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258823033 | pes2o/s2orc | v3-fos-license | JetSeg: Efficient Real-Time Semantic Segmentation Model for Low-Power GPU-Embedded Systems
Real-time semantic segmentation is a challenging task that requires high-accuracy models with low-inference times. Implementing these models on embedded systems is limited by hardware capability and memory usage, which produces bottlenecks. We propose an efficient model for real-time semantic segmentation called JetSeg, consisting of an encoder called JetNet, and an improved RegSeg decoder. The JetNet is designed for GPU-Embedded Systems and includes two main components: a new light-weight efficient block called JetBlock, that reduces the number of parameters minimizing memory usage and inference time without sacrificing accuracy; a new strategy that involves the combination of asymmetric and non-asymmetric convolutions with depthwise-dilated convolutions called JetConv, a channel shuffle operation, light-weight activation functions, and a convenient number of group convolutions for embedded systems, and an innovative loss function named JetLoss, which integrates the Precision, Recall, and IoUB losses to improve semantic segmentation and reduce computational complexity. Experiments demonstrate that JetSeg is much faster on workstation devices and more suitable for Low-Power GPU-Embedded Systems than existing state-of-the-art models for real-time semantic segmentation. Our approach outperforms state-of-the-art real-time encoder-decoder models by reducing 46.70M parameters and 5.14% GFLOPs, which makes JetSeg up to 2x faster on the NVIDIA Titan RTX GPU and the Jetson Xavier than other models. The JetSeg code is available at https://github.com/mmontielpz/jetseg.
Introduction
Semantic segmentation is one of the critical computer vision tasks for autonomous systems. It requires a complete understanding of the analyzed scene to recognize and divide each object comprising the image, video, or 3D data. Semantic segmentation aims to classify each pixel into its corresponding class label to understand the image's objects. The significance of a scene depends heavily on the destined application that may range from topics such as humanmachine interaction [1], medical imaging [2], and autonomous driving methods [3]. For instance, systems directed toward autonomous vehicles must be able to segment roads and detect objects and traffic signs, which must operate under real-time conditions and, often, limited computing capabilities.
In robotics, real-time semantic segmentation must reach breakneck running speeds for quick interaction and responses to aid decision-making. One of the earliest networks for real-time semantic segmentation was ENet [6], which implemented a bottleneck module in a residual layer that reduced it to up to 1% of SegNet [5] parameters.
Other proposals rely on pyramid feature extraction for real-time segmentation, such as FPENet [7] that produces an efficient feature pyramid for multi-scale context encoding, ESPNet that adopts efficient spatial pyramid dilated convolutions [8], and DFANet [9] that mixes multibranch and spatial pyramid pooling, and reduces complexity by reusing features enhancing feature representation.
Although these methods can achieve real-time inference speed, the significant loss of features hinders their performance considerably, sacrificing accuracy for efficiency. Due to this, few works have attempted to obtain a favorable trade-off between high-quality results and low-inference times for embedded system applications. One of these models is ADSCNet [10], a lightweight Asymmetric depthwise separable convolution network that connects sets of dilated convolutional layers using dense dilated convolution connections; this model achieves an mIoU of 67.5% on the Cityscapes dataset at 76.9 FPS while performing 21.1 MFLOPS and having around 20K parameters while running on a GTX 1080 Ti GPU. Similarly, ESPNet V2 [11] implements group pointwise and depth-wise dilated separable convolutions for representation learning. Experimentation was carried out on a GTX 1080 Ti GPU and an NVIDIA Jetson TX2, reaching an mIoU of 66.2-66.4% by performing 2.7 B FLOPs for the Cityscapes dataset and 67-68% mIoU with 0.76 B FLOPs respectively. In recent work, MFENet [12] uses modules for Spatial and Edge Extraction with a Laplace Operator to improve low-level feature extraction, a Context Boost Module for high-level features, and a Selective Refinement Module that combines their information. This network obtains an mIoU of 76.7% on the Cityscape dataset performing 12.5 GLOPs at a speed of 47 FPS, and 73.5% on the CamVid dataset when executed on an NVIDIA Titan Xp card. DSANet [13], a two-branch model that employs channel split and shuffle to reduce computation and attain high accuracy. Its dual attention and channel attention modules help with pixel-wise label prediction using multi-level feature maps simultaneously. The experiments on a GTX 1080 GPU for the CamVid and Cityscapes datasets achieved mIoU of 69.9% and 71% at speeds of 75.4 FPS and 34.04 FPS, respectively. MiniNetv2 [14] was developed for low-power systems, improving the MiniNet efficient real-time semantic segmentation. It attains an mIoU of 70.4% performing 12.89 GFLOPs and 6.45 GMACs at 50 FPS, having 0.52 M parameters and a memory footprint of 2.02 MB for a resolution of 1024 x 512 of the CityScapes benchmark while running on an NVIDIA Titan Xp card. Furthermore, several works regarding embedded system implementation show promising results when compared to workstation devices, such as ThunderNet [15] is an efficient and lightweight network with a minimal ResNet18 backbone that unifies PSPNet pyramid pooling and a decoding phase that achieves an mIoU of 64% at 96.2 FPS on a Titan Xp GPU and 20.9 FPS on a Jetson TX2. Another instance is WFDCNet, a network of full-dimensional continuous separation convolution modules (FCS) and lateral asymmetric pyramid fusion modules (LAPF) that enable it to attain high accuracy without hindering inference speed, reaching 73.7% of mIoU having 5.7 GFLOPs at 102.6 FPS and 0.5 M parameters on an RTX 2080 Ti GPU for the Cityscapes dataset while achieving a speed of 17.2 FPS on a Jetson TX2. EFNet [16], a modified version of ShuffleNet V2 , aims to further improve accuracy-speed trade-off by reducing its downsampling operations, stride, and deconvolutions. Two thinner convolutions replace its large convolution channel, and the final downsampling and upsampling channels are substituted by more elaborate modules that boost efficiency. As a result, it reaches an mIoU of 68% at 99 FPS and 0.18 M parameters on an RTX 3090 GPU for the Cityscapes dataset. However, when run on a Jetson Nano, the model achieves similar accuracy results but significantly lower inference speeds of 0.9 FPS. Nevertheless, it was tested on a novel memristor-based computing-in-memory accelerator to demonstrate its efficiency on embedded systems further, obtaining 40.8 FPS and comparable accuracy results.
Additionally to the trade-off between speed and accuracy of current real-time semantic-segmentation models, there is limited research on execution time and memory footprint for real-time models. Therefore, due to the necessities mentioned above, our work focused on developing an efficient model capable of achieving real-time semantic segmentation in low-power embedded devices; specifically, we contribute to state-of-the-art with the following: • We introduce a novel, light, and efficient architecture called JetSeg that can compete with state-of-the-art faster and bigger models, outperforming them in most cases. JetSeg contains the novel encoder JetNet, the novel block JetBlock, the novel operation JetConv, and features an original loss function named JetLoss, which contributions will be explained.
• JetConv is a novel convolutional operator designed for lightweight and efficient feature extraction in real-time, low-power embedded systems. It enhances spatial information representation, increases the receptive field, and captures long-range dependencies without adding parameters. In addition, by combining dilated asymmetric and non-asymmetric convolutions, JetConv maintains spatial symmetry and enables balanced feature extraction in all directions.
• JetNet is an encoder that can extract information without compromising speed. It consists of convolutional blocks comprised of JetConvs. By combining dilated asymmetric depthwise convolutions and non-asymmetric traditional convolutions, the encoder can acquire large amounts of context information on fine and large dependencies without drastically increasing the number of parameters. • JetBlock is an efficient unit for feature map extraction, achieving a balance between inference time, memory usage, and model abstraction. It consists of multiple stages for optimal performance, estimates the number of group convolutions to prevent overfitting, and improves generalization. In addition, batch normalization and lightweight activation functions like TanhExp maintain representation while minimizing inference time. • JetLoss is a novel loss function for improved semantic segmentation. It combines Precision, Recall, and IoUB losses, enhancing overall performance. Adaptive weightings based on pixel counts per class are included to further enhance effectiveness by focusing on challenging classes. As a result, JetLoss enables faster convergence, efficient training, and reduced computational requirements. Experimental results on the CamVid [17] benchmark dataset show its edge over traditional loss functions, positioning JetLoss as a strong competitor in semantic segmentation.
JetSeg
According to the revised related works, most real-time semantic segmentation models are unsuitable for low-power embedded devices. Therefore, our work was to design an efficient and lightweight model capable of running on resource-constrained devices, such as the NVIDIA Jetson Nano and NVIDIA Jetson AGX, due to most embedded devices possessing GPU as hardware accelerators [18].
Our proposed model JetSeg, see Figure 1, is an efficient real-time semantic segmentation model for low-power GPU embedded systems, specifically, NVIDIA Jetson devices. This model comprises two architectures: JetNet, our proposed encoder, and an enhanced version of the RegSeg [19] decoder; hence the name JetSeg for our model. JetSeg was designed for memory and operation efficiency while preserving a competitive mIoU and FPS to the state-of-the-art (SOTA) models for low-power real-time semantic segmentation without model optimization or compression. The JetSeg model has three configurations, one for the workstation, one for the NVIDIA Jetson AGX, and one for the NVIDIA Jetson Nano. Input features are first expanded and later reduced when nearing the output layers, which are processed in different phases depending on the stage they are in. Four cases dictate the stage and operations applied to the input features. S0 and S1 focus on high-level feature extraction, using three main operations: channel shuffle to reduce the number of parameters; JetConv, which captures low and high-level features without increasing model complexity; and a convolutional block (CBAM) [20] that enables the model to learn more discriminative and robust features. S0 also includes residual layers just as S2; however, S2 replaces the CBAM with the SAM [20], which allows the model to capture long-range dependencies and enhances spatial awareness. S3 presents half the residual layers compared to S2 and also includes an Efficient Channel Attention Module [21] that highlights relevant channel-specific information.
JetConv
The JetConv novel operator is for lightweight and efficient feature extraction in real-time low-power embedded systems. It was inspired by the EESP (Extremely Efficient Spatial Pyramid) module [11]. The dilated Depthwise Convolutions have been improved by introducing Combinated Dilated Depthwise Convolution (CDDC), which combines dilated asymmetric convolution followed by a non-asymmetric and non-dilated convolution. This method enables a more effective and balanced spatial information representation. The receptive field is also significantly increased, which allows capturing long-range dependencies without increasing the total number of parameters and balancing between power and computational efficiency. Asymmetric convolution limitations are compensated through non-asymmetric convolutions, which preserve spatial symmetry and allow equal feature extraction in all directions. This symmetry is beneficial when dealing with objects lacking directional properties and ensures balanced information among spatial dimensions. JetConv improves contextual understanding without sacrificing spatial symmetry or robustness by integrating the two types of convolutions, allowing it to capture long-range dependencies and global spatial patterns critical for semantic segmentation. Figure 2 shows the structure of the JetConv. This balanced approach benefits objects without directional properties, ensuring comprehensive spatial information. JetConv achieves improved contextual understanding, capturing critical long-range dependencies and global spatial patterns for semantics while preserving spatial symmetry and robustness. Figure 2 is a residual block composed by five layers. In the first layer, there are three CDDConv blocks; each CCDConv block is composed of two different convolution operations. The first JetConv configuration consists of a depthwise convolution (DConv) [22], ADDConv (Asymmetric Dilated Depthwise Convolution) [23], and the novel convolution operation name ADDConv; they can configure different size kernels with and without dilatation rate, they are: 3 × 3, 5 × 5 and 7 × 7 for non-asymmetric, and for dilated asymmetric (3 × 1 + 1 × 3), (5 × 1 + 1 × 5) and (7 × 1 + 1 × 7). The second is an additive layer that adds the features obtain in the first layer, depending on the number of CDDConv blocks we have. The objective of the third layer is to concatenate the result of the second layer, i.e., we stack the obtained results of each CCDConv block to combine the feature extraction of each configuration of CCDConv. The GPConv in the fourth layer red reduces the overfitting, memory usage, and inference time. The objective of the last layer is to perform feedback of the GPConv result to the first layer to complete the residual calculus. The JetConv block allows three basic modifiable configurations: a) the first one contains one CDDConv block in the first, layer followed by the four layers shown in Figure 2, b) the second configuration adds a CCDConv block to the first configuration, c) the third one adds a third CCDConv block the second configuration.
JetBlock
JetBlock is a new efficient unit for feature map extraction comprised of several stages that allow it to achieve an equilibrium between inference time, memory usage, and model abstraction. First, the convenient number of group convolutions is estimated, preventing overfitting and improving model generalization. Group convolutions are followed by batch normalization and activation functions suited for lightweight architectures, being TanhExp used to reduce inference time while maintaining capture and representation. The second stage focuses on computational efficiency through the channel shuffle operator, which optimizes memory access patterns, enhances hardware resource management, promotes feature diversity through layer interconnections, and speeds up computation. The third stage extracts high-level features using the JetConv layer and distinguishes between the important elements with the attention module layer.
Afterward, the block finishes with an activation function and a grouped pointwise operator, reducing the computational cost and memory requirements. The composition of the JetBlocks is defined in Figure 3.
JetNet
JetNet is an encoder architecture that heavily relies on a set of efficient convolutional blocks called JetConvs. It has four stages as S0 to S3, as shown in Figure 1. The Stage S0 is composed of one configurable JetBlock followed by a Batch Normalization (BN) and an Activation Function REU (Rectified Exponential Units) [24] as shown in 3(a). The Stage S1 is a residual block composed of four configurable JetBlocks as shown in 2(b); in this stage, the four blocks are similar in structure. Its first layer is composed of a GPConv of 1 × 1 that expands the number of input channels in order to extract more features, followed by BN, REU, Channel Shuffle (CS), JetConv, Attention Module (AM), REU and 1 × 1 GPConv in order to reduce the number of features. Note that the AM layer is a CBAM module. Stage S2 is similar to stage S1; the difference is the configuration, particularly in the AM module, and the number of features used. We used only the spatial attention module in the AM module because the image size was reduced. Stage S3 is similar to stage S2; the block of this stage where reduced to improve the processing speed of JetNet. In this stage, the attention module was also changed; it used ECAM instead of SAM.
The main goal of the JetConv layer is to extract feature maps with as few operations as possible. The JetConvs are the main blocks for the two JetNet block variations; see Figure 3. The input block consists of a JetConv, a batch normalization layer, and an activation function. The subsequent stages of JetSeg are more intricate. The beginning and end of these blocks are similar to the input block; there, we replace the JetConv with a 1 × 1 convolution to reduce computational complexity. After that, we continue with channel shuffle as proposed in [25] followed by another JetConv, then an attention module, an activation function, and we finish by a 1 × 1 convolution. The attention module can be any of the following: a spatial attention module (SAM) [20], a CBAM, or an efficient channel attention module (ECAM) [19]. Finally, we use a skip connection to concatenate the residual for each block.
JetLoss
The JetLoss function is a novel loss function inspired by the work of Tian [26], designed to enhance model performance in semantic segmentation. JetLoss integrates three loss functions into one, combining elements from Precision [27], Recall [26], and IoUB [28] losses to improve the overall performance, particularly in terms of recall.
To further improve the effectiveness of JetLoss, adaptive weightings based on the number of pixels per class, following Cui's approach [29], are incorporated. These adaptive weightings assign importance to each class during training, enabling the model to focus on challenging classes and achieve better overall performance. As a result, using JetLoss promotes faster model convergence, efficient training, and reduced computational resource requirements. Moreover, it ensures a fair evaluation of the semantic segmentation model's performance.
The experimental results on the CamVid [17] benchmark dataset demonstrate the advantages of using JetLoss compared to conventional loss functions, highlighting its potential as a robust contender in semantic segmentation loss function proposals.
The components of the JetLoss function are described by the following set of equations 1: In the equations above, R loss represents the recall loss, P loss represents the precision loss, and B loss represents the IoUB loss. T P c , F N c , F P c , and W c denote true positives, false negatives, false positives, and class-specific weights, respectively. The overall JetLoss is computed as the sum of the three component losses.
Experiments and results
The following metrics are used to evaluate JetSeg and are generally employed to gauge the performance of real-time semantic segmentation models. The computational complexity is essential when working with limited hardware resources; hence the FLOPs metric is fundamental for evaluating a CNN model. It measures the total number of arithmetic computations performed in a process, emphasizing additions and multiplications. According to authors such as [30], several factors play a part in FLOPs, for instance, the expense in the computation of convolutions (F conv ), fully connected layers (F f c ), activation functions (F act ), as well as batch normalizations (F bn ). Equation 2 shows the total FLOPs formula broken down, where K w , K h , H in , and W in represent the kernel size, input height, and input width of the convolution layer, respectively; input and output channels are represented by C in and C out ; H in , W in , H out and W out represent the input height, input width, output height and output width of the fully connected layer; activation function cost is represented by F act and computed with the number of activation functions in the input tensor given by C in × H in × W in , as well as the number of operations needed per activation; and F bn represents batch normalization and is calculated based on the number of activations in the input tensor, which is given by C in × H in × W in . The fundamental metric to evaluate semantic segmentation performance is the mIoU [28], which refers to the total percentage of pixels belonging to the ground truth class that the model was able to classify, meaning the overlapping degree between the ground truth and model prediction. Equation 3 describes the computation.
The variables T P c , F P c , and F N c represent the counts of True Positive, False Positive, and False Negative pixels, respectively, for class c. The set D = 1, 2, ..., C refers to all the categories present in the dataset, while P represents the set of all pixels within the dataset.
This section describes of the experimental setup and results analysis used to evaluate our proposed JetSeg model. We conducted our experiments using the widely recognized dataset CamVid, commonly used as a benchmark dataset for Autonomous Driving Semantic Segmentation. CamVid [17] is a database for understanding road and driving scenes initially from five videos, which add to a total of 701 frames divided into 32 classes.
We conducted two phases of experiments: a classification evaluation and a real-time embedded system implementation. We utilized the CamVid dataset for both. A split of 67% of the data for training (468 samples) and 33% for testing (233 samples). Additionally, we divided the training set into 78% (367 samples) for training and 22% (101 samples) for validation.
The hardware used for training and evaluation consisted of an NVIDIA TITAN RTX GPU. We employed GPU-equipped embedded system boards to evaluate the real-time embedded implementation: the NVIDIA Jetson Nano and the NVIDIA Jetson AGX. To develop this work, we utilized a variety of software tools. Firstly, we employed PyTorch [31], the currently most widely used framework for deep learning. To ensure the reproducibility of our experiments, we utilized Docker containers [32] for all our hardware, including both our workstation and NVIDIA Jetsons. 1 The following section presents our evaluation of the JetSeg model in a real-time setting, showcasing the model's efficiency on embedded devices with limited resources. The evaluation was conducted on NVIDIA Jetson GPU-Embedded Devices using NGC Docker containers to ensure the reproducibility and generalizability of the experiments. For Jetson AGX Xavier, we utilized JetPack version 5.0.2 and the NGC Docker container nvcr.io/nvidia/l4t-ml:r35.1.0-py3. For Jetson Nano 4GB, we used JetPack 4.6.1 and the NGC Docker container nvcr.io/nvidia/l4t-ml:r32.7.1-py3. These configurations allowed us to effectively demonstrate the performance of JetSeg in real-time scenarios with constrained resources.
The experiments are divided into two categories: workstation and embedded devices. During the training phase, the model is trained from scratch for 15 epochs, without any pre-training, model compression, or data augmentation techniques. Table 1 presents the performance metrics of the model on the CamVid dataset with an input size of 512 × 512. The model achieves a considerably fast speed of 158 FPS, performing 1.125 G FLOPs with only 0.00003 G parameters. These results highlight that JetSeg is a compact and high-speed model for semantic segmentation.
When evaluated on embedded devices, the NVIDIA Jetson AGX demonstrates real-time performance, achieving a speed of 39.9 FPS. Despite its low hardware requirements, the Jetson AGX performs 1.125 G FLOPs with 0.00003 G parameters, making it suitable for real-time semantic segmentation in embedded system applications. Moreover, the model also runs successfully on the NVIDIA Jetson Nano, showcasing its lightweight nature and capability to operate on low-memory devices.
Discussion
This work proposes a novel model called JetSeg for the task of semantic segmentation for embedded devices in real-time, presenting an innovative, efficient, and lightweight architecture comprised of an encoder called JetNet, new model blocks named JetBlocks, original convolution operations called JetConv, and a new loss function called JetLoss, which allows it to run on embedded devices such as the NVIDIA Jetson AGX in real-time. This architecture lets the model extract low and high-level features while reducing computational complexity, maintaining spatial symmetry and balanced feature extraction thanks to the JetConv convolutional operator. JetNet combines dilated asymmetric depthwise convolutions and non-asymmetric traditional convolutions to extract context information from fine and large dependencies. JetBlock comprises JetNet and is an efficient unit for feature extraction, preventing overfitting while minimizing inference time. Lastly, JetLoss improves semantic segmentation performance by integrating three losses: Precision, Recall, and IoUB, which enhances overall performance through adaptive weighting based on pixel counts per class. This enables faster convergence and efficient training with reduced computational requirements. JetSeg can be executed in three configurations suited for workstations, such as the NVIDIA Titan RTX GPU, the NVIDIA Jetson AGX, and NVIDIA Jetson Nano embedded devices achieving high FPS, such as 148 FPS on the Titan RTX GPU and 39.1 FPS on the Jetson AGX, inference speeds suited for real-time implementation. The comparative experiment results showed that JetSeg outperformed the tested state-of-the-art models in inference speed up to 2× while executing less than 5× GFLOPs, as well as preserving a small number of parameters without model optimization, compression techniques, pretraining, or data augmentation.
Authorship Note
The contribution for the realization of this paper is as follow: The idea and project development are attributed to Miguel Angel Lopez Montiel and collaborators Daniel Lopez Montiel and Oscar Montiel Ross. In the submitted paper to NeurIPS2023, the authors Yoshio Rubio and Cynthia Olvera were included to participate in the writing and document review; however, they could not participate due to time constraints preventing them from fulfilling these roles; hence, they do not appear in this paper. | 2023-05-22T01:15:41.079Z | 2023-05-19T00:00:00.000 | {
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258138001 | pes2o/s2orc | v3-fos-license | THE INFLUENCE OF KNOWLEDGE, MOTIVATION AND WORKLOAD ON IMPLEMENTATION OF MEDICAL COMPLIANCE IN SIRS
Based on the Law of the Republic of Indonesia Number 44 of 2009 concerning Hospitals Article 32 it is stated that every Hospitals are required to record and report on all Hospital management activities in the form of a Hospital Management Information System. The effect of knowledge, motivation and workload in the implementation of SIRS with medical personnel compliance as an
The issuance of the Regulation of the Minister of Health of the Republic of Indonesia Number 82 of 2013 Article 3 states that every hospital is obliged to implement a Hospital Management Information System (SIRS). According to Yusof (2006), SIRS is a collection of processes that are implemented to help improve the efficiency and effectiveness of health organizations in carrying out their functions and achieving their goals. It aims to improve the quality of health services in hospitals.
Based on data from the Ministry of Health through the Hospital Information System (SIRS), guidelines for hospitals to carry out routine recording and reporting, until the end of November 2016 it was found that 1257 of 2588 (or about 48%) hospitals in Indonesia already have a Hospital Management Information System. (SIRS) which is functional. There are 128 hospitals (5%) that report having a Hospital Management Information System (SIRS) but not running functionally, 425 hospitals (16%) do not yet have a Hospital Management Information System (SIRS) , and 745 hospitals (28%) that did not report having a Hospital Management Information System (SIRS) or not. Based on the 2016 SIRS processed data, the number of functional Hospital Management Information Systems (SIRS) is mostly found in type C hospitals (597 hospitals) followed by type B hospitals (267). mostly found in type A hospitals (79%) and type B hospitals (73%). (Wu, Chen, & Greenes, 2009) According to research by Lacrum, H. & Ellingsen, G. (2001) in conducting research on the use of three electronic medical record systems with the aim of comparing them (DIPS, DocutiveEPR, Infomedic). The results of this study, more doctors use the electronic medical record system in carrying out their duties, because it is much more efficient than the previous system. SIRS is very necessary to be implemented in hospitals, this is in line with the demands of the community who require increasingly quality health services, because SIRS can offer the advantage of preventing medical errors through three mechanisms, namely (1) prevention of adverse events, (2) having a fast response, after the occurrence of adverse events, and (3) tracking and having feedback on adverse events The RSDC Wisma Atlet Kemayoran Hospital has been operated as a field hospital since March 23, 2020 to be able to provide services for Covid-19 patients with mild to moderate disease severity (Lu et al., 2020). Referring to its status as a field hospital, it has an impact on the management system with the concept of disaster (Takain & Katmini, 2021). Unlike other definitive hospitals in general, the Wisma Atlet Kemayoran Hospital cannot manage the hospital independently. Hospital management is still using collaborative resources between several elements and institutions (TNI, POLRI, government agencies, private sector, and other volunteers).Wisma Atlet Kemayoran is the largest field hospital for handling Covid-19 in Indonesia (Chaudhry et al., 2006). SIRS implemented by hospitals is considered less attention by related parties and has not prepared everything related to SIRS, such as many officers who do not understand and understand, this is because there is no support, supervision and evaluation from management and technology that has not run optimally. In this previous study, SIRS had not run smoothly because officers did not understand and understand the implementation of SIRS (Sari & Ramadani, 2021).
