 enzymes. Different amounts of WT enzyme were added to a catalytic assay with 14 Clabeled LPC. Released free fatty acids were separated on TLC and quantified by phosphorimager. The graph shows a linear relationship between enzyme concentration and fatty acid release. In experiments examining the effects of mutations associated with dystonia-parkinsonism, the amount of enzyme added to the assays was equivalent to 2 ml WT PLA2G6 enzyme in the above experiment. | 2014-10-01T00:00:00.000Z | 2010-09-23T00:00:00.000 | {
"year": 2010,
"sha1": "12c7e89ac425123dc698524c3b990d28ba682005",
"oa_license": "CCBY",
"oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0012897&type=printable",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "17436c8ecf66204fede151ae00fa77bb2e7a47d0",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
44007921 | pes2o/s2orc | v3-fos-license | How Many Donor Colonies Should Be Cross-Fertilized for Nursery Farming of Sexually Propagated Corals ?
Coral reef restoration approaches have often utilized adult colonies as sources for fragments (i.e. clones) to be transplanted. Although restoration through this method is fast and cheap, it has been pointed out that it may reduce genetic diversity of the restored population. Low genetic diversity is a concern for reef restoration when seed fragments are raised asexually from only a few donor colonies. This can lead to lower fertilization rates among seed fragments, and reducing the longterm benefits of reef restoration in particular areas. Additionally, low genetic diversity can compound the effects of increased ocean temperature and other environmental stressors, further jeopardizing the health of a reef. An alternative approach through sexually propagated coral cultures and out-plantings can alleviate this problem. Sexually produced offsprings are more genetically diverse. They can be produced in far greater numbers than coral fragments and do not imply destructive methods. Ongoing research at the Akajima Marine Science Laboratory in Okinawa, Japan has helped to improve the production and maintenance of sexually propagated larval cultures. Our results show that crosses between gametes from 6 or more colonies will provide the highest fertilization rate (>95%). Based on the results, we suggest the use of 6 or more donor colonies for practical gamete fertilization in sexually derived coral culture.
Introduction
Currently, the most common approach to coral reef restoration is through the transplantation of fragments (i.e.clones).However, restoration practices that use a limited number of donor colonies can reduce gene flow [1].Many broadcasting coral species do not self-fertilize [2] [3], and gametes from clone-mates are incapable of fertilizing each other.Furthermore, there is a significant negative relationship between fertilization rate and genetic similarity in branching corals [4].It is ecologically important to maintain genetic/genotypic diversity within a coral population (see [5]).Low genetic diversity is a concern for reef restoration when seed fragments are raised asexually from only a few donor colonies.This can lead to lower fertilization rates among seed fragments, and reducing the long-term benefits of reef restoration in particular areas.Additionally, low genetic diversity can compound the effects of increased ocean temperature and other environmental stressors, further jeopardizing the health of a reef.
An alternative approach through the farming of coral offspring from larval cultures can alleviate these problems.The method involves the collection and cross-fertilization of gametes from gravid coral colonies during the spawning period, and the subsequent maintenance of the larval culture.Despite the potential cost, in addition to time that is required for this approach, it is a key solution that it could produce far greater numbers of offsprings than coral fragments and do not imply destructive methods which can impair donor coral population.Sexually produced offsprings are more genetically diverse, and provide long-term benefits than asexually produced coral fragments.As a result, higher fertilization rates can be expected from out-planted populations produced from sexually propagated offsprings than those from asexually raised fragments.
During past production of recruits using sexual propagation of acroporid corals, low fertilization rates were occasionally experienced when only a few donor colonies were used for the crosses.Although the reason for this is unknown, it was suspected that this occurred due to: 1) higher genetic similarity between donor colonies, 2) presence of morphologically indistinguishable cryptic species, or 3) the release of unhealthy gametes.Therefore, based on our experience, mixing the gametes from more than 3 donor colonies [6] or a minimum of 6 colonies [7] has been recommended to achieve higher fertilization rates (>95%).
In the present study, fragments of gravid donor colonies from two locations in Kerama Islands, Okinawa, were collected and fertilization rates of crosses were compared by changing the number of colonies from l to 6 in order to corroborate our previous (above stated) recommendation experimentally.
Methods
In 2011, coral fragments (ca.16 -22 cm in length) from 13 gravid, donor colonies of Acropora tenuis were collected from the northern side of Yakabi Island (26˚13.070'N,127˚14.274'E)on June 5 and the northeastern side of Kuba Island (26˚10.711'N,127˚14.582'E)on June 11 (Figure 1).Distance between the two locations was approximately 5 km.The donor colonies were distributed within a 150 m 2 area at each location.Their fragments were collected using a hammer and chisel, and maintained in cages on the seafloor at approximately 2 m depth near Majanohama (Figure 1) in Aka Island before spawning.They were then brought to the laboratory in Aka Island on June 19 when bundles emerged at the mouth of the polyps and placed in separate 20 L buckets.Spawning occurred from 19:35 to 20:10 hr on the same night.The positively buoyant bundles were collected by gently skimming the water surface using a 100 mL polyethylene cup.The bundles were gently agitated in a small volume of filtered seawater to dissociate the egg and sperm.The eggs floating on the surface were collected with a pipette and rinsed twice with 10 μm filtered seawater.The sperm suspensions were diluted to adjust their concentration.Three donor colonies (fragments)each from two locations were chosen for the cross-fertilization experiment.Egg and sperm collected from the 3 fragments from Kuba were designated A, B, C and a, b, c, respectively, while those from the 3 fragments from Yakabi Island were designated D, E, F, and d, e, f, respectively.Gametes were maintained in separate containers.
The gametes were then mixed in a pair-wise combination within glass vials, containing 50 mL seawater each.Sperm adjusted to a concentration of 10 5 /mL along with 300 -470 eggs were added to the vial for fertilization.The fertilization success can be influenced by competition between spermatozoids of distinct parents.Therefore, the concentration-adjusted sperm from 2 or more donor colonies was pooled together prior to mixing with eggs.
All combinations of crosses were conducted once within 3 hours after spawning.The gametes were allowed to fertilize for approximately 20 minutes in a room kept at approximately 25˚C, and percent fertilization was obtained by examining at least 100 eggs per cross under the dissecting microscope.Counts were obtained within 2(a)).The possibility of self-fertilization was also determined for comparison.The fertilization rate of 3-colony crosses between egg and sperm from Kuba Island (ABC × abc) and of 6-colony crosses between eggs from Kuba Island and sperm from Yakabi Island (ABC × def) were also compared.Finally, all gametes from the 6 colonies were mixed together (ABCDEF × abcdef).
Because of shortage of time, the experiment in 2012 was carried out using 6 colonies of A. tenuis from only Kuba Island (the same sampling location as 2011).The fragments were collected on June 1, and spawning occurred on June 11 (19:35 hr).The eggs (G, H, I, J, K, L) and sperm (g, h, i, j, k, l) from the 6 colonies were maintained in separate vials, and then mixed in a pair-wise combination following the procedure from the previous experiment.In addition to the six self-fertilization crosses, fertilization of eighteen 2-colony cross combinations, two 3-colony crosses (GHI × ghi and JKL × jkl), and three 6-colony crosses (GHI × jkl, JKL × ghi and GHIJKL × ghijkl) were conducted (Figure 2(b)).
Results
Results of the cross-fertilization using 6 donor colonies in 2011 are shown in Figure 2(a).When gametes from 2 colonies were crossed, the fertilization rate varied considerably from 38.4% to 100%.The fertilization rate was 77.2% when the gametes of 3 colonies from Kuba Island (ABC × abc) were mixed, whereas, the cross between eggs of 3 colonies from Kuba Island and sperm of 3 colonies from Yakabi Island (ABC × def) resulted in much higher rate (98.2%).It is possible that crosses between gametes from the 3 additional donor colonies, which were separated by greater geographical distance, provided additional genetic variation to the pool, thereby providing gametes with greater fertilization affinities.This was confirmed when gametes of all 6 colonies were Instead of mixing gametes from donor colonies from 2 distinct sites in 2011, gametes from only Kuba Island were mixed in 2012.However, the fertilization rates between gametes from 2-, 3-, and 6-colony crosses were higher than those from the initial experiment (Figure 2(b)).All self-crosses resulted in 0% fertilization rate except the G × g cross which recorded a 1.9%.Among fertilization rates between multiple colonies, the lowest percentage (86.9%)was recorded in a 2-colony cross (H × k).The 3-colony crosses yielded a fertilization success of approximately 97%, whereas all three 6-colony crosses resulted in the rates exceeding 97%.
In 2011 most of the embryos developed to or beyond the 128-cell stage between 5.5 and 7.0 hours after fertilization (Figure 3).However, five out of nine 2-colony crosses contained earlier stages, suggesting delayed embryogenesis.Additionally, two of 2-colony crosses contained abnormally developing embryos.Result of the experiments from 2012 was similar to that of 2011.Out of eighteen 2-colony crosses, nine contained developmentally delayed or abnormal embryos (data not shown).
Discussion
Genotypes of donor colonies used for the cross-fertilization were not determined in the present study.Use of colonies of unknown genotypes may reduce the potential for true crosses between distinct genotypes and may result in lower fertilization success.This may be the explanation for higher success in the 2012 experiment vs. the 2011 experiment.However, measurement of genetic similarity before fertilization is impractical for nursery farming of sexually propagated corals.The present cross-fertilizations were only conducted once for each combination, and therefore, were unable to confirm the results statistically.Lack of replicates may be criticized, but for the scope of this study, grasping general tendencies of the variation in fertilization rates was more meaningful than detailed analysis of the variation.It is important that the following similarities in outcome were obtained between the two years.1) With 2-colony crosses, the fertilization rate varied widely; 2) with 3-colony crosses the fertilization rate increased but some showed relatively lower rates; and 3) with 6-colony crosses the rate was consistently higher than 95%.A rarefaction analysis of population genetic datasets conducted by Shearer et al. [8] showed that >50% of the allelic diversity of a population could be retained by using gametes of 10 randomly sampled donor colonies, while 35 colonies would retain 90% of the original diversity.In practice however, it would be difficult and impractical to mix gametes from 35 gravid donor colonies to establish sexually propagated coral cultures.Since 2006, juvenile colonies of Acropora tenuis cultured after mixing gametes from 6 or more donor colonies were out-planted at Majanohama (Figure 1).They spawned initially at 4-and 5-year-old [9].Spawning has continued every year since 2009.Fertilization rates among these 6 cultured colonies (5-year-old A. tenuis) were >98.4% that was similar to those measured in crosses of 3 wild colonies [10].
In the present experiment, the number of donor colonies was limited to 6.It lacks replication and statistics, but shows evidence that fertilization rate increases with the number of donor colonies, as if there exists a threshold where the relationship reaches an asymptote.The purpose of the present study was not to assess how genotypic differences between the donor colonies affected fertilization rate, but aimed to investigate how many donor colonies are necessary to consistently achieve high fertilization rate.If the importance of allelic diversity of a population is taken into consideration, cross-fertilization with 10 colonies or more may be needed, but for practical nursery farming of sexually propagated corals, cross-fertilization with 6 or more colonies could be recommended in the laboratory.
Figure 1 .
Figure 1.Map of Kerama Islands.Sampling locations of wild donor colonies (Kuba Island and Yakabi Island: red spots) and cultured colonies (Majanohama in Aka Island: blue spot).5.5 hours of fertilization.In addition to the fertilization rate, the developmental stages of the embryos were scored.The presence of abnormally developing embryos, if any, was also recorded.Cross-fertilization between 2 colonies were arranged as follows: A × b, A × c, A × d, B × c, B × d, B × e, C × d, C × e, C × f (Figure2(a)).The possibility of self-fertilization was also determined for comparison.The fertilization rate of 3-colony crosses between egg and sperm from Kuba Island (ABC × abc) and of 6-colony crosses between eggs from Kuba Island and sperm from Yakabi Island (ABC × def) were also compared.Finally, all gametes from the 6 colonies were mixed together (ABCDEF × abcdef).Because of shortage of time, the experiment in 2012 was carried out using 6 colonies of A. tenuis from only Kuba Island (the same sampling location as 2011).The fragments were collected on June 1, and spawning occurred on June 11 (19:35 hr).The eggs (G, H, I, J, K, L) and sperm (g, h, i, j, k, l) from the 6 colonies were maintained in separate vials, and then mixed in a pair-wise combination following the procedure from the previous experiment.In addition to the six self-fertilization crosses, fertilization of eighteen 2-colony cross combinations, two 3-colony crosses (GHI × ghi and JKL × jkl), and three 6-colony crosses (GHI × jkl, JKL × ghi and GHIJKL × ghijkl) were conducted (Figure2(b)).
Figure 2 .
Figure 2. Comparison of fertilization ratio (%) between different combinations of 2-, 3-, 6-colony crosses.(a) Experiment in 2011; (b) In 2012.Number in each frame provides rates for 2-colony crosses.The rates for 3and 6-colony crosses are given in the margin.mixed, which yielded high fertilization rate.Instead of mixing gametes from donor colonies from 2 distinct sites in 2011, gametes from only Kuba Island were mixed in 2012.However, the fertilization rates between gametes from 2-, 3-, and 6-colony crosses were higher than those from the initial experiment (Figure2(b)).All self-crosses resulted in 0% fertilization rate except the G × g cross which recorded a 1.9%.Among fertilization rates between multiple colonies, the lowest percentage (86.9%)was recorded in a 2-colony cross (H × k).The 3-colony crosses yielded a fertilization success of approximately 97%, whereas all three 6-colony crosses resulted in the rates exceeding 97%.In 2011 most of the embryos developed to or beyond the 128-cell stage between 5.5 and 7.0 hours after fertilization (Figure3).However, five out of nine 2-colony crosses contained earlier stages, suggesting delayed embryogenesis.Additionally, two of 2-colony crosses contained abnormally developing embryos.Result of the experiments from 2012 was similar to that of 2011.Out of eighteen 2-colony crosses, nine contained developmentally delayed or abnormal embryos (data not shown).
Figure 3 .
Figure 3. Composition of developmental stage of embryo originated from cross-fertilization between different donor colonies. | 2018-02-10T07:57:38.513Z | 2014-07-31T00:00:00.000 | {
"year": 2014,
"sha1": "13f7139f06813aafd45a14676047ed3d872c6e6b",
"oa_license": "CCBY",
"oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=48443",
"oa_status": "GOLD",
"pdf_src": "ScienceParseMerged",
"pdf_hash": "13f7139f06813aafd45a14676047ed3d872c6e6b",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology"
]
} |
186929830 | pes2o/s2orc | v3-fos-license | Design and Application Research of VR/AR Teaching Experience System
This paper is based on the current situation of teaching, such as the lack of contextual interaction in the learning experience, the single situation, and the poor teaching experience and utilizes virtual reality (VR) and augmented reality (AR) technology to solve these problems. With the immersive, interactive and imaginative features of virtual reality technology, the virtual somatosensory virtual learning space with human-computer interaction is designed. The superposition of augmented reality technology is used to create an AR superposition learning space that combines real world and virtual space. According to the extension of VR/AR teaching thinking dimension, the teaching experience system design framework is constructed, and the physical model of experience system is proposed. Through data mining and teaching feedback, the learning experience is adjusted. Then design a personalized teaching experience system which can enhance learning experience and improve learning interest.
Introduction
With the continuous development of computer software and hardware technology, the combination of new technology and teaching is getting closer and closer, but the application of new technology has not solved the problems caused by traditional teaching. The current problems in school teaching mainly focus on the lack of interaction in the teaching process. Most of the teaching environment lacks a real contextual learning environment in the classroom. The teaching methods are single and it is difficult to inspire students' association and creativity. Application of the teaching process and teaching results are poor. So it is difficult to meet the task requirements of real jobs. The root cause of these problems is the poor teaching experience, which cannot provide learners with a real learning environment, intuitive and three-dimensional spatial experience.
The maturity of VR technology and AR technology, as well as the learner-centered, emphasis on teaching experience and the urgent need for adaptive learning, have driven the appearance of the VR/AR teaching experience system. VR achieved a brand-new state of human-computer interaction. It can obtain intuitive and real perceptions such as sight, hearing and touch by operating objects in the virtual world [1]. By combining virtual objects and the real world, AR can simultaneously display the information of the real world and the virtual world, enabling learners to use 3D models to enhance the visual perception ability of real-world situations [2].VR enhances sensory interactivity by constructing a simulated virtual world. The main features are: immersion, interactivity and imagination. Immersion allows learners to eliminate external disturbances and immerse themselves in virtual reality to gain an immersive feeling. Interactivity is based on the learner's head, hands, eyes, language and body movements to adjust the image and sound presented by the system. Imagination is to acquire visual, auditory, tactile, kinesthesia and other perceptions simultaneously in the virtual environment, enhance the learner's perception of the learning content, the high sensitivity and rational understanding of the cognitive content, so that to make the user to deepen the concept and sprouts new association, and motivate the learner's creative thinking. AR is a bridge connecting virtual world and real world. It is characterized by superposition and openness. It superimposes virtual information in the real world, enhances visual, auditory and tactile sense, and enables learners to experience the combination of real world and virtual world in the senses. The VR/AR teaching experience system compensates for the problems that appeared in traditional teaching. Figure 1 shows the VR/AR features.
VR/AR Teaching Experience System Model Design
VR/AR teaching experience system model design mainly focuses on how to implement the experience system, how to design the system physical structure. The experience system is divided into five levels, from the bottom to the top, VR/AR base layer, VR/AR space layer, VR/AR logic layer, teaching data mining layer, teaching adjustment layer. The implementation process is to use the underlying physical device to enter the VR/AR learning space, and learners enter the database to intelligently retrieve the experience environment and experience context according to the needs in the learning space. The learner learns the state and data in the learning space and analyzes it through the teaching data mining layer, and then feeds back to the teaching adjustment layer to achieve the dynamic update of the learner's learning experience. Figure 2 shows the VR/AR teaching experience system model design.
VR/AR base layer
The VR/AR base layer is the bottom part of the entire teaching experience system, and is also the physical device carrier and platform that enters the learning space, such as HMD, VR glasses, mobile phones, simulation devices, iPad, and so on. The learner enters the virtual platform through the physical device, and the virtual platform is a fusion of one or more learning spaces to ensure the reliability of the learner entering the VR/AR space layer.
VR/AR space layer
The VR/AR space layer contains various learning spaces of the system, including VR direct interaction space, AR superposition space, shape space, and virtual and real interlaced space [3]. In these spaces, learners can choose their own learning context according to their own learning situations, or they can choose intelligently through fixed and creative modes to understand the learners' knowledge storage and learning abilities, so that learners can be clear about themselves. In order to choose the best, most suitable learning situation and enhance the sense of learning experience.
VR/AR logic layer
The VR/AR logic layer achieves the learning of methods, processes and skills in the virtual space according to the characteristics and needs of the learners [4]. This layer implements the physical introduction and logical introduction of space. The physical device enters the learning context created by the VR/AR space layer, and sets the contactor. When the learner touches the contactor during the learning process, the logic introduction will start. The learning situation increases the difficulty node and the mission, and the learner completes the task by learning the knowledge and skills provided in the space to be able to enter the next contactor.
Teaching data mining layer
The teaching data mining layer mainly analyzes the learner's feedback data learning process and learning state in the VR/AR space layer, including the proficiency of the learners' knowledge when in the contactor or upgrade and the number of times that learner asks for help when encountering difficulties. Through the analysis and mining of these contents, the best learning path between learners and learning trajectories can be calculated and then be fed back to the teaching data adjustment layer to dynamically optimize the learner experience situation.
Teaching adjustment layer
The teaching adjustment layer is mainly divided into teaching strategy adjustment and teaching experience adjustment, mainly through the feedback of teaching data mining layer to adjust the learning strategy and adjust the learning situation. The teaching strategy adjustment is to personalize the teaching methods, set suspense or story plots, and attract learners to explore in the learning space. The teaching experience adjustment is to automatically complete the learners' experience scene in learning space according to the plots of teaching strategy adjustment.
VR / AR Learning Space
The VR/AR learning space is the core content of the teaching experience system. The learner goes to the virtual space for experience learning through the first viewpoint of VR, and enters the external perspective from the internal perspective [7]. The AR uses the third viewpoint to experience learning. The AR is a bridge between the virtual world and the real world. The learner enters the virtual world of the internal perspective from the external perspective in the real world to improve the interactive experience. The establishment of the two-way viewpoint of the VR/AR learning space enables the learner to switch back and forth between the internal perspective and the external perspective to achieve a fusion of virtual and real learning spaces and enhance the learning experience. The VR/AR learning space is mainly designed from database resources and JDK and SDK platforms. Figure 3 shows VR/AR learning space design. The database resources are mainly used to develop and store the models and scenarios required in the learning space. The Unity 3D is mainly used to develop the VR model system and the AR model system for the development tools. Using 3dsmax production model and UV finishing, using Photoshop for post-laying drawing, then outputting textures and models into Unity3D to implement interactive programs through C#, and then making AR and VR separately [6]. Database resources are then passed to the JDK and SDK platforms for interaction through the management server.
JDK, SDK development platform
The JDK and SDK development platforms mainly implement resource mutual transfer between the VR model system and the AR model system. The JDK and SDK development platforms use the management resource server to acquire database resources, and implement resource packaging and interaction functions through Unity, and finally form a VR model system. The AR model system uses HIAR as an augmented reality development tool. HIAR provides cloud image recognition services in the form of REST and API, and uses HIAR's background management tools to implement AR content editing and management [5]. Through the JDK and SDK development platform, the VR model system and the AR model system are mutually called to form a VR/AR learning space with interactive functions.
Design of VR/AR Teaching Experience System
The VR/AR teaching experience system is an adaptive dynamic adjustment experience system. It can enable learners to experience the learning situation and collect the activity data generated by the learner in the learning space through the switching between the VR learning space and the AR learning space. The data is transmitted to the storage server, and analyzed by the data mining technology, and the analysis result is fed back to the computing server, thereby achieve the teaching experience and teaching strategy adjustment of the learning space. Figure 4 shows VR/AR teaching experience system design.
VR/AR learning space entry
The VR/AR learning space needs to be accessed from the VR/AR base layer, and corresponding hardware devices such as VR glasses, HMD, smart phones, and emulation devices are required. These devices can provide direct or indirect access to the learning space. The selected learning space can be a single space or two mixed spaces.
VR/AR learning space conversion
VR/AR learning space conversion is mainly based on virtual reality interactive platform. Through the database resources and JDK, SDK development platform, the fusion of VR model system and AR model system is implemented. According to the needs of the learners, different resources are used to transform and interact among the learning spaces. The combination of virtual and reality and superposition of each other greatly enhances the sense of reality of the teaching experience.
VR/AR learning spatial data analysis
According to the learning goal, the learner enters the corresponding VR/AR space by means of VR or AR, and can select different learning modes depended on the proficiency of knowledge mastery in the space. The system records the learner's entire learning trajectory and learning action data and then submits it to the storage server for analysis and production of the results through data mining techniques.
VR/AR learning space teaching adjustment
Through the feedback of the teaching data mining layer, the computer server selects the most suitable learning situation and learning task for the learner according to the result, so that besides the reality, the learner can also experience the comfort in the learning situation, and increase the enthusiasm of the learner to learn, encouraging learners to continue to learn.
VR/AR Experience Output
Experience output refers to the experience and feelings that learners have gained in completing the learning process. Learners enter the learning space, choose the learning situation, complete the learning tasks based on the learning objectives, and the difficulties encountered in the learning space can be analyzed, fed back, adjusted by the system, and finally reach the optimal experience path.
Conclusion
The introduction of VR/AR has changed the traditional teaching thinking, increased the control of teaching problems and teaching quality, and made the VR/AR teaching experience system an achievable reality. The system is designed based on the problems existing in traditional teaching. The experience of learners switching between the VR learning space and the AR learning space is the key point. It tracks the learning path and learning activity data in the learning space in real time, changes the teaching strategy and teaching experience, and provides an optimal learning experience environment for the learners. | 2019-06-13T13:24:19.390Z | 2019-04-01T00:00:00.000 | {
"year": 2019,
"sha1": "b8db6efa92f0292f298026554b5988d9ec1d54bd",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1742-6596/1187/5/052079",
"oa_status": "GOLD",
"pdf_src": "IOP",
"pdf_hash": "65e866c7283d5fba18d79ca6d97d8dbd322d0d02",
"s2fieldsofstudy": [
"Computer Science",
"Education"
],
"extfieldsofstudy": [
"Physics",
"Computer Science"
]
} |
195428075 | pes2o/s2orc | v3-fos-license | Recounting the FANTOM Cage Associated Transcriptome
Long non-coding RNAs (lncRNAs) have emerged as key coordinators of biological and cellular processes. Characterizing lncRNA expression across cells and tissues is key to understanding their role in determining phenotypes including human diseases. We present here FC-R2, a comprehensive expression atlas across a broadly-defined human transcriptome, inclusive of over 109,000 coding and non-coding genes, as described in the FANTOM CAGE-Associated Transcriptome (FANTOM-CAT) study. This atlas greatly extends the gene annotation used in the original recount2 resource. We demonstrate the utility of the FC-R2 atlas by reproducing key findings from published large studies and by generating new results across normal and diseased human samples. In particular, we (a) identify tissue specific transcription profiles for distinct classes of coding and non-coding genes, (b) perform differential expression analyses across thirteen cancer types, providing new insights linking promoter and enhancer lncRNAs expression to tumor pathogenesis, and (c) confirm the prognostic value of several enhancers in cancer. Comprised of over 70,000 samples, the FC-R2 atlas will empower other researchers to investigate functions and biological roles of both known coding genes and novel lncRNAs. Most importantly, access to the FC-R2 atlas is available from https://jhubiostatistics.shinyapps.io/recount/, the recount Bioconductor package, and http://marchionnilab.org/fcr2.html.
Introduction
Long non-coding RNAs (lncRNAs) are commonly defined as transcripts devoid of open reading frames (ORFs) longer than 200 nucleotides, which are often polyadenylated. This definition is not based on their function, since lncRNAs are involved in distinct molecular processes and biological contexts not yet fully characterized 1 . Over the past few years, the importance of lncRNAs has clearly emerged, leading to an increasing focus on decoding the consequences of their modulation, studying their involvement in the regulation of key biological mechanisms during development, normal tissue and cellular homeostasis, and in disease [1][2][3] .
Given the emerging and previously underestimated importance of non-coding RNAs, the FANTOM consortium has initiated the systematic characterization of their biological function. Through the use of Cap Analysis of Gene Expression sequencing (CAGE-seq), combined with RNA-seq data from the public domain, the FANTOM consortium released a comprehensive atlas of the human transcriptome, encompassing more accurate transcriptional start sites (TSS) for coding and non-coding genes, including numerous novel long non-coding genes: the FANTOM CAGE Associated Transcriptome (FANTOM-CAT) 4 . We hypothesized that these lncRNAs can be measured in many RNA-seq datasets from the public domain and that they have been so far missed by the lack of a comprehensive gene annotation.
Although the systematic analysis of lncRNAs function is being addressed by the FANTOM consortium in loss of function studies, increasing the detection rate of these transcripts combining different studies is difficult because the heterogeneity of analytic methods employed. Current resources that apply uniform analytic methods to create expression summaries from public data do exist but can miss several lncRNAs because their dependency on a pre-existing gene annotation for creating the genes expression summaries 5,6 . We recently created recount2 7 , a collection of uniformly-processed human RNA-seq data, wherein we summarized 4.4 trillion reads from over 70,000 human samples from the Sequence Reads Archive (SRA), The Cancer Genome Atlas (TCGA) 8 , and the Genotype-Tissue Expression (GTEx) 9 projects 7 . Importantly, recount2 provides annotation-agnostic coverage files that allow re-quantification using a new annotation without having to re-process the RNA-seq data.
Given the unique opportunity to access lastest results to the most comprehensive human transcriptome (the FANTOM-CAT project) and the recount2 gene agnostic summaries, we addressed the previous described challenges building a comprehensive atlas of coding and non-coding gene expression across the human genome: the FANTOM-CAT/recount2 expression atlas (FC-R2 hereafter). Our resource contains expression profiles for 109,873 putative genes across over 70,000 samples, enabling an unparalleled resource for the analysis of the human coding and non-coding transcriptome.
Results
Building the FANTOM-CAT/recount2 resource The recount2 resource includes a coverage track, in the form of a BigWig file, for each processed sample. We built the FC-R2 expression atlas by extracting expression levels from recount2 coverage tracks in regions that overlapped unambiguous 2/18 exon coordinates for the permissive set of FANTOM-CAT transcripts, according to the pipeline shown in Figure 1. Since recount2's coverage tracks does not distinguish from between genomic strands, we removed ambiguous segments that presented overlapping exon annotations from both strands (see Methods section and Supp. Methods). After such disambiguation procedure, the remaining 1,066,515 exonic segments mapped back to 109,869 genes in FANTOM-CAT (out of the 124,047 starting ones included in the permissive set 4 ). Overall, the FC-R2 expression atlas encompasses 2,041 studies with 71,045 RNA-seq samples, providing expression information for 22,116 coding genes and 87,763 non-coding genes, such as enhancers, promoters, and others lncRNAs.
Figure 1.
Overview of the FANTOM-CAT/recount2 resource development. FC-R2 leverages two public resources, the FANTOM-CAT gene models and recount2. FC-R2 provides expression information for 109,873 genes, both coding (22,110) and non-coding (87,693). This latter group encompasses enhancers, promoters, and others lncRNAs.
Validating the FANTOM-CAT/recount2 resource We first assessed how gene expression estimates in FC-R2 compared to previous gene expression estimates from other projects.
Specifically, we considered data from the GTEx consortium (v6), spanning 9,662 samples from 551 individuals and 54 tissues types 9 . First, we computed the correlation for the GTEx data between gene expression based on the FC-R2 atlas and on GENCODE (v25) gene model in recount2, which has been already shown to be consistent with gene expression estimates from the GTEx project 7 , observing a median correlation 0.986 for the 32,922 genes in common. This result supports the notion that our pre-processing steps to disambiguate overlapping exon regions between strands did not significantly alter gene expression quantification.
Next, we assessed whether gene expression specificity, as measured in FC-R2, was maintained across tissue types. To this end, we selected and compared gene expression for known tissue-specific expression patterns, such as Keratin 1 (KRT1), Estrogen Receptor 1 (ESR1), and Neuronal Differentiation 1 (NEUROD1) (Figure 2). Overall, all analyzed tissue specific 3/18 markers presented nearly identical expression profiles across GTEx tissue types between the alternative gene models considered (see Figure 2 and S1), confirming the consistency between gene expression quantification in FC-R2 and those based on GENCODE.
Figure 2.
Tissue specific expression in GTEx. Log2 expression for three tissue specific genes (KRT1, NEUROD1, and ESR1) in GTEx data stratify by tissue type using FC-R2 and GENCODE based quantification. Expression profiles are highly correlated and expressed consistently in the expected tissue types (e.g., KRT1 is most expressed in skin, NEUROD1 in brain, and ESR1 in estrogen sensitive tissue types like uterus, Fallopian tubes, and breast). Correlations are shown on top for each tissue marker. Center lines, upper/lower quartiles and Whiskers represents the median, 25/75 quartiles and 1.5 interquartile range, recpectively.
Tissue-specific expression of lncRNAs
It has been shown that, although expressed at a lower level, enhancers and promoters are not ubiquitously expressed and are more specific for different cell types than coding genes 4 . In order to verify this finding, we used GTEx data to assess expression levels and specificity profiles across samples from each of the 54 analyzed tissue types, stratified into four distinct gene categories: coding mRNA, intergenic promoter lncRNA (ip-lncRNA), divergent promoter lncRNA (dp-lncRNA), and enhancers lncRNA (e-lncRNA). Overall, we were able to confirm that these RNA classes are expressed at different levels, and that they display distinct specificity patterns across tissues, as shown for primary cell types by Hon et al. 4 , albeit with more variability 4/18 likely due to the increased cellular complexity present in tissues. Specifically, coding mRNAs were expressed at higher levels than lncRNAs (log2 median expression of 6.6 for coding mRNAs, and of 4.1, 3.8 and 3.1, for ip-lncRNA, dp-lncRNA, and e-lncRNA, respectively). In contrast, the expression of enhancers and intergenic promoters was more tissue-specific (median = 0.41 and 0.30) than what observed for divergent promoters and coding mRNAs (median = 0.13 and 0.09) ( Figure 3A). Finally, when analyzing the percentage of genes expressed across tissues by category, we observed that coding genes are, in general, ubiquitous, while lncRNAs are more specific, with enhancers showing the lowest percentages of expressed genes (mean ranging from 88.42% to 41.98%, see Figure 3B), in agreement with the notion that enhancer transcription is tissue specific 10 .
Differential expression analysis of coding and non-coding genes in cancer
We analyzed coding and non-coding genes expression in cancer using TCGA data. To this end, we compared cancer to normal samples separately for 13 tumor types, using FC-R2 re-quantified data. We further identified the differentially expressed genes (DEG) in common across the distinct cancer types (see Figure 4). Overall, the number of DEG varied across cancer types and by gene class, with a higher number of significant coding than non-coding genes (FDR < 0.01, see table 1). Importantly,
Despite we did not identify any cancer association for common e-lncRNAs, one among those we identified, RP5-965F6, has been previously reported to be up-regulated in late-onset Alzheimer's disease 24 . Furthermore, the enhancers lncRNA class also yielded the lowest number of genes in common among all cancer types, reinforcing the concept that enhancers are expressed in a tissue specific manner (See Figure 3A and Supplementary Table S4).
7/18
Finally, we focused more in depth on prostate cancer (PCa) as a prototypical example, and we were able to confirm previous findings for both coding and non-coding genes (see Supplementary Figure S2). For coding genes, we confirmed differential expression for known markers of PCa progression and mortality, like ERG, FOXA1, RNASEL, ARVCF, and SLC43A1 25,26 .
Similarly, we also confirmed differential expression for non-coding genes, like PCA3, the first clinically approved lncRNA marker for PCa 27, 28 , PCAT1, a prostate-specific lncRNA involved in disease progression 29 , MALAT1, which is associated with PCa poor prognosis 30 , CDKN2B-AS1, an anti-sense lncRNA up-regulated in PCa that inhibits tumor suppressor genes activity 31,32 , and the MIR135 host gene, which is associated with castration-resistant PCa 33 .
Enhancer expression levels hold prognostic value
The number of lncRNAs involved in cancer development and progression is rapidly increasing, we therefore analyzed the prognostic value of the lncRNAs we identified in our gene expression differential analysis in TCGA, as well as those previously reported in other studies. To this end, Chen and collaborators have recently surveyed enhancers expression in nearly 9,000 patients from TCGA 34 , using genomic coordinates from the FANTOM5 project 35 , identifying 4,803 enhancers with prognostic potential in one or more TCGA tumor types. We therefore leveraged the FC-R2 atlas to identify prognostic coding and non-coding genes using Univariate Cox proportional hazard models, comparing our results for e-lncRNAs with those reported by Chen and colleagues.
When we considered e-lncRNA expression, we identified a total of 5,382 prognostic e-lncRNAs (FDR 0.05), and no single one was associated with survival across all cancer types. Overall, the number of significant prognostic e-lncRNAs varied across tumor types, ranging from 3 in head and neck cancer to 3,850 in kidney cancers (see Supplementary Table S6). Notably, two (out of three) e-lncRNAs from our differential gene expression consensus list across all tumor types were also prognostic.
Higher expression of CATG00000107122 gene was associated with worse prognosis in kidney cancer (Supplementary figure S4b) Overall, despite differences in annotation and quantification (see Supplementary Table S5), we were able to confirm prognostic value for 2,765 e-lncRNAs out of the 4,803 reported by Chen et al 34 , including "enhancer 22" (ENSG00000272666), which was highlighted as a promising prognostic marker for kidney cancer (Supplementary Figure S3).
Finally, we analyzed the prognostic value for dp-lncRNAs, ip-lncRNAs, and mRNAs (See Supplementary Tables S7, S8, and S9, respectively), and assessed the survival prognostic potential of our consensus genes across tumor types. Thirty-seven of the 41 coding mRNAs, 22 of the 28 differentially expressed dp-lncRNAs, and two out of the four DE ip-lncRNAs, respectively, were found to be prognostic (See Supplementary Tables S10, S11, S12, and S13). Kaplan-Meier survival curves for one selected DE gene on each RNA subtype evaluated here are shown in supplementary figure S4.
Discussion
The importance of lncRNAs in cell biology and disease has clearly emerged in the past few years and different classes of lncRNAs have been shown to play crucial roles in cell regulation and homeostasis 36 . For instance, enhancers -a major category of gene regulatory elements, which has been shown to be expressed 35, 37 -play a prominent role in oncogenic processes 38,39
8/18
and other human diseases 40,41 . Despite their importance, however, there is a scarcity of large-scale datasets investigating enhancers and other lncRNA classes, in part due to the technical difficulty in applying high-throughput techniques such as ChIP-seq and Hi-C over large cohorts, and to the use of gene models that do not account for them in transcriptomics analyses.
Furthermore, the large majority of the lncRNAs that are already known -and that have been shown to be associated with some phenotype -are still lacking functional annotation.
To address these needs, the FANTOM consortium has first constructed the FANTOM-CAT meta-transcriptome, a comprehensive atlas of coding and non-coding genes with robust support from CAGE-seq data 4 In order to validate our resource, we have compared the gene expression summaries based on FANTOM-CAT gene models with previous, well-established gene expression quantifications, demonstrating virtually identical profiles across tissue types overall and for specific tissue markers. We have then confirmed that distinct classes of coding and non-coding genes differ in terms of overall expression levels and specificity patterns across cell types and tissues. Furthermore, with this approach, we were also able to identify mRNAs, promoters, enhancers, and other lncRNAs that are differentially expressed in cancer, both confirming previously reported findings, and identifying novel cancer genes exclusively annotated in the FANTOM-CAT gene model, which have been therefore missed in prior analyses with TCGA data. Finally, we also analyzed the prognostic value of the coding and non-coding genes we identified, and confirmed survival associations in TCGA for measurable enhancers.
Collectively, by confirming findings reported in previous studies, our results demonstrate that the FC-R2 gene expression atlas is a reliable and powerful resource for exploring both the coding and non-coding transcriptome, providing compelling evidence and robust support to the notion that lncRNA gene classes, including enhancers and promoters, despite not being yet fully understood, portend significant biological functions. Our resource, therefore, constitutes a suitable and promising platform for future large scales studies in cancer and other human diseases, which in turn hold the potential to reveal important cues to the understanding of their biological, physiological, and pathological roles, potentially leading to improved diagnostic and therapeutic interventions.
Finally, all results, data, and code from the FC-R2 atlas are available as a public tool. With uniformly processed expression data for over 70,000 samples and 109,873 genes ready to analyze, we want to encourage researchers to dive deeper into the study of ncRNAs, their interaction with coding and non-coding genes, and their influence on normal and disease tissues. We hope this new resource will help paving the way to develop new hypotheses that can be followed to unwind the biological role of the transcriptome as a whole.
9/18
Methods Figure 5. Processing the FANTOM-CAT genomic ranges. This figure summarizes the disjoining and exon disambiguation processes performed before extracting expression information from recount2 using the FANTOM-CAT gene models. A) Representation of a genomic segment containing 3 distinct, hypothetical genes: gene A having two isoforms, and genes B and C with one isoform each. Each box can be interpreted as one nucleotide along the genome. Colors indicate the 3 different genes. B) Representation of disjoint exon ranges from example in Panel A. Each feature is reduced to a set of non-overlapping genomic ranges. The disjoint genomic ranges mapping back to two or more distinct genes are removed (crossed grey boxes). After removal of ambiguous ranges, the expression information for remaining ones is extracted from recount2 and summarized at the gene level.
Data and pre-processing.
The FANTOM CAT gene catalog (permissive set) was obtained from the FANTOM consortium within the frame of the FANTOM6 project (Ramilowski J, et al., manuscript in review 42 ). This catalog accounts for 124,245 genes supported by CAGE peaks and it includes those described by Hon et al. 4 . In order to remove ambiguity due to overlapping among exons from distinct genes, the BED files containing the coordinates for all genes and exons were processed with the GenomicRanges R/Bioconductor package 44 to obtain exon coordinates disjoining. To avoid losing strand information from annotation we processed data using a two-step approach by first disjoining overlapping segments on the same strand and then across strands ( Figure 5). The genomic ranges (disjoint exons segments) that mapped back to more than one gene were discarded. The expression values for these ranges
Thus, the FC-R2 atlas provides expression information for coding and non-coding genes (including enhancers, divergent promoters, and intergenic lncRNAs) for 9,662 samples from the Genotype-Tissue Expression (GTEx) project, 11,350 samples from TCGA, and over 50,000 samples from the Sequence Read Archive (SRA).
Correlation with other studies.
To test if the pre-processing steps used for FC-R2 had a major impact on gene expression quantification, we compared our data to the published GTEx expression values obtained from recount2 (version 2, https://jhubiostatistics. shinyapps.io/recount/). Specifically, we first compared the expression distribution of tissue specific genes across different tissue types and then computed the Pearson correlation for each gene in common across the original recount2 gene expression estimates based on GENCODE and our version based on the FANTOM-CAT transcriptome.
Expression specificity of tissue facets.
We analyzed the expression level and specificity of each gene stratified by RNA class (i.e. mRNA, e-lncRNA, dp-lncRNA, ip-lncRNA) using the same approach described by Hon et al. 4
Identification of differentially expressed genes.
We analyzed differential gene expression in 13 cancer types, comparing primary tumor with normal samples using TCGA data from the FC-R2 atlas. Gene expression summaries for each cancer type were split by RNA class (coding mRNA, intergenic promoter lncRNA, divergent promoter lncRNA, and enhancer lncRNA) and then analyzed independently. A generalized linear model approach coupled with empirical Bayes moderation of standard errors 46 was used to identify differentially expressed genes between groups. The model was adjusted for the three most relevant coefficients for data heterogeneity as estimated by surrogate variable analysis (SVA) 47 . Correction for multiple testing was performed across RNA classes by merging the resulting p-values for each cancer type and applying the Benjamini-Hochberg method 48 .
Prognostic analysis.
To evaluate the prognostic potential of the genes in FC-R2, we performed univariate Cox proportional regression analysis separately for each RNA classes (22106 mRNAs, 17,404 e-lncRNAs, 6,204 dp-lncRNAs, and 1,948 ip-lncRNAs) across each of the 13 TCGA cancer types with available survival follow-up. Genes with FDR equal or less than 0.05, using Benjamini-
11/18
analyzed the prognostic value of the consensus genes we identified comparing tumors to normal samples by intersecting the corresponding gene lists with those obtained by Cox proportional regression. Finally, in order to compare the results from prognostic analyses, we obtained data on enhancers position and prognostic potential from Chen et al. original publication 34 and performed a liftover to the hg38 genome assembly to match FC-R2 coordinates.
Data Availability
All data is available from http://marchionnilab.org/fcr2.html. Expression data can be directly accessed through https://jhubiostatistics.shinyapps.io/recount/ and the recount Bioconductor package (v1.9.5 or newer) at https://bioconductor.org/packages/recount as RangedSummarizedExperiment objects organized by The Sequence Read Archive (SRA) study ID. The data can be loaded using R-programming language and is ready to be analyzed using Bioconductor packages or the data can be exported to other formats for use in another environment. | 2019-06-26T13:59:48.736Z | 2019-06-04T00:00:00.000 | {
"year": 2019,
"sha1": "425fb9ad734bb7b8680dc00efc28ac7da7bdc1a2",
"oa_license": "CCBYNC",
"oa_url": "https://genome.cshlp.org/content/30/7/1073.full.pdf",
"oa_status": "GREEN",
"pdf_src": "BioRxiv",
"pdf_hash": "51b6ec90eb1f4a390b48b5eac0c78fa9ac4ef878",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
222224100 | pes2o/s2orc | v3-fos-license | Basti Chikitsa for Healthy Life in Present Scenario : A Review
In a present era, the human being is prone for numerous degenerative issues, because of modern lifestyle, professional stress, food habits etc. 1,2 Lifestyle related disorders like hypertension and diabetes, reported especially in young adults are a prior concern. 3-7 Association between different diseases like cardiac dysfunction and chronic liver diseases have also been reported. 6,8 In such disorders Ayurveda management is very useful and satisfactory because of its long-lasting effects and multisystemic regenerative actions without any harm. 6 Panchakarma is a comprehensive system of knowledge and practice to purify the body from the degenerative influence of toxins and restore it to balance with natural laws.
INTRODUCTION
In a present era, the human being is prone for numerous degenerative issues, because of modern lifestyle, professional stress, food habits etc. 1,2 Lifestyle related disorders like hypertension and diabetes, reported especially in young adults are a prior concern. 3-7 Association between different diseases like cardiac dysfunction and chronic liver diseases have also been reported. 6,8 In such disorders Ayurveda management is very useful and satisfactory because of its long-lasting effects and multisystemic regenerative actions without any harm. 6 Panchakarma is a comprehensive system of knowledge and practice to purify the body from the degenerative influence of toxins and restore it to balance with natural laws.
In Ayurveda, there are two types of Karmas Shaman Karma and Shodhana Karma. In Shodhana Karma there are five types of Karma such as Vaman, Virechana, Basti, Nasya and Raktamoshana. Among the Shodhana Karma, Bastikarma is considered as prime as it can provide relief in Koshtagata, Dhatugata, Marmagata andSarvashareergatavikara . 9 Basti is one of the major treatment modality that comes under Yuktivyapashraya Chikitsa. As Vata is responsible for disjunction and conjunction of faeces, urine, bile etc. with their receptacles, there is no remedy other than Basti for the pacification of Vata when it is aggravated severely. Hence Basti is said as half medicine or even whole medicine by Acharyas. The rectal absorption can prove the good alternative route of drug administration as it provides partial avoidance of first portal pass metabolism. It has been demonstrated that the rectal route is more efficient than even the intravenous route. 6,10 view, Basti is used not only for the diseased person but it used in a healthy person. It seems to be very effective concerning immunity. Basti provides equilibrium of Dosha, Dhatu, Malas, provided person should follow Dinacharya and Ritucharya. Shodhana in the form of Basti possesses benefits like disease preventive, curative and health promotive. Thus, this is the relevance of Basti in healthy person for its immunity. Keeping this in view, this article has highlighted the relevance of Basti Karma in diseased person as well as healthy persons to cures the disease and maintains health respectively.
AIMS AND OBJECTIVE
• To discuss, evaluate and elaboration of Bastichikitsa in a healthy person. • To discuss, evaluate and elaboration of Bastichikitsa in diseased persons.
Material and Methods:
Ayurvedic classical text such as Charak Samhita, Sushrut Samhita, Ashtanghrudaya etc. and other literature were reviewed critically and scientifically to compile the concept of Basti. Modern scientific literature describing the mechanism of absorption was reviewed critically. Further, data available data on web-based sources and article published on the internet were compiled.
Literary review of Basti
Basti karma means enema which is medicated. In this process, medicated decoctions and oils are administered via anus in the body with the aid of instruments specially designed for the process of Basti. Basti karma's place of action is Pakwashaya which is Vata Dosha's main site. 11 Hence it is the major treatment modality for Vata Dosha. It is the major treatment modality for Vata Dosha. Vāta is considered as the cause of all type of movements of the body and is the main etiological factor in the pathogenesis of various diseases. 12 Apan Vayu is responsible for the elimination and retention of faeces, urine and other excreta. 13 Vata is mainly located in the large intestine.
Basti sustains the age, improves strength, digestive fire, intellect, voice, and complexion and provides a happy life. The body performs all its functions smoothly. 14 It is very beneficial for all age groups. The bioavailability of the drug i.e. the part of the drug increases when given as Basti. According to Acharya Sushrut, Basti works as plant watered at its root and then root circulate it in all branches. 15 It has also been described as Ardha Chikitsa i.e. half treatment for the management of diseases. 16 The age of administration of Basti starts from one year.
Classification of Basti
According to the consistency of the drugs, Basti is broadly divided into two types Varsharutu is the best time for Basti because in this season Vataprakopa is present in the body as well as in the environment. 21 Usually, NiruhaBasti is given in the morning because evening time is the period of Vataprakopaka and Niruha Basti causes Vataprakopa. This is the reason why snehadravya is added to NiruhaBasti. AnuvasanaBasti can be given in the evening. Basti can also be given according to age, Rutu, Kala, etc.
Contraindication-
Krusha(very emaciated), Amatisara(Acute diarrhea), Chhardi (vomiting), Kasa (cough), Shwas (asthma), Madhumeha (Diabetes), Shoonapayu (inflamed anus), Kritahara (immediately after taking food) Table 1. Basti-Any substance present in the body which is not conducive to health is considered as mala. 29 Vagbhata said that if one ignores to evacuate this Mala properly, it may lead to various diseases such as Prameha, Arsha, Grahani, Medoroga etc.In healthy and empty rectum the absorption is more and in diseased condition and the presence of stool delays absorption . 30 Hence proper excretion of Mala is very essential which is done by Basti.
Bastidravya given through rectal route reaches Pakwashaya which is considered as the main site Vata. As Vata is pacified the disease itself get cured because it is a major causative factor in the disease. Other two Doshas do not have existed without Vata.
VataSthana and so it can alleviate the Vataat.
According to Ayurveda, water given at the root of the plant gives nutrition to the whole plant similarly the Basti functions. 31
DISCUSSION
Vāta is considered to be the cause of all type of movements of the body and plays a major role in the pathogenesis of many diseases. It aggravates due to Dhātuksaya (diminution of Dhatu) or by Margavarana(occlusion of its channel by other tissues). 35 Most of the neurodegenerative disorders occurring in present scenario show Vata dominant symptoms where Vata primarily gets aggravated due to Margavarana (occlusion), which in turn leads to Dhātuksaya (diminution of Dhatu) leading to the increased provocation of Vāta. Due to this critical mechanism, neurodegenerative diseases become almost incurable, if they are treated late.
Basti administered in the Pakvashaya affects the whole body by its Virya. According to modern science, its action of active principles of drug act on receptors in the gastrointestinal tract which is similar to the enteric nervous system. ENS (Enteric Nervous System) is a considerable group of neurons, it is accomplished with Autonomous reflex without the influence of the central nervous system. More than 500 million neurons are present in the ENS (Enteric Nervous System). Hence it's also called "the second brain". 36 There are several similarities between CNS -ENS concerning cellular structure, neuropeptide secretion and specific functions and recent studies have shown that there is a great impact of CNS and ENS on each other. 37 Basti may act on the receptors of the ENS to stimulate the CNS causing secretion of obligatory hormones or other chemicals. It is recognized that the enteric nervous system has a unique capability to arbitrate reflex activity independently of input from the brain or spinal cord. 38 This suggests that the ENS comprises sensory receptors, primary afferent neurons, interneurons and neurons of the motor. The events regulated, at least in part, by the ENS are numerous, involving motor activity, secretion, absorption, blood flow and interaction with other organs, such as the gall bladder or pancreas. 39 ENS produces several hormones and around 40 neurotransmitters of the same classes as found in the brain. Moreover, neurons in the gut are supposed to generate as much dopamine as those in the head. Serotonin produced in the gut comes into the blood, it is involved in mending damaged cells in the liver and lungs there. 40 Basti Basti is effective on Asthivaha and Majjavaha Srotas also. Purishadhara Kala, the colon membrane, is considered as Asthidhara Kala, the membrane of the bone tissue. 41 Asthi is important sites of Vata Dosha. Hence, medications are given rectally affect all the tissues up to bone tissue. A significant increase in serum calcium was seen after the course of Basti.
Though serum calcium decreased after 90 days, it was still higher than the baseline level. Basti Chikitsa exerts a modifying influence on immune responses by regulating proinflammatory cytokines, immunoglobulins and functional properties of T-cells. 42 In Rickets, there is decreased absorption of calcium and phosphorus from the intestine, leading to bending and softening of the long bones. The large intestine contains a large number of Ca-Na ion channels, which take up calcium from the extracellular fluid. 43 Basti in pregnancy-Nowadays the ratio of normal labour is decreasing rapidly because of lifestyle changes. Administration of Matrabasti during the ending of 8 th month and in 9 th month of pregnancy helps in the induction of normal labour.
In children-Basti is like nectar in child patient. It is very effective in the development of the baby as it develops paraneuron which stimulates the nervous system. Significant improvement occurs in the case of cerebral palsy. When the channels of the body are cleaned by Niruha Basti it provides complexion and strength. Anuvasana Basti destroys roughness, lightness and coldness of Vata. Basti provides clarity of mind, energy, and strength to the body. 44 All the Dhatus gets nourished by Basti enhancing the body's Dhatwagni to maintain Dhatusamya (homeostasis) and increase the immunity of the body towards the invasion of the disease.
CONCLUSION
Basti is one of the most important and useful treatments for a diseased and healthy person. There are so many types of Basti according to diseases, Rutu, Bala, Kala. So we can consider these factors while adopting the Basti in any person. Basti is not only curative but also disease preventive and health promotive. Basti helps to maintain the equilibrium of Vata dosha in our body. It increases Shukra(potency), Oja (vital energy) and Agni (digestive fire). In this modern era, as per our daily routine, health management is very difficult. So, by the use of Basti Karma, we can manage our hectic lifestyle and make it a healthy life. | 2020-10-09T00:12:51.593Z | 2020-01-01T00:00:00.000 | {
"year": 2020,
"sha1": "5fe18e0503a92fe2e6e9df9ec8870a97e895a175",
"oa_license": null,
"oa_url": "https://doi.org/10.31782/ijcrr.2020.121820",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "5fe18e0503a92fe2e6e9df9ec8870a97e895a175",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
256037310 | pes2o/s2orc | v3-fos-license | The simplest massive S-matrix: from minimal coupling to black holes
In this paper, we explore the physics of electromagnetically and gravitationally coupled massive higher spin states from the on-shell point of view. Starting with the three-point amplitude, we focus on the simplest amplitude characterized by matching to minimal coupling in the UV. In the IR, for charged states this leads to g = 2 for arbitrary spin, and the leading deformation corresponds to the anomalous magnetic dipole moment. We proceed to construct the (gravitational) Compton amplitude for generic spins via consistent factorization. We find that in gravitation couplings, the leading deformation leads to inconsistent factorization. This implies that for systems with Gauge2 = Gravity relations, such as perturbative string theory, all charged states must have g = 2. It is then natural to ask for generic spin, what is the theory that yields such minimal coupling. By matching to the one body effective action, we verify that for large spins the answer is Kerr black holes. This identification is then an on-shell avatar of the no- hair theorem. Finally using this identification as well as the newly constructed Compton amplitudes, we proceed to compute the spin-dependent pieces for the classical potential at 2PM order up to degree four in spin operator of either black holes.
JHEP04(2019)156
Recently a new formalism for four-dimensional massive scattering amplitude was introduced by one of the authors [1] that manifests the covariance of the SU(2) massive Little group. Through such formalism, many fundamental properties of interacting systems become manifest, including Weinberg-Witten theorem, limits on the spin of fundamental point like particles, as well as Higgs mechanisms as the natural infrared unification. Furthermore, the new formalism also allows one to streamline computations such as anomalous magnetic dipole moment as well as classical electric and gravitational potentials [2,3]. Given its utility in making physical properties manifest, it is natural to pose the following question to such formalism: what is the simplest massive scattering amplitude? A similar question was posed for the massless case long ago [4], for which remarkable JHEP04(2019)156 properties of N = 8 supergravity amplitudes were unmasked. Here we expect the lessons to be equally, if not more, interesting. For one, the space of massive theories is much richer than that of massless ones. It includes not only fundamental particles, but monopoles, BPS states, and infinite tower of string resonances. Indeed, recently such on-shell approach was utilized for extremal (half-BPS) black holes in N = 8 supergravity [5], which demonstrated the absence of perihelion precession.
We answer this question by starting with the three point amplitude describing a spins state coupled to either a photon or a graviton. As discussed in [1], this is given by an {2s}⊗{2s} symmetric SL(2,C) tensor, with 2s+1 distinct structures. Assigning the massive legs to be 1 and 2 with equal mass, the general three point amplitude is parametrized by λ 3 (along with αβ shares the responsibility of carrying the SL(2,C) indices) and x, defined as: and m is the mass of the massive legs. The simplest amplitude then corresponds to that comprises of x and αβ solely. This amplitude is identified as minimal coupling in the sense that in the high energy limit, the amplitude matches the minimal massless amplitude that has the least number of derivatives. For the case of charged particles this also matches with that of classical magnetic dipole moment 2 for any spin, and deformation of the form λ 2 3 represents g−2.
Interestingly when extended to gravitational coupling λ 2 3 deformations are forbidden on the grounds of general covariance. Note that for systems in which the gravitation coupling is given by the square of vector couplings, such as perturbative string theories, this immediately leads to the conclusion that the charged particles must have g = 2.
Given that the minimal coupling has special properties both in the UV and IR, it is natural to ask for generic spin-s, which theory leads to such minimal coupling. Naïve expectation would be the leading trajectory states of open and closed string theories, since from the world-sheet CFT point of view, their vertex operators are the simplest. It turns out, the answer is quite the contrary, as we demonstrate that the leading trajectory massive spin states are the maximal non-minimal coupling, reflected in the fact that all 2s+1 tensor structure are present. Allowing ourselves to take the classical values of spin, i.e. s 1, we show by matching to the one-body effective action of a point particle coupled to gravitational background, minimal coupling matches on to that of a Kerr black hole. Thus the matching between minimal coupling and Kerr black hole, is the on-shell way of stating a consequence of the no-hair theorem.
Given the importance of minimal coupling, we explore the four-point (gravitational) Compton amplitude for general spin, by constructing an ansatz whose residues match that of products of minimal coupling. This leads to potential polynomial ambiguities. For s ≤ 2, such ambiguities are identified as finite size effects, as they are accompanied with additional 1 m factors. For s > 2 the polynomial terms in general can be of the same order in 1 m as the pole terms, reflecting the inherent non-fundamental nature of such higher spin particles. We also consider four-point amplitudes with deformations from minimal coupling, demonstrating that consistent factorisation bans λ 2 3 terms in the three-point coupling with where the massive legs (1 and 4) are of spin-s 1 and s 4 respectively and the massless legs 2 and 3 have helicity h 2 , h 3 . The curly bracket indicates that one is symmetrizing over the 2s SU (2) indices I, J, taking value in 1, 2.
Since amplitudes are covariant quantities, it should be a function of objects that are not singlets under the Little group, i.e. objects that carry little group indices. In the usual textbook approach, one introduces external line factors or polarization tensors which serve the purpose of converting Lorentz representations into Little group representations. Since except for scalars the size of the two representations are distinct, doing so introduces large amount of redundancy, which is the underlying reason for the complexity in the usual Feynman diagram approach. In contrast, the spinor helicity formalism introduces bosonic spinor variables that transform under the fundamental representation of the Little group, while directly comprising the kinematic data, the momenta. This allows us to remove the redundancy and dramatically reduce the complexity of the final answer. Furthermore, as we will see, such on-shell approach will render many physical properties, such as high the energy behaviour, transparent and straightforward.
The massless/massive spinor helicity formalism
We begin by introducing SL(2, C) representations. A Lorentz vector, such as the momenta, is written as a bi-fundamental tensor under SL(2, C): where α,α = 1, 2. The usual Lorentz invariant inner products are then mapped to the contraction of these tensors with the 2 × 2 Levi-Cevita tensor: From the above one sees that p 2 = detp αα . Thus for massless momenta, the 2 × 2 tensor p αα is of rank one and one has: 1 p αα = λ αλα . (2.4) The relation between the bosonic spinor variables and the momenta is invariant under the following transformation: λ → e −i θ 2 λ,λ → e i θ 2λ (2.5) Note that this is precisely the definition of the Little group! Thus we identify the spinors λ,λ as having (− 1 2 , + 1 2 ) Little group weight respectively. Using these bosonic spinors it is JHEP04(2019)156 then convenient to define the following Lorentz invariant, Little group covariant building blocks: ij ≡ λ α i λ β j αβ , [ij] ≡λ iαλ jβ αβ .
(2. 6) In terms of these blocks, the usual Mandelstam variables are given as 2p i · p j = ij [ji]. For massive momenta, p αα has full rank and we have p αα = λ I αλ Iα , (2.7) leaving behind a function that is symmetric in SL(2, C) indices instead. We will refer to this representation as the chiral basis, reflecting the fact that we are using the un-dotted SL(2, C) indices. One can equally use the anti-chiral basis, and the two can be converted to each other by contracting with p αα m . This separation will be useful when considering suitable basis for all possible three-point interactions as we will now see.
General structure of the three-point amplitude
We now consider the most general form of the three-point amplitude for one massless and two equal mass legs with spin s. Without loss of generality, the momenta p 1 and p 2 can be taken to be massive, and the amplitude takes the form: where h is the helicity of the massless leg. Now we have a {2s} ⊗ {2s} SL(2, C) tensor, and we are interested in the general structure of all possible couplings. This entails the need of a basis to span the two-dimensional space. It is preferable to use the kinematic variables of the problem to serve as a basis, thus it is natural to introduce λ 3α , αβ (2.12) as the expansion basis.
JHEP04(2019)156
Since λ 3 carries helicity weight − 1 2 of the massless leg 3, in order to represent general amplitudes, one should also have a variable that carries positive weights. This variable is introduced by noting that for equal mass kinematics, 2 2p 3 · p 1 = 3|p 1 |3] = 0 , (2.13) and hence the spinor λ α 3 must be proportional toλ 3α pα α 1 . Through this proportionality, we introduce a new variable x defined as: where p 2 1 = m 2 . Note that the above equality tells us that x is dimensionless and carries +1 helicity of leg 3. Using an auxiliary spinor ξ, we can represent x as The above shows that x can be nicely written in terms of polarization vectors: with the polarization vector ε (+) αα = √ 2λ 3α ξα 3ξ , and the auxiliary spinor is identified with the reference spinor of the polarization vector.
Equipped with the new variable, we can write down the general structure of a three point amplitude for two spin s and a helicity h state: where the 2s ⊗ 2s separately symmetrized SL(2, C) indices are distributed across the Levi-Cevita tensors and λ 3 s. Thus we see that there are in total 2s+1 structures for spin s states, and we've normalized the couplings such that the g i s are dimensionless. Note that the above classification is purely kinematic in nature, and does not correspond to the classification of local operators in the usual derivative expansion. Indeed in the usual Lagrangian language, there may be a large number of operators at a given derivative order simply due to the different ways the derivative can contract. Furthermore, operators at the same derivative order may behave very differently in the high-energy limit. For example, consider the following Lagrangian for a charged spin-s field: where φ (s) is the short hand notation for a rank s field, the Lorentz indices of φ is contracted withφ, and · · · represents additional terms needed to ensure that through equations of JHEP04(2019)156 motion, φ (s) andφ (s) are symmetric, traceless and transverse. 3 Consider the three-point amplitude from the leading term, given by: To convert to our chiral basis, we strip-off the polarization tensors and convert the dotted indices into un-dotted indices, by contracting with p m : Using the identity m , we find that in the chiral basis the leading coupling in eq. (2.18) written as: Here we have all g i = 0 for all i ≤ n. In other words, a single local operator in the Lagrangian is expressed as a sum of many terms in such on-shell basis. The reason that there is such dramatic difference is because the on-shell basis is completely determined from kinematics, and thus each term in the expansion is distinct in a purely kinematic way. On the other hand, operators in a Lagrangian can often be related through integration by parts or field redefinitions, and each operator can contain several kinematically distinct pieces.
In fact, as we will see in the next section, by expressing the three-point amplitude on such on-shell basis, we will be able to cleanly separate terms that behave poorly in the UV, allowing us to define in a physically meaningful way what minimal coupling is. It will be convenient to make connection with the amplitudes computed from the usual Feynman diagram approach. For this, we simply put back the λ I i factors that was pulled out that defined the chiral basis in eq. (2.10). For example, As an illustration of how these interaction arrises from terms of a local Lagrangian, consider the coupling of Maxwell field to Dirac spinors; L int = eA µΨ γ µ Ψ. The 3pt amplitude for this interaction term with the convention Ψ(p 1 ) incoming, A µ (k 3 ) incoming positive helicity, andΨ(p 2 ) outgoing is where we have used the eq. (2.25) for the last equality. This gives the second term of the expansion eq. (2.22), with s = 1 2 .
Classical spin-operators from amplitudes
It will be useful to view the three-point amplitude as an operator acting on the Hilbert space of SL(2, C) irreps. More precisely, since M +h,s,s is basically a {2s} ⊗ {2s} Lorentz tensor contracted between 2s λ 1 and 2s λ 2 s, it can be viewed as an operator that maps the spin-s representation in the Hilbert space of particle 1 to that of particle 2. In other words, schematically we have: We know that the operator O {α 1 ,··· ,α 2s },{β 1 ,··· ,β 2s } is a linear combination of polynomials in αβ and λ 3α λ 3β . The former naturally can be identified as the identity operator, while the latter is the spin-operator which we now show.
Let's start with the spin vector S µ defined as the Pauli-Lubanski pseudo-vector S µ = − 1 2m µνρσ p 1ν J ρσ . For the Lorentz generator J µν , we will be interested in its action on SL(2, C) irreps. For spin-s, we write: α i+1 · · · δ β 2s α 2s , and the last equality reflects the fact that the irreps are symmetric tensors of 2s indices. Using this, we find that (2.32) Finally, dotting into the massless momenta p 3 we arrive at: (2.33) From this result we see that the operator O {α 1 ,··· ,αs},{β 1 ,··· ,βs} is comprised of identity operators and the spin vector operator, projected along the direction of the massless momenta. For example, for s = 1, we have The simplest three-point amplitude In the previous section, we've seen that for a massive spin-s particle, whether it is fundamental or composite, the emission of a photon or graviton can in general be parameterized by eq. (2.22). This parameterization is unique in the sense that the expansion basis is defined on kinematic grounds unambiguously. The expansion is organized in terms of powers of 1 m , with higher order terms hinting at potential problems in the UV, i.e. the massive amplitude does not have a smooth m → 0 limit. In other words, this parameterization manifests the high energy behaviour for a given interaction. To illustrate this feature in more detail, take for example the Lagrangian in eq. (2.18) with spin-1, which is known to lead to four-point amplitudes that violate tree-unitarity at high energies and is not removable via the presence of an extra Higgs. Indeed this can be seen already at the three-point level, where in our parameterization is given as: We see that while in the Lagrangian the interaction is given by a single local operator, in our on-shell parameterization, it is comprised of two pieces, with the latter behaving worst in the high-energy limit compared to the first. Indeed, if we consider the three-point amplitude of a photon with W-bosons, we will only find the leading piece at tree level. Consider an amplitude with only the leading term in eq. (2.22). The above discussion would indicate that not only is the amplitude simple in the number of terms involved, but is also simple in the sense of having the best UV behavior. At high energies we only have massless states, and we can ask what amplitude in the UV does this pure x-piece matches JHEP04(2019)156 to. Note that simply "unbolding" the spinors might lead to ill defined limit, as while the denominator of 1 m tends to zero in the limit, the angle brackets in the numerator can tend to zero as well even in complex kinematics. Thus we would like to have a controlled way of approaching the high energy limit for eq. (2.22). To this end, let us decompose the massive spinors onto the helicity basis of the massless limit: where IJ ξ +I ξ −J = 1 and λη = [λη] = m. The SU(2) spinors ξ ±I are the eigenstates of spin-1 2 for J z in a given frame. In the m → 0 limit we see that the finite contribution correspond to taking the ξ ±I of the two massive legs to have opposite helicity. In other words we the two massive spin-s states will translate into a +s and −s helicity state separately at high energies. To avoid a singular piece we must have λ 1 ∼ λ 2 ∼ λ 3 . Choosing leg 1 to be the positive helicity, we then have Since the λ i s are proportional to each other, we introduce proportionality factors y 1 , y 2 defined via λ 1 = y 1 λ 3 and λ 2 = y 2 λ 3 . Momentum conservation then fixes: We see that in the high-energy limit, the pure x-piece will become that of the minimal coupling: the minimal mass dependence for a three point amplitude with h 3 > 0 and |h 1 | = |h 2 | = s states. 4 One can straightforwardly check that subleading terms in eq. (2.22) match to higher derivative couplings in the UV. Thus minimal coupling in the UV uniquely picks out from all possible low energy couplings. For this reason it is natural to refer to the choice of setting all coupling constants except g 0 (ḡ 0 ) to zero as minimal coupling. Once again, we stress that our minimal coupling is defined through kinematics solely. As we will see in the next section, this will be minimal in a very precise sense in the IR as well! In the following, we will study this simplest amplitude in more detail for photons and gravitons separately.