Method
In this study, an analytical observational method was used with across-sectional The approach departs from a theoretical framework, the ideas of experts, as well as the understanding of researchers based on experience, then developed into problems and proposed solutions to obtain justification or rejection in the form of empirical support in the field. The independent variables are (Knowledge, Motivation, Workload) (X)) while the dependent variable is the application of SIRS (Y) while the intervening is the compliance of medical personnel (Z). Respondents who have low knowledge in the compliance group non-compliant with SIRS (50.0%) and compliance with SIRS (50,0%). Meanwhile, respondents who have high knowledge in the SIRS low compliance group (38.3%), than those who have high knowledge of adherence (82.1%). The results of the Chi Square test showed that there was a significant relationship between SIRS knowledge and compliance (P−value 0.000). The results of the PR calculation show that respondents who have low knowledge are 4 times less likely to comply with the implementation of SIRS (95% CI 2,250-9,358). Respondents who had low motivation in the SIRS non-compliant group (52.9%) and high motivation in SIRS compliance (47.1%). Meanwhile, respondents who have high motivation in the SIRS low compliance group (15.1%), than those who have high motivation to comply (84.9%) (Putra & Vadriasmi, 2020). The results of the Chi Square test showed that there was a significant relationship between SIRS user motivation and compliance (P−value 0.000). The results of the PR calculation show that respondents who have low motivation 6 times are not compliant with the implementation of SIRS (95% CI (3,024-13,325). Respondents who have a low workload in the compliance group do not comply with SIRS (47,2%) and a high workload on compliance SIRS compliance (52.8%). Meanwhile, respondents who have a high workload in the SIRS low compliance group (18.0%), than high workload in compliance (82.0%). Chi Square test results show that there is a significant relationship between the workload of SIRS users and compliance (P−value 0.000) The results of the PR calculation show that respondents who have a low workload are 4 times not compliant with the implementation of SIRS (95% CI (2.2003-8.319).
Results and Discussion
Respondents who have low SIRS application in the group SIRS non-compliance (17.5%) and SIRS implementation were high on SIRS compliance (82.5%), while respondents who had high SIRS application were in the low SIRS compliance group (44.4%), than those who had SIRS implementation were low. high adherence to compliance (55.6%). The results of the Chi Square test showed that there was a significant relationship between the implementation of SIRS and compliance (P−value 0.000). Relationship Effect of Knowledge, Motivation, Workload in the Implementation of SIRS. (2022) Respondents who have low knowledge in the group do not apply SIRS (39.4%) while the application of SIRS (60.6%). Meanwhile, respondents who have high knowledge in the group do not apply SIRS (56.8%), than those who have high knowledge on the application of SIRS (43.2%). Test Chi-Square show that H0 failed to be rejected, which means that there is no significant relationship between knowledge on the implementation of SIRS and the obtained value (P Value 0.29)> (0.05).
Respondents who had low motivation in the group did not apply SIRS (41.2%) and high motivation in the application of SIRS (58.8%). Meanwhile, respondents who have high motivation in the group do not apply SIRS (55.9%), than those who have high motivation to apply SIRS (44.1%). Test Chi-Square showed that H0 failed to be rejected, which means that there was no significant relationship between motivation to the implementation of SIRS and obtained a value (P Value 0.65)> (0.05).
Respondents who have a low workload in the group without SIRS application (48.6%) and high workload in the SIRS application (51.4%). Meanwhile, respondents who had a high workload in the group that did not apply SIRS (50.6%), than those who had a high workload in the group that applied SIRS (49.4%). Test Chi-Square show that H0 failed to be rejected, which means that there is no significant relationship between workload and the implementation of SIRS with the obtained value (P Value 0.806)> (0.05).
Respondents who had low SIRS compliance in the compliance group did not comply with the implementation of SIRS (28.0%) and adhered to the application of SIRS (72.0%). Meanwhile, respondents who had high SIRS compliance in the group that did not apply SIRS (59.5%), than those who had high SIRS implementation in the noncompliant group (40.5%). The results of the Chi Square test showed that there was a significant relationship between compliance with the implementation of SIRS (P−value 0.000). Hospital is one of the organizations engaged in the field of health services, which is very complex and professional, technology-intensive, and rule-intensive (Uswatun Hasanah, 2022). As one of the organizations in health services, hospitals often experience difficulties in processing information for both internal and external needs, so it is necessary to improve the management of information that is efficient, fast, easy, accurate, and safe. information technology through the use of a computer-based Management Information System. The results showed that the results of the Chi Square test showed that there was a significant relationship between compliance with the implementation of SIRS (P−value 0.000). The conclusion is that based on the variables of knowledge, motivation, workload on SIRS compliance at RSDC Wisma Atlet Kemayoran there is a relationship (P−value 0.000).
Relationship Effect of knowledge, motivation, and workload on the implementation of SIRS with medical personnel compliance as an intervening variable at RSDC Wisma Athlete Kemayoran.
The Hospital Management Information System is known as SIRS. SIRS is an application program or computer software created to assist hospital management in performing data entry, processing data and making patient data reports. Hospital management information system is an inseparable part of overall hospital services, and is even one of the main joints in daily activities (Sutanta, 2003). Application of Hospital Management Information System.
The results of the research from Hendri 2018 are known that the success in implementing SIRS RSUD Dr. Sudirman Kebumen is influenced by factors of system quality, service quality, system use, user satisfaction and benefits. User satisfaction is the variable that has the greatest influence.
The Relationship of the Effect of SIRS Knowledge in Improving Compliance Medical Personnel
Knowledge is the result of remembering something, including recalling events that have been experienced either intentionally or unintentionally and this occurs after people make contact or observations of a certain object (Notoatmodjo, 2007).
Knowledge or knowledge is the result of human sensing or the result of knowing someone about an object through their five senses (Ananta & Dirdjo, 2021). The five human senses to sense objects are sight, hearing, smell, taste and touch. At the time of sensing to produce knowledge is influenced by the intensity of attention and perception of the object. A person's knowledge is mostly obtained through the sense of hearing and the sense of sight (Notoatmodjo, 2014).
The results of the Chi Square test showed that there was a significant relationship between SIRS knowledge and medical personnel compliance (P−value 0.000). The results of the PR calculation show that respondents who have low knowledge 4 times experience disobedience to the application of SIRS. The results of previous research from (Regester & Larkin, 2008). There are factors that most influence the behavior of nurses in using SIRS is the knowledge factor, the respondent's understanding of SIRS is not related to the training obtained but associated with how often they use SIRS in documenting all activities related to patients.
The Relationship of the Effect of Motivation in Improving Compliance of Medical Workers
Motivation is very important because with motivation it is expected that every employee will work hard and be enthusiastic to achieve high work productivity. Motivation will provide inspiration, encouragement, morale for employees so that good working relationships are established between employees and leaders so that organizational goals can be achieved optimally (Ahmad, 2014).
The results of the Chi Square test showed that there was a significant relationship between SIRS user motivation and compliance (P−value 0.000). The results of the PR calculation show that respondents who have low motivation 6 times do not comply with the implementation of SIRS (Regester & Larkin, 2008).
The results of previous research from Muhammad Husni found that motivation had a positive and significant effect on adherence to writing diagnoses on the patient's medical resume (p values 0.000 and 0.000; R20.562 and 0.574). Knowledge, attitude and motivation jointly affect compliance (p value 0.000; R2 0.679). Zahirah Hospital should continue to support the working environment at the hospital so that the doctor's compliance in writing disease diagnoses on the patient's medical resume increases. Doctors at Zahirah Hospital should be able to write a disease diagnosis on a medical resume in a timely manner.
The results of previous research from Dani 2019. found that the implementation of SIRS at the TPPRJ had run smoothly, with the SIRS at the TPPRJ it really helped them in their work and was more time efficient from manual to system. However, in its application there are some officers who have not been responsible and disciplined. This is because there is no support and motivation from management specifically for officers not yet.
Relationship Effect of Workload in Improving Compliance of Medical Personnel
Workload can be defined as a difference between the capacity or ability of workers and the demands of the work that must be faced. Given that human work is mental and physical, each has a different level of loading. The level of loading that is too high allows excessive energy consumption and overstress occurs, on the contrary Loading intensity that is too low allows boredom and saturation or understress. Therefore it is necessary to strive for the optimum level of loading intensity that exists between the two extreme limits and of course differs from one individual to another.
According to (Di Gennaro et al., 2020) every job is a burden for the perpetrator. The burden depends on how the person works so it is called workload. So the definition of workload is the ability of the human body to accept work. Based on an ergonomic point of view, every workload received by a person must be appropriate and balanced both to the physical abilities, cognitive abilities and limitations of humans who receive the load. The burden can be in the form of a physical burden or a mental burden. Physical workload can be in the form of heavy work such as caring, transporting, lifting, and pushing. While the mental workload can be in the form of the extent to which the level of expertise and work performance possessed by individuals with other individuals (Indonesia, 2020).
The results of the Chi Square test show that there is a significant relationship between the workload of SIRS users and compliance (P−value 0.000). The results of the PR calculation show that respondents who have a low workload 4 times are not compliant with the implementation of SIRS (Khairani, Soviyant, & Aznuriyandi, 2018).
Conclusion
Knowledge, motivation, and workload have a significant effect on the compliance of health workers in implementing SIRS as an intervening variable at RSDC Wisma athletes Kemayoran. because the presence of these three variables has an impact on the implementation of SIR on the compliance of health workers. Knowledge has a positive effect in increasing the compliance of health workers. with good knowledge of health workers on SIR can have an impact on the use and utilization of the. Motivation system has a positive effect in increasing compliance of medical personnel. With the motivation given by the hospital leadership, it will have a positive impact on organizational culture so that health workers have the responsibility to carry out the tasks that are given responsibility for the use of SIR. Workload has a positive effect on increasing the compliance of medical personnel. in accordance with the workload with the number of health workers will make it easier to use SIR because with the high workload automatically health workers are less focused on the use of SIR. Knowledge has a negative effect on the implementation of SIRS at the Wisma Atlet Kemayoran Hospital. with the influence of high or low knowledge does not affect the application of SIR because in the hospital system it is always recommended to apply SIR in accordance with the regulations. Motivation has a negative effect on the implementation of SIRS at the RSDC Wisma Atlet Kemayoran. with low motivation, it cannot affect the application of SIR because in hospitals it is always recommended the application of SIR, but its application is not optimal. Workload has a negative effect on the implementation of SIRS at RSDC Wisma Atlet Kemayoran. because in hospitals it is always recommended to apply SIR even though the number of human resources is not in accordance with the workload experienced by health. workers. the presence of competent health workers can affect compliance with the application of SIR so that SIR can facilitate existing services at the hospital. | 2023-04-15T15:19:03.550Z | 2023-02-25T00:00:00.000 | {
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55325952 | pes2o/s2orc | v3-fos-license | Analysis of critical states of composite drive shafts with two reduced masses – selection of the right model
Carbon fiber drive shafts are nonlinear structures of complex character, difficult to establish “a’priori”. The only way of modeling is to make identification based on the results of measurements of real objects. In previous works, Authors describe the process of building a mathematical model of such a system and its identification on the basis of experimental results but for only one reduced mass. On this basis, however, it is not possible to write a two-mass model because the identified function describing the resilience characteristics also ‘covers’ errors of other model parameters. So, Authors in this paper showed the process of selection of right model which can show the effects of passing shaft’s critical speeds by system with two reduced masses. In conclusion, future model identification procedure for nonlinear phenomena was announced.
Introduction
It is well known that carbon fiber constructions are very flexible, anisotropic and have nonlinear characteristics of elasticity.For this type of material, Hook's law is not satisfied either.The carbon composite is also not essentially a material in the present meaning of the word, since it acquires its full properties only after shaping the finished product.It is therefore difficult, on the basis of classical strength tests usually performed on a flat beam sample, to conclude on the properties of an actual structure operating under complex load conditions.These features make the use of traditional calculation methods not applicable here.Such a design would usually be too large in relation to the requirements and often it may not be able to meet the basic strength requirements.
Because of the lack of dynamic computation methods, Authors in some previous works [1][2][3][4][5] proposed methodology of building mathematical model based on some experimental results.The results obtained in these works made it possible to develop the accurate model of the shaft, that simulates critical acceleration and deceleration.The model was based on several simplifications.Firstly, the shaft was reduced to a single rotating disc and massless shaft.Secondly, we take into account only transverse vibrations.Such simplifications allow to reduce the model to the system with one degree of freedom with linear damping and vibration forced by harmonic force.
Authors decided to use obtained experimentally the elasticity characteristic and damping coefficient for identification and model tuning.This also simplified the measurements, since only the displacement of the shaft centre with reduced mass was considered.
Authors decided to use as the identification base, the movement of the centre of the shaft.The displacement measurements were carried out by the laser displacement sensor.Experiments were performed for various velocities of accelerations and decelerations of the shaft.Obtained results [3][4][5] indicated very strong dependence on acceleration velocity (deceleration) of the shaft.
Equation of motion model obtained in this way will take a form: where the elasticity force is represented by the dependence:
Proposition of model based on two rotating disks and massless shaft
The reduced mass model illustrated in point 1, allows a very good and rapid identification of low complexity systems.Unfortunately, it does not allow generalization of inference and transfer of results to more complicated systems [6][7][8].For this purpose, consider a model with two masses concentrated.The fundamental difference with one mass model is that we can't use it to identify the elasticity characteristics obtained experimentally.Assuming a symmetric mass distribution, we must accept at least 2 different stiffness values.This cause that the previously adopted "correction" taking into account the influence of velocity on the elasticity characteristics can't be applied in the same way.In order to obtain correct and physically interpretable model identification, we must assume the existence of at least two independent correction factors.A separate problem is damping in the system.Assume in analogy to one mass system its linear character, assume initial value obtained experimentally and equally for all three sections of the shaft.
Fig. 1. The model of carbon fiber shaft -two disks with massless beam
The model shown at (Fig. 1) is a classic model with two degrees of freedom.The introduction of non-linear characteristics of the elasticity dependent in addition to the velocity of vibration requires considerable attention and diligence in the analysis of the results, so that it can be identified with experimental results.An additional impediment to the task is that the force exerted by and is in fact the force-induced harmonics dependent on the rotation frequency of the Ω.
Therefore, consider the identification of the model shown in Figure 1 in three steps.Equations of motion model written in accordance with d'Alembert principle will be as follows: In the first step, we assume that the force inducing and = 0 and damping = = 0, we obtain free vibrations for which the predicted solution of the system Eq.(3) will be given by the expression: where the natural frequencies and depend solely on the mass and elasticity distribution and the amplitude ratios will be formulated by the following expressions: ( The values of and will determine the forms of vibration.
As we can see, we see the damping exponents ℎ and ℎ , the frequency of self-damped vibrations as well as the forms of their own vibrations.
The most complicated model, we will consider in the third stage.In addition to damping, it will include the harmonic force ( ) = cos( ) = : Considering the forced vibrations established (after the free vibration has been extinguished due to the activation of the force), we can assume:
Conclusions
As you can see from these brief reflections, choosing the right model for identification is not a trivial matter.It is safe to say that at the mathematical modelling stage, it is difficult to answer the question of which model will best pass the test for identification with experimental results.Currently, the authors are at the stage of performing an active experiment and choosing the right model.The effect of these works will be presented in the future articles. | 2018-12-06T22:11:15.339Z | 2017-09-26T00:00:00.000 | {
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18418398 | pes2o/s2orc | v3-fos-license | Long-term impact of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on body weight and glycemic control in Japanese type 2 diabetes: an observational study
Background Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, has been shown to possess pleiotropic effects including body weight reduction. However, long-term effect of liraglutide on body weight and glycemic control has not been elucidated in Japanese type 2 diabetes (T2D) subjects. Present study investigates whether liraglutide treatment maintains the body weight-decreasing and glucose-lowering effects for 2 years in Japanese T2D subjects. Methods The enrolled subjects were 86 T2D patients [age; 59.8 ± 12.8 years, duration of diabetes; 15.8 ± 9.5 years, glycated hemoglobin (HbA1c); 8.5 ± 1.5%, body mass index (BMI); 27.3 ± 5.4 kg/m2 (15.8 - 46.5 kg/m2), mean ± SD]. Among 86 subjects, liraglutide was introduced in 25 inpatients and 61 outpatients, and 46 subjects were followed for 2 years. Clinical parameters were measured at baseline and 3, 6, 9, 12, and 24 months after liraglutide introduction. The increase in liraglutide dosage and the additional usage of glucose-lowering agents depended on each attending physician. Results At 1 year after liraglutide introduction, 69 patients (80.2%) decreased body weight and 58 patients (67.4%) improved glycemic control. Body mass index (BMI) was changed 27.3 ± 5.4 kg/m2 to 25.9 ± 4.8 kg/m2 and percent reduction of body weight was significant and maintained over 4% at 2 years after liraglutide introduction. HbA1c was significantly decreased from 8.5 ± 1.5% to 7.7 ± 1.2% for 2 years. Liraglutide treatment tended to ameliorate lipid profile and hepatic enzymes. Stepwise regression analysis demonstrated that baseline BMI and previous insulin dose were positively associated with body weight reduction and baseline HbA1c was positively associated with reduction of HbA1c at 2 years after liraglutide introduction. Conclusions Long-term liraglutide treatment effectively maintained the reduction of body weight and the fair glycemic control, and also improved lipid profile and liver enzymes in Japanese T2D subjects.
Introduction
Recently, systematic analysis for the global prevalence of overweight and obesity has reported that the proportion of overweight adults gradually increased between 1980 and 2013 from 28.8% to 36.9% in male and from 29.8% to 38.0% in female [1]. The prevalence of type 2 diabetes has also increased worldwide [2] and similar increase was observed in Asia including Japan [3]. Evidently, obesity, especially visceral fat obesity, is located upstream of type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis [4]. Obese type 2 diabetes subjects are more liable to cardiovascular diseases. Indisputably, the effective and efficient therapeutic strategy for obese type 2 diabetes should be developed and promoted. It is clear that the weight reduction is a therapeutic basis for obese type 2 diabetes. However, the management for obese type 2 diabetes often encounters difficulty in the control of appetite and body weight. In addition, treatment with insulin, sulfonylurea, and thiazolidinedione sometimes increase adiposity and such anti-diabetic treatments incidentally result in poor glycemic control because of weight gain.
Glucagon-like peptide-1 (GLP-1) is an incretin hormone with a potent blood-glucose lowering action only during hyperglycemia by inducing insulin secretion and reducing glucagon secretion in a glucose-dependent manner [5]. Beyond glucose-lowering effect, GLP-1 delays gastric emptying and induces satiety, leading to weight reduction. The mechanism is partly explained by the combination effects of GLP-1 on the gastrointestinal tract and the brain [6]. Native GLP-1 is immediately degraded and its elimination half-life is 1-2 min, whereas liraglutide has a long half-life, around 13 hours, and can be administered once a day [7]. A series of Liraglutide Effect and Action in Diabetes (LEAD) study showed the significant effect of liraglutide on weight reduction as well as glycemic control mainly in Caucasian diabetes subjects. Recently, long-term efficacy of liraglutide on weight reduction has been demonstrated in the subanalyses of LEAD study [8,9]. Our group has indicted the beneficial effects of liraglutide on visceral fat adiposity, body weight, and eating behavior until 6 months after liraglutide initiation in Japanese obese type 2 diabetes subjects [10,11]. However, it has not been elucidated in Japanese type 2 diabetes subjects whether the effect of liraglutide on body weight and glycemic control would be maintained for longer period.
In Japanese type 2 diabetes subjects, we herein investigated the effects of liraglutide on body weight and glycemic control, and analyzed the association of clinical parameters and changes in body weight and HbA1c for 2 years after liraglutide introduction.
Subjects
Present study was an observational study and the inclusion criteria were the subjects who were type 2 diabetes, introduced with liraglutide therapy at Osaka University Hospital (Osaka, Japan) between November 2010 and December 2012, and continued liraglutide treatment over one year. Number of subjects who were initiated with liraglutide during the indicated period was 136 diabetes patients. Among 136 patients, 22 patients returned to their home doctor, and thus 114 patients were followed up by our hospital. The 28 patients discontinued the liraglutide treatment within one year because of the following reasons: 11 patients suffered complications (digestive symptoms, severe loss of appetite, headache, and vertigo), 2 patients significantly improved glycemic control and discontinued liraglutide treatment, 14 patients deteriorated glycemic control and changed liraglutide treatment to other anti-diabetic agents, and 1 patient was diagnosed colon cancer. We finally analyzed 86 subjects who continued liraglutide treatment over one year, and 46 subjects were followed for 2 years among them. Among 86 subjects, 25 patients were introduced liraglutide during hospitalization and 61 patients were initiated liraglutide as an ambulatory treatment.
Insulin (%) 56
Data are mean ± SD or number of subjects. LDL-C; low-density lipoproteincholesterol, HDL-C; high-density lipoprotein-cholesterol, BG; biguanide, SU; Sulfonylurea, αGI; α-glucosidase inhibitor, TZD; thiazolidinedione, DPP4i; dipeptidyl peptidase-4 inhibitor. circumference was measured at the umbilical level in the late expiration phase at standing position by using a non-stretchable tape. Blood pressure was examined by using an aneroid sphygmomanometer at sitting position under resting condition in the consultation room. The increase in liraglutide dosage (0.3 mg/day, 0.6 mg/day or 0.9 mg/day, representing the maximum dose used in Japan) and the additional usage of glucose-lowering agents was depended and decided by each attending physician. The study protocol was approved by the human ethics committee of Osaka University and was registered with the University hospital Medical Information Network (Number: UMIN 000004192), and the informed consent was obtained from study subjects.
Questionnaire for eating behavior
Eating behavior was assessed in part of patients before liraglutide treatment by using the questionnaire of The Guideline For Obesity issued by the Japan Society for the Study of Obesity. As reported previously [10], this questionnaire consists of 55-item questions of seven major scales as follows: 1) Recognition for weight and constitution (e.g., 'Do you think it is easier for you to gain weight than others?'), 2) External eating behavior (e.g., 'If food smells and looks good, do you eat more than usual?'), 3) Emotional eating behavior (e.g., 'Do you have the desire to eat when you are irritated?'), 4) Sense of hunger (e.g. 'Do you get irritated when you feel hungry?'), 5) Eating style (e.g., 'Do you eat fast?'), 6) Food preference (e.g., 'Do you like meat?'), 7) Regularity of eating habits (e.g., 'Is your dinner time too late at night?'). All items were rated on a four-point scale ranging from 1 (seldom) to 4 (very often).
Statistical analysis
Data of two groups were compared by the Student's ttest or the Mann-Whitney test. The frequencies were compared between two groups by the χ 2 test. The correlations between body weight, HbA1c and other parameters were first analyzed by simple regression analysis and then by multivariate stepwise analysis. In all cases, p values <0.05 were considered statistically significant. All analyses were performed with the JMP Statistical Discovery Software 8.0 (SAS Institute, Cary, NC).
Results
Characteristics of participants Table 1 shows the baseline characteristics of 86 subjects before liraglutide introduction. The mean age and body mass index (BMI) were 59.8 years and 27.3 kg/m 2 , respectively, and BMI ranged from 15.8 to 46.5 kg/m 2 . Fifty-nine subjects (69%) were obesity in Japanese criteria (BMI ≥ 25 kg/m 2 ). The duration of diabetes was 15.8 years and the mean hemoglobin A1c (HbA1c) was 8.5%. Eighty-six percent of patients had hypertension Patients were introduced with liraglutide treatment from 0.3 mg/day and its dosages were gradually increased according to glycemic control level by attending physicains. The number of patients treated with liraglutide at 0.3, 0.6, and 0.9 mg/day was 7, 24, and 55 patients at 1 year after liraglutide introduction, respectively, and such number of patients was 2, 8, and 36 patients at 2 year after liraglutide introduction, respectively.
Changes of body weight and HbA1c at 1 year after liraglutide introduction
In Figure 1, the individual changes in body weight and HbA1c from baseline to 1 year are shown as a scatter plot. The number of patients who reduced body weight was 69 cases (80.2%), and that of who improve glycemic control was 58 patients (67.4%). Forty-six patients (53.5%) were located in the lower left quadrant, i.e. both body weight and HbA1c were decreased in these patients. Table 2 summarizes correlation analyses for the reduction of body weight or HbA1c from baseline to 1 year with baseline clinical parameters. Body weight reduction was associated positively with baseline BMI, aspartate aminotransferase (AST), alanine aminotransferase (ALT), baseline insulin dose, and previous insulin use. Stepwise regression analysis demonstrated that baseline BMI (P = 0.007) and previous insulin use (P = 0.027) were significantly and independently correlated with body weight reduction at 1 year. Reduction of HbA1c was associated positively with baseline HbA1c and negatively with insulin use for previous treatment. Lipids such as low-density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) tended to be correlated with change in HbA1c. Stepwise regression analysis demonstrated that baseline HbA1c (P < 0.001) was correlated significantly and independently with the reduction of HbA1c.