JHEP04(2019)156
3.1 Photon minimal coupling and g = 2 Let us first consider the case where the minimal coupling involves the massive states coupled to a photon, |h| = 1. The coupling we are interested in will then be: where we've included the charge e and made an overall sign choice for a better interpretation as operators. Since we are considering coupling to photon that is sensitive to its spin, a natural quantity of interest would be its magnetic dipole moment. Recall that in the nonrelativistic limit, the magnetic dipole moment is defined through the Zeeman coupling: In the rest frame of the charged particle with momentum p 1 , the magnetic field B can be written in the following Lorentz covariant form: The expression for the Zeeman coupling then has the following Lorentz invariant form: 5 into the Zeeman coupling equation eq. (3.10) for s = 1 2 in the dotted frame. For plus helicity photon this results in: For general s we simply have: To compare with our three-point amplitude we contract the SL(2, C) indices with massive spinor helicity variables, yielding (3.13) 5 The normalisation factor of √ 2 may seem unconventional, but introduction of this factor simplifies the analysis: the scalar potential coupling VS = eφ can be covariantly written as P ·A m , and setting Aµ = √ 2ε ± µ results in V + S = ex and V − S = ex.
JHEP04(2019)156
Now let us compare with our minimal coupling amplitude. We will use the anti-chiral representation in eq. (2.23) with h = 1. Since all g n vanishes except for g 0 which we set to electric coupling,ḡ 0 = g 0 = e, andḡ 1 = 2s. The three-point amplitude then takes the form M +1,s,s 3 = emx [21] 2s m 2s + 2s [21] 2s−1 [23][31] m 2s + · · · . (3.14) Compared with the Zeeman coupling in eq. (3.13), we immediately see that our minimal coupling leads to g = 2 for arbitrary spin. Thus the simplest amplitude with photon coupling is also characterized by the classical magnetic dipole moment being 2! Note that terms denoted as · · · corresponds to higher multipole moments as they are of higher order inλ 3 , indicating higher derivative terms on the field strength. Since minimal coupling is also related to good high energy behaviour, this indicates that for an isolated charged spin-s particle with good UV behaviour, the classical magnetic moment must be 2. By isolated we are referring to the case where there are no other states with similar mass. Indeed the classical value for g is 2 for massive vectors arising from Higgs mechanism, and it is known that when constrained to operators with two derivative couplings, tree-level unitarity requires g = 2 for isolated massive spinning particles [23].
Finally, from eq. (2.26) we see that the only terms affectingḡ 1 is g 1 (with g 0 set to e), which correspond to a non-zero λ 2 3 coupling in the chiral basis. Since we already have g = 2 when g 1 = 0, the presence of g 1 indicates (g − 2) contributions. Indeed as one finds that the coupling parameterized by g 1 is generated at one loop [1].
Gravitational minimal coupling
We now turn to gravity. The minimal three-point coupling for a positive helicity graviton is , (3.15) Following our photon discussion, we can ask whether there is a gravitational analogue of Zeeman coupling for gravitomagnetic interactions, and the minimum coupling correspond to a particular value for the gravitomagnetic dipole moment. Indeed one can consider a Kaluza-Klein decomposition of the metric: where Φ will be identified with the gravitational potential and A the vector potential for gravitational version of magnetic field. The full gravitational potential then takes the form: Note that in contrast to the photon case, here α is fixed by the requirement that the resulting Hamiltonian reproduces the correct evolution of the spin operator S, which is dictated from general covariance. This fixes α = − 1 2 , where we leave the details to appendix B. Thus we seem to have a potential contradiction: since the gravitomagnetic dipole moment is completely fixed from general covariance, minimal coupling is inconsistent if it doesn't
JHEP04(2019)156
reproduce the correct value. However, from an on-shell point of view, there is no apparent sickness either in its high energy behaviour or consistent embedding in a four-point amplitude, as we will see in section 5. Not surprisingly, we will find that minimal coupling exactly reproduces the correct result!
The discussion above indicates that the gravitomagnetic dipole moment should be universal on grounds of general covariance. 6 Since the dipole moment is associated with minimal coupling as well as the coefficient of λ 2 3 , this implies that one can simply consider an arbitrary diffeomorphism invariant action, and read off the latter coefficient. The result would be universal! Again introducing a scalar like kinetic term for general spin-s field for integer s, we start with the on-shell action: where the sign factor (−1) s is there to make sure that the kinetic term for physical degrees of freedom have the right sign. Note that while additional terms are generally needed to impose tracelessness and transversality condition [25], such terms cannot generate non-zero g 1 and have been neglected. This can be seen by noting that such terms can be recast into linear combinations of D µ φ µν 2 ···νs D ρ φ ρν 2 ···νs and φRφ via integration by parts identities, where the latter is a schematic representation with index contractions suppressed. The former term vanishes due to transverse tracelessness of the polarisation tensors, while terms involving the Reimann tensor yields g i terms with i > 1 [26]. Expanding around the flat metric, terms linear in graviton can be separated into two terms: where we've separated the piece that stems from expanding Γ λ µν as G µνλ . The stress tensor is then given as T µν =T µν −∂ λ G µνλ . These two sources will contribute to the 3pt amplitude as following terms. (3.20) Using eq. (2.24), we convert the expression into pure chiral form: (3.21)
JHEP04(2019)156
Note that there are no λ 2 3 couplings, so g 1 = 0! Thus it appears that general covariance simply tells us that λ 2 3 couplings are forbidden. In section 5, we will present an alternative on-shell view point of why nonzero g 1 is prohibited, this time under the constraint of consistent factorisation.
Finally we comment that the action in eq. (3.18) yields deviations from minimal coupling that begins at λ 4 3 , with coefficient − s(s−1) 2 . Indeed as was pointed out in [27], such action leads to violation of tree-level unitarity for longitudinal scattering. For s < 3 this can be completely resolved by introducing a new coupling to the Reimann tensor with h set to 1. We see from the above, this is precisely the requisite choice to cancel the λ 4 3 term, consistent with the conclusion that terms beyond minimal coupling lead to bad UV behaviours. Note that string theory in general has h = 1, as discussed in [28], where it evades the UV unitarity disaster by introducing an infinite tower of states whose mass scale is the same as the state in question.
Universality of g for perturbative string states
The requirement that g 1 = 0 for gravitational couplings has important implications for systems in which the three-point coupling to a graviton is given by the square of the coupling to a photon. More precisely, the spin-2s spin-2s coupling to a graviton is given by the square of three-point amplitude of spin-s, spin-s and photon: where we've used bolded numbers to indicate the massive legs, and their exponent indicating their spin. An immediate example is perturbative string theories, where type II closed string amplitudes are given by the square of type I, and similarly closed bosonic string is given by the square of open string. In such case, if g = 2 for the charged states, which implies g 1 = 0, then the cross terms in the double copy procedure will lead to g 1 = 0 in the gravitation sector. Thus we conclude that for systems with double copy relation between gauge and gravity three point amplitudes, the charged states in the gauge theory sector must have g = 2. This is applicable to not only leading trajectory, but also all daughter trajectory states. Indeed such result was found previously in [23].
4 Black holes as the s 1 limit of minimal coupling In light of the discussion in the previous section, we see that if we consider the "simplest" three-point amplitude with g i = 0 for i > 0, we have the bonus simplicity in the UV: it matches minimal coupling in the UV and has the best high energy behavior. For spin-1 2 , 1, this is precisely the couplings for particles in the standard model. It is then natural to ask the following: are there particles in nature with s > 1 that have such minimal couplings?
JHEP04(2019)156
Given the good UV behaviour of string theory, one might expect that the higher spin string resonances would be a perfect candidate. Interestingly it is quite the contrary. For example, the three-point coupling between a photon and the leading trajectory states in open bosonic string theory is given by: (4.1) One sees that the coupling is "maximally complex" in that all g i = 0 except for g 1 . Note that this does not violate our discussion with regards to the violation of UV unitarity, since at the energy level where the 1 m factor becomes singular, we are at the string scale and the infinite string resonances now come into play.
Instead of looking to the UV, we consider the IR. For a most general approach, we consider the one body effective action of a point particle coupled to gravity, introduced by Goldberger and Rothstein [29] and generalised to cases involving spin by Porto [30]. 7 This is an effective action where the internal degree of freedom in the object is integrated away, and shows up as "higher dimensional operators" multiple moments. This is given by the following world-line action: where u µ ≡ dy µ dσ , S µν correspond to the spin-operator and Ω µν is the angular velocity. The first two terms correspond to minimal coupling and are universal, irrespective of the details of the point-like particle, while the terms in L SI correspond to spin-interaction terms that are beyond minimal coupling, and depend on the inner structure of the particle. The angular velocity Ω µν is defined as Ω µν := e µ A De Aν Dσ , where e µ A (σ) is the tetrad attached to the worldline of the particle. The defining relation for this tetrad is η AB e µ A (σ)e ν B (σ) = g µν . Generalising the quadrupole moment operator introduced in [31], the non-minimal spininteraction terms can be parameterized as [32]: where E and B are the electric and magnetic components of the Weyl tensor defined as: Note that here the Riemann tensors are taken to be linear perturbations around flat space, and the information with regards to non-trivial backgrounds is encoded in the Wilson 7 The authors would like to thank Rafael Porto for an explanation on the historical development of the subject.
JHEP04(2019)156
coefficients C # . For generic astrophysical objects the Wilson-coefficients are obtained by matching with the multipole moments used in numerical simulations. For Kerr black-holes the coefficient is 1, which we review in appendix D.
Universal part of the one body EFT
We first consider the terms besides L SI in eq. (4.2) which are universal for all particles. The spin-independent part of minimal coupling is given by Keeping only linear order in h µν = 2ε µ ε ν , 8 and identifying x = √ 2(ε + · u), this term simply yields the scalar three-point interaction: Next we consider the minimal spin coupling − 1 2 S µν Ω µν , given as [30,33], As usual the spin connection ω A µ B is defined as ω A µ B = e Bν ∂ µ e Aν + e Bν Γ ν µλ e Aλ . Since we are only interested in the three point amplitude with one graviton, the derivative on the tetrad will not contribute, and we have: In classical mechanics, the spin S µν can be defined as the difference between the angular momentum that the orbital part: However, this separation is ambiguous without a reference frame to define the origin and hence X. The choice of the origin can be translated into an additional constraint on S µν known as spin supplementary condition (SSC). Of the various choices for SSC known in the literature, one that can be generalised to curved space without any ambiguity is the covariant SSC S µν p ν = 0, also known as Tulczyjew SSC or Tulczyjew-Dixon SSC. Adoption of this condition can be met by the following choice of S µν , where the vector u µ is defined as u µ = p µ m .
Note that this spin operator can be cast in the form Pauli-Lubanski psuedo-vector eq. (A.54). The 3pt amplitude can be computed by adopting 8 The extra factor of 2 was inserted to make equations simpler.
JHEP04(2019)156
the following definitions and replacement rules: (4.11) Combined with three particle kinematics, eq. (4.8) becomes where we've used the spin-1 2 representation of the Lorentz generator in the chiral representation, and eq. (2.33) to obtain the rightmost expression. Generalisation to higher spin follows from eq. (A.63), which gives − p 3 ·S m with the understanding that, (4.14) A caveat with using this form is that higher degree of this operator must be evaluated from the definition of Lie algebra eq. (A.62). For example, when symmetrization is taken into account.
Thus the universal piece of the 1 body EFT translates into the following three-point interaction: where both the operator I and p 3 ·S are defined to act on the Hilbert space of SL(2, C) irreps.
The three-point amplitude from L SI
We now consider the three-point amplitude arising from the Wilson operators in eq. (4.3). The electric and magnetic components of the Weyl tensor are converted to: When the graviton is chosen to have negative helicity, the sign of p3 · S term flips.
JHEP04(2019)156
The one-body EFT Lagrangian eq. (4.3) then translates to the following form for threeparticle kinematics: 10 The Wilson coefficients C S n are defined as C S 2m = C ES 2m for even n = 2m and C S 2m+1 = C BS 2m+1 for odd n = 2m + 1. It is possible to add the universal pieces in eq. (4.16), so that the sum starts from n = 0, with the definition C S 0 = C S 1 = 1.
We will be interested in the three-point scattering amplitude of a spin-s particle emitting a graviton described by the effective action eq. (4.2). Again the incoming and out going momenta will be p 1 , p 2 , while the graviton being p 3 . The polarization tensor for a spin-s particle is given by: where the total symmetrization of the Little group indices ensures the transversality of the polarization tensor. As the polarization tensors are contracted with the operators in the effective action, terms with spin-operator of degree n, with n ≤ s, will contribute. Furthermore, for each fixed n, we sum over the all possible distributions of the n spin operators between the chiral and anti-chiral indices of the polarization tensor. This results in the following three-point amplitude: 11 where the + subscript indicates that this is the plus helicity graviton amplitude, and we denote the combinatoric factors asc s a,b for reasons that will be clear shortly. In a sense this provides an alternative parameterization for the general three-point amplitude, where the information of the specific interaction is encoded in the Wilson coefficients C S a+b . Again for Kerr black holes they are unity.
The matching to minimal coupling
We are now ready to recast our minimal coupling to the above EFT basis. While minimal coupling for the positive helicity graviton is simple in the chiral basis, the EFT basis in 10 The sign of k3 · S term in the last line flips when negative helicity is chosen for the graviton, which is consistent with the sign flip in eq. (4.16). 11 The irrelevant overall factor of 2 s has been neglected.
JHEP04(2019)156
eq. (4.20) is in the symmetric basis. To convert the chiral basis into the symmetric basis, we use the following identity, This relation can be readily generalised to integer higher spin.
Note that the ratio of c s a,b with respect toc s a,b yields the Wilson coefficients for the minimal coupling. The coefficient c s a,b can be readily computed by identifying it as simply the coefficients of (1 + x + y + 2xy) s , Note that if 2 c in c s a,b was substituted by 1, which is equivalent to using (1 + x + y + xy) s to evaluate c s a,b , then we would simply havec s a,b = c s a,b ! This observation can be used to derive the following formula.
Sincec s a,b tends to s a+b a!b! for asymptotically large s, each term in the series is subleading in powers of 1 s for fixed set of a and b. In other words, There are no 1/s corrections when either a or b of c s a,b is zero; c s a,0 =c s a,0 and c s 0,b =c s 0,b . It is worthy of note that since C S 1 is fixed to be unity, c s 1,0 =c s 1,0 and c s 0,1 =c s 0,1 ; these conditions imply that introduction of M 2+ 3 ⊃ x 3 21 2s−1 23 31 term in the graviton 3pt amplitude, or introduction of non-zero g 1 , is forbidden in this context as well.
Thus we see that in the s 1 limit, the minimal coupling reproduces the Wilson coefficient of a Kerr black hole! The fact that one should take the large spin limit is not surprising since the spin of a black hole takes a macroscopic value. The reader might wonder that since the matching is occurring at the large spin limit, it may very well be that deviation from minimal coupling is subleading in s and hence suppressed. In such case, the matching of minimal coupling to black holes is simply a reflection of it being the leading contribution in the limit. We now show this is not the case.
JHEP04(2019)156
The simplest deformation from minimal coupling is introducing λ 4 coupling to the 3pt amplitude. The three-point amplitude then becomes The Wilson coefficients C S n for asymptotic s is then given as The natural value for g 2 can be deduced from eq. (3.21) to be ∼ s 2 in the large s. Thus we see that introducing terms that generate deviations to minimal coupling does indeed modify the Wilson coefficients from the black hole value. Note that this is consistent with the intuition that the terms beyond minimal couplings represent finite size effects that indicate deviation from point particle. In other words, the fact that black holes are given by minimal coupling is a kinematic way of saying that it has no "hair".
Compton amplitudes for arbitrary spin
Consistent factorization at four-points often imposes new constraints for the underlying theory that are not visible at three-points. For example, the color algebra associated with non-abelian theories can be recovered by simply enforcing that the residue from one factorization channel can be made consistent with that of another [1,34]. For massive amplitudes, the application of such consistency condition has been initiated in [1], which led to bounds on the spin of isolated massive particles. Here we will systematically construct the Compton amplitude, as well as its gravitational counterpart, for general massive spin-s particle, utilizing consistent factorizations. The gravitational Compton amplitude will later serve an important ingredient in extracting the spin-dependent piece of the 2 PM potential.
Let us first give an over view of our strategy. We will start from gluing the known 3pt amplitudes on s-channel together. Putting the result on an s-channel propagator gives a putative ansatz for the four-point amplitude: Without loss of generality, we take legs 1 and 4 to be the massive spin-s state, and legs 2 and 3 to be either photons or gravitons. For minimal couplings, the gluing on a specific channel is not local, reflecting the presence of another factorization channel. Thus we will need to check whether the factorization constraint on the other channel is also satisfied. If s channel gluing in our Compton amplitude carries u-channel information, 12 correct factorization in
JHEP04(2019)156
the u-channel is guaranteed if the s-channel residue is given in a form symmetric under (1 ↔ 4) exchange. In general for photon Compton amplitude, we will find: where f u can be deduced from f s via 1 ↔ 4 symmetry. For graviton Compton amplitudes, we will find: This procedure fixes the four-point amplitude up to polynomial terms, which do not have poles and therefore are not subject to previous constraints. Importantly, for (photon) graviton couplings s ≤ (1)2 the possible polynomials must be of higher order in 1 m suppressions, which reflects the fact that these are finite size effects. For s > (1)2, the order of 1 m for such ambiguity is of the same order as terms in the Ansatz. Thus the result given here are "correct" only up to polynomial ambiguities for charged spinning particles with s > 1, and gravitationally coupled spin states with s > 2.
Photon
The minimal coupling 3pt amplitude of a photon with 2 massive spin s particles is given by:
Photon Compton amplitude with s ≤ 1
s-channel gluing gives: with P as the momentum of the s-channel propagator and the second equality in eq. (5.5) comes from solving the conditions: and by the definition of the x-factors:
JHEP04(2019)156
Since there's no 3-photon interaction to be considered in the t channel, we identify the t in the denominator as −(u − m 2 ). Putting back the (s − m 2 ), we obtain an ansatz for photon Compton amplitudes: for s ≤ 1 this is precisely the Compton amplitudes. On the other hand, for s > 1, there will be spurious poles 3|p 1 |2] in the denominator and the ansatz ceases to be local. Then we conclude for s ≤ 1,
Photon Compton amplitude with s > 1
For higher spin charged particles, we need to more work to find a completely local ansatz.
The assumption that went into eq. (5.9) was that we used a representation of P such that it matches both the s and u-channel residue. This anticipates the fact that the s-channel residue sits on top of a u-channel pole as well. However, it is also possible that part of the s-channel residue is in fact local, and thus do not need to satisfy any u-channel constraint.
Thus the task becomes that of separating the residue into local and non-local pieces. Again starting with the s-channel residue: We rewrite the term in the parenthesis, which is simply 1|P |4], as where F is written in a way that is symmetric under angle square exchange for the massive legs. Now, the F term is completely local and satisfies the correct spin-statistics property under (1 ↔ 4), with the price of introducing extra factors of m in the denominator. In expanding (F + B) 2s , one of the B factor in the B dependent terms will cancel the u pole, since −t = u − m 2 when s = m 2 , and its spurious pole will be canceled by the prefactor. Thus these terms will be pure s-channel terms. The remaining B factors still contain unphysical pole, but can be removed by imposing s-channel kinematics: We now see that only the F 2s term carries both s and the u channel poles. The pure u-channel term will be fixed by (1 ↔ 4) symmetry.
JHEP04(2019)156
Now we conclude that the photon Compton amplitude for s > 1 to be: where we dropped the (s − m 2 ) terms in eq. (5.13) since it would not contribute to the residue at any poles.
Graviton
The minimal coupling 3pt amplitude of a graviton with 2 massive spin s particles is given by:
Graviton Compton amplitude for s ≤ 2
We again start out with s-channel gluing of the graviton Compton amplitude, where eq. (5.6) and eq. (5.7) is applied in the second equality in eq. (5.16). The double pole t 2 in the denominator can be identified as one massive u-channel pole and one massless t channel pole comming from the 3-graviton interaction. So the ansatz for graviton Compton amplitude is Note that now we should also check that this ansatz correctly factorizes in the t channel.
Here, we should match both the MHV ([23] = 0) and anti-MHV ( 23 = 0) t-channel residues: which is indeed the case. Now that our ansatz eq. (5.17) consistently factorizes in all three channels and that it contains no other poles for s ≤ 2, it gives us the graviton Compton amplitude: Again there will be spurious poles when s > 2 and thus need to be further taken care of.
Definition of variables
The variables we'll be using are defined as follow: The F is defined such that it remains invariant under (F1 ↔F1) and (F2 ↔F2). Also, for the pure t-channel terms, we'll be needing: We'll be taking C = C 23 for gA(n) and hA(n), C = C [23] for gS(n) and hs(n), with g(n) satisfying g(n ≥ 2s − 1) = 0. They will be used in the numerator of the pure-t channel:
Graviton Compton amplitude for s > 2
Just as in section 5.1.2, for s > 2 we will relax the constraint that the s-channel residue sits on both the t-and u-channel poles. Instead the s-channel residue will be converted into one that has both t and u-channel poles, one that only has either t or u-channel poles, and one that is completely local. The u-channel image will be fixed by (1 ↔ 4) again. Simply doing so still wouldn't give us a consistently factorizing amplitude, since we need to ensure that the ansatz also matches that of the t-channel pole. We will find that the t-channel residue of our ansatz differs from eq. (5.18) and eq. (5.19), by local polynomial terms, and hence the mismatch can be removed by a pure t-channel term.
JHEP04(2019)156
We again go back to the s-channel gluing eq. (5.16) and apply the identity eq. (5.12). Putting back (s − m 2 ) and fixing the u-channel by spin statistics, our ansatz become Taking the t-channel residue of eq. (5.21) for both MHV and anti-MHV poles, we find that it yields eq. (5.18) and eq. (5.19) plus additional pure polynomlias. All we need to do is subtracting them off, adding minus the polynomial terms over t: We conclude that the graviton Compton amplitude for s > 2 is: which is consistent with the ansatz eq. (5.3).
JHEP04(2019)156
We can see that F , F 1 and F 2 carry inverse powers of m, and do not have a healthy high energy behaviour. So we can again conclude that massive particles with s > 2 cannot be elementary.
The Compton amplitude for non-minimal coupling
In previous sections, we've seen that minimal coupling can always be embedded into a local consistent four-point amplitude, and no constraint other than possible high energy sickness was revealed. In this subsection, we proceed and investigate the case of nonminimal couplings. Recall that we've argued through general covariance, that λλ couplings are forbidden for gravitational couplings. We will see this constraint as a consequence of inconsistent factorizations for the four-point amplitude.
λλ deformation
We again start with the s-channel gluing of the three point amplitudes. With the λλ deformation, the 3pt amplitudes are Expanding the expression, we find that need to consider 4 contributions here. One is the pure minimal contribution which is already known from previous sections, which will not be repeated in this section. The other three are The x-factors of the both sides' non-minimal gluing cannot be completely absorbed in the graviton case and is the reason causing inconsistent factorization. Let's start with photons, where the minimal coupling 3pt amplitude is given by eq. (5.4) and the (λλ) defomation 3pt amplitude is given by: s-channel gluing of case (i) yields:
JHEP04(2019)156
where the tilde denotes that it is a partial contribution to the full non-minimal coupling amplitude. For higher spin, we follow the procedure of dealing with spurious poles demonstrated in section 5.1.2, leading to the following result Finally, the completely local contribution (c) is: So, we have obtained a λλ deformed photon Compton amplitude that consistently factorizes in all channels: is the Compton amplitude derived in the previous section. One thing worth mentioning is that for the mixed contribution, x-factors in the s channel gluing that cannot be absorbed by λ orλ via: becomes a (u − m 2 ) pole by using the identity eq. (5.7). On the other hand, x factors in the (c) contribution can be completely absorbed because we have enough λ andλ to use eq. (5.31) so that it is completely local. This discussion will be important to see the inconsistent factorization of λλ deformed Compton amplitude. Let's now turn to the non-minimal graviton Compton scattering. The minimal coupling 3pt amplitude is given by eq. (5.15) and the (λλ) deformation is: The mixed coupling (a) + (b) contribution for spin 1 2 is we find that there is further t-channel singularity. Note that this mismatch is not local, and thus cannot be removed by modifying the expression by pure t-channel contributions. Thus we have failed to obtain a local amplitude that correctly factorizes in all channels. Note that the source of this can be traced back to the excess x-factors. There is a factor of x 12 x 34 left in the gluing procedure that gives the extra t channel in the (c) contribution due to eq. (5.7). Thus, we conclude that λλ coupling is forbidden for gravity. This is consistent with the previous results shown in the 3pt amplitude. That is, worse than both (a) and (b) contributions when 0 < s ≤ 1, but not for higher spin charged particles.
(λλ) 2 deformation
For (λλ) 2 deformations, we should in general consider where g 1 = 0 for gravitons. In this section, we'll be mostly ineterested in the contributions We again start with photons. From our experience above, we will only be interested in (a ) and (b ) contributions to the deformed Compton amplitude since this is the only structure that causes the 1 m counting differ from that of 3pt counting in higher spin. The (λλ) 2 coupling of photon is given by:
JHEP04(2019)156
with O(m −2 ) in HE. And for s > 1, we'll need to deal with spurious poles. The (a ) contribution to the non-minimal photon Compton amplitudes for S > 1 is: which at least scales as O(m −6s+3 ) in HE.
Now we apply the same analysis for gravitons. Since (λλ) coupling is forbidden for gravitons, we will elaborate more on the (λλ) 2 coupling. The (λλ) 2 graviton 3pt amplitude is: The 4pt mixed coupling (a ) + (b ) contribution to the amplitude for 1 ≤ s ≤ 2 is given by: So the mixed coupling contribution to higher spin Compton amplitude is: Finally, for the (λλ) 2 ⊗ (λλ) 2 contribution to the amplitude, the x-factors can be completely absorbed by the λ's, so the gluing is going to be completely local. For spin 1, the amplitude is: The full amplitude containing the (λλ) 2 non-minimal coupling is the sum of the mixed one and the pure non-minimal one. The mixed one has already matched the t-channel residue and there are no t channel poles to be considered in the pure non-minimal piece because the x-factors are completely absorbed.
UV behaviour of the 4pt amplitudes
It is natural to ask the following question. Given that the minimal coupling 3pt amplitude has the best high energy behavior among all possible structures, does the Compton
JHEP04(2019)156
amplitude obtained from gluing the minimal 3pt amplitudes together automatically has better high energy behavior than the non-minimal contributions? The naive answer to this question is yes, because the 1 m counting in the minimal coupling 3pt amplitude for a given spin is always lower than the non-minimal couplings. This is true for s < 1 photon Compton amplitudes and s < 2 graviton Compton amplitudes, but it no longer holds for higher spin Comptons. The procedure of removing the spurious poles introduces various powers of 1 m . This can make the HE behaviour of the minimal Compton amplitude worse than the non-minimal ones. Now, we summarize the HE behaviour of all the Compton amplitudes we have obtained until now.
Photon
• The pure minimal Compton amplitude has no 1 m factors for 0 ≤ s ≤ 1 and thus has a good HE behaviour and scales at least as O(m −6s+1 ) at HE for s > 1. • The (a) and (b) contribution from (λλ) 2 coupling deformation scales at least as O(m −6s+2 ).
Graviton
• The pure minimal Compton amplitude has no 1 m factors for 0 ≤ s ≤ 2 and thus has a good HE behaviour and scales at least as O(m −6s+2 ) at HE for s > 2.
Finally we conclude that the HE behaviour predicted by 3pt amplitudes only holds for lower spins. For higher spins, the powers of 1 m are determined by the number of the spurious poles cancelled. One factor of 1 3|p 1 |2] cancelled raises the inverse mass factor counting by 1 m 2 more.
Computing the classical potential (1 PM)
Now that we have identified the s 1 limit of minimal coupling three-point amplitudes as that describing Kerr black holes from the one body EFT framework, we can utilize this fact to compute the spin-dependent part of the classical potential between two black holes, which is defined as Fourier transform of the non-relativistic centre of mass amplitude as in [10]. When we talk about the classical potential, we are referring to a long range effect which in momentum space corresponds to the zero momentum limit. The standard JHEP04(2019)156 textbook setup is then to consider the amplitude for a massless exchange between two massive states, gravitons p 2 p 4 p 3 p 1 .
As we will be interested in long range effects, this requires taking the q 2 = ( q) 2 → 0 limit of the above process, where q is the momentum transferred in the center of mass frame. Thus the classical potential can be extracted by taking the four-point amplitude and extracting the t = (P 1 − P 2 ) 2 = −q 2 channel massless pole and discontinuity, schematically given as: The computation can be organized in terms of powers of Newton constant G, which corresponds to post Minkowskian (PM) expansion. At leading order (1 PM), the classical potential is given by the residue of the massless pole in the tree-amplitude. At (2 PM), the contribution to the classical potential arrises from the t-channel triangle integral in the scalar integral basis. There the relevant factor would then be the scalar triangle integral coefficient.
Note that these contributions are on-shell in nature: the residues in a tree-level amplitude are simply products of lower point amplitudes, and at one-loop the integral basis coefficients are computable by generalized unitarity cut methods [12,13], which again are given by products of tree-amplitude. Thus in principle, one should be able to bypass the need for the full four-amplitude, and compute the potential using these on-shell building blocks. There is one obstruction, however, in that in the centre of mass (COM) frame the transfer momentum is space-like, and the t → 0 limit is only reachable by taking the limit of vanishing transfer momentum. On the other hand, to fully utilize the on-shell nature, we need to have light-like exchange momentum.
Cachazo and Guevara [2,3] demonstrated how this can be circumvented by utilizing the "holomorphic classical limit" (HCL), which keeps the total exchanged (complex) momenta to be light-like and then perform a non-relativistic expansion. This expansion is in √ The general procedure for obtaining the classical potential then proceeds as follows: • (A) One first computes the Leading Singularity (LS) for the wanted order in G in the HCL. At tree-level the LS is just the t-channel residue which is the product of three-point amplitudes on both sides of the pole. For one-loop it is the pole at infinity for a specific parametrisation of the triangle cut, which yields the triangle coefficient.
JHEP04(2019)156
Since for the HCL the kinematic setup is essentially that of two three-point on-shell kinematics, the LS can be expanded as: and the spinors |λ] and |λ] are defined from the null exchanged momenta in the HCL.
Here the LS is written with free SL(2, C) indices, as 2s a and 2s b external massive spinors have been stripped. The coefficientsà i,j will be functions of the external momenta, and have an expansion in √ r 2 − 1.
• (B) Next one considers a set of local Lorentz invariant operators also evaluated in the HCL. Although the number of operators are possibly infinite, the number of combinations actually used to form the basis is not high due to the observation that number of spin operators and number of momentum transfer vector appearing in the classical potential are closely related [3]. These operators will in general have different powers of √ r 2 − 1 in the HCL, and thus be unambiguously mapped to the LS. We thus have a representation of the LS in terms of local operators.
• (C) Divide the LS obtained above by the additional factor of 1 4EaE b . This additional factor changes the normalisation of density of particles from one particle per volume ∝ m |E| , relevant for relativistic scattering, to one particle per unit volume, relevant for non-relativistic scattering. The classical potential is then simply the non-relativistic expansion of this result.
where the last two lines indicate the extra contributions that arises from the effect of putting back the polarization tensors. Appendix E outlines how the non-relativistic results for the last two lines were worked out.
Note that from eq. (6.3), we see that for a given scattering of particle with spins s a and s b , we can compute terms in the potential that is up to degree 2s a in S a and 2s b in S b . For example for spin-{1, 1 2 } we can compute terms with Importantly, a given operator in the potential may appear in many different choice of {s a , s b }, and they all must give identical results. For example the operator S a · S a , should emerge from the LS of spin-{
JHEP04(2019)156
Before starting with explicit examples, as we will be interested in the spin-dependent part of the potential, some comments for the spin supplementary condition (SSC), discussed around eq. (4.9), is in order. The covariant condition S µν p ν = 0 used in former sections was used in [30] and [35] to compute leading order (LO) gravitational spin-orbit interactions, which also have been reproduced in the following sections. However, this choice of SSC is in conflict with canonical Poisson bracket relations [36]. To get canonical variables, another choice called Newton-Wigner (NW) SSC is needed [35,36], the choice referred to as baryonic condition in [30]. In curved space NW SSC can be formulated as S µν (p ν + me 0 ν ) = 0 where e 0 is the time-like vielbein, and following sections will be mostly concerned with this choice. In the following we will use the tree-level LS (1 PM) as detailed examples to illustrate the details of this procedure, while reproducing known results in the literature. In the next section, we will present the corresponding 2PM results, which will be mostly new.
Kinematic variables
Kinematic variables will be taken to have the following parametrisation in the centre of mass frame, which is the parametrisation adopted by Guevara in [3]; 13 The HCL corresponds to the limit β → 1, which is equivalent to the limit β → 1. Since the "approaching speed" of the limit is the same for both cases, the limit β → 1 will be used to denote the HCL. Note that this on-shell limit has been reached by complex momenta K.
The usual definitions for the Mandelstam variables, s = (P 1 + P 3 ) 2 and t = (P 1 − P 2 ) 2 , has been adopted. In this frame t = −q 2 , where q 2 = ( q) 2 . All external momenta are taken to be on-shell; P 2 1 = P 2 2 = m 2 a and P 2 3 = P 2 4 = m 2 b . The spinor brackets are taken to be constrained by the conditions λη = [λη] = m a and λη = [λη] = m b . The variables u, v, and r are defined as follows; (6.9) 13 While it was implicitly assumed that β = β in [3], unless ma = m b this does not hold true in general.
JHEP04(2019)156
In the HCL, the variables u and v tend to the values u → m a m b x 1x3 and v → m a m bx1 x 3 . Following relations can be derived from kinematic constraints. [ (6.10) To compute the classical potential, an expansion in r or = √ r 2 − 1 is needed. This expansion is obtained by utilising the following relations that hold in the HCL. (6.11)
Lorentz-invariant combination of operators
The independent four-vector kinematical variables are P 1 , P 3 , S a , S b , and K. Some examples of non-trivial invariants (in the COM) that can be constructed from these variables are; However, not all such invariants are of interest. The invariants relevant for computing the classical potential should reduce to powers of Sp a and Sp b defined in eq. (6.4) when HCL is taken. This removes the candidacy of the last two terms in (6.12). Indeed the first two terms in (6.12) in the HCL takes the form: The last two results are obtained from the definitions of u and v, which are consistent with eq. (B.4) of [3] up to phase. These Lorentz-invariant combination of operators constitute the basis on which the computed LS is expanded, so that classical potential can be read out from the results.
Symmetric basis for polarisation tensors
As undotted spinor index and dotted spinor index can be interchanged freely by the "Dirac equation relations" eq. (2.9), all LS computations can be simplified by expressing all operators to act on dotted indices. The end result can be expressed as eq. (6.3) where definitions for Sp a and Sp b are the same as in eq. (6.4).
As the above notation indicates, we've expressed the LS in purely dotted basis, i.e. the external massive spinors (wave functions) have been stripped off. Note that eq. (6.14) tells us that the SL(2, C) indices of the external wave functions are contracted either with Sp a,b s or the Levi-Cevita tensors αβ . We stress that this simplification is a consequence of the HCL. The normalisation factors N sa,s b i,j are the combinatoric factors that appear due to Lie algebra properties as in eq. (4.15).
While it is tempting to match Sp a and Sp b in the above expansion to Lorentz-invariant combination of operators discussed in section 6.1.2, the basis used to computeà i,j does not treat dotted and undotted indices democratically at all. It is natural to relate the operators considered in section 6.1.2 to one-body effective action operators introduced in section 4, and the natural basis on which these operators act should treat dotted and undotted indices equivalently. The expansion eq. (6.14) needs to be massaged so that LS is expanded in one-body effective action operators.
An educated guess that could be made from the identity eq. (2.24) for three-point kinematics is that half of Sp a and Sp b in eq. (6.15) do not encode the dynamics but come from kinematics, therefore this kinematical contribution which is irrelevant to the dynamics should be factored out from the computed coefficientsà i,j . A trick 14 that can be used is to repackage the coefficients by constructing power series in variables x a and x b in the following way.
The multiplication by the factor e −xa/2 e −x b /2 has the effect of factoring out the kinematical (or non-dynamical) contributions fromà i,j coming from eq. (2.24) when expressing the LS in the anti-chiral (or purely dotted index) basis. Thus, the data of dynamics is encoded by the series expansion The authors would like to thank Justin Vines for helpful discussions. which can be viewed as the expression for LS in the vector (or the symmetric) basis. The Sp a and Sp b in the above expression are operators which act on all indices, where their action on anti-chiral indices is given by eq. (6.15). While their action on chiral indices is not specified, eq. (6.15) and eq. (6.13) can be compared to match Sp a and Sp b with HCL operators (K · S a ) and (K · S b );
JHEP04(2019)156
Reduction to the form K · S in the HCL does not mean that the operator is necessarily the operator K · S. The exact matching onto Lorentz-invariant combination depends on the power of √ r 2 − 1 that may appear; e.g. the following matching rules can be devised from inspecting the list eq. (6.13) closely. (6.19)
Matching at leading order (1 PM)
The leading order contribution corresponds to the tree-level amplitude. The LS at this level is just a product of two 3pt amplitudes, multiplied by the massless graviton propagator 1 t as represented in figure 1; Following Guevara [3], this LS can be cast into a purely anti-chiral form. Adopting the definitions in 6.1, this expression simplifies to [3] where Sp a = |λ][λ| ma and Sp b = |λ][λ| m b , as defined above.
JHEP04(2019)156
A note of caution is that allà i,j coefficients of LSs were computed as if LSs were operators acting from left to right to match the sign choices in [3], i.e. the polarisation tensors for the incoming particles are put to the left while that for the outgoing particles are put to the right. The factor e −xa/2 e −x b /2 in eq. (6.16) must be changed to e xa/2 e x b /2 in this convention to eliminate the non-dynamical kinematical factors, since the sign appearing in the middle of eq. (2.24) will be flipped. Also, the rule eq. (6.6) will have a sign flip due to this convention. The rules (modified) eq. (6.6), eq. (6.18), and eq. (6.19) are then applied to yield the expression for the classical potential.
Spin-independent Newtonian gravity
Tree-level computation of LS yields the following result for A 0,0 , the term responsible for V = − GM m r of Newtonian gravity.
Since only this term can contribute to the spin-independent part of the LS, this part determines the spin-independent part of the classical potential. The classical potential to leading and subleading orders in PN can be read out by multiplying a factor of 1 4EaE b , which is consistent with the results of [10]. (6.23)
Spin-orbit contributions
The result for A 1,0 up to subleading order in √ r 2 − 1 is the following. (6.24) Of the two set of rules eq. (6.18) and eq. (6.19), only the second rule matches the order in √ r 2 − 1. Thus it can be concluded that LS| S 1 to leading PN order where eq. (6.12) was used to evaluate non-relativistic expression which is consistent with the known results; eq. (48) of [30] and eq. (71) of [35]. This result corresponds to the choice of covariant SSC S µν p ν = 0.
Spin-spin interactions
The computation result up to subleading order in √ r 2 − 1 is the following: Thus, applying the rules eq. (6.18) to the LS makes the LS to take the form and the classical potential to leading PN order is consistent with the results eq. (6.9) of [33] and eq. (90) of [10]. Note that contributions due to eq. (6.6) do not change the potential in the leading order in PN, since additional dependence on p i makes the potential subleading in powers of v 2 c 2 .
Quadratic in spin effects
A 2,0 is relevant for this computation.
This is the first two leading terms in expansion over √ r 2 − 1. The LS and the classical potential up to leading PN order then takes the form also consistent with the results eq. (6.10) of [33], provided that C 1(ES 2 ) = 1. Similar to spin-spin interaction term, the application of eq. (6.6) does not change the potential at this order.
Cubic in spin effects
There are two terms to consider; A 3,0 and A 2,1 . Their first two leading terms in the √ r 2 − 1 expansion are given below.
Computation of S 3 a -term is straightforward, since there are no ambiguities.
This leading PN order expression matches the terms proportional to C 1(BS 3 ) in eq. (3.10) of [37] when it is set to unity. When eq. (6.6) is taken into account, there is an additional term generated from eq. (6.32) that contributes to this potential which matches the terms proportional to C 1(ES 2 ) when it is set to unity.
At first sight, computing the contribution from A 2,1 seems complicated by the fact that there are two combinations that reduce to the same factor ( in the HCL. Nevertheless, it is possible to write this LS as a linear combination of the two by introducing an arbitrary real parameter α. (6.39) Computing the classical potential up to leading PN order requires taking the non-relativistic limit.
JHEP04(2019)156
However, the following vector identity [37] can be used to relate the different combinations.
and since there is an overall factor of q −2 in the amplitude already, changes in α is reflected in the classical potential as derivative delta-like interaction which does not affect the long-distance behaviour; α is a free parameter that can be tuned arbitrarily without affecting the long-distance behaviour. The non-relativistic limit takes the following form in position space.
(6.44)
When α is set to unity, this expression matches the sum of first two terms proportional to C 1(ES 2 ) in eq. (3.10) of [37] provided C 1(ES 2 ) is set to unity. Note that there are two sources that can contribute to this potential through eq. (6.6); the first is the contribution from A 2,0 which is and the other is the contribution from A 1,1 which is In position space, these two contributions take the following form.
Adding up eq. (6.44), eq. (6.47), and eq. (6.48) gives an expression that matches with eq. (3.10) of [37] when C 1(ES 2 ) is set to unity. Note that using eq. (6.43), the final potential can be written in the following form in momentum space.
There are no ambiguities for matching the LS from these results.
Computing the classical potential to leading PN order is straightforward, which is consistent with the results eq. (4.4) of [37] when C 1(ES 2 ) , C 2(ES 2 ) , C 1(BS 3 ) , and C 1(ES 4 ) are all set to unity. Note that eq. (6.6) does not induce any corrections at this order. Based on dimensional analysis, it can be argued that two particle irreducible (2PI) diagrams with only one massive internal leg per loop contribute to the classical potential for the case of non-spinning particles [18]. The only topology that meets this criteria at one loop is the triangle topology. More precisely, since in four-dimensions one-loop amplitudes can be cast into a scalar integral basis involving box, triangle and bubble integrals [38,39], the statement is that only triangle scalar integrals are relevant for contributions to the classical potential.
To understand why note that the one-loop integrals that are relevant to our problem always contains two massless graviton propagators: This implies that the result will have non-analyticity in q 2 = −t, reflecting the presence of the massless cut. There are two types of such non-analyticity, The first corresponds to classical contribution and the second quantum [6]. It is then straightforward to march through the scalar integrals, and find that only scalar triangle yields the desired non-analyticity [9]: where p will be the momenta of one of the external lines. Thus to extract the classical result at 2 PM amounts to computing the integral coefficient for the scalar triangle. The integral coefficients can be readily computed using generalized unitarity methods [12,13]. As the triangle integral has three propagators, one can explore the kinematic regime of the loop momenta where all three propagators become on-shell, and the "residue" simply becomes the product of two three-point and a four-point on-shell amplitude, as shown in figure 2. This is termed the triangle cut. Note that the triangle integral is not the only basis integral that contributes to the triangle cut. Box integrals with one extra propagator can contribute as well. The challenge is then to separate these two contributions.
This problem was beautifully solved by Forde [40], which parameterize the loop momenta in terms of four complex variables, and can be fixed as propagators go on-shell. For the triangle cut, the loop momenta has only one complex variable left, and the cut can be viewed as a function of this variable with poles at finite values as well as infinity. The finite poles represents extra propagator becoming on shell, and hence the presence of box integrals. Thus the contribution from the scalar triangle simply corresponds to the pole at infinity.
We can again simplify things by evaluating the triangle coefficients in the HCL limit, and match with a preferred local operator basis, after which one performs the nonrelativistic expansion to recover the classical potential just as what was done in the 1 PM case. In summary the 2PM result are obtained as follows: • Compute the following Leading Singularity in the triangle cut where D i = L 2 i − m 2 i represents the three propagators that were put on-shell, and ∞ indicates that we are picking the contribution at infinity for the remaining integration variable.
• Due to solving the delta functions, the above generates a Jacobian factor J. Thus to get the triangle coefficient, we need to multiply the LS by J −1 .
• Finally we multiply the resulting triangle coefficient to the loop integral and perform the q 2 → 0 expansion, and picking out the relevant classical piece, which from eq. (7.2), is given by
JHEP04(2019)156
Thus the final 2PM result is given by: The Jacobian factor can be computed explicitly and in the HCL limit, yielding J = − 1 32m √ q 2 , which cancels the last term in the above product! Thus the 2PM classical potential is simply reproduced from the LS along, as pointed out by Cachazo and Guevara [2,3].
From the previous sections it is clear that the three-point amplitudes that should enter the cut would be that of minimal coupling. For the four-point amplitude, we use the Compton amplitude that was constructed from matching the three-point minimal coupling on the residue. This however, leaves us with polynomial ambiguities as discussed previously. For s ≤ 2, the polynomial ambiguities come with additional 1 m factors, which was absent from the answer constructed from residues, and thus can be argued as finite size effects. Such separation is no-longer true for s > 2. Thus for now, we will constrain ourselves to using the Compton amplitude of s = 2, which in practice, means we will be limited to terms in the potential that are at most degree 4 in the each particle's spin operator.
When computing the 1-loop scattering amplitude in the non-relativistic limit, there are terms that diverge as COM average momentum vanishes. These terms have an interpretation as second order perturbation theory effects from the 1 PM potential, or second Born approximation terms. Such terms are artifacts of iterating the 1 PM potential, and they must be subtracted to compute the correct 2 PM contributions to the potential; when these terms are not subtracted, the amplitude computed from the classical potential will double-count such contributions and lead to a wrong answer. It can be shown that such iteration terms only consist of singular terms in COM momentum p 0 := | p a | when nonrelativistic propagator is used [10], 15 so the following simple prescription for subtracting the iteration terms will be adopted; when there is a divergent term in the expression for LS, all poles in p 0 will be interpreted as coming from iteration and will be subtracted. The remaining finite pieces will be interpreted as the 2 PM potential. 16 In this section we compute the spin dependent pieces of the 2 PM classical potential. The analysis is similar to 1 PM case, but 2 PM computations require separation of iterated 1 PM contributions, which is usually referred to as the second Born approximation term [10].
Parametrisation and computation of the LS
The parameterisations used in [3] was used to compute the LS in this manuscript. The details of the parameterisation apart from the ones given in section 6.1.1 will be presented 15 While it is also noted in [10] that using non-relativistic propagators to separate iteration terms do not lead to a potential which is useful for computing equations of motion, this prescription will be adopted for its simplicity. A method is provided in the appendix of [10] which computes the iteration terms from propagators with relativistic energy-momentum dispersion relations. 16 The conclusion depends on the order of p0 pole subtraction and flux normalisation; taking the nonrelativistic flux normalisation first and then subtracting p0 poles gives the result which matches that of [20], while subtracting p0 poles first and then taking the non-relativistic flux normalisation gives the result which matches that of [10]. The latter is adopted in this manuscript.
JHEP04(2019)156
here. Consider the triple-cut diagram in figure 2, which is referred to as the b-topology.
The loop momentum L runs through the massive internal leg, and massless internal legs are parameterised as k 3 = −L + P 3 and k 4 = L − P 4 . The parameterisation for the loop momentum L = L(z) is; Imposing the triple-cut conditions k 2 3 = k 2 4 = L 2 − m 2 b = 0 fixes ω = − 1 z , and A(z) and B(z) as rational functions of z and β. Defining y = − z (β−1) 2 as in [3], the LS from this topology is computed to be where Γ LS is taken to be the contour enclosing the pole at y = ∞. The product of onshell amplitudes that constitute the integrand need to be interpreted as operator products, detailed procedure being given in [3]. The choice for internal momenta spinor-helicity variables are given below. (7.10) After having computed the b-topology LS, the result is added to the computed result for a-topology LS which can be evaluated from the b-topology LS by reflection, e.g. u ↔ v, m a ↔ m b , etc.
Results for the classical potential
In our computations, we are interested in the classical potential up to quartic order in spin, so all the results presented in this section are calculated from spin-2 particle scattering with M 4 (P 1 , −P 2 , k h 3 3 , k h 4 4 ) in eq. (7.9) given by the lower spin Compton amplitude eq. (5.20). 17 Recall that from eq. (6.3), the LS for minimally coupled {s a , s b } particles can capture terms in the potential that is up to degree 2s a in S µ a , and 2s b in S µ b . Here we have verified that 17 Here we only take the mixed helicity Compton amplitude contribution into account because the same helicity Compton amplitude has zero residue at y → ∞. Take the (++) channel Compton amplitude for example, the integrand 1 y M4(P1, −P2, k + 3 , k At the end of the current section we will discuss how to utilize this fact to fix ambiguities associated with higher spin scattering.
Spin-independent
is needed to compute 2 PM contributions to the spin-independent 2PM contribution to the classical potential.
The resulting leading PN order classical potential is consistent with the results given in [10]. (7.12)
Spin-orbit interaction
The coefficient A 2 PM 1,0 , which is the 2 PM counterpart to A 1,0 computed in section 6.2.2, up to first three terms in the √ r 2 − 1 expansion takes the following form.
(7.13)
This is the first of numerous terms that include 1 PM potential iteration pieces; in the stationary limit r → 1 this expression diverges due to the factor 1 √ r 2 −1 in the first term. Using the following formula this expression can be converted to Laurent series in p 0 , and dropping poles in p 0 gives the following expression.
All subleading √ r 2 − 1 expansion pieces were dropped in the above expression. Combined with the contributions from A 2 PM 0,0 due to eq. (6.6), the following expression is obtained for leading PN 2 PM spin-orbit coupling. This is consistent with the results eq. (57) in [10].
2qm a (m a + m b ) ( S a · p × q) .
Quadratic order in spin
Up to first three terms in √ r 2 − 1 expansion, the terms relevant for quadratic order in spin are; The leading terms after p 0 poles are subtracted out are and becomes in the non-relativistic limit The latter can be compared with eq. (95) of [10]; The two expression match up to terms proportional to q 2 S a · S b , which are subleading in the HCL. While this subleading HCL contributions did not affect the long-distance behaviour for LO, this is no longer true for 2 PM; q 1 in momentum space is roughly equivalent to r −4 in position space. It is not possible at the moment to compute subleading HCL contributions so the answers provided above cannot be complete, but the directional dependence on relative orientation of the bodies ( S a · r)( S b · r) can solely be attributed to JHEP04(2019)156 non-vanishing HCL contributions and they can be computed by the methods provided in this manuscript.
Taking such HCL equivalence classes into account, the potential at this order will have the following form in momentum space.
O refers to an unknown operator that is vanishing in the HCL. The corrections induced by eq. (6.6) does not affect the potential at this order.
Cubic order in spin
The leading PN corrections at 2PM order for cubic order spin interactions are formally classified as 4.5PN corrections, and according to [41] they were not known in the literature. The coefficients up to first three terms in √ r 2 − 1 expansion that will be relevant are the following. .
(7.29)
After subtraction of p 0 poles, leading PN terms take the following form.
The S 3 a -term has no ambiguities, apart from HCL-vanishing contributions.
Taking the non-relativistic limit gives
The other cubic spin interaction term suffers from an ambiguity that was elaborated in section 6.2.5. Since this ambiguity can be absorbed into the unknown HCL-vanishing contributions, this ambiguity will be ignored in this section. The interpretation for A 2 PM 2,1 reg is then (7.35) and in the non-relativistic limit it takes the form Taking effects from eq. (6.6) into account, 2 PM S 2 a S 1 b potential takes the following form. (7.37)
Quartic order in spin
Formally, leading PN corrections at 2PM order at quartic order in spin is classified as 5PN corrections, which were also not known in the literature according to [41]. At quartic order in spin, the following coefficients computed up to first three terms in √ r 2 − 1 expansion
Subtraction of poles in p 0 yields the following leading term expression.
Proceeding as in former examples, the relativistic LS takes the following form which, with non-relativistic flux normalisation, yields the following expression for the potentials.
Since the terms introduced by eq. (6.6) are subleading in PN expansion, they do not need to be considered.
Partial results for higher order in spin
Though we cannot obtain the full results for higher spin effects due to the polynomial ambiguities of the higher spin Compton amplitude eq. (5.24), we can still obtain partial results from LS computations of spin 2 particle scattering. We present them in the following list: • Fifth order in spin: • Sixth order in spin: • Seventh order in spin: • Eighth order in spin:
Fixing the local polynomial term at S 4 from consistency condition
In the beginning of this section, we mentioned that certain terms in the potential can be computed with more than one way of assigning the spin to the two particles, and the result from each assignment should be identical. More precisely, for a specific order of spin operator, the coefficient should be independent of which external spins were chosen to extract the JHEP04(2019)156 potential. Indeed in derivation of the potential, we have verified that the same result has been reached with different choices of spin assignment. In this subsection we will explore the possibility of using this consistency condition to fix the polynomial ambiguity. Recall that the Compton amplitudes were derived from matching the factorization poles, which leaves us open to polynomial ambiguities. As we will show the polynomial ambiguities can only enter at the quartic order in spin operator in the HCL limit.
In general, a candidate polynomial term needs to satisfy the little group weights and spin-statistics relations. Since we are talking about the one contributing to the classical potential, the contact terms should also survive the HCL. So we require the following conditions: The following list exhausts all possible spinor combinations that survive the HCL limit: Spa m a We can see that the only combination that provides the correct helicity weight of the gravitons is K 4 . Since this term contains 4 SU(2) indices for both particle 1 and particle 4, contact terms starts to affect the classical potential at quartic order in spin. Universality of spin effects demands that all S 4 potential extracted from particles with s > 2 should also take the same form. However, one would find that using eq. (5.24) in the LS calculation to extract quartic order spin effects for s > 2 particles will yield a result different from the one we presented in section 7.2.5. This difference exactly comes from the polynomial ambiguities. Now we propose the following ansatz for the contact terms: S 5 effects and higher. As an example, one can compare the A 5,0 coefficient of the LS calculated from eq. (5.24) and eq. (5.20). We found that they differ by: One might think that they just differ by a polynomial term. But a polynomial term of the Compton amplitude of particle a should not carry any information of particle b. That is, the difference must only carry powers of m a and no powers of b in the denominator while eq. (7.60) carry (m a + m b ) 5 in the denominator. So, such difference is definitely not from the polynomial ambiguity of the Compton amplitude. Thus we conclude that the spurious Compton amplitude is not applicable for higher spin effects.
Conclusion and outlook
In this paper we systematically study charged and gravitationally coupled higher spin particles. We focus on "minimal couplings", where the UV limit matches to the minimal derivative coupling. We identify that these interactions can also be characterised in the IR through various physical properties such as g = 2, and the absence of finite size effects. For spins-1/2 and 1, this corresponds the usual minimal couplings for Dirac fermions as well as W bosons. We also derive the (gravitational) Compton amplitude, up to polynomial ambiguities, for the minimal coupling with arbitrary spin. These are derived through the requirement of consistent factorisation. Applying the same criteria for non-minimal couplings, we find that λ 2 interactions are forbidden for gravitational coupling. We argue that the absence of λ 2 deformations, or anomalous gravitomagnetic dipole moment, is a reflection of general covariance. For theories whose gravitational coupling is the square of gauge couplings, this implies that the charged states must have g = 2, consistent with string theory.
Having equipped with the Compton amplitude, we proceed to utilize it to compute the spin dependent piece of the classical gravitational potential. We follow the work of Cachazo and Guevara, where the 2PM potential is computed by evaluating the one-loop triangle leading singularity in the holomorphic classical limit, and matched to local Lorentz invariant operators. Using the spin-2 Compton amplitude, we derive the spin-dependent parts of the potential up to degree four in the spin operator of either black holes. We also discuss to which extent the polynomial ambiguities of the higher spin Compton amplitudes can be fixed by requiring that the resulting classical potential yields the same coefficient for the spin operators as that for the lower spins.
As alluded to in the paper, the leading trajectory states in string theory do not yield the simplest coupling. In fact, it is the most complex, as all allowed deformations except for the (gravito)magnetic dipole moments are turned on. It would be interesting to see if the couplings for the subleading trajectories are simpler. This would be in line with the expectation that the large degeneracy for subleading trajectory states become the dominant contribution for black hole microstates, which we know are simple. It would be fascinating
JHEP04(2019)156
if this is the case, as vertex operators for subleading trajectories are generally much more complicated than the leading one, and yet it yields a simpler amplitude, providing further evidence that the worldsheet point of view can often be misleading.
An immediate task is to identify what is the theory that gives minimal coupling for spins ≥ 2. To construct the corresponding Lagrangian, one starts with the quadratic term in eq. (3.18), and successively adds terms linear in the Reimann tensor to remove the spin-operator pieces that are induced by eq. (3.18), characterizing the deviation from minimal coupling. This is not only of theoretical interest, but it will resolve the polynomial ambiguity in the gravitational Compton amplitude, allowing one to extract spin effects beyond quartic order.
The fact that the infinite number of Wilson coefficients of the one body effective action is reproduced by the minimal coupling which is simply an x 2 in our on-shell parameterization, lends one to wonder if further simplification can be achieved by reformulating all computations in the one body EFT approach in terms of computations involving x 2 . A tantalising example would be the fact for charged black holes, one also has g = 2 [42], and one can conjecture that x gives the correct Wilson coefficient for the electricmagnetic couplings. This would be the simplest example of double copy for classical objects.
Finally, an important question is whether the relation between minimal coupling and black holes persists through quantum corrections. It is well known that quantum effects generate (g−2) for charged particles. On the other hand the gravito-magnetic moment is argued to be universal and thus should be protected. It would be interesting to see why λ 2 terms are not generated by loop corrections. Furthermore, whether minimal coupling states in gravity stays minimally coupled quantum mechanically, in that all deformations are never turned on.
JHEP04(2019)156
To relate the (connected part) of the Lorentz group SO(1, 3) and its double cover SL(2, C), we follow a widely adopted convention for spinors and gamma matrices, where σ denote Pauli matrices in the standard convention. Complex conjugation exchanges undotted and dotted indices. It is easy to check that forms a representation of the algebra eq. (A.1). Spinor indices are raised and lowered by the invariant tensor of SL(2, C) satisfying For example, λ α = αβ λ β andλα = αβλβ . For any (momentum) 4-vector, the bi-spinor notation is defined by
A.2 Massless momenta
For massless momenta, p αα as a (2 × 2) matrix has rank 1, so it can be written as For a real momentum, the spinors satisfy the reality condition, The Little group U(1) acts on the spinors as The spinors for p and those for (−p) must be proportional. We fix the relation by setting It is customary to introduce a bra-ket notation, which leads to the Lorentz invariant, Little group covariant brackets, The massless Mandelstam variables, which are both Lorentz invariant and Little group invariant, can be expressed as
A.3 Massive momenta
For massive momenta, the on-shell condition in the bi-spinor notation is given by The massive helicity spinor variables are defined by 14) The index I indicates a doublet of the SU(2) Little group. The reality condition reads SU(2)-invariant tensor. Given a matrix representation of the doublet of SU (2), the two defining properties of SU (2) can be written as where the SU(2)-invariant tensor IJ shares, by convention, the first three properties in eq. (A.4). Just like spinor indices, the Little group indices are raised and lowered by IJ and IJ . It follows from eq. (A.17) that the two variables below transform in the same way. BOLD. For a fixed massive particle, the SU(2) Little group is always completely symmetrized. Ref. [1] introduced the BOLD notation, which suppresses the SU(2) little group indices by means of an auxiliary parameter for each particle. For instance, for particle 1, It is clear how to reinstate the SU(2) index if needed. The Dirac equation eq. (A.21) can be written in the BOLD bra-ket notation as In the main text, we define the x factor for a 3pt amplitude: Decomposing the massive momenta into the spinor helicity variables, we can derive A.4 High-Energy limit Definition. Consider a system of massive particles whose masses are equal or similar to some fixed m. As in the scattering problem of the main text, we assume that the particle number is conserved and the mass of each particle is also conserved. Let p i be the incoming momenta, and γ ij = p i · p j /m i m j (i = j be the Lorentz invariant measure of the pairwise relative velocity. The High Energy (HE) limit is defined such that all γ ij 's grow arbitrarily large while the ratios γ ij /γ kl remain fixed.
Frame dependence. In the center of momentum (COM) frame among all incoming momenta, it can be shown that p 0 = E m holds for each particle in the HE limit. Suppose in the COM frame. The two diagonal matrix elements are well-separated in the HE limit, where we suppressed corrections of order O(m/E) 4 .
JHEP04(2019)156
Unlike the definition of the HE limit, the relation E m depends on the Lorentz frame; it does not hold in the particle's own rest frame. We can specify the frame dependence in a Lorentz covariant way. Let u µ be the time-like unit vector of the COM frame. Introduce In the COM frame, where u µ = (1, 0, 0, 0), uα α is the identity matrix. So, both (p|u) and (u|p) have the same matrix elements as p αα in eq. (A.32). But, now (p|u) and (u|p) are Lorentz covariant operators acting on spinors. Their eigenvalues, which coincide when E ± p in the COM frame with p µ = (E, 0, 0, p), can now be regarded as Lorentz invariant quantities.
Bearing in mind the frame dependence, we decompose each massive momentum as The first piece corresponds to the large eigenvalue (E +p) and the second piece to the small one (E − p). It is often convenient to rescale the sub-leading piece by (η ,η ) = m(η,η), 36) or, to discuss many particles at once, (A.37) In this notation, the definition of the HE limit can be rewritten as [1] ij Explicit form of helicity spinor variables. The spinor helicity variable λ α I is defined up to actions of the SL(2, C) Lorentz group and the SU(2) Little group. For numerical computations, it might be useful to have a prescription to fix both group actions.
JHEP04(2019)156
In terms of these SU(2) spinors, we may write Comparing it with the Lorentz covariant expression (with I ∈ {+, −}), leads to the identification To make contact with the HE limit, we make simple replacements to recover eq. (A.36): HE limit of 3pt amplitudes. We use the decomposition eq. (A.37) to examine the HE limit of the 3pt amplitudes with two massive particles of the same mass and spin coupled to a massless particle. Without loss of generality, we assume that the massless particle has positive helicity. It is well-known that the 3pt amplitude for three massless particle can be non-vanishing only if the momenta are complex valued and either |1 ∝ |2 ∝ |3 or |1] ∝ |2] ∝ |3] holds. We cover the two cases separately.
JHEP04(2019)156
The spin operator for multiple spinor indices follows from the Lie algebra, α i+1 · · · δ β 2s α 2s . When acting exclusively on the totally symmetric representation, the spinor operator is effectively proportional to the spin, A similar equivalence works for the dotted spinors as well.
A.6 Polarisation
Massless case. We take the following definitions for the polarisation vectors of photons, where ζ parametrises the gauge redundancy. The polarisation vectors satisfy Alternatively, in the bi-spinor notation, The polarisation tensors for higher-spin particles are constructed as symmetric products of eq. (A.64).
Massive case. For a massive spin 1 particle, we adopt the following definition for the polarisation vector:
JHEP04(2019)156 B The normalization of gravitomagnetic Zeeman coupling
It is expected that the full gravitational potential V will have "scalar potential" coupling mΦ and Zeeman-like coupling α S · B with gravitomagnetic field B := ∇ × A.
V := mΦ + α S · B (B.1) The coefficient α will be fixed by requiring that the correct time evolution of the spinoperator S will be reproduced by the corresponding Hamiltonian. The natural evolution of spin vectors in general relativity required by the equivalence principle is described by what is known as the Fermi-Walker transport: 18 The vector u µ is the tangent vector of the curve γ(s) along which S µ is transported, and is normalised by u µ u µ = = ±1. The acceleration vector a µ is defined as a µ := u ν ∇ ν u µ . Setting u = ∂ 0 , Fermi-Walker transport for spin vector gives the following equation.
The Christoffel symbols up to O(h) are given by, assuming stationary solutions, i.e. ∂ 0 = 0,
JHEP04(2019)156
C Some details of the t-channel matching of the higher spin graviton Compton amplitude Let's go back to eq. (5.21), and take the t-channel residue. 19 to cancel as much (s − m 2 ) and (u − m 2 ) as possible until there is no more F 2 in the leading term of each of the summation. Then we identify the last term in the first line of Eq (C.1) as P oly and the piece that only carries the (s − m 2 ) pole as P ole s , so that: Now, we are free to write F 1 in the terms other than P oly and P oly s as:
Res[Ansatz]
such that the first term in the expansion of − repeatedly until there is no more 3|p 1 |2] to be absorbed. On the other hand, since r = 1 in the summation of P ole s is zero, we can further apply eq. (C.2) to P ole s once more. We end up with: where f S (n) is defined by: until f (n ≥ 2s) such that the residue is completely local. 20 F 2s 1 +P oly+P oly [23] (C. 10) which is just the correct residue eq. (5.19) plus pure polynomial terms. For [23] = 0, we do not need to do the calculations again. Since we are using the variables F , F 1 and F 2 that are symmetric under F 1 ↔F 1 and F 2 ↔F 2 , we can just 20 The recursion relation eq. (C.9) is obtained by And there are (u − m 2 ) present to cancel with the ones in the denominators, leaving only local terms.
D Wilson coefficients for black holes
The Wilson coefficients C # for coupling of spin degrees of freedom to spacetime curvature have an interpretation as gravitational multipole moments generated by spin effects. For this purpose it is convenient to introduce the vector a µ := 1 m S µ . The terms linear in h µν in the one-body effective action can be recast as follows.
C BS 2n+1 (2n + 1)! − (−a · ∂) 2 n u (µ ν)αβγ u α a β ∂ γ h µν + (u · ∂) [· · · ] + O(h 2 ) (D.1) The notation C ES 0 = C BS 1 = 1 has been adopted to simplify the equations, and covariant SSC was used to express S µν as S µν = m µνλσ u λ a σ . The round brackets on µ and ν indices on the second line indicates symmetrisation, i.e. A (µν) := 1 2 (A µν + A νµ ). When integrated on the worldline, the terms with (u · ∂) can be converted to boundary terms which becomes irrelevant when trying to interpret this Lagrangian as the source term for h µν . Upon integration by parts, this Lagrangian reduces to the following source term expression. 21 3) x wl (s) is the worldline of the particle, parametrised by s. 21 The coupling constant κ has been absorbed into definition of hµν .
h µν (x) = 4G d 4 y G ret (x − y)T µν (y) (D.7) Note that integration by parts identity dyK(x − y) d dy f (y − z) = d dx dyK(x − y)f (y − z) for vanishing boundary contributions can be applied to pull out the derivatives on the source term. Setting the worldline of the particle to lie at the origin, i.e. x wl (s) = (s, 0), the following expression for the trace-reversed graviton field is obtained.
It is known that trace-reversed graviton field for exact Kerr geometryh Kerr µν can be put in the following form [43]. , q µ = P µ 1 − P µ 2 . In terms of average momentum and momentum transfer, the polarisation tensors can be expressed as follows.
JHEP04(2019)156
Since polarisation tensors are defined in some reference frame and then extended to arbitrary momentum by boosts for massive particles, the polarisation tensor ε(p) can be schematically be written as follows.