To exclude the effect of other anti-diabetic agents on body weight and glycemic control, we collected 35 patients whose anti-diabetic agents except liraglutide were not altered from baseline to 1 year ( Table 3). Reduction of body weight was correlated significantly and positively with baseline BMI, ALT and the scores of the questionnaire for eating behavior, and was correlated negatively with systolic blood pressure. Stepwise regression analysis demonstrated that baseline BMI tended to be associated with body weight reduction. Reduction of HbA1c was correlated positively with baseline BMI, HbA1c, LDL-C, TG, and ALT, and negatively with baseline HDL-C and previous insulin use. However, there were no significant associations with the reduction of HbA1c and the baseline clinical parameters by stepwise regression analysis.
Changes in body weight, HbA1c, lipid profile and hepatic enzymes for 2 years The 46 patients who continued to be treated with liraglutide over 2 years were next analyzed. BMI at 0, 3, 6, 9, 12, and 24 months were 27.3 ± 5.4, 26.3 ± 4.9, 25.8 ± 5.1, 25.9 ± 5.9, 25.8 ± 5.0, and 25.9 ± 4.8 kg/m 2 , respectively. Percent change in body weight indicated that body weight was significantly reduced and its reduction was maintained over 4% for 2 years (Figure 2A). HbA1c was significantly reduced at 3 months (7.4 ± 1.1%), and tended to increase at 6, 9, 12, and 24 months (7.7 ± 1.3%, 7.7 ± 1.3%, 7.8 ± 1.4%, and 7.7 ± 1.2%, respectively). Significant decrease of HbA1c was observed until 2 years after liraglutide introduction, compared to baseline HbA1c ( Figure 2B). Lipids ( Figure 3) and hepatic enzymes ( Figure 4) were also measured. LDL-C level was significantly reduced at 6 and 12 months, while high-density lipoprotein-cholesterol (HDL-C) tended to increase at 12 months (P = 0.098 versus 0 month). There were no significant changes of TG level from 0 to 24 months. AST level was significantly reduced at 3 months and tended to decrease at 12 months (P = 0.095 versus 0 month). ALT level also tended to Correlation of baseline clinical parameters with the reduction of body weight or HbA1c at 2 years Table 4 summarizes correlation analyses for the reduction of body weight or HbA1c from baseline to 2 years with baseline clinical parameters. Reduction of body weight was positively correlated with baseline BMI, waist circumference (WC), and insulin dose. Stepwise regression analysis demonstrated that baseline BMI and insulin dose was significantly associated with body weight reduction for 2 years. Reduction of HbA1c was correlated positively with baseline HbA1c and LDL-C, and it was correlated negatively with sulfonylurea use for previous treatment. Stepwise regression analysis demonstrated that baseline HbA1c was significantly associated with the reduction of HbA1c.
Discussion
We demonstrated that liraglutide treatment decreased body weight and visceral fat adiposity, and ameliorated eating behavior until 6 months after liraglutide initiation in Japanese obese type 2 diabetes subjects [10,11]. However, further long-term effect of body weight reduction and glycemic control by liraglutide has not been examined in Japanese type 2 diabetes subjects. Present study for the first time demonstrates the efficacy of liraglutide on weight reduction and fair glycemic control over 2 years in Japanese type 2 diabetes subjects. There are few reports describing the effect of liraglutide on body weight over 2 years. The LEAD-3 sub-analysis showed the significant weight reduction by liraglutide treatment for 2 years [8]. As shown in Figure 2A, liraglutide treatment for 2 years resulted in the 4.7% weight reduction (decrease of body weight: −3.4 kg from baseline). Present study suggests that over 4% of long-term body weight reduction can be expected by the low dose liraglutide treatment for Japanese patients, even with the additional other anti-diabetic agents causing weight gain such as insulin and sulfonylurea.
Evidently, the control of food intake cannot be ignored in the treatment of obese type 2 diabetes, but it is often difficult to control appetite and reduce adiposity in obese subjects. GLP-1 delays gastric emptying and induces satiety, which is probably related to the combined effect of GLP-1 on the gastrointestinal tract and the brain [12,13]. Peripheral administration of liraglutide or lixisenatide can cross the blood brain barrier and enhanced cAMP level in brain, suggesting that GLP-1 receptor agonists directly acts on central nerve system [14]. Gastrointestinal and central nervous effect of GLP- 1 receptor agonists synergistically exhibits the decrease of energy intake and body weight [15,16]. Our previous studies demonstrated that liraglutide improved eating behavior in obese type 2 diabetes subjects until 6 months after liraglutide introduction [10,11]. As shown in Table 3, reduction of body weight was correlated positively with the baseline scores of eating behavior in patients whose combined anti-diabetic agents were unchanged for 1 year. Present study suggests that liraglutide effectively reduces body weight especially in obese patients with the disordered eating behavior. As in Figure 1, 46 patients (53.5%) resulted in the amelioration of both body weight and glycemic control. However, 23 patients (26.7%) revealed weight reduction but deterioration of glycemic control, while 12 patients (14.0%) showed improvement of glycemic control but weight gain. These results may imply that liraglutidemediated ameliorations of body weight and glycemic control are independent and possibly by different mechanisms in GLP-1 actions. Fadini et al. recently reported in 166 patients (the average follow-up time: 9.4 months, the average daily dose of liraglutide at 16 months: 1.37 mg/day) that significant determinants of weight reduction or glycemic control were baseline BMI, or baseline HbA1c and previous insulin use, respectively [17]. Similar to their report, in the current study, significant determinants for the reduction of body weight or HbA1c from baseline to 2 year were baseline BMI and insulin dose, or baseline HbA1c, respectively (Table 4). Recent and current studies show that baseline BMI and HbA1c may be important predictors for weight reduction and glycemic control, respectively, before liraglutide therapy. Aiming at body weight reduction, especially in obese subjects with insulin therapy, the switch to liraglutide treatment may be more effective and beneficial in clinical use. Several pleiotropic effects of GLP-1 receptor agonists have been demonstrated. GLP-1 receptor activation has revealed the cardiovascular protection [18,19] and thus GLP-1 receptor agonist may be considered a promising new agent for the treatment of cardiovascular diseases linked to obese type 2 diabetes [20,21]. GLP-1 receptor agonists have been shown to suppress glucagon secretion in mice and human [22]. Glucagon-mediated hyperglycemia may be attenuated by liraglutide treatment, but glucagon level was not monitored in present study. GLP-1 receptor agonists have been also shown to improve other cardiometabolic parameters such as lipid profile and blood pressure following 5-10% weight reduction [23]. Similar to previous reports [23,24], in present study, LDL-C was significantly reduced at 6 and 12 months even under statin treatment in 55% of patients. By using questionnaire for eating behavior, we previously showed the significant reduction of preference for fat at 6 months after liraglutide introduction, suggesting that the reduction of dietary fat intake partly contributed to the decrease of LDL-C level. Liraglutide treatment also showed the slight reduction of hepatic enzymes (Figure 4), which may indicate the improvement of fatty liver.
Present study has several limitations. This study is an observational study, not a randomized clinical trial (RCT) study. Dosage of liraglutide and combination with other glucose-lowering agents were depended on the attending physicians.
In summary, long-term treatment with liraglutide effectively reduced both body weight and HbA1c, and also improved lipid profile and liver enzymes in Japanese patients with type 2 diabetes. Significant independent determinants of weight reduction were baseline BMI and previous insulin therapy, suggesting that effect of liraglutide on weight reduction is further expected in obese type 2 diabetes patients with previous insulin therapy. Longer-term randomized clinical trials are warranted to more thoroughly elucidate the effect of liraglutide on body weight and complications of metabolic syndrome. | 2017-06-30T15:24:07.477Z | 2014-09-08T00:00:00.000 | {
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248931639 | pes2o/s2orc | v3-fos-license | The role of green finance in eradicating energy poverty: ways to realize green economic recovery in the post-COVID-19 era
Realizing green economic recovery and eradicating energy poverty have become China’s strategic priorities in the post-COVID-19 era. In the context of the active advocacy of green finance, to empirically investigate whether green finance can help eradicate energy poverty, this study utilizes provincial sample data to explore the energy poverty eradication effect of green finance. Our study also examines the regional heterogeneity and mediating effect. The main findings are as follows: (1) Green finance is a powerful weapon to alleviate China’s energy poverty and accelerate green economic recovery, indicating that the green evolution of financial institutions is effective means to facilitate green economic recovery in the post-COVID-19 era; (2) green finance only eradicates energy poverty in low energy poverty regions and the eastern areas, and green finance can alleviate energy poverty in both high and low green finance areas; and (3) improved green finance not only directly eradicates China’s energy poverty, but also alleviates current energy poverty by accelerating technical innovation and optimizing the industrial structure. Following the above main findings, this study advances a series of policy implications in terms of facilitating the green transition of the financial industry and realizing green economic recovery.
Introduction
In 2015, all members of the United Nations adopted the 2030 Sustainable Development Agenda which incorporates Sustainable Development Goals (SDGs) to ensure universal access to affordable, reliable, and modern energy services until 2030 (UN 2020). However, according to statistics from the International Energy Agency (IEA), approximately 670 million people worldwide cannot obtain sufficient electricity by 2030 under the current policies (IEA 2021). At the same time, the 26th United Nations Climate Change Conference (COP26) held in Douglas UK in November 2021, discussed in-depth global actions to reduce emissions (Dwivedi et al. 2022). Calls for a renewable energy transition are growing as commitments to net-zero emissions increase around the world to achieve the 1.5 °C target set by the Paris Agreement. Therefore, to meet the SDGs and emission-reduction initiatives as well as achieve the green economic recovery in the post-COVID-19 era across the globe, finding ways to establish clean renewable energy to produce electricity has become the focus of global future development Taghizadeh-Hesary et al. 2021). On this basis, according to the requirements of SDGs, the IEA (2021) defines energy poverty as a lack of clean fuels such as electricity and cooking facilities, and highly relying on traditional solid biomass and firewood.
As the world's largest energy consumer and most populous country, China has faced severe energy poverty in the past century. However, with the continuous emission-reduction efforts of the Chinese government, preliminary mitigation results have been achieved (Dong et al. 2020;Cheng et al. 2021;Ren et al. 2022). From 2000 to 2019, the ratio of people obtaining electricity in China rises from 98.6% to 100%, and the residents using clean cooking have also increased from 46.8% to 71.3% (IEA 2020; Zhao et al. 2021a). China's per capita electricity consumption has exceeded that of average upper-middle income countries. However, due to China's huge population, the large gap between rich and poor, and uneven regional development, the country still suffers from energy poverty (Lin and Wang 2020). According to data from China Family Panel Studies (CFPS), in 2014, 41% of households in rural China still depended on firewood for cooking fuel (CFPS 2022). As the COVID-19 continues to evolve, actions such as layoffs or quarantines have significantly reduced income levels, further exacerbating energy poverty. Accordingly, energy poverty is still the top priority in China's poverty alleviation efforts.
In view of the important influence of energy poverty in China, many scholars have explored different ways to reduce energy poverty, such as low-carbon energy transition (Dong et al. 2021a, b), energy security , off-farm work (Lin and Zhao 2021), renewable energy consumption (Zhao et al. 2022), and addressing income inequality (Igawa and Managi 2022). However, some scholars have pointed out that green finance has a tendency to reduce energy poverty, which is a reaction to insufficient renewable energy investment (Markham et al. 2014;Mohsin et al. 2021;Yang et al. 2021). On the one hand, green finance is recognized as an effective tool to achieve green economic growth and structural transformation, and the use of green finance can actively facilitate green transition, especially in the post-COVID-19 era . On the other hand, green finance also contributes to the development of clean energy technologies and investments, and enhances technical innovation intensity, thereby reducing energy poverty (Taghizadeh-Hesary and Yoshino 2020; Li et al. 2021). Under the United Nations Framework Convention on Climate Change, full implementation of the Paris Agreement which aims to achieve the 1.5 °C target will require 1.5 trillion dollars in green financing annually by 2030 (Azhgaliyeva and Liddle 2020). Therefore, it is imperative to examine whether developing green finance is an effective way to address energy poverty and facilitate green economic recovery in the post-COVID-19 era. Accordingly, in this paper, we will examine the following three questions in detail: (1) Does green finance have the potential to eradicate China's energy poverty and accelerate green economic recovery in the post-COVID-19 era? (2) Does green finance have heterogeneity impacts on energy poverty? (3) What is the mechanism by which green finance affects energy poverty? To solve the above issues, this study explores whether green finance has the potential to eradicate energy poverty by using balanced sample data in 30 provinces in China for the period 2004-2018. Furthermore, we examine the heterogeneity and mediating mechanisms in the green finance-energy poverty nexus.
Consequently, this paper contributes to the existing literature in the following three ways. First, we empirically discuss whether developing green finance has the potential in alleviating energy poverty and facilitating green recovery of the economy in the post-COVID-19 era; this exploration has implications for the Chinese government and even many governments in developing countries, and provides a valuable reference for them to formulate energy mitigation policies in the future. Second, this paper investigates the heterogeneity of green finance development on energy poverty, which has important benefits for China in narrowing the uneven urban-rural energy development level and the energy poverty gap. Third, we also examine the mechanism through which green finance alleviates energy poverty. This discussion provides a reference direction for the government to develop policies and measures related to fuel poverty by considering the roles of technical progress and industrial adjustment.
We present the remainder of this paper in the following sections. The next section provides the relevant literature on green finance and energy poverty, followed by the theoretical framework built in Sect. 3. Section 4 represents the methodology and data. Estimated findings of this study are illustrated in Sect. 5. Section 6 further explores the mediating role of technical progress and industrial adjustment in the green finance-energy poverty nexus. Section 7 concludes our study and develops a series of policy suggestions.
Research on green finance
To the best of our knowledge, green finance refers to financial instruments that provide environmental benefits. Traditional financial markets focus on the profitability of investment projects, and ignore the impact on the environment. As a result, green finance drives the transition of massive investments from those that result in high pollution to those that promote resource conservation (Yang et al. 2021). Cowan (1999) points out that green finance is concerned with the practical issues related to paying for the level of environmental protection rather than how or why or what the society decides. In 2000, the American traditional dictionary summarized the definition of green finance. The dictionary states that as part of environmental economy, green finance should facilitate the evolution of environmental finance and environmental industry through various financial instruments, so as to realize the protection of the environment and biodiversity .
Based on the above scholars' definition of green finance and the vigorous publicity and widespread popularization of green concepts in the global financial industry, a growing body of scholars has conducted in-depth explorations on green finance (Weber and ElAlfy 2019;Zhang et al. 2019b;Akomea-Frimpong et al. 2021). Since China became the world's largest energy consumer and greenhouse gas emitter, and also the largest green bond issuer between 2016 and 2019 (Azhgaliyeva and Liddle 2020), the evolution of its green finance has received increasing attention (Lv et al. 2021;. For example, Yin and Xu (2022) explore the relationship between green finance and economic level in China using a coupling coordination degree model. Their results indicate that green finance is lagging behind economic growth. use the grey correlation method to empirically test the relationship between green finance and the upgrading of China's industrial structure. Their results imply that green finance produces the greatest driving effect on tertiary industry, facilitating the rapid development of tertiary industry and promoting upgrading of the industrial structure. Yang et al. (2021) quantitatively explore the linkage between green finance, financial technology, and high-quality evolution. Their findings provide strong support for the effective role of green finance in promoting high-quality economic evolution. However, green finance requires funds not only to invest in projects such as green industries and environmentally friendly infrastructure, but also to consider potential environmental impacts (Khan et al. 2021;Yin and Xu 2022). Zhou et al. (2020) empirically check the role of green finance in the financial industry in the coordinated development of the economy and environment by using Chinese provincial data from 2010 to 2017, and point out that green finance can achieve a win-win situation for both environmental quality and economic development. Muganyi et al. (2021) apply the semi-parametric difference-in-difference method to find that the relevant policies of green finance generally reduce industrial gas emissions in China.
Research on the green finance-energy poverty nexus
Some scholars believe green financing will help alleviate energy poverty. This is because green finance can speed up the energy transition and economic development (Hafner et al. 2021;Ngo et al. 2021), make modern energy affordable in countries living at lower levels of development, and alleviate energy shortages. For example, Setyowati (2020) points out that the Indonesian government should mobilize the private sector to provide large-scale climate finance for renewable energy generation, which is critical for Indonesia to achieve its renewable energy goals and provide electricity to people living in energy poverty. After examining the relationship between factors such as energy poverty and economic development in seven South Asian countries, Amin et al. (2020) find that increasing the proportion of capital investment in renewable energy-related sectors is conducive to the rapid growth of economy and the mitigation of energy poverty. Azhgaliyeva and Liddle (2020) point out that investment in fossil fuel still dominates energy investment, which could threaten the expansion of green energy needed to deliver energy security and meet climate and clean air goals. Aassouli et al. (2018) also indicate that achieving the SDGs and sustainable energy inclusion in the sub-Saharan region requires them to focus on investment in green projects, so green finance is a good channel to address energy poverty. Moreover, energy access is critical for meeting a wide range of development challenges, including poverty reduction (Gujba et al. 2012; Ansu-Mensah and Kwakwa 2022). Therefore, many scholars have also explored the impact of green finance on energy access. Duppati et al. (2021) identify the size of the funds committed to energy access initiatives in the African region as critical in ensuring a positive relationship between green finance and electricity access. Okesola (2021) proposes bridging the energy supply gap and improving electricity supply by creating a federal green bank to promote investment in renewable energy.
Literature gaps
Although many scholars have systematically summarized green finance and its impact on the economy and environment, knowledge gaps still exist. First, a growing body of scholars has measured green finance from different perspectives; few scholars have assessed the current development situation of green finance, especially in China. Second, the potential effects of the evolution of green finance are explored from various aspects by many studies (see Sect. 2.1), relevant research on the green finance-energy poverty nexus is still in an exploratory stage, and related studies in China are still rare. Whether developing green finance in China can help facilitate green economic recovery in the post-COVID-19 era is also worth exploring. Third, the mechanism by which green finance affects energy poverty is still unclear, especially the typical ways of affecting technical progress and industrial adjustment. Filling the gaps of the above studies can help scholars and governments formulate green financial solutions to alleviate energy poverty more clearly.
Theoretical framework
In recent years, the connotation of green finance has been expanding, and a green financial service system with commercial banks as the core has been gradually formed (Taghizadeh-Hesary and Yoshino 2019). Green finance adopts financial instruments such as green stocks, green bonds, green insurance, and carbon finance to provide capital base for green and low-carbon economic transition. The construction of green financial system, the development of green financial products, and the introduction of relevant policies have laid favorable market conditions for the green development of different industries, especially the clean and renewable energy industry (Rasoulinezhad and Taghizadeh-Hesary 2022). Through the leverage of capital market, green finance can effectively redirect capital flow, restrain loans of polluting production projects, and actively guide the transfer of capital to green technology projects in the energy industry (Muganyi et al. 2021); more specifically, the government will introduce relevant policies to improve green investment bank, actively guide financial institutions to invest in the facility construction and technology research of renewable energy industries such as wind energy and solar energy, increase the total power supply, reduce the cost of clean energy, and gradually replace the use of traditional solid biomass and polluting fossil energy, thereby alleviating energy poverty.
In addition, the increasing introduction of green finance policies, on the one hand, will help support the implementation of green innovation systems (Molla et al. 2019;Zeng et al. 2022). Financial methods such as green credit and green bonds can effectively promote enterprises' investment in environmental protection equipment, strengthen the development of clean technology, and facilitate enterprises to become the backbone of green innovation. Furthermore, green finance is an effective marketoriented means for local governments to gather social forces to serve the environmental protection industry. The research and development (R&D) of environmental protection technology has the characteristics of large investment and long cycle (Huang et al. 2021). It is necessary to reduce the cost and risk of the development of enterprises through financing means, and maintain the enthusiasm and determination of enterprises and society to develop green technologies. On the other hand, the advocacy of green finance effectively reduces the capital accumulation of highpolluting industry (Zhang et al. 2021). Through the guiding role of capital, polluting and backward production capacity can be phased out in accordance with the principles of green, efficient, intensive, and safe, industrial optimization and upgrading can be accelerated. As Zhao et al. (2021a, b) and Wang et al. (2022) stress, the vigorous promotion of technical innovation and rapid upgrading of industrial structure can effectively facilitate the evolution of renewable energy industry, promote the optimization of energy system, adjust the energy supply and demand pattern, and thus alleviate energy poverty. To this end, we develop the following two hypotheses: Hypothesis 1 Green finance is a powerful weapon to eradicate energy poverty.
Hypothesis 2 Green finance can help eradicate energy poverty through industrial structure upgrading and technological innovation.
Model construction
Our paper aims to investigate whether green finance is a powerful weapon to eradicate energy poverty and promote green economic recovery in the post-COVID-19 era; that is, whether the increased evolution of green finance can eradicate China's energy poverty. In doing so, the empirical econometric model is constructed in the following equation: where the subscripts i and t refer to China's specific provinces and the sample period (i.e., 2004-2018), respectively. EPI represents the energy poverty composite index, GFI indicates the green finance index, Tec denotes technical process, Ind means industrial structure adjustment, Pgdp stands for economic level, Edu represents education level, FDI refers to foreign investment level, and Wag indicates residents' wage level. 0 is the intercept term, and k (k = 1, 2, ..., 7) are the coefficients that need to be estimated. i , v t , and it refer to the provincial effect, time effect, and error term, respectively. In this equation, we expect the coefficient of green finance (i.e., 1 ) to be negative.
Considering the potential dynamic effect of the green transformation of financial institutions to eradicate energy poverty, we introduce the lag term of energy poverty into Eq. (1) for econometric regression; the specific equation is illustrated as follows: where 0 represents the intercept term, and k (k = 1, 2, ..., 8) refer to the coefficients of the variables that need to be assessed.
Regarding the estimated method for the dynamic panel model, the generalized method of moments (GMM) technique, which performs the first difference on Eq.
(2), is used to solve potential endogeneity within the econometric equation; Eq.
(2) in the first-order difference is shown as follows: (1) where Δ refers to the first-order difference form of the variables, and Z denotes a vector consisting of Tec, Ind, Pgdp, Edu, FDI, and Wag. Two main types of GMM estimation method-differential GMM (Diff-GMM) and system GMM (Sys-GMM)-are applied in the econometric regression in our study. It is worth noting that the latter (i.e., Sys-GMM) incorporates Eqs. (2) and (3) into a unified framework for systematic analysis, which can more effectively address potential endogeneity issues and provide consistent estimates compared to the former (i.e., Diff-GMM) (Pan et al. 2021;Zhao et al. 2021b); therefore, this study employs the estimated results of the Sys-GMM strategy as the benchmark regression.
Explained variable
Energy poverty (EPI). In recent years, China has achieved remarkable results in energy poverty (Zhang et al. 2019a;Dong et al. 2021b); thus, to effectively evaluate the current situation of energy poverty in China, this study constructs a comprehensive index system of energy poverty by referring to the relevant studies of Wang et al. (2015) and Zhao et al. (2021a), which mainly include four aspects: energy access capability, energy cleanliness, energy infrastructure completeness, and energy efficiency & resident affordability; the specific measurement, code, and property of the indicators are presented in Table A1 in the Appendix. In this indicator system, significant differences exist in the dimensions of each indicator. Thus, we utilize the improved entropy method to gauge China's energy poverty. The specific calculation steps of this technique can refer to the work of Zhao et al. (2021a). We also calculate the sub-index of the four aspects, i.e., energy access capability index (EP_1), energy cleanliness index (EP_2), energy infrastructure completeness index (EP_3), and energy efficiency and resident affordability index (EP_4). The relevant data of the indicators are obtained from the China Statistical Yearbook (CSY 2021) and the China Energy Statistical Yearbook (CESY 2021).
After calculating the composite index and sub-indexes of energy poverty, we first draw the time trend chart of average energy poverty to describe the actuality of energy poverty eradication (see the line chart in Fig. 1). Clearly, China's energy poverty has been significantly reduced over the sample period. Furthermore, the spatial pattern of energy poverty in 2004, 2009, 2014, and 2018 is presented in Fig. 2 due to the space limitations of the article. We find that significant regional heterogeneity exists across different provinces. For example, energy poverty in the northern provinces is much worse than that in the eastern coastal provinces.
Explanatory variables
Green finance (GFI). As a product of green economy, energy finance can effectively facilitate the flow of capital to the field of environmental protection and green technological innovation, so as to prevent environmental degradation. Similar to energy poverty, we also build a comprehensive system for China's energy finance that incorporates economy, finance, and the environment (Jiang et al. 2020) (see Table A2).
To be specific, three indicators are included in economy and eight indicators are included in finance, respectively, and environment consists of six indicators. By Fig. 3 Spatial distribution of China's green finance index in selected years and China Regional Financial Operation Report provide data on the environment and finance, respectively. The time trend of average green finance is shown in the histogram in Fig. 1. We find that the average composite index of green finance shows a significant trend of fluctuation during the sample period and reaches its peak in 2011, and green finance has been on the rise from 2016 to 2018. More importantly, the spatial distribution of green finance is shown in Fig. 3. Notably, the green development level of the financial industry in the prosperous eastern coastal provinces and northern provinces is obviously better than it is the central provinces.