ε(p) = G(p; p 0 )ε(p 0 ) (E.3) Thus, the derivative on polarisation tensor can be represented as Using NW SSC S µν (p ν + mδ 0 ν ) = 0, the following relation can be derived for S i0 ; S i0 (p 0 + m) = −S ij p j = ijk p j S k (E.8) Therefore, the derivative can be represented as follows in the non-relativistic limit.
ε * (P 4 )ε(P 3 ) = ε * (p b ) 1 1 + i 2m 2 b ( p b × q) · S b + · · · ε(p b ) (E.14) 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. | 2023-01-21T14:44:10.086Z | 2019-04-01T00:00:00.000 | {
"year": 2019,
"sha1": "708827ad8087090cb2ad81328b40e84b43c33ffd",
"oa_license": "CCBY",
"oa_url": "https://link.springer.com/content/pdf/10.1007/JHEP04(2019)156.pdf",
"oa_status": "GOLD",
"pdf_src": "SpringerNature",
"pdf_hash": "708827ad8087090cb2ad81328b40e84b43c33ffd",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": []
} |
155749797 | pes2o/s2orc | v3-fos-license | Context Matters: Economic Voting in the 2009 and 2014 European Parliament Elections
Using the 2009 and 2014 European Election Studies (EES), we explore the effect of the economy on the vote in the 2009 and 2014 European Parliament (EP) elections. The paper demonstrates that the economy did influence voters in both contests. However, its impact was heterogeneous across the two elections and between countries. While assessments of the economy directly motivated voters in 2009 by 2014 economic appraisals were conditioned by how much responsibility voters felt the national government had for the state of the economy, implying a shift in calculus between the two elections. The analysis suggests that voters in 2009 were simply reacting to the economic tsunami that was the Global Financial Crisis, with motivations primarily driven by the unfavourable economic conditions countries faced. But in 2014, evaluations were conditioned by judgments about responsibility for the economy, suggesting a more conscious holding to account of the government. Our paper also reveals cross-country differences in the influence of the economy on vote. Attribution of responsibility and economic evaluations had a more potent impact on support for the government in bailout countries compared to non-bailout countries in 2014. Our findings demonstrate the importance of economy on vote in EP elections but also highlight how its impact on vote can vary based on context.
Introduction
Between 2008 and 2014 the advanced industrial world faced its greatest economic challenge since the Great Depression of the late 1920s. The Global Financial Crisis (GFC) saw unemployment across the European Union (EU) rise to unprecedented levels, prolonged periods of negative economic growth, a series of banks come close to collapse forcing national and EU institutions to step in and preserve them, national deficits spiral, and the true indebtedness of many EU member states become evident. Such were the scale of eco-nomic problems that serious questions were raised regarding the ability of the Euro currency to survive (e.g. Eichengreen, 2013;Hotten, 2011). Eight member states (Cyprus, Greece, Hungary, Ireland, Latvia, Portugal, Romania and Spain) were forced to seek so-called 'bailouts' between 2008 and 2013, where the International Monetary Fund (IMF) and the EU provided finances to these countries to enable them to keep their ships of state afloat. Support came with the proviso that these countries would implement a series of austere economic measures, including salary cuts and reduced public services. Austerity became the economic orthodoxy of most member states with the GFC also having a number of political repercussions including the ejection from office of many governments in power at the time the GFC hit (LeDuc & Pammett, 2013), the development of new anti-establishment political movements across Europe, and a dampening of enthusiasm towards the EU (Treib, 2014). Taken together, all of this ensured the economy has been the dominant preoccupation of both citizens and governments alike over the past eight years and in this context, it is reasonable to assume economics has been a key issue, if not the key issue, on voters' minds as they went to the ballot box during this period.
Economics have also been shown to shape attitudes towards European integration (e.g. Gabel, 1998;Tucker, Pacek, & Berinsky, 2002) and preferences in EU referendums (e.g. Doyle & Fidrmuc, 2006;Elkink, Quinlan, & Sinnott, 2015). However, its influence on vote choice in European Parliament elections has been explored much less. Traditionally, EP elections have been considered 'second-order', with voter behaviour conditioned by attitudes to the incumbent government (e.g. Hix & Marsh, 2011;Marsh, 1998;Reif & Schmitt, 1980;Schmitt & Teperoglou, 2015). More recently, there has been an appreciation that other reasons also influence voters in EP contests (e.g. de Vries, van der Brug, van Egmond, & van der Eijk, 2011;Hobolt & Spoon, 2012). Our claim is that economics are another crucial component, especially in light of the Global Financial Crisis. Therefore, our paper focuses on how the economy motivated vote choice in the 2009 and 2014 European elections.
There has never been a more apt time to explore the motivations underlying citizen behaviour in EP elections given the increasing powers of the European Parliament (Hix, 2013;Hobolt, 2014). EP elections are among the few means citizens have to pass judgment on the EU and thus, exploring what motivates voters in these contests is valuable. Studying behaviour in European elections is all the more interesting considering the 2014 elections marked an attempt by the EU to make European elections "different" from previous EP contests by introducing spitzenkandidaten, where parties at the European level proposed rival candidates for the Presidency of the European Commission. It was hoped by offering voters the opportunity of voting for an executive office and providing a link between voter preferences and selection of the Commission might create more interest in the elections and add a greater 'European' dimension to contests that have been classically characterised as 'second-order' (Hobolt, 2014). However, if vote choice is motivated by attitudes to the national economy as we suspect, this would undermine the idea that European elections were anything but elections where voters were motivated by domestic matters. Accordingly, assessing the influence of the national economy on vote is relevant. Additionally, few studies of EP elections have focused on the link between economics and vote (for an exception see Tilley, Garry, & Bold, 2008) and those that have done have done so through the prism of the 'second-order' model and in periods of general economic calm. But the 2009 and 2014 contests where held in the midst and aftermath of the Crisis. Couple this with the prominent role European institutions have played in shaping member states responses to the GFC, there is merit in reassessing its impact in these circumstances.
We develop a set of expectations about how the economy influenced vote choice in the 2009 and 2014 EP elections. In line with previous scholarship which has highlighted the importance of context (e.g. Anderson, 2000;Powell Jnr. & Whitten, 1993;Whitten & Palmer, 1999), we argue the impact of economy on vote is heterogeneous across both elections and countries. We expect that in 2009 economic perceptions directly influenced vote as the poll took place as the GFC was taking root and the effects were only becoming obvious. It also offered the first opportunity for most European citizens to have their say at the ballot box in a national contest, and thus a direct link is anticipated. By 2014, we expect the economy still influences vote but voter calculus might have shifted. We suggest voters' economic assessments will be conditioned by how much responsibility for economic performance they attribute to the national government. Building on a large literature that shows voter ascriptions of responsibility matter (e.g. de Vries, Edwards, & Tilman, 2011;Hellwig & Coffey, 2011;Marsh & Tilley, 2009;Tilley & Hobolt, 2011), we expect this change of calculus to be driven by a mixture of factors including that governments in power at the time of the GFC hit had been dismissed in many countries (LeDuc & Pammett, 2013), the initial shock of the GFC had dissipated with voters now well accustomed to the economic realities post-crisis, and voters by this point were now adjudicating on their government's response to the economic crisis, as much as responding to the economic context themselves. Thus, we expect the more a government is perceived to be responsible for economic performance, the stronger economic voting will be.
Our third expectation relates exclusively to the 2014 elections. We posit the impact of the economy on the vote might vary between countries based on whether a state received external financial aid or not in the preceding six years. The reasoning is simplebailout and non-bailout countries had different economic experiences with the crisis more pronounced in the former compared with the latter, with 'bailout states' having been more constrained as they were subject to scrutiny from external institutions. Consequently, we suggest the impact of economics on vote in 2014 will be more potent in countries that received a bailout.
Using a series of multivariate models based on data from the 2009 and 2014 European Election Study (EES), our expectations are largely borne out. Our analysis advances our understanding of economic voting and European elections in numerous ways. First, we demonstrate that during the economic crisis and its aftermath, economic perceptions played an important role in determining vote in the European Elections, challenging previous research suggesting economic voting in EP contests were minimal (Tilley et al., 2008). Second, we show economic perceptions influence on vote in EP elections are conditional on context, with perceptions being a direct motivator of vote choice in 2009 but economic assessments impact on vote conditioned by ascriptions of responsibility in 2014. This highlights the extent to which economic voting, while robust and clearly evident, is conditioned by context. Third, the idea that the 2014 EP elections were any different from past European elections is severely undermined. Clearly, EP elections retain a distinct and strong national flavour.
We structure our article as follows: we begin by reviewing the economic voting literature, defining our conception of economic voting, and then developing three hypotheses to test its impact on vote in the 2009 and 2014 EP elections. In section 3, we describe our research strategy and data. Section 4 details our empirical results while section 5 provides a summary of our results and their implications.
Defining the Mechanisms of Economic Voting in EP Elections
"It's the economy, stupid!"-the phrase coined by Bill Clinton's campaign team during his run for the American Presidency in 1992 stresses the importance political campaigns credit to the economy's impact on voters. And they do not appear to be wrong for the economy has been shown to influence vote time and again crossnationally (e.g. Duch & Stevenson, 2008;Lewis-Beck & Stegmaier, 2013;Lewis-Beck & Paldam, 2000;Lewis-Beck, Nadeau, & Elias, 2008;Singer, 2011). Economic voting comes in three forms: valence, positional, and patrimonial (Lewis-Beck, Nadeau, & Foucault, 2013). Traditionally, the valence model has gained most attention and is based on the premise that when voters consider the economy to be doing well, they are more likely to vote for the government, and conversely, when it is perceived to be underperforming, they are more likely to vote against them. This reward-punishment axiom has led Anderson (2007, p. 277) to note that "given citizens limited willingness and capacity to process complex information about politics, rewards and punishment should most easily be detectable with regard to the performance of the economy-after all, the economy is perhaps the most perennially talked-about issue". The economy is especially likely to be salient during an economic crisis (Dassonneville & Lewis-Beck, 2014;Singer, 2011). If we couple this with the fact that ideologically motivated voting has been declining, fewer citizens now have a predisposition to vote for a particular party (e.g. Dalton, 2006), and valence issues are more prominent than ever (e.g. Clarke, Sanders, Stewart, & Whiteley, 2009), we expect the economy will impact vote.
While an abundance of literature suggests a strong link between economy and vote, critics argue partisan bias heavily distorts voters' economic perceptions. In the words of Bartels (2002, p.138) "partisan loyalties have pervasive effects on perceptions of the political world". Thus, some scholars (e.g. Evans & Anderson, 2006;Wlezien, Franklin, & Twiggs, 1997) have contended political predispositions contaminate attitudes towards the economy and that economic assessments have no independent effect of their own. Assertions of endogeneity have been met with vigorous counterclaims that even when it can be fully teased out (for example using panel data and an instrumental variable approach), there is persuasive evidence to showing economics have a direct effect on vote, and if anything, cross-sectional analysis may suppress the true impact of economic voting (e.g. Fraile & Lewis-Beck, 2014;Lewis-Beck, Nadeau, & Elias, 2008). Our room for manoeuver in this analysis is limited as we only have access to cross-sectional data. However, we are buoyed by scholarship that shows even when endogeneity concerns can be conclusively addressed, the strong impact of economy on vote persists. As means of allaying concerns to the extent we can, we do control for partisanship in our models and do draw inferences across two cross-sectional samples rather than one. While not circumventing the endogeneity issue completely, this does allow us to have greater confidence in our conclusions than we otherwise might.
Much debate also rages about the mechanisms underlying economic voting. Existing research recognizes two different facets. First is whether voters are motivated by sociotropic or egocentric rationales, and second whether voters base their opinions on retrospective or prospective judgments. We deal with each in turn below.
Sociotropic motivations assume voters act according to their perception of the overall macroeconomic situation in their country while egocentric motivations are predicated on 'personal' utility with voters deciding on the basis of their personal economic gain or loss (Nannestad & Paldam, 1994). While we do not discount the possibility some voters in EP elections might be egocentric, we suspect most are motivated by sociotropic utility. We come to this view not only because most research suggests sociotropic considerations drive economic voting more (e.g. Anderson, 2000;Lewis-Beck & Stegmaier, 2013), but also because of the nature of EP contests. As elections are taking place simultaneously across the EU, we suspect benchmarking (Kayser & Peress, 2012) is more likely to take hold, where citizens compare the economic performance of their countries to others, and in doing so, are more likely to be making sociotropic rather than pocketbook comparisons. In any event, our measure of economic performance only allows us to explore sociotropic motivations.
Another area of debate is whether voters base their judgments on retrospective or prospective evaluations. Retrospective assessments assume voters' decisions are based on the past performance of the government and follow the premise that politicians are held accountable for their actions (Woon, 2012). On the other hand, such an assumption is incompatible with the idea that voters are forward-looking (e.g. Ashworth & Bueno de Mesquita, 2008;Gordon & Huber, 2007). Consequently, many argue that when casting a vote, the electorate are thinking about how politicians will handle the economy in the future (Woon, 2012). Besides the stronger evidence of retrospective voting (e.g. Duch & Stevenson, 2008;Lewis-Beck & Stegmaier, 2013), we assume that the characteristics of EP elections, particularly those of 2009 and 2014, makes retrospective voting much more likely. Voters in EP elections are not voting for a government and consequently are unlikely to judge the prospect of future economic dividends accruing from the election of individuals to the European Parliament, especially as the EU's economic power is distributed across a range of institutions from the Commission to the European Central Bank. 1 In sum, we expect economic voting in EP elections to be sociotropic and retrospective.
Economic Voting in the European Parliament Elections of 2009 and 2014
Our interest lies in deciphering the impact of the economy on vote in the 2009 and 2014 European elections. Traditionally European elections have been studied from the 'second-order' perspective with vote choice considered to be primarily influenced by attitudes to the incumbent government (e.g. Hix & Marsh, 2011;Reif & Schmitt, 1980;Schmitt, 2005). In recent years, other reasons beyond 'second-order' stimuli such as attitudes towards integration and citizens level of sophistication have been shown to influence voters as well (e.g. de Vries, van der Brug, van Egmond, & van der Eijk, 2011;Hobolt & Spoon, 2012). Nonetheless, EP elections continue to have a 'second-order' dimension, with elections campaigns dominated by domestic issues, low voter turnout, and established parties and incumbent governments losing votes (e.g. Cordero & Montero, 2015;Quinlan & Okolikj, 2016;Schmitt & Teperoglou, 2015). Accordingly, we would expect to find support for valence economic voting in EP elections with voters judging government performance on a domestic issue (the national economy).
Yet few studies have explored economic voting in EP elections. A notable exception is Tilley et al.'s (2008) analysis of economic voting in the 2004 contest. They concluded economic voting was limited and only observable among sophisticated voters and in countries that had single party governments in power. A more comprehensive assessment of economic voting using European election data comes from van der Brug, van der Eijk, and Franklin (2007). Using data from the European Election Studies between 1989 and 1999, they found that while the economy matters it is just one factor among many that influences vote. Further, its impact depends on its saliency as an issue, leading them to conclude that while the economy matters, its impact is not quite as important as we might have assumed. However, a re-assessment of the economy's impact on vote in EP elections is in order considering the GFC, which has put the economy front and centre of political debate since 2008 and seen EU institutions take a prominent role in dealing with the fallout from the Crisis. Given these circumstances, it can be expected the economy might have influenced vote choice to a greater extent in 2009 and 2014.
But how might the economy have shaped vote in 2009 and 2014? An abundance of research has previously demonstrated that context conditions economic voting (e.g. Anderson, 2000;Powell Jnr. & Whitten, 1993). We suppose context will also mediate the impact of economy in EP elections too and that its influence will vary across both elections and countries. Let us first take the differences between 2009 and 2014 polls. We suspect economic perceptions will have directly influenced vote in 2009 considering that the Crisis was still evolving, with the ramifications of the GFC only becoming gradually clear. The fallout consumed voters as unemployment rose, GDP declined, member states debt levels increased, and significant majorities of citizens across the Union judged their national economic circumstances as poor (see Figure 1). The 2009 EP elections also represented the first opportunity for most voters to pass judgment on the Crisis in nationwide elections. Of the twenty-seven member states, only in Lithuania had voters been to the polls since the GFC's critical tipping point of September 2008. 2 Given these circumstances, we expect economic perceptions directly influenced vote in 2009.
We infer that the economy still matters in 2014 but as the contextual circumstances were different at this point, we posit voter calculus will have shifted. By this stage, six years had elapsed since the GFC took hold and while many countries were still dealing with the fallout from it, the global economy had stabilized to an extent. In many member states, the economic outlook was looking better both at a macro level and in terms of citizen perceptions (for e.g. see Figure 2). Furthermore, many governments in power when the Crisis took hold had been dismissed. Thus, while we still expect economic perceptions to matter, we expect voters will take a wider view and incorporate how responsible they felt the government to be for the economic circumstances in 2014. A large literature has highlighted that ascriptions of responsibility matter (e.g. de Vries, Edwards, & Tilman, 2011;Hellwig & Coffey, 2011;Hobolt & Tilley, 2014;Marsh & Tilley, 2009;Powell Jnr. & Whitten, 1993). We contend responsibility attribution matters in 2014 because voters will have had time to absorb the shock of the GFC, and having already dismissed many governments in power at the time of the GFC, evaluations of new governments' handling of the 2 While the active phase of the GFC can be dated to early 2007, September 2008 remains an important crunch point in its evolution with the collapse of Lehman Brothers Bank in the United States and inter-bank lending seizing up, triggering a global economic shock which resulted in a number of European banks failing, stock indexes plummeting, and governments being forced to intervene in the economy in unprecedented ways. economy should become more prominent. Hence, we expect perceptions of economic responsibility (PER) will matter in 2014, and that they will condition economic voting. The more responsibility voters credit to the government for the economy, the more likely they are to vote for/against them, depending on whether they assess the economy to be performing good or bad.
Two critiques might be levelled at our suppositions. The first relates to the methodological issue of restricted variance regarding individual economic perceptions. Given the devestating effects of the GFC, there is an expectation that as everybody thought the economy was performing badly, at least in 2009, there might be little variation to explore (for more see Fraile & Lewis-Beck, 2014). However, inspection of citizens' views about economic performance at the time shows a more nuanced picture. Figures 1 and 2 detail the distribution of economic perceptions by country. In 2009 ( Figure 1) sufficient numbers of citizens in most member states had an alternative view to the conventional wisdom that the economy was performing poorly, although in some countries this respresented a small proportion of the electorate. Our cross-national strategy aids us here as with evaluations varying between countries, there is suficient variance overall to explore economic perceptions legitimately. Thus, we do not believe the restricted variance problem is something that plagues our analysis to a sufficient extent. Additionally, these distributions support our idea that as voter perceptions of the economy have changed between 2009 and 2014 (which inspection of Figures 1 and 2 by country illustrate they clearly have), this might play into economic voting being different between the two elections and between countries. The second critique is our focus on attributions of responsibility by voters. Critics suggest they might be contaminated by pre-existing political views (e.g. Bisgaard, 2015;Tilley & Hobolt, 2011). An alternative strategy might have been to look at responsibility delineation from an aggregate perspective using an index based on institutional criteria including a country's electoral and party system, majority status of government, and opposition influence, as previous work has done (e.g. Anderson, 2000;Powell Jnr. & Whitten, 1993). However, this assumption requires us to believe voters have detailed information as to how the political system operates and where the responsibility for power really lies, which is debatable. In any event and in the vein of many other studies (e.g.: Hobolt & Tilley, 2014;Marsh & Tilley, 2009;Sanders, 2000), we believe subjective perceptions are key. Controlling for the respondent's ideological distance from the ideological position of the party of the incumbent head of government on the left-right scale circumvents this problem to an extent. Further, we are buoyed by Lavine, Johnston and Steenbergen's (2012) work showing ambivalent partisans-voters who can suspend their partiality, do exist, and that these voters judge based on evidence. We also know suspension of partisanship is all the more likely to occur when there is sizeable consensus on an issue, such as a severe economic crisis (Parker-Stephen, 2013;Stanig, 2013) or when economic conditions are improving. Thus, while we accept that our measure is not ideal and thus a caveat to our conclusions, we are confident the issue is not as problematic as might first appear.
In sum, our expectations can be summarized as follows: H1: Economic assessments will directly influence vote choice in the 2009 EP elections.
H2: Voter attributions of responsibility to the national government for economic performance will condition the impact of economic assessments on vote choice in the 2014 EP elections.
We also expect the impact of the economy on vote choice in 2014 to vary by country. Specifically, we assume economic voting might differ between bailout and non-bailout countries. We classify 'bailout' countries as EU member states that received external financial assistance to avert sovereign defaults between 2008 and 2012. Eight states fall into this category: Cyprus, Greece, Hungary, Ireland, Latvia, Portugal, Romania and Spain. 3 Our supposition is the economic crises in bailout countries have been markedly worse compared to nonbailout countries and the consequences felt much more profoundly, especially as bailout countries were subject to much more stringent austerity measures, with their government's economic decisions were under external scrutiny from institutions like the EU and the IMF. Table 1 contrasts the positions of the two sets of countries on four prominent indicators of economic performance for the year 2014. As we can see, the eight countries who received external financial assistance performed significantly worse on average on three indicators: unemployment, youth unemployment, and government debt as a percentage of GDP. Only for economic growth are the bailout and nonbailout countries similar in performance. But if we remove the extreme outlier among the bailout countries for 2014, namely Ireland which recorded economic growth of 5.2% in 2014, the difference is much greater (1.34% growth for bailout countries versus 1.93% for non-bailout countries). In sum, there is clear evidence to suggest sizeable differences in economic performance between the two sets of countries and this might play into how economics influenced vote in the 2014 EP elections. Thus we suggest that: H3: Economic assessments and voter attributions of responsibility for economic performance will have a greater impact on vote choice in the 2014 EP elections in countries that have received bailouts from external bodies compared to those who have not.
Data and Primary Variable Classifications
Our data comes from the 2009 (van Egmond, van der Brug, Hobolt, Franklin, & Sapir, 2013) and 2014 (Schmitt, Popa, & Devinger, 2015) European Electoral Studies (EES), two cross-sectional comparative postelection surveys administered in all EU member states. The data includes identical questions posed to respondents that tap voters' assessments of the economy, who they perceive to be responsible for it, as well as other relevant political and demographic variables. Our dependent variable is binary and captures whether a respondent voted for the national governing party/coalition (coded 1) or another party/coalition (coded 0) on the basis of the question: "Which party did you give your vote to in these recent European Parliament elections?" Respondents who reported ab-staining were excluded from the analysis. 4 We have two primary independent variables. The first is perceptions of economic performance. Our measure is sociotropic and retrospective and is based on the question: "Compared to 12 months ago, do you think that the general economic situation in [our country] is…" In common with previous studies of economic voting, we recoded this measure into a binary variable, with a 1 capturing those who rated the economy as "Is a lot better" and "Is a little better" and 0 representing respondents who said "Is a little worse" and "Is a lot worse". 5 Our second independent variable of interest is responsibility attribution for the national economy. We capture this with responses to the question: "thinking about the economy, how responsible is [incumbent] government for economic conditions in [our country]". We refer to this as perceptions of economic responsibility (PER), which is scaled from 0 to 10, with a score of 0 indicating a voter does not hold the government responsible for the economy and a score of 10 representing voters who deemed the government fully responsible.
Our total N at the micro level across the two election cycles is 26,728 observations: 19,878 for the 2009 elections and 6,850 for the 2014 EP elections, with all country samples across both waves having a minimum of 1,000 initial observations and being representative of the national population. The difference in number of observation between 2009 and 2014 sample is a result of fewer observations available in 2014 and due to our classifications. 6 Our macro observations are 27 for both elections. 7
Modelling Strategy and Covariate Selection
The hierarchical nature of the EES data calls for a multilevel strategy. When observations within a sample are clustered, the data violates the assumption of independence of observations. Failure to take account of this could result in the incorrect estimation of standard errors, which can increase the probability of Type-I errors (Gelman & Hill, 2007). Multilevel modeling allows us to estimate separate variance components for both the micro and macro levels. For this study, we define two levels of analysis: citizens (micro-level) that are nested in countries (macro-level). 8 We estimate multilevel models with random effects for country and economy, as we expect the impact of economy will vary between countries.
We face difficulties in taking a multilevel approach in testing H3: the difference between bailout and nonbailout countries in terms of economy's impact on vote in the 2014 elections. With bailout countries only accounting for eight observations, a multilevel approach is infeasible because multilevel models with an inadequate number of macro observations have been noted for producing biased estimates (e.g. Maas & Hox, 2004;Stegmueller, 2013). One might assume we would circumvent this by estimating a multilevel (or indeed simple logit) model by just including a three-way interaction in our model. However, given the noted difficulties in interpreting interactions in logit models (Brambor, Roberts Clarke, & Golder, 2006), we suggest a threeway interaction is too convoluted and a more comprehensive interpretation is achieved from this strategy. Accordingly, Models V and VI in Table 3 are based on regular logit models with robust standard errors.
The remainder of our model is based on the wellestablished specification for testing economic effects in a comparative analysis (e.g. Duch & Stevenson, 2008;Lewis-Beck & Nadeau, 2012). Therefore, we control for religious service attendance as a proxy of cleavage and ideology. Our ideological measure captures the ideological distance of the respondent from the ideological position of the party of the incumbent head of government on the left-right scale. 9 To this, we add the in-For robustness, each model was re-estimated omitting each individual country to establish if a specific country was driving our results. We identified no substantive deviations, with the alternative models in line with our theoretical expectations. 8 We also estimated our models in the classic pooled analysis fashion and we identified no substantive deviations from our theoretical expectations. 9 We calculated ideology-distance variable as follows: we took the mean of respondents' placements of the head of government party on a left-right scale for each country and each election. Then we subtracted this mean from the individual respondent's self-placement on the left-right scale and took the absolute value of the results, which created the ideological distance variable. teraction term 10 between economic evaluations and perceptions of government responsibility in line with our theoretical expectations, to arrive at the following model: Vote = f (Cleavage, ideological distance, economy * perceptions of government responsibility) Summary statistics for each variable are included in the appendix (see Tables A2-A4).
Empirical Analysis
Our empirical analysis consists of three parts. First we are interested in establishing the direct effect of evaluations of the economy on vote choice. Models I (2009) and II (2014) of Table 2 test this. Second we add an interaction component to the basic model to test our second hypothesis-our expectation being the impact of the economy on vote might be mediated by how much responsibility is attributed to the national government for economic performance. We expect to observe heterogeneity between the elections-i.e. perceptions of economic responsibility will mediate the impact of economy on vote in 2014 but not in 2009. Models III (2009) and IV (2014) of Table 2 investigates this. Third, we explore whether the impact of economy and responsibility attribution is the same in 2014 between countries. We assume different effects will take hold in bailout compared to non-bailout states, with economy and the interaction between economy and responsibility attribution having a stronger impact in bailout states. We test this in Models V (bailout) and VI (non bailout) of Table 3.
In models I and II of Table 2, we see the economy variable is positive and statically significant in both models (p<0.01). This suggests that for respondents who judge the economy has improved in their country in the preceding twelve months before the election, the likelihood of voting for the government increased substantially. Such an effect is hardly surprising and confirms the potency of economics in shaping vote, even in a second-order election like the EP elections. 10 It should be noted that the magnitude and statistical significance of interaction effects in models with binary dependent variables can vary by observation (Ai & Norton, 2003). As a robustness check, we estimated the interaction effects following Norton, Wang and Ai (2004) and observed no significant deviations from our reported analysis. However, our supposition is the 'true impact' of economy only reveals itself when perceptions of responsibility for economic performance (PER) are accounted for. Therefore, we add an interaction term to our models. We detail the results in models III and IV of Table 2. It is evident that the inclusion of responsibility attribution and the interaction with economic assessments results in some important changes. First, PER on its own has an influence in both elections. In both 2009 and 2014, the more citizens thought the economy was the responsibility of the government, the more likely they were to vote against the government, hardly surprising given the economic circumstances in many states. Second, the strong direct effect of economic perceptions on vote persists: the more voters thought the economy was performing well (incidentally few did), the more likely they were to vote for the incumbent administration, and this effect remains independent of responsibility attribution. However, the interaction term in 2009 does not reach statistical significance. 11 This is in line with our expectations-economic perceptions were the primary driver of economic voting in the 2009 poll.
We see a different pattern for 2014 (Model IV). Here, the addition of attributions of responsibility results in the direct impact of the economy reaching statistical significance at p<0.05 level and still having a positive impact. However, the interaction term is positive and significant implying the impact of the economy is conditioned by voter perceptions of responsibility attribution. When the economy was considered to be doing well and the government were perceived to be re- 11 We were conscious that the magnitude and statistical significance of interaction effects in models with binary dependent variables can vary by observation. For robustness, we reestimated the interaction using Ai and Norton's (2003) approach and found that the significance levels and sign of the coefficients to be in same direction as those detailed in our analysis.
sponsible for it, voters were more likely to have supported the incumbent administration. To gain an idea of the magnitude of this effect, Figure 3 (for the 2009 EP election) and Figure 4 (for the 2014 EP election) plots the interaction effect between economy and perception of economic responsibility. Looking at Figure 4, the upper right plot indicates that when perception of economic responsibility is at the highest level, the probability to vote for incumbent government is around 60 percent for respondents who thought that the economy has improved in the last 12 months prior to the election. Yet, for those respondents who were more sceptical about the economy, the probability to vote for the governing party or coalition drops to around 30 percent. However, this difference is not so evident when the perception of economic responsibility is at its lowest (bottom left plot Figure 4). Here the probability to vote for the incumbent party between those respondents who answered that economy was better was approximately 50 percent compared to those who believed that the economy was worse in the past 12 months (approximately 40%). In sum, we deduce the following from our analysis. There is support for H1-the economy had a direct impact on vote choice in 2009. Voter assessments of the economy were enough to influence vote choice with those perceiving the economic situation to be good voting for the incumbent administration and those classifying it as worse voting against it. While economics also mattered in 2014, its impact on vote was conditioned by how much responsibility voters credited to the government for economic performance, with the more responsibility assigned to the government and the better the economic circumstances, the more likely voters were to vote for the government. The key point though is the direct effect of economy in 2014 was partially channelled through responsibility ascriptions, providing support for H2.
Regarding the impact of the economy and responsibility attribution on vote in 2014, we anticipated this effect may not be universal across the EU bloc but that it might have differed across countries dependent on whether a state had received a bailout or not. To test this, we divided our sample into 'bailout' and 'nonbailout' countries and devised two separate logit models to explore the differences between the two sets of nations. We expect in countries that have received a financial support from the EU/IMF, we will observe a greater level of economic responsibility: the perceptions of responsibility will have a stronger and negative effect on vote compared to non-bailout countries. This is because the economic situation in bailout countries was much worse compared to the non-bailout countries. Therefore, we would expect that the attribution of responsibility for the economic circumstances would have larger negative effect on the incumbent government among bailout countries.
Our results are detailed in Models V and VI of Table 3. Perceptions of the state of the economy do not have a significant effect on the vote in either set of countries, which fits with our overall theoretical argument that economic voting in 2014 was channelled through perceptions of responsibility. Instead, the differences between the two sets of countries are seen with respect to the impact of perceptions of responsibility and perceptions of responsibility when interacted with economic assessments. First, attribution of responsibility on its own had a significant effect in bailout countries-when governments were held responsible for the performance of the national economy, voters were more likely to vote against it. However, we do not have evidence of the same effect taking hold in non-bailout countries. We suggest this significant difference reflects the poorer state of bailout countries' economies.
The interaction effect in both models is positive and significant which is line with our expectations-the better the economy is and the more governments are considered to be responsible, the greater the likelihood of voting for the incumbent government. We conclude there is support for H3: voters in bailout countries approached the 2014 elections differently to the nonbailout. First and foremost, attribution of responsibility on its own mattered in these countries, while it did not in non-bailout states. Further, the impact of the economy was conditioned on this basis but also was shown to be stronger in bailout countries than non-bailout countries. Our overall results show that economic voting is present in both the 2009 and 2014 EP elections. However, this does mask clear differences as to how economic voting shaped vote choice between the two elections and cross-nationally.
Discussion and Conclusion
While much ink has been spilt exploring the influence of economy on the vote in national elections and referendums, and on attitudes towards European integration, few studies have analysed the effect of economic perceptions on vote in European Parliament elections. Yet the onset of the Global Financial Crisis (GFC), its domination of the political agenda over the past seven years, and the fact its marked effects are still very much felt, calls for a re-assessment. Our contribution explores the economy's impact in the 2009 and 2014 contests respectively.
Our study shows that like other elections, citizens' views about the economy do influence their vote in European elections. This is somewhat contrary to the conclusions of Tilley et al. (2008), who found minimal evidence for economic voting, and argued that existing political preferences contaminate economic perceptions. In a cross-sectional sample, endogeneity is difficult to rule out but we are confident that convincing evidence has been presented which shows that even when endogenous concerns can be conclusively addressed, economic voting exists. Accordingly, we suggest the differences in our findings compared to theirs are best explained by the different contexts. The Global Financial Crisis and its profound implications on citizens have ensured economics has been front and centre of the political agenda for the past seven years. As such, it became the central preoccupation of citizens and this translated into it becoming more important in shaping vote choice at the European level.
However, in line with previous scholarship which has found economic voting depends on context (e.g. Anderson 2000;Powell Jnr. & Whitten 1993), we found economics shaped vote in different ways across the two elections. In 2009, with the GFC in its infancy, and its consequences becoming gradually apparent, voters, many of whom were getting their first opportunity to go to the ballot box since the eruption of the crisis, were directly motivated by their perceptions of how the economy was performing. In 2014, economic perceptions still mattered. However, voter calculus shifted and the potency of economic voting depended on how much responsibility voters assigned to the national government for economic performance. The more responsibility they felt the government had, the more likely economic voting was to take hold. These assessments were seen to be stronger in countries that had received external financial assistance in the preceding six years, hardly surprising considering the GFCs impact on these states was more manifest than others. This underlines that economic voting, while prevalent in the European context, is conditional both on time and space. It also suggests that voters' response to economic crisis evolves, with economic perceptions initially enough to shape vote choice, but as time goes on, economic voting becomes motivated by other considerations including who is deemed to be responsible for the circumstances.
Our study also cast doubt on the idea that the 2014 European elections were much different compared to EP elections of the past, in spite of the characterisation of 'This time it's different' (see Hobolt, 2014). For one thing, economics mattered in both elections, albeit differently. But more fundamentally, voters in both remained strongly motivated by national considerations with domestic issues such as national economic perceptions playing a key role. Therefore, we can deduce the EP elections remain classic 'second-order' contests (for similar conclusions see Quinlan & Okolijk, 2016;Schmitt & Teperoglou, 2015).
Interesting avenues of research remain. While our study has focused on voters' ascriptions of economic responsibility to national governments, the attributions voters ascribe to the European Union remains underdeveloped (an exception is Costa-Lobo & Lewis-Beck, 2012). Additionally, economic voting is multidimensional. Here we have concentrated on the conventional valence reward-punishment model but other dimensions merit exploration. Gaining traction is the idea that there is a patrimonial aspect to economic voting with citizens' wealth has been found to shape voter preferences in Denmark, France, and Portugal (e.g. Costa-Lobo, 2013;Stubager, Lewis-Beck, & Nadeau, 2013). Given its existence at the national level, it might be that patrimony also influences voters in European contests and future studies should explore this. Note: Standard error in parenthesis; *p<0.1; **p<0.05;* **p<0.01. The upper right plot indicates that PER is at the highest level. The lower left plot indicates that PER is at the lowest level. | 2019-05-17T14:40:31.480Z | 2016-02-29T00:00:00.000 | {
"year": 2016,
"sha1": "6617cbbd74dc0bb59c5aee9a2e29c4320459ef2c",
"oa_license": "CCBY",
"oa_url": "https://www.cogitatiopress.com/politicsandgovernance/article/download/458/458",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "567f0a23b4c4e85d67df34c4f95e97d535fb1e27",
"s2fieldsofstudy": [
"Economics"
],
"extfieldsofstudy": [
"Economics"
]
} |
226363434 | pes2o/s2orc | v3-fos-license | A Dynamic-Bayesian-Networks-Based Resilience Assessment Approach of Structure Systems: Subsea Oil and Gas Pipelines as A Case Study
Under unanticipated natural disasters, any failure of structure components may cause the crash of an entire structure system. Resilience is an important metric for the structure system. Although many resilience metrics and assessment approaches are proposed for engineering system, they are not suitable for complex structure systems, since the failure mechanisms of them are different under the influences of natural disasters. This paper proposes a novel resilience assessment metric for structure system from a macroscopic perspective, named structure resilience, and develops a corresponding assessment approach based on remaining useful life of key components. Dynamic Bayesian networks (DBNs) and Markov are applied to establish the resilience assessment model. In the degradation process, natural degradation and accelerated degradation are modelled by using Bayesian networks, and then coupled by using DBNs. In the recovery process, the model is established by combining Markov and DBNs. Subsea oil and gas pipelines are adopted to demonstrate the application of the proposed structure metric and assessment approach.
Introduction
Resilience of engineering systems in the face of the variety of disruptive events has been attracted an enormous amount of research with the increasing of complexity and size of the systems. Structure system, made up of various mechanical components, is almost the most important part in an engineering system. Under the unanticipated natural disasters, any failure of the structure components may cause the crash of the entire structure system. Building resilience into structure system is the key to protecting against the deleterious influences of system disruption because of natural disasters. It is necessary to apply a comprehensive framework to assess the resilience of structure systems through a quantitative approach from a macroscopic perspective.
For certain specific systems, quantitative resilience assessment approaches have been proposed and reported. In the field of engineering systems, for instance, Henry et al. (2012) proposed generic metrics and its corresponding approaches to quantitatively evaluate the resilience of a road network; Shirali et al. (2013) proposed a novel approach for quantitative assessment of the resilience for the engineering system by using PCA and NT approaches; Hosseini and Barker (2016) quantified the infrastructure resilience with the example of an inland waterway port; Francis and Bekera (2014) contributed a novel metric incorporated adaptive capacity, absorptive capacity, and recoverability to assess the resilience of the engineering system and infrastructure system; Specking et al. (2019) proposed an analysis approach based on multiple objective decision to assess the engineering resilience for systems with multiple performance measures; MacKenzie and Hu (2019) proposed a resilience framework that is based on the value-driven design to en-able the assessment of engineering system resilience; Yodo et al. (2018) presented a resilience modelling and analysis approach through using the fundamental control theory to assess the resilience of complex engineering systems; Cai et al. (2018) proposed an engineering resilience metric based on the availability and its corresponding assessment methodology to assess the engineering resilience; Feng et al. (2019) proposed a resilience metric to describe the additional capacity of an engineering system, evaluate and design the system, and a case study on offshore wind farm is used to demonstrate the application.
Although various resilience metrics and corresponding assessment methodologies are developed, they are only suitable for assessing the resilience of the entire engineering system instead of the structure system. The failure mechanism of structure system is different from that of the entire engineering system. That is because the failure of the structure system is caused by the sudden changes of the fatigue mechanical properties, which is related to the mechanical properties of the structural components. The structural failure is usually caused by multiple factors which influence the structure components at the same time. For an engineering system, it usually consists of various subsystems, such as structure systems, control systems, and electrical systems. The failure mechanism of the engineering systems is complex. The evaluation metrics for engineering systems usually incorporate adaptive capacity, absorptive capacity, and recoverability, which are not related to failure mechanism. The performance modelling for the degradation process and recovery process of the engineering structure system is difficult. To the date, how to evaluate the structure resilience is still a challenging task.
Bayesian networks (BNs) can be described briefly as a directed acyclic graph, and it can graphically represent complex relationships between variables. Meanwhile, in the probabilistic knowledge representation and reasoning fields, it is one of the most useful modelling approaches. BNs are widely used in various fields, such as the assessment of system reliability, safety, risk, and resilience. For example, John et al. (2016) proposed an assessment approach through using BNs to enhance the seaport system resilience; Fu and Khan (2019) proposed a probabilistic framework to evaluate the operational risk quantitatively by using BNs; Cai et al. (2012) evaluated the reliability of the subsea blowout preventer control system by using BNs; Abimbola and Khan (2019) used the dynamic object-oriented BNs approach to assess the resilience of engineering systems. It is hard to model the transformation process between multiple states only using BNs when modelling the system performance changing process. The modelling problem can be solved by combining BNs with Markov model. For instance, Dhulipala and Flint (2020) proposed a semi-Markov process model to capture the interdependencies among events in recovery process when the infrastructure system is attacked by successive hazard events; Rebello et al. (2018) proposed an approach for assessing the system functional reliability by combining dynamic Bayesian networks (DBNs) and hidden Markov model.
The work aims to develop a novel resilience assessment metric and a novel resilience assessment approach of structure systems based on the remaining useful life (RUL) of key components. DBNs and Markov are combined to establish the resilience assessment model. The rest of the paper is organized as follows. Section 2 proposes the resilience assessment approach. Section 3 provides a case study of subsea oil and gas pipelines to demonstrate the application of the proposed metric and approach. Section 4 summarizes the work.
Modelling methodology for structure resilience
A new structure resilience assessment metric and its corresponding assessment methodology are proposed. The RUL of structural components is taken as the new structure resilience assessment metric. The resilience assessment methodology of the structure system is shown in Fig. 1. This methodology consists of three main steps: degradation process modelling, recovery process modelling, and resilience value calculation. In the degradation process modelling, the influences of single factors, such as natural factors and external factors, are modeled by BNs, respectively. The influences of natural factors are caused by nature environment, and the influences of external factors are caused by external disasters. The DBNs model is developed through coupling the single factors with each other to simulate the performance degrad-ation of the system. In the recovery process modelling, the DBNs model is developed by combining BNs with Markov model to simulate the performance recovery of the system. In the resilience calculation, the RUL changing trend is simulated according to the performance changing trends; and then the structure resilience is calculated based on the trend of the RUL.
Degradation process modelling
The factors that affect the performance degradation of structure systems are divided into two parts, namely natural factors and external factors. Natural factors refer to the factors which come from the nature environment, such as corrosion, sand erosion and so on. External factors are aroused by random external disasters, such as typhoon, earthquake, internal wave and so on. Assume that there are two factors which influence the system performance, named A and B, in which A is the natural factor and B is the external factor. The degradation models are shown as follows: (1) where P 1 is the system performance under the influence of natural factor A; P 2 is the system performance under the influence of external factor B; a 1 , a 2 , a 3 , b 1 , b 2 , and b 3 are variables. Firstly, when establishing the degradation model of the structure system, the degradation process of system performance is divided into natural degradation process and accelerated degradation process according to the sources of influencing factors. The performance models of the structure system with the impact of the different single factors are modelled by BNs, respectively. Then the degradation model for structure system with the impact of multi-factors is modelled by coupling the natural degradation model with the accelerated degradation model. The coupled multi-factor model is extended to a DBNs model according to time variation. At last, the performance degradation trend of structure system is obtained through the result of the DBNs model. The coupling equation is shown as follows: where P is the structure system performance with the impacts of multiple factors.
Recovery process modelling
All information which is required to predict a state at time t+∆t is contained at time t, and information about earlier time is not required. That is, the recovery process of the structure system follows Markov law. Markov law is used to describe the system that has multiple states and model the transition relationships among the multiple states. In the recovery process of the structure system, Markov law is used to describe the transition relationships, and the DBNs model is established according Markov model. The transition probabilities among time slices are depended on the failure rates of structure components. As shown in Fig. 2a, the system performance degrades from the working state S 0 to failure state S 1 under the influence of various degradation factors, and then the system performance can recover from S 1 to S 0 by taking the complete maintenance approach. When taking the incomplete maintenance approach, the performance can recover from S 1 to an acceptable state S 2 , as shown in Fig. 2b. According to the state transition diagrams of the system, it can be known that the state transition matrixes of the system are as follows:
Modelling of DBNs
DBNs' modelling is divided into two parts, namely structure modelling and parameter modelling. In the structure modelling process, the structure model is transformed according to the physical model. The nodes in the BNs represent variables in the physical model, and arcs represent the causal relationships among variables. Take the degradation process of the structure system under the influence of multiple factors as an example, In a time slice, the performance variables a 1 , a 2 , a 3 , b 1 , b 2 , b 3 … affect the physical performance nodes P 1 and P 2 , so the variable nodes and performance nodes are linked by using arcs. The physical performance nodes P 1 and P 2 affect the system performance node P, then these nodes are linked by using arcs. Finally, the structure of the static BNs is finished. For DBNs model, it is replication of static BNs from time t to t+(n−1)∆t. The variables, like a 1 , a 2 , a 3 , b 1 , b 2 , b 3 ... affect their state at the next moment, so the corresponding nodes between adjacent time slices are connected by arcs. The structure model of the DBNs model is shown in Fig. 3.
The parameter modelling of DBNs also includes two parts, namely intra time slice parameter modelling and inter time slice parameter modelling. Firstly, the prior probabilities of the root nodes are required to be determined when establishing the intra time slice parameter model. The prior probabilities or distributions are usually obtained from expert experience and historical data. However the trends of prior probabilities are usually the same for structure systems, but the quantitative values are different. According to the actual situation, the prior probabilities can be exact numbers, and the prior distributions can be exponential distribution, normal distribution, etc. Next, the conditional probabilities among child nodes and parent nodes need to be determined. For nodes that have a definite causal relationship, the conditional probabilities among them can be represented by standard values, such as 0 and 1. If there are exact expressions among nodes, the expressions can be transformed into conditional probabilities. When establishing the inter time slice parameter model, the transition probabilities among time slices also need to be determined. The transfer relationship among time slices follows certain rules, such as Markov law, or laws given by physical models.
Resilience calculation
The performance changing trend of the structure system can be integrated according to the DBNs models of the degradation process and the recovery process. The structure performance is determined by the estimated objects. For example, the performance can be reliability, safety, availability of structure systems, or crack depth. The structural performance changing trend is used to calculate the system RUL values. As shown in Fig. 4, the system is attacked by an external disaster at time t 1 , and the RUL degrades over time. At time t 2 , the structure system starts to be repaired and the RUL of the system is gradually increasing. Until time t 3 , the RUL of the structure system returns to a new stable value. The time period from time t 1 to t 2 is the degradation process, the structure system degrades from the working state to the failure state, and the time period from time t 2 to t 3 is the recovery process of the structure system, and it recovers from the failure state to a new stable state. The degradation time and recovery time are determined according to the expert experience and historical data. During the degradation process, a series of activities such as configurations of maintenance equipment and allocations of main-tenance personnel are in need to be completed. The degradation time is longer than the effect time of external shocks, and it may need two days. Recovery time refers to the period from the beginning to the end of the maintenance activity. In this process, the failure components need to be diagnosed, repaired, and installed to original location. The recovery time may need three days. The system resilience is calculated as the area ratio of the shaded part to the rectangular part which is enclosed by the dotted line and abscissa axis. The calculation equation is shown as follows: 3 Case study
Introduction of subsea oil and gas pipelines
To the date, subsea production system gradually becomes a mainstream mode of deep water oil and gas resources development with the development of offshore oil and gas industry extending to deep water. Subsea pipelines are important in the connection among various subsea equipments. What is more, subsea pipelines are also critical for the structure safety of the offshore oil and gas industry. Under the harsh working condition of the subsea, the subsea pipelines are often attacked by various external disasters, such as earthquake and internal waves. At the same time, the subsea pipelines can also be influenced by natural factors from the ocean environment, such as sand erosion and corrosion. The comprehensive influences of natural factors and external factors should be considered in the resilience assessment for the subsea pipelines. The RUL of the subsea pipelines is an important metric to assess the resilience. Crack is the main failure mode of the subsea pipelines, and it determines the RUL primarily. To quantitatively assess the resilience of the subsea pipelines with the impact of external disasters, the crack depths need to be evaluated firstly.
Corrosion and sand erosion are the main natural factors that cause the occurrence and growth of the crack. There are two types of the pipeline corrosion, namely external corrosion and internal corrosion. The subsea oil and gas pipelines are more susceptible to internal corrosion. For the internal corrosion, it is the electrochemical process which is formed through the presence of contaminants, like carbon dioxide and hydrogen sulfide. The internal corrosion starts in a local part, and it develops slowly. The corrosion rate is influenced by the concentration of carbon dioxide, temperature and pressure. For the external corrosion, the surrounding environment of the pipelines determines its mechanism. The increasing of the concentration of the dissolved oxygen on the metal surface, the increase of current velocity, and the decreasing of seawater temperature can significantly increase the speed of external corrosion (Yang et al., 2017). Sand erosion is another important reason that causes the occurrence of the crack. Sand erosion can arise severe crack in the internal wall for the pipelines, particularly for elbows and tees. The sand that included in the fluid can damage the protective film in the pipeline, so the failure rate of the pipeline is accelerated. The external factors are determined by the types of external disasters. Earthquake is one of the most typical environmental excitations during the service life of the subsea pipeline (Zhang et al., 2019). Many researchers focus on studying the influences of earthquake on the engineering systems. For example, Mazumder et al. (2020) presented a framework to evaluate risk and resilience of water distribution systems with considering both the influence of earthquake and time-variant corrosion of pipelines; Lin and El-Tawil (2020) simulated the resilience of lifeline systems in a test bed subjected to a series of seismic events. Under the influence of the earthquake, the ranges and numbers of cyclic stresses that suffered of the pipeline increase significantly. This phenomenon can lead to the sudden changes of the fatigue mechanical properties, resulting in rapid expansion of cracks and the failure of subsea pipelines.
The proposed resilience assessment metric and methodology are demonstrated through using the subsea oil and gas pipelines (elbows unless otherwise specified). At the same time, the combined influences of natural factors and external factors are studied in the case. Crack depth is selected as the system performance, and the RUL of the subsea oil and gas pipelines is taken as the assessment metric to calculate the resilience value.
3.2 Resilience assessment model of subsea oil and gas pipelines (1) Modelling of earthquake The pipelines are affected by wave load when they are influenced by the earthquake, which can lead to strong vibration of the pipelines. The physical model to demonstrate the influence of vibration is the Paris law (Luque and Straub, 2016;Straub et al., 2009), shown as follows: where dD(n)/dn is the crack growth rate; n is the number of cyclic stresses; C and M are empirically determined material parameters, and the value of C is related to M; D(n) is the crack depth; ΔS e is the equivalent stress range per cycle, which can be empirically represented as: λ where Γ() represents the Gamma function; is the scale parameter of Weibull distribution; K is the shape parameter of Weibull distribution.
Assume that the initial depth of the oil and gas pipelines is D 0 , the crack depth at the n-th stress cycle is calculated as: The physical model of the earthquake is mapped into a BNs model, and the structure model is shown in Fig. 5. The distributions and corresponding values of the variable nodes are obtained through the expert knowledge and historical data, as shown in Table 1. The performance changing trends of the pipelines under the influence of different strengths of external disasters can be modelled by changing the number and range of stress cycles. (2) Modelling of corrosion The corrosion of the subsea pipelines is mainly caused by CO 2 corrosion. Many companies have developed various corrosion prediction models in the oil and gas industry. The most widely used semi-empirical model is the De Waard95 model (Nešić et al., 2003). This model considers the comprehensive impact of temperature, partial pressure of carbon dioxide, pH value, dynamic process of corrosion, mass transfer process and so on. The corrosion rate is mainly related to the reaction rate and the mass transfer rate, shown as follows: where V corr is the corrosion rate; V r is the reaction rate; and V m is the mass transfer rate. V r and V m are calculated as (De Waard et al., 1991): +0.58 log P CO 2 −0.34(pH act − pH CO 2 ); (13) where t is the temperature of the fluid; P CO2 is the pressure of CO 2 ; pH act is the actual pH value; pH CO2 is the pH value of the CO 2 saturated solvent; U is the liquid flow velocity; d is the pipe diameter; n C is the fraction of CO 2 in the gas phase; p o is the operating pressure.
The physical model of the corrosion is mapped into a BNs model, and the structure model is shown in Fig. 6. According to the expert knowledge and historical data, the value of n C is 0.5 and the value of p o is 52. The corresponding values of other variable nodes are shown in Table 2. (3) Modelling of sand erosion Sand erosion is caused by the particles contained in the fluid, and the particles have sufficient momentum to impinge the pipe wall. Many parameters can affect the rate of the sand erosion, such as the liquid flow velocity, the fluid density, the size of the sand and the dimensions of the subsea pipeline. So it is hard to forecast the rate of the sand erosion exactly, and the prediction usually relies on semiempirical models. For the subsea oil and gas pipelines, one of the most used physical models is the Tulsa model, as shown below (Vieira et al., 2016), where R e is the rate of the sand erosion; K is the empirical parameter that is related to the material of the pipeline; F s is the particle shape coefficient, and it is equal to or smaller than one for sharp, semi-round and fully-round sand; V p is the particle impingement velocity; n is an empirical con-θ stant; f( ) is the empirical function for incorporating the particle impingement angle, and it is represented as: where is the characteristic value of the particle impingement angle.
The physical model of the sand erosion is mapped into a BNs model, and the structure model is shown in Fig. 7. According to the expert knowledge and historical data, the value of K is 2×10 −9 , and the value of F s is 0.02. The corresponding values of the other variable nodes are shown in Table 3. (4) Entire DBNs for degradation process of subsea oil and gas pipelines The three BNs models for the degradation process of the subsea oil and gas pipelines under the impacts of natural factors and external factors are integrated, and the integrated model is extended to construct the total DBNs model, as shown in Fig. 8. The performance degradation is affected by various factors, and the effects are quantitatively described by the physical models, like the Paris law, De Waard model, and Tulsa model. In the DBNs model, the transition probabilities between adjacent time slices are de- termined by the physical models. When integrating the entire model, the performance indexes of the three sub-networks need to be unified. In other words, the same time scale should be used to measure the variation of crack depth. Under the influence of the earthquake, the crack depth is calculated as the product of the crack growth rate and the number of cyclic stresses. Under the influence of the corrosion and the sand erosion, the corrosion rate V corr and the sand erosion rate R e need to be transformed into crack depth, and the value is calculated as the product of rate and time. In the integrated BNs model, the structure model is constructed by linking the performance nodes D n , V corr and R e in the three sub-network to the total performance node C. The parameter model is constructed by the relationships among D n , V corr , R e and C. In this case, the performance is represented by the crack depth. Crack is the main failure mode of the structure system, and it is closely related to structural mechanics principles. For the structure system, the crack depth can directly reflect the system performance. The subsea oil and gas pipelines are vulnerable to environmental assisted crack under the harsh working conditions. Environmental factors, such as corrosion and sand erosion, are the main natural factors that cause the occurrence and growth of the crack. RUL is selected to be the middle variable to develop a new resilience metric because it is influenced directly by the failure mechanism of structure systems. The RUL is calculated based on the total crack depth of the elbows, and then it is used to quantitatively assess the resilience of the elbows. Finally, the assessment model of the degradation process representing the performance changing trend is obtained by expanding the integrated model into a DBNs model .
(5) Modelling of recovery process of subsea oil and gas pipelines Maintenance measure is one of the most critical operations in operational activities of the subsea pipelines. According to the operation position, maintenance measure of subsea oil and gas pipelines can be divided into two parts, namely above-water operation method and underwater operation method. For the above-water operation method, it repairs the pipelines after lifting the damaged segment to the support vessel. The technology of this method is more mature and repair time is shorter. For the underwater method, the maintenance activity needs to be completed on the seafloor, and it can be divided into two parts, namely the repair in a dry-type cabin and the repair in water. The maintenance method is decided by the damage degree and the mechanism of the subsea pipelines, pipeline materials and so on.
Markov law is adopted to demonstrate the transition laws among different states when modelling the recovery process of the subsea pipelines. DBNs and Markov are combined to model the recovery process. The structure model is constructed according to the relationships among the sys-tem components; the parameter model is developed according to Markov law. Through considering the actual situation of the maintenance activities for the subsea pipelines, the pipelines are maintained in the 40th hour after the occurrence of the external disaster. A series of activities such as configuration of maintenance equipment and allocation of maintenance personnel are both needed to be completed after the occurrence of the external disaster. When modelling the DBNs model of the maintenance process, the prior probabilities are determined by the final state of the degradation process. For the transition relationships between adjacent time slices, they are represented by Markov law as: μ where p is the transition probability of nodes; is the maintenance rate, and it is determined by the maintenance time of the pipelines.
Structure resilience under different strengths of earthquake
According to the developed DBNs model of the degradation process, the distributions and corresponding probabilities for the crack depth of the elbows are shown in Fig. 9. It can be seen that the peak of probabilities for the crack depths move to the right over time under the combined impacts of natural and external factors. In other words, the crack depths of the elbows are gradually increasing as time goes on. After taking the maintenance activities, the distributions and corresponding probabilities for the crack depth in the recovery process are shown in Fig. 10. It can be seen that the peaks of probabilities for the crack depths move to the left over time, that is, the crack depths of the elbows gradually decrease as time goes on.
In the case study, the performance of the subsea oil and gas pipelines is represented by the crack depth. The RUL value is calculated through quantifying the crack depth of elbows. The value of the crack depth is calculated through the sum of products of the crack depths and the correspond- ing probabilities. The failure threshold is selected as 20.00 mm in the current case.
The structure resilience values under the influence of high, medium and low strength are respectively studied. The changing trends of the crack depths under different earthquake strengths are plotted in Fig. 11. It can be seen that the crack propagation speed becomes faster and the crack depth reaches to the threshold earlier with the increasing of the earthquake strength. In the later period, the crack depth increases almost exponentially, because the earthquake has greater effect on the elbows than others. Since the recovery process of the subsea pipelines follows Markov law, the maintenance time is longer if the crack depth is deeper, and the pipelines need more time to reach a new state. According to quantized crack depth of the elbows, the changing trends of the RUL with different earthquake strengths are calculated, as shown in Fig. 12. The RUL of the pipelines decreases rapidly with the increasing of the earthquake strength. The RUL value degrades to 0 only after 36 hours under the impacts of the high earthquake strength, that is, the pipelines reach a complete failure state. The pipelines will remain in the complete failure state if the maintenance measures are not taken by operators. The RUL of the pipelines will gradually recover to a stable state by taking the maintenance measures. However, the RUL of the pipelines cannot reach the original level because of the ex-istence of the natural factors, such as corrosion and sand erosion.
According to the resilience calculation method in Fig. 4, the resilience of the elbows under the influence of different earthquake strengths are calculated, and the results are shown in Fig. 13. They are 77.17%, 73.61% and 72.02%, respectively. The resilience gradually decreases with the increasing of the earthquake strength. It can be seen that the resilience values are not significantly different. The impact of the earthquake on the subsea pipelines is enormous, and the pipelines can reach the complete failure state in a short time period. The strength changing has little impacts on the pipelines, so that the resilience values are not significantly different.
Structure resilience with different initial crack depths
It can be seen from Fig. 14 that the crack depth grows more rapidly with the increasing of the initial depths. When the initial crack depth of the elbows is 0.1 mm, the crack depth grows very slowly in the early stage of the degradation process, and the crack depth only increases to 5 mm in the 30th hour. In the later stage, the crack depth grows rapidly over time, and the changing trend is almost exponential. When the initial crack depths of the elbows are 0.15 mm and 0.2 mm, the degradation trends are almost similar. The growth rate of the crack is obviously increased in the early period, and the crack depth grows to 5 mm in the 18th hour. In the later period, the crack depth grows very rapidly, and it increases almost exponentially. The RUL changing trends with different initial crack depths are shown in Fig. 15. The RUL of the elbows decreases more rapidly with the increasing of the initial crack depth. The recovery trends are totally the same because the elbows all reach the complete failure state during the degradation process. According to the changing trends of the RUL, the resilience values for the elbows with different initial crack depths are calculated, and the results are shown in Fig. 16. They are 78.43%, 73.61% and 73.22%, respectively. The structure resilience of the elbows gradually decreases with the increasing of the initial crack depth. When the initial crack depth of the elbows are 0.15 mm and 0.2 mm, the degradation trend and recovery trend are almost similar, so the resilience values have no big difference.
Structure resilience for different pipeline components
Elbows, tees and straight pipes are three significant components of subsea pipelines. In this part, crack depth growths, RUL changing trends and resilience values of tees and straight pipes are studied, and the results are respectively compared with the elbows. It can be seen from Fig. 17 that crack depth of elbows is the largest over time, the crack depth of straight pipes is the smallest, and the value of tees is in between. That is because the effect of the sand erosion on the straight pipes is the smallest, and the tees are generally made of materials which are resistant to corrosion and sand erosion.
The changing trends of the RUL for the different parts of the subsea oil and gas pipelines are shown in Fig. 18. The degradation speed of the RUL for the straight pipes is the slowest, the degradation speed of the elbows is the fastest, and the degradation speed of the tees is in between. These are corresponding to the crack growth trends for the elbows, tees and straight pipes. At the same time, the recovery trends are totally consistent for the elbows, tees and straight pipes because they all reach the complete failure state during the degradation process. CAI Bao-ping et al. China Ocean Eng., 2020, Vol. 34, No. 5, P. 597-607 According to the changing trends of the RUL, the resilience values of the elbows, tees and straight pipes are calcu-lated, and the results are shown in Fig. 19. They are 73.61%, 74.8% and 75.99%, respectively. The resilience for the elbows is the lowest. The overall pipeline system will fail if the elbows are in failure, so the overhaul cycle for the elbows should be shortened to enhance the resilience of the overall pipeline system.
Parameter sensitivity analysis
Parameter sensitivity analysis is the analysis of how sensitive the results of a belief update is to variations of the value of a parameter of the model. It is measured according to the variance of beliefs and variance reduction. The parameters sensitivity analyses for the different nodes are conducted in the 1st hour, 20th hour and 40th hour, and the results are shown in Fig. 20.
In the early period of the degradation process, i.e. the 1st hour and 20th hour, the corrosion has a great influence on the crack depth of the pipelines. In the later period, i.e. the 40th hour, the earthquake has a great influence on the pipelines. In the early period, the initial depth of the crack is small, and the depth is more sensitive to the mass transfer velocity. The fluid velocity can considerably affect the corrosion and the sand erosion, so the corrosion and sand erosion contribute greatly in the crack growth. When the initial crack depth of the elbows is big, the influence of the earthquake can quickly deteriorate the degradation process, so it dominates the crack depth propagation in the later period. Other variables, such as pipe diameter, operating pressure, and fluid temperature have small sensitivities on the crack depth growth.
Conclusions
This paper proposes a new resilience assessment metric and its corresponding methodology based on the RUL of the key components of the structure systems. The weakest parts, that is, the elbows of subsea oil and gas pipelines are used to demonstrate the application of the metric and methodology. The influences of the strengths of the external disasters, the depths of initial crack, and the types of pipelines on the structure resilience are researched, and the parameter sensit-ivities are analyzed. The results show that the assessment metric and methodology can be used to calculate the resilience of structure systems. Under the integration impact of natural factors and external factors, subsea oil and gas pipelines can reach the complete failure state within 40 hours. The system resilience decreases with the increasing of the strengths of external disasters, and corresponding resilience values are all below 80%. The initial depth also affects the degradation speed greatly. When the depth of the initial crack for the elbows is larger than 0.1 mm, the pipelines are totally failed within 36 hours. Therefore, the initial crack requires to be strictly detected and controlled. The pipelines are sensitive to the mass transfer velocity in the early period, that is, the corrosion has a larger effect on the pipelines, and they are sensitive to the stress range in the later period, that is, the effect of earthquake is larger. | 2020-10-29T09:04:22.770Z | 2020-09-01T00:00:00.000 | {
"year": 2020,
"sha1": "a2f2b81171df11935ceb7add0c9ca631a6c469cc",
"oa_license": "CCBY",
"oa_url": "http://www.chinaoceanengin.cn/article/doi/10.1007/s13344-020-0054-0",
"oa_status": "HYBRID",
"pdf_src": "SpringerNature",
"pdf_hash": "3fa2df04ac15589150db62174319ccb296db6c00",
"s2fieldsofstudy": [
"Engineering",
"Environmental Science"
],
"extfieldsofstudy": [
"Computer Science"
]
} |
86525192 | pes2o/s2orc | v3-fos-license | Adding Support for Automatic Enforcement of Security Policies in NFV Networks
This paper introduces an approach toward the automatic enforcement of security policies in network functions virtualization (NFV) networks and dynamic adaptation to network changes. The approach relies on a refinement model that allows the dynamic transformation of high-level security requirements into configuration settings for the network security functions (NSFs), and optimization models that allow the optimal selection of the NSFs to use. These models are built on a formalization of the NSF capabilities, which serves to unequivocally describe what NSFs are able to do for security policy enforcement purposes. The approach proposed is the first step toward a security policy aware NFV management, orchestration, and resource allocation system—a paradigm shift for the management of virtualized networks—and it requires minor changes to the current NFV architecture. We prove that our approach is feasible, as it has been implemented by extending the OpenMANO framework and validated on several network scenarios. Furthermore, we prove with performance tests that policy refinement scales well enough to support current and future virtualized networks.
I. INTRODUCTION
Currently two technologies seem to have the power to significantly change computer networks: Network Functions Virtualization (NFV) [1] and Software-Defined Networking (SDN) [2].NFV proposes a virtualized infrastructure where network functions are implemented by software appliances, named Virtual Network Functions (VNF), that are decoupled from physical network devices.SDN introduces the possibility to dynamically reconfigure the network, by selecting arbitrary paths and redirecting traffic through specific middleboxes.These technologies are in synergy.NFV fosters flexible and programmable network function deployment and ease of scaling, and its adoption is expected to reduce administration tasks, response times, and TCO (Total Cost of Ownership).SDN enables a more efficient use of resources, as selected traffic can be dynamically redirected to virtual and physical network functions, e.g., per-user and per-flow basis.
Flexible management is one of the main advantages of these new networking paradigms.However, in NFV flexibility cannot be extended to security controls, named (virtual) Network Security Functions (NSFs) 1 , as the enforcement of security policies requires careful adaptation every time a change is made.In fact, while networking elements and protocols are designed to automatically adapt to changes, dynamic adaptation is not always a design principle for NSFs.Therefore, in C. Basile, A. Lioy, F. Valenza are with the Politecnico di Torino, Dip.Automatica e Informatica; e-mail: {first.last}@polito.it, A. Pastor, D. R. Lopez are with Telefónica I+D; e-mail:{first.last}@telefonica.com,This work has been partly supported by the SECURED and SHIELD project (grant agreements no.611458 and 700199) , co-funded by the European Commission.
1 https://datatracker.ietf.org/wg/i2nsf/charter/this paper we propose to take advantage of the flexibility of networking management to also achieve flexible enforcement of security policies.The research in this paper addresses two important challenges, as highlighted by works literature [1], [3].First, it supports the automatic enforcement of security policies.Given a target network and a set of security requirements expressed with a high-level language (i.e., the security policy), our model supports the configuration2 of all the security functions in the target NFV network so that the security policy is enforced.Our model identifies and reports non-enforceability issues when the security policy requires security functions not available in the target network (e.g., to filter URLs without having a HTTP filter).Second, we support dynamic adaptation to network changes.We enable administrators to react and maintain the security policy enforced in case of any topological change of the network, like NSF failure or substitution of a NSF with another one that implements the same security functions (e.g., substitution of a firewall with a different one from another vendor with equivalent filtering capabilities), and any addition/removal of VNFs or NSFs.More precisely, given 1) a set of security requirements expressed with a high-level security policy language, 2) an initial network already configured to enforce this policy, and 3) a target network (different from the initial one), our model allows the configuration of all the NSFs so that the security policy is enforced in the target network.Non-enforceability issues are reported in this case too.
The long term objective of our research is to support a broadest scenario: security policy aware network management and orchestration (SPA-MANO), where only the functional and security requirements are specified, while the network topology and the security functions that optimally implement the security policy are automatically decided and allocated in a way that is transparent to the administrators.
We address the two challenges above in NFV networks by adding a new component, the Security Awareness Manager (SAM), as a part of the Orchestrator.Indeed, the two scenarios are very similar and we addressed them by means of policy refinement 3 .The SAM is in charge of executing the policy refinement when the network is initialized and every time a change is detected into the network or into the security policy.Our policy refinement approach also includes a decision phase where an optimization model allows selecting the best way to refine the policy in the target network, according to (network and device) performance, and security.Although other works exist on policy refinement, to the best of our knowledge there are no initiatives that propose to support automatic security policy enforcement in NFV with policy refinement.
In addition to the refinement and optimization models, this paper presents other contributions.To make policy refinement feasible we have developed a solution to a known open issue 4 : to find an unequivocal method to represent the security features that an NSF could provide for policy enforcement purposes.We propose the capability model, which is presented in Section III, where capabilities are a compact yet precise way to determine which policies an NSF can implement, as they describe the actions that can be enforced and the conditions for their enforcement.Moreover, capabilities describe other policy-related aspects that a refinement system must know when generating configurations for target NSFs (like the supported resolution strategy or how to set the default action).Other works related to a capability model exist, however, they address a similar issue but only for traffic flow control [6], or model virtual resources for allocation purposes [7].Instead, we specifically model NSFs capabilities.
On top of the capability model, we have built the Medium-Level Policy Language (MLP), an abstract language to represent NSF configurations with a vendor-independent but capability-aware format (see Section IV-C), another known open issue in literature [1].
Finally, we have developed a High-Level Policy language (HLP), which allows easy specification of security requirement at a high-level of abstraction.The HLP has been defined to specify the security policies useful in our use cases.Other high-level languages exist in literature, mainly for access control policy specification (see Section VIII-C), but, as in our case, they only solve problems related to specific use cases.
We tested the validity of our approach by specifying security requirements and automatically enforcing them in NFV networks deployed with OpenMANO [8].First, we proved that adding the SAM in a NFV architecture is straightforward and effective, as we easily included the SAM in OpenMANO.Then, we used the SAM to enforce security requirements into a set of target networks, by refining policies into configurations for all the NSFs.Both security requirement and target networks have been taken from the use cases of the EU project SECURED, thus we proved the applicability to real cases (see Section VII-B).Moreover, we have performed in-depth scalability testing on synthetic-yet-realistic networks and we have found promising results (see Section VII-C).
Note that, we don't think that our models can be directly adopted by working groups or used in practice as they are, as several other low-level and management aspects need to be considered at large.However, they were essential to prove that using policy refinement for easy management of VNF is feasible, and it can be done by using a capability model and an abstract representation of the policy.
The paper is structured as follows.Section II introduces our approach, the SAM, and shows how to implement it in the current NFV architecture.Section III presents the foundations of our capability model and the algebra for composing capabilities.Sections IV and V discuss the models to refine HLP policies and allow optimal selection of NSFs to use for policy enforcement.Section VI sketches how dynamic adaptation to network changes is managed with our approach and Section VII describes the implementation of our approach and the results of the performance analysis.Section VIII presents the most important related work in three areas, NFV management and orchestration, resource optimization, and policy refinement.Finally Section IX draws conclusions, discusses on the applicability of this approach and depicts the next steps towards a security policy aware MANO.
II. APPROACH
We now discuss that supporting automatic enforcement of security policies and dynamic adaptation to network changes is a feasible task that requires minor changes to the current NFV architecture.
A. Background
ETSI has defined the Management and Orchestration Framework (MANO) to provide the required functionality for provisioning and configuring VNFs as well as the configuration of the infrastructure where these functions must run.MANO also provides orchestration and life cycle management of VNF resources.We shortly sketch here the main features, while further details, which are not needed for this treatment, can be found in a survey from Mijumbi et al. [1].
The main NFV MANO components are the Virtual Infrastructure Manger (VIM), the VNF Manager (VNFM), and the NFV Orchestrator (NFVO).The VIM manages and controls a NFV Infrastructure (NFVI).NFVIs are combination of physical and virtual resources where VNFs are deployed.A NFV architecture may contain many VIMs, if there are more NFVIs provided by more infrastructure providers.The VNFM ensures the correct life cycle management of VNFs, which include instantiation, scaling (increase or reduce the resources associated to a VNF), and termination.The VNFM may resort to a (legacy) component, the Element Management system (EM), to manage selected VNFs.NFVO orchestrates resources and services.Moreover, it may allocate end-to-end services by composing VNFs [9].
The MANO may access information from ad hoc repositories that store data needed for its operations (e.g., the Network Service catalogue (NS) and the VNF catalogue).However, it may also collect additional data as Network Service Descriptors (NSDs).A NSD consists of static information that describes deployment flavours of Network Service and potential dependencies, like the VNF Forwarding Graphs, Virtual and Physical Network Functions, and Virtual Links.
Even if the current NFV specifications state that VNF Forwarding Graph information elements contain Network Forwarding Path elements that are used to route traffic "which in turn include policies (e.g.MAC forwarding rules, routing entries, etc.) and references to Connection Points (e.g.virtual ports, virtual NIC addresses, etc.)" [9], there are no examples of Network Forwarding Path in the standard appendices.Moreover, the tools that we have examined, namely OpenMANO and OPENBATON, do not support forwarding information.That is, the network services that can be specified are chains of VNFs.ETSI proof of concept 28 (POC#28) on NFV is looking for effort towards SDN Controlled VNF Forwarding Graph, but it is an ongoing activity.Since we internally use abstract service graphs to represent networks for policy refinement purposes, this lack of available standard representations does not affect our work.
For our experimentations, we have specified target networks as a set of NSDs, each one describing a chain, connected by means of external SDN routing information by OpenFlow rules.These rules have also been used to route the traffic of our sample client nodes towards the network services.
B. Reference scenario
For the sake of clarity, we introduce a simplified scenario to be used whenever an example is needed to contextualize our approach.
A Network Service Provider (NSP) provides network and security services for its customers.The NSP uses five categories of NSFs: packet filters (PF), web application firewalls (WAF), parental controls (PC), virtual private networks (VPN), and network security monitors (MON).The NSP uses a single application/control for each category of NSFs, namely: PF is implemented with Iptables; WAF is enforced by Squid; PC is implemented by E2gardian; VPN uses IPsec+IKE features implemented by a Strongswan instance; MON is implemented by Bro.NSFs are provided as service function chains.
A company buys the network and services offered by the NSP and wants three security requirements enforced, expressed here as HLP-like statements: 1) "Enable malware detection in PDF attachments in corporate email"; 2) "Allow employees to connect to social networks at lunch"; 3) "Protect confidentiality of traffic between Turin and Madrid offices".
Additional information about this scenario will be discussed in Section VII-B, where we present the validation of our approach, and in the supplementary downloadable material, where we report all the input and output data necessary to validate our approach on this example in the NFV context.
C. The Security Awareness Manager architecture
To have a security policy aware orchestration, we propose the use of a new component, which we name Security Awareness Manager (SAM).The SAM transparently enforces user security requirements by providing an additional layer between the administrator and the NFV orchestrator that performs the following key features: 1) interpretation of the security policy and identification of the NSFs that may enforce the policy, 2) generation of the configurations of the NSFs that will implement the security policy, and 3) invocation of the standard MANO management operations to deploy the generated configurations.
In our implementation, the SAM is a component that provides additional features to the NFVO but whose functionality logically pertains it.However, in the future, the SAM could be part of the NFVO.In practice, the SAM is a component that has to be connected to the NFVO to provide its policy-related services, but it is immaterial, with the current NFV standard, whether it is an internal module of a security-aware NFVO or a separate module that is part of an external framework.
The SAM requires a dedicated repository to perform its operations: the Policy Repository (PR).In principle, the PR will be in charge for storing all the policy abstractions needed for security policy awareness purposes, including the input security policies and the output NSF configurations.We will show in the next sections that in our policy refinement approach, the PR stores the input policies (expressed with HLP), the abstract NSFs configurations (in MLP), and the concrete configurations of all the needed NSFs (expressed in their own configuration languages).We also store in the PR all the intermediate data structures in the NSDs (used for policy enforceability purposes) and the reports produced by the refinement process (to inform the administrators in charge for configuring the NFV architecture and enforcing the policy).Furthermore, the SAM accesses an internal Knowledge Base (KB) to store the semantic of high-level concepts, that is, the mappings between high-level HLP concepts to the network-related low-level information needed to implement the policy (e.g., network topology info, addresses, black lists).For instance, in our example, we have introduced mappings to associate "social networks" with the URLs of social network web sites, "Turin offices" with the public IP addressed of the Turin offices, and "corporate email" is associated to MIME objects from/to the mail server defined with IP, ports and protocols.Identifying these mappings is a laborious yet easy task.To implement our prototype, we followed a bottomup approach; we have written the configurations of several security controls able to enforce the statements allowed by the HLP, found the involved concepts, and added into the KB the proper mappings.Unfortunately, every time that a new HLP concept or new types of statement is introduced in the HLP, new relations could be needed by the KB.However, we expect open source initiatives or companies interested in commercializing products to perform these maintenance operations.Analogously, while we manually populated the KB for our experiment (i.e., by defining all the mappings), we expect that, in future, filling in the KB will be (almost completely) performed by including external sources of information, such as corporate directory services, DNS data, virus DB, and URL lists 5 .Finally, the SAM retrieves the network and VNF informations from the VNF repository.
We have integrated and tested the SAM in OpenMANO, an open source project led by Telefónica, which provides one of the most up to date open source implementations of the NFV MANO reference architecture [8].OpenMANO has been preferred due to its modular architecture, which has allowed us a smooth insertion of the new functionalities.Furthermore, OpenMANO is actually used by Telefónica to manage, deploy and test different types of VNFs in their labs, and currently evolving into a complete MANO solution supported by a wide community through the OSM project.Thus OpenMANO is a working software, not only a prototype, and it is currently maintained and extended by Telefónica.Moreover, recently OpenMANO has become the starting point of Open Source MANO, the official ETSI NFV reference architecture [10].The integration of the SAM to OpenMANO has shown a promising impact on policy-based NFV management.The results in Sections VII show that adding support for policy awareness does not significantly delay the NFVO operations.
D. The Security Awareness Manager workflow
Fig. 1 shows the SAM and the new storages (in gray) together with the ETSI NFV components (the other white boxes), and the interactions among them.The adoption of our approach imposes additional steps to the ETSI NFV workflow, which are represented as dotted arrows (the solid ones are already foreseen by NFV).Further details on the steps are presented in the next sections.
The workflow starts with a user writing his own security requirements as an HLP policy (step 1), which is stored in the PR.The creation of the user policy triggers the following steps.The SAM, after being notified about the availability of a new policy, retrieves the HLP policy (step 2).The SAM, by using the information in the KB (and information about target network, that is the NSDs and SDN information as OpenFlow rules), identifies the capabilities NSFs needed to enforce the HLP policy then it selects the best set of NSFs in the target network to enforce the identified capabilities.If the network does not contain NSFs able to enforce the policy, the user is informed about the non-enforceability issue and a log is generated.If the policy is enforceable, the SAM first refines the HLP policy into a set of abstract MLP policies for all the selected NSFs (with the mapping stored in the KB) then it translates them into the corresponding NSF configurations (step (3), Sections IV-D, IV-E, and V).The SAM stores the MLP and the configurations in the repository (step (4a)), and notifies the NFVO that new information is available into the PR ((step (4b))).The NFVO retrieves the NSF configurations from the PR and the NSD from the standard repositories (step (5)).The NFVO contacts the NVFM to instantiate the selected NSFs through the VIM and passes it the configurations (step (6)).The actual instantiation is performed by the NFVI and VIM (steps 7a and 7b).The actual configuration of the network functions is pushed by the NFVM through the Element Managers (EM) of each involved function (step ( 8)).
Note that, if our approach is not used, the configurations for all the NSFs need to be manually written and pushed through Operation and Business Support Systems (OSS/BSS) by the administrators.
E. Advantages of the SAM
Currently, only the resources to be assigned to the each NSF (e.g., memory size, processor utilization, storage) and available in the VNF descriptors are considered for the allocation of VNFs.However, a security policy aware MANO, given the information generated during the refinement process by the SAM, may use algorithms that allocate the NSFs of all the users (and customers) that work better than the existing ones, because more data are available to base their decisions.For instance, if users need to perform packet filter, a SPA-MANO may decide to add the rules that enforce the user policy into an existing Iptables NSF and forward traffic with SDN, or if users ask (and pay) for isolation, it may allocate a separate NSF.This decision may be led by the resources available at the nodes, performance considerations, on the network delays introduced to forward the traffic, the workload of the NSF (which will also include the number and type of rules to enforce).Moreover, a SPA-MANO may decide based on more precise performance models of security functions, which may also consider the number of the rules they have to implement (which are currently available for some packet filters [11]) and the condition types of each rule (e.g., if rules use conditions as regular expressions [12]).As an example, this information can be used to determine how many rules to insert before deciding to allocate an additional NSF, as for some functions, performance is stable until a threshold is reached, then it dramatically increases.Even if it is not in the scope of this paper, optimized allocation algorithms that also consider policy-related information is an interesting research area we are considering for future work.
Finally, another important aspect that is outside the scope of this research paper but needs to be addressed before using this refinement system in the current NFV implementations, is determining the proper order of configuration of the NSFs.Indeed, the glitches arising from transition from a policy configuration to a new one can lead the target network to inconsistent states that may have devastating effects on both functionality and security.We are not aware of existing systems able to perform this task, research should focus on extending the features of the VIM.
III. CAPABILITY MODEL
As human beings, administrators and network security experts understand each other when referring to security controls by just naming categories.For instance, experts unequivocally refer to "packet filters" as stateless devices able to drop packets based on conditions on source and destination IP addresses, source and destination ports, and IP protocol type fields [13].Moreover, it is known that packet filter rules are prioritized 6and it is often possible to specify a default action.
However, more information is needed to better clarify the behaviour of, for instance, stateful firewalls or application layer filters.Controls that fall in these categories may show substantial differences (e.g., depending on the vendor/product) on how they classify packets and communications to determine when to apply decisions, maintain the stateful information, and process protocols.That is, they are in the same category but they cannot be used interchangeably.Also communication protection protocols are usually considered as a single category.Indeed, all of them protect packets 7 with symmetric algorithms whose keys could have been negotiated with asymmetric cryptography, and have a clear behaviour defined by standards.However, each protocol has its own peculiarity, e.g., it works at a different ISO/OSI layer and support different encryption algorithms, and authentication, key agreement and negotiation abilities.Moreover, implementations of these protocol show further minor differences.Therefore, even in this case, no interchangeability is ensured.
For this reason, a capability model is needed to precisely and unequivocally clarify what a security function can do in term of security policy enforcement.With such a model, automatic systems, like the refinement engine we are proposing, and human beings can understand 1) whether a function is suitable or not for implementing a high-level policy and 2) how it can be configured to enforce such high-level security policy.
To build our capability model8 , we have analysed several NSFs that fall in different categories, including packet filter, URL filter, HTTP filter, VPN gateway, anti-virus, antimalware, content filter, monitoring, anonymity proxy.Moreover, we have also analysed common extensions of the generic NSFs, like optionally loadable modules (e.g., iptables supports the addition of modules with the -m option, some module is standard "state" and included in the base distribution, some needs to be explicitly downloaded and installed, like "time").Therefore, based the geometric model of policies [14], we have modelled the capabilities of a NSF in two parts: 1) rule-based capabilities, which describe how rules can be written in terms of actions and conditions (see Section III-A); 2) policy-based capabilities, which describe how to write a coherent policy given a set of rules specified according to the rule-based capabilities (see Section III-B).We have identified a list of rule-based and policy-based capabilities (presented in the the supplemental material provided with this paper).Nevertheless, our model is extensible as additional capabilities can be added if required to describe new security functions.Our capability model is provided with a set operations to manipulate capabilities by means of a capability algebra (see Section III-C).
A. Rule based capabilities
Security functions are able to enforce actions and own traffic classification features.Actions (A) describe the operations that a security function can perform on packets/flows, like filtering or encrypting traffic, and operations that are not performed directly on the traffic, like logging matching rules or notifying events.We assume that all the actions available at a security function are well known and organized in the NSF action set.For instance, the action set of a generic packet filter will include the Allow and Deny actions.Communication protection functions will have action sets that depend on the technology.For instance, NSFs based on IPsec will have action sets that include actions expressing the protocol (AH vs. ESP), and the mode (tunnel vs. transport mode).
Classification features (C) concern the possibility of the security function to identify target packets/flows on which the actions could be enforced.Classification features are specified by means of conditions clauses that are logical formulas of conditions [14].Conditions are typed predicates defined over given selector.A selector is an abstract representation of the values that a protocol field may take, e.g., the IP source selector is the set of all possible IP addresses, and the part of the packet where the values come from, e.g., the IP source selector refers to the IP source field in the IP header.Moreover, selectors may imply a list of allowed values, such as the states associated to a traffic (like the NEW, ESTABLISHED, RELATED, INVALID states in iptables), or implicitly determine the type of data (like the strings of characters for HTTP protocol fields).In practice, a condition on a given selector matches a packet/flow if it is evaluated to true with the values extracted from the packet/flow.A condition clause matches a packet/flow if its formula is evaluated to true.Since in most cases formulas are represented in disjunctive normal form, a condition clause matches a packet/flow if all the conditions are true, otherwise a Boolean expression needs to be evaluated.
We have categorized the types of selectors in exact match, range-based, regex-based, and custom-match [12].
Exact match selectors are (unstructured) sets: elements can only be checked for equality, as no order is defined on them.As an example, the protocol type field of the IP header is a unordered set of integer values associated to protocols.
Range-based selectors are ordered sets, thus easily mapped to integers, where it is possible to naturally specify ranges.As an example, the ports in the TCP protocol are well represented using a range-based selector (e.g., 1024-65535).We include in the range-based selectors all the category of selectors that have been defined by Al-Shaer et al. as prefix match [13].These selectors allow the specification of ranges of values by means of simple regular expressions.The typical case is the IP address selector (e.g., 10.10.1.*).There is no need to distinguish between prefix match and range-based selectors as 10.10.Another category of selector types includes the regex-based selectors, where the matching is performed by using regular expressions.This selector type is frequent at the application layer, where data are often represented as strings of text.The regex-based selector type also includes as subcase the string-based selectors, where matching is evaluated using string matching algorithms (SMA).Indeed, for our purposes, string matching can be mapped to regular expressions, even if in practice SMA are much faster.For instance, Squid, a popular Web caching proxy that offers various access control capabilities, allows the definition of conditions on URLs that can be evaluated with SMA (e.g., dstdomain) or regex matching (e.g., dstdom_regex).
Finally, we introduce the idea of custom check selectors to model conditions that do not provide details about logic of the matching process.For instance, custom check selectors describe features of malware analysis and IDS tools that look for specific patterns and return a Boolean value if the pattern is found.In order to be properly used by high-level policy based processes (like reasoning systems, refinement systems), custom check selector need (at least) to be described as blackboxes, that is, by means of the list of fields that they take in input in order to return the Boolean verdict.
B. Policy-level capabilities
Besides the rules, other "technicalities" need to be specified in order to properly manage and configure security functions, and a sound capability model must also be able to describe them.According to the geometric model, a policy is specified by means of: 1) resolution strategy (R), which abstracts the decision criteria for the action to apply when a packet matches two or more rules; 2) external data, such as priority, identity of the rule creator, and creation time, which are associated to each rule (but not part of the rule itself) and used by the resolution strategy to decide; 3) default action (D), which is the action applied if no rules match the packet/flow.
We have ignored in this work options to generate configurations that may have better performance (e.g., with chains or ad hoc structures [11]).Adding support to these forms of optimization is certainly feasible with a limited effort but it was outside the scope of this paper, that is, to show that adding security awareness to NFV management and orchestration features is possible.It is one of the task for future work.
C. Formal model and algebra of capabilities
Formally, the capabilities associated to a NSF X are defined by a 4-tuple: cap X = (A; C; R; D) ⊆ (A; C; R; A).Where A is the set of all the possible actions and A ⊆ A are the actions available from X, C is set of all the existing conditions types and C ⊆ C are the ones available from X, R is the set of all the existing resolutions strategies and R ⊆ R are the ones contained in X, and D ⊆ A lists the actions supported by X that can be used as default action (∅ if the default action is not configurable).
Moreover, capabilities can be added and subtracted, given Note that addition and subtraction do not alter the resolution strategy and the default action method of the first operand, as our main intent was to model addition of modules (that enable conditions and actions but do not change the core behaviour of the NSF).The operations only merge the rule based capabilities and leaves untouched the policy capabilities.
As an example, we report how to define the capabilities of a generic packet filter that supports the FMR, supports explicit default actions and a another packet filter that also supports time-based conditions:
D. Use of the capability model
Our capability model has several applications.First of all, it can used to precisely asses security policy enforceability, as needed by our policy refinement approach.If the security function can enforce the actions required by the policy and can identify the packets/flows to which the security policy wants the action enforced, then the security control is capable of enforcing the policy, and a manager can use it.Note that, in some cases, a single function may not be able to enforce a policy (incapable), however, a set of security functions can be capable of enforcing that policy.In both cases, a simple capability matching algorithm is enough.
Then, the capability model entails information about the valid MLP policies for a given NSF.Information about actions and classification features is actually used to determine how to write valid rules, policy-based capabilities allow setting up the remaining details.Indeed, it is not possible to use specific conditions or actions when an NSF that does not own the proper capabilities.
Furthermore, we have used the capability model to describe generic categories of security functions and specific products in unambiguous way.We used the model to describe the categories of security controls we have analysed.
We have created a set of generic security functions (g-NSFs), one for each category, and we have associated them to a set of core capabilities owned by (almost) all the controls in a category.The purpose is to remove ambiguity, as with our formalization the behaviour of each g-NSF is well understood, and use security function with the same clearness as network components (i.e., a switch is a switch and experts know how to use it even if it may have some vendor-specific functions).In our idea, vendors and developers should describe their products starting from the g-NSFs.That is, generic functions become templates that can be customized by adding and removing capabilities by means of the capability algebra in Section III-C.
IV. REFINEMENT
The most significant achievement in this paper is the definition of the policy refinement model.
For the definition of the refinement model needed for automatic enforcement of security policies and dynamic adaptation to network changes, we assume the following design principles: a) the refinement process translates high-level policies into configurations directly usable by the NSFs; b) the refinement must warn users against cases of non-enforceability of the input policy on the target network.Moreover, we added another requirement, the vendor independence, that is, it should be easy to substitute an NSF with another one able to enforce the same types of policies.
Practically, the final goal of our approach is to generate concrete configurations for the NSFs into the target NFV network, which are the rightmost element in Figure 2. The syntax and expressiveness of the concrete configurations of the actual NSF is not under our control.We don't have the possibility to ask for new features nor to change the specific languages to cope with our needs.Hence, for our purposes, low level languages are constraints.
A. Approach and SAM workflow
We propose to use two refinement steps and an intermediate abstract language to translate high-level policies into low level configurations (see Fig. 2).This is, the internal workflow performed of the SAM.The High-to Medium-level Refinement Service (H2M Service) first performs the semantic interpretation of the HLP policies to determine the capability needed to enforce them and infer the NSFs available in the network that can enforce them.Then, it outputs the set of all the MLP policies for all the NSFs in the NFV network.If the NSFs in the target NFV network do not satisfy these requirements, the user is provided with a detailed non-enforceability report (NER) and a proposed remediation strategy.The semantic interpretation of HLP policies, its relations to the capability model and the model-driven generation of valid MLP policies is presented in Section IV-D.
When the MLP policies are generated, the Medium-to Low-level Refinement Service (M2L Service) is in charge of translating the abstract policies in the concrete configurations for the selected NSFs, as presented in Section IV-E.
B. High-level policy language
We defined the HLP language [15], [16] to express the security requirements collected from various use cases, such as parental control, corporate environments, user-driven paradigms, and IoT.The HLP has been designed as an authorization language that follows the subject-action-objectattributes paradigm (also referred to as target-effect-condition) [17].HLP policies are composed of HLP statements in the form: [sbj] action obj {(field_type,value),...} where attributes are (field_type,value) pairs.HLP statements map to equivalent user friendly sentences, close to natural language, which are preferred for interactions with administrators.Examples of security requirement are "scan email for malware", "block access to gambling sites", "only allow Internet traffic from 18:30 to 20:00 for Alice".
It is worth noting that this language is an example to prove the validity of our approach in our use cases thus we don't claim that this is the universal language for enforcing security policies, although it can be extended to support more complex scenarios.Nevertheless, the scope of our language allows the specification of high-level policies for a number of security controls, such as packet filtering, application firewall, antimalware, spam detector, VPN gateway, traffic anonymizer, IDS, DPI, file and traffic analyzer.Thus, as it stands, the HLP can be useful in daily security administrators' tasks as well as for normal users.The detailed list of security control categories that are supported can be found in the supplemental material together with examples of HLP statements.
C. Medium-level policy language
The MLP language has been designed to abstract the configurations settings of NSFs.Even if in the practice these settings are conveyed with different languages, the capability model allows us to easily know, in an abstract way, all the features owned by each NSF.Therefore, the MLP language is specified with a general model that defines the abstract concepts (i.e., policies, rules, conditions, actions, resolution strategies) and the required associations among them (e.g., rules are aggregations of conditions and actions, a policy is association to its default action).However, when instantiating a policy for a specific NSF, only instances of concepts present in the capabilities of the NSF can be used.That is, a condition on URL cannot be used if the NSF does not own a URL-related classification feature.
D. H2M Service
The refinement process performed by the H2M Service can be split into two logical tasks: selecting the NSFs that will enforce the policy, and deriving configurations for them.
The decision of the NSFs is performed in two steps.First, the H2M Service interprets the HLP statements to determine the capabilities to enforce the policy.To have greater flexibility and to easily adapt to other contexts, this interpretation is not static, it is performed by using the information (i.e., the semantic mappings) in the Knowledge Base.Examples of association rules and inference rules are reported in the supplemental material.Specifically, the H2M Service performs the following steps.1) It maps the values HLP subject, object and fields fields to low-level concetps by means of direct associations in the KB.For example, 'Employees' maps to a set of IP addresses, 'Madrid Subnet' to a subnet, 'lunch time' is a time period and 'gambling sites' is a set of IP and URLs.After this phase, each HLP statement becomes an enriched HLP statement.2) It uses a set of inference rules, also stored in the KB, to associate concepts from each enriched HLP statement to the rule-based capabilities (i.e., action and classification futures) needed to enforce them.For example, for the HLP "Employees are authorized to access Internet traffic (time period, {12:30-13:30 UTC})", the H2M Service first deduces that an Allow filtering action is needed, because 'are authorized to' is associated to 'Internet traffic' which maps to 'any IP address'.Then it deduces that the target NSF must be able to enforce a condition on time (timestart).Note that the system also deduces that filtering on IP destination is not needed as 'any IP address' is not needed if condition clauses are in DNF form.Moreover, it deduces that all the other communications not explicitly allowed by the HLP policy must be denied (i.e., the default action is Deny).The result is the set of the required capabilities.3) It generates, with a process driven by the MLP policy model, a set of policy rules where the MLP concepts are instantiated from the low-level parameters in the enriched HLPs and the inferred rule-base capabilities.
After this semantic interpretation, the H2M Service identifies the NSFs in the target that can be used to enforce the HLP policy.This is performed by matching the required capabilities with the ones owned by the NSFs.However, according to (the action implied by) the HLP policy, there are different ways to select the NSFs and perform the matching, which we have categorized in single function enforcement, coupled functions enforcement, and path functions enforcement.As a result of the application of these three selection ways, all or a subset of the NSFs in each of the paths will be configured.
A policy requires single function enforcement when the activation of a single NSF in the target network is enough to enforce the policy.As an example, having a security control that performs anti-malware checks over the corporate email attachments is enough to enforce policies that ask for verification of email attachments even if network flows may need to be redirected to the selected NSF with SDN features.Therefore, checking the capabilities of individual NSFs is enough to determine capable functions.
A policy requires coupled functions enforcement when the policy enforcement requires the coordinate configuration of two security functions.As an example, configuring a VPN tunnel between two corporate subnets that communicate through the Internet (e.g., the Madrid and Turin ones) requires the use of two gateways.Moreover, the H2M Service also checks that the two coupled NSFs are also compatible.For instance, both IPsec-and TLS-based controls could be used to enforce a VPN policy, but both gateways must speak the same protocol.Also in these cases, network flows may need to be redirected to the selected NSFs (e.g., the gateways).Here network topology info is used to explore the neighbours of the entities involved by the policies and find the ones having the capabilities to be coupled (e.g., a VPN concentrator at the same security level as the connecting entity).
A policy requires path functions enforcement when the policy enforcement requires the simultaneous configuration of all the security functions with specific capabilities in the communication path.Communication paths are determined by processing topological information.Moreover, routing information is used to establish the actual communication paths from the alternative ones.As an example, the configuration of an allowed communication requires the configuration of all the filtering devices (e.g., packet filter or HTTP filter) in the path from the source to the destination.In these cases, the decision is not on the security control to use, rather on the path to choose, therefore, all the security controls in the path must own the required capabilities.Also in these cases, network flows may need to be redirected to the selected NSFs (e.g., the gateways).
If some required capability is not available or it cannot be properly coupled, the H2M Service generates the NER.It is not interesting discussing the format of the NER.Briefly, it contains the reason for non enforceability, that is, the policies that cannot be enforced, the missing capabilities in the target NSFs, or the impossibility to find one or more devices to couple to enforce the policy (like VPN gateways) in the set of NSFs that can be selected for a given user, together with a list of NSFs that can be added in the network (and where) to make the policy enforceable.
In case of availability of several NSFs that can enforce the HLP statements, the distribution of the generated policy rules to the available NSFs can be optimized.We have developed an optimization model that performs this distribution also based on network and device performance and security metrics (see Section V and VI).
Finally, the H2M Service generates the MLP policy for each one of the NSFs selected by the optimization process by 1) collecting the associated policy rules, and 2) finalizing the policy gathering from the KB the policy-based capability information, collecting each policy rules with the same rule base capability, specifying the resolution strategies and the default action (retrieving these information in the KB) .
The derivation of the configurations in MLP is a simpler task.Once the security controls have been identified, generating the configuration is quite straightforward, as the required rules are determined when processing the HLP statements.
E. M2L Service
The M2L Service performs the syntax change to adapt the policy specified in MLP into actual NSFs configurations directly usable by the target NSFs.As an example, it is in charge to translate the MLP policy for a generic packet filter into a valid iptables configuration file.As it bridges the gap to real NSFs, it is a crucial component for the adoption of our refinement approach towards the automatic enforcement of security policies and the dynamic adaptation to network changes.It was also needed to validate and test our approach in OpenMANO.Nonetheless, its design did not require to address peculiar research issues.
We have implemented an M2L service as a framework that dynamically loads (from a remote repository) the plug-ins that translate the MLP into the NSF-specific syntax.Indeed, each NSF typically has a different configuration language, thus, an NSF-specific translation module proved to be the most convenient way to perform the mapping.Since the MLP policy already includes all the concepts that will be needed at the lowest level, every module has to perform a change of syntax and add the necessary fixed information (e.g., initialization, global options).Note that, supporting future NSFs that provide new capabilities does not pose issues to the MLP.Indeed, changes to the MLP are model-driven from the capability descriptions, that is, it is just required to extend the KB to support the new policies that can be enforced by these NSFs, as described in Section II-C.
V. OPTIMIZATION
As anticipated, the optimization phase performed by the H2M Service selects the NSFs to use to enforce the HLP policy, when alternatives security functions can be used.The idea is that an optimization phase can help in improving the performance and achieving a better use of the available resources, especially for large networks where multiple choices are often available.Instead of developing ad hoc allocation strategies, we have preferred tackling the decision problem formally, with an optimization model (in standard form).The optimization model is an instance of the set covering problem, which is NP-Complete in worst case but it is solved with good performance by standard solvers (there is also a greedy algorithm that works well in practice).Moreover, our formulation of the problem can be can be easily customized to support a large set of optimization scenarios by simply adding constraints and customizing the parameters, as shown below.
Let P = {p 1 , ..., p t } be the MLP policy rules derived from the refinement of the input HLP policies to enforce, and let S = {s 1 , ..., s n } be the NSFs in the target network, we define two functions to map them to capabilities, namely γ p : P → C returns the capabilities needed to enforce a policy, and γ s : S → C returns the capabilities owned by a NSFs.
A set of Boolean variables x i,j = {0, 1} reports if the NSFs s i ∈ S is selected to enforce the policy rule p j ∈ P.
A set of metrics has been proposed to generalize the target function used to choose of the NSFs.That is, we assume that there are l metrics µ 1 , . . ., µ l that associate each NSF s to a real value r depending on the policy rule p it has to enforce: µ i : S × P → R (s, p) → r Note that this definition also covers, as subcase, metrics that do not depend on the policy to enforce.
A generic function K, named cost, computes the aggregated cost associated to the use of the NSF s to enforce the policy rule p, which is expressed as a real number and defined as a linear combination of different metrics values µ i computed over a NSF s: where w i weights the importance of the metric µ i .
For our prototype we have defined several sample single objective target functions by customizing the cost functions and defining the corresponding metrics.We have introduced metrics to estimate the (economic) costs associated to buying or using a NSF, metrics that capture user rating of NSFs, and experts trustworthiness expectations (i.e., by trivially counting the number of patches and averaging them with their severity), and security evaluation of NSFs.
Moreover, we have considered metrics to quantify performance, based on the information available in the VNF descriptors (see the supplemental material provided with this paper for examples of VNF descriptors).For instance, the delay metric estimates the time needed by a NSF to process traffic and by links to transfer it.The difficult part was modelling the performance profiles associated to NSFs.Indeed, while for virtual resources the performance degrades (linearly) with the number of rules, physical NSFs may use ad hoc hardware to have constant time processing regardless of the rules [11].Unfortunately, models that estimate performance degradation based on the configured policies are not linear and needed a linearization to be solved efficiently.Furthermore, we have introduced metrics to estimate the "distance" between solutions, which weight the differences among where policy rules are enforced.These metrics are used to add penalties to solutions that are too different from an initial configuration and have been used to support change management (see Section VI).As a future work, we planned to introduce new metrics to capture CTO estimation.Finally, multi-objective criteria have been defined to trade-off competing objectives by combining single objective target functions.
Our model also includes several constraints, some of them are configurable by the users.The first set of constraints avoids that the NSFs allocated on each physical node exceeds the node resources.The only resources that we have considered in our optimization models are the virtual CPU count, needed RAM, and required disk storage, that is, the only data available in the VNF descriptors (see the supplemental material for examples of OpenMANO VNF descriptors).For instance, there are constraints to cope with the different NSF selection strategies.When enforcing a single function enforcement policy p j , the model excludes from the decision space all the security functions s i that do not own the required capabilities: Capable functions are OR-ed with additional equations that limit the number of functions to use (i.e., at most one).For instance, defence-in-depth is achieved by playing with these equations.Then, when looking for NSF to associate for coupled functions enforcement, only the neighbours of the end nodes implied by the policies are considered (e.g., VPN gateways are selected at the border of the network to protect).Thus, the model excludes all the NSFs that are not in the neighborhood and do not own the required capabilities:x i,j = 0 ⇔ s i / ∈ N (e 1 (p j )) ∪ N (e 2 (p j )).where e 1 (p j ) and e 2 (p j ) are the functions that determine the endpoints (determined by the refinement process performed by the H2M Service) and N (•) is the function that abstracts the selection of the neighborhood of endpoints to consider (e.g., in the same network or broadcasting domain, or the part of the network at the same security level).
Supporting path functions enforcement also adds constraints.The NSFs in the same path to activate are ANDed.Given the paths {π k (e 1 (p j ) , e 2 (p j ))} k between the endpoints implied by a policy, we introduced a set of variables v k = x i,j (for s i ∈ π k ) to force the configuration of all the nodes in the path π k .Also in this case, equations allow the selection of at leat one path ( v k = 1), exactly h path ( v k = h) , or all the paths (∀k, v k = 1).In case of static routing, there is only one path to consider, the other possible NSF are therefore excluded.The optimization model is instantiated by the H2M Service that automatically generates optimization programs that instantiate these logical formulas with values from the target network and for the input policies.Then, the H2M service solves the generated models with standard solvers.We used the MOEA framework, as we planned support for multi-objective optimization, however, any ILP solver can be used.
VI. MANAGEMENT OF CHANGES
We have considered several scenarios to deal with changes.We have addressed dynamic adaptation to network changes, a first step towards security policy aware network management and orchestration.This is still an ongoing research, however, the preliminary results are promising.
First, with our approach, substituting a NSF X with a new one Y owning the same capabilities as X can be managed with a quick procedure.Indeed, after the refinement, the H2M Service outputs an MLP policy for a generic NSF that exploits (some or all) the capabilities of X.The MLP policy M is then translated to be usable by X by the M2L Service.Hence, switching to Y just requires that the M2L Service translates the policy M for the syntax of Y .More generally, the quick procedure can be used with any NSF that owns at least the capabilities of X exploited in M (vendor independence).
The general case of dynamic adaptation to network changes, that is, changes that happen in the network and are not just substituting a NSF with an equivalent one, can be dealt with a complete refinement of the HLP policy onto the new network.However, this approach may originate too many changes and lead the network to a temporary inconsistent state, as a subset of NSFs may enforce the new policy while the others NSFs still behave as required by the previous policy.Therefore, to reduce the issues related to changes, we have introduced the distance metrics, anticipated in Section V, which favour solutions that are maybe a bit less optimal from the performance point of view, but minimize the impact on the network.A simple metrics counts the NSFs whose policy needs to be updated, others count the number of coupled functions that need to be reconfigured to re-establish secure channels and the paths that are affected by the changes.Linear combinations of distance metrics are then used in the target functions.However, these distance metrics and the target functions built on them will need further research effort to guarantee a better stability of the network over time, not only on the single change.
Also the management of dynamic changes of the policy can be dealt with a complete refinement of the new HLP policy, as for dynamic adaptation to network changes.However, we are investigating faster refinement algorithms that only process the differences between the old and the new policy (delta refinement), which could reduce the likelihood of negative impacts of the refinement performance.Indeed, the management of changes is essential for the practical adoption of our approach.Unfortunately, the consequences of only considering the differences have not yet been extensively investigated.Indeed, inconsistencies may arise when mixing configuration rules from different refinement processes.Currently, we are researching criteria that characterize the differences that do generate inconsistencies to determine when the complete refinement can be avoided.Indeed, an a posteriori inconsistency checking would render useless the performance improvement of the delta refinement.
Another ongoing research concerns algorithms to automatically assess the changes to the input policy and the target network and decide the best way to push the new configurations.The idea is to develop advanced configuration deployment strategies that decide how and when to push configuration of NSFs to allow seamless transition between solutions, i.e., network states (i.e., topology and NSFs configurations).These strategies should avoid the inconsistencies (and related consequences) that may arise when passing from a network state to a different one as a consequence of changes.
VII. IMPLEMENTATION AND VALIDATION
We have integrated our solution into the OpenMANO project and we have used OpenMANO to validate our approach on virtualized scenarios.The virtualized scenarios have been selected in the context of EC funded project SECURED.The SECURED use cases were perfect to show that it is possible to perform a policy-based management and orchestration of virtualized networks.Indeed, these use cases depict NSPs enforcing security policies requested by residential users (divided in classes of services) and/or SMEs by means of one or more chains of network security services formed by one or more NSFs.Use cases were representative of medium size networks but did not allow us to measure the scalability of our approach, which was one of the critical points for its adoption.Therefore, we have also designed a set of tests where the SAM was used as a black-box policy refinement engine to derive configurations from HLP for network that were only represented with their service graphs, i.e., without actual deployment.Hence, our validation is composed of two parts: validation of the policy-based configuration process and testing on synthetic networks with very large number of policies.
A. Implementation
The OpenMANO project has developed three components: 1) Openmano, the northbound interface, based on a REST APIs, which exposes a set of orchestration and management tasks (such as creation and deletion of VNF instances and network services); 2) Openvim, the NFV Virtualised Infrastructure Manager that directly interfaces with an OpenFlow controller and a compute nodes via a REST API; 3) Openmano-gui, the web interface to invoke the Openmano API.
To prove the validity of our approach we have extended Openmano and Openmano-gui.The SAM has been implemented as a new Openmano module.The SAM refinement services are exposed by means of a REST API, which is used to pass as input the HLP and receive in output the MLP, the NSD and the NSF configuration to store in the PR.The SAM has been developed as a single threaded Java 1.7 application that relies on two Java-based open source frameworks: Drools and MOEA.Drools is a Rule Engine that we have used to implement an Expert System that reasons about capabilities for HLP policy enforcement purposes.Drools uses the Rete-OO algorithm whose performance is excellent in practice, also considering the expressiveness of the inference rules, and whose complexity is presented in depth in a past work [18].
The MOEA framework is a Java library for developing multi-objective optimization programs and solve them with evolutionary algorithms.We have used to implement the optimization models (Section V).
We also implemented the PR (using the Django REST framework) and added it to the OpenMANO repositories.The PR API allows the addition, deletion, and update of all the artifacts needed by our approach that were not available in the OpenMANO distribution.These artifacts include information about HLP and MLP policies, and NSF configurations.
We extended the openmano-gui to allow users to specify the input HLP policies and visualize the all the artifacts used by our refinement approach.
Finally, to perform the experiments, we have also implemented one Element Manager for each of the NSFs we have used for validation purposes in order to to push configurations and manage their lifecycle (start, stop, restart).
B. Validation with use cases
The validation on use cases aimed at proving that our approach is actually able to select and configure the NSFs in a policy-driven manner.It consisted in the following phases: 1) selecting a use case network (with all the information about the available NSFs, including the capabilities) and specifying security requirements for that network; 2) drawing the network in OpenMANO with the openmano-gui; 3) specifying the corresponding HLP policy with the extension to the openamano-gui; 4) deploying and configuring the network with the SAM-extended OpenMANO and measuring the performance of every phase; 5) manually inspecting the generated NSFs policies at the MLP level (i.e., not the low-level configurations); 6) testing with probe packets and ad hoc built policy violations to verify, also with traffic inspection, that the selected security requirements were actually enforced.
We only report here the entire validation results of the network service example presented in Section II-D.However, we have considered several use cases from the SECURED project that include parental control, corporate network protection, and user-centric policy enforcement 9 .
The input policies in HLP of the example were: H1 : enable email scanning {(mimeType, pdf)} H2 : Developers are authorized to access Internet {(time period, 13-15 UTC), (specific URL, www.facebook.com)}H3 : protects integrity and confidentiality {(source, TurinNet),(target, MadridNet)} The SAM correctly inferred the capability required to enforce the HLP policies then determined that H 1 can be enforced by Bro, H 2 can be enforced by Squid, and H 3 can be enforced by strongSwan.The SAM first generated the MLP policies for three generic anti-malware, application layer filtering, and IPsec channel protection security functions (that own a subset of the capabilities owned by the three selected NSFs).Then, the SAM translated the MLP policies into the NSF configurations.
We have performed further advanced testings to prove an effective reaction to changes.First we have modified the attributes in the input policies (time period from 13-15 to 8-12, specific URL from www.facebook.comto www.tweeter.com,and from specific URL=www.facebook.comto specific IP=10.0.0.1) and verified that the SAM only updated the squid MLP and low level configuration and did it correctly.Finally, we have changed squid with privoxy, another application layer filter that owns the capabilities to enforce H 2 .As expected, the SAM only generated the configuration of privoxy from the same MLP policy used before for squid.
C. Scalability testing
We have generated synthetic networks by means of an algorithm developed to connected chains of network services composed of one or more NSFs to form a tree (whose root was intended as the connection to the "outside") then added connections among other tree nodes to simulate a proper number of redundant paths.Chains of network services were available in a catalogue and explicitly designed by us (i.e., chains are not randomly generated).A number of users, i.e., the target of the policies, were connected at the beginning of each chain.Other creation rules ensured that the generated networks were close to reality, for instance, increasing the concentration of security services at the root or selected intermediate nodes to simulate border security, providing some security services with a centralized or distributed approach, and ensuring that enough capabilities were available at each path to reduce nonenforceability issues.Finally, HLP policies were randomly generated for the inserted users starting from a set of template policies that covered the whole HLP expressiveness.
We have performed our tests on an Intel i7-3630QM @ 2.4 GHz laptop with 16 GB RAM.Each test was run on each scenario 50 times; results have been averaged.
We have first measured the time spent to perform the refinement of the HLP policy into configurations, which is the sum of the time to translate HLP policies into MLP policies for all the NSFs in the network (by the H2M Service) and the time to translate all the MLP policies into configurations (by the M2L Service).We have generated networks with a fixed number of NSFs, while we have increased the number of HLP policies (ranging from 10 to 500.000. Figure 3a depicts the results obtained respectively with 5 and 20 NSFs.As expected, there is a more than linear dependency with the HLP policy count.We noted that for a small number of HLP policies, the initialization time of the two frameworks (Drools and MOEA) gave an important contribution that has a minor impact when more than 500 rules are considered.This test gave us the most important result, as it proved that the approach scales well enough to be used in practice, despite the limited hardware resources used.We also measured that the time to generate configurations from MLP linearly depends on the number of rules in the MLP policy but is negligible.In the work case, the translation of five thousands rules (for Squid) took less than 100 ms.Furthermore, the MLP translation can be easily parallelized, an advantage when the number of NSF to configure is huge.Moreover, we have measured the time required by the Element Manager to configure the NSFs. Figure 3a plots the results obtained when measuring the time to push a configuration depending on the number of rules it contains (ranging from (c) Time to instantiate a service chain.
Fig. 3: Results of the scalability testing.
10 to 1000 rules).The bottom line represents the results of Iptables, the NSF that has shown the best performance, and the top line Squid, the NSF that required more time to be configured.The configuration time is nearly constant and lasts a few tenths of second.Thus it can be considered negligible for scalability analysis purposes.
Finally, even if it is not directly related to our contributions, it interesting reporting here the time Openvim spent to instantiate a network service chain from its NSD, depending on the number of NSFs. Figure 3c depicts the results obtained to instantiate up to 20 NSFs.Also in this case, the time to setup the network grows almost linearly, as expected since the virtual machines are deployed in order by OpenMANO.This result confirmed the feasibility of our approach.Given the current NFV technology, the optimized policy would be ready before the MANO completes the instantiation of the virtualized network.
A. NFV management, orchestration and modelling
In addition to the ETSI working group, which has defined the standard, a great number of researchers analysed in depth the NFV challenges and benefits.The most significant papers are authored by Mijumbi et al. who proposed several unexplored NFV research topics.Authors stated that management and orchestration of NFV-based networks will become more challenging in future, the most promising proposal being the MANO framework by ETSI [9].Mijumbi et al. also highlighted the importance of formally modelling network and security functions to exploit the NFV's ability to deliver high levels of automation and flexibility.Since the resources and functions in NFV will be provided by different entities, the availability of well understood, open and standardized descriptors for multi-vendor resources, functions, and services will be key to large-scale NFV deployment.
ETSI and others working groups have provided a possible set of information and data models for NFV resource and function modelling.Examples are OVF, TOSCA, YANG and SID.However these models are only used in the definition of software/hardware resource (their components, relationships, and the processes that manage them), but, at the best of our knowledge, none seems to address issues related to NSFs management and support.Giotis et al. [19] proposed a preliminary architectural model for policy-based VNF orchestration, which use an Information Model to abstracts network resources and VNFs capabilities.The main limitation is that the model only addresses access control and forwarding policies.Shen et al. proposed vConductor, a MANO architecture to support E2E Virtual Network Integration as a Service (VNIaaS) [20].vConductor is a NFV-based service that provides a multiobjective resource scheduling and NFV-oriented inventory management capabilities.However, it does not support policy refinement, the configurations of NSFs are directly written by network administrators.Finally, Spinoso et al. proposed the use of functional descriptions of VNFs to correctly integrate and configure third-parties VNFs in NFV NSP networks [5].
B. Optimization
The provisioning of NFV brings up the resource allocation problem, also known as Virtual Network Function Placement (VNF-P).Moens et al. [21] proposed an Integer Linear Programming (ILP) model to minimize the number of used servers in mixed physical and virtualized environments.Yoshida et al. [22] proposed to optimize the resource allocation when enforcing stakeholder policies on a network infrastructure.Conflicting objectives are dealt with a Multi-objective Genetic Algorithm (MOGA), which produces approximate solutions in a reasonable computation time.Gember et al. [23] present a network-aware orchestration layer for Middleboxes (MBs), named Stratos.Stratos allows tenants to specify middlebox deployment by using a simple logical topology abstraction and three features, application-aware scaling, rack-aware placement, and network-aware flow distribution.Mehraghdam et al. [24] proposed an optimization model for building chains of VNFs that satisfy requirements of the tenants and the operator expressed with a formal language.These work are complementary to our optimization model.Their optimization criteria could be used, after extension and adaptation, to improve our optimization model.
C. Refinement
Policy refinement has received great attention in literature.Many models and methods have been proposed to efficiently deal with both management and enforcement of security policies.However, the interest from the theoretical viewpoint has not yet shown real advancements in the practice.The main reasons are related to the lack (and in most cases of the unavailability) of formal representations of the huge amount of data that are needed to actually perform the refinement.
NFV networks, and in general, virtual environments, where orchestration and management have access to a detailed representation of the running infrastructure, are a promising field to finally see the refinement dream to come true.According to Weise and Martin [25], a security policy must be implementable through system administration procedures (e.g., publishing of acceptable use guidelines) and enforceable with security tools or controls, where appropriate, and with sanctions, where actual prevention is not technically feasible.Unfortunately, in literature, enforceability analysis has received little or no attention and it has not been investigated in-depth.For instance, in a real scenario, some policies may be less precisely enforceable in some systems than in others or in worst case, completely non-enforceable.As suggested by [26], the access control on traditional UNIX systems is much less granular when compared with ACLs on modern implementations and some access control requirements may be not fully supported.Schneider proposed an approach to determine when a security policy is enforceable with Execution Monitoring [27].A policy is non-enforceable when its set of executions is not a safety property and the Execution Monitoring does not have an enforcement mechanism.This concept has been improved by Bauer et al. who extended the model the Schneider's to support new classes of security policies [28], and Basin et al. who proposed Execution Monitor able to distinguish actions that are controllable and those that are only observable [29].We tried to overcome current limitations by proposing the capability model, which allows precise identification of nonenforceability issues.
Refinement models exist for firewalls.Bartal et al. proposed a solution, named Firmato, for the refinement of highlevel filtering requirements into packet filter configurations [30].Firmato uses a knowledge base, formalized with an entity-relationship model that describes high-level filtering requirements and network topologies, and a translator, which refines high-level filtering requirements into packet filter rules.However, Firmato has been validated on a network with a single border firewall, hence its applicability to large and heterogeneous scenarios has not been proven.Verma et al. used a similar approach to develop FACE, a firewall analysis and configuration engine [31].FACE takes as inputs the network topology and a global security policy written in a high-level language, and outputs the packet filter configurations.
Valenza et al. proposed the use of ontologies to capture the semantics of high-level filtering and channel protection policies (e.g., IPsec) [32].By means of ontology inferences, high-level concepts (such as users and services) are mapped to low-level network concepts (such as IP addresses, ports, protocols) so that high-level security policies can be translated into configurations settings for target security controls.Our approach shares with this work the idea of an inference engine to perform HLP policy refinement.However, our approach is much more powerful, as ontology proved to be very difficult to use and extend, failed to capture concepts (like connections) in a simple way, and their performance scaled quite slowly.
IX. CONCLUSIONS
This paper presented the first steps towards a Security Policy Aware NFV MANO (SPA-MANO) by supporting two innovative scenarios: automatic enforcement of security policies and dynamic adaptation to network changes in NFV networks.We have developed refinement models that allow the transformation of high-level security requirements into configuration settings for the Network Security Function (NSF) in the target NFV network.These models also support optimal selection processes, which permit the selection of the NSFs to use in case of alternative functions.To build such an approach, we have defined an algebra of capabilities useful to describe the security functions implied by the security policy requirements and the functions actually available and the NSFs.We have also proved that supporting security awareness in NFV networks is feasible and requires limited changes in the current NFV architecture.Moreover, we have integrated the new component in the OpenMANO framework.
It is important to highlight that this approach cannot be used as it stands now in production networks.Indeed, policy languages need to be extended to support more security requirements and to be adapted to the various scenarios where this new paradigm needs to be applied.Also the capability model needs to be further detailed to be able to express a larger set of NSFs.However, both for policies and capabilities, an incremental effort is needed, whose purpose is support of a broader range of cases.Indeed, we have analysed large but not exhaustive set of security controls, most of them are traditional security controls, which can be virtualized, but we have not addressed specific NSFs that are natively virtual.Thus, limited changes to the capability model can be expected.
Presently, scalability of this approach has been proved very promising also on off-the-shelf hardware.Advantages are expected when using more powerful resources, like the ones available at the data centers where NFV networks will be deployed.However, improvements to the refinement algorithms can further reduce the impact of the policy refinement.
However, the most important future works concentrate on the definition of the security aware resource allocation, that is, on the design of the missing parts to have a SPA-MANO that also performs optimized allocation based on policy information.This will add more degrees of freedom to the decision process, thus rendering the optimization problem more complex.On the other hand, this optimization can lead to a better use of available resources and a save of the expenditures at the NSP, especially if integrated with target functions that better support changes.Also the management of dynamic changes in the high-level policy is an interesting research topic.Indeed, only refining the differences between the old and new policy may consistently optimize the refinement process, as the whole security policy will not change frequently.Therefore, we are investigating criteria that ensure that a partial refinement does not introduce any inconsistency in the globally enforced policy.As future application case, we want to extend also Open Source MANO, which has been selected as the new reference implementation of NFV by ETSI.Since, Open Source MANO is built on OpenMANO, we expect limited effort.
Time to configure the NSFs.
Cataldo
Basile received a M.Sc.(summa cum laude) in 2001 and a Ph.D. in Computer Engineering in 2005 from the Politecnico di Torino, where is currently a research assistant.His research is concerned with policy-based management of security in networked environments, policy refinement, general models for detection, resolution and reconciliation of specification conflicts, and software security.Fulvio Valenza received the M.Sc.(summa cum laude) in 2013 and the Ph.D. (summa cum laude) in Computer Engineering in 2017 from the Politecnico di Torino, Italy.His research activity focus on network security policies, access control policies, orchestration, management and automatic configuration of network security functions in the context of SDN/NFV-based networks.Antonio Lioy is Full Professor at the Politecnico di Torino, where he leads the TORSEC cybersecurity research group.His research interests include network security, policy-based system protection, trusted computing, and electronic identity.Lioy received a M.Sc. in Electronic Engineering (summa cum laude) and a Ph.D. in Computer Engineering, both from the Politecnico di Torino.Diego R. Lopez Diego Lopez received his MS from the University of Granada in 1985, and his PhD degree from the University of Seville in 2001.Diego joined Telefónica I+D in 2011 as a Senior Technology Expert on network infrastructures and services after several years in the academic sector.His current interests are related to network infrastructural services, new network architectures, and network programmability and virtualization.Antonio Pastor received the MSc.degree in industrial engineering from the Carlos III University of Madrid (UC3M), Spain, in 1999.Since then, he has been with Telefónica I+D, where he works on the research and engineering of different worldwide Telefonicas networks and where he is currently Network Security Expert. | 2023-07-22T15:41:39.682Z | 0001-01-01T00:00:00.000 | {
"year": 2019,
"sha1": "d29e9f2056eca87b28d2a9d0a6ead5f7c0d4097c",
"oa_license": "CCBY",
"oa_url": "https://zenodo.org/records/3266876/files/security_policies_main.pdf",
"oa_status": "GREEN",
"pdf_src": "Anansi",
"pdf_hash": "6b9c48309b69dc5e93980797c05cd88cc7f27b44",
"s2fieldsofstudy": [
"Computer Science",
"Engineering"
],
"extfieldsofstudy": []
} |
9589770 | pes2o/s2orc | v3-fos-license | Use of a Web Forum and an Online Questionnaire in the Detection and Investigation of an Outbreak
A campylobacteriosis outbreak investigation provides relevant examples of how two web-based technologies were used in an outbreak setting and potential reasons for their usefulness. A web forum aided in outbreak detection and provided contextual insights for hypothesis generation and questionnaire development. An online questionnaire achieved a high response rate and enabled rapid preliminary data analysis that allowed for a targeted environmental investigation. The usefulness of these tools may in part be attributed to the existence of an internet savvy, close-knit community. Given the right population, public health officials should consider web-based technologies, including web fora and online questionnaires as valuable tools in public health investigations.
Introduction
Web-based technologies are increasingly being used in a wide range of public health applications such as disease surveillance, outbreak detection, research and communication of public health messaging [1][2][3][4]. Still, much research remains to be done to determine when and how web-based tools can be most effectively utilized. Here we outline the role that two web-based technologies played in the detection and investigation of an outbreak and potential reasons for their usefulness.
The Study
A large campylobacteriosis outbreak affecting 225 people was associated with a June 16, 2007 mountain bike race in British Columbia (BC), Canada. The investigation identified ingestion of contaminated mud as the likely source of illness [5]. Two web-based resources were used in the outbreak investigation: an existing mountain biking web forum, and an online questionnaire.
The Web Forum
There was a pre-existing web forum for mountain biking in BC with a discussion thread specifically related to the bike race of interest. The first postings regarding ill racers appeared on June 18, two days after the race [ Figure 1]. Further postings about ill racers prompted the race organizer to contact the local health unit on June 20th. The first reports of laboratory confirmed Campylobacter jejuni among racers were received by the local health unit on June 25, at which point an investigation was initiated.
Between June 18 th and July 6 th 2007, there were 100 race related posts. Of these, 58 entries posted by 34 individuals specifically discussed the issue of ill racers. Prior to the design of the online questionnaire, the web forum was reviewed to identify potential exposures of interest. Racers discussed inadvertent mud consumption, foods consumed, and the use of a common cloth to wipe riders' faces. Additional useful information posted to the forum included the fact that some racers did not complete the race but became ill, some used fenders and others did not, and that there were different types of water delivery systems used, including water bottles and personal hydration packs. Additional postings and photos also informed investigators of the conditions of the race course and the extreme mud coverage of racers' hands and faces.
A qualitative analysis of the web forum discussion thread was conducted to explore possible reasons why it was a useful source of information in this investigation. Some individuals who participated in the race-specific discussion joined the forum as early as 2002, revealing that they had been part of a virtual community for some time. The analysis also revealed that the thread was not moderated and that participants were willing to share personal information such as their symptoms, photos and knowledge of illness in fellow racers. For example, one rider said "Did anyone else who raced get sick yesterday/today? … Symptoms are (and they are ugly) non stop washroom visits that are far from normal, extreme fatigue…unable to eat, drink and sleep".
The Online Questionnaire
An online questionnaire was designed as part of a cohort study to investigate the outbreak. The questionnaire took less than 48 hours to design, set-up on the web using Ultimate Survey Enterprise.NET (v3.0.7) software and pilot. As racers required an email address for race registration, a web link to the online survey was sent by email to the 787 race participants.
Race participants were predominantly male (84%) and 78% were less than 45 years of age. Most participants were Canadian (94%), many of whom were from the community in which the race was held. A total of 549 participants (70%) completed the questionnaire before the site closed, of which 537 were included in the cohort study and 225 (attack rate: 42%) met the ill case definition [5]. Persons completing the questionnaire were not significantly different when compared to all race participants with respect to age, sex and residence.
Responses were given a time stamp: within two hours, 69 responses (13%) were received and in 48 hours more than half of the total responses (n=293) (53%) were submitted. An increase in responses was seen on July 5 th following an email reminder sent to race participants [see Figure 1]. Data were quickly extracted and preliminary analysis conducted to direct environmental sampling.
Discussion
In the present investigation, the feeling of comfort and community in the well-established, nonmoderated web forum potentially contributed to the sharing of personal and detailed information, which made the forum useful to investigators. The impact of an outside moderator in virtual communities is unclear [6,7]. Moderation could possibly impact participation and the type and amount of information shared.
The nature of the web forum may have led individuals who would not have sought medical attention and laboratory testing for their illness, to still discuss their symptoms in the forum. The discussion of illness in race participants in web forum postings brought the outbreak to the attention of the local health unit five days prior to notification of any C. jejuni positive laboratory results. Although in this instance the investigation was not initiated earlier, it demonstrates the capacity of web fora to aid in early outbreak detection.
In this outbreak, web forum postings also aided in hypothesis generation by suggesting possible routes of C. jejuni exposure and providing useful information regarding the race and mountain biking that investigators may not have originally considered. To the knowledge of the authors a web forum providing this type of contextual insight into informing an investigation has not previously been reported.
Online questionnaires have been used previously for outbreak investigations [8][9][10][11][12][13]. They can be efficient tools to rapidly reach a large proportion of a population. A study comparing the response rates of online questionnaires to those administered by telephone revealed no significant differences in median response rates.
However, telephone administered questionnaires took significantly more time to complete [14]. Here, the use of an online questionnaire eliminated the time and public health resources required to conduct phone interviews and enter data. The resulting rapid preliminary analysis enabled a targeted environmental investigation.
Several factors may lead to a high response rate to online questionnaires. Internet access and comfort with online tools are prerequisites [14]. The target population in our study had features suggesting a familiarity with internet tools. All race participants had active email addresses as evidenced by the requirement for email registration for the race. The existence of and participation in a race-specific discussion thread on a web forum also suggests that many racers were internet savvy. The ease of completing the online questionnaire may also have contributed to the high response rate.
A strong sense of community combined with high attack rates amongst members of that community may increase interest and participation in an outbreak investigation. A high response rate (96%) to an online questionnaire was seen in a norovirus outbreak linked to a dinner reunion where the attack rate was 73% and attendees likely also had a strong sense of community [12]. In the present investigation, the majority of racers were from BC, with many living in the race community, and many racers were part of a virtual community. The relatively high attack rate (42%) in this outbreak also meant that many individuals were either personally affected or knew someone who was. In contrast, in an investigation of a salmonellosis outbreak following a sporting event involving athletes and spectators from numerous states and therefore likely less sense of community, coupled with a low attack rate (22%), the response rate to the online questionnaire was only 34% [10].
Limitations
The usefulness of web fora and other social media sites in outbreak investigations is clearly dependent upon the ability of public health professionals to identify and utilize these resources in a timely way. Although it was found to be very useful, one limitation of the web forum in this investigation was that it was only one source of information from a small number of participants. Our assessment of potential reasons for the success of the online questionnaire was limited by the fact that we did not ask respondents what motivated them to complete the questionnaire. Additional limitations of our online questionnaire, compared to phone based interviews, include the fact that respondents could not ask for or be prompted for clarification. In order to help ensure completion we also limited the length of the questionnaire compared to what may have been created for a phone based interview. Analysis of the online questionnaire response times was limited due to technical difficulties. We were only able to capture time stamp information for 506 of the 549 completed questionnaires.
Conclusions
Internet technologies can play a role in the identification and investigation of outbreaks. Public health officials should consider web fora potential sources of not only early signals of outbreaks but also resources that can provide contextual insights to inform investigations. With the proliferation of social media in most facets of daily life, public health professionals need to be cognizant of their application in investigating and responding to adverse community health events such as outbreaks. The highly accessible and scalable nature, as well as immediate reach provides an essential platform for public health communication in this era. In this situation the web forum was not developed or owned by public health and public health specialists chose not to directly intervene. In certain situations identifying or developing public health informatics applications to alert a community at risk using web forums and other forms of social media could be useful. It is also important that public health professionals have the tools available to rapidly create and utilize online questionnaires when needed. Online questionnaires can successfully expedite data gathering, especially among an internet savvy cohort where there is a sense of community and a high attack rate, thus making them a valuable tool for public health investigations. | 2017-04-06T18:34:32.981Z | 2011-06-22T00:00:00.000 | {
"year": 2011,
"sha1": "3953d7a28343c6280d2cbeaf23930216637f0ba4",
"oa_license": "publisher-specific license",
"oa_url": "https://journals.uic.edu/ojs/index.php/ojphi/article/download/3506/3009",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "3953d7a28343c6280d2cbeaf23930216637f0ba4",
"s2fieldsofstudy": [
"Computer Science",
"Environmental Science",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
260714824 | pes2o/s2orc | v3-fos-license | Novel Enhanced Recovery After Surgery Pathway Reduces Length of Stay and Postoperative Opioid Usage in Adolescent Idiopathic Scoliosis Patients Undergoing Posterior Spinal Fusion
Purpose: The goal of this study was to compare our institution’s recently implemented enhanced recovery after surgery (ERAS) protocol to previous post-operative management for adolescent idiopathic scoliosis patients undergoing posterior spinal fusion, specifically assessing length of stay, opioid consumption, and pain scores. Methods: This is a retrospective analysis that compares the length of stay, opioid consumption, and pain scores of patients undergoing posterior spinal fusion for adolescent idiopathic scoliosis. Patients were analyzed prior to the implementation of our ERAS protocol, deemed the traditional pain pathway (TPP), to those who underwent the ERAS pathway. All patients undergoing posterior spinal fusion for adolescent idiopathic scoliosis were included. Patients were excluded if they weighed less than 40kg, had significant comorbidities, or had non-idiopathic causes of scoliosis. Results: We examined 22 patients in the TPP cohort and 20 in the ERAS cohort. Length of stay in the ERAS cohort was significantly reduced compared to the TPP by 1.7 days (P<0.01). Overall opioid consumption was also significantly reduced in the ERAS with 1.4 ± 0.7 morphine equivalents (ME)/kg compared to the TPP 2.4 ± 1.1 ME/kg (P < 0.01). We found no difference in pain scores between the two groups. Conclusion: Implementation of an ERAS pathway at our institution significantly reduced length of stay and opioid consumption in adolescent idiopathic scoliosis patients undergoing posterior spinal fusion. These outcomes reduce morbidity and costs associated with posterior spinal fusion and provide an overall improvement in the quality of care for our patients.
Introduction
The postoperative period for adolescent idiopathic scoliosis (AIS) patients undergoing posterior spinal fusion (PSF) is fraught with challenges, including adequate postoperative pain control and prolonged hospitalization. Inadequate postoperative pain control can contribute to prolonged hospital stays and even intensive care unit (ICU) admissions. While intravenous opioid medications are the mainstay of postoperative pain management for PSF patients, other techniques include epidural analgesics, intrathecal opioids, systemic ketamine, and gabapentinoids [1]. Despite varying methods for pain management, patients' pain often remains difficult to control.
Enhanced recovery after surgery (ERAS) protocols were originally developed to improve overall outcomes in surgical patients [2]. In the early 2000s, colorectal surgeons first implemented ERAS protocols, and their patients experienced reduced morbidity and decreased length of hospital stay (LOS) [3]. One aspect of ERAS emphasizes scheduled multimodal pain medication regimens that allow patients to achieve earlier postoperative milestones, resulting in a shorter LOS [4]. Orthopedic surgeons have more recently adopted ERAS protocols for adult patients undergoing total joint arthroplasties and spine operations, which has led to a significant reduction in LOS, postoperative pain, and overall cost [5,6]. Although ERAS pathways are now commonplace in the adult realm, they remain relatively understudied in the pediatric surgical population [7,8].
Studies examining ERAS outcomes in AIS patients undergoing PSF are few [9][10][11][12][13]. The goal of this study was to compare our institution's recently implemented ERAS protocol to previous post-operative management for AIS patients undergoing PSF, specifically assessing length of stay, opioid consumption, and pain scores.