Control variables
In addition to energy poverty and green finance, a series of control variables are introduced in our econometric model, and the specific measures are as follows: Technical progress (Tec) is assessed by the number of domestic patent applications authorized, industrial structure adjustment (Ind) is calculated by the added value of tertiary industry divided by the added value of secondary industry; economic level (Pgdp) is assessed by per capita gross domestic product (GDP); education level (Edu) is evaluated by the ratio of the number of high school students of the total population; foreign investment level (FDI) is assessed by the registered capital of foreign-invested enterprises (in million US dollars); and wage level (Wag) is calculated by the average wage (yuan) of urban staff and workers on duty. The provincial data on technical progress, industrial structure adjustment, economic level, education level, foreign Table 1 lists the definitions and descriptive statistics of all the variables used.
Preliminary analysis
Prior to the benchmark estimates, we first check the correlation between variables (see Table 2). Obviously, the coefficients of green finance, technical progress, economic level, education level, foreign investment level, and wage level are all negative. Also, Fig. 4 shows the scatter plot between green finance (i.e., lnGFI) and energy poverty (i.e., lnEPI), which implies a negative role of green finance in eradicating energy poverty. In summary, the above correlation analysis provides preliminary findings for the positive energy poverty eradication effect of green finance development in China. Notably, to effectively assess the potential role of green development of China's financial industry in eradicating energy poverty, it is necessary to select more complicated and accurate empirical econometric models. Notably, before analyzing the benchmark findings, we find that the autocorrelation and overidentification tests are passed due to the significance of AR (1), AR (2), and the Sargan test. In this table, regardless of the static panel estimates or dynamic panel estimates, the coefficient values of green finance are significantly negative. This provides a robust regression result for green finance to help eradicate energy poverty. More specifically, the estimated results of Sys-GMM indicate that the coefficient of green finance (i.e., lnGFI) is − 0.308, which emphasizes that improved development of green finance is negatively associated with energy poverty; put differently, increased green finance is a powerful weapon to eradicate energy poverty. The reasons may be that the green transition of the financial industry, on the one hand, can effectively guide investments to support the infrastructure establishment of natural gas and clean energy, strengthen energy supply, and improve the availability of energy consumption for residents. In addition, the gradual evolution of green finance facilitates the development of the non-thermal power industry, and strengthens the transformation of the current energy structure towards modernization, thus accelerating the eradication of fuel poverty (Rasoulinezhad and Taghizadeh-Hesary 2022). In the post-COVID-19 era, as all industries are affected, the gradual investment of financial industry in environmental technologies has obviously stimulated the greening and modernization of the economy, and the increasingly clean energy sector has provided a realistic guarantee for the green economic recovery. On the other hand, while promoting the growth of the clean energy industry, the green investment of financial institutions can also strengthen the completeness of management facilities of related industries, facilitate green innovation, and mitigate the deterioration of the environment, thus alleviating energy poverty and facilitating the green recovery of the economy.
Benchmark estimates
Regarding the control variables, industrial structure adjustment, technical progress, and improved economic level can help eradicate energy poverty; this is basically consistent with the findings of Dong et al. (2021b). Adjusting the current industrial structure can effectively accelerate the transition from the secondary industry dominated by heavy industry to a green high-tech secondary and tertiary industry, which will not only strengthen the concentration of the population and resources, but also stimulate the development of the renewable energy industry. More importantly, technical Table 3 Estimated results of the impact of green finance on energy poverty *** , ** , and * refer to statistical significance at the 1%, 5%, and 10% levels, respectively; the values in parentheses indicate the z-statistics progress can significantly eliminate outdated polluting technologies, reduce fossil energy use, and improve energy efficiency, thus alleviating energy poverty (Tu and Rasoulinezhad 2021). In addition, the strengthening economic level can lay a solid economic foundation for the rapid evolution and technological innovation of the country's clean energy industry. However, rising levels of education and household wages are not conducive to eradicating energy poverty, which is contrary to our expectations. For education, the zero effect may reflect the fact that the education does not include sufficient sustainability-related elements. For wages, the positive wage-energy poverty nexus might be linked to the so-called rebound effect.
Robustness test 1: estimation of the four indexes of energy poverty
To empirically check the robustness of the negative green finance-energy poverty nexus, we first re-estimate Eq.
(2) by applying the four indexes of energy poverty (i.e., lnEP_1, lnEP_2, lnEP_3, and lnEP_4) to replace the composite index of energy poverty (i.e., lnEPI) by using the Sys-GMM technique; the corresponding estimates are shown in Table 4. The coefficient of green finance in the first, third, and fourth columns of Table 4 is significantly negative, while regarding lnEP_2, the coefficient of green finance is positive. This implies that the rapid development of green finance can alleviate energy poverty in terms of energy access capability, the completeness of the energy infrastructure, and energy efficiency & resident affordability, but has little effect on poverty alleviation in terms of energy cleanliness. In addition, the coefficients indicate that the positive alleviating effect of green finance on poverty in terms of availability, management completeness, and affordability can offset the inhibiting effect on cleanliness. Thus, local governments should strongly support the development of green finance to achieve the effect of killing two birds with one stone, that is, the coordinated development of green finance and energy poverty eradication. This verifies the robustness of the benchmark conclusion: green finance can effectively help alleviate energy poverty.
Robustness test 2: alternative measures of energy poverty
The widespread use of natural gas and the vigorous advocacy of electricity consumption are effective measures for addressing the problem of energy poverty Zhao et al. 2021a). Thus, in this section, we proceed to check the reliability of the estimated results by employing the alternative measures of energy poverty alleviation-replacing the composite index of energy poverty (i.e., lnEPI) with per capita electricity consumption (i.e., EC) and per capita natural gas consumption (i.e., NG) for econometric regression based on the FGLS and Sys-GMM techniques.
The results are illustrated in Table 5. It can be found that the green evolution of the financial industry can facilitate the increase of household electricity and natural gas consumption, and the stimulus effect on natural gas is obviously better than that on electricity consumption. This finding also supports the negative linkage between green finance and energy poverty.
Robustness test 3: alternative estimation technique
In this section, we further verify the robustness by using the alternative estimated technique-the instrumental variable (IV) strategy proposed by Lewbel (2012). It is worth noting that this method mainly examines the causal relationship between green finance and energy poverty in China by constructing an instrumental variable based on heteroscedasticity in the random disturbance term (i.e., it ). We report the regression results of Lewbel's (2012) IV method in Table 6, and find that the potential effect of green finance on energy poverty and its four sub-indexes are consistent with the benchmark regression results, which significantly proves the robustness and reliability of the negative green finance-energy poverty nexus.
Provincial heterogeneous analysis
The provincial spatial distribution of green finance and energy poverty shows that significant regional heterogeneity exists in China's green finance and energy poverty across different provinces. Thus, to comprehensively and accurately assess the differential impact of China's green finance in eradicating energy poverty, we first divide the whole sample into high energy poverty (high EPI) areas, low energy poverty (low EPI) areas, high green finance (high GFI) areas, and low green finance (low GFI) areas according to the mean values of energy poverty and green finance in every province in 2018; the specific provinces of high EPI, low EPI, high GFI, and low GFI are shown in Table A3. After the division of areas, we further evaluate the potential effect of green finance in eradicating energy poverty in different areas, and present the corresponding results in Table 7. In the high EPI area, the coefficient is 0.093, while in the low EPI area, the coefficient is − 0.225. This implies that the green transformation of financial institutions can effectively alleviate energy poverty in low EPI areas, but the effect on energy poverty eradication in high EPI areas is not obvious enough. From the spatial pattern of energy poverty, the low EPI areas are distributed mainly in economically prosperous provinces. The strong economic foundation can guarantee an improved financial system in these provinces and contribute to the alleviation of energy poverty. However, Hansen J 0.000 0.000 0.000 0.000 0.000 Table 7 Estimated results of the regional heterogeneous analysis *** , ** , and * refer to statistical significance at the 1%, 5%, and 10% levels, respectively; the values in parentheses indicate the z-statistics in high EPI areas, economic development is backward, the financial systems are relatively incomplete, and the alleviating effect of energy poverty is limited. In addition, green finance has a positive correlation with energy poverty alleviation in both high and low GFI areas, and the alleviating effect of energy poverty in high GFI areas of China is more significant than that in low GFI areas.
In addition, we divide the full sample into eastern, central, and western regions based on geographical location with reference to the classification standards of the National Bureau of Statistics of China; the estimated results are reported in the last three columns of Table 7. We find that green finance can effectively contribute to energy poverty alleviation only in the eastern provinces. Against the current background, it is clear that the huge economic base, complete financial institutions, superior geographical location, and convenient transportation conditions of the eastern provinces can provide a sufficient basis and reasonable opportunities for energy poverty alleviation.
Model construction
After conducting the benchmark estimates on the green finance-energy poverty nexus and analyzing the regional heterogeneity, we examine in greater depth how green finance alleviates energy poverty, which can provide new ideas for further discussion on green economic recovery in the post-COVID-19 era from the perspective of green finance. In the benchmark estimates, technical progress and industrial structure adjustment have a particularly significant stimulating effect on fuel poverty eradication; accordingly, we attempt to discuss the mediating role of these two variables in affecting the green finance-energy poverty nexus. In this regard, the mediating effect model is a wise choice to deal with such problems, and the specific equations of the mediating effect model are constructed as follows: where 0 and 0 refer to the constant terms, and k (k = 1, 2, ..., 6) and k (k = 1, 2, ..., 6) represent the coefficients that need to be assessed. M indicates technical progress and industrial structure adjustment. The variables and parameters are consistent with Eq. (2). Notably, only when green finance significantly affects ln EPI it = 0 + 1 ln EPI i,t−1 + 2 ln GFI it + 3 ln Tec it + 4 ln Ind it energy poverty, that is, the coefficient of green finance in Eq. (4) (i.e., 2 ) is significant, can we further test the direct and indirect effects in the mediating effect model. Furthermore, the coefficient of green finance in Eq. (6) refers to the direct role of green finance on energy poverty, and only when the coefficients of green finance in Eq. (5) and technical progress as well as industrial structure adjustment in Eq. (6) are all significant can the existence of indirect effect be confirmed. The product of the coefficients (i.e., 2 3 and 2 4 ) indicates the magnitude of the indirect effect of technical progress and industrial structure adjustment, respectively.
Results and discussion
By applying the Sys-GMM strategy, we estimate Eqs. (4)-(6) and report the empirical results in Table 8. In the first column of this table (i.e., Model (1)), we find that Table 8 Estimated results of the mediation effects *** , ** , and * refer to statistical significance at the 1%, 5%, and 10% levels, respectively; the values in parentheses indicate the z-statistics
Variable
Model (1) Model (2) Model ( the coefficient of green finance is -0.383, which indicates the empirical results of the total effect of green finance on eradicating energy poverty. Furthermore, Model (2) presents the potential effects of green finance on technical progress and industrial structure adjustment. In these two columns, the estimated coefficients of green finance in technical effect and structure effect are 0.370 and 0.020, respectively. This implies that: (1) the green transformation of the financial industry can strengthen regional technical innovation activities and accelerate the process of industrial structure adjustment; (2) the stimulating effect of green finance on technical innovation is significantly greater than that on promoting industrial transformation. Against the background of the steady progress of green finance policies, the rapid development of the green finance industry actively invests in economic activities that prevent environmental degradation and conserve resources, effectively guides the transfer of limited resources from high-polluting industries to low-polluting industries, and accelerates the process of technical activities and industrial structure adjustment. The coefficient of green finance in Model (3) is − 0.308, which denotes the direct effect of green finance on energy poverty. Furthermore, the estimated coefficients of technical progress and industrial adjustment are − 0.063 and − 0.219, respectively. Thus, 2 * 3 = 0.370 * (−0.063) = −0.0233 and
Conclusions and policy suggestions
To empirically investigate whether green finance vigorously advocated by local governments is conducive to alleviating China's current energy poverty and promoting the green economic recovery in the post-COVID-19 era, we examine the dynamic green finance-energy poverty nexus by applying provincial sample data for the period 2004-2018. Furthermore, this study further explores the potential regional heterogeneity across different regions and the mediating effects of technical progress and industrial structure adjustment. The primary findings are presented as follows: First, the main conclusion of this study is related to the negative green finance-energy poverty nexus. The estimated results of the benchmark regression report that the coefficient of green finance is − 0.308, which suggest that the rapid promotion of green finance can effectively facilitate poverty eradication and accelerate green recovery of the economy in the post-COVID-19 era. This finding is also verified by a series of robustness tests.
Second, the analysis of provincial heterogeneity shows that green finance is negatively related to energy poverty in low energy poverty areas, while in high energy poverty areas, improved green finance cannot contribute to energy poverty eradication. A significant negative correlation between green finance and energy poverty exists in high and low energy poverty areas. In addition, green finance can only eradicate energy poverty in the eastern regions.
Third, from the analysis of the mediating effect model we find that green finance not only helps eradicate China's energy poverty directly, but also indirectly facilitates energy poverty alleviation by accelerating technical innovation activities and adjusting industrial structure.
The discussion on the green finance-energy poverty nexus in this study provides an effective reference for alleviating existing energy poverty and facilitating green economic recovery. Based on the negative relation between green finance and energy poverty, we develop the following policy implications.
First, the primary finding highlights the important role of China's green finance in facilitating energy poverty alleviation and accelerating the green economic recovery in the post-COVID-19 era. Accordingly, continuing to vigorously advocate the green transformation of financial institutions and formulate relevant policies and regulations to promote the green evolution of the financial industry is crucial. Currently, green finance has become a vital pillar of the green economy. To effectively achieve a win-win situation of continuous green evolution of finance and energy poverty eradication, on the one hand, local governments should set up provincial green The influence mechanism between green finance and energy poverty finance-development institutions. For instance, Gansu and Guizhou provinces have successively established green finance innovation and development-leading groups. Establishing a strong regional leadership center can effectively enhance high-level design. In addition, the formulation of a construction plan for a green finance system can send a positive signal to society that the government focuses on green finance, so as to clarify the goals and tasks of relevant departments and institutions in green finance. Financial regulators should also provide basic guarantees for increasing the growth of green finance. A positive incentive mechanism to facilitate the development of green finance, establishing and improving relevant systems of green credit and green insurance, and strengthening theoretical research and publicity related to green finance are all effective strategies to facilitate the green evolution of the financial industry.
Second, our results highlight the regional heterogeneity of green finance in energy poverty alleviation. In other words, significant differences in the effects of green finance exist across different regions. Therefore, when local governments actively formulate relevant strategies to promote the green evolution of finance, they should implement them in more detail, according to the local actual situation. For example, in the prosperous eastern coastal provinces, provincial governments should strengthen theoretical research on green finance and set an example for other provinces. However, in the underdeveloped central and western regions, strengthening the infrastructure construction of green finance and improving the financial institution system are more effective measures.
Third, the mediating role of technical progress and industrial optimization between green finance and energy poverty can improve the current situation of energy poverty in China. Therefore, local governments should provide financial and policy support to enterprises actively carrying out green technical innovation, and effectively solve the financial and institutional obstacles in the current process of enterprise transformation. At the same time, local governments should vigorously advocate and publicize green economic transformation and facilitate the increasing infiltration of the concept of green economic development. In addition, strengthening the rapid growth of the clean energy industry will not only facilitate the optimization and upgrading of the industry, but will also reduce the dependence of residents and enterprises on highly polluting fossil energy, thus achieving a win-win situation of green finance and energy poverty alleviation.
The current research on the green finance-energy poverty nexus only assesses the potential role of the evolution of green finance in eradicating energy poverty in China, research gaps still exist. One is associated with the research sample. In our study, we only check whether the development of green finance can help eradicate energy poverty in China, it is necessary for us to explore the nexus in other economies, especially in the global case. Another shortcoming is related to influencing mechanism. The underlying effects of technical progress and industrial adjustment are explored by using the mediation effect model; other influencing channels such as energy consumption structure and financial structure also deserve further discussion. | 2022-05-21T15:19:47.592Z | 2022-05-19T00:00:00.000 | {
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221571217 | pes2o/s2orc | v3-fos-license | Impact of Albumin and Omeprazole on Steady-State Population Pharmacokinetics of Voriconazole and Development of a Voriconazole Dosing Optimization Model in Thai Patients with Hematologic Diseases
This study aimed to identify factors that significantly influence the pharmacokinetics of voriconazole in Thai adults with hematologic diseases, and to determine optimal voriconazole dosing regimens. Blood samples were collected at steady state in 65 patients (237 concentrations) who were taking voriconazole to prevent or treat invasive aspergillosis. The data were analyzed using a nonlinear mixed-effects modeling approach. Monte Carlo simulation was applied to optimize dosage regimens. Data were fitted with the one-compartment model with first-order absorption and elimination. The apparent oral clearance (CL/F) was 3.43 L/h, the apparent volume of distribution (V/F) was 47.6 L, and the absorption rate constant (Ka) was fixed at 1.1 h−1. Albumin and omeprazole ≥ 40 mg/day were found to significantly influence CL/F. The simulation produced the following recommended maintenance doses of voriconazole: 50, 100, and 200 mg every 12 h for albumin levels of 1.5–3, 3.01–4, and 4.01–4.5 g/dL, respectively, in patients who receive omeprazole ≤ 20 mg/day. Patients who receive omeprazole ≥ 40 mg/day and who have serum albumin level 1.5–3 and 3.01–4.5 g/dL should receive voriconazole 50 and 100 mg, every 12 h, respectively. Albumin level and omeprazole dosage should be carefully considered when determining the appropriate dosage of voriconazole in Thai patients.
Introduction
Patients with hematologic diseases that receive chemotherapy are at high risk for invasive fungal infection, which is a severe illness with high mortality [1][2][3]. Voriconazole is a broad-spectrum triazole antifungal that is highly effective against a wide range of yeasts and filamentous fungi [4]. In the treatment of invasive aspergillosis, voriconazole was shown to improve survival and have less
Population PK Model Development
The PK profile of voriconazole can best be described using the one-compartment model with first-order absorption and elimination (see the model code in the Supplementary Materials). The two-compartment model did not adequately fit the data. Additionally, nonlinear (Michaelis-Menten) elimination did not improve the model fit. The apparent oral clearance (CL/F), the apparent volume of distribution (V/F), and the absorption rate constant (Ka) were estimated. The estimated Ka was not reliable, therefore, we decided to fixed it with a value from a previous population PK study (1.1 h −1 ) [25]. The sensitivity analysis of various Ka was shown in Table S1 in Supplementary Materials. The interindividual variability (IIV) of CL/F was described using an exponential model. The addition of IIV for V/F failed to fit the data, and estimates were near parameter boundaries. The objective function value (OFV) of allometric scaling was not significantly different from the base model. Thus, the variability in Ka and V/F was not estimated. The residual variability (RUV) was described using an additive model. The shrinkage value of CL/F was 11%, indicating enough data available for each patient to estimate the individual parameters reliably.
Covariate analysis showed that clearance was significantly affected by albumin and dose of omeprazole ≥ 40 mg/day. CYP2C19*2/*3 and sulfamethoxazole/trimethoprim were found to be significant only during forward selection. Age, gender, weight, CYP2C19 genotypes or phenotypes, AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase), TB (total bilirubin), DB (direct bilirubin), and co-medication, including omeprazole 20 mg/day and hormones, were not found to be significant covariates.
As the only two significant covariates, albumin and omeprazole ≥ 40 mg/day were included in the final model. These factors resulted in a 15.4% drop in the IIV of CL/F from the base model, and a drop in the OFV of 22.657 points (p < 0.001). The equation used in the final model is shown, as follows: where ALB is the plasma albumin (g/dL), and OME = 0 for patients receiving omeprazole 20 mg/day or less, and 1 for patients receiving a dose of omeprazole ≥ 40 mg/day. Table 2 shows the analysis that compared the 1000 bootstraps with the final model. The parameter estimates obtained from bootstrap were similar to the NONMEM estimates, which demonstrates the stability of the parameter estimates from the final model. The goodness-of-fit (GOF) evaluation was performed by plotting the corresponding individual predictive values (IPRED) and population predictive values (PRED) with the observed values, as well as the time and PRED with conditional weighted residual errors (CWRES). These plots showed good scattering with all points located within ±4, and most points within ±2 (Figure 1). Prediction-corrected visual predictive check (pcVPC) and visual predictive check (VPC) were used to test the predictability of the final model. The results of pcVPC indicated that the final model had adequate predictive properties. Figure 2 shows the median, 5th percentile, and 95th percentile of the observed plasma concentrations that were included in the range of the 95% confidence interval (CI) of these percentiles for the simulated data. Those findings suggested the sufficiency of the model's predictive power. Figure S1 in Supplementary Materials shows the traditional VPC plot.
Simulation
Monte Carlo simulations were performed using the final model to determine the optimal dosing regimens for Thai patients. Steady-state trough or minimal concentrations (C min, ss ) of voriconazole were simulated using the final model that included the significant covariates for CL/F. Sixteen thousand (16,000) replicate C min, ss of voriconazole using the dosage regimens of 50, 100, 150, 200, 250, 300, 350, and 400 mg twice daily (2000 replicates per group) were simulated for each covariate subgroup. The simulated C min, ss were randomized according to various plasma albumin levels 1.5-4.5 g/dL (all plasma albumin levels ranged from 1.6 to 4.5 g/dL; median: 3.2 g/dL, IQR: 2.78-3.6 g/dL), and then these were classified into two omeprazole dosing groups as follows: 1000 replicates for non-omeprazole and omeprazole 20 mg/day, and 1000 replicates for omeprazole ≥ 40 mg/day. The simulated C min, ss in different subgroups are shown in Figures 3 and 4. . Simulated steady-state trough or minimal concentrations (C min, ss ) in the non-omeprazole group or omeprazole 20 mg/day group stratified by plasma albumin (Alb) and a maintenance dose of voriconazole administration (50, 100, 150, 200, 250, 300, 350, and 400 mg orally every 12 h of voriconazole). The round, square, and triangle lines show the percentage that achieved a therapeutic range (C min, ss = 1-5 mg/L), a toxic range (C min, ss > 5 mg/L), and a sub-therapeutic range (C min, ss < 1 mg/L), respectively.
For example, in the group that included non-omeprazole or omeprazole 20 mg/day and albumin 1.5-2 g/dL, the maintenance dose of voriconazole, 150 mg orally every 12 h, yielded the percentage of C min, ss of voriconazole that achieved a therapeutic range of voriconazole (1-5 mg/L) of 51.45%, whereas the percentage that achieved a toxic range (C min, ss > 5 mg/L) was 45.65%, and the percentage that achieved a sub-therapeutic range (C min, ss < 1 mg/L) was 2.90%. In the group of omeprazole ≥ 40 mg/day and albumin 1.5-2 g/dL, the maintenance dose of voriconazole, 150 mg orally every 12 h, yielded a percentage of C min, ss of voriconazole that achieved a therapeutic range of voriconazole (1-5 mg/L) of 22.86%, while the percentage that achieved a toxic range (C min, ss > 5 mg/L) was 76.43%, and the percentages that achieved a sub-therapeutic range (C min, ss < 1 mg/L) was 0.71%.
Discussion
To our knowledge, this is the first population PK study of voriconazole in Thai adults with hematologic diseases. We investigated factors that influence PK parameters of voriconazole, and we performed Monte Carlo simulation to determine dosing recommendations for Thai patients. In the present study, first, we established the lower albumin level and the higher dose of omeprazole ≥ 40 mg/day that were able to describe the variability of CL/F. In addition, optimal voriconazole dosage recommendations were established according to these covariates.
The model which best described the PK profile of voriconazole was the one-compartment model with first-order absorption and elimination, which was consistent with previous studies [14,16,18,19,21,25,31]. However, Mangal et al. characterized PK with one compartment with first-order absorption and Michaelis-Menten elimination [17]. Alternatively, a study by Han et al. described the voriconazole data using the two-compartment model with first-order absorption and elimination [22], whereas Dalton et al. [13], Liu et al. [15], and Friberg et al. [23] employed a two-compartment model with first-order absorption, a lag time, a mixed linear approach, and Michaelis-Menten elimination. We explored the Michaelis-Menten elimination and two-compartment models, but these models did not fit our data. The discrepancy in the PK of voriconazole could be explained by blood sampling time data, a difference in the proportion of sample characteristics, and co-administration with CYP2C19 inhibitors. In our study, most patients received PPIs (omeprazole 20 mg/day 44.62%, omeprazole ≥ 40 mg/day 26.15%, esomprazole 3.08%, and rabeprazole 1.54%). In contrast, in the Mangal et al. study, most patients received pantoprazole 70.6% [17]. Han et al. studied the population PK of lung transplant recipients, and collected full intensive PK data from the second dose injection of an initial dose 6 mg/kg of intravenous (IV) form, and 5th-35th dose 200 mg of oral form voriconazole [22]. Dalton et al. [13] studied 3352 blood samples from 63 healthy volunteers and 177 patients that were collected 1-29 times/dose of single or multiple dose, that varied in dose IV or oral form. Liu et al. [15] studied subjects (305 subjects, 965 blood samples) whose collected samples tended to have a peak concentration, middle concentration (time between peak and trough), and trough concentration of voriconazole on day 3, day 7, and day 14, post dose, with dosing of voriconazole of 6 mg/kg IV q 12 h 2 doses, then 4 mg/kg IV q 12 h, switching to 300 mg (150 mg for body weigh <40 kg) for oral form q 12 h when patients could feed/eat. Friberg et al. [23] studied both pediatric and adult patients, which was different from our study. Covariate analysis revealed that albumin level and omeprazole ≥ 40 mg/day have a significant impact on CL/F, and therefore they were included in the final model. The PK parameters were generally well estimated in the final model. The estimations had acceptable precision, and the goodness-of-fit plots indicated a reasonable fit to the data. The results of bootstrap analysis showed the robustness of the model, and prediction-corrected visual predictive checks showed the model to be adequate for describing the data.
Since there were a limited number of samples drawn during the absorption phase, we fixed the Ka at 1.1 h −1 [25]. Interpatient variability in the V/F could not be estimated due to inadequate blood samples during the distribution phase. The estimation of CL/F was 3.43 L/h, which was within the range of previously reported values in population analysis (range from 2.88 to 11.2 L/h) [14,16,18,19,21,22,25]. The estimation of V/F was 47.6 L, which was lower than the values observed in other populations. However, the value of V/F was within the range reported (22.47-248 L) in previous studies [14,16,18,19,21,25,31,32]. In addition, our study could not identify body weight as a covariate since we performed allometric scaling.
Plasma albumin was found to be a factor that significantly influenced the CL/F of voriconazole. A study by Vanstraelen et al. found a positive relationship between voriconazole plasma protein binding and plasma albumin concentration, which could be due to a higher unbound voriconazole concentration with decreasing albumin concentration [33]. Nevertheless, an explanation of low plasma albumin level associated with the reduction of CL/F of voriconazole was still inconclusive. In our opinion, an increase in voriconazole metabolism is related to an unbound form. However, voriconazole is a low extraction ratio (0.09-0.39) [34], the saturated enzyme for drug metabolism limited the clearance of voriconazole. Moreover, since almost all of the patients in our study took the same dose of voriconazole, the Michaelis-Menten equation could not be clearly demonstrated. Dote et al. [29] and Hirata et al. [35] found hypoalbuminemia to be associated with decreasing clearance of voriconazole, therefore, toxic vigilance is important in patients with low albumin.
Concurrent administration of PPIs is common in patients receiving voriconazole. A physiologically based PK study showed that the impact of PPIs on the PK of voriconazole was related to the dose and inhibitory effect of CYP2C19. The area under the curve (AUC) of voriconazole was increased by 39%, 18%, 12%, and 1% with co-administration of omeprazole 40 mg/day, esomeprazole 40 mg/day, lansoprazole 30 mg/day, and rabeprazole 20 mg/day, respectively [36]. A study by Wood et al. Investigated the effect of omeprazole 40 mg/day on the steady-state PK of voriconazole in 18 healthy male volunteers. Their study found that C max and AUC were increased by 15% and 41%, respectively [37]. The mechanism behind the interaction with CYP450 inhibitors, omeprazole, and voriconazole is via CYP2C19 and CYP3A4 inhibitors [4,38,39]. On the one hand, the effect of PPIs on the PK of voriconazole depends on the dose and kind of PPIs, and how these factors affect various capabilities of CYP2C19 [12,36]. On the other hand, a recommendation specific to omeprazole 20 mg/day stated that this drug and dosage did not significantly affect voriconazole levels, which was similar to our study [4,40].
Conflicting findings relative to the impact of CYP2C19 polymorphisms on voriconazole clearance have been observed among some population PK analyses. Previous studies have found the poor metabolizer group (11.3-19%) [16,18,19] to be adversely affected relative to the clearance of voriconazole. In our study, the data showed a poor metabolizer group of 10.77% (CYP2C19*2/*2 7.69%, and CYP2C19*2/*3 3.08%), which indicates CYP2C19 polymorphisms in the analysis, but we were not able to find any influence of gene polymorphism. This observed absence of association is likely due to the modest effect on clearance of voriconazole among extensive, intermediate, and poor metabolizer, in each group. Furthermore, it is possible that the effect of CYP2C19 expressers could be minimized by omeprazole ≥ 40 mg/day and plasma albumin effects, which could mask the effect of gene polymorphisms.
Final model-based simulations were performed to compare each dosage regimen with the percentages of C min, ss of voriconazole that achieved therapeutic range (1-5 mg/L) [2,3,9,10]. Several dosage regimens of a maintenance dose of voriconazole after loading (50, 100, 150, 200, 250, 300, 350, and 400 mg orally every 12 h) were used to predict C min, ss under different covariate combinations and conditions. This is the first study to establish the influence of plasma albumin level and co-administration of omeprazole and voriconazole on dose-adjusted concentration of voriconazole in patients with hematologic diseases. The results of dosing simulations are presented in Figures 3 and 4. In the non-omeprazole or omeprazole 20 mg/day group (Figure 3), patients with albumin 1.5-3, 3.01-4, and 4.01-4.5 g/dL that received a dose of voriconazole 50, 100, and 200 mg orally every 12 h, respectively, had the highest proportion of patients that reached the therapeutic concentration (C min, ss 1-5 mg/L) at steady state. Among patients who received omeprazole ≥ 40 mg/day (Figure 4), those with albumin 1.5-3 g/dL who received a dose of voriconazole 50 mg orally every 12 h had the highest percentage of patients who reached the therapeutic target. In contrast, patients with albumin 3.01-4.5 g/dL who received omeprazole ≥ 40 mg/day and a dose of voriconazole 100 mg orally every 12 h had the highest number of patients that achieved a therapeutic range. Moreover, the optimal regimens of voriconazole should lower the percentages of patients that achieve toxic or sub-therapeutic range. The application of our simulation (Figures 3 and 4) for predicting C min, ss was found to be appropriate for a plasma albumin range of about 1.6-4.5 g/dL.
Some limitations of this study should be noted. Firstly, the time of blood collection varied from 0.75-12 h. However, most of the blood samples that were collected in this study had a trough level that was consistent with the predicted trough concentration of voriconazole. Secondly, data specific to the absorption phase (0-2 h) and distribution phase were limited. Thus, Ka and V/F and their variability could not be well estimated. Finally, the effect of simulation dose on clinical outcome and toxicity was not evaluated in this study. Further studies should be conducted to investigate this effect.
Patients and Ethics
A PK study of oral voriconazole was conducted in Thai adult patients with hematologic diseases at Siriraj Hospital, which is a 2300 bed university-based national super tertiary referral center that is located in Bangkok, Thailand.
Patients with hematologic diseases were treated with voriconazole for prevention or treatment of invasive aspergillosis following the guidelines of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group: EORTC and the National Institute of Allergy and Infectious Diseases Mycoses Study Group: MSG or EORTC/MSG 2008 [41]. The inclusion criteria were Thai adult patients aged ≥ 18 years with hematologic diseases. Voriconazole was administered orally according to patient's body weight. The loading dose was 400 mg every 12 h (2 doses in all 65 patients) on the first day, followed by a maintenance dose of 200 mg every 12 h for weight ≥ 40 kg. In patients with weight < 40 kg, the maintenance dose depended on doctor's discretion (see the information in Supplementary Materials). Blood samples were collected in 18 subjects on the seventh day of voriconazole (7 blood samples; 0 (pre-dose), 1, 1.5, 2, 4, 8, and 12 h) during August 2016 to October 2018, and a trough blood level at different times was collected in another group of 47 subjects from the 7th to the 40th day of maintenance dose of voriconazole. Patients who were pregnant, unable to take or tolerate voriconazole until the end of the specified date, had severe hepatic disease (Child Pugh C), or had voriconazole hypersensitivity were excluded.
The protocol for this study was approved by the Siriraj Institutional Review Board (SIRB) of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA numbers 610/2558 (EC1) and 104/2554 (EC2)). Written informed consent was obtained from all patients.
Measurement of Voriconazole Plasma Concentrations
Blood samples were collected in an ethylene diamine tetra-acetic acid (EDTA) tube and kept at −80 • C until drug concentration analysis. Plasma voriconazole concentrations were measured by validated method with high-performance liquid chromatography (HPLC) assay, as previously described [42]. All samples were analyzed by the Infectious Laboratory, Department of Preventive and Social Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University. The lower limit of quantification was 0.2 mg/L (the linearity ranged from 0.2 to 20 mg/L). The correlation coefficient value was 0.98. The CV% of voriconazole concentrations had an intra-day variability range of 0.78-3.01%, and an inter-day variability of 1.52-4%. The ranges for accuracy and extraction recovery were 99.3-101% and 99.2-101%, respectively [42].
Data Collection
Patient data that were collected from medical records included the following: demographic data (i.e., age, gender, comorbidity, and body weight); source of fungal infection; drugs taken concurrently with voriconazole, such as PPIs, sulfamethoxazole/trimethoprim, hormones, and steroids; and laboratory data, including white blood cell, absolute neutrophil count, hemoglobin, hematocrit, platelets, blood urea nitrogen, serum creatinine, AST, ALT, ALP, DB, TB, albumin, globulin, gene polymorphism of CYP2C19, and plasma voriconazole concentrations.
CYP2C19 Genotype Analysis
DNA was extracted from a buccal swab using a Gentra Puregene Blood Kit (QIAGEN, Hilden, Germany).
Population Pharmacokinetic Modeling Method
A population PK model was established using nonlinear mixed-effects modeling. The PK parameters and their variabilities were estimated using first-order conditional estimation with interaction (FOCE-I) method. The software packages used for modeling included NONMEM (version VII; Icon Development Solutions, Ellicott City, MD, USA), Perl-speaks-NONMEM (PsN) Toolkit (version 3.7.6: http://psn.sourseforge.net/), and Pirana (version 2.8.1.). The graphical analysis was performed using R package (version 3.1.2, R Development Core team; http://www.r-project.org) and Xpose program (version 4.5.0).
The PK characteristics of oral voriconazole were investigated for fit to the data using oneand two-compartment model with first-order absorption with and without absorption lag time, and linear or nonlinear (Michaelis-Menten) elimination.
The inter-individual variability (IIV) was described with an exponential model; thus, a log-normal distribution of the PK parameter was assumed: CL/F = θ × (EXP) η i , where CL/F is the individual apparent oral clearance (CL/F) of voriconazole, θ is the typical value in the population CL/F, and η i is the IIV which is assumed to be normally distributed with a mean of zero and variance of ω 2 .
where C obs,ij is the ith observed concentration of the jth individual; C pred,ij is the ith predicted concentration of the jth individual; ε ij is the RUV, which was assumed to be normally distributed with a mean of zero and a variance of σ 2 ; ε1 is the RUV with proportional function; and ε2 is the RUV with additive function. The influence of body weight on apparent clearance (CL/F) and the volume of distribution (V/F) were explored using allometric scaling as follows: CL/F = θ 1 × (WT i /WT std ) 0.75 and V/F = θ 2 × (WT i /WT std ) 1 , where θ 1 and θ 2 are the mean parameters to be estimated for CL/F and V/F, respectively; WT i is the individual body weight (kg); and WT std is the standard body weight, which is the median weight of the population.
The influence of clinical and genetic factors on PK parameters was explored graphically to identify potential relationships, and then covariate analysis was performed using a stepwise approach. The difference in OFV > 3.84 (χ 2 , df = 1, p < 0.05) and 6.63 (χ 2 , df = 1, p < 0.01) was used as cut-off criteria for forward inclusion and backward deletion, respectively.
The effect of covariates on PK parameters was evaluated using different functions based on the type of covariate. Continuous covariates (e.g., age, body weight, albumin level, AST, ALT, ALP, TB, and DB) were centered by their median and were explored with linear, power, and exponential model. Categorical covariates (e.g., gender, CYP2C19 genotypes or phenotypes, dosing of omeprazole (20 mg/day and ≥40 mg/day), sulfamethoxazole/trimethoprim, and hormones) were explored with additive, fractional, and exponential model.
Model Evaluation and Validation
Model selection was guided by the difference in objective function value (OFV), improvement in the goodness-of-fit (GOF) plots, and the precision of parameter estimates (%RSE). Model evaluation was performed using prediction-corrected visual predictive checks (pcVPC) and bootstrap method (n = 1000 samples with replacement from the original dataset).
Simulation
Monte Carlo simulation was performed to determine optimal dosing regimens of voriconazole. A simulation of minimal voriconazole concentrations (C min, ss ) at steady state was conducted using the parameter estimates from the final model. A total of 2000 replicates of C min, ss were simulated for each dosage regimen, and the influence of each significant covariate was evaluated. A C min, ss of voriconazole between 1 and 5 mg/L represents the therapeutic target of drug efficacy [2,3,9,10].
Statistical Analysis
Patient characteristics were analyzed by STATA software (version 14.2) (StataCorp, LLC, College Station, TX, USA). Categorical data are reported as frequency and percentage, and continuous data are summarized as mean ± standard deviation (SD) and range or median and interquartile range (IQR).
Conclusions
The population PK model of voriconazole has been developed, and its PK profile has been characterized in Thai adult patients with hematologic diseases. The final model indicated that plasma albumin level and omeprazole ≥ 40 mg/day significantly influenced the clearance of voriconazole. The simulations suggested a decreased dose of voriconazole in patients who receive omeprazole ≥ 40 mg/day. In addition, patients with a lower albumin level should receive a lower dose of voriconazole than the dose that would be given to patients with a normal albumin level. Voriconazole should be used with caution in hypoalbuminemia patients who receive omeprazole ≥ 40 mg/day. Thus, therapeutic drug monitoring can aid dosage adjustment in patients who are treated with voriconazole. The results of this study could be applied to design a dosing optimization model of voriconazole that included significant covariates identified in Thai population.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2079-6382/9/9/574/s1, Figure S1: A visual predictive checks (VPC) plot of the final model. Open circles show observed plasma concentrations, and the solid line and dashed line show the median and 95% confidence interval (CI) of observations, respectively. The red shaded area and the blue shaded area show the 95% CI of the median and the 5th and 95th percentiles of the consequence of 1000 simulations of the final model, respectively, S1: Code of the one-compartment model, S2: Patients with body weight <40 kg, Table S1: The sensitivity analysis results by base model showed that the clearance estimates were consistent across different values of the absorption rate constant (Ka) 0.163-1.38. | 2020-09-10T10:23:10.004Z | 2020-09-01T00:00:00.000 | {
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11781580 | pes2o/s2orc | v3-fos-license | High Prevalence of West Nile Virus in Domestic Birds and Detection in 2 New Mosquito Species in Madagascar
West Nile virus is an arthropod-borne zoonosis transmitted by a large number of mosquito species, and birds play a key role as reservoir of the virus. Its distribution is largely widespread over Africa, Asia, the Americas and Europe. Since 1978, it has frequently been reported in Madagascar. Studies described a high seroprevalence level of the virus in humans in different areas of the island and a human fatal case of WNV infection was reported in 2011. Despite these reports, the epidemiology of WNV in Madagascar, in particular, viral circulation remains unclear. To explore the transmission of WNV in two rural human populations of Madagascar, we investigated local mosquitoes and poultry for evidence of current infections, and determined seroprevalence of candidate sentinel species among the local poultry. These 2 areas are close to lakes where domestic birds, migratory wild birds and humans coexist. Serological analysis revealed WNV antibodies in domestic birds (duck, chicken, goose, turkey and guinea fowl) sampled in both districts (Antsalova 29.4% and Mitsinjo 16.7%). West Nile virus nucleic acid was detected in one chicken and in 8 pools of mosquitoes including 2 mosquito species (Aedeomyia madagascarica and Anopheles pauliani) that have not been previously described as candidate vectors for WNV. Molecular analysis of WNV isolates showed that all viruses detected were part of the lineage 2 that is mainly distributed in Africa, and were most closely matched by the previous Malagasy strains isolated in 1988. Our study showed that WNV circulates in Madagascar amongst domestic birds and mosquitoes, and highlights the utility of poultry as a surveillance tool to detect WNV transmission in a peri-domestic setting.
Introduction
West Nile virus (WNV) is a zoonotic arbovirus affecting humans, horses and wildlife. The virus is widely distributed in Africa, Asia and Europe and spread over the last 2 decades to North and South America [1]. WNV is a flavivirus described as 8 lineages but only lineages 1 and 2 are involved in important human outbreaks [2]. Lineage 1 includes viruses from Saharan Africa, Europe, Asia, Australia and North, Central and South America while lineage 2 was mainly detected in Africa and Madagascar [3,4]. Although in humans, WNV associated disease is more often characterized by a febrile stage with myalgia, arthralgia and lymphadenopathy, a less common manifestation is neurological disease, which can be fatal [5,6]. WNV is maintained within a bird-mosquito-bird transmission cycle. Some bird species, like American crow (Corvus brachyrhynchos) and house sparrow (Passer domesticus) develop a sufficient viremia titer to transmit the virus to mosquitoes, while others like the domestic rock pigeon (Columba livia) and the barn owl (Tyto alba) do not [7,8]. These birds are considered as amplifiers with a key role in the epidemiology of the virus and can as act reservoirs. Domestic birds like chickens do not develop sufficient viremia to permit a transmission cycle and so are considered as dead end hosts. Poultry species have been used as surveillance sentinels in many geographic regions [9] but their role of WNV transmission and surveillance value has not been well-investigated at this time.
In Madagascar, WNV lineage 2 was first isolated in 1978 from an endemic parrot species (Coracopsis vasa) [10]. To date, ten mosquito species are considered to be the main WNV vectors in Madagascar. The last inventory of WNV amongst Malagasy mosquitoes performed from 1978 to 1988 showed that 12 species were infected with WNV including 2 Anopheles species, 4 Aedes species and 6 Culex species [11]. The possible involvement of Anopheles species is quite surprising. However, to date, 23 Anopheles species are already described in Madagascar with a highly zoophilic behavior, including on birds, and could occasionally feed on humans (unpublished data).
In 1990, a serosurvey study conducted amongst people aged from 5 to 20 years-old in Madagascar showed that 29.9% of sera tested positive for WNV antibodies [12]. A second serosurvey conducted in 1996 on children younger than 15 years-old in the highlands, and in 1999 in the north-western coast showed that 2.1% and 10.6% tested positive, respectively [13]. These data suggested the virus has been circulating for years in various areas of Madagascar. More recently, in 2011 a woman returning from Madagascar died on Reunion Island from WNV neuroinvasive disease [6]. Subsequently, our study's purpose is to evaluate WNV exposure in candidate sentinel species among local poultry species by determining seroprevalence, a measure of past infection, and investigating local mosquitoes as its candidate vectors, thus exploring its potential circulation in two human populations in rural Madagascar.
Study sites
Two study areas were selected in two Western regions of Madagascar: Mitsinjo and Antsalova districts. These districts correspond to ecotypes in which human population, domestic and wild migratory wild birds coexist, and the presence of potential mosquito vectors and WNV circulation has been previously reported (i.e., positive serological results and viral detection).
In Mitsinjo district, the Kinkony Lake represents the second biggest lake of Madagascar and is a stopover site for migratory birds and is also home to resident waterfowl. Three villages were investigated in November and December 2012: Marofandroboka, Ankisaosy and Mahakary close to the Kinkony Lake In Antsalova district, three major lakes composed a national park where wild birds are present year-round. Four villages, belonging to the Masoarivo municipality, were investigated in July 2013: Antsakoramby and Ankirangato close to Soamalipo lake, and Masoarivo and Mananga around the Antsamaka Lake.
Domestic birds sampling
Serum samples (400μl) were collected by venipuncture in the wing vein on all domestic healthy birds brought by villagers (i.e., chicken (Gallus gallus domesticus), duck (Cairina moschata), turkey (Meleagris gallopavo), goose (Anser anser domesticus), and guinea fowl (Numida meleagris). All these animals were resident in the villages without travel history outside the village. Birds are considered as juvenile when the farmer reported it as younger than 5 months old. All serum samples were centrifuged in the field, transferred in liquid nitrogen to the laboratory, and stored at -80 C until use.
Ethics statement
Bird trapping, handling, and sampling were implemented with the approval of national veterinary authorities and did not involve endangered or protected species. Farmers in each zone gave a verbal consent to participate into our study and gave permission for the blood sample collection from birds on their property. Our study protocol and procedures were approved by the committee of the Livestock ministry of Madagascar which is the sole relevant authority for animal care in Madagascar. The ethical committee number is: 2012/WN/Minel/3. We followed the European guidelines (European directives EU 86/609-STE123 and 2010/63/EU) for animal handling. Sampling was exclusively done by veterinarians and the animals were blood-sampled without suffering and were subsequently released. No animals were sacrificed during the study.
Entomological survey
During the same period of time in which domestic birds were sampled and within the same study sites, mosquitoes were collected for testing. Mosquito traps were placed near the lake shore and in the villages. Mosquitoes were captured in CDC light traps (BioQuip Products, Inc., Rancho Dominguez, USA), net traps baited with chickens, BG-Sentinel baited with chickens (Biogents, Regensburg, Germany) [14], human landing, or a backpack aspirator (BioQuip Products, Inc., Rancho Dominguez, USA), and identified and stored as previously described [15]. In Mitsinjo district, four villages were investigated: Marofandroboka, Amboanjo, Morafeno and Mahakary. In Antsalova district, two villages were investigated (Masoarivo and Antsakoramby) as well as in the vicinity of the Antsamaka lake.
Serological analysis
Bird serum samples were tested using IDScreen West Nile Competition Multi-species ELISA (enzyme-linked immunosorbent assay) (IdVet, France) according to manufacturer's guidance, to detect immunoglobulins M and G (IgM and IgG) [16].
Virological analysis
Viral detection was carried out on pools containing 10 sera from 10 different individual birds. Viral RNA was extracted from 140 μl of a pool of 10 sera (14 μl of each serum), by using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. Positive pools were further analysed individually with the aim of identifying individual positive sera.
Molecular analysis was performed on mosquito pools that contained 1 to 34 monospecific unfed female mosquitoes homogenized in 750 μl of cell culture medium (MEM) (containing 40% fetal bovine serum, 2 mM l-glutamine, 1000 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotericin B). Grinding was performed by shaking the pools twice at 25 Hz frequency for 2 min with a 5-mm stainless steel ball (Dejay Distribution Ltd., Crowborough, United Kingdom) in TissueLyser II (Qiagen, Crawley, United Kingdom). Viral RNA was then extracted from 140 μl of the supernatants using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The 60 μl eluted RNA was stored at -80°C.
The molecular detection of the virus was performed on a T3000 Biometra thermocycler using a semi-nested RT-PCR. The first round of PCR used primers WN132 and WN240 described by Berthet et al. [17] giving a 328 bp product and the second round used the forward primer WN132 and the in-house degenerated primer WN ESN: 5'-CTCCAKGGSAGGTTS AGRTCCAT-3' giving a 228 bp product. Briefly, RT-PCR and the first round of PCR were performed in a one-step process on 4 μl of resuspended RNA template using the SuperScript III One-step RT-PCR reaction mix (Invitrogen). The second round of PCR was performed on 1μL of primary PCR product in a final volume of 25 μL containing 2X GoTaq 1 Hot start Mastermix (Promega, Madison, USA) and 500nM of primers WN 132 and WN ESN. Cycling conditions of the second round of PCR were as follows: 95°C Ã #x2013;5min, then 25 cycles of 95°C-45 sec / 53°C-45 sec / 72°C-45 sec, followed by a final elongation step at 72°C-12 min. When a sample tested positive, a second extraction and RT-PCR from the original sample was processed to confirm the result.
Sequence and phylogenetic analysis
Positive amplicons were purified on a 1% (w/v) agarose gel by electrophoresis with the QIAquick gel extraction kit (Qiagen), and sequenced commercially (Macrogen, Korea).
For the determination of WNV genotype of the Malagasy strains, a total of 42 WNV Gen-Bank partial sequences of 228 bp of the envelope gene and 1 partial sequence of JEV were selected according to their origin (Asia, Europe, Africa and America), their host (human, birds, mosquitoes) and their lineage. Basic Local Alignment Search Tool (BLAST) alignments were generated using MUSCLE [18] through the Mega 5 software [19]. A phylogenetic tree was generated by neighbour-joining distance analysis with node values generated by 1000 bootstraps replications, using the Mega 5 software [19].
Statistical analysis
Data were recorded and analyzed statistically with the R software package (version 3.0.1) [20]. The importance of main factors (sex, species, age (adult or juvenile) of animals, district and commune of origin) to the outcome of animals seroprevalence were evaluated with a regression analysis with multiple variables.
Field collection
A total of 589 animals were sampled and bled, 300 in Mitsinjo and 289 in Antsalova districts respectively. In Mitsinjo district, most of the birds were sampled in Mahakary (n = 150) and Marofandroboka (n = 120) villages. In Antsalova district, animals were mainly sampled in Masoarivo (n = 100) and Antsakoramby (n = 97) villages. Ducks (n = 343), chickens (n = 195) and turkeys (n = 43) comprised the majority of birds sampled in both districts. In Antsalova district, geese and guinea fowls were also sampled. Sampling information is presented in Fig 1. The mosquito sampling results are briefly described below and a study on the efficacy of the various techniques utilized is presented elsewhere [15].