Materials And Methods
In this study, we assessed the efficacy of a newly implemented ERAS protocol for patients with AIS undergoing PSF. Primary outcomes included LOS, opioid usage, and pain scores. All patients included in this study had a PSF performed by one of two orthopedic surgeons from December 2013 to April 2018. All patients who underwent PSF for AIS were included in the study. Patients were excluded if they weighed less than 40 kg, had significant comorbidities, or had non-idiopathic causes of scoliosis. The traditional pain pathway (TPP) cohort included patients who had their operations between December 2013 and April 2017. The ERAS cohort included all patients from May 2017 to April 2018, after the implementation of the ERAS protocol. This change served as a surrogate for intervention in our study allowing us to analyze the differences between these two groups. We collected all data retrospectively from our institution's electronic medical records and recorded the LOS from the date of surgery until discharged from the hospital. Postoperative opioid usage was recorded and all medications were converted to morphine-equivalents (ME). ME per kg (ME/kg) was then calculated. Nurse charting of pain levels using the visual analog scale (VAS) was reviewed and the daily average and highest pain score was recorded for each patient. We also reviewed each group for any potential complications including urinary retention, neurologic deficits, local wound pathology, prolonged ventilation, or return to trip to the operating room within three months. Our institutional review board reviewed and approved this study.
Traditional pain pathway (TPP)
The TPP included surgeon-administered intraoperative intrathecal morphine, a first postoperative night stay in the pediatric intensive care unit (PICU), and highly variable postoperative pain medication administration. Without a standardized protocol, much of the post-operative pain management was left to the discretion of different medical teams. Teams that managed postoperative pain during the TTP varied between the pediatric medicine team and the primary surgical team without consistency. Furthermore, members of these teams frequently changed personnel which resulted in a wide variety of opioid and nonopioid medications utilized. These included morphine, oxycodone, and hydromorphone. Providers proved a range of frequency and dosages of pain medication administration ranging from patient-controlled to PRN.
ERAS pathway
Our ERAS protocol emphasized multimodal pain control. The acute pain service (APS) managed all postoperative pain medications in order to decrease variability in treatment. Preoperatively patients received gabapentin, celecoxib, and midazolam. Intraoperatively, they received total intravenous anesthetic of propofol, remifentanil, and ketamine, while receiving methadone, dexamethasone, ondansetron, acetaminophen, and methocarbamol for pain control. Since the use of intrathecal morphine was abandoned, patients no longer required monitoring in the PICU on the first night postoperatively. They went from the post-anesthesia care unit (PACU) to the general post-surgical floor. On postoperative day 0 (POD0), patients were placed on hydromorphone, methadone, acetaminophen, ketorolac, methocarbamol, ondansetron, diazepam, and a bowel regimen. On POD1, gabapentin and oxycodone were added. Ketorolac and diazepam were discontinued and ibuprofen was started on POD2. On the morning of POD3, hydromorphone was discontinued and patients remained on an oral pain regimen for the remainder of their stay comprising oxycodone, acetaminophen, ibuprofen, methocarbamol, ondansetron, gabapentin, and a bowel regimen. See Appendix - Figure 4, for the full summary of the ERAS protocol. Additionally, our ERAS pathway emphasized the achievement of early postoperative milestones, including early transition from IV to oral pain medications and prompt return to activities of daily living.
Patient selection
We identified all patients undergoing PSF performed by two different surgeons from December 2013 to April 2018 (n = 75). Of the cases reviewed, we excluded 33 patients due to non-idiopathic causes of scoliosis as shown in Figure 1.
Statistical analysis
All statistical analysis was performed in Statistical Analysis Software (SAS) 9.4 (SAS Institute, Cary, NC), with significance set to α=0.05. Baseline preoperative characteristics of the two groups were compared using Wilcoxon-Mann-Whitney tests for interval data, and Fisher's exact test for nominal frequency data. Postoperative differences in LOS, total postoperative opioid consumption, opioid consumption by POD0-3, and average pain from POD0-3 were compared between groups using Wilcoxon-Mann-Whitney tests. Effect sizes (Cohen's d, and common language effect size: CL) for these comparisons were also computed. Differences between groups in changes in opioid dose and pain from POD0 through POD3 were analyzed using repeated measures linear mixed models. The focus of those analyses was on the group by POD interaction effect, which tests for differences between groups in the magnitude of changes in opioid dosage or pain over the postoperative study period. Where those interactions were non-significant, we removed them from the models and tested for the main effects of group and day. The sample analyzed is a convenience sample of all available eligible procedures during the designated study period. No prior power analysis was performed.
Results
We examined 22 patients in the TPP cohort and 20 patients in the ERAS cohort. Patient characteristics for each group are shown in Table 1. Patients managed with the ERAS pathway experienced a significantly shorter length of hospitalization than the TPP patients. LOS in the ERAS cohort was 1.7 days shorter than in the TPP cohort (ERAS 3.3 + 0.6 days, TPP 5.0 + 1.9 days, P < 0.01), as shown in Figure 2. Within the ERAS group, 100% of patients were discharged by POD5 and 95% stayed four days or less. Of those in the TPP group, 82% were discharged on POD5 or earlier, and four patients required hospitalization until POD7.
FIGURE 2: Comparison of the total morphine equivalents per kilogram between the traditional and ERAS pathways.
ME/kg: morphine-equivalents per kg, ERAS: enhanced recovery after surgery, TPP: traditional pain pathway When comparing overall opioid consumption between the two groups, we found that ERAS patients required significantly fewer opioids postoperatively. Corrected for weight, total opioid consumption in the ERAS cohort was 1.4 ± 0.7 ME/kg compared to the TPP 2.4 ± 1.1 ME/kg (P < 0.01, Figure 2).
Further analysis compared daily opioid consumption on POD0-3. On POD0, both groups required similar amounts of opioid analgesics (ERAS 0.04 + 0.05 ME/kg, TPP 0.02 + 0.03 ME/kg, P = 0.06). Though not statistically significant, the ERAS group trended towards using fewer opioids when comparing ME/kg on POD 1, 2, and 3 ( Figures 3, Table 2). Regarding complications, we found that neither group had difficulty with urinary retention or prolonged ventilation. However, the TTP group did have two patients that required a return to surgery. The first developed wound dehiscence and leg weakness that required a repeat trip to surgery and hospitalization for IV antibiotics. The other had a large subfascial hematoma causing sensory changes in their leg, which required a return to surgery for evacuation. Both patients' neurologic deficits returned fully by their first post-operative visit and neither had further wound complications.
Discussion
The goal of any ERAS pathway is to improve overall patient outcomes using a multimodal, evidenced-based approach [14]. The use of ERAS in adult spine and reconstructive arthroplasty surgeries has benefited patients by delivering shorter hospitalizations, improved pain scores, and substantial cost savings [5]. Implementation of ERAS for our pediatric AIS patients undergoing PSF resulted in a significant reduction in LOS and opioid use while maintaining satisfactory postoperative pain control.
Increased LOS is correlated with morbidity and serves as the surrogate of postoperative milestones. In adolescent scoliosis patients undergoing surgical fixation, prolonged LOS is directly related to increased risk for surgical site infections, sepsis, and readmission [15][16][17]. In pediatric ERAS protocols, early mobilization is thought to be a contributing factor to decreased LOS and has been shown to improve physical, emotional, and social outcomes in patients [18,19]. Implementation of our ERAS pathway for AIS patients undergoing PSF resulted in a significant reduction in LOS by 1.7 days (p < 0.01) without complications. With discharge criteria at our institution including patients ambulating with minimal assistance, pain adequately controlled with oral pain medication, and the ability to perform activities of daily living independently, we can correlate a reduced LOS to improved patient postoperative outcomes and reduced morbidity.
In addition to enhanced outcomes, ERAS protocols also provide cost savings for patients, their families, and the healthcare system overall [10,20]. PSF for the treatment of AIS is one of the most expensive pediatric surgeries, with charges for a single inpatient POD averaging over $17,000 [21]. Additionally, utilization of an accelerated discharge protocol corresponds to an approximate 22% decrease in postoperative hospital charges after PSF [10]. Implementation of our ERAS protocol resulted in bypassing a PICU admission and a significantly shorter total LOS; therefore, patients presumably also profit from substantial cost savings.
While opioids are frequently prescribed postoperatively, it is not without the potential for significant risk to patients' health. Short-term complications include acute respiratory depression, constipation, and altered sensorium, while long-term complications include risk for opioid use disorders [22,23]. The adolescent population is at 3-5 times greater risk for serious medical complications related to opioid use compared to younger children, and all of our PSF patients fall into this higher-risk age demographic [23]. Compared to patients using our previous methods for pain control, our ERAS patients demonstrated a significant reduction in opioid consumption by 0.99 ME/kg (P< 0.01). We attribute this reduction to the multimodal nature of our ERAS pathways, leading to more reliance on non-opioid analgesics for postoperative pain control.
Given the accelerated nature of the ERAS pathway with an emphasis on early ambulation, one might anticipate an increase in daily opioid analgesic requirements. We found that the TPP patients used slightly fewer opioids on POD0, but that trend quickly changed to the ERAS group requiring less on POD 1, 2, and 3, as shown in Table 2 and Figure 3.
We attribute the lower opioid consumption on POD0 in the TPP group to the use of intra-operatively administered intrathecal morphine, which was not included in the postoperative opioid utilization calculations. Conversely, the ERAS cohort did not receive intrathecal morphine and instead received postoperative methadone, which was calculated in opioid consumption totals. By removing intrathecal morphine and administering methadone, patients were judged to be at a lower risk for postoperative respiratory depression and therefore able to avoid an overnight stay in the PICU for monitoring.
We must consider the limitations of this study. First, our sample size is relatively small, which is likely due to stringent exclusion criteria. We placed an emphasis on only including healthy patients with adolescent idiopathic scoliosis in order to limit confounding variables. Secondly, the numeric pain scale is fairly subjective and highly variable between patients, and therefore may not be the best way to measure pain. Lastly, since this was an unblinded retrospective study, surgeons were aware of the ERAS pathway implementation and may have introduced bias when discussing expectations with families prior to surgery. Overall, while additional patients would increase the power of the study, we are satisfied that our new ERAS pathway provided significant results.
Conclusions
The ERAS pathway at our institution significantly reduced LOS and opioid consumption in AIS patients undergoing PSF. We hope this pathway can serve as a model to reduce the overall utilization of opioids for AIS patients, thus reducing the associated risks. Additionally, we feel that this ERAS pathway better controls patients' post-operative pain allowing for earlier rehabilitation resulting in reduced LOS. While this study only focuses on immediate post-operative care in the hospital setting, further studies would be necessary to assess the effect of the ERAS pathway on recovery as a whole. This could include long-term follow-up with patient-reported outcomes after PSF surgery. Overall, we feel this ERAS pathway has improved recovery after PSF surgery for AIS and continues to be utilized at our institution.
Appendices
have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. | 2023-08-09T15:07:49.515Z | 2023-08-01T00:00:00.000 | {
"year": 2023,
"sha1": "02f0a8e5b2943c5ad906c0af0cc65f6af33b992c",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "ScienceParsePlus",
"pdf_hash": "1c938c9e157845889574bb72dbf587b7b69767eb",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
48361209 | pes2o/s2orc | v3-fos-license | Engineering a nanolab for the determination of lysosomal nitric oxide by the rational design of a pH-activatable fluorescent probe† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc04415d
A pH-activatable fluorescent probe, Rhod-H-NO, was designed and synthesized for the determination of lysosomal NO in living cells and in vivo.
Introduction
Nitric oxide (NO), a small uncharged free radical, which is one of the mediators of cellular responses, produced by nitric oxide synthases, plays a very important role as a signaling molecule in a variety of physiological and pathological processes that take place in the cardiovascular, nervous, and immune systems. 1 In a recent study, it was discovered that lysosomal functions are subtly regulated by NO. 2 These functions include the degradation of a cells own components to supply energy and nutrients for its growth through the lysosomal machinery, 3 and can cause various diseases (such as Fabry, Gaucher, or Danon diseases) due to lysosomal disorders. 4 Recently, numerous uorescent probes for intracellular NO have been developed and applied to intracellular sensing and imaging, 5,6 which has signicantly enriched our knowledge about NO homeostasis and the crucial roles of NO in many biological processes. It is worth noting that obtaining convincing evidence about the interrelation between the variations of NO levels in lysosomes and different physiological processes remains a challenge, because available probes for the selective tracking of NO in the lysosomes of living cells have rarely been reported hitherto. One of the bottlenecks is the lack of organelle-specicity, which results in a high background signal from cytosol and other organelles, meaning that the probes cannot provide high spatial resolution in lysosomes. To tackle this challenge, Xiao et al. conjugated a lysosomal-specic morpholine moiety as the guiding unit with a NO probe to target the subcellular organelle recently, 7 but this strategy suffers from a serious problem: the morpholine moiety exhibits an alkalizing effect on the lysosomes so that longer incubation times with these probes can induce an increase in lysosomal pH and result in cell death. 8 In addition, existing research shows that organic probes are susceptible to acidic pH and interferences from other intracellular species, and are even prone to being degraded by substantial hydrolase in lysosomes, 9 which makes the detection unsatisfactory for application to living cells. Therefore, the development of an ideal uorescent probe with lysosomal targeting is highly desirable for quantifying the variations of NO levels in real time and in a dynamic range of concentrations.
Mesoporous silica nanoparticles (MSNs), popular smallmolecule drug reservoir systems which show excellent biocompatibility, ease of functionalization and are nontoxic to cells, have attracted widespread interest for drug delivery purposes within the past decade. 10 In particular, MSNs can undergo cellular uptake into acidic lysosomes by endocytosis when the nanoparticles are below 200 nm in diameter, 11 which could provide a potential way to study lysosomal tracking and explore its role in invasion with high-resolution spatial images.
Recently, we have developed two uorescent probes for the detection of lysosomal H 2 S and Cu 2+ based on simultaneous target and location activation. 12,13 One of the targeting strategies is via entrapping the uorescent probe into the nanopores of MSNs. The results were inspiring to us, showing that the intact probe molecule can be stored in the nanopores without interference and degradation and then automatically accumulated in lysosomes, which successfully realized the detection of lysosomal Cu 2+ and also provided an efficient method to address these challenges in the determination of lysosomal NO.
As a part of our ongoing interest in uorescent probes for application in lysosomes, we herein fabricate a nanolab for the determination of lysosomal NO by engineering a pH-activatable uorescent probe into the nanopores of MSNs (Scheme 1). Firstly, a pH-activatable uorescent probe, namely Rhod-H-NO, was designed and synthesized for the determination of NO. To avoid the unpredictable uorescence signals from other organelles and to make full use of the acidic conditions of lysosomes, we adopted a strategy to lock the widely applied NO recognition site moiety of o-phenylenediamine by a pH-sensitive imine bond. 14 The presence of the imine bond shows a silent uorescence response to NO in neutral and alkaline conditions, while the lysosomal pH (4.0-6.0) of which mediates hydrolysis of the imine group affords a large, rapid, NO-induced uorescence response. Then, in order to realize the accumulation of probes in lysosomes, MSNs were applied as protective nanocoats to entrap Rhod-H-NO in the nanopores, which prevented the probes from interference and degradation, and thus provided a reaction lab for the determination of NO. Finally, b-cyclodextrin (b-CD), a widely reported "gatekeeper" for closing the gates of the pores of MSNs, 15 was conjugated on the surface of the MSNs to stop external species. This strategy holds the promise of real application in biological research and medical diagnosis.
Results and discussion
Rhod-H-NO was synthesized according to Scheme 1A. The reaction between rhodamine B and o-phenylenediamine led to the spiro compound Rhod-NO, and further condensation reaction between Rhod-NO and propionic aldehyde afforded the probe Rhod-H-NO in 65.2% yield. The puried product was fully characterized by NMR spectroscopy and ESI-MS to conrm the structure (Fig. S1-S3 †). With the probe Rhod-H-NO in hand, we rstly veried the feasibility of the above-mentioned design that NO couldn't "switch-on" the uorescence of Rhod-H-NO unless being activated by acidic pH. As shown in Fig. S4, † Rhod-H-NO exhibits no absorption features in the visible region and is essentially non-uorescent in neutral aqueous solution (pH ¼ 7.0) owing to the spirolactam structure. Upon the addition of NO, there is no signicant absorption or uorescence change indicating that the spirolactam structure remained intact. When Rhod-H-NO was treated with an acidic buffer (pH ¼ 5.0), a strong absorption band centered at 552 nm (3 ¼ 6.50 Â 10 4 M À1 cm À1 ) and a bright uorescence emission with peak maximum at 590 nm (f F ¼ 0.63) were observed obviously, indicating rhodamine ring-opening via a NO-induced reaction in acidic medium. HPLC analysis further demonstrated that the reaction of Rhod-H-NO and NO in acidic medium was identical with the proposed mechanism ( Fig. S5 †). Based on this, we anticipate that the activation response model is benecial towards fabricating an effective lysosome-targetable molecular tool for exploring NO biology.
To ensure that Rhod-H-NO can accumulate effectively and exist stably in lysosomes without interference and degradation, a novel nanolab was fabricated by engineering the probe into the nanopores of MSNs and using b-CD as the gatekeeper of the nanopores, which allows NO to pass through the cavities of b-CD and enter the nanopores to react with Rhod-H-NO. The fabrication process is shown in Scheme 2. MSNs with typical MCM-41 hexagonal arrangements were rst prepared according to the literature protocol. 16 Then the surface of the MSNs was functionalized with chloride groups by treatment with (3-chloropropyl)triethoxysilane to afford MSN-Cl. The azide terminated MSN-N 3 was obtained from the reaction of as-prepared MSN-Cl and sodium azide. Aer removing the surfactant template (n-cetyltrimethylammonium bromide, CTAB) from MSN-N 3 , Rhod-H-NO was then entrapped in MSN-N 3 by a diffusion experiment. Subsequently, the CD-alkyne, which was synthesized according to a previously reported method ( Fig. S6 †), 17 was attached to the MSN surface using a click chemistry approach, giving the nanolab Rhod-H-NO@MSN-CD.
Transmission electron microscopy (TEM) images showed an obvious border aer b-CD was conjugated to the surface of the MSNs, but the average diameter did not show a signicant difference ($80 nm) (Fig. 1A). In addition, MSN-Cl only showed the silica framework vibrations, whereas MSN-N 3 exhibited the characteristic azide stretch signal at 2110 cm À1 , but this band was strongly reduced in intensity upon modication with b-CD (Fig. 1B). The zeta potential values of MSN-Cl, MSN-N 3 and MSN-CD were +18.5 mV, À20.6 mV, and +30.2 mV respectively (Fig. S7 †). These results conrmed that the surface of MSNs was well modied with b-CD. When Rhod-H-NO was loaded into the nanopores of MSN-CD, XRD patterns showed that the low-angle reections indexed as (110) and (200) had disappeared ( Fig. S8 †), and nitrogen adsorption-desorption isotherms and pore size distributions showed that the average pore size decreased with an increase in surface density and inlling ( Fig. S9 †). Fig. 1C further demonstrates that Rhod-H-NO is effectively entrapped in the nanopores without being released for more than 48 h, which is attributed to the size of Rhod-H-NO (1.84 nm  1.29 nm  0.96 nm, calculated by Gaussian 09 programs) which is larger than the nanopore gates aer modi-cation with b-CD. The loading efficiency of Rhod-H-NO in MSN-CD was estimated to be $15.6 wt%. Owing to the protective effect of the MSNs and the enhanced water-solubility of b-CD, Rhod-H-NO@MSN-CD exhibited an improved long-term stability without degradation in cell lysate compared with free Rhod-H-NO (Fig. 1D). Most importantly, NO molecules could freely diffuse into the nanolab through the cavities of the b-CD rings to react with Rhod-H-NO (Fig. S10 †).
The real-time kinetics of Rhod-H-NO@MSN-CD and its response toward NO in a buffer solution with different pH environments further conrmed that Rhod-H-NO@MSN-CD is pH-activatable for NO determination ( Fig. 2A). Aer changing the neutral solution of Rhod-H-NO@MSN-CD to acidic conditions, there was slight, quick, increase in the uorescence intensity which then remained steady over tens of minutes. However, the uorescence intensity dramatically increased and reached a plateau in $30 min aer being subsequently incubated with NO. When NO was rst added into the neutral solution of Rhod-H-NO@MSN-CD and the solution was then adjusted to acidic pH, the uorescence change further affirmed the pH activation characteristics (Fig. S11 †). Then, we got the optimal pH range by measuring the uorescence intensity enhancements of Rhod-H-NO@MSN-CD towards NO. As seen in Fig. S12, † a signicant enhancement of F/F 0 was observed over the pH range from 6.0 to 4.0, which is in good accord with lysosomal pH conditions, while Rhod-NO@MSN-CD was pH insensitive for NO response, suggesting that Rhod-H-NO@MSN-CD is a good candidate for the detection of lysosomal NO.
Next, we performed uorescence titration studies of Rhod-H-NO@MSN-CD for NO in a buffer solution (pH ¼ 5.0). As shown in Fig. 2B, the addition of NO with increasing concentrations from 0 to 20.0 mM NO, elicited a gradual enhancement of the emission band at 590 nm. The uorescence enhancement (F/F 0 ) reached a maximal value (26-fold) in the presence of 20.0 mM NO. Moreover, there was an excellent linear correlation between F/F 0 and NO concentration in the range 0.2 to 6.0 mM with a detection limit (3s/slope) of 100 nM, indicating that Rhod-H-NO@MSN-CD would be a potential tool to monitor endogenous NO in lysosomes. Moreover, Rhod-H-NO@MSN-CD exhibited high selectivity for NO with various biologically relevant species in an aqueous buffer solution at pH ¼ 5.0 (Fig. S13 †). The presence of reactive oxygen (H 2 O 2 , $OH, O 2 À , 1 O 2 , ClO À ), reactive nitrogen (NO 2 À , NO 3 À , ONOO À ), reactive sulfur (SO 3 2À , Cys, Hcy, GSH), and some common metal cations (K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Cu 2+ ), didn't cause an observable uorescence enhancement, which was due to the specicity of the o-phenylenediamine receptor for NO.
Rhod-H-NO@MSN-CD was then investigated for the ability to both target the lysosomes and respond to NO in living cells. The cytotoxicities of Rhod-H-NO@MSN-CD on living cells were rst evaluated by employing standard cell viability protocols (MTT assay) (Fig. S14 †). Aer being cultured for 24 h, the cellular viability of HeLa cells was over 95% and no signicant difference in the morphology was observed even when the concentration of Rhod-H-NO@MSN-CD was increased up to 100 mg mL À1 , showing a very low cytotoxicity. Next, co-localization experiments were performed by co-staining HeLa cells with LysoTracker Green, MitoTracker Green and Rhod-H-NO@MSN-CD. From Fig. 3 and S15, † it can be seen that HeLa cells stained with Rhod-H-NO@MSN-CD for 2 h at 37 C in the presence of 100 mM exogenous NO displayed signicant red uorescence, which merged well with the image from staining with LysoTracker Green but not with MitoTracker Green. Moreover, the intensity proles of the linear regions of interest across the HeLa cells stained with Rhod-H-NO@MSN-CD and LysoTracker Green vary in close synchrony (Fig. S16 †). The Pearson's colocalization coefficient and overlap coefficient were 0.865 and 1.528 respectively, as calculated using Autoquant X2 soware, indicating that Rhod-H-NO@MSN-CD existed predominantly in the lysosomes and was activated by the coexistence of acidic pH and NO. TEM provides further additional physical evidence for the nanoparticles residence in the lysosomes (Fig. 3d). In view of the Rhod-H-NO probe selectively responding to the coexistence of H + and NO in vitro, HeLa cells were pretreated with 1,8-bis(dimethylamino) naphthalene (DMAN), a common proton sponge, to promote lysosomal damage due to the proton-sponge effect. 18 As expected, no obvious uorescence was observed aer incubation with Rhod-H-NO@MSN-CD in the presence of exogenous NO (Fig. S17 †) because the probe escaped into the cytoplasm when the lysosomes were disrupted, further proving that the intracellular response behaviour of Rhod-H-NO@MSN-CD happened in acidic lysosomes rather than in cytoplasm and other organelles with neutral environment.
Next, further experiments were performed to verify the viability of Rhod-H-NO@MSN-CD to detect variations of NO level in living RAW 264.7 macrophages, which are well-known as NO producing cells. As shown in Fig. 4, the image of the probe-loaded macrophages gave barely detectable uorescence ( Fig. 4A(a)). Nevertheless, a signicant increase in the intracellular uorescence was visualized aer further treatment with the widely used NO donor diethylamine NONOate (Fig. 4A(b)), and the intensity presented a tendency toward increased uorescence over time and reached a maximum aer 20 min (Fig. S18 †), indicating that NO released from NONOate was able to diffuse into the lysosomes and then enter into the nanolab to react with Rhod-H-NO. According to the spontaneous NO releaser which in principle releases two equivalents of NO, 19 the linearity between the relative uorescence intensity and NO concentration was established, as shown in Fig. 4B and C, by pretreating the cells with different concentrations of NONOate, which demonstrated that the probe could quantitatively evaluate the intracellular NO content. Thus, to initiate physiological NO production, the well-known external stimuli, bacterial endotoxin lipopolysaccharide (LPS), L-arginine (L-Arg) and pro-inammatory cytokine interferon-gamma (IFN-g), were pretreated with cells for 12 h to promote NO production by inducible nitric oxide synthase (iNOS), 20 and Rhod-H-NO@MSN-CD was subsequently introduced to the cells, showing a strong red uorescence (Fig. 4A(c)). According to the linearity established in Fig. 4C, the average basal NO levels were estimated as ca. 8.36 mM. It should be conrmed that the uorescence increase shown in Fig. 4A(c) was caused by NO generation and not by environmental changes. A NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3oxide (PTIO), 21 was further introduced to the LPS and IFN-g treated cells, which lead to the uorescence being weakened obviously (Fig. 4A(d)). Moreover, we further quantitatively evaluated the endogenous NO in individual cells on a large scale using ow cytometry analysis. Fig. 4D demonstrates the intracellular accumulation of NO where an increase in intracellular uorescence is indicated by a shi in the uorescence signal measured in the FITC channel, which shows the same trend as the microscopy imaging of Fig. 4A. These results indicate that uorescence imaging using Rhod-H-NO@MSN-CD as a nanolab is an effective tool for measuring different NO levels produced in lysosomes.
With an excellent lysosomal-targeting nanoprobe in hand, we used Rhod-H-NO@MSN-CD to investigate the produced NO level in lysosomes in an inamed mouse model. 200 mL of LPS (1 mg mL À1 ) was subcutaneously injected into the le rear leg of a mouse to cause inammation for 12 hours, while the right rear leg was treated with saline simply as the control. From in vivo imaging (Fig. 5A), one can see that strong uorescence was observed from the le leg but not from the right leg aer Rhod-H-NO@MSN-CD was intravenously injected, suggesting that the NO level increased signicantly during inammation. To further examine whether the signal came from the lysosomes of macrophage cells, the le leg skin was then sectioned to get the inammation tissue. As shown in Fig. 5B, the uorescence signal from Rhod-H-NO@MSN-CD was consistent with the results of the immune-staining histological sections with macrophage marker CD11b. From the results we came to the conclusion that the lysosomal NO level of macrophage cells during inammation could be detected by Rhod-H-NO@MSN-CD.
Conclusions
In conclusion, we have presented a novel strategy to fabricate a nanolab for the determination of intracellular bioactive molecules by engineering the highly selective and sensitive small molecular probe into mesoporous nanomaterials. The nanolab was capable of detecting lysosomal NO changes in the presence of exogenous or endogenous NO in living cells and in vivo. Signicantly, this paper successfully addresses some challenges in intracellular imaging analysis. On the one hand, the location-activatable approach could effectively eliminate false signals and improve spatial resolution. On the other hand, the hermetical nanolab can protect the stability of the functional probe and avoid interference from a complex biological environment. In addition, the modication with b-CD on the surface of MSNs can improve the stability and biocompatibility of the nanomaterials in a physiological environment. In view of these merits, we anticipate that this design strategy would open up a new train of thought for the development of efficient molecular tools for bioanalytical and biomedical applications. | 2018-06-15T00:17:35.087Z | 2015-11-30T00:00:00.000 | {
"year": 2015,
"sha1": "ffa8e9a153aa22a2cc737662bbd6bccca62f3895",
"oa_license": "CCBY",
"oa_url": "https://pubs.rsc.org/en/content/articlepdf/2016/sc/c5sc04415d",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "e542c0823ae13df019fb12653befeac305390323",
"s2fieldsofstudy": [
"Chemistry",
"Engineering"
],
"extfieldsofstudy": [
"Medicine",
"Biology"
]
} |
49908087 | pes2o/s2orc | v3-fos-license | Moving forward: why responding to migration, mobility and HIV in South(ern) Africa is a public health priority
Abstract Introduction Global migration policy discussions are increasingly driven by moral panics – public anxiety about issues thought to threaten the moral standards of society. This includes the development of two Global Compacts – agreed principles to guide an international response – for (1) “Refugees” and (2) “Safe, Regular and Orderly Migration.” While the need to address migration and health is increasingly recognized at the global level, concerns are raised about if this will be reflected in the final Compacts. The Compacts focus on securitization, an approach that aims to restrict the movement of people, presenting potentially negative health consequences for people who move. Globally, concern is raised that migration‐aware public health programming initiatives could be co‐opted through a global health security agenda to further restrict movement across borders. This is particularly worrying in the Southern African Development Community (SADC) – a regional economic community associated with high levels of migration and the largest population of people living with HIV globally; this case is used to explore concerns about the health implications of the Global Compacts. Discussion Current HIV responses in SADC do not adequately engage with the movement of healthcare users within and between countries. This negatively affects existing HIV interventions and has implications for the development of universal HIV testing and treatment (UTT) programmes. Drawing on literature and policy review, and ongoing participant observation in policy processes, I outline how Global Compact processes may undermine HIV prevention efforts in SADC. Conclusions The global health imperative of developing migration‐aware and mobility‐competent health responses must not be undermined by moral panics; the resultant international policy processes run the risk of jeopardizing effective action at the local level. Globally, migration is increasingly recognized as a central public health concern, providing strategic opportunities to strengthen public health responses for all. Without mainstreaming migration, however, health responses will struggle. This is particularly concerning in SADC where HIV programmes – including UTT initiatives – will struggle, and key health targets will not be met. Globally, contextually appropriate migration‐aware responses to health are needed, including and a specific focus on HIV programming in SADC.
| INTRODUCTION
While health has long been considered an essential component of human and economic development, the health of migrants has remained in the shadows of key global health, migration, and development dialogues and processes, and many migrants still lack access to affordable health services. (IOM, 2017: p. 4) In spite of recent calls at the global level to improve responses to migration and health, and the development of a global research agenda to support this [1][2][3][4], the "unfinished agenda of migrant health for the benefit of all" [5,6] remains a glaring gap in current global, regional and national policy discussions. To further complicate this situation, attempts to develop interventions on migration and healthat all levelsmay be undermined by the global migration policy terrain. Following the 2016 "New York Declaration on Refugees and Migrants" [7], two Global Compacts are due to be finalized and released in the second half of 2018the "Global Compact on Refugees, " and the "Global Compact on Safe, Regular and Orderly Migration. " Global Compacts refer "to an agreement between states on matters of common interest of concern, " and provide opportunities for determining how member states will "conduct themselves in the future"in this case in relation to the management of migration [8]. Two discrete motivations for engaging with migration at a global level currently existone from a right to health, wellbeing, and public health perspective [1,3], and the other from a securitization and restriction of movement approach [8,9]. The resulting tensions within the global community present multiple challenges for the development of improved responses to migration and health.
In this commentary, I explore the implications of the current global migration policy terrain for the Southern African Development Community (SADC), a regional economic community made up of 15 member states that is associated with high levels of both internal (within country) and cross-border (between country) migration and a high communicable disease burden, including the largest population of people living with HIV globally [10][11][12][13]. With the aim of offering suggestions for ways to mobilize a regional response to migration and HIV in SADC, I outline the challengesand strategic opportunitiesthat result from the current global migration policy terrain. This is done by drawing on a review of research and policy associated with migration and HIV in SADC (13 and Table 1) and my ongoing participant observation within various global, regional and national policy processes.
| A global policy crisis?
As evidenced by the current Global Compact processes, we find ourselves in a world increasingly concerned with securitizing national borders and restricting the movement of people between nation states. Much of this focus on security is driven by moral panicspublic anxiety about issues thought to threaten the moral standards of societyassociated with migration, including human trafficking and the independent movement of women [9], and the so-called "Migration Crisis" in Europe [14][15][16]. Internationally, discussions on migration tend to ignore long-established population movements within Global South contexts where forced migration and movement in search of improved livelihood opportunities are commonplace and outnumber similar movements in the Global North; the Southern African region is no exception [17]. The current global discourses surrounding population mobilitythat are fuelling morally panicked policy discussionshave negative impacts both for those who move, and for the development of improved responses to migration and health. Centrally, this includes the implementation of increasingly restrictive immigration policies, including further securitization of the borders of nation sates. In relation to health and wellbeing, historical perceptions of the migrant as the "diseased body"; as a carrier and transmitter of infectious diseases, particularly HIV; and, consequently, as a burden on the welfare state of receiving countries, are reemerging [18][19][20][21]. We need to remain vigilant and ensure that the re-emergence of this discourse is not used to support securitization agendas as health status may (once again) be used to mediate the ability to legally cross national borders. Particularly worrying is that this may include an unwelcome return to a focus on the HIV status of people crossing borders.
The "draft zeros" of the two Global Compacts were released in early 2018 [22,23] and, while they do acknowledge health, concerns remain that healthand other social justice issueswill be left off the final agenda [9,24]. As a result, the final Global Compacts run the risk of calling for actions that ignore or may even be in contravention ofexisting approaches aimed at improving health for all, including the Sustainable Development Goals ( Table 1 summarizes the key international policy processes that have been engaging with migration and health, and the more recent processes associated with the Global Compacts. From a review of these processes, participation in both the 1st and 2nd Global Consultations on Migration and Health in 2010 and 2017 [3,28], and more recent engagement in global migration and health research initiatives, it becomes apparent that the progressive agenda being developed around migration and health stands the risk of being undermined by the Global Compacts process. With a clear focus on securitization and the restriction of movement, health-related issues are being side-lined in current global discussions. In addition, and particularly important for SADC, concerns have been raised that the loudest voicesthose associated with the anti-immigrant agendas of northern Europe, are driving the Global Compact processes, resulting in a global agenda calling for further restrictions on international migration which will be detrimental to other regions of the world, including the African continent [29-31]. To this end, the African Union developed a draft Common African Position, that involved regional dialoguesincluding within SADCin an attempt to ensure that the contextual realities of the continent are engaged with in the finalization of the Global Compacts [32]. This Position paperwhich makes reference to the importance of ensuring access to healthcare for migrantswas presented in draft form at the international preparatory meeting on the Global Compacts held in Mexico in December 2017, and has since been finalized and approved [33]. How effective these interventions are, and whether health-related concerns are emphasized in the final Compacts, remains to be seen.
| Migration, mobility and HIV
In spite of the SADC region being associated with a long history of diverse population movements, current public health responses to HIV still fail to adequately engage with migration and the movement of healthcare usersboth within and between countries [10][11][12]. The result is a range of negative outcomes with serious implications for population health and HIV prevention, including challenges in initiating treatment, ensuring treatment continuity, and the associated risks for defaulting and drug resistance [34]. In addition, the absence of evidence-informed and migration-aware responses to HIV has led to the continued scapegoating of migrantsparticularly non-nationalsas the carriers of HIV; the "diseased" migrant body is a long-standing trope, and one that conjures up the idea of the migrant as a disease vector, whose movements are solely responsible for the spread of new HIV infections [18]. Such imaginings conveniently absolve the state from its own shortcomings in terms of inadequate healthcare, health promotion and HIV prevention strategies. Rather, the state and their healthcare institutions blame internal and cross-border movements for placing an excessive burden on the state.
Globallyand in SADCkey population groups currently targeted for HIV prevention initiatives are often highly mobile, including sex workers and men who have sex with men [13]. Recent UNAIDS Gap Reports make the case for developing migration-aware responses to HIV, acknowledging the importance of developing cross-border initiatives and mainstreaming migration into national HIV strategic plans [13,[35][36][37]]. This has implications for the development and implementation of effective HIV prevention programming, including universal HIV testing and treatment (UTT) and pre-exposure prophylaxis (PreP). A key challenge relates to how to do this: migration is a politically sensitive issueassociated with anti-foreigner and xenophobic rhetoric, in spite of internal mobility being far Table 1. An overview of key global and regional migration and health policy processes (expanded on from [3,12] more prevalent-and HIV (and health more broadly) is also a politically sensitive concern. Bringing together the interconnected concerns and agendas relating to migration and HIV is challenging and innovative approaches to this are required; migration is a central public health concern thatif approached carefullycan provide a strategic opportunity to strengthen responses to HIV for the benefit of all. For example, framing the development of responses to migration and HIV as a key entry point to improving health for all, for addressing health inequities, and for working towards universal health coverage, could assist in obtaining the political support needed to fund and support migration-aware responses to HIV in SADC. This will have further positive impacts, namely in supporting activities for achieving global health targets, including the SDGs [26] and the UNAIDS 90-90-90 targets [38]. However, a key challenge existsevidence suggests that SADC remains poorly equipped to initiate and manage the political discussions within and between member states that are required to develop appropriate regional responses to migration, mobility, and HIV [11,39]. This is partly due to the historical preference for the development of individual bilateral agreements between member states rather than regional responses [39]. However, as presented in Table 1, some regional processes have been initiated. For example, the SADC Framework for Population Mobility and Communicable Diseases was developed in 2009 [40] but remains in draft form with several member states refusing to ratify the Framework. The associated exercise to explore financing mechanismsat the request of member stateswas eventually completed by SADC in 2015, but remains unpublished (Oxford Policy Management, unpublished work). In 2010, the "SADC HIV and AIDS Cross Border Initiative" was awarded funding from the Global Fund to establish a regional cross-border HIV programme involving the establishment of 32 clinics offering HIV testing and treatment, alongside primary care, in border areas and along transit routes to serve migrant and mobile populations, and local migration-affected communities; 12 mainland member states signed Memorandums of Understanding (MoUs), agreeing to participate [41]. However, progress is painfully slow: phase 1 involved just 12 clinics being opened, and the second phasewith a further 20 clinics due to open was initiated at the end of 2017, with the anticipation that all 32 clinics will be handed over to member states in the first half of 2018. The slow process and challenges associated with this give a good indication of the challenges facedboth political and logisticalin developing and implementing crossborder, migration-aware HIV interventions at a regional level. The only regional health and migration policy process that has been implemented is the 2012 Declaration on Tuberculosis in the Mines [42]; ratification of this Declaration happened quickly, and appears to be the result of any associated financial burden being the responsibility of the private sector, and not member states who are unwilling to commit to regional responses associated with migration and health.
| A local lens: South Africa's engagement with migration and health
Within the SADC region, South Africa is the recipient of the largest number of cross-border migrants (most of whom come from other SADC states), has a long history of internal migration, and a continued high prevalence and incidence of HIV [11,13]. However, responses to HIV that engage with population mobility are noticeably absent, in spite of calls being made for migration-aware responses to HIV [10,12,43,44]. In addition, South Africa has not ratified the 2009 "Draft SADC Framework on Population Mobility and Communicable Diseases"in spite of the development of financing models to support equitable cost-sharing for regional responses to HIV. . In 2014, a task team was established to explore HIV, migration and mobility within the National Strategic Plan for HIV, STIs and TB. However, responses to migration in the national HIV plan remain limitedand no framework has been developed for their implementation [12]. South Africa is in the process of developing a National Health Insurance (NHI) whichin many waysis a progressive development. However, current iterations of the NHI present a possible regression in the rights of non-nationals to access healthcare, including ART [52].
| CONCLUSIONS
There is an urgent need to develop migration-aware [13] and mobility-competent [3] responses to health globally. Particular concerns are raised around the need to develop appropriate responses to migration and HIV in SADC. While gains have been made in the response to HIV in SADC, multiple challenges remainparticularly in the era of UTTas current responses do not adequately engage with migration and the movement of healthcare users [13,53]. HIV prevalence and incidence remain high, and it has been suggested elsewhere that it is the lack of migration-aware programming that has undermined HIV prevention efforts at the regional level, where diverse internal and cross-border population movements are prevalent [13]. Currently, public healthcare systems in the southern African region are not designed to ensure continuity of care for migrant and mobile populations and prevailing xenophobic and anti-foreigner sentiments present additional barriers to cross-border migrants [11,12,34]. The development of increasingly securitized migration management initiatives results in some crossborder migrants struggling to access the documentation required to be in a country legallywhich is often required to access healthcare [12]. As a result, people moving both within countries and across national borders face barriers when trying to access HIV prevention, treatment and care [12,53]. The established evidence is clear: delayed testing and/or treatment initiation has negative impacts for infected individuals and for the populations with which they interact [34]. By not engaging with migration, programmes currently designed to support HIV testing, antiretroviral treatment initiation and treatment continuity are jeopardized, with potentially, devastating consequences for HIV prevention programmingparticularly in relation to UTT initiatives.
Concerns about how the Global Compact processes may undermine efforts to improve responses to health and migration globally should not be taken lightly. Vigilance is required to ensure that migration-aware public health programming is not co-opted to support securitization agendas that place the health and wellbeing of people on the move at risk. Globally, contextually appropriate migration-aware responses to health are required; migration is a central public health imperative that provides a strategic opportunity to strengthen public health responses for allincluding universal healthcare coverage [10,12]. In the SADC context, HIV prevention and treatment programmes will continue to struggle if migration is not mainstreamed, and key health targets will not be met [12]. There is an urgent need to implement a regional strategy for the development of contextually appropriate migration-aware responses to HIV in SADC, particularly in the UTT era. Efforts must be made to ensure that local-level health programmingincluding HIV programming in SADCis not undermined by current global moral panics, and resultant policy discourses.
A U T H O R ' S A F F I L I A T I O N S
African Centre for Migration & Society, University of the Witwatersrand, Wits, South Africa; Centre of African Studies, University of Edinburgh, UK
C O M P E T I N G I N T E R E S T S
The author has no conflicts of interest to declare.
A C K N O W L E D G E M E N T S
The author thanks the Wellcome Trust for funding an Investigator Award held by the author (WT104868MA) that facilitated the research and writing of this article. | 2018-08-06T12:56:23.153Z | 2018-07-01T00:00:00.000 | {
"year": 2018,
"sha1": "6781a1b2dd5a4beb578b6cdd7de5ed2cd98fdc24",
"oa_license": "CCBY",
"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jia2.25137",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "6781a1b2dd5a4beb578b6cdd7de5ed2cd98fdc24",
"s2fieldsofstudy": [
"Political Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
240540752 | pes2o/s2orc | v3-fos-license | Language processing in glioma patients: speed or accuracy as a sensitive measure?
ABSTRACT Background Glioma (brain tumour) patients can suffer from mild linguistic and non-linguistic cognitive problems when the glioma is localised in an eloquent brain area. Word-finding problems are among the most frequently reported complaints. However, mild problems are difficult to measure with standard language tests because they are generally designed for more severe aphasic patients. Aims The aim of the present study was to investigate whether word-finding problems reported by patients with a glioma can be objectified with a standard object naming test, and a linguistic processing speed test. In addition, we examined whether word-finding problems and linguistic processing speed are related to non-verbal cognitive abilities. Methods & Procedures We tested glioma patients (N=36) as part of their standard pre-treatment clinical work-up. Word-finding problems were identified by a clinical linguist during the anamnesis. Linguistic processing speed was assessed with a newly designed sentence judgment test (SJT) as part of the Diagnostic Instrument for Mild Aphasia (DIMA), lexical retrieval with the Boston Naming Test (BNT), presence of aphasia with a Token Test (TT), and non-verbal processing with the Trail Making Test A and B (TMT). Test performances of glioma patients were compared to those of healthy control participants (N=35). Outcomes & Results The results show that many glioma patients (58%) report word-finding problems; these complaints were in only half of the cases supported by deviant scores on the BNT. Moreover, the presence of reported word-finding problems did not correlate with the BNT scores. However, word-finding problems were significantly correlated with reaction times on the SJT and the TMT. Although there were no significant differences between the patient and control group on the SJT, a subgroup of patients with a glioma in the frontal lobe of the language-dominant hemisphere was slower on the SJT. Finally, performance on the SJT and TMT were significantly correlated in the patient group but not in the control group. Conclusions Linguistic processing speed appears to be an important factor in explaining reported word-finding problems. Moreover, the overlap between speed of language processing and non-verbal processing indicates that patients may rely on more domain-general cognitive abilities as compared to healthy participants. The variability observed between patients emphasises the need for tailored neuro-linguistic assessments including an extensive anamnesis regarding language problems in clinical work-up.
Gliomas
Gliomas are the most common type of primary brain tumour. The World Health Organization categorises gliomas into four grades. High-grade gliomas (HGG, grades III-IV) are more aggressive and more common than low-grade gliomas (LGG, grades I-II; Sanai & Berger, 2012). Gliomas are often located in eloquent areas of the brain (Duffau & Capelle, 2004;Gerritsen et al., 2019). In these cases, surgery is aimed at resecting the tumour whilst preserving cognitive functions (Ilmberger et al., 2008;Sanai & Berger, 2008;De Witte & Mariën, 2013). Due to the preferential localisation of gliomas in eloquent areas of the brain, patients may experience neurological and cognitive impairments that can have serious consequences on their quality of life.
Sensitivity of assessments
It is of central importance for quality of life to investigate how patients subjectively experience (loss of) abilities, such as language (Cruice, Worrall, & Hickson, 2006). Word retrieval difficulties are among the most common complaints of people with a glioma (Racine et al., 2015). Importantly, performance on objective cognitive tests may not necessarily reflect the patients' complaints (Gehring et al., 2015;Racine et al., 2015;Taphoorn & Klein, 2004;Van der Linden et al., 2020). More specifically, patients appear to report language complaints that are not supported by lower scores on standard language measures, demonstrating insufficient sensitivity of those tests (Brownsett et al., 2019;Satoer et al., 2012).
For instance, Satoer et al. (2012) compared scores on the Aphasia Severity Rating Scale (ASRS; Goodglass et al., 2001) to the self-reported problems and found that more patients reported issues in daily communication than were shown to have impairments based on the ASRS (57% vs. 39%, respectively). This shows the value of combining standardised tests with an evaluation of self-reported complaints in the assessment of cognitive abilities of glioma patients (Taphoorn & Klein, 2004).
A potential reason for the discrepancy between subjectively experienced language difficulties and objective test performance, is that aphasia assessments are generally designed for patients who suffered a stroke. Glioma patients, with comparable lesion size and location, typically have milder language and/or cognitive deficits (Anderson et al., 1990). These mild impairments can be the result of neural reorganisation (i.e., compensation for loss of function) due to the slow growth rate of gliomas as compared to the sudden onset of neurological damage caused by stroke (Duffau, 2008(Duffau, , 2014. The subtlety of cognitive-linguistic impairments in glioma patients poses a problem for the assessment of their cognitive functions.
Processing speed
Including a measure of response speed in the assessment of glioma patients may increase sensitivity of standard measures and provide a way to objectively measure self-reported word-finding problems. Language processing in patients with a glioma was investigated by Moritz-Gasser et al. (2012), who studied the correlation between naming capacities and the ability to return to work after surgery. They found that naming speed, rather than accuracy, significantly predicted return to work, an important marker for quality of life. Importantly, none of the patients in their study was classified as "aphasic" according to the Boston Diagnostic Aphasia Examination (BDAE; Goodglass & Kaplan, 1972), a test battery originally designed for stroke patients. Another recent study has shown that patients with gliomas are significantly slower on a speeded naming test compared to healthy participants (Ras et al., 2020). This difference could not be explained by naming ability measured with the Boston Naming Test (BNT; Kaplan et al., 2001).
As for non-verbal processing speed, previous studies have shown that glioma patients performed significantly worse than a healthy control group (Habets et al., 2014;Wefel et al., 2016). Interestingly, it even appeared to be the most-often impaired cognitive ability in this patient group (Ek et al., 2010). These studies typically operationalise non-verbal processing speed with a Symbol Digit Modalities Test (Smith, 1973) or the Trail Making Test Part A (TMT-A; Army Individual Test Battery, 1944).
Including an assessment of processing speed may not only increase the sensitivity of measures, but may also bear a direct relationship with communicative difficulties experienced by patients in everyday conversations. Everyday communication requires the conversational partners to process information quickly and respond to it in an appropriate manner, and speedy processing of linguistic information is crucial (e.g., Carragher et al., 2012). Subjectively experienced word-finding problems may therefore be the result of not only a lexical retrieval problem, but may also be due to slowed processing.
Domain generality of processing speed
The finding that patients with a glioma are slower on both linguistic (Moritz-Gasser et al., 2012;Ras et al., 2020) and non-linguistic tasks (Ek et al., 2010;Habets et al., 2014;Wefel et al., 2016), raises the question whether slowed performance of a language test is specific to language processing, or whether it has a more domain-general origin. This topic has been investigated in people with aphasia due to stroke. For example, individuals with aphasia (and individuals with left-hemispheric lesions without aphasia) were found to have lower processing speed both within and outside the language domain (Yoo et al., 2021). Moritz-Gasser et al. (2012) and Ras et al. (2020), on the other hand, found that naming speed of patients with a glioma could not be explained by non-verbal processing speed measured with the TMT-A. Their findings suggest that there is a discrepancy between naming accuracy, naming speed, and general processing speed. However, these two studies investigated processing speed in the production of language, leaving receptive linguistic processing speed of patients with a glioma open for investigation. It is not self-evident that the influence of processing speed is the same in both language modalities, as a discrepancy between deficits in language production and reception has been described (De Witte et al., 2015b).
From this discussion of the literature it has become clear that the subjectively experienced communication difficulties are not always supported by impaired performance on standard language measures. Measuring processing speed may be useful in objectively assessing subjectively experienced word-finding problems, not only because information processing speed has often been found to be impaired in patients with a glioma, but also because everyday communication relies on speeded integration of linguistic information. Problems in everyday communication may therefore be the result of slower linguistic or non-linguistic processing abilities.
Present study
We aim to investigate whether including a measure of response speed in a receptive language test is a sensitive measure for self-reported word-finding problems in patients with a glioma. The presence of self-reported word-finding problems was correlated with lexical retrieval, receptive linguistic processing speed, and non-verbal cognitive abilities. We compared a group of patients with a glioma to a group of age-and educationmatched healthy control participants. Individual patients were also described and compared to norm groups. The following research questions were investigated: RQ1: To what extent can self-reported word-finding problems of patients with gliomas be explained by: Based on previous findings in the literature, we hypothesised a discrepancy between the subjectively experienced word-finding problems and the objectively measured abilities of patients (Brownsett et al., 2019;Satoer et al., 2012). The addition of reaction time measures to a sentence judgment test is expected to lead to a sensitive measure that can explain anamnestic complaints (Moritz-Gasser et al., 2012;Ras et al., 2020). Finally, if the word-finding problems and slower language processing are the result of a more global cognitive impairment, we expect that patients will also exhibit longer reaction times on a non-linguistic task.
Participants
The study group consists of glioma patients (N = 50) who have undergone awake surgery (between March 2015 and November 2017) at the Erasmus MC University Medical Centre. All patients diagnosed with a glioma, regardless of the hemispheric localisation, were included in the study, as previous research has shown that patients with a glioma in the right hemisphere may also experience language difficulties (Vilasboas et al., 2017;De Witte et al., 2015c). Fourteen patients were excluded due to a recurrent tumour with second or third surgery (N = 10) 1 ; too many missing data (N = 2); or co-occurring developmental dyslexia (N = 1) or Noonan Syndrome (N = 1). This resulted in 36 participants in the patient group. All patients were native speakers of Dutch.
Healthy native speakers of Dutch (N = 35) constituted the control group of the study. They were matched to the patient group on age and education but not on gender, as gender has generally not been shown to influence performance on standard language tests (e.g., Snitz et al., 2009;De Witte et al., 2015b). They were included if they had no (history of) cardiovascular, neurological, psychiatric, or developmental language disorders; no toxic substance abuse; normal vision and hearing; no sleep medication, psychotropic, or neuroleptic drugs. The demographic information of the patients and control participants is given in Table 1. None of the participants was financially compensated for his/her participation. The Ethical Committee of the Erasmus MC approved of the study and all participants gave their informed consent.
Word-finding problems
Information on word-finding problems in patients was based on complaints reported during the preoperative anamnesis. The information in the anamnesis is gathered in an interview with the patient by a clinical linguist, using a standard set of questions about encountered problems with language, memory, attention, and executive functioning. The word-finding complaints were labelled as follows: 0: no complaints; 1: mild complaints, if the patients only reported difficulties after more targeted questions, if they indicate that they "sometimes" experience problems, or if their partner reported word-finding difficulties; 2: clear complaints, if the patient presented their word-finding complaints centrally in the anamnesis, or with modifiers such as "often", or "severe". The same coder re-coded the data at a later timepoint and the intra-coder reliability was assessed using an intra-class correlation analysis. The intra-class correlation estimate was based on a single-rating, absolute-agreement, two-way mixed-effects model. The results show that agreement between these two timepoints was good-excellent (intraclass correlation coefficient = .89, p < .001).
An example of a mild complaint
"Patient does not report cognitive problems. After additional questions, he reports subtle word-finding difficulties. Handwriting is also a bit messier."
An example of a clear complaint
"Patient reports word-finding difficulties that result in avoiding talking to people. Word is in mind but cannot be pronounced. Patient also fails in writing and typing. In addition, there are sound changes, and articles and function words that are forgotten."
Standard language tests
The Boston Naming Test (BNT; Kaplan et al., 2001), a standard test to assess anomia in individuals with aphasia was administered. Patients also completed the shortened Token Test (TT;De Renzi & Faglioni, 1978), a standard test to measure aphasia severity.
Linguistic processing speed
The Sentence judgment Test (SJT), a subtest of the Diagnostic Instrument for Mild Aphasia (DIMA; Satoer et al., 2021), was used to test comprehension and language processing on the semantic, syntactic, and phonological level. The SJT was administered in E-Prime 10 (26) software (Psychology Software Tools, 2012) or in Praat (Boersma & Weenink, 2018). The SJT consists of 30 sentences, half of which contain errors in three different linguistic domains. The phonological items aim to assess phonological awareness by including pseudo-words (Example 1). The syntactic items contain errors in verb inflection (tense and agreement), word order, or pronouns (Example 2), and the semantic items include sentences with semantic anomalies (Example 3).
Example 2 Linda zingt gisteren een lied.
Linda sing-AGR.PRES yesterday a song "Linda sings a song yesterday".
Example 3 De loodgieter repareert de regenboog.
The plumber repair-AGR the rainbow "The plumber repairs the rainbow".
The participants read the sentences on a computer screen and rated their correctness by pressing the keys "F" for fout "wrong" and "J" for juist "right" on the keyboard. Reaction times (RTs) in milliseconds and accuracy were measured. RTs were operationalised as the time between the start of the stimulus presentation and the manual response of the participant. Items were presented in randomised order, and the test contained four practice items to familiarise participants with the procedure.
Non-language tests
Nonverbal cognitive abilities of the participants were assessed using the Trail Making Test A and B (TMT-A and -B; Army Individual Test Battery, 1944). In the TMT-A, the participant connects numbers (1-25) in an ascending order on a paper sheet. The TMT-B requires the participant to connect alternating numbers and letters (i.e., 1-A-2-B-3 etc.). The score on both tasks consists of the time in seconds it takes to finish. Visuoperceptual speed underlies performance on the TMT-A, while TMT-B relies more heavily on updating and concept-shifting abilities (Sánchez-Cubillo et al., 2009). The difference score TMT-BA, operationalised as the ratio score B:A, provides a relatively pure measure of cognitive flexibility.
Procedure
The clinical staff at the Erasmus MC University Medical Centre collected the data of the patients. An elaborate neuro-linguistic test protocol was administered as part of the standard clinical work-up, and the tests we report on in the present study are part of this protocol. The results of the preoperative assessment were compared to the performance of healthy control participants, who were tested in a private setting. The BNT, SJT, and TMT were administered in a random order. The entire procedure lasted approximately 15 minutes.
Data analysis
All statistical analyses were carried out in R (R Core Team, 2019) and the graphics were created using R-package ggplot2 (Wickham, 2016). The results on the SJT, TMT, TT, and BNT constitute the dependent variables. The data were analysed using regression models in the R package lme4 (Bates et al., 2015) and lmerTest (Kuznetsova et al., 2017) to retrieve p-values. The accuracy scores and RTs of the SJT were analysed with a (generalised) linear mixedeffects regression model with random slopes for participants and items. The outcomes of the TMT, TT, and BNT were analysed using a linear regression model. The scores on the TMT were log-transformed to meet the model criteria. We adhered to an α-level of 0.05.
The main predictor in each model was group (patients vs. control participants), and covariates age and education level were included in all models. Within the patient group, the effects of tumour grade (LGG vs. HGG) and hemisphere (left vs. right) and the interaction effect between these factors were estimated. The output of the statistical models is included in the Appendix.
The analysis of the SJT results was carried out with the anomalous sentences. 2 Linguistic levels (semantics, syntax, phonology) and trial-by-trial sequence (i.e., the position of each item in the test) were included as additional within-participant predictors. We removed outliers before the group analysis of the RTs of the SJT. Items with an RT below 500 milliseconds were removed as it is assumed that participants need at least 500 milliseconds to properly assess an item, so shorter RTs are likely due to slips of attention. In addition, items with an RT above 10 seconds were removed, as the E-Prime experiment included a time limit and any responses longer than 10 seconds were classified as null responses. This led to the exclusion of 13 trials (0.7%). Thereafter, outliers per participant were calculated and removed from the dataset using the trimr package (Grange, 2015). An outlier was defined as an RT value of 2 SD above or below the mean for each participant. This led to the exclusion of 87 trials (5%). The remainder of the RTs were log transformed to normalise the data and meet the model criteria. The log-transformed RTs provided a good fit for the raw data (ρ = .96, p < .001).
To estimate the relationship between the anamnestic complaints and the scores on the objective measures, the correlation between these measures was calculated using Pearson's correlation coefficient.
The scores on the shortened Token Test did not correlate with the reported wordfinding difficulties (ρ = −.07, p = .71). The mean score of the patient group was 33.8 out of 36 points. Adhering to the cut-off score of 29 (De Renzi & Faglioni, 1978), only one patient showed a deviant score on the Token Test. The Token Test scores were not significantly influenced by tumour grade (ß = 1.79, SE = 1.08, p = .11), or hemispheric localisation (ß = −1.38, SE = 1.86, p = .47). And there was no significant interaction effect between these variables (ß = 1.43, SE = 2.19, p = .52).
Linguistic processing speed
At the group level, there were no significant differences for RTs between patients and control participants (ß = −0.02, SE = 0.08, p = .84), but the difference between the two groups on accuracy scores in all linguistic domains combined was significant (ß = 1.16, SE = 0.41, p = .01). The differences between the groups are presented in Figure 1. Tumour grade, hemispheric localisation, or the interaction between these factors, did not significantly affect RTs nor accuracy scores. The reported word-finding problems were strongly correlated with the RTs on the SJT averaged over all linguistic domains (ρ = .64, p < .01), and with each linguistic level separately (syntax: ρ = .61, p = .003; semantics: ρ = .64, p = .002; phonology: ρ = .55, p = .01). However, the word-finding complaints did not correlate significantly with the accuracy scores on the SJT over all linguistic domains (ρ = −.23, p = .31).
At the individual patient level, it appears that there is a subgroup of patients with deviant RTs in the SJT compared to normative data (Satoer et al., 2021). Six out of twentyone (29%) patients had slightly deviant RTs (≥1.5 SD from population mean) in at least one of the three linguistic domains (phonology, semantics, syntax). All six patients with deviant RTs on the SJT had a glioma in the left hemisphere, and all but one (83%) in the frontal lobe. One patient (17%) had a grade-II glioma, two patients (33%) a grade-III glioma, and three patients (50%) had a grade-IV glioma. Eight out of twenty-one (38%) patients showed deviant accuracy scores on the SJT. Nine out of 35 control participants (26%) had deviant RTs on one of the language domains of the SJT, and two control participants (6%) showed deviant accuracy scores.
Non-verbal cognitive measures
At the group level, there was no statistically significant difference between the patient group and the control group on the TMT-A and B (ß = 0.08, SE = 0.08, p = .30 for TMT-A and ß = 0.13, SE = 0.09, p = .16 for TMT-B). However, patients had a larger difference score on the TMT-BA compared to healthy participants (ß = 0.56, SE = 0.19, p = .004). The differences between the groups are presented in Figure 2. Within the patient group, patients with an HGG were slower to finish the TMT-B (ß = −0.51, SE = 0.22, p = .03) and had a larger ratio score on TMT-BA (ß = −0.96, SE = 0.45, p = .04) compared to patients with an LGG. This was not the case for the TMT-A (ß = −0.22, SE = 0.16, p = .17). There were no significant main effects of hemispheric localisation or interaction effects between these factors on any of the TMTs. In the patient group, performance speed on the TMT-A and -B strongly correlated with the RTs on the SJT (ρ = .61, p = .01 for TMT-A and ρ = .74, p < .001 for TMT-B), indicating that longer RTs on the SJT were accompanied by longer completion time on the TMT-A and -B. The ratio of the difference score TMT-BA also correlated moderately with the RTs on the SJT (ρ = .59, p = .01), indicating a shifting component in the SJT independent of speed. Interestingly, in the control group significant correlations between the RTs on the SJT and the TMT-A and -B were absent (ρ = .20, p = .256 and ρ = .10, p = .58, respectively). In the control participants, the ratio score of the difference TMT-BA also did not correlate significantly with the RTs on the SJT (ρ = −.07, p = .682). These correlations are presented in Figure 3.
There was a weak but significant correlation between the reported word-finding problems and the TMT-B (ρ = .44, p = .01) and a marginally significant correlation between the word-finding problems and the TMT-A (ρ = .34, p = .052). The correlation between word- finding problems and the TMT-BA was not significant (ρ = .32, p = .07). Model comparison showed that the linear regression model with only the RTs in the SJT as a predictor (and no TMT measure) yielded the best fit for the word-finding problems (Figure 4).
Eleven out of 33 patients (33%) had an impaired score on at least one of the subcomponents of the TMT (Tombaugh, 2004). Three patients (9%) scored >1.5 SD from the normal score on both the TMT-A and B. Three patients (9%) had problems with the TMT-B and a deviant difference score TMT-BA (cut-off ratio score >3, Arbuthnott and Frank, 2000). Five out of 33 patients (15%) had a selective problem with concept shifting (cognitive flexibility), exemplified by a deviant difference score TMT-BA.
Linguistic processing speed and word-finding complaints
We aimed to examine whether assessment of processing speed in a receptive language test would provide a sensitive measure to objectively determine reported language problems, and whether it is related to non-verbal processing speed. First, we showed that 58% of glioma patients experience word-finding difficulties in daily life, but that their reported problems were supported by deviant BNT scores in only 50% of the cases. The reported word-finding problems also did not correlate with the accuracy scores on the SJT. The Token Test proved to be insensitive to detect language problems in this patient group; only one patient scored below the cut-off level and the scores were not correlated with the reported word-finding problems. At the group level, however, glioma patients scored significantly worse on the BNT and had worse accuracy scores on the SJT compared to the control group.
The discrepancy between reported language complaints and scores on objective language measures has been described in previous research (Satoer et al., 2012). In addition, there is evidence that impaired linguistic variables found in spontaneous speech of glioma patients do not correlate with performance on standardised language tests (Satoer et al., 2018(Satoer et al., , 2013. At the same time, Brownsett et al. (2019) found that after surgery, 58% of glioma patients reported communication difficulties, which did not correspond with the Aphasia Quotient of the Western Aphasia Battery-Revised (Kertesz, 2006, 27% of patients scoring below normal cut-off), but could be explained with the scores on the Comprehensive Aphasia Test (Swinburn et al., 2004, 77% of patients scoring below normal cut-off). The inconsistency between the BNT scores and the language complaints we found in the current study could indicate that the word-finding problems originate from an issue other than a pure lexical retrieval deficit.
We investigated the relationship between the reported complaints and linguistic processing speed. Previously, productive language tasks with a time constraint have been reported to be difficult for glioma patients, illustrated by longer response times on naming tasks (Moritz-Gasser et al., 2012;Ras et al., 2020). In contrast to our expectations, the results of the current study did not show deviant group-level performance on the RT measure of the SJT, which assesses speed of receptive language processing. However, glioma patients had significantly lower accuracy scores compared to the control group. This could be due to a deviant speed/accuracy trade-off, in which higher response speed is favoured over accurate responding.
In a subsequent analysis, we looked at the individual RT scores in the SJT and found that all patients with long RTs had a glioma in the language-dominant hemisphere, mostly in the frontal lobe. This seems to suggest that assessing speed of language processing in patients with left frontal damage may be particularly useful, although data from more patients is necessary to further investigate this observation.
In contrast to the absence of a significant correlation between the word-finding problems and BNT and accuracy scores, more severe word-finding complaints were accompanied by longer RTs on the SJT. We found that the presence of word-finding problems was significantly correlated with the overall RTs in the SJT, but also with each linguistic level separately. The commonalities between the different linguistic levels may point to a shared underlying attentional component required to perform this task. This is in accordance with the finding that the reported complaints also correlated with performance of the TMT. Although the TMT is not a perfectly matched non-verbal equivalent of the linguistic processing speed task, it provides a measure of visuoperceptual speed and relies on attention. Therefore, our findings could imply that domain-general attentional mechanisms underlie experienced word-finding problems. This aligns with previous research in which attentional deficits were observed in persons with self-reported mild anomia, who performed within normal limits on standard language assessments (Hunting-Pompon et al., 2011).
At the same time, it must be noted that the observed correlations between reported word-finding problems and the TMT were weaker than the correlations with the SJT. A model with linguistic processing speed as a sole predictor best fit the word-finding complaints of the patients, compared to models also including scores on the TMT as predictors. This indicates that, despite an important role for more domain-general processing abilities in lexical retrieval, there appears to be an indispensable linguistic factor to word-retrieval difficulties.
The word-finding problems may be the most salient issue that glioma patients experience in everyday communication. Dialogues require conversational partners to process verbal information quickly and respond to it promptly in an appropriate manner. This entails the integration of a range of different abilities, which may be challenging for individuals with aphasia. For example, they have been shown to experience more difficulties with language production on a story retelling task, when they have to perform another task simultaneously (Harmon et al., 2019). Apart from linguistically meaningful and grammatically correct output, other cognitive functions, attention and executive functioning in particular, have been shown to play a crucial role in the successful everyday communication of aphasic speakers (Fridriksson et al., 2006;Olsson et al., 2019). This may be an explanation for the relationship between word-finding complaints and slower processing of both linguistic and non-linguistic tasks. Given the characteristics of functional communication, their experienced word-finding problems could be the result of slowed processing rather than lost function.
Underlying mechanism of linguistic processing speed
The significant correlation between performance of the TMT and the presence of reported word-finding difficulties could imply that there is a domain-general attentional basis for the experienced language difficulties. This is corroborated by the significant correlation between RTs on the language task (SJT) and performance on the non-verbal tasks (TMT-A and B), indicating that longer completion time on the TMT co-occurred with longer reaction times on the SJT. The cognitive abilities known to underlie performance speed on the TMT are visuoperceptual speed (TMT-A and -B) and concept shifting (TMT-B and -BA).
Remarkably, a significant correlation only existed in the patient group and was absent in the control group. This suggests that linguistic and non-linguistic functions are more heavily interconnected in glioma patients as compared to healthy participants. In addition, the contribution of domain-general abilities in performing language tasks could explain why the outcomes on the BNT and SJT were not influenced by hemispheric tumour localisation. If patients recruit domain-general cognitive abilities to perform language tasks, lesions in the left or right hemisphere may lead to impairments.
These results show that the receptive linguistic processing speed partially constitutes a more general cognitive speed. This is in accordance with the literature on persons with aphasia due to stroke. For example, Yoo et al. (2021) found that persons with aphasia show domain-general cognitive slowing, as indicated by slower processing speed on linguistic and non-linguistic tasks. However, our finding is in contrast with Ras et al.'s (2020) and Moritz-Gasser et al.'s (2012) results for patients with a glioma, who did not find a significant correlation between the RTs on a rapid naming test and overall processing speed measured with the TMT-A.
One potential explanation for this discrepancy lies in the difference between modalities of the used language tests. In the present study, we measured receptive reading abilities, whereas Ras et al. and Moritz-Gasser et al. administered a speeded naming test, assessing language production in a more isolated manner. As Sánchez-Cubillo et al. (2009) noted, the TMT-A mainly relies on visual search and perceptual speed. Therefore, a comparison between a reading task such as the SJT (both perceptual and visual) and the TMT-A may result in stronger relationships than with a naming task. Importantly, Moritz-Gasser et al. did find naming speed to be highly correlated with executive tasks that require lexical access (fluency and the Stroop test), and argue that the decreased naming speed, in absence of impaired naming accuracy, is due to the cognitive functions involved in language processing.
We found that linguistic processing speed was correlated with the ratio score of the TMT-BA, a measure of concept shifting. This could be because multiple linguistic levels are combined in the SJT. The participants assessed correct sentences and sentences that contain a semantic, syntactic, or phonological error. The correct and incorrect items are presented in a randomised order. It could thus be argued that there is constant task switching within the SJT, placing a higher demand on cognitive flexibility (Rubinstein et al., 2001) and explaining the significant correlation with the ratio score of the TMT-BA. Combining various tests and presenting them in a rapidly alternating way has previously been shown to be a good way to assess brain tumour patients (De Witte et al., 2015b). The SJT requires the participant to simultaneously integrate various processes, such as sentence processing, sentence evaluation, and task switching.
Limitations of the present study
A first limitation is that there was missing information on the language lateralisation via fMRI for the left-handed patients (N = 8). All left-handed patients had a glioma in the left hemisphere. Previous research has shown that while language lateralisation is more mixed, the majority of non-right-handed people nevertheless show typical language lateralisation in the left hemisphere (Szaflarski et al., 2002). Secondly, we could not perform analysis on the specific tumour location and its effects on linguistic and non-linguistic functions due to small group sizes. This is an important direction for future work. Thirdly, although the reported word-finding problems were coded twice at different timepoints, allowing for an intra-coder reliability analysis, having multiple independent coders assess the complaints would have further increased the reliability of the scoring. A fourth limitation is the task choice of the present study. Considering that data collection took place in a clinical context, we were bound by the tasks that are part of the standard clinical work-up. While the TMT and SJT are good measures of visuoperceptual processing speed and linguistic processing speed, respectively, and both tasks rely on attentional processes, the two tasks are not perfectly matched verbal and non-verbal variants. A final limitation of the study is that a pure reading task was not part of the test protocol. Consequently, we could not verify whether reading issues interfered with performance on the SJT. While this should be addressed in future studies, previous research has shown that reading performance is generally unaffected in glioma patients (Satoer et al., 2014(Satoer et al., , 2012.
Clinical implications and future directions
In clinical practice, demands for brevity generally compete with needs for sensitivity (e.g., Ek et al., 2010). Therefore, critical evaluation of the sensitivity of tests can guide the selection of materials for a patient group. The SJT is part of the DIMA (Satoer et al., 2021), which is designed to be both short and sensitive enough to detect mild language difficulties in patients with neurological diseases. The finding that deviant RTs in the SJT were most often observed in glioma patients with a lesion in the frontal lobes of the dominant hemisphere suggests that the task may be particularly suitable for this patient group. This is in accordance with De Witte et al. (2015b) who also suggest the administration of sentence judgment tests in patients with gliomas in the frontal and temporal (sub)cortical areas. Including measures of RTs, as was done for the SJT in the DIMA, could further increase the value of such judgment tests.
The finding that, despite a significant correlation between the TMT and the RTs on the SJT, not all patients with deviant scores on the SJT show impaired performance on the TMT (or vice versa) is an indication that both tests are necessary for a reliable interpretation of cognitive functioning. Additionally, considering that at the group level, patients do not show significantly lower processing speed than healthy control participants, demonstrates the need for elaborate anamnesis and assessment tailored to the individual patient.
Our results imply that administering the SJT could be beneficial for patients who report word-finding problems, but do not show deviant scores on Token tests or standard naming tests. Assessing linguistic processing speed provides a way to objectively assess these complaints. The finding that word-finding problems were significantly, but weakly correlated with the TMT-A and -B, shows that lexical retrieval has a general processing speed component but cannot be fully explained by this. This is an important observation that deserves attention in the clinical setting. Clinicians could try to gain additional information on the distinction between delayed and failed lexical access by administering a naming test under time pressure. The anamnesis is another valuable source of information; clinicians could ask patients more targeted questions about word retrieval. Patients differ in how they present their complaints during the anamnesis, which emphasises the importance of asking more thorough questions. Examples of such questions are whether difficult words surface eventually or not at all, or whether there are specific circumstances (noisy environments, time pressured conversations, etc.) under which word-finding problems are more prominent.
Finally, investigating the relationship between the performance of the SJT and nonlinguistic functions in populations with different neurological diseases, such as stroke or traumatic brain injury, is a potential direction for future work. The result that response speed of the SJT only correlates with visual search speed and concept shifting in the patient group, and not in healthy participants, suggests that patients may recruit a wider network to perform language tasks. It is interesting to see if similar relationships can be observed in patients with other neurological impairments. Moreover, this finding can serve as a starting point for therapy. Previous work on cognitive rehabilitation of glioma patients has found that in-person training (Locke et al., 2008), and telerehabilitation (Van der Linden et al., 2018) of cognitive functions is feasible and evaluated positively. Cognitive rehabilitation has short-term positive effects on subjective cognitive functioning and longer-term objective benefits for attention and verbal memory (Gehring et al., 2009). However, detailed individual assessment of the patient's impairments should guide the choice of therapy.
Conclusions
This research studied the linguistic processing speed in glioma patients and investigated whether these abilities could be a more sensitive measure to capture word-finding complaints. We found that patients' reported word-finding problems were not correlated with the BNT, a well-known test to assess lexical retrieval difficulties, nor with accuracy scores on the SJT. However, the word-finding problems were correlated with linguistic processing speed, operationalised as response speed in the SJT. At group-level, apart from patients with a glioma in the frontal lobe of the dominant hemisphere, response speed of the SJT was not deviant in glioma patients compared to the healthy control group. Furthermore, a relationship between linguistic processing speed and non-verbal functioning was found in the glioma patients but not in the healthy control group, suggesting that patients rely on more domain-general abilities to perform the task. These results indicate that the SJT, a time-constrained task assessing receptive language abilities, appears to be influenced by non-verbal processing speed, and that processing speed may contribute to subjectively experienced problems. This demonstrates the importance of administering tasks that assess language as well as nonverbal cognitive processing speed for the interpretation and dissociation of impairments.
Notes
1. Patients with a recurrent tumour are excluded because it is impossible to attribute their preoperative impairments to the presence of the tumour alone, as their impairments may also be the result of the previous surgery. 2. An analysis including both correct and incorrect target items showed that there was a significant main effect of correctness of the item on the reaction times across both groups (ß = 0.25, SE = 0.05, p < .001). Participants responded significantly faster to anomalous sentences than to correct target sentences.
Appendices
Outcomes of the statistical models.
Influence of participant characteristics
The demographic factors age and education level were included in the statistical models as covariates. These factors contributed significantly to the outcomes of the BNT, TMT, and the RT measures of the SJT. The effect of age and education level on these tests has been corroborated in earlier studies (Snitz et al., 2009;Tombaugh, 2004;De Witte et al., 2015b). In addition, significant interaction effects between age, education, and group on the SJT RTs, TMT-B, and TMT-BA, seem to suggest that older patients with lower education are more affected by their glioma than younger patients with a higher education when it comes to linguistic processing speed, visuoperceptual speed, and concept shifting. | 2021-10-19T16:24:26.727Z | 2021-09-21T00:00:00.000 | {
"year": 2022,
"sha1": "e9f80e9d987bb041f9b343a29bf81edab3784286",
"oa_license": "CCBY",
"oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/02687038.2021.1970099?needAccess=true",
"oa_status": "HYBRID",
"pdf_src": "TaylorAndFrancis",
"pdf_hash": "25616320c995c0394de52711dd0a6695eb38c979",
"s2fieldsofstudy": [
"Medicine",
"Psychology"
],
"extfieldsofstudy": [
"Psychology"
]
} |
16644650 | pes2o/s2orc | v3-fos-license | WHO/INRUD prescribing indicators and prescribing trends of antibiotics in the Accident and Emergency Department of Bahawal Victoria Hospital, Pakistan
A descriptive, retrospective and cross sectional study was conducted to assess the prescribing practices and antibiotic use patterns in the Accident and Emergency department of the Bahawal Victoria Hospital, Bahawalpur, Pakistan. A sample of 4320 prescriptions (systematic random sampling) was drawn out of a total of 1,080,000 prescriptions written during the period 1st January–31st December 2014. The standard World Health Organization/International Network for Rational Use of Drugs prescribing indicators were used to determine the prescribing practices of physicians. Published ideal standards for each of the indicators were used to identify irrational drug use. We also utilized an additional indicator to report the percentage share of antibiotics prescribed. The average number of drugs prescribed per encounter was 2.3 (SD = 1.3) (optimal value 1.6–1.8). Drugs prescribed by generic name occurred 83.1% of the time (optimal value 100%). Antibiotics and injections were prescribed 52.4% (optimal value 20.0–26.8%) and 98.0% (optimal value 13.4–24.1%) of the time respectively. Drugs prescribed from the Essential Drugs List equated to 81.5% (optimal value 100%). Out of 52.4% (n = 2262) prescriptions with antibiotics prescribed, 77.7% (n = 1758) had one antibiotic, 22.1% (n = 499) included two antibiotics, and 0.2% (n = 5) had three antibiotics. Cephalosporins were the most commonly prescribed class of antibiotics (81.5%) followed by penicillins (6.4%) and fluoroquinolones (6.2%). Among the individual antibiotics, ceftriaxone contributed the highest percentage share at 71.8% followed by cefotaxime (5.6%) and metronidazole (4.7%). The most frequently prescribed antibiotic combination was ciprofloxacin with metronidazole (52.1%). Irrational prescribing practices were common. Continuous education and training of physicians is required to ensure rational prescribing at Bahawal Victoria Hospital in the future.
Background
The importance of assessment and evaluation of quality in healthcare is gaining global recognition and medicines play a pivotal role in the healthcare delivery system. The appropriate use of medicines is a crucial element in the quality of health outcomes and in the appropriate medical care provided to patients (World Health Organization 1993). The most common causes of irrational medicine use are; self-medication, polypharmacy, inappropriate use of antibiotics, overuse of injectable medicines and the prescribing of medicines without following relevant clinical practice guidelines (World Health Organization 2002). Furthermore, there are numerous factors that influence irrational prescribing including; patients, healthcare professionals (HCPs), the working environment, the drug supply system (including industrial impacts), legal regulations, information and Atif et al. SpringerPlus (2016) 5:1928 misinformation about medicines, and profiteering intentions by selling more medicines (Geest et al. 1991;Spurling et al. 2010).
The fundamental step to limiting the irrational use of medicines is to quantify the extent to which this is occurring. It is particularly important with antibiotics as resistance continues to climb and the armamentarium of new antibiotics coming to market is not on the increase. In the 1990s, the World Health Organization (WHO) in collaboration with the International Network of Rational Use of Drugs (INRUD) developed a set of indicators to measure the performance of healthcare facilities related to the utilization of drugs (World Health Organization 1993). In developing countries, assessment of drug use patterns through the WHO/INRUD indicators is on the increase which is promising (Hogerzeil et al. 1993) with the indicators having been successfully implemented in more than 30 developing countries (Laing et al. 2001).
With regard to the rational use of antibiotics, the WHO has defined this as 'the cost-effective use of antibiotics which maximizes clinical therapeutic effect while minimizing both drug-related toxicity and the development of antimicrobial resistance (AMR)' (World Health Organization 2001a, b). The supervision of antimicrobial use has environmental, economic and clinical implications (World Health Organization 2012). Between 30 and 50% of all hospitalized patients are prescribed at least one antibiotic and this class constitutes more than 30% of the hospital budget (Vlahovic-Palcevski et al. 2000). According to another estimate, 20-50% of antimicrobial utilization is inappropriate (Čižman 2003) and this has a significant effect on the quality of health services provided (Shao-Kang et al. 1998), treatment expenses (Segade 2000) and frequency of adverse drug reactions (ADRs). It has also been estimated that around one quarter (25%) of total ADRs can be attributed to antimicrobial use (Beringer et al. 1998). Despite these concerns, the most alarming problem associated with irrational prescribing of antimicrobials is the development of resistance (Dellit et al. 2007). This problem is exacerbated by the limited number of new antimicrobials coming through the development pipeline of large pharmaceutical companies (O'Neill 2015).
In the United States (US), the economic burden of AMR amongst outpatients is estimated to be between $400 million and $18.6 billion. The costs associated with inpatients are thought to be much higher (Okeke et al. 2005). According to the WHO, AMR costs in Europe are estimated to be €9 billion per annum (World Health Organization 2011). Unfortunately there is a scarce literature on the fiscal burden of AMR in developing countries such as Pakistan. The expenses associated with AMR could inflict significant fiscal damage, not only for the governments but also for patients living in developing countries. Thus, monitoring and supervision of antimicrobial utilization has significant benefit to society through the saving of valuable resources (World Health Organization 2012).
The aim of this study was to assess the prescribing practices in the Accident and Emergency (A & E) department of the Bahawal Victoria Hospital (BVH), Bahawalpur, Pakistan. We also report the antibiotic use patterns in this same context. The results of this study could assist the hospital administrators to develop and implement appropriate interventions to improve rational prescribing of medicines in the A & E department of the BVH. The study findings may also be applicable to other hospitals that are suspected to have similar drug use practices.
Study setting
This study was carried out in the A & E department of the BVH, Bahawalpur, Pakistan. The BVH is a 1600 bed tertiary care hospital with established surgical and medical facilities. A total of 350 physicians, 10 pharmacists, 400 nurses and 3000 paramedical staff serve an average of 90,000 patients per month. The A & E department was established in 2005 with a capacity of 100 beds. The staff includes 25 doctors, one pharmacist and 54 nurses who provide care for between 2000 and 2500 patients per day.
Study design and outcome measures
A retrospective, cross-sectional study design was employed to evaluate the prescribing practices for all medicines, as well as antibiotic utilization patterns. The optimal values for the prescribing indicators were adopted from previous studies (Desalegn 2013;Atif et al. 2016b). The prescribing indicators include; the average number of drugs prescribed per encounter (optimal value 1.6-1.8), the percentage of drugs prescribed by generic name (optimal value 100%), the percentage of encounters where an antibiotic was prescribed (optimal value 20.0-26.8%), the percentage of encounters where an injection was the route of administration (optimal value 13.4-24.1%), and the percentage of drugs prescribed from the Essential Drugs List (EDL) or some other recognized formulary (for which the optimal value is 100%). Another indicator was used to determine antibiotic prescribing patterns at the A & E department of the hospital.
Inclusion All prescriptions written for the patients attending the A & E department of the BVH from 1st January 2014-31st December 2014 irrespective of age, gender and diagnosis were included in the study.
Sample size and data collection method
The sample size for this study was 4320 prescriptions collected retrospectively from 1,080,000 written during the 1-year period of study. The prescriptions written each month were separated and then 360 prescriptions from each month were selected using a systematic random sampling technique.
The data relating to the prescribing indicators was noted for each prescription and then recorded into a standard WHO prescribing indicator form (World Health Organization 1993). A data collection form was developed to capture antibiotic utilization patterns and data was recorded into it. The Anatomical Therapeutic Chemical (ATC) classification system was used for the classification of antibiotic (WHO Collaborating Centre for Drug Statistics Methodology 2014). The data was collected by four trained research associates during the months of January-March 2015.
Statistical analysis
Statistical Package for Social Sciences (IBM SPSS Statistics V21.0) was used for the analysis of data. Descriptive statistics such as frequencies, percentages, averages and standard deviations (SD) were used to present data.
Prescribing indicators
The average number of drugs per encounter was 2.3 (SD = 1.3). The percentage of drugs prescribed by generic name was 83.1% (SD = 1.1). The percentage of encounters with an antibiotic prescribed was 52.4% (SD = 0.5) and 98% (SD = 0.3) of drugs were given by the injectable route. Over three quarters (81.5%) of drugs were prescribed from the EDL or some other formulary (SD = 1.1) ( Table 1).
Discussion
Irrational prescribing practices exist all over the world and eventually they lead to unwanted effects in patients (Akl et al. 2014). In this study, WHO/INRUD prescribing indicators were used to determine current prescribing practices and antibiotic use patterns in a tertiary care hospital in Pakistan. The findings from this study are important to the health system of Pakistan because they help to assess whether the BVH is following a set norm of practices to ensure optimal medication use. Only a limited number of studies are available from Pakistan on this topic and therefore our findings provide a source of baseline information for continuous monitoring of drug therapy and process improvement at the institutional level.
A & E departments are often the first point of contact with the healthcare system and in Pakistan there is a considerable throughput of patients in these departments. Turnover of patients in this setting is higher than general wards and one should not underestimate the importance of the appropriate prescribing of antibiotics to ensure current levels of resistance do not continue to climb. In addition to the contribution made in the Pakistani context, our findings may also be relevant to other countries with similar drug use practices or with similar health care systems. This study may provide the impetus for academics, clinicians and hospital administrators in those countries to begin to assess the status of their own nations prescribing within A & E departments; especially as it relates to antibiotics.
Prescribing indicators
The contents of a prescription are influenced by a prescribers' training, their attitude towards the disease being treated and the type of healthcare system within which they work. The results of the current study revealed that the average number of drugs per prescription were 2.3 (SD = 1.3) (Table 1). This value is higher than the admissible range of 1. Our results revealed that antibiotics were prescribed on over half the prescriptions (52.2%) (optimal value 20.0-26.8%) ( Table 1). This would suggest that either every second person who presents at the A & E department has an infection related issue, or that there is excessive and inappropriate prescribing of antibiotics occurring in this hospital department. To compare internationally, this value was relatively lower in other developing countries such as Bangladesh (25%) (Guyon et al. 1994) and Brazil (28.8%) (Holloway and Henry 2014). In a few countries, antibiotic prescribing was higher such as in Kenya (73.4%) (Holloway and Henry 2014), Timor-Leste (70%) (Stanley Chindove and Martins 2012), and Sudan (70.4%) (Holloway and Henry 2014). Unnecessary prescribing of antibiotics is a worldwide problem that eventually leads to ADRs and frequent hospital admissions (Beringer et al. 1998).
In our study, 98% of prescriptions included at least one injectable product (optimal value 13.4-24.1%) ( Table 1). This value was much higher than the studies conducted in Afghanistan (17%) (Ahmad et al. 1995) and Kuwait (9.1%) (Awad and Al-Saffar 2010). We conducted this study in the A & E department where the excessive use of injectables may be attributed to the patients' condition such as emergency and unconscious cases where the oral route for drug administration is often not possible. Nevertheless, excessive use of injections may lead to a higher probability of blood borne diseases (World Health Organization 2002) and injections are always more expensive than the equivalent oral formulation (Akl et al. 2014). With regard to drugs prescribed from the EDL, our findings were comparable to other studies conducted in the Lao People's Republic (86.2%) (Holloway and Henry 2014) and Bangladesh (85%) (Guyon et al. 1994). Rational prescribing includes the optimal use of drugs selected from the EDL which are issued by the WHO. These agents are older, time tested and available at lower cost than the originator branded drugs (Akl et al. 2014).
Antibiotic usage patterns
There is some evidence to support the notion that antibiotic consumption is much higher in developing countries as compared to developed countries (Knobler et al. 2003;Center for Disease Dynamics Economics & Policy 2015). According to one study, 35-60% of the patients were prescribed antibiotics and less than 20% of antibiotics were prescribed appropriately (World Health Organization 2001a, b).
In this study, out of the 52.4% (n = 2262) prescriptions containing antibiotics, 77.7% (n = 1758) contained one antibiotic, 22.1% (n = 499) included two antibiotics and 0.2% (n = 5) had three antibiotics. These findings can be compared with a study conducted in Jordan (Al-Niemat et al. 2014), which showed that out of 85% prescriptions with antibiotics, 88% prescriptions had one antibiotic, 11% had two and 1% had three antibiotics. Likewise in a Turkish study involving 39.4% prescriptions with antibiotics, 73.6% prescriptions had one antibiotic, 19.6% had two, 5.7% had three and 1.1% had four antibiotics (Erbay et al. 2003). Similarly, results of a study conducted in Nepal revealed that 21% prescriptions included one antibiotic, 37% prescriptions included two, 28% prescriptions included three, 10% included four and 4% of the prescriptions included five and above antibiotics (Palikhe 2008).
Our study demonstrated that the most frequently prescribed classes of antibiotics were cephalosporins (81.5%), penicillins (6.4%) and fluoroquinolones (6.2%) ( Table 2). A study performed in Saudi Arabia revealed that cephalosporins (31.9%), penicillins (24.9%) and macrolides (9.7%) were the most frequently prescribed classes of antibiotics (Mohajer et al. 2011). In a similar manner, a study from Turkey reported that cephalosporins (19.9%) were the most commonly prescribed class followed by penicillins (19.1%), aminoglycosides (11.7%) and quinolones (11.1%) (Erbay et al. 2003). Another study showed that cephalosporins were the most frequently prescribed antibiotics (34%) followed by penicillins (33%), aminoglycosides (16%) and fluoroquinolones (6%) (Palikhe 2008). A study from Jordan revealed that penicillins (46%) and macrolides (39%) were the most frequently prescribed antibiotic classes (Al-Niemat et al. 2014). With regard to individual antibiotics, the findings of our study revealed that ceftriaxone (71.8%) contributed the highest percentage share amongst all the antibiotics followed by cefotaxime (5.6%), metronidazole (4.7%), amoxicillin (4.7%), ciprofloxacin (4.2%) and moxifloxacin (1.9%). An Indian study showed that the highest prescribed antibiotics were cefixime (37.98%), ceftriaxone (7.97%), azithromycin (6.33%) and gentamicin (6.25%) (Khan et al. 2011). In another study, azithromycin contributed the highest percentage share (97%) of the total antibiotics (Al-Niemat et al. 2014). It is clear from this body of literature that the extent of antibiotic prescription is high but that there is considerable variation in the number and types of antibiotics selected to prescribe. What is concerning from our study is the selection of very high powered cephalosporins first line. This study provides the platform to implement policy that should result in a change in this prescribing behavior. In this regard the study is very relevant and has implications for policy and practice that can make a difference in Pakistan.
The increasingly growing threat of AMR and the lack of a significant pipeline of new antibiotics in development has drawn attention toward multi-drug therapies (Golan et al. 2011). In the majority of cases infections are usually cleared within a few days of antibiotic treatment with a single agent, but severe and complicated infection may require longer treatment with a combination of multiple antibiotics (Nicolle and Committee 2005). We found that the most commonly prescribed antibiotic combinations were ciprofloxacin with metronidazole (52.1%) and ceftriaxone with metronidazole (38.8%). In the field of medicine, the multi-drug antibiotic therapies are usually sought to achieve broader antibacterial spectrum (Ejim et al. 2011). Different occasions where these combination therapies have been shown to be more effective include; synergism; prevention of AMR; and as empirical therapy for poly-microbial infections. The most commonly anticipated drug-drug interactions with antibiotic combinations are antagonism, addition and synergism (Rybak and McGrath 1996). In most cases, synergistic effects are considered for treatment failure cases or when the incidence of AMR development is most likely (Cremieux and Carbon 1992). But some recent studies have reported that antimicrobial synergistic combinations may speed up the process of AMR development (Pena-Miller et al. 2013).
There are certain other risks and adverse effects associated with the use of antibiotics and antimicrobial combinations. The major associated risks include development of super-infections, augmented toxicity and greater cost. The well described adverse effects include hypersensitivity reactions, diarrhea, nephrotoxicity and coagulopathy (Rybak and McGrath 1996). Published studies have emphasized the rational use of antibiotics (alone or in combination) to prevent the AMR development (McGowan 1983), improve quality of patient care (Shao-Kang et al. 1998) and to minimize the cost of therapy (Segade 2000).
Conclusion and recommendations
The current study demonstrates irrational prescribing practices in the A & E department of a tertiary level hospital in Pakistan. The findings in terms of prescribing indicators are divergent with the expected norms. Cephalosporins contributed the highest percentage share amongst all the antibiotic classes, while ceftriaxone was the most commonly prescribed antibiotic. The extent of antimicrobial prescribing in this setting raises concerns of AMR and highlights an ignorance of doctors towards their prescribing behavior.
This study contributes to the international literature by addressing medicines use in the context of the A & E department; a point of first contact with the health system in many developing countries. Further, this study adds to the body of knowledge surrounding antibiotic use in this A & E setting and compares use in Pakistan with other developing countries. The study adds further evidence that policy-makers, hospital administrators, doctors, nurses and pharmacists should all be wholly concerned about the developing world's ongoing contribution to AMR. There are few antibiotic medicines in the drug development pipeline and therefore few therapeutic solutions when current agents stop being effective. The strategy must immediately be focused on curbing the current rise in AMR.
The study findings support a recommendation that continuous training and educational programs for medical staff concerning rational use of injections and antibiotics should be implemented and monitored so that the required changes in prescribing become sustainable. A feedback monitoring system on doctors' antibiotic prescriptions would be expected to significantly improve their prescribing behavior. Knowledge and compliance with updated clinical guidelines is also recommended to enhance the degree to which generic prescribing occurs.
Further studies are encouraged to find the reasons for irrational use of medicines and to demonstrate the factors driving the irrational prescribing of antimicrobials by the physicians at BHV. Appropriate interventions regarding rational prescribing of antimicrobials should be designed and implemented in the hospital and an evaluation is required to determine whether there has been any improvement in the prescribing practices of physicians.
Limitations of the study
There are limitations with pharmaco-epidemiological approaches that compare studies across countries where the hospital context might be different. However, the authors are left with few options when the literature is scarce and limited within the context of A and E antibiotic prescribing in developing countries. In addition to international comparisons the findings from this study should not be generalized to the whole of Pakistan; but they do give a robust indication of what is happening in one hospital A and E department and may precipitate other hospitals in Pakistan to begin an audit which will give a sense of whether the high use of IV ceftriaxone is a national phenomenon. Based on the fact that a uniform healthcare policy is implemented throughout the country, and medical graduates from various institutions are working in Bahawalpur city the practices may be similar to A & E departments of other tertiary care hospitals in Pakistan. This needs to be further instigated and if it is the case then a national policy will need to be developed to address this issue. In this study, we did not explore the reasons for irrational drug use, which could be considered in future studies. | 2018-04-03T02:16:04.379Z | 2016-11-08T00:00:00.000 | {
"year": 2016,
"sha1": "cac419eeef0774326bb136b9d1f406d7bccaf3df",
"oa_license": "CCBY",
"oa_url": "https://springerplus.springeropen.com/track/pdf/10.1186/s40064-016-3615-1",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "cac419eeef0774326bb136b9d1f406d7bccaf3df",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
44074531 | pes2o/s2orc | v3-fos-license | Crystal structure of a palladium(II) complex containing the wide bite-angle bis(selenium) ligand, cis-[(tBuNH)(Se)P(μ-NtBu)2P(Se)(NHtBu)]
A palladium(II) complex containing a bis(selenium) ligand based on cyclodiphosph(V)azane, cis-[(tBuNH)(Se)P(μ-NtBu)2P(Se)(NHtBu)] has been synthesized and structurally characterized. The crystal structure revels chelation of ligand through selenium donors with a natural bite-angle of 110.54 (1)°
Chemical context
Cyclodiphosph(III)azanes are four-membered P III -N ring systems with general formula, cis-[RP(-N t Bu) 2 PR]. The planar nature of the four-membered ring favors a bridging bidentate coordination mode through phosphorus donors rather than chelation, to afford structurally interesting macrocyclic and polymeric complexes (Balakrishna, 2016). The main-group chemistry of the corresponding P V analogue cyclodiphosph(V)azanes, cis-[R(E)P(-N t Bu) 2 P(E)R] (E = O, S, Se, and Te; R = NH t Bu) and its amide derivatives has been studied extensively by Stahl (2000) and Briand and co-workers (Briand et al., 2002). While examples of coordination of cyclodiphosph(V)azanes with transition metal ions are scarce, the sulfur and selenium derivatives are especially interesting as they have a special affinity for soft metals and have the potential to form complexes with wide natural bite-angles through chelation (Chivers et al., 2001). Several late transitionmetal complexes containing wide natural bite-angle chelating ligands (L-M-L = 100-134 ) have been developed over the years and have shown promising catalytic activity for several reactions (Kamer et al., 2001). The majority of these wide biteangle ligands are phosphorus and/or nitrogen donor ligands (Motolko et al., 2017;Czauderna et al., 2015). Herein we report the synthesis and crystal structure of the palladium(II) complex (II) with a wide bite-angle selenium ligand based on cyclodiphosph(V)azane cis-[( t BuNH)(Se)P(-N t Bu) 2 P(Se)-(NH t Bu)], (I).
Supramolecular features
In the crystal, the molecules are connected through weak N-HÁ Á ÁCl and C-HÁ Á ÁCl hydrogen-bonding interactions (Fig. 2, Table 1). Interestingly, in the solid-state structure II, the exocyclic nitrogen substitutents are arranged in an endo, endo fashion, whereas in ligand I they are arranged in exo, endo orientations . An overlay plot of the free ligand molecule I with the ligand fragment of II is shown in Fig. 3. This conformational change upon coordination is possibly caused by the formation of intermolecular hydrogenbonding interactions. A similar conformational change influenced by hydrogen-bonding interactions has previously been noted (Chandrasekaran et al., 2011).
A dichloromethane solution (10 mL) of [Pd(COD)Cl 2 ] (100 mg, 0.35 mmol) was added dropwise to a solution of cis- Perspective view of palladium complex II with displacement ellipsoids drawn at the 50% probability level. All H atoms have been omitted for clarity except at N3 and N4. Only the major occupancy component of the disordered t-butyl group is shown. Table 1 Hydrogen-bond geometry (Å , ). (7) for 6 h. The solution was then concentrated to 10 mL, diluted with 10 mL of pentane, and stored at 248 K for a day to afford the analytically pure orange crystalline product. X-ray quality crystals were obtained by slow evaporation from a DMF solution at room temperature. Yield: 76% (206 mg, 0.067 mmol), m.p. 455-457 K.
Refinement
Crystal data, data collection and structure refinement details are summarized in Table 2. All H atoms attached to carbon were placed in calculated positions (C-H = 0.98 Å ), while those attached to nitrogen were placed in locations derived from a difference-Fourier map and their coordinates adjusted to give N-H = 0.91 Å . All were included as riding contributions with U iso (H) = 1.2-1.5 times those of the parent atoms. The t-butyl group attached to N4 was modeled as rotationally disordered over two sites of approximately equal population. These were refined with restraints so that the geometries of the two components of the disorder are comparable. Overlay of the uncoordinated ligand I (gray) with the coordinated ligand fragment in complex II (purple). Computer programs: APEX2 and SAINT (Bruker, 2013), SHELXT (Sheldrick, 2015a), SHELXL2014 (Sheldrick, 2015b), Mercury (Macrae et al., 2006) and SHELXTL (Sheldrick, 2008).
Special details
Experimental. The diffraction data were obtained from 3 sets of 400 frames, each of width 0.5° in ω, collected at φ = 0.00, 90.00 and 180.00° and 2 sets of 800 frames, each of width 0.45° in φ, collected at ω = -30.00 and 210.00°. The scan time was 10 sec/frame. Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2sigma(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. Hatoms attached to carbon were placed in calculated positions (C-H = 0.98 Å) while those attached to nitrogen were placed in locations derived from a difference map and their coordinates adjusted to give N-H = 0.91 Å. All were included as riding contributions with isotropic displacement parameters 1.2 -1.5 times those of the attached atoms. The tbutyl group attached to N4 is rotationally disordered over two sites of approximately equal population. These were refined with restraints that the geometries of the two components of the disorder be comparable. | 2018-06-05T06:45:07.778Z | 2018-01-19T00:00:00.000 | {
"year": 2018,
"sha1": "6598b07f01320780850471158d4ebf3890e759c8",
"oa_license": "CCBY",
"oa_url": "https://journals.iucr.org/e/issues/2018/02/00/nk2239/nk2239.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "6598b07f01320780850471158d4ebf3890e759c8",
"s2fieldsofstudy": [
"Chemistry"
],
"extfieldsofstudy": [
"Chemistry",
"Medicine"
]
} |
254406138 | pes2o/s2orc | v3-fos-license | Bile acids as inflammatory mediators and modulators of intestinal permeability
Bile acids are critical for the digestion and absorption of lipids and fat-soluble vitamins; however, evidence continues to emerge supporting additional roles for bile acids as signaling molecules. After they are synthesized from cholesterol in the liver, primary bile acids are modified into secondary bile acids by gut flora contributing to a diverse pool and making the composition of bile acids highly sensitive to alterations in gut microbiota. Disturbances in bile acid homeostasis have been observed in patients with Inflammatory Bowel Diseases (IBD). In fact, a decrease in secondary bile acids was shown to occur because of IBD-associated dysbiosis. Further, the increase in luminal bile acids due to malabsorption in Crohn’s ileitis and ileal resection has been implicated in the induction of diarrhea and the exacerbation of inflammation. A causal link between bile acid signaling and intestinal inflammation has been recently suggested. With respect to potential mechanisms related to bile acids and IBD, several studies have provided strong evidence for direct effects of bile acids on intestinal permeability in porcine and rodent models as well as in humans. Interestingly, different bile acids were shown to exert distinct effects on the inflammatory response and intestinal permeability that require careful consideration. Such findings revealed a potential effect for changes in the relative abundance of different bile acids on the induction of inflammation by bile acids and the development of IBD. This review summarizes current knowledge about the roles for bile acids as inflammatory mediators and modulators of intestinal permeability mainly in the context of inflammatory bowel diseases.
Introduction
Bile acids are synthesized in the liver from cholesterol and secreted into the intestine where they serve to emulsify and facilitate the absorption of dietary lipids. Approximately 5% of the bile acid pool is eliminated in the stool and serves as a major pathway for eliminating cholesterol from the body (1). Most bile acids are reabsorbed in the distal ileum and are transported back to the liver for re-secretion, establishing their enterohepatic circulation. In addition to being lipid emulsifiers in the intestinal lumen, bile acids also activate specific receptors with varying physiological effects in several organ systems throughout the body (2). Different receptors for bile acids have been identified in various immune cells throughout the gastrointestinal tract. Strong evidence suggests that the activation of bile acid receptors in immune cells modulates the immune responses and alters inflammatory reactions (3). Also, bile acids have been shown to trigger specific signaling pathways that disrupt barrier function and increase intestinal epithelial permeability. The leaky gut condition induced by bile acids could be attributed to the pathophysiology of inflammation in inflammatory bowel diseases (IBD) (4).
The bile acid pool is highly conserved and is composed of different types of bile acids, each having a specific affinity to certain bile acid receptors. Interestingly, secondary bile acids are generated in the intestine and therefore the gut microbiota is crucial for expanding the metabolic profile of the bile acid pool (5). The abundance of certain bile acids and the overall composition of the bile acid pool ultimately determines its functional capacity. Indeed, there is strong evidence to suggest that gut dysbiosis in inflammatory bowel disease (IBD) leads to alterations in the bile acid pool that may affect the severity of the disease and the development of associated pathologies (6). Over the years, this topic has generated extensive interest leading to many seminal studies investigating the roles of bile acid homeostasis and bile acids in the pathophysiology of IBD. In this review, we will highlight recent advances that have enhanced our understanding about the physiological roles of bile acids and how they contribute to IBD by altering the immune responses and epithelial barrier function in the intestine.
Bile acid homeostasis 2.1 Bile acid synthesis
The liver is the primary site for bile acid synthesis in most vertebrates. There are exceptions, however, such as basal jawless vertebrate sea lampreys (Petromyzon marrinus) where bile acid synthesis occurs in the liver and intestine (7). The two primary bile acids synthesized in the liver are cholic acid (CA) and chenodeoxycholic acid (CDCA). The synthesis of CA and CDCA involves several cytochrome P450 (CYP) enzymes, with CYP7A1 acting as the rate-limiting enzyme of the process (8). Following their synthesis, bile acids are conjugated to taurine or glycine, a process mediated by the bile acid-coenzyme A ligase (BAL) and the bile acid-coenzyme A:amino acid N-acyltransferase (BAT) (9). Bile acids undergo several transformations by bacteria in the colon, including deconjugation and the formation of secondary bile acids. The removal of amino acids is controlled by a family of bile salt hydrolases (BSHs), while the generation of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is mediated by 7a-dehydroxylation enzymes (10).
The average bile acid pool in healthy humans is 3-5 grams (11). Approximately 95% of the bile acid pool is actively reabsorbed in the distal ileum with approximately 5% of the pool (~0.5 g) being excreted into the feces.
The hormone-like protein Fibroblast growth factor 19 (FGF19 in humans, FGF15 in rodents) plays a key role in bile acid homeostasis by serving as a negative regulator of bile acid synthesis. Following active absorption in the ileum, bile acids activate the intestinal farnesoid X receptor (FXR), leading to the induction of FGF19 expression. FGF19 is then secreted into the portal blood stream and eventually suppresses CYP7A1 and bile acid synthesis in the liver (12). Interestingly, studies in primary human hepatocytes have shown that treatment with CDCA following knockdown of FGF19 is sufficient to downregulate CYP7A1 expression, suggesting that bile acids also can regulate their own synthesis without the negative feedback of FGF19 (13).
While bile acids are preserved in the enterohepatic circulation, a small percentage enter the systemic circulation where they modulate the function of different organ systems, including the central nervous system (CNS) (14). Indeed, bile acids can cross the blood-brain barrier (BBB) and are found in both mouse and human brains. In this regard, bile acids have been recently characterized as having both neuroprotective and cytotoxic effects in the brain, with the 12a-hydroxylated bile acids cholic acid (CA) and deoxycholic acid (DCA) shown to be cytotoxic (15). Furthermore, recent studies have implicated bile acids in the pathophysiology of several neurological diseases, including Alzheimer's, Parkinson's, and Multiple Sclerosis (MS) (16).
Bile acid structure
It is important to note the differences between the human and mouse bile acid pool. While most of the human intestinal bile acid pool is composed of CA, CDCA, DCA and LCA along with their conjugated forms (17), the most highly abundant bile acids in the mouse pool are the primary bile acids a-muricholic acid (MCA) and b-MCA, which are generated from CDCA via CYP2c70 (18). These differences in the bile acid pool should be considered when studying changes in bile acid synthesis and homeostasis. For example, the mouse pool of bile acids is more hydrophilic and less potent as detergents compared to the human pool, and depletion or expansion of certain bile acids may have differential effects (19). Steps have been taken to account for this challenge of making the mouse bile acid pool more like the human pool. For example, de Boer et al. generated a liver-specific CYP2c70 knockout mouse model and found these mice to have a more hydrophobic bile acid pool similar to the human pool (20).
Bile acid transport
Both primary and secondary bile acids are actively reabsorbed in the distal ileum via the Apical Sodium-Dependent Bile Acid Transporter (ASBT) before circulating back to the liver (2). ASBT function is critical for maintaining the enterohepatic circulation of bile acids. In addition to ASBT, there are several other proteins in the intestine and liver that are crucial for proper bile acid transport. For example, following active transport via ASBT, bile acids bind to the intracellular bile acid binding protein (IBABP) and then exit the enterocytes by the organic solute transporters OSTa and OSTb.
The hepatic secretion of bile acids is controlled by the ATPbinding cassette transporters bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP2). While humans mainly use BSEP to efflux bile acids into the bile, mice use both BSEP and MRP2 (21). Bile acids are transported into hepatocytes mainly by the hepatic Sodium-taurocholate cotransporting polypeptide (NTCP). Additionally, the human organic aniontransporting polypeptides (OATP) OATP1B1 and OATP1B3 transport specific types of bile acids in the liver while excluding others. Specifically, CA, CDCA, and DCA are transported by these polypeptides, while the secondary bile acids LCA and UDCA are not (22). Furthermore, kinetic analyses revealed that conjugated bile acids are the preferred substrates as compared to unconjugated bile acids (23).
Bile acids and the gut microbiome
There are many dynamic interactions between gut bacteria and bile acids that depend largely on the expression of functional bacterial proteins, including bile acid transporters and modifying enzymes. For example, several proteins encoded by the bile acid inducible (bai) operon are involved in 7a -dehydroxylation of bile acids (24). Furthermore, the protondependent bile acid transporter (encoded by baiG gene) is responsible for the entry of primary bile acids into the bacterium, and the rate limiting step of 7a-dehydroxylation (25) is mediated by the bile acid 7a-dehydratase (baiE). Gut bacteria possess several other enzymes, such as the bacterial hydroxysteroid dehydrogenases (HSDs): a-HSD and b-HSD (26), which are responsible for oxidation and epimerization of bile acids at 3-, 7-and 12-positions in the steroid ring. The bacterial proteins known to interact with bile acids are widely distributed among different bacterial communities (27). Bile salt hydrolases, as well as oxidation and epimerization processes, have been identified in four different genera, including Bacteroides, Clostridium, Bifidobacterium, and Listeria (28).
There is an intricate and dynamic relationship between bile acid homeostasis and the gut microbiome that, when disrupted, can lead to several physiological complications. For example, the dietary supplementation of DCA in mice was shown to reduce the abundance of BSH-producing bacteria and increase the abundance of bacteria in the Parabacteroides and Bacteroides genera (29).
Recently, a novel class of human bile acids known as microbially conjugated bile acids (MCBAs) has been discovered (30). Unlike traditional conjugated bile acids, MCBAs are conjugated to the amino acids phenylalanine, leucine, and tyrosine (30). While the main species thought to produce these bile acids are Enterocloster bolteae and Clostridium bolteae, the underlying mechanisms behind the production of MCBAs have yet to be fully elucidated. The identification of these different bile acid species suggests that the gut microbiota plays a critical role in bile acid homeostasis and that alterations in bacterial composition significantly alter what species are present in the bile acid pool. Interestingly, the levels of MCBAs were shown to be elevated in patients with IBD and cystic fibrosis (30). However, the exact physiological roles of these newly discovered conjugated bile acids and how they contribute to diseases remain largely unknown.
Bile acids in IBD
Inflammatory bowel diseases (IBD) are multifactorial disorders of chronic intestinal inflammation consisting of two subtypes, Ulcerative Colitis (UC) and Crohn's Disease (CD) (31). Ulcerative colitis is characterized by continuous lesions in the rectum and colon with inflammation localized to the mucosa and submucosa (32,33). Crohn's Disease, is characterized by skip lesions anywhere across the intestinal tract, most commonly affecting the terminal ileum, with full thickness inflammation (34,35). Bile acids have been found to be altered in patients with IBD because of gut microbial dysbiosis (6). Generally, the observed trend is an increase in host derived bile acids, with a decrease in microbial derived secondary bile acids (6). Also, several lines of evidence support the notion that bile acid receptors modulate the immune response in IBD. The following sections review existing literature on the alterations in bile acids due to IBD as well as the directs effects of bile acids on IBD.
Bile acid receptors and immune responses
Bile acids are known to activate different receptors that were recently shown to affect immune responses as they relate to IBD (Table 1).
FXR
FXR is expressed in cells of myeloid lineage including monocytes, macrophages, dendritic cells (DCs), and natural killer T-cells (NKTs) with different effects upon activation (52). FXR signaling in dendritic cells results in antiinflammatory effects through decreases in IL-6, IL-1b, and TNF-a (36,37). NKT cell-mediated activation of FXR also exhibits anti-inflammatory effects with decreases in interferongamma (IFN-g) and TNF-a, and increases in IL-10 (36,37). This signaling also shifts NKT cells towards an invariant NKT cell known as NKT10 cells, which produce IL-10 (39). These cells have been shown to decrease anti-tumor immune activity while also preventing autoimmunity in a mouse model of experimental autoimmune encephalomyelitis (EAE) (39). Furthermore, FXR activation in NKT cells has been shown to inhibit the production of osteopontin, a pro-inflammatory cytokine, through SHP (40). Generally, FXR utilizes small heterodimer partner (SHP) to bind to and sequester pro-inflammatory proteins (53). More specifically, SHP has been shown to inhibit the production of pro-inflammatory cytokines by decreasing NF-kB activation and mitogen activated protein kinase (MAPK) (54). FXR signaling in hepatic and intestinal macrophages exerts anti-inflammatory effects with decreases in the activation of IL-1b, TNF-a, NLPR3 inflammasomes, and caspase 1 (36,37). One study utilized peripheral blood mononuclear cells (PBMCs) from healthy subjects, separated them into monocytes and dendritic cells, stimulated them with LPS, and treated them with INT-747, an FXR agonist, and found decreased TNF-a production (55). Overall, there is strong evidence that supports antiinflammatory effects of FXR signaling.
These findings have been corroborated in various models of intestinal inflammation. A previous study utilized DSS and TNBS induced colitis in wild type and mice lacking FXR and showed that mice were treated with FXR agonist INT-747 exhibited decreased severity of colitis (55). This study also utilized lamina propria leukocytes from subjects with both ulcerative colitis and Crohn's disease and found decreased secretion of IFN-g, and TNF-a after treatment with INT-747. Follow up studies showed that obeticholic acid (OCA), a semisynthetic bile acid used as medication and potent FXR agonist, prevented DC migration from the spleen to the colon in
Innate lymphoid cells (ILCs) RORgt
• ↑ differentiation and function of ILC3 subtypes (3,50) response to DSS-colitis with an increase in plasma IL-10 levels and IL-6 levels (38). It is interesting to note that IL-6 was also increased and that while less DC migration and increased IL-10 levels correspond with decreased inflammation, the increases in IL-6 demonstrate complex inflammatory responses resulting from FXR activation rather than binary pro-or antiinflammatory effects. A retrospective study performed in a cohort of CD patients found that females carrying an FXR-1GT genotype had three times less FGF19 serum levels and had a higher risk of needing surgery, showing that impaired FXR signaling can be detrimental in IBD (56).
GPBAR1/TGR5
G-protein bile acid receptor 1 (GPBAR1), also known as transmembrane G-protein-coupled receptor 5 (TGR5) is broadly expressed in various tissues and is also expressed in monocytes, macrophages, dendritic cells (DCs), and natural killer T-cells (NKTs) (57). TGR5 activation leads to decreases in pro-inflammatory IL-6, IFN-g, and TNF-a, and increases in IL-10 (36,37). TGR5 activation also promotes differentiation of macrophages from classically activated pro-inflammatory phenotypes to alternatively activated anti-inflammatory phenotypes (36,37). In dendritic cells, TGR5 activation leads to decreased TNF-a and IL-12 production (36,37). As with FXR, when TGR5 is activated, it promotes the polarization of NKT cells towards NKT10 cells, which produce the anti-inflammatory cytokine IL-10 (39). Additionally, activation of TGR5 in NKT cells also leads to decreases in IFN-g, and TNF-a (36,37). This body of evidence demonstrates important anti-inflammatory effects of TGR5 signaling in immune cells.
One recent study has shown that administration of TGR5 agonists during inflammatory stress protected against inflammasome activation. Treatment with Genistein, a compound known to protect against DSS colitis, increased intracellular cAMP levels in LPS treated macrophage cell lines (THP and U937 cells) (41). Furthermore, coadministration of the TGR5 agonist INT-777 with an NLRP3 inflammasome activator (Nigericin) increased the levels of intracellular cAMP and decreased secretion of caspase-1 and IL-1b, providing evidence for beneficial anti-inflammatory TGR5 signaling in macrophages (41). Another study showed that TGR5 activation by BAR501 ameliorated TNBS and oxazolone colitis by polarizing macrophages towards the less inflammatory alternative phenotype (58). The beneficial effect from BAR501 was not observed in TGR5 null mice (58). Further study using raw 264.7 cells showed that IL-10 activity was dependent on TGR5 (58). Another previous study showed that macrophage colony-stimulating factor (MCSF) and interferon-gamma differentiated macrophages challenged by enterococcus faecalis or LPS produced less TNF-a when treated with a TGR5 agonist (59). Furthermore, this study showed lamina propria macrophages from subjects with active Crohn's disease had higher levels of TGR5 mRNA than those from healthy controls. The TGR5 agonist suppressed Enterococcus faecalis induced production of TNF a (59). Additional studies support a beneficial role for TGR5 signaling in the context of intestinal inflammation. TGR5 null mice showed increased susceptibility to severe colitis, and TNBS colitis mice treated with oleanolic acid, an TGR5 agonist, showed improvement (60). This evidence supports important roles for TGR5 signaling in inflammatory responses that should be further explored in additional clinically relevant models of inflammation. Collectively, it is interesting to note that activation of these two major bile acid receptors, FXR and TGR5, exerts anti-inflammatory response that could be exploited for therapeutic interventions for IBD.
S1PR2
Bile acids are known to activate the sphingosine 1 phosphate receptor 2 (S1PR2). Many studies indeed, have shown the roles of S1PR2 in the modulation of immune responses. In this regard, Wang X et al. examined the effects of S1PR2 activation through LPS in primary murine macrophages (42). S1PR2 was found to be responsible for the increased protein expression of downstream targets RhoA and ROCK1. Administration of S1PR2 and ROCK1 antagonists was found to reduce the expression of CD86 and MCP-1 in an acute DSS model of colitis, suggesting that S1PR2 promotes polarization of macrophages to an M1/pro-inflammatory phenotype. This was followed up with in vitro studies where they used these antagonists in the presence of LPS and found that blocking S1PR2 and ROCK1 prevented increases in CD86, MCP-1, TNFa, and iNOS mRNA expression in primary mouse macrophages providing further evidence that S1PR2 signaling in macrophages leads to a pro-inflammatory phenotype (42).
Additionally studies have shown that activation of S1PR2 results in a pro-inflammatory M1-like phenotype in human macrophages (61). Their data demonstrated treatment with an S1PR2 agonist led to an increase in the mRNA expression of proinflammatory M1 markers (nos2, cd80, cd86, il-1b, and il-12) in THP-1 derived macrophages challenged with Mycobacterium tuberculosis compared to those without S1PR2 activation. Furthermore, activation of S1PR2 in THP-1 derived macrophages during both Mycobacterium tuberculosis and Mycobacterium avium infections resulted in nitric oxide secretion as well as production of ROS. Regarding cytokine secretion, macrophages infected with Mycobacterium avium demonstrated increased IL-6 and decreased IL-10 secretion whereas those infected with Mycobacterium tuberculosis did not show differences in IL-6 secretion but did show increased secretion of TNF-a compared to untreated cells (61). Overall, this provides evidence that S1PR2 may play a role in response to infections. This provides an exciting opportunity to study the effects of bile acid mediated S1PR2 activation in the presence of infectious agents.
The roles of S1PR2 have also been explored in the context of murine cholestatic liver injury, where there is an increase in NLRP3 expression in bone marrow derived macrophages which translocated to the liver (43). Blocking S1PR2 with JTE-013 in bone marrow derived macrophages in vitro prevented an increase in the mRNA expression of NLRP3, pro-Il-1b, pro-IL-18, as well as IL-1b and IL-18 secretion. This was further supported by additional work by Hou L. et al (62) where it was found that activation of S1PR2 in murine bone marrow-derived macrophages led to the activation of the NLRP3 inflammasome. Specifically, S1PR2 activation with CYM-5520 led to secretion of IL-1b, and increased protein expression of NLPR3, pro-IL-1b, and pro-IL-18. This adds to the growing evidence that activation of S1PR2 in macrophages leads to a pro-inflammatory response.
There have also been extensive studies into the effects of S1PR2 signaling in neutrophils. Zhao X. et al. studied the roles of S1PR2 activation in neutrophils utilizing a neutrophil migration assay and found that inhibition of S1PR2 with JTE-013 in murine bone marrow neutrophils led to significantly less migration (44). S1PR2 antagonism also decreased neutrophil F-actin, suggesting that S1PR2 is important in neutrophil cytoskeletal remodeling. Similar results were found in the human dHL-60 cell line in which the same cell migration and cytoskeletal remodeling assays were performed. In vivo studies utilizing a mouse model of cholestatic liver injury showed that intraperitoneal (IP) administration of JTE-013, an S1PR2 antagonist, reduced neutrophil chemotaxis to the liver as measured through Lys6G mRNA expression and flow cytometry. Further work by Zhao X. et al. also found neutrophils can form neutrophil extracellular traps (NETs) in response to S1PR2 activation (45). The increased formation of NETs following S1P administration was confirmed in vitro through immunofluorescence of Cit-H3, neutrophilic elastase (NE), and myeloperoxidase (MPO). Furthermore, they applied S1P treatment in the presence of S1PR2 antagonist JTE-013 and found that the formation of NETs was blunted. In vivo, they administered S1PR2 siRNA in MCDHF (methionine and choline deficient high fat)-fed mice and found that blocking S1PR2 led to decreased levels of MPO-DNA complexes in the serum of these mice along with decreased protein expression of Cit-H3 in those livers, providing evidence for decreased neutrophil activation that was accompanied by decreased severity of liver injury. It was also found that S1PR2 was responsible for preventing spontaneous neutrophil apoptosis by administering S1PR2 antagonist JTE-013 and measuring the proportion of Annexin V positive cells (54). This finding suggests that S1PR2 activation in neutrophils also prolongs neutrophil life. Overall, this work adds to the growing body of evidence that S1PR2 is essential for pro-inflammatory neutrophil activation.
Another important aspect of S1PR2 signaling also involves intestinal epithelial cells as they possess important functions in immune regulation. Chen T. et al. examined the roles of S1PR2 signaling in intestinal epithelial cells utilizing intestinal epithelial cells (IECs) isolated from WT and S1PR2 KO mice both with and without acute DSS colitis (46). Co-culturing S1PR2 -/-IECs with CD4+ T-cells resulted in increased proliferation as compared to WT IECs. This suggests that in the presence of an inflammatory challenge, S1PR2 may be responsible for gatekeeping the amount of IEC mediated CD4+ T-cell proliferation. This was further supported with an increase in the mRNA expression of MHC-II in IECs from S1PR2 KO colitis mice compared to WT colitis mice suggesting that S1PR2 limits MHC-II expression and presentation to CD4+ T-cells. Interestingly, this study does not broadly agree with previous studies showing anti-inflammatory effects when S1PR2 was inhibited, however it suggests a more interesting phenomenon in which S1PR2 signaling exerts different effects depending on its cellular origin. In fact, two S1PR2 inhibitors, Etrasimod and Ozanimod have demonstrated potential as treatment options for individuals with ulcerative colitis, further highlighting the complexity of S1PR2 signaling in intestinal inflammation (63, 64).
PXR
Pregnane X receptor (PXR) has also been shown to activated by bile acids and the roles of this receptor in altering immune responses was investigated. Hudson et al., studied the effect of PXR activation in LPS-primed macrophages by treating both murine peritoneal macrophages and THP-1 derived macrophages (47). Both mouse and human macrophages exhibited increased production of IL-1b after activation by species specific PXR ligands. Also, in both mouse and human macrophages lacking NLPR3, there was no induction of IL-1b production or activation of cacspase-1 after PXR activation, suggesting that PXR activation in macrophages is mediated through NLPR3. There were similar findings in human macrophages by showing that THP-1 derived macrophages treated with PXR agonists released ATP. While there is limited evidence compared to other bile acid receptors, this justifies further investigation into inflammatory responses after PXR activation.
VDR
The Vitamin D Receptor (VDR) is not only highly expressed in the intestine but also in immune cells. Bile acids were shown to activate VDR, and this receptor is highly expressed in monocytes, macrophages, dendritic cells, and T cells. VDR activation in monocytes and macrophages generally results in antiinflammatory effects with decreases in IL-1, IL-6, IL-8, IL-12, and TNF-a (36,37). DC VDR activation results in the inhibition of the differentiation and maturation of dendritic cells. VDR activation in T-cells, results in the inhibition of T cell proliferation, and polarizes Th1 cells towards a Th2 phenotype (49). It also inhibits the differentiation of Th17 cells, which are known to play a role in the maintenance of the intestinal barrier in response to extracellular pathogens and increases the differentiation of tolerogenic Treg cells (3).
A recent study found that VDR activation by VitD3 suppressed MLPR3 inflammasome, IL-1b secretion, and caspase 1 activation in LPS challenged mouse peritoneal macrophages (48). This was confirmed via VDR siRNA silencing. Furthermore, VitD3 mediated VDR signaling reduced the severity of DSS colitis and found that it increased the ratio of M2 to M1 macrophages as well as inhibiting the differentiation of CD4+ T-cells into Th1 and Th17 T-helper cell subtypes. Wang et al. has also shown that VDR signaling protected from DSS colitis, providing additional evidence for VDR in immune signaling by shifting macrophages towards the M2 phenotype. He L., et al., utilized a TNBS model of colitis and found that mice lacking VDR in intestinal epithelial cells and specifically in colonic epithelial cells exhibited increased severity of colitis with increased Th1 and Th17 responses, and increased epithelial cell apoptosis with impaired gut permeability (65). Despite various studies demonstrating anti-inflammatory effects from VDR signaling, the direct influence of bile acids on VDR in the context of inflammation has not been clearly illustrated, providing an opportunity for important future studies.
RORgt
Both Isoallo-LCA and 3-oxo-LCA are inverse agonists of RAR-related orphan receptor gamma (RORyt). In innate lymphoid cells (ILCs), RORyt activation leads to increased differentiation and function of ILC3 subtypes, which play an important role in mucosal homeostasis (3,50). In T-cells, the activation of RORyt increases differentiation of Th17 cells and inhibits differentiation of Treg cells (51). In the case of the above mentioned oxo-bile acids, they bind RORyt as inverse agonists and in models of colitis, this results in decreased IL-17 and Th17 cells with a resulting improvement in inflammation.
A study with CD subjects showed that BI119, an RORyt antagonist inhibited the expression of Th17 genes in PBMCs challenged with bacterial antigens while simultaneously favoring expression of Treg genes (66). Furthermore, there was a decrease in pro-inflammatory mediators IL23R, CSF2, XCL1, CXCL8, and S100A8 and an increase in DEFA5, an antimicrobial protein, in ileal CD biopsy samples. RORyt inhibition in a T-cell transfer model of colitis also attenuated colitis. This offers evidence that RORyt has important roles in inflammatory responses, specifically in the intestines. While there is strong evidence that bile acids are playing key roles in RORgt signaling, the direct effects of specific bile acids on RORgt signaling remains understudied and thus remains an appealing area for future study.
Bile acid homeostasis
Several studies have extensively reported on alterations in bile acid signaling and bile acid levels in IBD. These studies are summarized in Table 2. Wilson A. et al., examined the changes in serum bile acid composition to study previously reported decreases in PXR and FXR activity in IBD patients (74). Both FXR and PXR reporter cell lines were treated with bile acid mixtures with the same composition found in human subjects (74). At a concentration of 75 mM, bile acid reporter activity was significantly decreased in the compositions corresponding to patients with CD for both FXR and PXR (74). 4bOHC was measured in their subjects as a marker of PXR mediated CYP3A4 activity and found them to be significantly lower in participants with CD (74). To look at FXR activity, FGF19 serum concentrations were measured and found to be significantly decreased in those with CD (74). Overall, this study provided strong evidence that the composition of the serum bile acid pool is significantly altered in Crohn's Disease with a simultaneous alteration in bile acid homeostasis as evidenced by decreases in FXR and PXR signaling (74).
Another key aspect of bile acid homeostasis is the diversity of the bile aicd pool which is heavily influenced by bacterial transformation. Das P. et al. performed shotgun metagenomics to study bile salt transformation genes on the phyla level in the gut microbiota of healthy subjects and those with IBD (27). Subsequently, they investigated how these changes corresponded to levels of fecal bile acids. The abundance of bile salt biotransformation genes (BSBGs) was significantly lower in subjects with CD compared to healthy subjects. This was further examined by mapping each gene to its corresponding taxonomic lineage and found that the majority of BSBGs corresponded to the phylum Firmicutes with consistent decreases in the abundance of Firmicutes in subjects with IBD.
Another study examined bile acid metabolism directly related to the gut microbiota by applying pure bile acid solutions to stool homogenates followed by direct measurement of chemically modified bile acids. The microbiota of healthy subjects was able to perform deconjugation, transformation, and desulphation on bile acids, whereas those capabilities were diminished in the IBD samples. These findings demonstrate how IBD induced gut microbial dysbiosis directly affects bile acid composition (84).Rutgeerts P. et al. examined colonic CD subjects and found that the bile acid pool size was significantly decreased in CD subjects when compared to healthy subjects and to subjects with UC (77). Another study from the same group found that CD subjects with ileal involvement maintained a normal level of fasting total bile acids, whereas those with additional colonic involvement did not, suggesting that the colon played a role in the maintenance of the total bile acid pool and that this process was disrupted in CD with colonic involvement (85). Koga T. et al., showed that in patients with CD, the administration of diet higher in fat led to greater excretion of fecal bile acids, providing evidence for bile acid malabsorption caused by CD (86). Rutgeerts P. et al., showed that subjects with CD experiencing bile acid malabsorption experienced an increase in duodenal cholesterol saturated bile (78). HDCA ↑ indicates an increase; ↓ indicates a decrease; ↔ indicates no change.
Another important area of study has been measuring changes in metabolism of bile acids in IBD. Tougaard L. et al., examined the turnover rate of orally administered [14]-C labeled TC by measuring fecal excretion of CA, CDCA, DCA, and LCA in order to study bile acid metabolism in CD subjects with terminal ileal involvement (87). There was increased turnover of CA and DCA in CD subjects, along with decreases in CA and DCA in unoperated subjects. Those who had undergone ileal resection showed normal levels and presumably underwent compensatory increases in the synthesis of CA and DCA.
Furthermore, most patients with IBD are on medical treatment, a variable that is often confounding, but Roda G. et al., specifically looked at serum bile acid levels in IBD subjects who were treated with anti-TNF agents (88). Based on a bile acid profile of up to 15 different bile acids, various patient populations can be distinguished. PCA analysis showed separation between anti-TNF treated and untreated CD subjects, UC subjects, and healthy controls. CD patients on anti-TNF treatments showed an increase in total serum bile acids compared to CD patients that did not receive those treatments. This was driven by an increase in secondary bile acids. Ultimately, anti-TNF agents used to treat IBD may reestablish physiological levels of bile acids that are disturbed during IBD.
There are additional variables which need to be considered in the study of bile acid homeostasis disruptions in IBD such as age. Jacobs J.P. et al., recruited families with pediatric IBD cases to study gut microbial structure and associated metabolic changes (89). The bile acids CA, 7-ketoDCA, sulfated CDCA, and 3-sulfoDCA were associated with CD. Martin F.P., et al. studied urinary bile acid profiles in pediatric subjects with IBD and found that subjects with IBD excreted less urinary GHDCA (90). IBD subjects also excreted higher levels of urinary THCA, UDCA, TUDCA, and B -UCA. In general, there are few studies on pediatric cohorts with IBD and additional rigorous studies are warranted to determine if there are age specific alterations in bile acid homeostasis. Collectively, changes in bile acids in IBD patients are mainly mediated by the associated dysbiosis.
Conjugated and unconjugated bile acids
As previously mentioned, a key aspect in bile acid homeostasis involves the conjugation of bile acids and several changes in conjugation have been reported in IBD. Gnewuch C. et al. found decreases in total serum unconjugated bile acid levels in UC (67). Those with CD also exhibited a higher ratio of serum conjugated to unconjugated bile acids when compared to healthy subjects (74). Duboc, et al., found increases in conjugated bile acids in those with IBD, and an increase in sulphated forms of bile acids only in those with active disease when compared to healthy controls (84). Vantrappen G et al. found a decrease in the total size of the bile acid pool with an increased ratio of glycine conjugated bile acids to taurine conjugated bile acids in CD subjects when compared to healthy controls (83). The reduction in bile acids was related to the activity of the disease. Linnet K., et al. studied the effects of CD on fasting and postprandial serum bile acids (91). After a meal, the concentrations of taurine conjugated bile acids were lower in CD than in healthy controls. On the other hand, there was no difference in glycine conjugated bile acids. Additionally, the ratio of total glycine to taurine in serum bile acids was increased in CD subjects than in healthy controls in both fasting and postprandial states. Tanida N. et al. also found that there was an increase in the fecal excretion of total bile acids in both wet and dry fecal compartments in UC subjects (92). Additionally, glycine and taurine conjugated bile acids were higher in diseased subjects compared to healthy controls. Further, unconjugated and sulfated bile acids were decreased in UC feces compared to healthy controls. These changes were exacerbated in those with increased disease severity. Ejderhamn J. et al. examined fecal bile acid excretion in a pediatric population of IBD (70). Bile acids in the feces and in fecal water were measured and demonstrated that subjects with IBD had increased levels of total bile acids, unconjugated bile acids, as well as glycine and taurine conjugated bile acids in their feces. In the fecal water, total bile acid excretion was increased, including increases in taurine conjugated bile acids with concentrations 2 to 5-fold higher in IBD subjects. In subjects with clinical remission of their disease, increased taurine conjugated bile acids in both the feces and fecal water compartments were observed. Overall, these findings suggest a decrease in bacterial Bile Salt Hydrolase (BSH) activity in IBD patients leading to a decline in the relative abundance of unconjugated bile acids.
Serum
Several studies have measured the levels of unconjugated bile acids in the serum of subjects with IBD under various states (Table 2). One study observed that in CD subjects who had undergone ileocecal resection, serum CA and CDCA were increased when compared to controls and CD subjects that had not undergone surgery (67). Heuman R. et al. measured fasting and postprandial serum levels of CA and CDCA conjugates in IBD (68). The average postprandial increase in CA and CDCA was significantly decreased in subjects with CD compared to healthy subjects. Additionally, subjects who had undergone surgical resection for their disease exhibited a more subdued postprandial increase in CA with no differences in CDCA. These studies suggest that there is a lack of absorption of bile acids due to ileal resection and damage from CD. Furthermore, this may lead to a compensatory increase in the hepatic production of CA and CDCA.
In order to explore why patients with IBD typically have less PXR and FXR activity, Wilson A. et al., examined their changes in serum bile acid composition (74). They found no differences in serum proportion of CDCA and decreased proportions of LCA between healthy controls and subjects with inactive or active CD.
They also found no differences in serum DCA levels between healthy subjects and those with CD (74). Gnewuch C. et al.
(2009) found a decrease in serum LCA in both a CD and UC cohort (67). LCA and UDCA were increased in UC subjects with hepatobiliary issues compared to those without. This is a very interesting finding that warrants further mechanistic inquiry. Overall, a decrease in LCA levels in subjects with IBD falls in line with our understanding that microbes are needed to transform primary bile acids and those microbes can be lost during IBD.
In a study of pediatric IBD, Ejderhamm J. et al. found that subjects with active UC exhibited significantly higher levels of CA and CDCA in their serum, four hours after food consumption when compared to healthy, age-matched subjects (69). These findings suggest either increased absorption of primary bile acids in the small intestine perhaps as a compensatory mechanism for impaired colon function or a decrease in the bacterial transformation of these bile acids due to microbial dysbiosis, however this is purely speculative and there may be additional physiologic variables due to this being a pediatric cohort.
Overall, these studies demonstrate how changes in bile acid during IBD are context specific. While conducting such studies would be challenging, further clinical investigations with larger sample sizes and granular stratifications based upon age, severity of disease, surgical history, and intestinal region affected, are warranted to allow for better understanding of how unconjugated bile acids specifically are being altered in both UC and CD.