Serological analysis
Differences in seroprevalence rates were observed among villages, as well as differences in species and age (Fig 1). In Mitsinjo district, WNV seroprevalence in birds in Mahakary village was significantly lower than the prevalence in Maforandroboka and Ankisaosy villages (Fig 1A). In the Antsalova district, Antsakoramby and Ankirangato villages showed lower seroprevalence than in Masoarivo and Mananga villages (Fig 1B).
Overall, in both areas, turkeys presented the higher seroprevalence rate than other species sampled. No statistical difference was observed between ducks and chickens. In the Antsalova
Mansonia uniformis 407 15
Uranotaenia alboabdominalis 1 0 district, geese sampled also presented a high seroprevalence rate but the number of animals tested was low (n = 7). Only one guinea fowl was assessed and tested negative. In the two study sites, seroprevalence rates increased with age of animals despite non-significance. A slightly higher seroprevalence is observed in males than in females in Antsalova district.
Molecular analysis
One of 60 pools of avian sera tested positive for WNV RNA; this pool contained sera from adult chickens from Marofondroboka, part of the Mitsinjo district. Eight of 121 mosquito pools tested positive for WNV nucleic acid.
In the Antsalova district, one pool of Anopheles pauliani and four pools of Mansonia uniformis were WNV-positive. In the Mitsinjo district, three pools of mosquitoes were WNV-positive: one pool of Aedeomyia madagascarica and two pools of Anopheles coustani.
Phylogenetic analysis
A partial sequence of 228 bp was obtained for the 9 Malagasy isolates (8 from mosquito pools and 1 from a chicken serum). The 9 sequences are deposited in the Genbank database under the accession numbers: KP099553, KP099554, KP099555, KP099556, KP099557, KP099558, KP099559, KP099560 and KP099561. The topology showed two distinct lineages. The 9 Malagasy isolates belonged to lineage 2 (Fig 2). They also branched with the strains already detected in Madagascar (99% nucleotide homology), forming a distinct Malagasy clade (including strains isolated for the first time in 1978), with a high node support value (posterior probability pp = 0.95).
Discussion
Our results confirm domestic poultry are exposed to WNV in Madagascar and describe two new mosquito species as potential vector for WNV. The diversity of species trapped is described by Boyer et al. (2014) and many of these species (especially Culex and Anopheles species) were previously described as WNV potential vectors [15]. The two study sites were characterized by different abundances and species composition of mosquitoes. Differences in the abundance and the diversity of wildlife around villages could be one of the numerous factors influencing the variation in WNV seroprevalence of poultry in Antsalova and Mitsinjo. Molecular analysis of vector pools indicated current circulation of the virus in mosquitoes in the west region of Madagascar. Viral RNA was identified in 4 mosquito species (Anopheles coustani, Anopheles pauliani, Mansonia uniformis, and Aedeomyia madagascarica). Anopheles coustani and M. uniformis have been detected with WNV in Israel and Ethiopia, respectively [21]; however, the present study describes the first detection of WNV in these species in Madagascar. Interestingly, Aedeomyia madagascarica and Anopheles pauliani, 2 endemic species have not been previously documented with WNV infection. Furthermore, the 4 mosquito species An. coustani, An. pauliani, Ma. uniformis, and Aed. madagascarica are already known as zooanthropophagous insects, increasing the possibility of WNV interaction among human, mosquito vectors, and other potential WNV amplifying hosts in these particular ecotypes. Aed. Madagascarica and An. pauliani are known to preferentially feed on avian blood while Ma. Uniformis and An. coustani generally prefer cattle in Madagascar.
Cross reactivity can be observed within flaviviruses family. However, the ELISA kit used in this study presented a good sensitivity and specificity [16]. Furthermore, excellent agreement was demonstrated between serum neutralization (SN) test (considered as the reference test) and this ELISA kit, further supporting the validity of ELISA results and the conclusions drawn for them [22]. A high seroprevalence was observed with geese and turkeys and it was significantly different from chicken and ducks, although the low number of specimens sampled limited the interpretation. Chicken and turkeys are not good amplifier hosts, with low viremia, not Genetic relatedness of geographically distinct WNV isolates determined by using the nucleotide sequence data from a 228 bp region of the E gene. The tree was constructed with the PAUP by using the neighbor-joining distance program of Mega 5 software. Node values were determined for 1,000 replicates. Isolates are labeled as follows: strain identification, country, date of isolation, genbank accession number. really good for mosquito contamination [23]. Turkeys were more likely to seroconvert than other poultry species in the village ecosystem, although this may be due to their older age compared to chickens, and therefore longer exposure to infected mosquitoes [12]. Consequently, turkeys and young chickens may be the best choices as sentinel birds to detect a current circulation. Moreover, viral RNA was detected in a chicken serum. This rare event has already been evidenced by Petrovic et al. [24].
We detected most of WNV positive mosquito pools from villages located close to the lakes where lakeshores with emergent vegetation may support mosquito breeding. However, we also found that some villages like Masoarivo, Mananga and Marofondroboka (that are not located at the lake edge) exhibited high prevalence rates in poultry, despite lower numbers of trapped mosquitoes in these regions during the same time frame. Different hypotheses could be proposed to explain such a situation; firstly, the villages are not so far from the lakes and temporary waterholes favorable to mosquitoes. Secondly, the timing of WNV infections of birds cannot be determined; birds over a year of age may have been infected in years prior to the study [12]. Thirdly, wild birds are found everywhere, and many are adapted to humanized ecosystems and mosquitoes have the ability to move from a site to another, increasing the risk of infection throughout these relatively small areas.
Phylogenetic analysis revealed that all isolates detected in our study belonged to lineage 2, in accordance with the global repartition of the different WNV lineages [3,4]. In Madagascar, all isolates achieved since 1978 have been closely related, suggesting local circulation of WNV presumably maintained by wild birds, acting as a reservoir of the virus, and transmitted by various potential mosquito vectors of the virus.
Finally, this study highlighted local exposure of WNV in domestic birds in the villages. Most of all, with 2 new identified WNV candidate vectors, the number of mosquito species that serve as potential vectors of the virus increases to 28 in Madagascar (Tantely, personal communication). A study is consequently in progress in the Mitsinjo district to assess the role of wild birds and mosquitoes in the virus transmission, identify a possible seasonality of WNV and evaluate the risk for humans. | 2018-04-03T00:26:23.239Z | 2016-01-25T00:00:00.000 | {
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4028199 | pes2o/s2orc | v3-fos-license | Direct and indirect effects of age on interoceptive accuracy and awareness across the adult lifespan
Various aspects of physical and mental health have been linked to an individual’s ability to perceive the physical condition of their body (‘interoception’). In addition, numerous studies have demonstrated a role for interoception in higher-order cognitive abilities such as decision-making and emotion processing. The importance of interoception for health and typical cognitive functioning has prompted interest in how interoception varies over the lifespan. However, few studies have investigated interoception into older adulthood, and no studies account for the set of physiological changes that may influence task performance. The present study examined interoception from young to very late adulthood (until 90 years of age) utilising a self-report measure of interoception (Study One) and an objective measure of cardiac interoception (Study Two). Across both studies, interoception decreased with age, and changes in interoceptive accuracy were observed which were not explained by accompanying physiological changes. In addition to a direct effect of age on interoception, an indirect effect of ageing on cardiac interoceptive accuracy mediated by body mass index (BMI) was found, such that ageing was associated with increased BMI which was, in turn, associated with reduced interoceptive accuracy. Such findings support and extend previous research demonstrating interoceptive decline with advancing age, and highlight the importance of assessing whether decreasing interoceptive ability is responsible for some aspects of age-related ill-health and cognitive impairment.
Given this body of evidence linking interoception to aspects of higher-order cognition and physical and mental health (Brewer, Cook, & Bird, 2016;Khalsa & Lapidus, 2016;Murphy et al., 2017), examining the stability of interoception across an individual's lifespan may be crucial. Notably, it has been found that cardiac interoceptive accuracy declines with age (Khalsa, Rudrauf, & Tranel, 2009), supporting earlier work reporting that the perception of other interoceptive signals, including thirst (see Silver, 1990), taste (Stevens, Cruz, Hoffman, & Patterson, 1995), temperature (Clark & Mehl, 1971) and pain (see Gagliese, 2009), is less accurate in later life (but see Garfinkel et al., 2016b). However, although a limited number of studies have therefore looked at developmental influences on the accuracy of interoception, to our knowledge no study has examined age-related changes in self-reported awareness of interoceptive signals (known as interoceptive sensibility under the framework of Garfinkel, Seth, Barrett, Suzuki, & Critchley, 2015), which is of interest as self-report measures of interoceptive awareness do not always correlate with objective tests of interoceptive accuracy (Garfinkel et al., 2015). Study One therefore aimed to examine interoceptive awareness across the lifespan.
Alongside the investigation of whether interoceptive awareness declines over the lifespan, there is a need to improve our understanding of changes in interoceptive accuracy. Studies examining changes in interoceptive accuracy across the lifespan typically use one of the two most commonly used measures of interoception: heartbeat tracking (Schandry, 1981) and discrimination procedures (Brener & Kluvitse, 1988;Whitehead, Drescher, Heiman, & Blackwell, 1977), which both assess perception of cardiac signals. In the former, participants are asked to count their heartbeats over a series of intervals whilst their heartbeat is objectively measured. The difference between the objective and subjective measurements acts as a measure of interoceptive accuracy. In the latter, participants are asked to determine whether a signal is in or out of sync with their heartbeat. In a study by Khalsa and colleagues (2009), age explained~40% of the variance in heartbeat discrimination performance in participants aged up to 63 years of age, such that increasing age was associated with poorer performance.
Examining cardiac interoception across the lifespan is complicated, however, by previous findings demonstrating that various physiological and psychological factors influence task performance, and have also been associated with ageing. For example, lower resting heartrate, reduced heartrate variability and body composition (e.g., lower BMI/body fat) have all been associated with better cardiac perception (Knapp-Kline & Kline, 2005;Rouse, Jones, & Jones, 1988), while reduced systolic blood pressure has been linked to worse cardiac perception (O'Brien, Reid, & Jones, 1998). Psychological factors such as participants' beliefs regarding heartrate have also been shown to strongly influence performance on objective tests of cardiac interoceptive accuracy (Ring, Brener, Knapp, & Mailloux, 2015;Ring & Brener, 1996;Windmann, Schonecke, Fröhlig, & Maldener, 1999) and time perception has also been linked with task performance (see Wittmann, 2013).
It is therefore unclear whether ageing is associated with changes in interoceptive accuracy independent of its effects on these psychological and physiological factors, or whether in fact these factors mediate the effect of age on cardiac interoceptive accuracy (Franklin et al., 1997;St-Onge, 2005;Turgeon, Lustig, & Meck, 2016;Umetani, Singer, McCraty, & Atkinson, 1998;Yashin et al., 2006). Examining whether changes in interoception across the lifespan are attributable directly to aging, or to factors co-varying with age, may be crucial for identifying the mechanism underlying interoceptive change with increased age. Uncovering this mechanism is of interest given the relationship between interoception and socio-cognitive abilities (Murphy et al., 2017), and the well-documented decline in these abilities in later life (e.g., Ruffman, Henry, Livingstone, & Phillips, 2008;Sparrow & Spaniol, 2016). Furthermore, although a decline in heartbeat discrimination performance with age has been demonstrated in participants aged up to 63 years of age (Khalsa et al., 2009), whether cardiac interoceptive accuracy continues to decline after this period into late, and very late, adulthood remains an unanswered question, and is the focus of Study Two.
The present pair of studies therefore aimed to quantify interoceptive awareness (Study One) and accuracy (Study Two) across the lifespan, from young adulthood to very late adulthood. Study One utilised self-report measures, whereas Study Two utilised objective measures whilst also measuring psychological and physiological factors which may mediate any association between cardiac interoception and age.
Participants
A total of 1008 participants took part in an online survey with a prize draw offered as incentive. Of these, 898 participants completed the survey as part of a larger study that included additional questionnaires, with the remainder (110 participants) only completing the interoceptive awareness questionnaire. Participants were recruited from pre-existing participant databases and via social media outlets. Of the 1008 participants, 345 participants fully completed the survey, reported no pre-existing psychiatric conditions and had English as their first language and were therefore included in the study (M age = 38.66, SD age = 17.59, Age range 18-89 years, 95 males, 0 other). Of the 345 participants, 235 completed the additional questionnaires. In line with the declaration of Helsinki, all participants gave informed consent and were debriefed upon task completion.
Measures and procedure
The online survey was conducted using Qualtrics Research Suite (Qualtrics, Provo, UT, USA). To quantify interoceptive awareness, the very short version of the Body Perception Questionnaire (BPQ; Porges, 1993;Kolacz et al., in preparation) was used. In this 12-item questionnaire, participants are asked to indicate on a 5point Likert scale (strongly disagree to strongly agree) whether they are aware of particular bodily sensations (e.g., their mouth being dry) during most situations. Prior to the questionnaire, demographic details were collected, including age (years) and sex (male, female, other). Additionally, participants were asked to indicate whether English was their first language and whether they had a diagnosis of any psychiatric conditions.
Results and discussion
BPQ scores ranged from 12 to 57 (M = 26.89, SD = 10.12) with high scores representing greater self-reported awareness of interoceptive signals. A Spearman's rank order correlation indicated a significant negative correlation between age (years) and BPQ scores, r(345) = -.337, p < .001 (two-tailed; Fig. 1). No sex differences were found, t(343) = .208, p > .250, d = .03, 95% CI for d (-0.211, 0.261). No differences in BPQ scores were observed between participants completing just the interoception questionnaire and those completing additional questionnaires when age was statistically controlled for, t(343) = 0.68, p > .250, d = .08, 95% CI for d (-.154, .317). A significant negative association between age and selfreported interoceptive awareness was found. These results suggest that interoceptive awareness deteriorates across the lifespan and continues to decline into late, and very late, adulthood. However, given previous findings of a dissociation between subjective tests of interoceptive awareness and objective tests of interoceptive accuracy (Garfinkel et al., 2015), it remains unclear whether the continuing decline of interoceptive awareness into very late adulthood would be accompanied by decreasing interoceptive accuracy as measured by objective tests. To examine this question, Study Two utilised an objective measurement of interoceptive accuracy to quantify interoception across the lifespan.
Participants
A total of 140 participants took part in this study in exchange for a small honorarium. Participants were selected on the basis that they had no known psychiatric or neurological conditions. To screen for cognitive impairment, all participants were administered the Mini Mental State Examination test (MMSE; Folstein, Folstein, & McHugh, 1975), with scores below 23 indicative of cognitive impairment (Tombaugh & McIntyre, 1992). Four participants were excluded (one scored below the typical threshold on the MMSE; two disclosed existing psychiatric/neurological conditions post-testing; and one was excluded due to equipment failure) resulting in 136 valid cases (M age = 55.10, SD age = 19.50; age range 20-90 years, 49 males). A minimum of 5 participants were present in each 5-year age bracket. In line with the declaration of Helsinki, all participants gave informed consent and were debriefed upon task completion. To minimise the effects of elevated heartrate on accuracy (Knapp-Kline & Kline, 2005), all participants were asked to refrain from caffeine for 6 h prior to testing. Fig. 1 A significant relationship between self-reported interoceptive awareness, as measured by the very short BPQ, and age was observed such that increasing age was associated with poorer interoceptive awareness
Interoception
The heartbeat tracking task (Schandry, 1981) was used to quantify interoceptive accuracy with timing ability employed as a control task (Ainley, Brass, & Tsakiris, 2014;Shah et al., 2016). Objective heartbeat was measured using a pulse oximeter (Contec Systems, CMS-50Dþ; Qinhuangdao, China) attached to the participant's right index finger. Each participant completed both the heartbeat tracking task and the timing task over four durations. Two sets of durations were used (either 25, 35, 45, 100 s or 28, 38, 48, 103 s) and the duration sets were counterbalanced across participants such that half of the participants completed the longer durations for the timing task and half completed the longer intervals for the heartbeat task. Additionally, task order was fully counterbalanced, and the order of individual durations was counterbalanced according to a Latin-square across participants. Task order did not affect performance in interoceptive accuracy, t(134) = 1.640, p > .05, d = 0.28, 95% CI for d (-0.057, 0.619) or timing accuracy, t(134) = 1.364, p > .05, d = 0.23, 95% CI for d (-0.104, 0.571).
During the task, participants were seated with both feet flat on the floor and both hands on the table. Participants were instructed that they would be asked to silently count their heartbeats over a period without physically measuring their heartbeat. With their eyes closed, they were asked to count their heartbeats from when the experimenter said Bstart^until they heard a beep, at which point they should indicate the number they had counted. They were explicitly told to only count heartbeats they felt and not to count seconds or guess. They were also told that if they did not feel anything they should give zero as their answer. Participants were then given 2 min to practice prior to the first heartrate trial; no feedback was provided. The timing task was identical to the heartbeat task except participants were asked to count seconds rather than their heartbeats.
Anthropometrics
For each participant body mass index (BMI) and blood pressure measurements were taken. Blood pressure was taken using an electronic upper arm monitor (Omron M2) whilst participants were seated.
Beliefs regarding heartrate
To quantify beliefs, participants were asked to estimate the average person's resting heartbeat. Specifically, participants were asked 'How many times do you think the average person's heartbeats, in 1 min when they are at rest?' Note that earlier studies sometimes required the participant to estimate their own heartrate (Ring, et al., 2015;Ring & Brener, 1996); this was avoided in the present study to avoid any effect of the estimation on the heartbeat tracking task or vice versa.
Interoceptive and timing accuracy
Interoceptive accuracy on the heartbeat tracking task was estimated on a scale from 0 to 400: Σ[1 -(|Actual number of heartbeatsparticipant's estimate|/Actual number of heartbeats)] × 100. Higher scores indicate better performance (Shah et al., 2016; see also Garfinkel et al., 2015). Timing scores were estimated similarly, Σ[1 -(|Actual number of secondsparticipant's estimate|/Actual number of seconds)] × 100. Again, high scores indicate better performance. Average ratio scores (participants' estimate/objective measure) were also computed with scores above one indicative of overestimation. Unsurprisingly, given the explicit task instructions (see Methods), >95% of participants underestimated the number of heartbeats and a similar pattern was observed for timing accuracy (>80%). This variable was therefore not considered further.
Beliefs regarding heartrate
The accuracy of participants' beliefs was calculated as a continuous variable calculated by taking the absolute difference between participants' estimates and the grand mean of resting heartrate reported in large studies of human physiology (Agelink et al., 2001;Ramaekers, Ector, Aubert, Rubens, & Van de Werf, 1998; grand mean = 72.26 beats per minute; bpm). As this variable indicates how far participants' estimates are from the grand mean, higher scores indicate more inaccurate beliefs about the average person's heartrate.
Heartrate physiology
A proxy of heartrate variability was calculated from pulse-rate signals. This method has been shown to be reliable when participants are at rest (Schäfer & Vagedes, 2013). For each participant, the root mean squared of successive differences (RMSSD) was calculated from the last 60 s of heartrate recording for the longest interval examined (100 or 103 s). RMSSD was favoured over other measures of heartrate variability due to evidence attesting to its better reliability over short durations (Munoz et al., 2015). From this same 60-s interval, mean resting heartrate (average bpm) was taken. For three participants, the recording for the 100-s interval was corrupted, and a 60-s recording from one of the other intervals was used as a replacement which was comparable to the 100-s recordings.
Analysis strategy
Zero-order correlations revealed the associations between ageing and cardiac interoceptive accuracy, and ageing and the timing control task. The size of these correlations were compared using Steiger's Z-test (Steiger, 1980) using the quantpsy web implementation (Lee & Preacher, 2013). Correlations were then used to examine the association between age and the possible psychological and physiological mediators of the effect of age on interoceptive accuracy, before similar analyses compared the relationship between these potential mediators and interoceptive accuracy.
The existence of direct and indirect effects of age on interoceptive accuracy was investigated using a parallel mediation model in which BMI, gender, systolic blood pressure, accuracy of beliefs about average heartrate, heartrate variability, mean heartrate, and timing accuracy were entered as potential mediators of the effect of age on interoception. Mediation modelling was carried out using the SPSS macro-script (Process) provided by Hayes (2013) and Preacher and Hayes (2008). For indirect effects, 90% (one-tailed) bias-corrected bootstrapped confidence intervals were calculated using 5,000 repetitions. This method was selected over the Sobel (1982) method as the former does not require the assumption of a normal distribution and simulation studies indicate higher power whilst controlling for Type one error rates (MacKinnon, Lockwood, Hoffman, West, & Sheets, 2002;Mackinnon, Lockwood, & Williams, 2004). As outlined by Preacher and Hayes (2004), an indirect effect is significant if the confidence intervals for the indirect effect do not include zero. For all analyses where directional hypotheses are made, one-tailed p values are reported. Standardized coefficients are reported in the mediation analysis.
Results and discussion
A small amount of randomly-distributed data were missing (0.74%; three blood pressure measurements, two belief estimates, and one measure of heartrate variability and resting heartrate) and these missing values were imputed using multiple imputation in SPSS. The Mersenne-Twister algorithm with a starting point fixed to 2,000,000 was utilised for random number generation. All variables were entered into the model, the automatic method was selected, and all variables were used as predictors. No participant had more than one missing data point.
Simple zero-order correlations revealed a significant negative association between interoceptive accuracy and age, r(136) = -.21 p = .008 (one-tailed). Correlations between interoceptive accuracy and timing performance, r(136) = .12, p > .05 (one-tailed) were not significant, but the correlation between age and timing performance approached significance r(136) = -.14, p = .05. When formally compared, the sizes of the correlations between ageing and interoceptive accuracy, and ageing and the timing control task, were not significantly different (z = 0.6, p = .55).
General discussion
This set of studies aimed to examine variation in interoceptive awareness and accuracy across the lifespan. In Study One, a negative relationship between age and interoceptive awareness was observed; older participants rated their interoceptive awareness as reduced. Building on this, Study Two examined cardiac interoceptive accuracy across the adult lifespan using an objective performance measure, demonstrating a negative relationship between age and interoceptive accuracy. These data confirm previously reported evidence suggesting a decline in interoceptive accuracy in older adulthood (Khalsa et al., 2009), and go further to suggest that this decline continues into both late and very late adulthood.
The results from Study Two highlight that the poor performance observed in late adulthood is due to both a direct effect of age on interoceptive accuracy and an indirect effect mediated by BMI, whereby age was associated with increased BMI, which in turn, predicted reduced interoceptive accuracy. Therefore, it appears that changes in BMI partially mediate the relationship between age and interoception. Other physiological and psychological changes such as heartrate variability, resting heartrate, and time perception, previously associated both with interoceptive accuracy (Knapp-Kline & Kline, 2005;O'Brien et al., 1998;Rouse et al., 1988;Wittmann, 2013) and increasing age (Franklin et al., 1997;St-Onge, 2005;Umetani et al., 1998;Yashin et al., 2006), were not significant mediators of the age-interoception relationship.
The partial mediation of the effect of age on interoceptive accuracy by BMI provides some indication as to the mechanism by which ageing negatively impacts interoceptive accuracy. However, the existence of a direct effect of age indicates at least one further mechanism to be identified. Although the results of Study One demonstrated reduced awareness of interoceptive information with increasing age, previous reports of a dissociation between self-reported interoceptive awareness and interoceptive accuracy (Garfinkel et al., 2015) mean that care must be taken in interpreting reduced awareness of interceptive signals as underlying the direct effect of age on interoceptive accuracy.
The relationship between age and performance on the interoceptive accuracy and timing control tasks is worthy of note. Whereas the zero-order correlations indicated a similar magnitude of the effect of ageing on performance of these tasks, the results of the mediation analysis demonstrate that the effects of ageing on interoceptive accuracy were not a product of reduced timing/counting ability (Turgeon et al., 2016;Wittmann, 2013), and are also likely not due to general problems with motivation or attention. Thus, although similar age-related declines were observed across these tasks, the effects of age were independent.
The finding that interoception declines throughout the lifespan raises important questions regarding the extent to which interoception is related to the decline in socioemotional competence and altered cognition observed in late adulthood (Murphy et al., 2017). For example, a body of research indicates poorer emotion recognition (Ruffman et al., 2008) and changes in risky decision making with advancing age (Sparrow & Spaniol, 2016). Crucially, these same abilities have been linked to interoceptive accuracy (Dunn et al., 2010b;Füstös et al., 2013;Schandry, 1981;Sokol-Hessner et al., 2015;Terasawa et al., 2013;Werner et al., 2009;Fig. 2 Depicts the results of the mediation analysis. Mediation analysis indicated a significant path from age to BMI, heartrate variability and time perception ability (a path; the individual relationships between the IVand the mediators; e.g., from age to BMI; Fig. 2 left), whilst significant paths to interoceptive accuracy from BMI, gender, heartrate variability beliefs and time perception ability were also observed (b path; the relationship between the mediators and the DV controlling for other mediators and keeping the IV constant; e.g., from BMI to interoceptive accuracy; Fig. 2 right). Most importantly, a direct effect of age on interoceptive accuracy was observed (c' path; the relationship between the IVand the DV controlling for the mediators; from age to interoceptive accuracy; Fig. 2 centre), in addition to an indirect path from age to interoception via BMI. The indirect path via BMI thus partially mediated the effect of age on interoception. *Denotes significance at the p < .05 level (one-tailed). BMI Body Mass Index; Gender Male or Female (0 or 1); Heartrate Variability variation in the time interval between heartbeats; Mean HR The average second-by-second heartbeat recorded over 60 seconds; Beliefs the error of participants' estimates regarding the average heartrate; Time Perception Performance on the time estimation task; Systolic Blood Pressure Taken whilst seated and measured in millimetres of mercury Wiens et al., 2000). Given the interrelatedness of these factors, determining the extent to which age-related changes in these abilities is predicted by interoception is an important aim for future research. Moreover, if interoception does underlie adverse age-related cognitive change, then such evidence may inform interventions, such as interoceptive training (e.g., Canales-Johnson et al., 2015;Schaefer, Egloff, Gerlach, & Witthöft, 2014;Schandry & Weitkunat, 1990) designed to ameliorate undesirable effects of ageing on cognition and socio-emotional competence.
In comparison to the only other study examining the impact of older age on cardiac interoceptive accuracy (Khalsa et al., 2009), the variance explained by age in the present study was modest. The difference in the size of the observed age effect could be because the present study quantified interoceptive accuracy using the heartbeat tracking task (Schandry, 1981), whereas Khalsa et al. (2009) utilised the heartbeat discrimination procedure (Brener & Kluvitse, 1988;Whitehead et al., 1977). Whilst small to moderate correlations have been observed between these two tasks (Garfinkel et al., 2015;Knoll & Hodapp, 1992; but see Phillips, Jones, Rieger, & Snell, 1999), the extent to which task differences impact the accurate measurement of interoceptive accuracy remains unknown. It is generally accepted, however, that the heartbeat discrimination task is more difficult than the heartbeat tracking task (possibly due to the requirement to integrate exteroceptive and interoceptive signals; see Pennebaker, 2012), and it is possible that agerelated differences are more apparent when tasks are more demanding. Also, the present study investigated interoceptive accuracy into later stages of the lifespan than Khalsa et al., (2009). It remains a possibility that with advancing age certain individuals pay increased attention to accurate measurements of bodily sensations (e.g., monitoring blood pressure). Such individual differences in health-related behaviour may account for the increasingly varied performance observed in later life.
Whilst the present set of studies investigated interoception across the dimensions of interoceptive accuracy and awareness, these facets of interoception were not examined together. Thus, the extent to which interoceptive awareness predicts interoceptive accuracy in late adulthood remains an unanswered question. However, examining changes in metacognition for interoceptive information (the extent to which confidence in one's interoceptive accuracy and awareness predicts interoceptive accuracy; Garfinkel et al., 2015) across the lifespan (Palmer, David, & Fleming, 2014;Vukman, 2005) may be crucial for identifying individuals at risk of illness associated with poor interoception. Whilst this study and others converge to suggest that interoceptive accuracy declines across the lifespan, if older adults are aware of this deterioration they may be more inclined to utilise external strategies for gauging interoceptive states (e.g., to ensure good hydration older adults may audit their liquid intake rather than relying on interoceptive feelings of thirst). In contrast, individuals with poor interoceptive metacognition-particularly those with inflated beliefs regarding their ability-may utilise unreliable interoceptive cues for gauging interoceptive states, placing them at a greater risk of adverse health outcomes (e.g., dehydration).
In conclusion, Study One demonstrated a negative relationship between age and interoceptive awareness. Study Two demonstrated that the decline in interoceptive accuracy across the lifespan continues into late and very late adulthood. A decline in interoceptive awareness and accuracy with increasing age highlights the importance of understanding the relationship between age-related changes in interoceptive ability and agerelated changes in cognition and physical and mental health. | 2018-04-03T02:48:48.708Z | 2017-07-06T00:00:00.000 | {
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259315013 | pes2o/s2orc | v3-fos-license | Arthroscopic cartilage regeneration facilitating procedure can modify the clinical course of knee osteoarthritis
Background The effectiveness of arthroscopic treatment for knee osteoarthritis (OA) has been controversial. This study compares the clinical outcomes of the arthroscopic cartilage regeneration facilitating procedure (ACRFP) and conservative treatment. Methods During the year of 2016, 524 patients (882 knees) who were older than 40 years of age and diagnosed with different stages of knee OA were scheduled for ACRFP under the protocol of knee health promotion option (KHPO) for knee OA. Of those, 259 patients (413 knees) eventually received ACRFP (the ACRFP group), and 265 patients (469 knees) didn’t receive ACRFP but received conservative treatment (the non-ACRFP group). A telephone questionnaire was used to evaluate the subjective satisfaction and the incidence of receiving arthroplasty for these patients. Results After the mean follow-up period of 61.6 months (SD 4.5), there were 220 patients (374 knees, 90.6%) in the ACRFP group and 246 patients (431 knees, 90.0%) in the non-ACRFP group completed the outcome study. The satisfactory rate was statistically higher for the ACRFP group (90.64%) than for the non-ACRFP group (70.3%) and the difference in subjective satisfaction was more obvious in patients with more advanced knee OA. As for the incidence of patients having subsequently received arthroplasty, it was higher (13.46%) in the non-ACRFP group than in the ACRFP group (4.28%). Conclusion Compared with conservative treatment, ACRFP could satisfy more patients with knee OA and modify their natural course by decreasing the incidence of subsequent arthroplasty.
Introduction
Knee osteoarthritis (OA) remains a tremendous public health concern, both in terms of health-related quality of life and financial burden of the disease.Conservative therapies, such as nonpharmacological interventions, systemic drug treatment and intra-articular therapies ranging from corticosteroids to hyaluronans to more recently platelet-rich plasma and even stem cells have been used for symptom relief before resorting to surgery, but conservative therapies have often provided inadequate disease control.There was no evidence to verify their effectiveness in reversing the natural course of knee OA till now.Arthroscopic intervention has been commonly used for many years to treat knee OA and to address degenerative articular cartilage and menisci, [1][2][3] however, several studies have verified that it is not an effective treatment for the majority of cases and should generally not be considered when managing knee OA. [4][5][6][7][8] Based on a series of studies highlighting medial abrasion phenomenon (MAP) as a cause of knee OA, [9][10][11][12][13][14] an integrated protocol for the treatment of knee OA called "knee health promotion option (KHPO)" was established. 15A concept of arthroscopic cartilage regeneration facilitating procedure (ACRFP) under the protocol of KHPO was also developed and reported in 2012 which pointed out that stress and inflammatory responses are the key events that lead to the onset and progression of knee OA caused by MAP and that the ACRFP procedure, by eliminating stress and inflammatory responses as well as comprehensively improving the environment of the knee joint for cartilaginous regeneration, will bring forth longterm favorable effects. 16Not only for medial compartmental OA, the effectiveness of ACRFP has also been validated for lateral compartmental OA, according to a recent study. 17Although the clinical effectiveness of this concept has been reconfirmed by other series, 18,19 the evidence has not been strong enough to support the popularity of the ACRFP due to a lack of control group in those studies.We therefore conducted a modified randomized controlled study to verify the clinical effectiveness of ACRFP in comparison with conservative treatment.
The protocol of KHPO for knee OA
The first step of KHPO for the treatment of OA knee is thorough evaluation of the patient's knee condition and establishing the clinical staging for each weight bearing compartment of the knee by both physical and roentgenographic examinations (Figure 1 and Table 1). 15The staging of OA of the knee is given as the most advanced stage of the two weight bearing compartments.Once the clinical staging has been made, the decision of treatment option for individual patient was made according to our guideline of treatment (Table 2). 15For stage I or II patients, all patients were recommended to encounter into the supervised conservative treatment which we call smart knee care for at least 3 months before the decision of receiving ACRFP was made.The sine qua non for a successful smart knee care is to make patients and the family completely understand our concept that the MAP is an important etiologic factor for knee OA.Daily activities, job and sport modification were tailored for individual patient focusing on the avoidance of the MAP.In general, activities and sports need repeated or quickly knee bending are regarded as harmful.Moreover, home exercises including muscle strengthening by straightleg-raising and stretching exercises to increase the flexibility by knee hug and knee press were emphasized (Figure 2).For stage III and IV patients, ACRFP is the best choice compared to osteotomy and arthroplasty if the deformity is less severe.However, if the deformity exceeds 7°, arthroplasty including high tibial osteotomy, unicompartmental arthroplasty or total knee arthroplasty with elimination of the MAP which we call precision arthroplasty would be recommended considering patients' preference and biopsychosocial condition.
The arthroscopic cartilage regeneration facilitating procedure
Either spinal or general anesthesia was used for this procedure.Throughout the procedure, patients were put in a supine position and bloodless surgical field was obtained by pneumatic tourniquet.The arthroscopic examination was performed through the inferolateral portal.The presence of medial plica related MAP and its sequelae 9 was first investigated and confirmed.Medial release was then performed step by step as described in previous publication. 16The inflammatory tissue occupying the space over the inferomedial region of the patella including ligamentum mucosum, fibrotic or inflamed synovium, capsule and distal part of the medial plica was first eradicated.Then, the tight and obliterated medial facet of patellofemoral joint was released by resection of the fibrotic synovium, capsule, and the proximal part of the medial plica.Sometimes, medial retinaculum including fibrotic fascia of pes anserinus and even medial patellofemoral ligament was released as needed.After the medial release, the patella would always deviate laterally.Lateral release was performed by inserting a No. 11 scalpel into the inferolateral portal and cutting the lateral retinaculum percutaneously.Finally, any focal synovitis or loose chondral flaps on the cartilaginous surface was removed as conventional arthroscopic debridement for knee OA.No bony procedure such as drilling or microfracture was performed.From the day of surgery, full range of motion, full weight bearing, and free ambulation were allowed as tolerated.A suction drain was used for 24-48 h.The patient was discharged 2 days after the operation.Home exercise programs, including quadriceps strengthening and passive range of motion, were emphasized.The compliance of patients regarding the home exercise program was strictly monitored by case managers.No supplementary treatment including oral glucosamine sulfate, steroid injections, or intraarticular injection of HA or PRP was given throughout the postoperative follow-up period
The patients
During the year of 2016, 524 patients (882 knees) who were older than 40 years of age and diagnosed with different stages of knee OA were scheduled for ACRFP after failed smart knee care according to the protocol of our KHPO (Figure 1).The clinical staging of the knee OA by both physical and roentgenographic examinations (Table 1) was given by the senior author.The knee injury and OA outcome score (KOOS) and the Knee Society score (KSS) were evaluated and recorded at their first visits by nursing specialists.These patients were recommended to receive ACRFP corresponding to our guideline for the surgical treatment of knee OA (Table 2).According to this guideline, all these scheduled patients were at the clinical staging of stages II, III, or IV.Since the waiting list for ACRFP in our center at that time was more than 1 year, we recommend them to encounter smart knee care including behavior modification and home exercise under the protocol of KHPO to diminish MAP while waiting for surgery.These patients were followed up and their outcomes were evaluated as part of an Institutional Review Board Registry.Eventually, there were 259 patients (413 knees) received ACRFP (the ACRFP group) and 265 patients (469 knees) didn't receive ACRFP (the non-ACRFP group).The demographic data of these two groups were shown in Table 3.The ACRFP group was older in age, in more advanced stage, and clinically more severe according to the parameters of KOOS and KSS.
Outcome study
A telephone questionnaire was used to evaluate the subjective satisfaction of these patients after a minimum of 5 years.Subjective satisfaction was derived from subjects' answers to direct questions using a categorical scale prepared for this study: (1) excellent, free of symptoms, no limitation in activities; (2) good, greatly improved, occasional pain, normal activities; (3) fair, same as the condition of the first evaluation, no improvements; and (4) poor, has received arthroplasty.The outcome was regarded as satisfactory if subjective satisfaction was rated as "excellent" or "good".The inquiries into subjective satisfaction were conducted by our nursing specialists.All investigations focused on individual knees in bilateral cases.
Statistical analysis
Statistical analysis was carried out using JMP, the Statistical Discovery Software (Version 5.0.1.2,SAS Institute Inc., Cary, NC, USA).All values were presented with means and standard deviations.Analyses for comparing demographic data, satisfactory rate, and incidence of patients received arthroplasty between the ACRFP and non-ACRFP groups were performed using Pearson's Chi-square test.A p-value <0.05 was considered statistically significant.
Results
In the ACRFP group, 220 patients (374 knees, 90.6%) completed the outcome study.There were 15 patients passed away, seven patients refused to answer the questionnaire, and 17 patients lost contact.During ACRFP, different severity of the sequelae of MAP described in the previous publication 9 was discovered after synovectomy and medial plica (or its remnant) resection in all patients.After medial release, all patellae would deviate laterally, and balance was restored by lateral release.In the non-ACRFP group, 228 patients (431 knees, 90.0%) completed the outcome study.The reasons of cancelling ACRFP for these patients were all related to the long waiting time.There were 10 patients passed away, eight patients refused to answer the questionnaire, and 19 patients lost contact.The mean follow-up period was 61.6 months (SD 4.5).As shown in Table 4, the satisfactory rate was statistically higher for the ACRFP group (90.64%) than that for the non-ACRFP group (70.30%) and the difference in subjective satisfaction was more obvious in patients with more advanced knee OA.As for the incidence of patients having subsequently received arthroplasty, it was significantly higher (13.46%) in the non-ACRFP group than in the ACRFP group (4.28%) and the difference was more obvious in patients with more advanced knee OA as shown in Table 5.
Discussion
In this report we present and compare the satisfactory rate and incidence of arthroplasty between knee OA patients who have received ACRFP and those who haven't received this procedure.Although the ACRFP group was older in age, in more advanced stage of knee OA, and clinically more severe according to the parameters of KOOS and KSS, after at least 5 years of follow-up, the satisfactory rate was significantly higher in the ACRFP group (90.64%) than in the non-ACRFP group (70.30%).The incidence of patients receiving arthroplasty was remarkably lower in the ACRFP group (4.28%) than in the non-ACRFP group (13.46%).The satisfactory rate in our ACRFP group is not only higher than that in the non-ACRFP group, but it is also significantly higher than what was shown in a previous study which reported that the medium-term outcome (mean follow-up: 49.2 months) was poor in about 71.7% of patients with isolated Kellgren-Lawrence grade 2 (definite osteophytes and possible narrowing of joint space) 20 medial-compartment knee OA who subsequently underwent arthroscopy. 21A meta-analysis of 30 papers for the effects of arthroscopic debridement in knee OA showed satisfactory outcomes in around 60% of all patients.The required conversion rate to arthroplasty increased as the follow-up interval increased: 1 year-6.1%, 2 years-16.8%,3 years-21.7%and 4 years-34.1%. 1 In comparison, our data disclosed that ACRFP could modify the natural course of knee OA.The incidence of patients receiving arthroplasty in our ACRFP group was 4.28% after 5 years follow-up.According to a report investigating the likelihood of TKA following arthroscopic surgery for knee OA, 22 the annual incidence of TKA was 2.62% after arthroscopic surgery for OA and is compatible with our 5 years incidence of 13.46% in the non-ACRFP group.This conversion incidence in our non-ACRFP group is compatible with some studies regarding conventional arthroscopic management or conservative treatment.A study found that compared with conservative treatment, arthroscopy did not decrease or delay TKA in a 5-years retrospective study of 382 patients with knee OA.Of the 214 patients treated with arthroscopy, 32 (15%) eventually underwent TKA, compared with 30 of the 168 patients (17.9%) treated conservatively. 23Another study found the incidence of TKA after corticosteroid and/ or HA injections for 5 years was 13.9%. 24A study conducted in patients who participated in a single 8-weeks multimodal knee OA treatment program consisted of five intra-articular knee injections of sodium hyaluronate, structured physical therapy, knee bracing, and patient education disclosed that TKA was performed in 22.8% of knees during mean follow-up of 3.7 years. 25ince there is no consensus in the pathogenesis of knee OA, the efficacy of arthroscopic debridement or lavage for knee OA was disputed after publication of several randomized controlled trials and meta-analysis. 4,6,7,26][30][31][32] Our report might shed some light on the confusing and negative impression about arthroscopic treatment for knee OA.Unlike the uncertain beneficial mechanism and the diverse outcomes of common arthroscopic techniques including lavage, debridement, abrasion arthroplasty, microfracture, and autologous chondrocyte implantation (ACI) for knee OA, the concept of ACRFP has precise rationale of treatment and more beneficial, reproducible outcomes.It was developed and conceptualized according to a series of studies regarding MAP as a cause of knee OA.The outcomes of previous studies affirm that, besides eradicating the MAP, the key to a successful ACRFP is to adequately decompress the tight patellofemoral joint and maintain this appropriate tension around the patella with skillful supervised post-operative home-rehabilitation.The immediate effect of ACRFP was obtained by releasing the tension around patella caused by chronically inflamed soft tissue and by eradication of the hypertrophied and inflamed synovium that may have caused pain in these osteoarthritic knees over the medial compartment and the patellofemoral joint.Furthermore, by eliminating these stress/inflammatory responses that are the key events on the onset and progression of OA, this procedure also brings forth to long-term favorable effects as a consequence of the global improvement of the environment of the knee joint for cartilaginous regeneration.Just like the release-handcuffs effect for the repeated injured skin on the wrist, by a purposeful and timely eradication of all prejudicial factors in the degenerative knee, the jeopardized cartilage could have a chance to regenerate by its innate healing response.
There are limitations in this study.First, this is not a standard randomized controlled study.The long waiting list phenomenon might produce some bias considering the different intention-to-treat effect between groups.The ACRFP group might have higher motivation to preserve their knees according to their understanding of MAP as a cause of knee OA.Second, the compliance of doing home exercise according to our KHPO protocol by the patients in the ACRFP group was monitored by case managers.On the other hand, the patients in the non-ACRFP group were in a status of loss-of-follow-up.This difference in the intensity of medical care might have affected their outcomes.Third, the decision making of performing arthroplasty by different surgeons might have produced biases for the incidence of arthroplasty in the non-ACRFP group.Fourth, by using telephone questionnaires as the investigation method, we could not perform clinical and radiological evaluation.However, the simple and clear questionnaire designed for this study might still obtain informative outcome data.Fifth, although we have cited some publications about conventional arthroscopic surgery for knee OA for comparison, a study using conventional arthroscopic procedure as the control group would have been more convincing.All these limitations warrant more precisely designed investigations.
In conclusion, compared with conservative treatment, ACRFP could satisfy more patients with knee OA and modify their natural course by decreasing their incidence of receiving arthroplasty.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Figure 1 .
Figure 1.Protocol of knee health promotion option for knee OA.
Figure 2 .
Figure 2. Three home exercises for muscle strengthening and increasing flexibility of the knee.
Table 1 .
Clinical staging for OA of individual compartment based on roentgenographic and clinical findings.
Table 2 .
Surgical treatment guideline for knee OA.
Table 3 .
Comparison of demographic data between ACRFP and non-ACRFP groups.
*Statistical significance, p value <0.05.MMC: medial compartment as the main involved compartment; KOOS: knee injury and osteoarthritis outcome score; ADL: activity of daily life; Sport/Rec: sports and recreation; QOL: quality of life; KSS: knee society score.
Table 4 .
Satisfactory rate of different stage of OA for ACRFP and non-ACRFP groups stratified by main involved compartment.
Table 5 .
Incidence of arthroplasty for ACRFP and non-ACRFP groups stratified by main involved compartment. | 2023-07-04T06:17:26.915Z | 2023-05-01T00:00:00.000 | {
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124590723 | pes2o/s2orc | v3-fos-license | The Extended CG Method for Non-Quadratic Models
Received on: 25 / 3 / 2007 Accepted on: 4 / 11 / 2007 ABSTRACT This paper investigates an interleaved algorithm which combines between the extended conjugate gradient with the hybrid method of Touati-Storey. This combined algorithm is based on the exact line search to solve a number of non-linear test functions with different dimensions. Experimental results indicate that the modified algorithm is more efficient than the original Sloboda algorithm.
Introduction
The problem to be solved by the Sloboda algorithm is to calculate the least value of a general differentiable function of several variables. Let n be the number of variables, x be the vector of variables and q(x) be the objective function and g(x) be the gradient of q(x). i.e.
Conjugate gradient algorithm does not require any explicit second derivatives and it is an iterative method. The sequences of points x1 , x2 , x3 , … are calculated by the successive iteration procedure and it should converge to the point in the space of the variable at which q(x) is least.
The conjugate gradient algorithm was first applied to the general unconstrained minimization problem by Fletcher & Reeves [7]. However, now there are several versions of the algorithm for this calculation. Let x1 be a given starting point in the space of the variable and let i denote the number of the current iteration starting with i=1. the iteration requires the gradient: gi = g(xi) , if i = 1, let di be the steepest descent direction, di = -gi (2) otherwise, for i > 1, we apply the formula: where βi and yi have the values: The vector norms being Euclidian and by searching for the least value of f(x) from xi along the direction di we can obtain the vector xi+1: Where i is the value of that minimizes the function of one variable.
This complete the value of the iteration, and another one is begun if f(xi+1) or gi+1 is not sufficiently small, [10].
Let A denote a symmetric and positive definite ii matrix. For i R x , we define: Let i F : R R denote a strictly monotonic increasing function and define: Such a function is called an extended quadratic function, Spedicato [12]. When a minimization algorithm is applied to f, the i th iterate is denoted by xi, the corresponding function value by fi and its gradient by Gi , the function value and gradient value of q are denoted by qi & gi , respectively and the derivative of
Non-Quadratic Sloboda Method
Sloboda [11] proposed a generalized conjugate gradient algorithm for minimizing a strictly convex function of the general form: This algorithm is as follows: ▪ Algorithm 1: Step 1: Set i = 1 ; d1 = -G1 ; Step 2: Step 3: Step 4: Test for convergence, if achieved stop. if not continue.
Step 6: Step 7: Compute the new search direction i i Step 8: Set 1 i + = i ; and go to step 2. Algorithm 1 terminates after i iterations in the case of a nonlinear scaled quadratic function using ELS.
A more general scaling has been considered by Spedicato [12], such a scaling transforms F into a new function where (9), is satisfied. He shows that the sequence of points generated is invariant with respect to nonlinear scaling if: However, this type of scaling also uses functions for which the analytic form is known apriority.
Al-Bayati [1] introduced another family of self-scaling VM-methods given by: where is again a free parameter; If an estimate of the inverse Hessian in maintained (rather than an estimate of the Hessian itself which is sometime preferred) then there is a strong motivation for However, it is possible to generalize Al-Bayati's family of self-scaling VMupdates (12) to be invariant to a nonlinear scaling by the following algorithm, Al-Bayati [2]. ▪ Algorithm 2: Step 1: Set i = 1; H1 = I ; d1 = -H1 G1 ; Step 2: Compute Step 3: Set Step 4: Test for convergence; if not continue.
Step 6: Compute Step 7: Set 1 i + = i and go to Step 2.
In this section we shall describe also another algorithm which effectively interleaves CG & VM-steps. It is also related to one given originally by Buckley [6], but our implementation differs in that we use the scaled quadratic model instead of the quadratic itself.
CG-algorithm 1. and the generalized VM-algorithm 2. The objective here is to show that, using Al-Bayati's self-scaling VM-update (12), the sequence of the generating points is the same in the generalized CG-algorithm 1.
Before making a few more observations we shall outline briefly the proposed strategy for the interleaved generalized CG-VM method Al-Bayati [2]. Here i is the index of the matrix updated only at restart steps and k is the index of iteration and the algorithm is not converged, until a restart is indicated.
Step 3: If a restart is indicated, namely that the Powell [9] restarting criterion is satisfied, i.e.
Then reset t to the current k , update Hi by: Step 4: Replace i by i + 1 and repeat from (13).
Hybrid Conjugate Gradient Method
Despite the numerical superiority of PR-method over FR-method the later has better theoretical properties than the formal see Al-Baali [3]. Under certain conditions FR-method can be shown to have global convergence with exact line search Powell [10] and also with inexact line search satisfying the strong Wolf-Powell condition. This anomaly leads to speculation on the best way to choose βi .
Touati -Ahmed and Storey in (12) proposed the following hybrid method: Step , otherwise go to Step 3.
Step 3: Here m, and user supplied parameters. This hybrid was shown to be globally convergence under both exact and inexact line searches and to be quite competitive with PR-and FR-methods.
Step 5:If PR Step 6: Compute k k Step 7: If a restart is indicated, namely that the Powell, restarting criterion is satisfied, i.e.: k-1 k *T *T k k |g g | 0.2 |g g | , then reset t to the current k, update Hi by: Step 8: Replace i by i+1 and repeat from (13).
Conclusions and Numerical results:
Several standard test functions were minimized (2 < n < 400) to compare the proposed algorithm with standard Sloboda algorithm which are coded in double precision Fortran 90. The proposed hybrid algorithm needs matrix calculation for 400400, this is the approximately the latest range for this computation of the matrix. The numerical results are obtained on personal Pentium IV Computer. The compete set of results are given in tables 5.1 and 5.2.