Feces
While there are a few studies looking at serum levels of unconjugated bile acids in IBD, there are several more doing so in the feces. Yang, Z.H., et al. found an increase in fecal CA with decreases in DCA, LCA, and 12-ketoLCA, in UC subjects compared to healthy controls, while also reporting trends towards increases in CDCA (71). Additionally, DCA, LCA, 6-ketoLCA, and 12-ketoLCA were negatively correlated with serum levels of IL-1a, IL-1b, TNF-a, and IL-6. These findings support mainstream understanding of bile acids in IBD where the abundance of primary bile acids is increasing either due to increased hepatic production, decreased intestinal absorption, and or a concurrent decrease in their microbial transformation to secondary bile acids. Further, increases in secondary bile acids during lower levels of pro-inflammatory cytokines also support less microbial dysbiosis during less inflammation.
An understudied area in the measurement of fecal bile acids involves colon fluid. Vertzoni M. et al. collected colon fluid endoscopically in UC subjects with UC in relapse and in remissions and analyzed the composition of the fluid (73). There were no significant differences in bile acids between subjects in relapse compared to those in remission. The authors surmised that the lack of apparent difference may have been due to low statistical power. Additionally, CA trended towards a decrease in subjects with a relapse in their UC compared to those in remission, although this was not significant. While these findings are unremarkable, there is less precedence for the use of colon fluid to measure bile acids in IBD subjects and models of IBD. Nevertheless, these findings are important to note and perhaps they will be augmented by future studies which may justify the use of colon fluid for bile acid measurements in IBD.
Jansson et al., performed non-targeted metabolic profiling in identical twin pairs including healthy subjects and subjects with CD and in the case of ileal CD, found increases in fecal 3a, 7a, and 12a-trihydroxy-5b -cholanate (72). Fiasse R. et al. analyzed fecal bile acid levels in subjects with ileal CD before and after ileal resection (82). DCA and LCA were decreased in unoperated ileal CD as compared to healthy controls, with a further reduction after surgery. The authors speculated that the decrease in DCA corresponded to decreased transit time in CD subjects and thus having a decreased exposure to bacterial 7a-dehydroxylase. It is also possible that the loss of microbes possessing 7adehydroxylase activity may contribute to these observations. Studies by Das P. et al. found that subjects with IBD had reduced levels of BSBGs and proceeded to examine fecal bile acid compositions in these subjects and found that CA was increased while LCA was decreased in IBD subjects (encompassing both UC and CD) (27). Ejderhamn J. et al. examined fecal bile acid excretion in a pediatric population of IBD (70). In the fecal water, CA and CDCA were increased with concentrations 2 to 5-fold higher in IBD subjects. Weng Y.J., et al. examined the interactions between diet, microbiota, and metabolites in patients with IBD and found that CDCA and LCA were significantly decreased in the feces of patients with both UC and CD (75). The species Fusobacterium nucleatum was also associated with CDCA and LCA levels in patients with CD. Franzosa E., et al., performed untargeted metabolomic and shotgun profiling in subjects with IBD and healthy controls (76). They found significant increases in CDCA in the stools of IBD subjects. They also found nonsignificant decreases in DCA and LCA in CD subjects. It is challenging to draw strong conclusions for the changes in fecal levels of unconjugated bile acids from the studies surveyed. This is due to several factors including small sample sizes which force the grouping of UC and CD subjects together even though both subsets of IBD have distinct pathophysiology. As with the studies in serum, rigorous studies with larger sample sizes across the various manifestations in IBD are needed. However, these will most likely require cross-disciplinary and multi-institutional collaborations with strong clinical implications for their findings to justify the studies in the first place.
Bile
A less investigated compartment for the measurement of bile acids in IBD is in the bile itself, due to the practical challenges of more invasive techniques needed for samples. Due to procedural challenges, these studies are not as common and are less current, but they offer an important insight, nevertheless. Many of these studies were performed by Dr. Rutgeerts and most were performed in subjects with CD.
In one study by Rutgeerts, studying cholic acid metabolism it was found that the metabolism of CA and CDCA was significantly increased in subjects with ileal CD when measured directly from the bile, and to those with both ileal and colonic involvement when compared to healthy subjects (85).
Nishida, T. et al., orally administered deuterated CDCA in subjects with CD and healthy volunteers (79). Using duodenal bile samples, they measured a significant decrease in the biological half-life of CDCA, the pool size of CDCA, and the total bile acid pool in the bile of subjects with CD. They also found a non-significant decrease in DCA and a non-significant increase in UDCA levels in CD when compared to healthy subjects.
Rutgeerts P. et al. examined duodenal bile in subjects with CD localized to the colon and found significantly decreased concentrations of DCA in subjects with CD compared to healthy controls (77). Another study by them found that in subjects with CD experiencing bile acid malabsorption, bile acid composition in bile had significant decreases in DCA when compared to healthy subjects (78).
Lapidus A. et al. found that bile acid composition in the duodenal bile of subjects with CD exhibited a significant decrease in the abundance of DCA and UDCA (80). In another study they found that in CD subjects with ileal resection, there was a significant decrease in DCA, but significant increase in UDCA levels in duodenal bile compared to healthy controls (81). Vantrappen G., et al., found that CD subjects had significant reductions of UDCA in their bile (83).
These studies highlight an exciting avenue for further investigation in pre-clinical models where the invasiveness of sampling bile does not pose the same concerns it does in humans even if it poses additional technical challenges. Few physiological studies have explored the changes in bile acid composition specifically in the bile in models of IBD. Such studies could provide important mechanistic insight into these clinical observations that have already been made.
Taurine conjugated bile acids 3.2.3.1 Serum
Overall, there are few studies examining the levels of taurine conjugated bile acids in the serum of IBD cohorts. Gnewuch C. et al. (2009) found a decrease in serum total bile acid tauroconjugates in a CD cohort, along with a general decrease in total bile acids (67). They also found an increase in serum TCA and TUDCA in UC subjects with hepatobiliary symptoms compared to UC subjects without hepatobiliary issues. Another study found that patients with CD also had significant decreases in the proportion of serum TCA, but no changes in TLCA (74). This limited evidence suggests that patients with CD have an impaired conjugation ability.
Feces
There are also few studies looking at fecal levels of taurine conjugated bile acids in IBD. Yang, Z.H., et al. found an increase in fecal TCA, but a decrease in TCDCA and TLCA in UC subjects compared to healthy controls (71). Furthermore, TCA was positively correlated with serum IL-a and TNF-a., while TLCA was negatively correlated with serum levels of IL-1a, IL-1b, TNF-a, and IL-6. Jansson et al., performed non-targeted metabolic profiling in identical twin pairs including healthy subjects and subjects with CD and in the case of ileal CD, found increases in fecal TCA (72). Weng Y.J., et al. found that TLCA was significantly decreased in the feces of patients with both UC and CD (75). While this is not extensive evidence, these findings suggest that fecal TCA may be associated with disease activity and its microbial transformation is diminished during IBD most likely due to gut microbial dysbiosis.
Serum
There are also limited studies on serum glycine conjugated bile acids in IBD. Gnewuch C. et al. (2009) found a decrease in serum total bile acid glycoconjugates in their CD cohort (67). In UC subjects with hepatobiliary symptoms, GCA and GCDCA was increased compared to UC subjects without hepatobiliary issues. Wilson A. et al., examined the changes in serum bile acid composition to study observed decreased in PXR and FXR activity in IBD patients, and found significant decreases in the proportion of GCDCA with significant increases in the proportion of GCA and GDCA in the in the serum of subjects with both inactive and active CD as compared to healthy controls (74). Suchy F.S., et al. looked at postprandial serum bile acid concentrations in subjects with ileal CD (68). There were greater increases in GCA and total bile acids after a meal in healthy subjects when compared to those with CD. These studies are important to note, but given their differences in context, it is challenging to reach a unified conclusion.
Feces
In the feces, Yang, Z.H., et al. found an increase and decrease in GCDCA and GLCA respectively in UC subjects compared to healthy controls (71). Furthermore, GCDCA was positively correlated with serum IL-a levels, while GDCA was negatively correlated with serum levels of IL-1a, IL-1b, TNF-a, and IL-6. They also found a trend towards an increase in fecal GCA in UC subjects compared to healthy controls, although it did not reach statistical significance (71). Also, GCA was positively correlated with serum IL-a. Jansson et al., performed non-targeted metabolic profiling in identical twin pairs including healthy subjects and subjects with CD and found that subjects with CD had higher levels of fecal GCDCA (72). These findings both demonstrate an increase in primary glycine conjugated bile acids in the feces of IBD subjects. This may be due to either decreased absorption and/or increased hepatic synthesis.
Primary and secondary bile acids
Another key variable to consider when examining alterations in bile acids during IBD is the difference in abundance between primary and secondary bile acids because of the gut microbial dysbiosis that is known to occur in IBD. Yang, Z.H., studied the gut microbial profiles in patients with UC and fecal bile acid levels (27). Briefly, 32 UC subjects and 23 age and sex matched healthy controls were recruited and stark differences in their microbiota was found. PCA analysis showed a clear separation between those subjects with UC and healthy controls. Additionally, measures for a diversity such as the Chao1, Shannon, and Simpson diversity indexes showed decreased diversity in those with UC. Further analysis demonstrated significant alterations in both phylum, genus, and species compositions. There was a significant decrease in the amount of overall secondary bile acids in the feces of patients with UC. Immunohistochemistry on mucosal biopsy samples demonstrated a significant increase in the expression of bile acid receptor TGR5 and a significant decrease in VDR in UC subjects. UC subject serum also had increased levels of IL-1a, IL-1b, TNF-a, IL-2, and IL-6. Overall, this study found that fecal bile acid profiles were strongly associated with gut microbial and serum cytokine profiles, demonstrating that bile acids are strongly impacted by the systemic inflammation and microbial gut dysbiosis that occurs in IBD and UC more specifically.
Kruis W. et al. found that the daily fecal bile acid excretion rate was increased in subjects with CD and decreased in UC compared to healthy controls (93). CD and UC subjects had significant increases in primary bile acids but decreased amounts of secondary bile acids as compared to healthy controls. At the time, the authors speculated that the decrease may have been attributed to faster gastrointestinal transit time in UC subjects and increased acidity in CD feces. However, given recent findings, it seems possible to speculate that these changes may also occur due to microbial dysbiosis.
Duboc, et al., studied the effects of IBD induced gut microbial dysbiosis on bile acid metabolism by measuring the levels of bile acids in both feces and serum in healthy subjects, active IBD, and IBD in remission (84). There was a decrease in secondary bile acids abundance in subjects with IBD in both serum and feces. Das P. et al. found that subjects with IBD had reduced levels of BSBGs and proceeded to examine fecal bile acid compositions in these subjects and found that the primary bile acid CA was increased in IBD subjects (encompassing both UC and CD), and LCA was low (27). Levels of conjugated bile acids GCA, TC, and TCDA were elevated in IBD subjects compared to healthy controls. They also found decreases in secondary bile acids in IBD subjects when compared to healthy controls.
Effects on bile acids on intestinal epithelial cells and intestinal inflammation
Most studies of bile acids and intestinal epithelial cells utilized cell lines which lack other cell types found in the intestines while also lacking some physiological aspects that are present in newer primary enteroid cell culture systems and thus the findings of such studies need to be carefully interpreted. Overall, studies examining the effects of bile acids on intestinal epithelial cells would be greatly improved with the utilization of more complex models such as primary intestinal cultures and primary intestinal cultures cocultured with immune cells to help delineate cell specific mechanisms that are often obscured in vivo but not captured with less complex in vitro models. There have been several studies examining the effects of individual unconjugated bile acids on intestinal epithelial cell TEER, apoptotic signaling, and cytokine secretion.
Unconjugated bile acids
One such study by Duboc H. et al., studied the effects of unconjugated bile acids on IL-1b induced IL-8 secretion in a Caco-2 cell culture model (84). While CA and CDCA did not alter IL-8 secretion, DCA and LCA inhibited the IL-1b mediated secretion of IL-8. LCA had a dose-response relationship with IL-1b mediated IL-8 secretion and treatment with sulfated LCA negated the anti-inflammatory effect. Another study utilized murine colonic epithelial cells to mimic wounds found in ulcerative colitis in an in vitro scratch model. Ultimately, CA, CDCA and LCA had no effect intestinal epithelial cell growth in response to the in vitro wound in the form of a scratch (94). However, DCA impaired epithelial cell growth in response to the insult. Sarathy et al. looked at the effects of bile acids on the production of inflammatory cytokine IL-8 in T84 cells. They found a five-fold increase in IL-8 production from CDCA, and an inflammatory cocktail led to a 21-fold increase (95). On the other hand, LCA led to a 50% reduction in IL-8 production in response to the inflammatory cocktail.
Conjugated bile acids
TUDCA, a secondary bile acid, has been shown to have antiinflammatory potential (Figure 1). Van den Bossche L. et al., studied the effects of TUDCA on TNF-a treated Caco-2 cells and TNF-a overexpressing mice CD with ileal involvement (96). TNF-a treated Caco-2 cells demonstrated decreased mRNA expression of PXR and OSTb with trends towards a decrease in the expression of FXR, VDR, ASBT, and OSTa. TNF-a also led to increased IL-8 secretion and decreased TEER. Administration of TUDCA to apical compartments prevented TNF-a induced reduction of PXR and OSTa expression and trended towards an increase in expression of FXR and ASBT. VDR and OSTba did not change in response to TUDCA. Surprisingly, TUDCA also resulted in a synergistic decrease in TEER.
The effects of TUDCA on intestinal epithelial cells have also been explored in additional cell lines. HT29 and T84 cells lines were challenged with TNF and co-administered TUCDA (97). TUDCA prevented a significant reduction in the induction of caspases 3, 8, and 9 in HT29 cells. In T84 cells, they found that TUDCA prevented the formation of apoptotic bodies as assessed by electron microscopy. They also found that co-administration of TUDCA with FasL in HT29 cells prevented FasL induction of caspase-3 activation. To find the pathway responsible for these findings, the role of the a5b1 integrin receptor (which can sense TUDCA in hepatocytes) was studied and found that blocking that peptide did not interfere with TUDCA protection against caspase-3 activation, keeping this mechanism unknown. 5 Bile acids as therapeutic agents for intestinal inflammation
Preclinical models
There is limited data on bile acids as therapeutics in human IBD, but several studies have been performed in preclinical animal models. These data as well as human studies are summarized in Table 3. In a model of indomethacin induced enteropathy, Shibuya N. et al. found that DCA exacerbated small intestinal inflammation (98). DCA increased expression of ICAM-1 and VCAM-1 which facilitated the adhesion and thus the migration of lymphocytes to the ileum. This effect was due to S1PR2 signaling as an S1PR2 antagonist diminished DCA induced increases in ICAM-1 and VCAM-1 expression in vitro Anti-inflammatory effects of TUDCA and UDCA in models of IBD. The enterohepatic circulation of bile acids transports bile acids synthesized and modified in the liver to the intestines through the bile duct. The rate limiting step of hepatic synthesis of bile acids is mediated by the CYP7A1 enzyme which is inhibited by bile acids and ileal FGF15/19. Gut microbes then convert primary bile acids into secondary bile acids. Gut microbial dysbiosis in IBD leads to a reduction in the secondary bile acid pool. Specifically, two key secondary bile acids have been shown to exert anti-inflammatory effects in IBD and relevant models, TUDCA and UDCA. Administration of TUDCA on cell culture and mouse models of IBD aided in bile acid homeostasis by preventing loss of OSTa expression as well as increased FXR and ASBT expression. Furthermore, TUDCA protected against colon shortening, weight loss, and led to improvements in histological score. TUDCA also led to the reduction of proinflammatory cytokines IL-1b, TNF, Cxcl2, and IFN-g as well as decreased apoptotic signaling. Furthermore, TUDCA led to decrease neutrophilic infiltration and activity, as well as general decreases in leukocyte infiltration. Administration of UDCA led to less diarrhea and weight loss as well as decreases in disease activity index and histology score. UDCA also led to decreased apoptotic signaling. Furthermore, UDCA led to decreases in IL-8, TNF-a, and Mad-CAM-1 in the colon, and less IL-17 and IL-23 in the serum of subjects with UC as well as increases in the abundance of Firmicutes and decreases in Proteobacteria. (101). Disease activity was assessed by measuring intestinal epithelial permeability through FITC-dextran uptake and by measuring apoptosis through capsapse-3 cleavage. LCA improved intestinal barrier function and reduced apoptotic signaling. Furthermore, histological scores were reduced by both LCA and UDCA However, additional parameters used to measure the severity of colitis such as weight loss and colon shortening were not significantly altered by LCA or UDCA. Similar findings were observed after LCA treatment during inflammation in a T84 colonic epithelial cell culture model treated with TNF-a and IFN-gamma. They found improvement in epithelial barrier integrity and apoptosis signaling in response to LCA and UDCA during inflammation. An interesting aspect of their studies was the use of a chemical analogue of UDCA which is not capable of being metabolized, 6-methyl-UDCA, and found this compound had no effect in any of their studies.
The role of secondary bile acids, specifically UDCA has also been explored in chemically induced models for IBD (Figure 1). Martinez-Moya P., et al., studied the effects of UDCA on a TNBS induced colitis rat model (102). Administration of UDCA at a dose of 50 mg/kg day through an esophageal catheter resulted in protective effects whereas there were no significant effects at lower doses. Rats treated with UDCA exhibited decreased alkaline phosphatase activity as well as a smaller affected area. However, UDCA treatment did lead to an increase in colonic IL-1b production compared to control rats where this was not observed with TNBS treated rats.
In a co-model for cholestatic liver injury and colitis in the form of multidrug resistance protein 2 (MDR2) knockout mice treated with DSS, it was found that the bile acid UDCA reduced the severity of colitis and downregulated colitis susceptibility protein mucosal addressin-cell adhesion molecule 1(MAdCAM-1) (103). Further, previous studies have also established UDCA and its precursor, LCA, protected against DSS colitis in vivo by preventing weight loss, restoring fecal pellet formation, and attenuating disease activity index (104). Similar effects we observed in vitro in cytokine treated colonic epithelial cells as measured by decreased TNF-a release.
The role of bile acid conjugation has also been studied specifically in the cases of taurine conjugated bile acids. Taurocholic acid (TCA), a conjugated primary bile acid has been shown to exert anti-inflammatory effects in chemically induced colitis. Yang Y., showed that oral gavage of TCA prevented TNBS weight loss and improved mortality (107). TCA also prevented colon shortening and increased colon weight due to TNBS colitis. Benefits were also observed on a histological level, where mice treated with TCA had a lower histological score on average than those without. TCA treatment during TNBS colitis also resulted in decreased colonic MPO, TNF-a, IL-1b, and INF-gamma levels. More recently, Wong, W.Y. et al. showed that increasing the amount of taurine conjugated bile acids through Lactobacillus case strain Shirota (LcS) mediated increases led to improvement in DSS induced colitis (111). Specifically, improvement in colon length, improvement in histological architecture, and decreases in CD45 expression in the colons of these mice as compared to untreated mice with DSS colitis. LcS treatment also improved expression of Occludin on the protein level, and Muc2 on the mRNA level. This treatment and this improvement coincided with serum increases in TCA and TCDCA in LcS treated mice compared to untreated DSS colitis mice. There were also serum decreases in aand b-MCA after LcS treatment. While this was not a direct treatment with bile acids, this study provides evidence for beneficial effects from taurine conjugated bile acids. This was evident as TCA and TCDCA level increases were accompanied by decreases in IFN-gamma and increases in IL-10 mRNA in the colons of DSS colitis mice treated with LcS as compared to untreated colitis mice. While there is evidence that taurine conjugated bile acids are protective against DSS colitis, there is additional evidence in other models for intestinal inflammation. TNF-a overexpressing mice given TUDCA in their water for eleven weeks showed benefits in their bile acid homeostasis (96). TUDCA treatment increased the mRNA expression of FXR with a trend towards an increase in VDR, and no change in PXR. TUDCA also resulted in increased mRNA expression of bile acid transporter genes ASBT, and OST-a, with no change in OST-b. Furthermore, they performed principal component analysis (PCA) utilizing the expression data from their selected bile acid homeostasis genes and found that TUDCA treated TNF ARE mice shifted closer towards wild type mice than TNF ARE mice who did not receive TUDCA. TUDCA treated mice demonstrated continued weight gain through 13 weeks of age as compared to TNF ARE mice which began to exhibit weight loss at 11 weeks of age. The histological score and leukocyte infiltration scores were also significantly decreased in TNF ARE mice treated with TUDCA. This study provides strong evidence that TUDCA provides some anti-inflammatory effects in a mouse model for Crohn's ileal inflammation while also restoring some aspects of bile acid homeostasis that are impaired in ileitis.
In a model of acute DSS colitis (7-day DSS treatment followed by 7-day recovery period) with Tollip (Tollinteracting protein) deficient neutrophils, Diao N., et al. showed that DSS colitis was more severe in mice with neutrophils lacking Tollip (toll-interacting protein) (108). They examined the roles of TUDCA on DSS colitis in WT mice because it is known to restore Tollip function. Mice treated with intraperitoneal TUDCA did not exhibit colon shortening or experience as much weight loss. TUDCA also reduced the amount of circulating activated neutrophils as measured through the cell surface expression of C14. This finding is in line with previous studies by Laukens D. et al., where they also demonstrated that TUDCA inhibited the effects DSS colitis (97). In this study, all mice treated with intraperitoneal TUDCA survived for a full two weeks after DSS administration. Mice treated with TUDCA exhibited a significant reduction in weight loss and a reduction in colon shortening 10 days post-DSS. Furthermore, there was significantly less IL-1b protein in the colon 10 and 14 days post-DSS. There was also significantly less TNF expression on post-DSS day 10 as well as significantly reduced Cxcl2 expression after 7, 10, and 14 days post-DSS.
TUDCA protected against intestinal epithelial barrier dysfunction by measuring less bacterial translocation to the spleen. Furthermore, they found that in colonocytes isolated from mice treated TUCDA, there was a significant decrease in Caspase-3 activity on day 3 and 14 post-DSS. There was also an increase in the expression of Bcl2 on days 3 and 10 post-DSS. Additionally, pre-treatment with TUDCA before challenge with LPS resulted in less intestinal barrier dysfunction as measured through FITC-dextran and less enterocyte apoptosis in the ileum.
The effects of TUDCA have also been studied in other models of colitis. Yang Y., et al., studied the effects of TUDCA on TNBS colitis and found that it protected against colitis during a seven-day treatment beginning 24 hours after rectal TNBS administration (23). Administration of TUDCA through oral gavage prevented TNBS colitis weight loss and improved survival rate. TUDCA also significantly improved histological score and resulted in a significant decrease in colon levels of TNF-a, IL-1b, IFN-gamma, and MPO. This study adds to the available evidence that TUDCA is protective against chemically induced colitis.
More recently, Long Y., et al., showed that treatment of TNBS colitis mice with TUDCA demonstrated improved colon length, and decreased histological score (109). To explore potential reasons for these beneficial effects from TUDCA, Grp87, an endoplasmic reticulum stress marker that they found was increased in patients with Crohn's Disease, was studied. Treatment with TUDCA prevented Grp78 upregulation in response to colitis through immunohistochemistry. This suggests TUDCA exerts its positive effects by relieving ER stress. The performed subsequent studies in vitro to show that ER stress can promote an inflammatory response through the activation of the p38 MAPK pathway, but they did not directly test the effects of TUDCA during those experiments.
Taurohyodeoxycholic acid (THDCA) was investigated by He J. et al. in a model of TNBS colitis (110). They used THDCA extracted from Pulvis Fellis Suis and administered it through oral gavage for seven days. THDCA alleviated weight loss due to colitis, improved macroscopic score, and improved histological architecture. There was a decrease in COX-2 expression assessed by immunohistochemistry in the colons of mice treated with THDCA. Furthermore, there were decreases in MPO activity in the colon, as well as decreased circulating TNF-a and IL-6 levels in the serum. Studies in other models of IBD may further elucidate the evidenced anti-inflammatory properties of THDCA.
Human subjects
While there have been extensive studies on bile acids as potential therapeutics for IBD in preclinical models, there are limited studies examining these effects in human IBD. Wang, Z. et al., has examined the roles of UDCA in conjunction with Mesalazine on patients with ulcerative colitis. In this prospective, single center study, participants with UC were randomly assigned to two arms: treatment with Mesalazine or treatment with Mesalazine and UDCA (105). Subjects in the two groups had comparable baseline Mayo scores, but one-week post treatment, those receiving UDCA had significantly lower Mayo scores. With regards to endoscopic Mayo score, those on Mesalazine alone did not show any changes even four weeks post treatment. However, those also receiving UDCA had significantly lower Mayo endoscopic scores four weeks post treatment compared to their baseline and compared to the first week post treatment. Subjects were also administered an IBD questionnaire to assess their quality of life. Four weeks post treatment, scores of social ability, emotional ability, systemic ability, and overall total score were increased in those receiving UDCA and Mesalazine compared to those receiving Mesalazine alone. Furthermore, serum IL-23 and IL-17 levels were also lower four weeks post treatment in those receiving UDCA and Mesalazine compared to those taking Mesalazine alone. Additional microbiota profiling revealed that those who received UDCA in addition to Mesalazine had higher abundance of Firmicutes and lower abundance of Proteobacteria. While this study offers promising evidence of UDCA exerting beneficial effects in human UC, this study showed limited information on the severity of the disease and most importantly was only conducted for four weeks, and thus longer-term data is vital.
While there is evidence for benefits in UDCA as an adjunct treatment for IBD, those benefits may come with unintended consequences. Matsui H., et al. reported on a case of a Crohn's disease patient who developed an enterolith after UDCA administration (106). This patient had achieved remission of her Crohn's disease with prednisolone, azathioprine, and infliximab, and maintained it with azathioprine and infliximab. After her diagnosis with Crohn's disease, the patient had been diagnosed with hepatic dysfunction for which she received oral UDCA. Ultimately, the enterolith was removed surgically with jejunal resection and it was found that 98% of the enterolith was made up of UDCA. While purely speculative, they suspect that the UDCA precipitated and in combination with intestinal stasis in the jejunum resulted in the formation of this enterolith. While this is a case study, this study is important to note.
Overall, care must be taken in analyzing these studies due to the nuances between the various models. In vitro studies modeling intestinal epithelial cells have shown that CDCA increased IL-8 secretion while LCA diminished IL-8 in two distinct human intestinal epithelial cell lines. However, to the best of our knowledge, CDCA alone was not explored in animal models and thus conclusions about CDCA remain limited. On the other hand, LCA appeared to improve intestinal barrier function and reduce apoptotic signaling in a chemical colitis model which coincides well with in vitro data. This was also the case with UDCA where there was strong evidence for antiinflammatory properties when considering studies performed in vitro, in preclinical animal models, and in humans ( Figure 1). Conversely, DCA showed a reduction in in vitro IL-8, but promoted lymphocyte migration and inflammatory signaling in mouse models of intestinal inflammation, highlighting the need for identification of cell specific sources of inflammation in such studies.
Furthermore, there is evidence that suggest antiinflammatory effects of taurine conjugated bile acids ( Figure 1). In several studies and various models including pro-inflammatory challenged intestinal epithelial cell lines, chemical induced colitis, and genetic models of enteropathy, there were overall decreases in the production of proinflammatory cytokines, weight loss and histological damage. These studies suggest a beneficial role for taurine conjugated bile acids in the context of intestinal inflammation that should be further explored as a therapeutic option.
6 Bile acids and barrier function/ intestinal permeability IBD are multifactorial disorders characterized by genetic susceptibility, immune cell overactivation, microbial gut dysbiosis, and changes in intestinal barrier permeability (112). The following section summarizes available literature on the studies of bile acids directly modulating intestinal barrier function in vitro, in animal models for IBD, and in human and animal intestinal explant studies using a Ussing chamber (Table 4). Overall, there is substantial evidence that for bile acids are modulating intestinal permeability through inflammatory changes, and alterations in tight junctions.
Unconjugated bile acids
Horikawa T., et al., looked at the effects of bile acids on intestinal barrier function in differentiated Caco-2 cells (113). While CA and UDCA had no effect and DCA induced a slight decrease in TEER, CDCA lead to a greater than 50% reduction in TEER with a concomitant increase in IL-8 secretion. This was further studied with time and dose-dependent studies and found decreases in TEER, increase in FITC-dextran measured permeability, and IL-8 secretion at their highest concentration of CDCA treatment. While the greatest reduction in TEER happened at 8 hours, the greatest increase in IL-8 secretion and mRNA expression occurred after 24 hours. To investigate mechanistically, they performed similar treatments in the presence of FXR inhibitor guggulsterone. While guggulsterone did not reverse CDCA mediated decreases in TEER, it did block CDCA induced release of IL-8, IL-6, and TNF-a, but not VEGF. FXR inhibition additionally prevented CDCA induced mRNA expression of IL-8 and IL-6. This work provides evidence that CDCA directly modulates intestinal barrier function at least partially through FXR signaling.
Lowes S. et al. studied the effects of cholic acid on human intestinal epithelial Caco-2 and T84 cells (114). Apical administration of cholic acid on Caco-2 monolayers did not affect TEER over time. However, basal administration of cholic acid led to a quick decrease in TEER and demonstrated dosedependent responses on TEER. This alteration was further characterized in intestinal barrier function by measuring mannitol flux and found that basal cholic acid increased mannitol flux through the Caco-2 monolayers. Furthermore, basal administration of DCA led to a quick decrease in TEER and demonstrated dose-dependent responses on TEER. They further characterized this alteration in intestinal barrier function by measuring mannitol flux and found that basal DCA increased mannitol flux through the Caco-2 monolayers. They also found that basal administration of CDCA reduced TEER and increased mannitol flux in both Caco-2 and T84 cells. Administration of sodium cholate led to an increase in permeability as assessed by lucifer yellow fluorescence. However, only some enterocytes were demonstrating this increase in permeability and that the brush border marker Aminopeptidase N (ApN) stayed localized to the apical surface and the Na+/K +-ATPase stayed localized to the basolateral membrane suggesting that there were no disturbances in the membranes in response to sodium cholate. This was further assessed by electron microscopy and found that while sodium cholate did not affect the columnar shape of enterocytes or the length of the microvilli in the brush border, there was a disruption of the dense hexagonal structures observed in untreated explants. Munch et al. studied the effects of DCA on intestinal permeability in human sigmoid colon biopsies with Ussing chambers and found that DCA and CDCA led to significant decreases in TEER (118). Furthermore, CR-EDTA was utilized as a marker of paracellular permeability and found that DCA demonstrated a dose-dependent response on CR-EDTA permeability. DCA and CDCA also increased the uptake of chemically killed E. coli.
Sarathy J. et al. studied the effects of CDCA in human intestinal epithelial T84 cells and found that CDCA decreased TEER (95). Furthermore, they studied the effects of CDCA with a pro-inflammatory cytokine cocktail consisting of TNFa, IL-1b, and IFNg. The inflammatory treatment led to a reduction in TEER, with a further reduction in combination with CDCA. Apical administration of CDCA increased paracellular permeability and in conjunction with the pro-inflammatory cocktail it led to a greater increase in paracellular permeability measured with 10kDA Cascade Blue dextran. LCA did not cause any changes in paracellular permeability alone, but it did attenuate the increases due to CDCA. Additionally, in combination with both CDCA and the pro-inflammatory cocktail, LCA also attenuated the increase in paracellular permeability observed during administration of the proinflammatory cocktail and CDCA. CDCA did not alter claudin-2 protein expression, but it decreased Occludin expression. Specifically, immunohistochemistry showed a disruption of Occludin localization patterns. LCA did not alter claudin-2 protein expression and treatment with LCA did not prevent the CDCA induced disruption on Occludin. Similar protective effects for LCA were observed by others. In this regard, Yao B. et al. examined the effects of LCA on intestinal barrier function in a human epithelial Caco-2 model challenged by TNF-a (122). Pre-treatment with LCA prevented TNF-a induced increases in FITC-Dextran permeability as well as TNFa induced decreases in TEER. LCA prevented TNF-a mediated decreases in the protein levels of ZO-1, E-cadherin, Occludin, and Claudin-1. Furthermore, they studied the localization of these tight junction proteins with immunofluorescence and found that LCA attenuated TNF-a mediated disruptions in tight junction protein localization. While UDCA had no effect, DCA exhibited a dose dependent response on the impairment of barrier function in both the jejunum and the colon as assessed by 4kDa FITC dextran flux. DCA also increased permeability to larger 20kDA FITC dextran in the colon and not in the jejunum. Although, coadministration of UDCA with DCA protected against DCA induced barrier dysfunction in the colon but not in the jejunum. DCA also altered cellular distribution of Occludin assessed through immunofluorescent imaging without altering the protein amount assessed through Western blot. Coadministration of LPS with DCA exacerbated DCA induced barrier function in the colon of both mice and rats while LPS alone had no effect on the flux of FITC dextran. In vivo, a diet supplemented with 0.1% DCA also impaired gut barrier function as assessed by serum FITC dextran.
Golden Jamie M. et al. studied the effects of UDCA on the intestinal barrier in a model of murine experimental peritonitis induced by intraperitoneal LPS (124). They found that UDCA significantly decreased LPS induced increases in gut permeability through a FITC-dextran assay. Additionally, they found that LPS treatment led to a shortening of villus height that was partially prevented with UDCA.
Liu L. et al. studied the effects of DCA in Caco-2 and IMCE (Immorto-Min colonic epithelium) cells (119). DCA led to decreases in TEER in Caco-2 cells. DCA also increased expression of pro-inflammatory cytokines IL-1b, IL-6, and TNF-a in both Caco-2 cells and IMCE cells. In mice, DCA led to a decrease in the expression of tight junction protein ZO-1 on an mRNA levels and through immunofluorescence. Zeng H. et al. (2022) found similar findings with DCA on Caco-2 cells (15). DCA led to increases in permeability of phenol red as well as a decrease in TEER. On a protein level, DCA led to a dose dependent decrease in Occludin expression. Furthermore, Occludin decreases were found from DCA treatment with immunofluorescence as well. Bile acids have also been shown to directly alter intestinal barrier function in the context of inflammatory bowel disease (IBD). One study utilizing a coculture of Caco-2 and HT29-MTX-E12 cells with LPS activated dendritic cells (DCs) in the basolateral compartment showed that activated DCs impaired barrier function through decreased TEER (120). Subsequently, LCA and sulfated DCA administration can marginally restore TEER. While there are several studies demonstrating how bile acids directly alter intestinal barrier function, a key factor in IBD, additional studies are warranted to better understand which bile acids are beneficial, which are not, and why they are leading to their respective alterations in intestinal permeability.
Bernardes-Silva et al. studied the effects of UDCA on intestinal permeability in a rat model of indomethacin induced intestinal inflammation (123). Intestinal permeability was assessed by measuring urine levels of chromium-51-ethylenediaminetetraacetate (42) (CR-EDTA) after oral gavage. Administration of UDCA in conjunction with the offending agent indomethacin prevented indomethacin induced increases in intestinal permeability to the point where no differences were found between rats who received UDCA and indomethacin versus the controls.
With the exception of LCA, most of the available literature points to unconjugated bile acids as being disruptors or having no effect on intestinal barrier function in both in vitro studies with models of intestinal epithelial cells and explants, and in vivo models of intestinal inflammation.
Taurine conjugated bile acids
Lowes S. et al. studied the effects of TCA and TDCA on human intestinal epithelial Caco-2 and T84 cells (114). Basal administration of TCA led to a quick decrease in TEER and demonstrated dose-dependent responses on TEER. This alteration in intestinal barrier function was assessed by measuring mannitol flux and found that basal TCA increased mannitol flux through the Caco-2 monolayers. Basal administration of TDCA also reduced TEER and increased mannitol flux. Furthermore, there were similar effects on T84 cells with T84 cells being more sensitive to bile acid mediated disruption of their barrier integrity.
Liu H. et al. studied the effects of CA supplementation on intestinal barrier integrity in rats (125). There was an accumulation of TCA at the ileum and subsequently found that TCA at concentrations found in CA fed mice increased ileal permeability in a Ussing chamber but not in other regions of the intestine.
There have also been important studies that have occurred in porcine models. Song M., et al. examined the effects of TUDCA specifically on the intestinal barrier function of weaned piglets (126). They administered TUDCA for 30 days to newly weaned piglets and found reduced incidence of diarrhea. Histological examination of the jejunum and ileum showed increases in the villus height to crypt depth ratio and in the proportion of PAS positive goblet cells per villus with TUDCA supplementation. There were increases in the expression of tight junction proteins Occludin and Claudin-1 but not ZO-1 at a protein level in the jejunum. Further, there were decreased levels of circulating LPS and diamine oxidase (DAO) in the serum. This evidence supports the finding that TUDCA directly influences intestinal permeability. Additionally, TUDCA exerted a beneficial effect on systemic and intestinal immunity. TUDCA reduced IL-1b and NF-kB while increasing IL-4 and IL-10 in the ileal mucosa. This was accompanied by an increase in serum IgG and secreted IgA in ileal mucosa. This provides direct evidence that TUDCA is directly modulating intestinal immunity in a porcine model. Song M., et al. also examined the effects of TUDCA in vitro in porcine intestinal columnal epithelial IPEC-J2 cells challenged with K88 E. coli to induce epithelial barrier dysfunction (126). Treatment with TUDCA reversed the E. coli induced decreased in ZO-1, Claudin-1, and Occludin protein expression while also reducing LDH activity in the cell culture supernatant. Additionally, TUDCA treatment with and without E. coli led to significant increases in the expression of bile acid receptor TGR5, but not in FXR. Subsequently, the beneficial TUDCA effects were mediated through TGR5 signaling by knocking down TGR5 and showing that without TGR5, TUDCA did not improve E. coli disruption of epithelial barrier function in vitro. This was also supported by their data showing that during the TGR5 knockdown, TUDCA did not lead to increased levels of cAMP. This was further explored by looking at the MLCK pathway. Intestinal epithelial cells challenged with E. coli had increased MLCK expression along with an increase in the ratio of phosphorylated to unphosphorylated MLC. TUDCA treatment prevented this MLCK pathway activation, but when TGR5 was knocked down, this did not occur further providing evidence that the beneficial effects of TUDCA on intestinal permeability are mediated by TGR5. This was further supported with a MLCK inhibitor and observed similar effects compared to TUDCA.
There is an interesting juxtaposition between the effects of taurine conjugated bile acids on intestinal barrier function and their effects on IBD or models of IBD. Most taurine conjugated bile acids showed beneficial effects towards intestinal inflammation. However, when relating to barrier function, the data suggests that taurine conjugated bile acids mostly impair intestinal barrier function. This is counterintuitive as intestinal permeability is thought of as a hallmark of IBD and as a marker of disease severity especially in pre-clinical models. These contradictions warrant further investigation and possibly even suggest that perhaps the field needs to rethink the way that intestinal permeability is linked to disease activity in IBD.
Conclusion
The roles of bile acids in modulating the immune responses are emerging. There is collective evidence from multiple studies and experimental approaches to support their disturbances in cases of intestinal inflammation. Unique alterations in the composition of bile acid pool are attributed to dysbiosis associated with IBD. Yet, the mechanisms by which bile acids contribute to the pathophysiology and/or the severity of intestinal inflammation are not fully understood. The complex nature of bile acid homeostasis and presence of several receptors expressed in different cell types that could be activated by bile acids make it challenging to delineate the causal link between bile acids and the inflammatory response in IBD. Future research in this field should leverage advanced bioinformatic and systems biology approaches to gain novel insights about their potential roles in the pathophysiology of IBD. Also, the profound differences in the composition of bile acid pool between humans and rodents raise serious concerns about the extent to which data obtained from these models could be extrapolated to humans. The generation of transgenic mouse models with bile acid pool similar to that of humans should offer excellent preclinical models to facilitate new discoveries related to bile acids and IBD. Further, better understanding of how gut microbiota shapes the response to a changing pool of bile acids remains a major interest of investigations. Since data suggest that a single bacterial modification of bile acids may rather involve communities of different bacteria from distinct species, future studies should focus on investigating the changes in specific microbial consortia in IBD and their effects on bile acid homeostasis. Additionally, investigating the novel roles of the newly discovered microbially conjugated bile acids will likely yield to novel findings that could further explain the potential roles of gut microbiota/bile acids axis in the development of intestinal inflammation. Overall, the increase in our knowledge about bile acids as inflammatory mediators will enhance our understanding of IBD and will define novel targets for improved therapeutic modalities for treatment of these debilitating gut disorders.
Author contributions
NC and SC performed initial literature screening. NC, SC, RKG, and WAA finalized the first draft. NC created tables and Figure. NC, SC, PKD, SS, RKG and WAA edited and approved final version. All authors contributed to the article and approved the submitted version. | 2022-12-08T17:55:35.972Z | 2022-12-07T00:00:00.000 | {
"year": 2022,
"sha1": "640c1b9693092c5c2a22cc5a39661381e59c140c",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Frontier",
"pdf_hash": "640c1b9693092c5c2a22cc5a39661381e59c140c",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": []
} |
15361758 | pes2o/s2orc | v3-fos-license | Clinical Ophthalmology Dovepress Dovepress Adjunctive Therapy Patterns in Glaucoma Patients Using Prostaglandin Analogs
Purpose: To analyze patterns of use of adjunctive therapies among new initiators of topical prostaglandin analogs (PGAs) in a managed care population. Methods: The study cohort included patients in a claims database who initiated PGA therapy between June 2007 and April 2011. Patients who had one or more adjunctive therapy prescriptions during 24 months of follow-up were included. Patterns of adjunctive therapy use were identified and compared between patients who had one or two fills of the initial adjunctive therapy and those who had three or more. Results: There were 16,486 eligible beneficiaries. Of these, 5,933 (36%) had one or more adjunctive therapies within 24 months from the start of the PGA, 82% of whom started adjunc-tive therapy within 12 months. About 28% of patients started adjunctive therapy with a fixed-combination product; 45% of these patients started within the first 30 days. Overall, a large number of patients (42%) required adjunctive therapy within 30 days. Twenty-five percent of patients had only one or two prescriptions of their initial adjunctive therapy; of these patients, 74% discontinued adjunctive therapy altogether. Conclusion: Approximately 30% of patients starting glaucoma therapy will require adjunc-tive therapy within 1 year, and many receive a fixed-combination product as initial adjunctive therapy shortly after starting glaucoma therapy. This suggests a prescribing trend toward earlier, more aggressive drug therapy to control pressure and minimize disease progression. We found that compliance with adjunctive therapy continues to be a problem for patients, which could be attributed to a number of treatment burden and economic factors.
Introduction
The benefits of achieving a low target intraocular pressure (IOP) in primary open-angle glaucoma (POAG) and ocular hypertension are well recognized. When acceptable IOP levels are not achieved with a single drug, adjunctive therapy with complementary mechanisms of action is recommended. The Ocular Hypertension Treatment Study (OHTS) found that, in eyes without significant optic nerve damage and at risk of developing glaucoma, more than 40% of patients needed additional medication within 5 years after starting treatment to reach a modest reduction in IOP of 20% or more long-term. 1 As many as 75% of patients with POAG may require adjunctive therapy within 24 months of starting glaucoma therapy in order to achieve the target pressure. 2 The Advanced Glaucoma Intervention Study (AGIS) found that, in patients with glaucoma, the number of patients requiring more than one drug might be as high as 80% when the disease is more severe. 3 The introduction of adjunctive therapies to a treatment regimen multiplies the negative influences on adherence (ie, complexity of regimen, likelihood of adverse events, increased preservative load). 4 Initial therapy for POAG and ocular hypertension is typically with a prostaglandin analog in the US. Outside the US, a beta-blocker is still frequently used as first-line therapy due to cost. 12 When a prostaglandin analog is insufficient to control pressure, there are many types of adjunctive therapies to add, including selective and nonselective beta-blockers, alpha-agonists, and carbonic anhydrase inhibitors (CAIs), as well as lesser-used therapies, such as miotics. There are also fixed combinations available that include a nonselective beta-blocker when more than one adjunctive therapy is needed to control IOP. A fixed combination of an alphaagonist and CAI is also now available for patients with a contraindication to a nonselective beta-blocker.
The use of a fixed-combination drug provides the opportunity to maximize pressure reduction by combining two complementary mechanisms of action in one bottle while minimizing the complexity of the treatment regimen. There is published evidence that multiple drops can be challenging to administer without assistance and there is also the increased risk of contamination from patients inadvertently touching multiple bottles to the eye. [13][14][15] Additionally, as topical medications are applied frequently and over a long period of time, preservative toxicity to the ocular surface becomes an important factor for a chronic disease such as glaucoma. [16][17][18] Previous studies suggest that the use of fixed-combination products for control of IOP has increased significantly over the past decade. [7][8][9] This current study adds to this body of evidence by looking at more recent trends in glaucoma prescription use and, in particular, at the use of fixed-combination products in the treatment regimen.
Methods
The patients included in this retrospective cohort analysis were receiving medical and prescription benefits and were included in the 2007-2012 MarketScan ® Commercial Claims and Encounters and Medicare Supplemental databases. The Commercial Claims and Encounters database covers more than 300 million lives and includes 12 health plans and approximately 100 employers and includes both employees and dependents (personal communication February 2013). The Medicare Supplemental and Coordination of Benefits database covers more than 2.5 million lives and includes patients 65 years and older with employer-paid or Medicare Part C coverage. Only services covered 100% by Medicare Part A or B are excluded. All data were de-identified in accordance with Protected Health Information standards under the Health Information Portability and Accountability Act so that no individually identifiable information was included in the study database. Therefore, review by an institutional review board was not required.
The study cohort included patients who first initiated therapy with one of three ophthalmic prostaglandin analogs (bimatoprost, latanoprost, or benzalkonium chloride (BAK)-free travoprost) between April 1, 2007 and April 30, 2011. To qualify, patients had to have 4 months of prior claims data in which there were no glaucoma therapy claims of any class and no claims for POAG (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] code 365.11). 19 The index date was defined as the first date on which a prostaglandin analog was filled. Glaucoma therapy claims were defined unique product identifiers for human drugs required as part of the Food and Drug Administration's listing process. Patients with two prostaglandin scripts, whether the same or different medications, on the index date were excluded, as it is unclear whether these were errors or drug trials. The study population was further defined by requiring patients to have at least 24 months of use of prostaglandin analogs following the index date (initial prostaglandin fill). This was operationalized as having at least one prescription for an ophthalmic prostaglandin analog during months 20-24 of the follow-up period; this could be the same medication as at the index date or a different prostaglandin. Patients were also required to be enrolled for 12 months following the first prescription for an adjunctive therapy. In some cases, where patients began adjunctive therapy more than 12 months after the index date, this extended beyond the original 24 months of continuous enrollment required; these patients were required to have additional follow-up beyond the 24 months. Patients with two adjunctive therapy prescriptions on their first date of adjunctive therapy were also excluded, as it was unclear how to classify these patients. Patients who did not meet these requirements (sufficient prior claims data, repeat use of a prostaglandin, use of one or more adjunctive therapies, sufficient follow-up after initiating adjunctive therapy) were excluded from the study database. Adjunctive medications were described by type (fixed combinations, beta-blockers, alpha-agonists, CAIs, plus other).
Participants were characterized in terms of available demographic characteristics (age and sex). Given previous evidence of patterns of discontinuation with nonselective beta-blockers, 20 patients were categorized by whether their initial adjunctive therapy was a nonselective beta-blocker (as part of a fixed combination or as a single agent) or another treatment (alpha-agonist, CAI, or other). Further, patients were classified based on whether they exhibited short-term use of the initial adjunctive therapy, defined as one or two prescriptions filled versus three or more. The adjunctive therapies, if any, following discontinuation of the first adjunctive agent were identified. Mean number of days to initiation of adjunctive therapy was calculated for each cohort; the proportion of patients with an initial adjunctive therapy in prespecified time periods was also calculated. The annual rate of surgical procedures for glaucoma (identified by Current Procedural Terminology (CPT ® ) codes 68566, 66170, 66172, 66180, 66185, 66625, 66630, 66710, 66711, 66761, and 66762) was also calculated. Statistical comparisons were conducted using Stata software (v 12.1; StataCorp LP, College Station, TX, USA).
Results
To provide context, existing studies on adjunctive therapy use were consulted. A review of the literature 7-9 demonstrates a trend toward increased use of fixed-combination therapies as the first-line adjunctive therapy over time, with a corresponding decrease in the use of most single-agent drugs. These previously published studies used the same inclusion criteria and identified patients with a prescription for a topical prostaglandin analog who had no glaucoma therapy claims in the 6 months prior and had at least 12 months of data available after the initial prostaglandin analog claim. Thus, these analyses are comparable to each other and the present study, as shown in Figure 1. Figure 2 shows the patient identification process for this analysis. In the present analysis, there were 16,486 beneficiaries with at least one prostaglandin analog prescription who met all requirements for eligibility. Table 1 shows that 5,933 (36%) had one or more pre-specified adjunctive therapies within 24 months from the start of the prostaglandin analog and had 12 months of follow-up available after the start of adjunctive therapy. For two-thirds of these patients (n=3,927), their initial adjunctive therapy was a nonselective beta-blocker, either alone (n=2,277) or as part of a fixed combination (n=1,650). The average age in all groups was just less than 70 years, and 51% of the patients were women.
Among patients who required adjunctive therapy, 25% had only one or two prescriptions of their initial adjunctive therapy. Of these patients, 74% did not have additional prescriptions for adjunctive therapies during the observation year, ie, for 12 months following the start of their adjunctive therapy (see Table 2). This ranged from 69% of patients with one or two prescriptions for an alpha-agonist to 82% of patients starting with a fixed-combination beta-blocker. Two of the largest shifts observed were patients who had one or two prescriptions for a fixed-combination beta-blocker switching to a single-agent beta-blocker, and vice versa (48.1% and 62.7%, respectively).
Among patients who had three or more prescriptions of their initial adjunctive therapy (75%), the vast majority (overall 93%, ranging from 89%-94% of patients by adjunctive therapy) continued long-term use of their initial therapy. For the 6%-11% who had a different adjunctive therapy for the fourth prescription, there was a wide range of agents used, with no clear pattern to suggest what was used next.
Time to initiating adjunctive therapies is shown in Table 3. Time to first adjunctive therapy was highest for patients starting adjunctive therapy with CAIs (mean 190 days) and lowest for those starting with an "other" medication (mean 63 days). However, the standard deviation was high for all groups. These are further broken down into categories that represent both data-driven and clinically meaningful time points. First, the proportions of patients who had a prescription for an adjunctive therapy on the same day (day 0) as their index prostaglandin analog prescription are shown. This varies from 25% for alpha-agonists to 44% for other medications. Across all therapies, this represents 30% of the patients identified in the study. The rate of starting adjunctive therapies on days 1-30 is much lower, at 12%, and the total for the first month is a notable 42%. The next category captures adjunctive therapies filled in months 2-6 after the index prostaglandin analog and includes 28% of adjunctive therapy start dates. The remaining categories for initiating adjunctive therapy are 7-12 months and greater than 12 months, and ranged from 12%-18% of adjunctive therapy start dates. The rate of surgical procedures for glaucoma, not shown here, was extremely low (ie, less than 0.001 annually per person during the observation period). Thus, the rate of surgeries was not a significant factor in this study, nor were there enough observations to compare across adjunctive therapy groups.
Discussion
In this analysis, treatment patterns were examined for patients based on their first adjunctive therapy initiated within 24 months after receiving an initial prescription for a prostaglandin analog for glaucoma. As shown in Figure 1, fixed combinations are being increasingly used as the first-line adjunctive therapy, in nearly 30% of glaucoma cases. This suggests a trend by providers toward earlier, more aggressive use of drug therapy to control pressure and prevent disease progression. Additionally, close to one-third of all adjunctive therapy prescriptions were filled on the same date as their first prostaglandin analog prescription. Same-day intervention of a fixed combination with a prostaglandin analog would be indicative of later-stage disease diagnosis as the patient's first diagnosis. The decision to provide a fixed-combination therapy initially versus an unfixed combination has been explored, with a recent meta-analysis suggesting lower rates of hyperemia with fixed combinations compared to either unfixed combinations or prostaglandin analog monotherapy. 21 Visual-field loss may already be present and be the reason for the patient seeking diagnostic services and treatment from a provider. Interestingly, the rate of surgical interventions was very low. We did not compare rate of surgical procedures by time to initiating adjunctive therapy; had the counts been higher, we would have considered such an analysis to identify whether starting adjunctive therapy earlier was indicative of more advanced disease.
1101
Patterns of adjunctive therapy use In Table 2, we show that 25% of the patients had only one or two fills of their initial adjunctive therapy and often discontinued therapy altogether; only 26% of these patients continued therapy with a different agent. Of note, in the group of patients with only one or two fills of their initial adjunctive prescription, 76% of those patients that started with a nonselective beta-blocker (either single agent or fixed combination) discontinued all adjunctive therapy. Use of a nonselective beta-blocker is contraindicated for patients with a diagnosis of cardiovascular and/or respiratory disease. [22][23][24][25] We found that 15% of the patients in the present study had a diagnosis for cardiovascular disease and 10% had a diagnosis for respiratory disease, but this could not be attributed directly to their discontinuation of adjunctive therapy.
Although discontinuation of nonselective beta-blockers did not appear to be linked to cardiovascular or respiratory care, patients could have described subclinical conditions to their ophthalmologists, which might have altered treatment patterns and possibly have led someone to switch from a fixed-combination or single-agent nonselective beta-blocker to adjunctive therapy without a nonselective beta-blocker. Still, the high rate of discontinuation of the adjunctive therapy rather than a switch to a different therapy cannot be explained given that these patients continued to use their primary prostaglandin analog therapy.
As with any claims database analysis, there are certain limitations that must be acknowledged. The use of a large claims database offers an important strength in terms of sample size but shortcomings remain. We cannot tell to what extent 'days to supply' guidelines, which may differ across patients and are not documented in the database, may influence refill patterns. Also, the patient population in any given database may not match the general population of patients with the condition in question, although, in this analysis, the age and sex of the population reflect that of the general glaucoma population. 26 Other characteristics, such as race, educational status, central corneal thickness, or self-reported health status, among others, are unknown. Although glaucoma is typically bilateral, the American Academy of Ophthalmology's guidelines suggest that eyes may respond to different medications. 27 Therefore, we cannot rule out the possibility that what we identify as adjunctive therapy might not, instead be a different treatment for the fellow eye. The population of prescribers may not accurately reflect national patterns, which could affect generalizability. Most claims databases have little or no data on the characteristics of prescribers, but as the data were from a large managed care organization, it is likely that there is a wide distribution of prescribers and practices. By their nature, claims databases only provide information on people with some type of insurance coverage, and those with coverage may be more likely to seek out and adhere to treatment. Finally, claims databases may contain coding biases or errors, although there is no reason to believe that these errors would be different across treatments; in addition, the claims in this database were reviewed and adjudicated before the database was prepared for this analysis.
The methodology for patient selection in this study is also subject to certain limitations. The study attempted to identify only patients who were new to therapy by requiring 4 months of glaucoma-free claims prior to the first prostaglandin analog prescription. However, there is no way to account for product samples, which one study identified as being received by 20% of patients, 6 and which could translate into patients who were already taking prostaglandin analogs before our study identified them or continuing on adjunctive therapies while appearing not to have refilled them. However, since we also required that patients had no glaucoma diagnosis in the four months prior to the index date, the likelihood of them having received glaucoma therapies prior to their index date is low. Also, based on this study methodology, patients could have had poor adherence but still have been included; patients in the cohort analysis were required to have a minimum of two prostaglandin analog prescriptions filled during the 24-month follow-up period, but no attempt was made to assess medication possession ratio. Interestingly, research suggests that patients who use both an adjunctive therapy and a prostaglandin analog may be more compliant than those without adjunctive therapy, although compliance at 12 months was still only about 60% in the relevant study. 28 However, other studies report that dosing errors are more common with adjunctive therapy 29 and that time between refills increases when adjunctive therapy is initiated. 30 This leaves the question as to why so many patients with glaucoma maintain prostaglandin use but discontinue adjunctive therapies when there is no obvious evidence of adverse events or surgical intervention contributing to the answer. Many studies [31][32][33][34][35][36][37] have evaluated compliance with glaucoma medications, which consistently report, much like in other therapeutic areas, that compliance decreases as regimens become more complicated. Patients may decide that their adjunctive therapies are not important enough to use regularly and physicians may not realize that patients are not using them as prescribed. Future studies that include analysis of a full electronic medical record so that physician notes can be reviewed may answer some of these questions.
Conclusion
This analysis of adjunctive therapy use patterns in POAG patients shows that these drugs are being prescribed early in the timeline of the patient's treatment. In light of trends across
1103
Patterns of adjunctive therapy use multiple studies, this suggests a prescribing trend towards earlier, more aggressive drug therapy for POAG. Related topics, such as the high rate of discontinuation from adjunctive therapies, could be explored through further analysis given additional robust data. | 2017-06-20T00:08:33.022Z | 0001-01-01T00:00:00.000 | {
"year": 2014,
"sha1": "7e42700bf6c9c7e46f6138e5eb881f71a5ab965a",
"oa_license": "CCBYNC",
"oa_url": "https://www.dovepress.com/getfile.php?fileID=20378",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "7e42700bf6c9c7e46f6138e5eb881f71a5ab965a",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
24155210 | pes2o/s2orc | v3-fos-license | SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington’s Disease Mice
Huntington’s disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies.
Introduction
Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin gene (HTT), which encodes for an expanded polyglutamine (polyQ) tract in the huntingtin protein (HTT) [1]. Symptoms usually appear in mid-life, comprise personality changes, problems with motor coordination and cognitive decline; disease duration lasts between 15 and 20 years and there are no disease-modifying treatments [2]. The neuropathology of HD is characterised by neuronal cell death in the striatum, cortex and other brain regions and the accumulation of cytoplasmic and nuclear aggregates [3].
Mouse models of HD include those that are transgenic for N-terminal fragments of HTT (e.g. R6/2) or the full length HTT protein or are knock-in models in which the HD mutation has been introduced into mouse Htt (e.g. HdhQ150) [4]. The R6/2 mouse is transgenic for an exon 1 HTT protein [5] and is a model of the aberrant splicing that occurs in HD [6]. The HdhQ150 model had a 150 CAG repeat knocked into the mouse Htt gene [7]. In addition to the full length protein, HdhQ150 mice express mutant exon 1 HTT through aberrant splicing [6] and many other N-terminal HTT fragments generated through proteolysis [8]. At late stage disease (14 weeks for R6/2 and 22 months for homozygous HdhQ150 mice) these models exhibit remarkably similar phenotypes [9][10][11][12][13][14] the main difference between these two models being the age of disease onset and rate of disease progression. SIRT1, a mammalian orthologue of the yeast Sir2 protein, is a NAD + -dependent deacetylase that plays a critical role in multiple biological processes including apoptosis [15], ageing [16], metabolism [17] and various stress responses [18]. It has been demonstrated that DBC1 (deleted in breast cancer 1) inhibits SIRT1 via a direct interaction with its catalytic domain [19]. This dynamic interaction is sensitive to the energetic state of the cell and involves the activity of AMPK (AMP-activated protein kinase), an important cellular energy sensor [20]. In circumstances of low cellular energy, AMPK stimulates compensatory processes, including the activation of SIRT1, resulting in the restoration of ATP levels [21]. However, the complexity of SIRT1 functions in the mammalian brain and the mechanisms involved in SIRT1 regulation are not fully understood.
SIRT1 has been shown to participate in neuronal protection and survival in various mouse models of neurodegenerative disorders through a number of substrates such as P53 [22] and HSF1 [23]. With relevance to HD, the activation of Sir2 was protective against mutant phenotypes in a C. elegans model [24]. Increased expression of Sirt1 attenuated neurodegeneration and improved motor function in N171-82Q and BACHD mice [25] and attenuated brain atrophy and reduced mutant HTT aggregation in R6/2 mice without prolonging lifespan [26]. More recently, SRT2104, a SIRT1 activator was reported to have beneficial effects in an HD mouse model [27] with the potential for interrogating SIRT1 activity in the clinic [28]. In contrast, a SIRT1 inhibitor, selisistat, has been reported to alleviate HD-related phenotypes in multiple HD models [29] and has been found to be safe in clinical trials [30]. Based on these findings, the mis-regulation of SIRT1 could have important implications in the development and progression of HD.
In this study we describe a striatum-specific phosphorylation-dependent regulatory mechanism that controls SIRT1 activity under normal physiological conditions that is impaired in HD. We show that SIRT1 activity is decreased in the brains of R6/2 and HdhQ150 mice, and that this is not caused by the sequestration of SIRT1 into HTT inclusions. We demonstrate that the presence of mutant HTT in the striatum and cerebellum of HD mice alters the phosphorylation status of SIRT1 and that these effects are related to the abnormal expression and cellular localization of AMPK-α1. Finally, we show that the ablation of DBC1, a negative regulator of SIRT1 [31] does not rescue the deficit in SIRT1 activity in HD mouse models. These results provide new insights into the mechanisms that regulate SIRT1 function and may lead to the development of new strategies by which SIRT1 can be manipulated for therapeutic benefit.
(CBA × C57BL/6) F1 background were generated by intercrossing Hdh Q150/Q7 heterozygous CBA/Ca and C57BL/6J congenic lines (inbred lines from Harlan Olac). R6/2 and Hdh Q150/Q150 homozygous mice were genotyped and the CAG repeat was sized as previously described [32]. The mean repeat size (± SD) for all mice used in the entire study was 165 ± 10 for Hdh Q150/Q150 homozygous mice and 204 ± 7 for R6/2 mice. Dbc1 heterozygous mice were obtained from the Eduardo Chini at the Mayo Foundation, Mayo Clinic College of Medicine, Rochester, Minnesota, USA. PCR conditions for genotyping Dbc1 knock-out mice have been previously described [19]. SirT1 floxed homozygous (SirT1 Fl/Fl) mice were obtained from the JAX Laboratory (Mouse Strain: B6;129-SirT1tm1Ygu/J) [33] and were bred with β-actin/Cre heterozygous mice to generate complete Sirt1KO mice. Sirt1 transgenic mice (CBA×C57BL/6J) [34] were obtained from David Holzman's laboratory at Washington University, Missouri, USA Animals were housed under 12 h light/12 h dark cycle, with unlimited access to water and food (Special Diet Service, Witham, UK) in a conventional Unit. Cages were environmentally enriched with a cardboard tube. R6/2 mice and all mice in phenotypic assessment trials were always given mash food consisting of powered chow mixed with water from 12 weeks of age until sacrificed. Upon sacrifice, dissected brain regions, whole brains or peripheral tissues were snap frozen in liquid nitrogen and stored at -80°C until use.
Mouse behavioural analysis
At 4 weeks of age, mice were weaned into cages of 5-6 animals. Each cage contained at least one representative of each genotype from mixed litters. The analysis of mice of different genotypes was distributed equally throughout the assessment period on any given day and all behavioural tests were performed blind to the investigator. Mice were weighed weekly and rotarod performance and grip strength were assessed as previously reported [35][36][37]. The statistical power of these tests was calculated as previously described [37]. The data were analysed by repeated measures general linear model ANOVA using SPSS software [37].
Protein extraction for SDS PAGE, Immunoblotting and Immunoprecipitation
Frozen mouse brain tissue was homogenized in 1 volume of ice cold NETN buffer (20 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, complete protease inhibitors and phosphatase inhibitors) using a polytron homogenizing probe. Samples were sonicated on ice with a vibracell sonicator (10 x 1 s 20 kHz pulses) and spun at 13,000 x g for 10 min at 4°C. The supernatant was retained and protein concentration was determined for each sample by the BCA assay (Thermo Scientific).
SDS PAGE and Immunoblotting
Protein lysates were diluted with 2x Leammli Buffer, denatured for 10 min at 95°C, loaded onto SDS polyacrylamide gels and subjected to western blot as previously described [8]. Membranes were blocked in 5% non-fat dried milk in PBS-0.2% Tween 20 (PBS-T) or 4% BSA for 2 h at RT. Primary antibodies were added overnight at 4°C in 5% non-fat dried milk in PBS-T (DBC1, SIRT1, HTT, AMPK-α1,) or 4% BSA (MpM2). β-actin, ATP5B, α-tubulin and histone pan-H3 were incubated for 20 min at RT in 5% non-fat dried milk in PBS-T. Blots were washed three times for 10 min in 0.2% PBS-T, incubated with the appropriate secondary antibody for 1 h at RT, washed three times for 10 min in 0.2% PBS-T and exposed to ECL according to manufacturer's instruction (Amersham). The signal was developed using Amersham hyperfilm and Xenograph developer. Densitometry of western blots was performed using a Bio-Rad GS-800 densitometer. Developed films were scanned and the average pixel optical density (OD) for each band was measured using QuantityONE software. The OD of an area devoid of bands was subtracted from the values obtained for bands of interest in order to normalize the OD against background. Relative expression was determined by dividing the normalized OD of bands of interest by the OD of the appropriate loading control for each sample. For full details of primary antibodies see S1 Table. Immunoprecipitation Protein lysates were prepared for immunoprecipitation (IP) as described above. For IP from striatal lysates, striata were pooled from two animals. IP reactions were performed in 1 ml of NETN buffer containing from 400 to 1000 μg protein and 1 μg of antibody and normal rabbit IgG (#2729; Cell Signaling) was used as a negative control. Reactions were left on a rotating wheel at 4°C for 90 min (AMPK-α1) or 4 h (SIRT1) and 15 μl of protein G-coupled Dynabeads (10004D; Life Technologies) were added for the last 45 min. Following IP, protein G-coupled Dynabeads were briefly spun at 13,000 x g for 30 sec, put on a magnetic rack, washed with 1 ml of NETN buffer (4x) and re-suspended in 15 μl of 2x Leammli buffer. Immuno-precipitated complexes were eluted from the beads by denaturation at 100°C for 10 min and immediately loaded for SDS-PAGE analysis.
Nuclear/cytoplasmic fractionation
All steps were performed on ice. Half brain tissue or liver was cut into small pieces and homogenized with a Dounce homogenizer in TKM buffer (0.25 M sucrose; 50 mM Tris-HCl, pH 7.4; 25 mM KCl; 5 mM MgCl2 and 1 mM PMSF) and nuclear and cytoplasmic fractions were prepared as previously described [19]. For the nuclear and cytoplasmic preparations from brain regions, striata were pooled from four animals, whereas a single cerebellum was used. The final pellet containing the purified nuclei was resuspended in 4% PFA for immunohistochemistry or in NETN buffer for protein analysis and protein concentration was determined by the BCA assay (Thermo Scientific).
Immunohistochemistry
The isolation of nuclei from brain or liver was as described above. Nuclei were extracted from 4 mice per genotype from half brain or liver and for brain regions, from two pools each containing specific regions from five mice. Samples were fixed on the slide for 30 min with 4% paraformaldehyde prepared in PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed 3X with PBS, and incubated for 1 h at RT in blocking buffer (PBS with 0.1% Triton and 1% BSA). Nuclei were incubated with the primary antibody in blocking buffer (DBC1, SIRT1, P53 and Ac-P53) overnight at 4°C, washed 3x with PBS at RT and then incubated with the secondary antibody and DAPI in PBS-0.1% Triton for 1 h at RT. Samples were mounted using VECTA-SHIELD mounting medium. Nuclei were visualized using a TCS SP2 Leica confocal microscope. Fluorescence intensity was quantified from 50 nuclei per sample imaged from 10 fields of view per slide using ImageJ. Ac-P53 levels were normalised to the P53 intensity level. Fluorescent intensity levels were presented as a fold change from WT levels as indicated in the figures. The direction of the fold change was inverted to depict the comparative deacetylase activity.
Fluor de Lys assay SIRT1 activity was determined with a SIRT1 Fluorometric Kit (BML-AK555) according to the manufacturer's instructions. Protein extraction was performed as described above. Homogenates were then incubated for 10 min at 37°C to allow degradation of any contaminant NAD + . 10 mM DTT was added to the medium, and homogenates were incubated again for 10 min at 37°C. The homogenates (20-30 μg protein/well) were then incubated in SIRT1 assay buffer in the presence of 50, 100 or 200 μM Fluor de Lys-SIRT1 substrate (Enzo Life Sciences), 5 μM TSA and 200 μM NAD + . After 0-, 20-, 40-and 60 minutes of incubation at 37°C, the reaction was terminated by adding a solution containing Fluor de Lys Developer (Enzo Life Sciences) and 2 mM nicotinamide. After 1 h the values were determined by reading fluorescence on a fluorometric plate reader (Spectramax Gemini XPS; Molecular Devices) with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
Taqman RT-qPCR RNA extraction, cDNA sysnthesis, Taqman RT-qPCR and ΔCt analysis were performed as described previously [38]. The Taqman qPCR assays were purchased from Primer Design and ABI. For a list of primers and probes, see S2 Table. Statistical Analysis Statistical analysis was performed with SPSS (repeated measures ANOVA General Linear Model) or Microsoft Excel (Student's t-test) software. p-values of <0.05 were considered significant. Graphs were constructed using Prism Ver.5.0b (GraphPad Software).
SIRT1 function becomes compromised in the brains of HD mice
There is considerable evidence to support the beneficial effect of SIRT1 manipulation in HD mouse models. However, the impact of mutant HTT on SIRT1 function has not been fully elucidated. As such, we set out to analyse SIRT1 activity and the mechanisms involved in its regulation in two different mouse models of HD: R6/2 transgenic and HdhQ150 knock-in homozygous mice.
SIRT1 regulates the activity of several transcription factors including P53 [39]. It deacetylates P53 on Lys382 thereby inhibiting its function [40]. There are a number of commercial kits that use the deacetylation of this P53 lysine residue to assess SIRT 1 activity. In order to have a direct measurement of SIRT1 activity, we applied the Fluor-de-Lys fluorometric activity assay (Enzo Laboratories). The specificity of the kit was evaluated on lysates from the brains of SIRT1 knock-out (Sirt1KO) [33] mice at 4 weeks of age, but unfortunately we found that this kit was not specific for SIRT1 in these brain lysates (S1 Fig). Therefore we tested an alternative published method to assess the steady-state levels of SIRT1 activity on endogenous P53 in mouse brains that makes use of nuclei purified from mouse tissues [19]. The genotypes of the mice used for the experiment were verified by western blot (Fig 1A). Nuclei were isolated from the brains of Sirt1KO and Sirt1Tg mice [34] at 4 weeks of age and immunostained for P53 and acetylated-P53 (AcP53) at Lys382, and counterstained with DAPI ( Fig 1B). P53 levels were equivalent between the Sirt1KO and Sirt1Tg lines and the corresponding wild type (WT) littermates ( Fig 1C). The acetylation of P53 Lys 382 was considerably increased in the Sirt1KO nuclei and decreased in those from the Sirt1Tg mice, consistent with a decrease in SIRT1 activity in the knock-out line and an increase in SIRT1 activity in the transgenic line respectively (Fig 1C), demonstrating that this approach could be used to monitor the steady-state level of SIRT1 activity in mouse brain.
To monitor the level of SIRT1 activity in HD mouse models, we isolated cell nuclei from the brains of R6/2 mice at 4, 9 and 14 weeks of age and HdhQ150 homozygous mice at 2 and 22 months together with their aged-matched WT littermates. Nuclei were immunostained for SIRT1, P53 and acetylated-P53 (AcP53), and counterstained with DAPI (Fig 2A and S2A Fig). We did not detect any variation in the intensity level of SIRT1 and P53 staining between HD mouse samples and their corresponding WT controls at each age of analysis ( Fig 2B and S2B Fig). In contrast, whilst we found that the acetylation levels of endogenous P53 were equivalent in HD as compared to WT littermate brains in presymptomatic mice (i.e. 4 week R6/2 and 2 month HdhQ150 homozygotes) (Fig 2A and 2B), the level of AcP53 was significantly higher (! 1.5 fold) in samples from early symptomatic R6/2 mice (9 weeks) and late stage symptomatic R6/2 (14 weeks) and HdhQ150 homozygous (22 months) mice (
SIRT1 does not co-localize with mutant HTT inclusions and is aberrantly phosphorylated in HD mice
Previous studies have shown that SIRT1 interacts with HTT in vitro [26]. To investigate whether the altered SIRT1 activity is caused by the sequestration of SIRT1 into HTT inclusions, we performed a double staining for SIRT1 and HTT (EM48) on nuclei isolated from the brains of 14-week R6/2 and 22-month HdhQ150 homozygous mice, together with their age-matched WT littermates. Interestingly, SIRT1 did not co-localize with HTT inclusions (Fig 3A). To further support this finding, the levels of SIRT1 protein were not decreased in HD brains as judged by western blot (Fig 3B).
The role of post-translational modifications (PTMs) in the regulation of SIRT1 activity has been the subject of several studies and phosphorylation has been described as a major control SIRT1 Phosphorylation, Impaired Activity and Huntington's Disease mechanism [41]. Therefore, to understand how mutant HTT reduces SIRT1 activity, we monitored the phosphorylation status of SIRT1 in HD mice. We performed SIRT1 immunoprecipitation from the brains of R6/2 mice at 9 weeks of age and HdhQ150 homozygous mice at 22 months and probed the phosphorylation level of SIRT1 by western blot using the mitotic phosphoprotein monoclonal 2 (MpM2) antibody [42]. This antibody detects the phosphorylation of serine and threonine residues when they are followed by a proline (S/T-P sites) and it is not specific for a SIRT1 phosphorylation site (S3 Fig). Interestingly, a higher level of phosphorylated SIRT1 was found in the brains of both R6/2 and HdhQ150 homozygotes as compared to their WT littermates ( Fig 3C). As previously shown in vitro [26], we were able to co-immunoprecipitate endogenous HTT from R6/2 lysates and mutant HTT and WT HTT from HdhQ150 homozygous and WT lysates respectively (Fig 3C). These results suggest that the impairment in SIRT1 function in the brains of HD mice is not related to its sequestration into HTT inclusions, but rather to an alteration in its phosphorylation profile.
SIRT1 phosphorylation becomes decreased in the striatum and increased in the cerebellum of HD mice
The analysis of total brain samples might mask or dilute any regional pathological changes. Therefore, we extended the analysis of SIRT1 phosphorylation to the striatum, cortex and cerebellum of R6/2 mice at 4, 9, and 14 weeks of age. We did not detect any difference in the phosphorylation status of SIRT1 at presymptomatic stages of the disease (i.e. 4-week-old R6/2) as compared to WT littermates in any brain region (Fig 4A and 4C and S4 Fig). In keeping with our functional data from total brain, the levels of phosphorylated SIRT1 were altered in the striatum and cerebellum of R6/2 mice by 9 weeks of age (Fig 4A and 4C). Surprisingly, the level of phosphorylation of SIRT1 remained unchanged in the R6/2 cortex at these later stages (S4 Fig), but notably was decreased in the striatum ( Fig 4A) and increased in the cerebellum ( Fig 4C) as compared to WT littermates. These data were replicated in the HdhQ150 homozygous mice: there was no difference in the SIRT1 phosphorylation level at 2 months of age (Fig 4B and 4D) whereas SIRT1 phosphorylation was decreased in the striatum and increased in the cerebellum of 22-month-old HdhQ150 homozygous mice (Fig 4B and 4D). Taken together, these results demonstrate that the presence of mutant HTT alters the phosphorylation status of SIRT1 in opposing directions for the striatum and cerebellum as the disease progresses.
Induction of SIRT1 activity is blocked in the striatum
Phosphorylation plays a central role in controlling protein activity, cellular localization and degradation [43]. To determine whether the differentially altered phosphorylation profile of SIRT1 in striatum and cerebellum corresponded to a compromised SIRT1 function in these brain regions, we immunostained for SIRT1, P53 and AcP53 in nuclei from the striatum and cerebellum of R6/2 and WT mice at 4, 9 and 14 weeks of age. Consistent with the total brain data, we did not detect a change in the intensity level of SIRT1 and P53 staining in either the striatum or cerebellum of R6/2 and WT mice, at any of the ages studied (S5A and S5B, S6A and S6B Figs). Interestingly, we observed a significant reduction in the level of AcP53 in the striatum of WT mice, corresponding to an increase in SIRT1 activity, between 4 and 9 weeks of age, which was absent in the striatum of R6/2 mice (Fig 5A and 5B). In contrast, when we analysed SIRT1 activity in the cerebellum, we detected no change in the level of AcP53 in WT samples at these ages and there was a significant increase in AcP53 in the cerebellum of R6/2 mice from 4 to 14 weeks of age (Fig 5C and 5D), corresponding to an impairment in SIRT1 activity. These data highlight that SIRT1 activity is regulated by different mechanisms in the striatum and cerebellum of WT mice between 4 and 14 weeks of age; SIRT1 activity is induced in the striatum between 4 and 9 weeks, whereas it remains constant in the cerebellum. The presence of mutant HTT can block this induction process in the striatum and cause a reduction in normal SIRT1 function in the cerebellum resulting in an impairment of SIRT1 activity in both brain regions (Fig 5).
SIRT1 induction in the striatum correlates with age-dependent phosphorylation
The comparison of SIRT1 activity in the striatum and cerebellum revealed that SIRT1 function is controlled by different mechanisms in these two brain regions in WT mice. In the striatum, SIRT1 is activated with age, a process that does not occur in the cerebellum. To monitor changes in the phosphorylation status of SIRT1 under normal physiological conditions we immunoprecipitated SIRT1 Phosphorylation, Impaired Activity and Huntington's Disease SIRT1 from striatal and cerebellar lysates of WT mice at 4, 9 and 14 weeks and immunoprobed with the MpM2 antibody. Notably, SIRT1 phosphorylation levels decreased in the striatum between 4 and 9 weeks of age (Fig 6A), a time at which the functional data revealed an increase in SIRT1 activity (Fig 5A and 5B). However, it then dramatically increased at 14 weeks (Fig 6A), a stage at which SIRT1 activity remains constant as compared to 9 weeks (Fig 5A and 5B). The MpM2 antibody detects phosphorylation on serine and threonine residues followed by proline (S/ T-P sites) and is not specific for a SIRT1 phosphorylation site; therefore, the increased phosphorylation signal at 14 weeks may correspond to the phosphorylation of different SIRT1 residues to those detected a 4 and 9 weeks of age. Conversely, SIRT1 activity remains constant during these ages in the cerebellum (Fig 5C and 5D) and this is reflected by a phosphorylation level that does not change (Fig 6B). Taken together these data provide a link between the phosphorylation status of SIRT1 and its function, suggesting that in the striatum changes in the SIRT1 phosphorylation with age might be related to the induction of SIRT1 activity.
The sub-cellular distribution of SIRT1 is not altered in R6/2 mice
Previous studies suggested that the phosphorylation of human SIRT1 can increase its nuclear localization and enzymatic activity [44]. To assess whether the mis-regulation of SIRT1 phosphorylation could affect its nuclear localization we prepared nuclear and cytoplasmic fractions from the striatum and cerebellum of R6/2 and WT mice at 9 and 14 weeks of age. Notably, we did not detect any difference in the distribution of SIRT1 at these ages between R6/2 and WT mice in either brain region (Fig 7A and 7B). However, the level of SIRT1 in the nuclear fraction was more pronounced at 14 weeks as compared to 9 weeks of age in the striatum and cerebellum of both R6/2 and WT mice (Fig 7A and 7B). We went on to analyse the phosphorylation level of SIRT1 in these two cellular compartments from the cerebellum by immunoprecipitation. This was not possible from the striatum due to limiting quantities of the extracts. Interestingly, a strong phosphorylation signal was detected in the nuclear fraction, that was absent from the cytoplasm, for both R6/2 and WT samples and, as previously shown on total lysates, the level of phosphorylation was much higher in R6/2 as compared to WT mice ( Fig 7C). These results demonstrate that the sub-cellular distribution of SIRT1 is not affected by the presence of mutant HTT and suggests, once again, that the phosphorylation levels might be directly linked to the regulation of SIRT1 activity.
Tissue specific alteration of the subcellular distribution of AMPK-α1 with disease progression
Previous studies showed that DBC1 directly interacts with the catalytic domain of SIRT1 inhibiting its activity both in vitro and in vivo [19]. This dynamic interaction is sensitive to the energetic state of the cell [19]. Activation of AMP-activated protein kinase-α1 (AMPK-α1), an important energy sensor in circumstances of low cellular energy, was recently shown to induce the activation of SIRT1 through the dissociation of SIRT1 and DBC1 [20,45]. To identify the possible role of AMPK-α1 and DBC1 in the molecular phenotypes described so far, we decided to study the interaction between these two opposing modulators of SIRT1 using co-immunoprecipitation. We immunoprecipitated AMPK-α1 from the striatum and cerebellum of 9-week R6/2, 22-month HdhQ150 homozygous and WT littermates and detected the co-immunoprecipitated DBC1. Interestingly, we observed a stronger interaction between AMPK-α1 and DBC1 in the striatum of HD as compared to WT mice (Fig 8A), whereas equivalent amounts of DBC1 were co-immunoprecipitated with AMPK-α1 from cerebellar extracts of HD and WT samples (Fig 8B). These data suggest two possible scenarios: either the increased interaction of AMPK-α1 with the SIRT1-DBC1 complex might attempt to promote SIRT1 activation in the striatum of HD mice through the dissociation from DBC1, or the inability to induce SIRT1 in R6/2 mice might be due to an inhibitory retention of AMPK-α1 via DBC1.
To gain insight into the molecular events involved in this process we examined the cellular distribution of AMPK-α1 and DBC1 in the striatum and cerebellum of R6/2 and WT mice at 9 and 14 weeks of age. Consistent with the phenotypes described so far, the distributions AMPK-α1 and DBC1 were different in the striatum and cerebellum. Interestingly, using western blots of nuclear and cytoplasmic fractions, we were able to detect DBC1 in both cellular compartments and, although at 9 weeks of age DBC1 was slightly more abundant in the striatal cytoplasmic fraction, this balance was reverted by 14 weeks of age for both R6/2 and WT mice ( Fig 8C). In contrast cerebellar DBC1 remained constant between the two cellular compartments at 9 and 14 weeks of age for both R6/2 and WT mice (Fig 8D). We next monitored the distribution of AMPK-α1 by immunostaining nuclei isolated from the striata of R6/2 and WT mice at 4, 9 and 14 weeks of age. At 9 weeks, AMPK-α1 was present in the nuclei from the WT striatum, whereas it could not be detected in nuclei from the striatum of R6/2 mice until 14 weeks of age ( Fig 8E). Conversely, cerebellar extracts showed an early nuclear accumulation of AMPK-α1 in R6/2 at 9 weeks of age as compared to WT mice, where AMPK-α1 could only be detected in the nucleus at 14 weeks of age ( Fig 8F). These data were confirmed by western blot (Fig 8C and 8D). The nuclear accumulation of AMPK-α1 at 9 weeks of age in the striatum of WT mice, in conjunction with an induction of SIRT1 activity, might indeed support a role for this kinase in the activation of SIRT1. This mechanism appears to be compromised in R6/2 mice and in this case, AMPK-α1 does not reach the nucleus until 14 weeks. Therefore, the increased interaction between AMPK-α1 and DBC1 might result in the retention of AMPK-α1 in the cytoplasm inhibiting the activation of SIRT1, and/or attempting to rescue SIRT1 activity by preventing DBC1 from binding to SIRT1. Conversely, the early nuclear accumulation of AMPK-α1 in the cerebellum of R6/2 at 9 weeks of age as compared to WT mice with the concomitant alteration in SIRT1 function might be an attempt to increase impaired SIRT1 activity. Taken together these data suggest that the inhibition of SIRT1 function in the striatum of R6/2 might arise through an altered functionality of AMPK-α1 and that AMPK-α1 might be involved in rescuing a deficient SIRT1 function both in the striatum and cerebellum, although through different molecular mechanisms. SIRT1, AMPK-α1 and DBC1 act as partners in the same regulatory circuit to control SIRT1 activity in the striatum Our data suggest that AMPK-α1 may play an active role in attempting to rescue SIRT1 deficiency in both the striatum and cerebellum of R6/2 mice. To obtain further evidence for a regulatory circuit involving these three proteins, we went on to compare the expression levels of SIRT1, DBC1 and AMPK-α1 in the striatum and cerebellum of R6/2 at 4, 9 and 14 weeks of age and HdhQ150 homozygotes at 2 and 22 months as compared to their WT littermates. Strikingly, there was a synchronised, statistically significant down-regulation (35-40%) of all three genes at the mRNA level from 4 to 9 weeks of age in the striatum of WT mice (Fig 9A). We detected the same significant reduction in the striatum of R6/2 mice for Dbc1 and Ampk-α1, and there was a weak trend for Sirt1 (Fig 9A). Notably, the presence of this regulatory circuit in the cerebellum was not supported by the same co-ordinated changes in expression levels, although the expression level of Sirt1 was significantly higher in WT mice at 9 and 14 weeks as compared to 4 weeks of age (Fig 9D). These mRNA changes did not result in concomitant alterations in the levels of the SIRT1, DBC1, and AMPK-α1 proteins (Fig 9B, 9C, 9E and 9F). The mRNA changes in the striatum may be the result of a stabilisation of these proteins between 4 and 9 weeks of age. Indeed, the levels of all three proteins were equivalent at 2 and 22 months in the HdhQ150 homozygotes and WT mice (S7A and S7B Fig). Interestingly, we observed a significant upregulation of AMPK-α1 in the striatum of WT mice between 4 and 9 weeks occurring in conjunction with the increase in SIRT1 activity, neither of which took place in R6/2 mice (Fig 9B and 9C). The only change that occurred in the cerebellum was a reduction in the level of SIRT1 in both WT and R6/2 at 14 weeks of age (Fig 9E and 9F).
Dbc1 ablation does not improve HD-related phenotypes
Our data indicate that brain region specific dysregulated cellular processes result in a reduction in SIRT1 activity in the brains of HD mouse models. We hypothesis that this is related to the phosphorylation status of SIRT1 and that the AMPK-α1 kinase attempts to rescue SIRT1 function. As DBC1 is a negative regulator of SIRT1, and Dbc1 knock-out mice are viable and healthy [19], we elected to use a genetic approach to ablate DBC1 levels in R6/2 mice and investigate whether the dissociation between SIRT1 and DBC1 could increase SIRT1 activity and improve HD phenotypes. We crossed R6/2 transgenic mice with Dbc1 heterozygous knock-out mice (Dbc1 +/-) to obtain Dbc1 +/-::R6/2 males that were then crossed with Dbc1 +/females to generate WT, Dbc1 +/-, Dbc1 -/-, R6/2, Dbc1 +/-::R6/2 and Dbc1 -/-::R6/2 mice. As predicted, genetic ablation of Dbc1 resulted in a significant decrease in Dbc1 mRNA and DBC1 protein levels, and we found that this did not alter the expression of SIRT1 (S8A Fig). To confirm that the removal of DBC1 resulted in an increase in SIRT1 activity, we immunostained nuclei extracted from the brains of WT, Dbc1 -/-, R6/2 and Dbc1 -/-::R6/2 mice at 9 weeks of age for SIRT1, P53, AcP53 and DBC1 and counterstained with DAPI. The absence of DBC1 did not affect the level and nuclear accumulation of SIRT1 and/or P53 (Fig 10A and 10B). As expected, the ablation of DBC1 in WT mice resulted in an increase in SIRT1 activity as indicated by a significant reduction (~65%) in the signal intensity for AcP53 in Dbc1 -/as compared to WT mice (Fig 10B). SIRT1 activity was decreased in R6/2 mice (consistent with Fig 2) and surprisingly the absence of DBC1 did not ameliorate this impairment (Fig 10). In line with these results, we did not detect improvements in the onset and progression of specific behavioural HD-related phenotypes such as body weight, grip strength and rotarod impairment (S8B, S8C and S8D Fig). Taken together these results suggest that the negative effect of mutant HTT on SIRT1 activity might be multifactorial and/or operate outside the inhibitory circuit controlled by DBC1.
Discussion
The involvement of SIRT1 in lifespan extension and cellular protection from aggregationprone proteins http://jcb.rupress.org/content/190/5/719.full-ref-91 has made it a promising therapeutic target for neurodegenerative disorders [46][47][48]. In the context of HD, the manipulation of SIRT1 activity has not generated results that are easy to interpret. On the one hand, over expression of SIRT1 has been shown to reduce mutant HTT-induced toxicity in HD mouse models, improving motor function and reducing brain atrophy [25,26]. In contrast to this, the pharmacological inhibition of SIRT1 has been shown to have beneficial effects in drosophila and mouse models of HD [29] and on the basis of these results, selisistat was assessed for safety and tolerability in a clinical trial aimed at the development of HD pharmacodynamic biomarkers [30]. Despite this interest, the integrity of SIRT1 function in HD has not been comprehensively investigated. In the present study, we have shown that SIRT1 activity is impaired in different brain regions from two distinct mouse models of HD and that this is linked to an altered SIRT1 phosphorylation status. Furthermore, we provide insights into the temporal tissue-specific regulation of SIRT1 activity in different brain regions from WT mice.
To monitor SIRT1 activity in the brain, we analysed P53 acetylation by performing immunohistochemistry on nuclei isolated from both R6/2 and HdhQ150 mice as compared to their WT SIRT1 Phosphorylation, Impaired Activity and Huntington's Disease littermates. We did not detect an alteration in SIRT1 function at the presymptomatic stage in either model. However, SIRT1 activity was overtly compromised by 9 weeks of age in the R6/2 mice with a comparable impairment at late stage disease in both models. This was not caused by SIRT1 sequestration into HTT inclusions and we did not detect any variation in either the level or sub-cellular distribution of SIRT1 between HD and WT mice. We also showed that this impairment occurred in liver and therefore extends to peripheral tissues. We would not expect the increase in P53 acetylation to be caused by the HD-related dysregulation of acetyltransferases as it has been shown that the P53 acetyltransferases: CREB binding protein, P300 and P300/CBP associated factor, are inhibited with disease progression in HD [49] and would therefore be expected to result in a reduction in P53 acetylation, the opposite to that observed in this study. We were unable to replicate these results using an independent measure of SIRT1 activity as the commercial kit that we tested was not specific for SIRT1 in mouse brain lysates.
The role of post-translational modifications in the regulation of SIRT1 activity has been the subject of several studies and phosphorylation has been described as a major control mechanism [41]. It has been shown that kinases such as JNK1 and CK2 can phosphorylate SIRT1 thereby increasing its nuclear deacetylase activity [44,50]. The phosphorylation of SIRT1 by JNK1 has also been shown to induce SIRT1 ubiquitination and proteasomal degradation [44]. In this study, although we detected an impaired SIRT1 activity in both the striatum and cerebellum of HD mice, the phosphorylation level of SIRT1 changed in opposite directions in these two brain regions, indicative of a tissue-specific SIRT1 regulation. Our further investigations led us to identify a striatum-specific phosphorylation-dependent induction of SIRT1 activity with age in WT mice, which does not occur in the cerebellum. Taken together, our findings suggest that it is the induction of SIRT1 function that is compromised by mutant HTT in the striatum of HD mice (Fig 11A), whereas in the cerebellum, mutant HTT impairs an already established SIRT1 activity.
In an attempt to rescue this SIRT1 deficiency, we crossed R6/2 mice with Dbc1 -/mice, as DBC1 negatively regulates SIRT1 via the direct interaction with its deacetylase domain [19]. Strikingly, despite a significant upregulation of SIRT1 activity in Dbc1 -/mice, the ablation of DBC1 from R6/2 mice had no effect on the impairment of SIRT1 activity, suggesting that mutant HTT alters key regulatory events that lie outside the inhibitory circuit controlled by DBC1. Consistent with this, the absence of DBC1 did not lead to improvements in the onset and progression of several behavioural HD-related phenotypes.
In contrast to DBC1, AMPK-α1 has been reported to positively regulate the activity of SIRT1 by inducing SIRT1 activation through its dissociation from DBC1 [20,45]. In addition, there is evidence to indicate that AMPK-α1 and SIRT1 can regulate each other [21]. Interestingly, our co-immunoprecipitation experiments revealed an increased interaction between DBC1 and AMPK-α1 in the striatum of HD mice, which might point to an attempt to rescue SIRT1 function. On the other hand, the nuclear accumulation of AMPK-α1 is delayed in the striatal nuclei of R6/2 mice and its retention in the cytoplasm through an interaction with DBC1 might impede SIRT1 activation. In contrast, the nuclear accumulation of AMPK-α1 in the cerebellum of R6/2 mice occurs earlier than in WT mice, indicating that it might be attempting to relieve the SIRT1 inhibition imposed by mutant HTT. In support of the existence of a striatum-specific regulatory circuit linking these three proteins in the induction of SIRT1 activity, we found that the downregulation of Sirt1, Dbc1 and Ampk-α1 is co-ordinated in WT mice between 4 and 9 weeks of age. The protein level of AMPK-α1 increases in WT mice during the same time frame, which correlates with the induction of SIRT1 activity, neither of which occurs in R6/2 mice. We propose a model whereby disease progression leads to an altered SIRT1 phosphorylation status. As a consequence, the decrease in SIRT1 activity leads to a reduction in the deacetylation of P53 and other SIRT1 substrates, modifications that may contribute to neuronal Proposed model for the striatum-specific regulation of SIRT1 via phosphorylation in WT mice and for the impairment in SIRT1 activity in HD brain. (A) The change in SIRT1 phosphorylation status in the striatum of WT mice between 4 and 9 weeks of age induces an increase in SIRT1 activity followed by a reduction in acetylated P53. The nuclear accumulation of AMPK-α1 in WT striatum at 9 weeks supports a role for this kinase in the activation of SIRT1 (AMPK-α1 is not the kinase involved in the change in SIRT1 phosphorylation detected here, as the MpM2 antibody only recognises Ser/Thr-Pro residues and AMPK-α1 does not phosphorylate Ser/Thr residues that are followed by proline). AMPK-α1 is present in the nucleus at 9 dysfunction ( Fig 11B). This would be consistent with previous data showing that the ablation of P53 from an HD mouse model had beneficial consequences [51]. In conclusion, our data provide two major new findings. First, we have shown that mechanisms controlling the tissuespecific regulation of SIRT1 activity differ between brain regions, and we have identified a novel striatum-specific phosphorylation-dependent mechanism of SIRT1 induction in WT mice. Second, we demonstrate that SIRT1 activity is impaired in two distinct HD mouse models. Given that SIRT1 plays a central role in metabolism, longevity and neurodegeneration, loss of SIRT1 activity may contribute significantly to disease progression in HD. These results provide new insights into the mechanisms that regulate SIRT1 function and may lead to the development of new strategies by which SIRT1 can be manipulated for therapeutic benefit. weeks and activates SIRT1 through a mechanism independent of DBC1. The down-regulation of Sirt1, Dbc1 and Ampk-α1 at the mRNA level between 4 and 9 weeks of age is consistent with these three proteins being partners in the same regulatory circuit. In the context of HD, the marked reduction in SIRT1 phosphorylation impedes the induction of SIRT1 activity. The greater interaction between AMPK-α1 and DBC1 may result in the cytoplasmic retention of AMPK-α1, inhibiting the activation of SIRT1, and/or promoting a futile rescue attempt by preventing DBC1 from binding to SIRT1. (B) The HD pathogenic process leads to an alteration in the phosphorylation status of SIRT1, resulting in an impairment in SIRT1 activity which modulated the function of SIRT1 targets that include P53 and may contribute to neuronal dysfunction. | 2016-05-12T22:15:10.714Z | 2016-01-27T00:00:00.000 | {
"year": 2016,
"sha1": "c71778d1b48c7e22a60338496b24168d130c6820",
"oa_license": "CCBY",
"oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0145425&type=printable",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "e699770a37a1c007aad974cf3c1ff7aeb75a2ec1",
"s2fieldsofstudy": [
"Biology",
"Chemistry",
"Medicine"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
52820984 | pes2o/s2orc | v3-fos-license | Fast stabilization of a high-energy ultrafast OPA with adaptive lenses
The use of fast closed-loop adaptive optics has improved the performance of optical systems since its first application. Here we demonstrate the amplitude and carrier-envelope phase stabilization of a high energy IR optical parametric amplifier devoted to Attosecond Science exploiting two high speed adaptive optical systems for the correction of static and dynamic instabilities. The exploitation of multi actuator adaptive lenses allowed for a minimal impact on the optical setup.
The use of fast closed-loop adaptive optics has improved the performance of optical systems since its first application. Here we demonstrate the amplitude and carrier-envelope phase stabilization of a high energy IR optical parametric amplifier devoted to Attosecond Science exploiting two high speed adaptive optical systems for the correction of static and dynamic instabilities. The exploitation of multi actuator adaptive lenses allowed for a minimal impact on the optical setup.
For a long time Ti:Sapphire amplified lasers, emitting around 800 nm, have been the only reliable sources of intense and ultrashort light pulses for numerous applications in Attosecond Science and strong-field Nonlinear Optics 1 . The last two decades have however witnessed an increased interest in Infrared Optical Parametric Amplifiers (IR-OPAs) as alternative sources for those applications [2][3][4][5][6][7][8] . OPAs have been extensively developed since the availability of intense pump lasers 9 and exploited also in low-energy applications like ultrafast spectroscopy [10][11][12] ; nowadays the scaling of these sources towards very high pulse energies by OPCPA 13 and high repetition rates 14 using fiber pump lasers is also achievable.
OPAs provide several benefits like huge spectral tunability and ultrashort pulse durations 15 , support of passive stabilization schemes for the Carrier-Envelope Phase (CEP) 16,17 and access to large photon and photoelectron energies in strong optical field phenomena such as high-order harmonic generation 18 and tunnel electron ionization 19 .
A limitation of IR-OPAs however comes from the requirement of a very good spatial quality and intensity stability of the pump beam that can hinder the scalability towards high pulse energies.
Furthermore, the performance of passive CEP stabilization schemes exploited in OPAs might be limited by the presence of phase-intensity noise coupling, that occurs whenever nonlinear optical processes are considered 20 ; this again calls for very stable pumping lasers.
System stability can be increased using Adaptive Optics (AO). For example, AO has been successfully used to increase the yield of non-linear phenomena exploiting iterative optimization algorithms [21][22][23] or to correct for wavefront aberrations induced by uneven thermal dissipation 24-26 . In the latter cases, the aberrations were corrected using a wavefront sensor and a closed loop control operating at 10 Hz.
In some cases however, instability shows a fast time varying behavior, that would require astronomical grade adaptive optics for an effective correction. Such a system implies a consistent complexity due to the need for a fast wavefront modulator and closed loop control operating at few hundreds of Hz requiring specific hardware, such as Field Programmable Gate Arrays (FPGAs) or Graphics Processing Units (GPUs). A drawback of AO systems in general is due to the use of deformable mirrors as wavefront modulators that implies important modifications to existing laser optical setup.
Here we report on the first application of fast closed loop adaptive optics based on adaptive lenses for the fast stabilization of a home-built high energy IR parametric source pumped by a 1-kHz, 10-mJ, 25-fs Ti:Sapphire laser. In particular, we were able to implement two separate and independent closed loop control systems operating at the same time in different positions of the system. This was made possible thanks to a new approach consisting in the use of multi actuator adaptive lenses 27 and a recently developed software package capable of working up to 500 Hz on a standard computer 28 . The closed loop control system was based on the use of a fast wavefront sensor (operating up to 500 Hz) triggered by the laser source. The multi actuator adaptive lens modulates the wavefront by changing its own shape. The lens is composed by two thin glass membranes and is filled with a BK7-dispersion-compatible liquid. Through the use of two ring shaped piezo electric actuators placed on the two faces of the lens, the wavefront can be modualted up to the 4th order of Zernike polynomials. The advantage of transmissive deformable optical elements is in the easiness of the installation that requires minimal changes to the existing optical setup 27 . While deformable mirrors can reach very high damage thresholds only with the use of dielectric coatings tuned on the laser wavelength, adaptive lenses can operate on intense laser beams over a broad spectral range (a discussion about lens transmission, working principle, chromatic aberration and damage threshold is reported in the section Adaptive Optics setup of the Supplementary Material).
The experimental setup is shown in Fig. 1: a portion of the pump laser beam undergoes spectral broadening and compression down to 10 fs exploiting a hollow-core fiber compressor (HCF) 29 ; an ultrashort IR seed is then obtained by intra-pulse difference frequency generation (DFG) driven by the compressed laser pulse in a BBO crystal. This scheme allows to generate an IR light pulse with a stable CEP 16 , which is a fundamental requirement for applications to the study of strong-optical-field phenomena in matter 1 . The seed is then amplified to the millijoule level in a two-stage OPA based on BBO crystals in type II phase-matching, that are pumped by the remaining fraction of the laser pump beam 18 . Since the overall OPA layout spans about 10 m, it is subject to environment instabilities such as temperature fluctuations, air fluxes etc.
We implemented two AO systems in the OPA layout: the first one (AO1) was placed in the collimated laser beam before the focusing section of the HCF, whereas the second AO system (AO2) was placed on the pump beam driving the second OPA stage (see Fig. 1). Each AO system is based on a Hartmann-Shack wavefront sensor (WFS) and a multi-actuator deformable lens 27 with 10-mm clear aperture, allowing to correct aberrations of the trasmitted laser beam up to the 4th order of Zernike polynomials. It is worth noting that nonlinear optical effects in the lenses, that might lead to phase front aberrations, are in this way corrected as well. The WFSs are optically conjugated to the deformable lenses by means of two lenses telescopes and analyze a tiny fraction of the beam that is extracted by exploiting the loss of two mirrors (see Fig. S1 of the Supplementary Material). The adaptive lenses used in this paper did not report any damage after two weeks of continuous operation (see again the Supplementary Material for the damage threshold characterization). Figure 2 shows the IR seed spectrum (red pattern) acquired by an infrared spectrometer as generated by DFG at the front end of the OPA. In a first set of measurements we characterized the stability of this seed by measuring the integral of its single-shot spectrum with an acquisition rate limited by the hardware to about 100 Hz; the time-series was then analyzed in the Fourier domain. Figure 3 shows the power spectral density of the acquired signal (blue curve) without any correction on the laser beam. The power spectrum shows significant noise components up to few Hz. By turning the AO1 system on, a significant improvement on the seed stability is achieved (red curve). A close inspection of the figure reveals some deterministic noise components at 3 Hz and its harmonics, that are related to the mechanical vibrations transferred from the cryogenic compressor mounted on the Ti:Sapphire power amplifier to the optical bench. A reduction of the IR seed fluctuations can be observed up to tens of Hz, although with less efficiency at high frequencies with respect to the low ones. However, the peaks of the deterministic noise components appear unaffected by the AO correction.
Results and Discussion
The analysis of the correction terms introduced by the AO1 system is reported in Fig. 4, where we show the standard deviation of the Zernike terms without (blue) and with (red) the AO1 system in operation. We observed a major influence of the environmental conditions on the first 5 Zernike terms that are the ones relative to the pointing stability (tip, tilt), defocus and astigmatism (0 and 45 degrees). The whole correction provided by the AO1 system improves considerably both the stability and the quality of the laser beam coupling to the HCF, as shown by the inset of Fig. 4 that displays the temporal evolution of the Strehl ratio of the laser beam (i.e. the ratio between the peak intensity of an aberrated wavefront and that of a perfect wavefront in the focal plane) determined at the HCF input over a period of 180 s. In particular, the Strehl ratio becomes very stable when the AO1 system is operating (red line), whereas shows large variations when no correction is applied (blue line). On the average, this parameter improves from 83.5% to 94.7%. This improves also the stability of the spectrum of the compressed laser pulses, that is transferred to the IR seed produced by DFG between the spectral tails of those pulses.
In a second set of measurements we performed a characterization of the performance of the IR-OPA source by exploiting both AO systems. In all the measurements we tuned the OPA around 1.45 μm by suitable setting of the BBO crystals orientation; Fig. 2 shows the corresponding pulse spectrum at the output of the OPA (blue pattern). In this condition the energy of the output pulses was about 500 μJ and the pulse duration, measured by a Frequency-Resolved Optical Gating (FROG) system, was about 25 fs. Even in this case, the stability of the source was evaluated by measuring the integral of the single-shot output spectrum at a 100-Hz acquisition rate. Figure 5 shows the evolution of the OPA output measured over 180 s; when the two AO systems are not operating, the r.m.s. fluctuation of the signal amounts to about 6% (blue curve); this fluctuation is halved by turning on both AO1 and AO2 (red curve). Both AO systems contribute to the improvement of the OPA stability; indeed the r.m.s. signal fluctuation increases to 4% when only AO1 is operating (Fig. 8 in the Supplementary Material), thus indicating an important role of the pump beam instability on the performaces of the OPA. The power spectral density of the OPA signal provides features similar to those observed in the seed case; in particular a significant improvement is observed on the slow noise components, whereas the correction becomes less effective at higher frequencies. It is worth pointing out that the AO systems can correct for beam aberrations, but are ineffective on fluctuations of the laser pulse energy; those fluctuation are responsible for the residual instability of the OPA output energy observed in Fig. 5.
A detailed investigation was also performed on the stability of the Carrier-Envelope Phase of the amplified pulses, that turned out to be affected by the instabilities of the pumping laser. The characterization of the CEP fluctuation was performed using a standard f-2f nonlinear interferometer coupled to a near-infrared spectrometer and based on white light generation in a thin fused silica plate followed by second harmonic generation in a BBO crystal 16 . In our experimental setup this CEP instability may have several sources: the first one comes from an instability in the coupling between the laser beam and the HCF, which translates in fluctuations in energy, spectral bandwidth and spectral phase of the compressed laser pulses at the HCF output. This noise is then transferred to the CEP of the seed IR pulses produced by DFG. A second noise source may come from the OPA stages, where the instability of pointing direction and intensity of the pump pulse may be translated to phase noise on the amplified seed by third order nonlinear effects 20 . As a last point, one has to consider that additional noise could be introduced by the instrumentation exploited for the CEP characterization, since f-2f interferometers are also prone to phase-intensity noise coupling 30 . In all those cases, the CEP fluctuations can be considerably reduced by improving the stability of the pump source. Figure 6(a) shows a scan of the spectral interference pattern acquired by the f-2f interferometer over 1 hour when both AO systems were operating; a similar scan was acquired when the two AO systems were not operating. The retrieved CEP evolution, determined from those scans by Fourier analysis, is reported in Fig. 6(b,c) in the cases without (blue curve) and with (red curve) the correction operated by the AO1 and AO2 systems. As shown in Fig. 6(c) the analysis restricted to a period of time of 3 minutes displays a considerable reduction of the CEP instability operated by the AO correction, with a decrease of the corresponding r.m.s. value from 256 mrad to 128 mrad. This improvement is still significant over the whole 1-hour acquired scan, corresponding to a decrease of the r.m.s. value from 236 mrad to 180 mrad. This noticeable result boosts the CEP stability performaces to a level comparable to active stabilization systems operating in standard high-energy Ti:Sapphire sources.
Conclusions
We reported on the first exploitation of deformable lenses in the stabilization of a high-energy IR parametric source designed for Attosecond Science applications. The combined use of adaptive lenses, in the place of deformable mirrors, and fast wavefront sensors allowed us an easy implementation of two adaptive optics systems operating at the same time on a high-energy laser source without any change to the optical setup. With respect to similar experiments reported in literature we have been able to correct for dynamic aberrations (sampling frequency up to 500 Hz) and to implement two independent aberration correction systems, placed in the most sensitive positions of the laser. The low absorption of the deformable lens allows to correct aberrations of mJ-level femtosecond pump beams, while the fast response of the AO system ensures the tracking of common instabilities in laser sources. In particular, beam pointing fluctuation and wavefront aberration induced by air turbulence and thermal instabilities can be promptly corrected. The significant improvement introduced by this AO system on the IR-OPA performances, in terms of both output energy and pulse CEP stability, is a further motivation towards the reliable long-term exploitation of high-energy few-cycle parametric sources in strong-optical-field Physics.
Data Availability
Data analysed during the current study are available from the corresponding author on reasonable request. Figure 6. Characterization of the CEP fluctuations at the IR-OPA output: (a) scan of the spectral interference pattern acquired by the f-2f interferometer over 1 hour when the AO systems were operating. (b,c) Retrieved CEP evolution without (blue curve) and with correction operated by the AO1 and AO2 systems (red curve) over 1 hour (b) and over a period of 3 minutes (c). | 2018-09-26T15:06:13.629Z | 2018-09-25T00:00:00.000 | {
"year": 2018,
"sha1": "604e8df18940767b2d5962834687c0fd81b13ec5",
"oa_license": "CCBY",
"oa_url": "https://www.nature.com/articles/s41598-018-32182-y.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "452bd1db292d792075e433eef1ff82886f1d3eff",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Medicine",
"Physics"
]
} |
1743978 | pes2o/s2orc | v3-fos-license | Groebner Bases for Everyone with CoCoA-5 and CoCoALib
We present a survey on the developments on Groebner bases showing explicit examples in CoCoA. The CoCoA project dates back to 1987: its aim was to create a mathematician-friendly laboratory for studying Commutative Algebra, most especially Groebner bases. Since then, always maintaining this"friendly"tradition, it has evolved and has been completely rewritten. CoCoA offers Groebner bases for all levels of interest: from the basic quick call in the interactive system CoCoA-5, to problem-specific optimized implementations, to the computer--computer communication with the open source C++ software library, CoCoALib, or the prototype OpenMath-based server. The openness and clean design of CoCoALib and CoCoA-5 are intended to offer different levels of usage, and to encourage external contributions.
§1. Introduction
The CoCoA project dates back to 1987 under the lead of L. Robbiano: the aim was to create a software laboratory for studying Commutative Algebra and especially Gröbner bases, and which is welcoming even to mathematicians who are wary of new-fangled computers, Since then the realm of applicability of Gröbner bases has continually expanded, so the researchers interested in using them now come from a broad palette of subject areas ranging from the theoretical to quite practical topics. So there are still the "pure" mathematicians as at the outset, but now also "programming" mathematicians, and statisticians, computer scientists, and so on. A factor crucial in making Gröbner bases relevant to practical problems is the interim progress in computer hardware and software techniques.
Since its beginning the CoCoA project has evolved and has been rewritten, and now comes in the form of a very flexible software combination CoCoA-5/CoCoALib, while maintaining its tradition of being user-friendly so it offers Gröbner bases for all levels of interest and programming ability: a Gröbner basis for everyone. This means that the "CoCoA experience" covers a wide range: from the basic, quick call in the interactive system CoCoA-5 [4], to the problem-specific optimized implementations, to the computer-computer communication with the open source C++ software library, CoCoALib [5], or with the prototype OpenMath-based server.
The importance that Gröbner bases have acquired derives from the fact they enable or facilitate so many other computational mathematical results. A natural consequence is that a Gröbner basis is almost never the final answer that is sought, but just a stepping stone on the way to the goal. It is very rare that anyone really wants to have a Gröbner basis, let alone actually look at one -usually they are frighteningly large beasts!
What is new in CoCoA-5? And what is not?
CoCoA-4 was widely appreciated for its ease of use, and the naturalness of its interactive language. However, it did have limitations, and several "grey areas". We designed the new CoCoA-5 language to strike a balance between backward-compatibility (hoping not to alienate existing CoCoA-4 users) and greater expressibility with a richer and more solid mathematical basis (eliminating those "grey areas").
So, what's not new? Superficially the new CoCoA-5 language/system closely resembles CoCoA-4 because we kept it largely backward compatible; at the same time it improves the naturalness and ease of use of the old system. We are very aware that a number of CoCoA users are mathematicians with only limited programming experience, for whom learning CoCoA was a "big investment", and who are reluctant to make another big investment -that is why we wanted to make the passage to CoCoA-5 as painless as possible.
So, if almost nothing has changed, what's new? The clearly defined semantics of the CoCoA-5 new language make it both more robust and more flexible; it provides greater expressibility and a more solid mathematical basis. In particular, it offers full flexibility for the field of coefficients: e.g. fraction fields and algebraic extensions, and even heuristically guaranteed floating point arithmetics with rational reconstruction (see Section 7.1).
However, under the surface, the change is radical since its mathematical core, CoCoALib, has been rewritten from scratch, to be faster, cleaner and more powerful than the old system, and also to be used as a C++ library.
How Do CoCoALib and CoCoA-5 differ?
The glib answer is: As little as possible! A more honest answer is that that is our aim, but there is more work still to do. In any case, using CoCoA-5 will not magically make you into a C++ programmer, so some differences will necessarily remain.
CoCoA-5 is an interactive, interpreted environment which makes it far better suited to "rapid prototyping" than the rigid, statically typed regime of C++. To keep it simple to learn, CoCoA-5 has only a few data types: for instance, a power-product in CoCoA-5 is represented as a monic polynomial with a single term, i.e. a ring element (of a polynomial ring); in contrast, in CoCoALib there is a dedicated class, PPMonoidElem, which directly represents each power-product, and allows efficient operations on the values (e.g. without the overhead of the superfluous coefficients).
CoCoALib [1] contains (practically) all the mathematical knowledge and ability while CoCoA-5 offers convenient access to CoCoALib's capabilities. Programming with CoCoALib tends to be more onerous than with CoCoA-5 largely because of C++'s demanding, rigid rules; the reward is typically faster computation (sometimes much faster). Also, of course, those who want to use CoCoALib's abilities in their own C++ program have to use CoCoALib, see Section 8. This is why everything which can be computed with CoCoA-5 should be just as readily computable with CoCoALib. Some CoCoA-5 functions are still implemented in CoCoA-5 packages; but they are being steadily translated into C++. (We don't say which ones, because the list is constantly shrinking) §2. Gröbner Bases with Ease The simplest context for Gröbner bases is for ideals in Q[x, y, z] or Z/(p)[x, y, z] with p a prime. These are also the easiest cases to give to CoCoA.
An essential ingredient in the definition of a Gröbner basis is the term-ordering: a total ordering on the power-products which respects multiplication, and where 1 is the smallest power-product. In CoCoA the term-ordering is specified at the same time as the polynomial ring; all Gröbner bases of ideals in that polynomial ring will automatically be computed with respect to that ordering -unless otherwise specified, the ordering is "degree reverse lexicographic", which is known to be the "best general choice". CoCoA has a fully general (matrix-based) implementation of term-orderings, with efficient handling of some common special cases.
Another well-known ordering is lex, which gives a Gröbner basis with a particular shape useful for solving polynomial systems (see, for example, the Kreuzer-Robbiano book [15], Sec.3.7). Their practical usefulness is limited by the fact that lex bases tend to be particularly big and ugly, and are frequently rather costly to compute.
In CoCoA the term-ordering is an intrinsic property of a polynomial ring: this means that Q[x, y, z] with lex is viewed as a different ring from Q[x, y, z] with StdDegRevLex. Here is an example of computing a lex Gröbner basis; note how the term-ordering is specified at the start along with the polynomial ring. /**/ use QQ[x,y,z], lex; // specify ordering together with ring /**/ I := ideal(x^3 +3, y-x^2, z-x-y); /**/ ReducedGBasis(I); // basis is wrt. lex ordering [z^3 +9*z -6, y -(1/4)*z^2 -(3/4)*z -3/2, x +(1/4)*z^2 -(1/4)*z +3 /2] In the last example above we used the command ReducedGBasis which computes a reduced Gröbner basis which is a "cleaned up" Gröbner basis with only non-redundant, monic, fully reduced elements -it is unique (up to the order of the elements).
Another family of computationally interesting rings in CoCoA is given by NewRingTwinFloat(BitPrec). It will be presented in detail in Sections 4 and 7.1.
§3. Gröbner fan and universal Gröbner bases
There is a notion of universal Gröbner basis which is a Gröbner basis for every term-ordering. The CoCoA function "UniversalGBasis" will compute one such basis; this function is based on the computation of the Gröbner fan (a richer structure, see below) which gives all possible reduced Gröbner bases: we can take the union of all of them to produce the universal basis.
The following example shows that the maximal minors of a 3 × 4 matrix of indeterminates form a universal Gröbner basis of the ideal they generate: d*g*i -c*h*i -d*e*k +a*h*k +c*e*l -a*g*l, d*f*i -b*h*i -d*e*j +a*h*j +b*e*l -a*f*l, c*f*i -b*g*i -c*e*j +a*g*j +b*e*k -a*f*k ] /**/ EqSet(-1*gens(I), ReducedGBasis(I)); true The Gröbner fan of an ideal was defined by Mora and Robbiano in 1988 ( [18]): it is a (finite) fan of polyhedral cones indexing the reduced Gröbner bases of the ideal. This has been implemented by Jensen in his software Gfan ( [16]) which he has recently linked into CoCoA; we note that CoCoA's fully general approach to representing term-orderings was essential in making this integration possible.
The Gröbner fan is useful because many well-known theoretical applications of Gröbner bases rely on the existence of a Gröbner basis of an ideal with prescribed properties, such as having a certain cardinality, or comprising polynomials of a specified degree, or all squarefree. For example, if an ideal I ∈ K[x 1 , . . . , x n ] has a Gröbner basis for some termordering comprising just quadrics, then the algebra K[x 1 , . . . , x n ]/I is Koszul.
The function GroebnerFanIdeals(I) returns all reduced Gröbner bases of the ideal I as a list of ideals: we chose to encode each basis in an ideal because in CoCoA an ideal I stores internally various pieces of information, including the term-ordering and the Gröbner basis, so putting the result in an ideal instead of a simple list of polynomials is more efficient for any subsequent operations.
The following ideal, Example 3.9 from Sturmfels's book [19], has 360 distinct reduced Gröbner bases: Storing all the possible different (reduced) Gröbner bases is practicable only for a small example; larger ideals may have thousands or even millions of different Gröbner bases. Typically we are interested only in those bases satisfying a certain property. So in CoCoA there is the function CallOnGroebnerFanIdeals which calls a given function on each Gröbner basis successively without having to store them all in a big list (if the computer's memory can handle it!). Using this CoCoA function needs a little technical ability, but might make the difference between getting an answer or not because the computer's memory filled up: define GBOfLen3(I) if len(GBasis(I))=3 then println; println OrdMat(RingOf(I)); println GBasis(I); endif; enddefine; /**/ use R ::= QQ[a,b,c]; /**/ I := ideal(a^5+b^3+c^2-1, b^2+a^2+c-1, c^3+a^6+b^5-1); /**/ CallOnGroebnerFanIdeals(I, GBOfLen3); matrix(ZZ, [ [3,7,7], [3,6,8], [[6, 7, 14], [6,5,15], [0, 0, -1]]) [c+b^2+a^2-1, -b^6-3*a^2*b^4-3*a^4*b^2+b^5+3*b^4+6*a^2*b^2+3*a^4-3*b^2-3*a^2, a^5+b^4+2*a^2*b^2+a^4+b^3-2*b^2-2*a^2] Here we see explicitly that CoCoA represents some term-orderings via matrices of integers. See Section 5 for an example of how to ask CoCoA to compute a Gröbner basis with a term-ordering given by a matrix. §4. Leading Term Ideals and "gin" . , x n ] be a polynomial ring over a field K, and let σ be a term-ordering on the power-products in P . If I is an ideal in P then we define its leading term ideal with respect to σ, written LT σ (I), to be the ideal generated by the leading power-products of all non-zero polynomials in I; some authors use the name "initial ideal" for this notion. A generating set for LT σ (I) may easily be obtained: we compute a reduced σ-Gröbner basis for I then collect the σ leading terms of the elements of the basis. Remarkably LT σ (I) captures some interesting "combinatorial" information about the original polynomial ideal I: for instance, its Hilbert series -so calling HilbertSeries(R/I) contains a "hidden" call to GBasis(I).
A more sophisticated tool in Commutative Algebra is the generic inital ideal of a polynomial ideal I. This is useful because it encodes more geometrical properties of I into a monomial ideal. It is defined by taking a generic change of coordinates g (i.e. g( . And here we have to admit that the acronym gin sounds nicer than gLT ! The definition of gin suggests an obvious algorithm for computing it (see Section 2.1 for an example with generic coefficients). However, it quickly becomes apparent that the coefficients in K(a i,j ) become unwieldy except for the very simplest cases; so the obvious approach is utterly hopeless. Instead we can pick an explicit, random change of coordinates h, and then compute LT σ (h(I)); the coordinate changes for which gin σ (I) = LT σ (h(I)) form a Zariski-open set. This approach can be used when K is infinite; if the random change of coordinates is chosen from a large set then LT σ (h(I)) will indeed be gin σ (I) with high probability. This is what CoCoA does.
While choosing random changes of coordinates with large coefficients increases the probability of getting the correct result, it also tends to produce large coefficients in the transformed polynomials. In this example the original polynomials have very small coefficients, but there is a coefficient with almost 50 digits in the tranformed polynomials: With coefficients like that, computing the Gröbner basis of the transformed ideal over the rationals would be quite expensive! Thus, when choosing random coefficients, one needs to strike a balance between a wide range, so the transformation is "generic enough", but not too wide, to limit coefficient growth during the computation.
The implementation for computing gin in CoCoA uses a special approximate floating-point representation for rational coefficients, namely twin-floats (see Section 7.1). The Gröbner basis of the twin-float transformed ideal will only have approximate twin-float coefficients, but this does not matter because we need only the leading power-products of the polynomials in the basis.
Twin-float numbers have fixed-precision (so do not grow in size the way rational numbers do), and employ heuristics to guarantee the correctness of results. This allows the implementation to make random coefficient choices from a wide range (in fact, integers between −10 6 and 10 6 ) without paying the price for calculating with transformed polynomials having complicated rational coefficients. If the chosen precision for the twin-floats is too low, this will be signalled; and the computation can be restarted choosing a higher precision.
CoCoA's function gin does this all automatically. Moreover, it tries a second random change of coordinates, just to make sure it gets the same leading term ideal. The internal workings can be seen via printed messages when using the option "verbose". -925894*x, 327379*x -729412*y, -945709*x +550455*y +499099*z ] --trying with FloatPrecision 64 ideal(x^5, x^4*y^16, x^3*y^18, x^2*y^20, x*y^22, y^24) §5. Elimination and related functions Elimination is a central topic in Computational Commutative Algebra (see for example the text book by Kreuzer and Robbiano [15], Sec. 3.4) and its applications are countless. Elimination means: given an ideal I ∈ K[t 1 , . . . , t a , x 1 , . . . , x b ], find a set of generators of the ideal I ∩ K[x 1 , . . . , x b ] where the indeterminates {t 1 , . . . , t a } have been "eliminated".
Given its usefulness, elimination is an operation offered in almost all Computer Algebra Systems. We can be sure that all such elimination functions internally compute a Gröbner basis with respect to an elimination ordering for the subset of indeterminates to be eliminated: with such an ordering the subset of polynomials in the Gröbner basis whose leading terms are not divisible by any of the t j are exactly the generators we seek for the ideal I ∩ K[x 1 , . . . , x b ].
In the example below we see the process we described and compare it with the actual output of CoCoA's own function elim. Note that in both cases the generators are not minimal, but they are indeed a Gröbner basis of the elimination ideal (wrt. to the restriction of the elimination term-ordering used). [-t +x, -x^2 +y, -x*y +z, y^2 -x*z] /**/ elim([t], I); ideal(-x^2 +y, -x*y +z, y^2 -x*z) /**/ MinSubsetOfGens(ideal(-x^2 +y, -x*y +z, y^2 -x*z)); [-x^2 +y, -x*y +z] The simple example above indeed shows a particular application of elim, the presentation of an algebra K[f 1 , . . . , f n ] ≃ K[x 1 , ..., x n ]/I. More precisely, let f 1 , ..., f n be elements in K[t 1 , . . . , t s ], where {t 1 , . . . , t s } is another set of indeterminates (viewed as parameters) and consider the K-algebra homomorphism φ : K[x 1 , ..., x n ] −→ K(t 1 , . . . , t s ) given by x i → f i for i = 1, . . . , n Its kernel is a prime ideal, and the general problem of implicitization is to find a set of generators for this ideal.
The Gröbner basis elimination technique consists of defining the ideal J = x 1 − f 1 , . . . , x n − f n in the ring K[t 1 , . . . , t s , x 1 , . . . , x n ] and eliminating all the parameters t i , as we saw in the example.
Unfortunately this extraordinarily elegant tool often turns out to be quite inefficent, resulting in long and costly computations. Knowing how to exploit special properties of a given class of examples might make a huge difference.
Toric
If the algebra we want to present is generated by power-products then the elimination can be computed by the CoCoA function "toric"; toric ideals are prime and generated by binomials.
Again we consider Q[t, t 2 , t 3 ], and also Q[t 3 , t 4 , t 5 ]: With a very slightly more challenging example we can clearly measure the advantage in using the specialized function "toric" over the general function "elim": The CoCoA function "toric" employs a non-deterministic algorithm: so the actual set of ideal generators produced might vary.
For further details on the algorithms implemented in CoCoA see Bigatti, La Scala, Robbiano [13]. The article describes three different algorithms; the default one in CoCoA is EATI (Elimination Algorithm for Toric Ideals).
For more details on the specific function "toric" type ?toric into CoCoA (or read the PDF manual, or the html manual in the web-site).
Implicitization of hypersurfaces
As mentioned earlier elimination provides a general solution to the implicitization problem, but this solution is more elegant than practical. We can do rather better in the special case of implicitization of a hypersurface. One immediate feature is that the result is really just a single polynomial since the eliminated ideal must be principal.
It is well-known that Buchberger's algorithm usually works better with homogeneous ideals (though there are sporadic exceptions). Yet the very construction of the eliminating ideal J = x 1 − f 1 , . . . , x n − f n looks intrinsically non-homogeneous. But with a little well-guided effort we can transform the problem into the calculation of a Gröbner basis of a homogeneous ideal.
If the f j are all polynomials then we take a new indeterminate (say h) and use it to homogenize each f j to produce F j . Now we do have a homogeneous ideal J ′ = x 1 − F 1 , . . . , x n − F n provided we give weights to the x i indeterminates by setting deg(x i ) = deg(f i ) for each i.
Since we are in the special case of a hypersurface, it can be shown that the (non-zero) polynomial of lowest degree in J ′ is unique up to scalar multiples; its dehomogenization is then the polynomial we seek! We get two advantages from the homogeneous ideal J ′ : we gain efficiency by using Buchberger's algorithm degree-by-degree, and we can stop as soon as the first basis polynomial is found -most probably there will still be many pairs to process. See Abbott, Bigatti, Robbiano [7] for all details and proofs, and also how to "correctly homogenize" parametrizations by rational functions. /**/ use P ::= QQ[s,t, x,y,z]; /**/ elim([s,t], ideal(x-s^2, y-s*t, z-t^2) ); ideal(y^2 -x*z) /**/ use R ::= QQ[s,t]; /**/ P ::= QQ[x,y,z]; /**/ ImplicitHypersurface(P, [s^2, s*t, t^2], "ElimTH"); ideal(y^2 -x*z) In the same paper we describe another algorithm which uses a completely different technique, a variant of the Buchberger-Möller algorithm (see Section 6), based on linear algebra. It is well-suited to low degree hypersurfaces.
/**/ ImplicitHypersurface(P, [s^2, s*t, t^2], "Direct"); ideal(y^2 -x*z) Both algorithms, in the case of rational coefficients, use modular methods computing the result for some primes, combining them using Chinese Remaindering, and reconstructing the rational coefficients of g using the fault-tolerant rational reconstruction described in Section 7.2. §6. Ideals of Points, 0-Dimensional Schemes Let X be a non-empty, finite set of points in K n , then the set of all polynomials in K[x 1 , . . . , x n ] which vanish at all points in X is an ideal, I X . One reason this ideal is interesting is because captures the "ambiguity" present in a polynomial function which has been interpolated from its values at the points of X. How best to compute a set of generators for I X , or a Gröbner basis, knowing the points X?
If X contains a single point (a 1 , . . . , a n ) then we can write down immediately a Gröbner basis, namely [x 1 − a 1 , . . . , x n − a n ]. If X contains several points we could just intersect the ideals for each single point, and these intersections may be determined via Gröbner basis computations; while fully effective and mathematically elegant this approach is computationally disappointing.
A more efficient method is the Buchberger-Möller algorithm [14]. Somewhat astonishingly this uses just simple linear algebra to determine the Gröbner basis. The original algorithm was closely analysed in [6], then later generalized to zero-dimensional schemes [11], where it turned out that it also incorporates the well-known FGLM algorithm for "changing term-ordering" of a Gröbner basis.
The BM algorithm is also amenable to a modular approach, a technique for achieving particularly efficient computation with rational numbers. The CoCoA implementation uses this approach. The simple use of linear algebra in the BM algorithm makes it a good candidate for identifying "almost-vanishing" polynomials for sets of approximate points: for instance, the points in the example above "almost lie on" a circle of radius 9.95 centred on the origin. The notion of Gröbner basis does not generalize well to an "approximate context" because the algebraic structure of a Gröbner basis is determined by Zariskiclosed conditions (i.e. the structure is valid when certain polynomials vanish); instead the notion of a Border Basis is better suited since its structure is valid dependent on a Zariski-open condition (i.e. provided a certain polynomial does not vanish). So long as the approximate points are not too few nor too imprecise the BM algorithm can compute at least a partial Border Basis, and this should identify any "approximate polynomial conditions" which the points the almost satisfy (see Abbott, Fassino, Torrente [10]).
We can ask CoCoA to allow a certain approximation on the coordinates of the points: It is well known that computations with coefficients in Q can often be very costly in terms of both time and space. For Gröbner bases over Q we are free to multiply the polynomials by any non-zero rational; so we can clear denominators and remove integer content. This does yield some benefit, but is not wholly satisfactory.
Sometimes the Gröbner basis has complicated coefficients (i.e. we mean big numerators and denominators), but more often the coefficients in the answer are reasonably sized, while the computation to obtain them involved far more complicated coefficients: this problem is known intermediate coefficient swell.
The phenomenon of coefficient swell is endemic in computer algebra, and many techniques have been investigated to tackle this problem. We illustrate what CoCoA offers.
TwinFloat
CoCoA offers floating-point arithmetic with a heuristic guarantee of correctness: the aim is to combine the speed of floating-point computation with the reliability of exact rational arithmetic -for a fuller description see the article [2]. Normally a twin-float computation will produce either a good approximation to the correct result or an indication of failure; strictly, there is a very small chance of getting a wrong result, but this never happens in practice.
To perform a computation with twin-floats the user must first specify the required precision; CoCoA will then check heuristically that the result of every computation has at least that precision. If the check fails then CoCoA signals an "insufficient precision" error; the user may then restart the computation specifying a higher precision. Although twinfloat values are, by definition, approximate, all input values are assumed to be exact (so they can be converted to a twin-float of any precision).
It is also possible to reconvert a twin-float value to an exact rational number. Like all other twin-float operations, this conversion may fail (because of "insufficient precision"). Printing out a twin-float automatically checks if the value can be converted to a rational as rationals are easier to read and comprehend. Twin-floats include a (heuristically guaranteed) test for zero; this means it is possible to compute Gröbner bases with twin-float coefficients. One reason for wanting to do this is that often the Gröbner basis over the rationals involves "complicated fractions" (i.e. whose numerator and denominator have many digits), and arithmetic with such complicated values can quickly become very costly. In contrast, with twin-floats the arithmetic has fixed cost (dependent on the precision chosen, of course). These characteristics are exploited in CoCoA for the computation of gin described in Section 4.
(Fault-tolerant) Rational reconstruction
A widely used technique for avoiding intermediate coefficient swell is to perform the computation modulo one or more prime numbers, and then lift/reconstruct the final result over Q. We call this the modular approach. There are two general classes of method: Hensel Lifting and Chinese Remaindering, the first is not universally applicable but does work well for polynomial gcd and factorization, while the second is widely applicable and works well in most other contexts.
The modular approach has been successfully used in numerous contexts, here are a few examples: polynomial factorization [20], determinant of integer matrices [9], ideals of points (see Section 6), and implicitization (see Section 5.2).
In any specific application there are two important aspects which must be addressed before a modular approach can be adopted, and there is no universal technique for addressing these issues.
• knowing how many different primes to consider to guarantee the result (i.e. find a realistic bound for the size of coefficients in the answer); • handling bad primes: namely those whose related computation follows a different route, yielding an answer with the wrong "shape" (i.e. which is not simply the modular reduction of the correct non-modular result). In the context of Gröbner bases we do not have good, general solutions to either of these issues. One of the first successes in applying modular techniques to Gröbner basis computation appeared in [12]. Finding good ways to employ a modular approach for Gröbner bases is still an active area. CoCoA does not currently use a modular approach for general Gröbner basis computations.
A vital complement to the modular computation is the reconstruction of the final, rational answer from the modular images. CoCoA offers functions for combining to two residue-modulus pairs into a single "combined" residue-modulus pair (i.e. using the chinese remainder theorem). It also offers functions for determining a "simple" rational number coresponding to a residue-modulus pair; this is called rational reconstruction. Correct reconstruction can still be achieved even in the presence of a few "faulty residues" (see [3]); this fault-tolerance was exploited in the functions for hypersurface implicitization (see Section 5.2).
Here we see how two modular images can be combined in CoCoA (using "CRTPoly" in this case), and then the correct rational result is reconstructed from the combined residue-modulus pair. This reconstruction is exploited in CoCoA for the computation of the implicitization of hypersurfaces described in Section 5.2. §8. Gröbner bases in C++ with CoCoALib As mentioned in Section 1.2, our aim is to make computation using CoCoALib as easy as using CoCoA-5. To illustrate this, here is the first example from Section 2 but in C++: ring P = NewPolyRing(RingQQ(), symbols("x,y,z")); ideal I = ideal(ReadExpr(P, "x^3 + x*y^2 -2*z"), ReadExpr(P, "x^2*y^3 -y*z^2") ); cout << GBasis(I); In comparison to CoCoA-5, this C++ code is more cumbersome and involved, though we maintain that it is still reasonably comprehensible (once you know that cout << is the C++ command for printing).
We have designed CoCoA-5 and CoCoALib together with the aim of making it easy to develop a prototype implementation in CoCoA-5, and then convert the code into C++. To facilitate this conversion we have, whenever possible, used the same function names in both CoCoA-5 and CoCoALib, and we have preferred traditional "functional" syntax in Co-CoALib over object oriented "method dispatch" syntax (e.g. GBasis(I) rather than I.GBasis()). This means that most of the CoCoA-5 examples given here require only minor changes to become equivalent C++ code for use with CoCoALib.
Naturally, a Gröbner basis is rare computed in isolation; it is usually just one step in a higher-level computation. We have designed CoCoALib to be easy to use, e.g. for preparing the generators of the ideals whose Gröbner bases are to be computed, and also for further processing with the Gröbner bases after they have been computed. To maintain the "friendly" tradition of CoCoA software, our design of CoCoALib follows these aims: • Designed to be easy and natural to use • Execution speed is good • Well-documented, including many example programs • Free and open source C++ code (GPL3 licence) • Source code is clean and portable (currently C++03) • Design respects the underlying mathematical structures (inheritance, no templates) • Robust (Motto: "No nasty surprises"), exception-safe, threadsafe §9. Conclusion The CoCoA software aims to make it easy for everyone to use Gröbner bases, whether directly or indirectly through some other function. The CoCoA-5 system is designed to be welcoming to those with little computer programming experience, while the CoCoALib library aims to make it easy for experienced programmers to use Gröbner bases in their own programs.
We hope this helps everyone to have their Gröbner basis! | 2016-11-22T14:10:27.000Z | 2016-11-22T00:00:00.000 | {
"year": 2016,
"sha1": "6cdbd84982901b09bcf595ee804de6545319aeac",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Arxiv",
"pdf_hash": "6cdbd84982901b09bcf595ee804de6545319aeac",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Mathematics",
"Computer Science"
]
} |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.