The linear search routine used was a cubic interpolation which use function and gradient values and it is adaptation of the routine published by Bunday [5].
We tabulate for all the algorithms; the number of functions evaluations (NOF) and the number of iterations (NOI). Overall totals are also given for NOF and NOI with each algorithm. Table 5.1 gives the comparison between the standard Sloboda algorithm and the proposed algorithm. Table 5.2 indicates that the suggested algorithm is more efficient than the standard Sloboda algorithm. Namely, there are an improvement of about (53 %) in both NOI and NOF according to our selected group of test functions. All the algorithms terminated when -10 min
Appendix
These test function are from general literature [8]. | 2019-04-21T13:04:10.861Z | 2011-06-28T00:00:00.000 | {
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216554281 | pes2o/s2orc | v3-fos-license | Remote Ischemic Conditioning for Intracerebral Hemorrhage (RICH-1): Rationale and Study Protocol for a Pilot Open-Label Randomized Controlled Trial
Background and rationale: Although many therapies have been investigated for intracerebral hemorrhage (ICH), none have succeeded in improving the functional outcomes. Remote ischemic conditioning (RIC) has been proven to promote hematoma resolution and improve neurological outcomes in an ICH model; whether it is safe and feasible in patients with ICH remains unknown. This trial aims to assess the safety, feasibility, and preliminary efficacy of RIC in patients with ICH and to plan for a phase-2 study. Methods: A proof-of-concept, assessor-blinded, pilot open-label randomized controlled trial will be carried out with patients with ICH within 24–48 h of ictus. All participants will be randomly allocated to the intervention group and the control group with a 1:1 ratio (n = 20) and will be treated with standard managements according to the guidelines. Participants allocated to the intervention group will receive RIC once daily for 7 consecutive days. Cranial computed tomography examinations will be performed at baseline, and on days 3, 7, and 14. Neurological outcomes will be assessed at baseline, and on days 1 to 14, 30, and 90. The primary outcome to be tested is safety. Secondary tested outcomes include changes of hematoma and perihematomal edema volume, incidence of hematoma expansion, functional outcomes, and frequency of adverse events. Discussions: This study will be the first proof-of-concept randomized controlled trial to ascertain the safety, feasibility, and preliminary efficacy of RIC in patients with ICH, results of which will provide parameters for future studies and provide insights into the treatment of ICH. Trial Registration: Clinicaltrials.gov, identifier: NCT03930940.
INTRODUCTION AND RATIONALE
Intracerebral hemorrhage (ICH) is a severe neurological disease and public health issue that accounts for 10-15% of strokes in European and American countries and 20-30% of strokes in Asian countries (1,2). Although the rate of ICH is far lower than that of ischemic stroke, which is more than 70% of all strokes, its mortality is as high as 50% at 30 days. Furthermore, only 20% of survivors regain functional independence (2). Over recent decades, many corresponding strategies, such as invasive and minimally-invasive hematoma evacuation, blood pressure control, hemostatic therapy, and osmotic treatment, have been investigated; however, few of them have succeeded in improving functional outcomes (3), leading ICH to be the least treatable type of stroke (4), and novel strategies and approaches for the treatment of ICH are urgently needed.
Remote ischemic conditioning (RIC) is a noninvasive systemic protective strategy in which several cycles of brief focal ischemia followed by reperfusion in arms or legs confer protection against more severe injuries in distant organs (5). At present, RIC if applied in arms has been found to benefit patients with acute ischemic stroke, intracranial atherosclerotic stenosis, cerebral small vessel disease, and those undergoing carotid stenting (6)(7)(8)(9). Although the underlying mechanisms are not fully understood, current evidence indicates that RIC could reduce inflammation, oxidative stress, and cerebral edema and have other positive impacts (10,11). Furthermore, in critically ill patients with aneurysmal subarachnoid hemorrhage, RIC applied in the legs has been demonstrated to be feasible, safe, and well-tolerated, with the potential to prevent delayed cerebral artery spasm and to reduce the incidence of both stroke and death (12)(13)(14).
In the ICH model, RIC has been found to reduce cerebral edema without exacerbating hematoma size within 72 h of ictus (15,16). More recently, an experimental study further demonstrated that 6 consecutive days of RIC could improve local cerebral blood flow perfusion, promote hematoma resolution, and improve neurological outcomes (17). However, it remains unknown whether RIC is safe, feasible, and effective in human patients with ICH.
We therefore designed the Remote Ischemic Conditioning for intracerebral Hemorrhage (RICH) study to investigate the safety, feasibility, and efficacy of RIC performed in unilateral arm in patients with ICH. RICH-1 is a pilot study, aiming to determine the safety and feasibility of RIC in patients with ICH and to plan for a prospective phase-2 study (RICH-2) that will investigate the efficacy of RIC.
Study Design
This is a proof-of-concept, assessor-blinded, pilot open-label randomized controlled trial that will be carried out in four centers with patients who suffered from ICH and will receive nonoperative treatment. The trial design flowchart is illustrated in Figure 1. Participants meeting the inclusion criteria but not the exclusion criteria will be randomly allocated to the RIC group or the control group. Both groups will be treated with the standard management according to the guidelines (4). Remote ischemic conditioning will be performed in the RIC group for 7 consecutive days after enrollment. Cranial computed tomography (CT) examination will be performed at baseline and days 3, 7, and 14. The National Institutes of Health Stroke Scale (NIHSS) score will be assessed by trained investigators blinded to the treatment assignment at baseline and days 1-14 after enrollment. Modified Rankin Scale (mRS) will be evaluated by trained investigators blinded at days 30 and 90.
All participants will be informed about the clinical study and the requirements to give informed consent. This study was approved by the ethical committee of each center and has been registered at Clinicaltrials.gov with NCT03930940.
Patient Population: Inclusion and Exclusion Criteria
Participants will be recruited from the wards. The inclusion criteria are (1) ≥18 and ≤80 years old; (2) diagnosis of supratentorial ICH confirmed by brain CT scan; (3) hematoma volume of 10-30 mL; (4) Glasgow Coma Scale (GCS) score >8; (5) ability to start RIC treatment within 24-48 h of ictus; and (6) informed consent provided by participant or legally authorized representative.
Exclusion criteria are as follows: (1) patients with suspected ICH secondary to tumor, coagulopathy, ruptured aneurysm or arteriovenous malformation, venous sinus thrombosis, cerebral infarction, and traumatic brain injury; (2) ICH concomitant with subarachnoid hemorrhage or intraventricular hemorrhage and planned surgical evacuation for the index hematoma prior to enrollment; (3) evidence of significant shift of midline brain structure (>10 mm) or herniation on brain imaging; (4) known pregnancy or breastfeeding; (5) concurrent participation in another research protocol investigating a different experimental therapy; (6) preexisting neurological deficit (mRS score >1) or psychiatric disease that would confound the neurological or functional evaluations; (7) life expectancy of fewer than 90 days due to comorbid conditions; (8) severe hepatic and renal dysfunction; (9) severe, sustained hypertension (systolic blood pressure >180 mm Hg or diastolic blood pressure >110 mm Hg); (10) contraindication to RIC because of severe soft tissue injury, fracture, or peripheral vascular disease in the upper limbs; (11) any condition which, in the judgment of the investigator, might increase the risk to the patient.
Randomization
All enrolled participants will be randomly assigned in a 1:1 ratio to the RIC group and the control group (n = 20 each). The randomization sequence will be made according to a predefined table generated by a computer program. Subsequently, the randomization sequence that indicated the group allocation will be concealed in sequentially numbered opaque closed envelopes. A research assistant, who will not be involved in the study, will prepare the envelopes prior to the study. After recording baselines measures, participants will be randomly allocated to either the RIC or the control group by the treating physicians who will open the sealed opaque envelopes.
Interventions
Participants in both groups will receive the standard management according to the guidelines, including blood pressure control, interventions for elevated intracranial pressure and mass effect, treatment of seizures, prevention of venous thromboembolism, maintenance of fluids, treatment of hyperglycemia, rehabilitation, and so on. In addition, participants allocated to the RIC group will undergo the RIC procedure once daily for 7 consecutive days after enrollment, and the interval between two RIC procedure is 24 h. In all the centers, the RIC procedure will be induced by using the same electric autocontrol device with cuffs placed on the nonaffected arm (Figure 2) and will consist of four cycles of arm ischemia with cuffs inflated to a pressure of 200 mm Hg for 5 min followed by reperfusion for another 5 min. The RIC procedure will be performed with assistance from a hospital-based nurse. For participants allocated to the control group, no RIC will be received.
Safety Outcomes Assessment
The safety is defined as any of the following: (1) neurological deterioration defined as an increase of four or more points in the NIHSS or a decrease of two or more points in the GCS score within 7 days of enrollment without a clear explanation; (2) death during the study period regardless of cause; (3) any adverse event that prolongs hospitalizations or requires medical treatment during the study period.
Efficacy Outcomes Assessment
The primary efficacy outcome is the change of hematoma volume to be detected on cranial CT images performed at baseline and days 3, 7, and 14. Axial noncontrast CT and images will be obtained at each participant's institution using standard local protocols. Cranial CT scans with 5-mm slice thickness reconstruction will be collected, and the hematoma volume will be measured according to the methods described in a previous study (18).
The secondary outcomes are the following: (1) The change of perihematomal edema volume to be detected on cranial CT images performed at baseline and days 3, 7, and 14: perihematomal edema volume will be measured according to the methods described in a previous study (18). (2) Incidence of hematoma expansion at day 3: hematoma expansion is defined as an increase in the volume of intraparenchymal hemorrhage of ≥33% as measured by image analysis on the 3-days CT scan as compared with the baseline CT scan; the cutoff for hematoma expansion is defined according to the standard set in Brott et al. (19), which is the change in size associated with significant neurological deterioration. The data of all subjects needed for outcomes measurements will be collected by the treatment physicians or their assistants. Each subject will be marked as a number according to the order they enroll in the trial. All outcomes measurements will be assessed by two observers who are not involved in the development of the clinical treatment plan of subjects and are blind to the treatment assignment; any disagreement will be resolved by reaching a consensus between the two, or if no consensus can be reached, another observer not involved in the development of the clinical treatment plan of subjects and blinded to the treatment assignment will have the final decision. Finally, data of outcomes and information of the group allocation will be collected by an investigator who will perform statistical analysis.
Data Monitoring
Database management and statistical analyses will be performed by an independent investigator blinded to the treatment assignment of this study. The treatment physicians will monitor compliance with training.
Sample Size Estimation
When estimating the sample size, no parameters could be referred to, as there is no completed clinical study of RIC in patients with ICH. Fortunately, Dobkin (20) has shown that 15 patients per group are often adequate for determining whether a larger multicenter trial should be conducted. Furthermore, Hertzog (21) has suggested that 10 to 20 patients in each research group are sufficient to evaluate feasibility in a pilot study. Therefore, our goal is to recruit 20 patients in each group. Results of this study should be able to determine the preliminary safety and feasibility of RIC in patients with ICH and will be used to estimate sample size and conduct a power calculation to plan a phase-2 trial.
Statistical Analyses
All analyses will be done according to the intention-to-treat principle, which will include all participants who enrolled in this trial. Per-protocol analyses, excluding patients who fail to complete the follow-up, will be managed as a supplement of the intention-to-treat analysis to further confirm the results. If the between-group difference in the change of hematoma volume at day 7 is not <12% in favor of RIC, we would move forward with a phase-2 trial.
Categorical variables including the incidence of hematoma expansion, the proportion of good functional outcomes, and the frequency of adverse events will be presented as counts and percentages. These will then be analyzed with χ 2 test, Fisher exact test, or continuity correction where appropriate. Continuous variables including NIHSS score, GCS score, and hematoma and perihematomal edema volume will be presented as mean and standard deviation or median and interquartile range. These will be analyzed with independent t-test or rank-sum test where appropriate. For missing data, we will conduct a sensitivity analysis and use multiple imputations to impute values for those with missing data as continuous data; for clinical events (e.g., death), we will regard the patients lost to follow-up as nonevents in both groups.
The statistical analyses will be conducted with SPSS statistics software for Windows version 20.0 (Armonk, NY, IBM Inc.).
DISCUSSION
Recently, the Minimally Invasive Surgery Plus Alteplase for Intracerebral Hemorrhage Evacuation III trial that evaluated the efficacy of minimally invasive evacuation followed by thrombolysis in ICH patients has completed, but it failed to improve functional outcomes despite a significantly decreased hematoma volume (22). Therefore, exogenous removal of hematoma may not be as effective as expected. Fortunately, the Intracerebral Haemorrhage Deferoxamine trial found that deferoxamine was safe in patients with ICH and merited further investigation in a phase 3 trial (23). Therefore, strategies that promote hematoma resolution and inhibit the secondary injury of hematoma appear to be promising in the future.
Remote ischemic conditioning has been found to attenuate perihematomal edema, improve cerebral blood flow, and promote hematoma resolution in ICH model (15,17). In this study, the safety and feasibility of RIC will be determined, and cranial CT will be performed at days 3, 7, and 14 to ascertain the potential effects of RIC on hematoma resolution and perihematomal edema, which will provide parameters for the design of a phase-2 study. Approximately 40% of patients experience hematoma expansion over the first 24 h after ictus (19), which further exacerbates the damage from ICH and is associated with neurological deterioration and worse outcomes (24). In this study, we exclude patients within 24 h of ictus and recruit patients at 24 to 48 h. One reason for this is to ensure safe implementation of the present study. Another reason is that the aim of the RICH trial is to evaluate the effects of RIC on hematoma resolution and edema instead of hematoma Frontiers in Neurology | www.frontiersin.org expansion. In addition, two other ongoing clinical trials (RESIST, NCT 03481777; SERIC-sICH, NCT03484936) are recruiting ICH patients within 4 and 12 h of ictus respectively and evaluating the effects of RIC on hematoma expansion.
There are limitations to this study. First, the RIC dose that will be used in this study is rather pragmatic and tailored to ICH patients, but it may not be the optimal one. In addition, a sham procedure is not planned in this study, and the participants will be not blinded; this may bias the study results. To alleviate potential bias, all outcomes raters will be blinded to the treatment assignment of this study.
In conclusion, the RICH-1 study is designed to identify the feasibility and safety of RIC in patients with ICH. Additionally, the preliminary efficacy results will provide parameters for the design of a future phase-2 trial.
ETHICS STATEMENT
The study protocol was reviewed and approved by the ethic committee of each center involved. The participants will provide their written informed consent to participate in the study.
AUTHOR CONTRIBUTIONS
XJ conceptualize the study and contribute to the study design and implementation as Principal Investigator. FJ and SL make substantial contributions to the design and content of the trial. ZG, HS, and YW contribute to the design of the trial from their area of expertise. CW, FG, QZ, and XG collaborate in the implementation of specific procedures. WZ contributed to the design, implementation, and writing of the protocol. All authors reviewed the manuscript and provided the final approval for the manuscript. | 2020-04-28T13:14:32.948Z | 2020-04-28T00:00:00.000 | {
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16321960 | pes2o/s2orc | v3-fos-license | Homogeneity of antimicrobial policy, yet heterogeneity of antimicrobial resistance: antimicrobial non-susceptibility among 108 717 clinical isolates from primary, secondary and tertiary care patients in London
Objectives We examined the 4 year trend in antimicrobial susceptibilities and prescribing across levels of care at two London teaching hospitals and their multisite renal unit, and for the surrounding community. Methods Laboratory and pharmacy information management systems were interrogated, with antimicrobial use and susceptibilities analysed between hospitals, within hospitals and over time. Results A total of 108 717 isolates from 71 687 patients were identified, with significant differences (at P < 0.05) in antimicrobial susceptibility between and within hospitals. Across the 4 years, rates of ESBL-/AmpC-producing Enterobacteriaceae ranged from 6.4% to 10.7% among community isolates, 17.8% to 26.9% at ward level and 25.2% to 52.5% in critical care. Significant variations were also demonstrated in glycopeptide-resistant enterococci (ward level 6.2%–17.4%; critical care 21.9%–56.3%), MRSA (ward level 18.5%–38.2%; critical care 12.5%–47.9%) and carbapenem-resistant Pseudomonas spp. (ward level 8.3%–16.9%; critical care 19.9%–53.7%). Few instances of persistently higher resistance were seen between the hospitals in equivalent cohorts, despite persistently higher antimicrobial use in Hospital 1 than Hospital 2. We found significant fluctuations in non-susceptibility year on year across the cohorts, but with few persistent trends. Conclusions The marked heterogeneity of antimicrobial susceptibilities between hospitals, within hospitals and over time demands detailed, standardized surveillance and appropriate benchmarking to identify possible drivers and effective interventions. Homogeneous antimicrobial policies are unlikely to continue to be suitable as individual hospitals join hospital networks, and policies should be tailored to local resistance rates, at least at the hospital level, and possibly with finer resolution, particularly for critical care.
Introduction
Antimicrobial resistance rates vary between countries, 1 and between the community and hospitals. 2 Variation within hospitals is also described; internationally, resistance rates are often highest in critical care, 3,4 but in Europe this varies by organism 1 and in the UK critical care reservoirs seem less apparent. 5 Robust benchmarking is, however, lacking despite advocacy towards the standardized collection of cumulative antimicrobial susceptibility test data 6 (the 'antibiogram'). 7 The identification of local variations in bacterial resistance between cohorts 2,8 and over time 9 enables informed decisions on empirical antimicrobial regimens and is becoming increasingly achievable as economic and political pressures create hospital networks where previously separate units, patient cohorts and their associated flora are now served by single large centralized laboratories. However, single antimicrobial policies are frequently adopted within these networks, often not adequately allowing for variations in bacterial resistance between and within the sites served. In this context, antimicrobial policies rarely account for the variations in carriage rates of resistant bacteria in relation to population structure and travel or migration patterns. 10 Defining patient cohorts according to locale, level of care and other acknowledged risk factors for antimicrobial resistance, with subsequent detailing of resistance trends, may facilitate more appropriate antimicrobial prescribing. A further concern is that highly standardized antimicrobial policies may concentrate selection pressure on particular agents, sequentially eroding their utility, exactly as has occurred with anti-gonococcal treatments. 11 This study analyses 4 years of bacterial susceptibility data and prescribing trends from two West London tertiary referral hospitals, their multisite renal unit and the surrounding community practices, all served by a single laboratory. The objectives were to identify and describe the fine-resolution variations in resistance rates and trends between the hospitals and within the hospitals at ward (NHS Level 0 and Level 1 beds) and critical care (NHS Level 2 and Level 3 beds) 12 levels, and furthermore to explore the relationships between resistance patterns and antimicrobial use.
Methods
The laboratory information management system (LIMS; Sunquest w , Misys) was interrogated for the seven cohorts of interest: Teaching Hospital 1 (27 critical care beds; 388 ward beds), Teaching Hospital 2 (26 critical care beds; 453 ward beds), the multisite renal unit (84 inpatient beds and 3100 dialysis and transplant outpatients) and community specimens (from over 50 local primary care practices and from outpatients attending clinics at Hospitals 1 and 2). Hospital 1 includes general medicine, cardiology and tertiary referral haematology, cardiothoracic surgery and hepatobiliary surgery. Hospital 2 includes general medicine, general surgery, trauma and orthopaedics and tertiary referral oncology and neurosurgery. All patients at Hospital 1, Hospital 2 and the renal unit were over 16 years of age. A third hospital within the hospital network utilized a different LIMS at the time of this study and was excluded. Infection advice for all sites is provided by an integrated team of infection specialists, with an established overarching antimicrobial policy and an active antimicrobial stewardship programme for all hospitals in the network. Off-policy prescription can occur under the direction of infection specialists.
All samples submitted for culture for clinical indications (as indicated by the clinician submitting the sample) were identified covering the four fiscal years from 2009 to 2013 (in the UK the fiscal year runs from April to March). They included blood and CSF, respiratory and ear/nose/throat, tissue and wound, genital and urine samples. Samples submitted for the purposes of cross-infection screening and MRSA screening were excluded, as variations in screening practice existed between and within the hospitals. The clinical criteria and sampling protocols to obtain diagnostic specimens for culture were uniform across the two hospitals in the corresponding ward and critical care cohorts. Results were de-duplicated for organisms repeatedly isolated within a 7 day period from the same patient. Laboratory operating procedures followed national standards for microbiological investigation; 13 isolates were identified using API w (bioMérieux) from 2009 to 2011 and by MALDI-TOF spectroscopy (Biotyper w , Bruker) from 2011 to 2013. Susceptibilities were determined by disc diffusion using BSAC criteria. 14 AmpC-and ESBL-producing Enterobacteriaceae were identified by standard methods. 13 Comparisons between sites and cohorts were carried out for glycopeptide-resistant enterococci (GRE), MRSA, Pseudomonas spp. and AmpC-and ESBL-producing Enterobacteriaceae (defined as including Citrobacter spp., Escherichia coli, Enterobacter spp., Hafnia spp., Klebsiella spp., Morganella spp., Pantoea spp., Proteus spp., Providencia spp., Raoultella spp., Serratia spp. and other lactose-fermenting coliforms, but excluding Salmonella spp. and Shigella spp.). Enterobacteriaceae were considered at family level rather than for each species separately as standard operating procedures stipulate identification to genus or species level only for isolates from specific sample types or with particular resistance patterns. Non-susceptibility (i.e. resistant and intermediate) proportions were calculated against the total number of isolates tested in each isolate group.
Antimicrobial usage data were sourced from the hospital network pharmacy system, and defined daily doses per 1000 occupied bed days (DDDs/1000 OBDs) were calculated. 15 Antimicrobial usage data is based upon antimicrobials distributed to wards for inpatient and stock supply and was available to the hospital level for Hospitals 1 and 2 (inpatients only) and for the renal inpatient practice. All antibacterials were included; antiviral, antileprotic, antimycobacterial and antihelminthic medications were excluded. Antimicrobial guidelines were reviewed for the relevant time periods to identify any policy shifts.
Variable referral practice from local primary care centres precluded an estimation of population-attributable rates of infection and resistance (i.e. an extrapolation of the observed rates in the study cohorts out to the wider population), but isolate frequency was calculated for inpatients based upon OBDs. CIs for non-susceptibility were calculated using the Wilson method with continuity correction. 16 Analysis was undertaken in Stata/ SE Version 11 w , with x 2 tests for inter-cohort comparison and for temporal trends, and with binomial regression analysis when these identified significant differences (P, 0.05, with the Bonferroni correction to account for multiple comparisons between years in each cohort).
Results
The LIMS extract yielded antimicrobial susceptibility results for 145 703 isolates. After excluding organisms not of interest to this study, 108 717 isolates from 105 319 samples and 71 687 patients remained.
Isolate frequency in relation to occupancy denominators
At ward level, little variation was observed between the two hospitals in terms of the frequency of isolation of the species groups reviewed (Table 1; expressed as isolates/1000 OBDs), with the exception of an upswing in Enterobacteriaceae isolates in the most recent year that was observed in both hospitals. The frequency of isolates from the renal inpatient cohort was comparable to that from the general ward areas. Marked year-by-year fluctuations were observed in isolate frequency in critical care but with a modest overall down-trend. Three features were notable: first, the high frequency of isolates of all species groups in Hospital 1 in critical care in 2009-10, not attributable to any discernible policy changes, case-mix or known outbreaks; second, a marked down-trend in the frequency of enterococci in both critical care units over the study period; and last, a year-on-year decrease in Enterobacteriaceae isolates from critical care patients at Hospital 2.
Resistance in Enterobacteriaceae
A total of 55 600 Enterobacteriaceae were identified ( Table 2). Significantly higher prevalence rates for Enterobacteriaceae with ESBL/AmpC phenotypes were seen in critical care versus general wards in Hospital 1 in three of the four years and in Hospital 2 in all four years (Table 3 and Figure 1a). The proportions of ESBL/ AmpC phenotypes were 1 in 5 Enterobacteriaceae from general wards and up to 1 in 2 in critical care. Fluctuating rates of Enterobacteriaceae with ESBL/AmpC phenotypes were seen at the two hospitals, with significant differences in these rates between the critical care areas in three of the four years, but only in the two most recent years in general ward cohorts. ESBL/AmpC rates among Enterobacteriaceae from the renal outpatient cohort were as high as at hospital ward level and, among renal inpatients, were as high as in critical care, peaking at 51.5% in 2012-13. The difference in prevalence of ESBL-/AmpC-producing Enterobacteriaceae between renal inpatients and renal outpatients was significant in all four years.
Non-susceptibility to ciprofloxacin among the Enterobacteriaceae varied surprisingly little between or within the hospitals (Table 3), with non-susceptibility rates clustered from 18.1% to 25.7% (Figure 1b). However, the renal unit showed a significant variation in ciprofloxacin non-susceptibility between the inpatient and outpatient cohorts in all four years. For renal outpatients, the prevalence of ciprofloxacin nonsusceptibility was almost double that among ward patients in Hospitals 1 and 2; that for renal inpatients neared triple those in Hospitals 1 and 2. There was no significant temporal variation in ciprofloxacin non-susceptibility in either hospital or in the renal unit. Non-susceptibility to ciprofloxacin in community isolates was approximately half that among inpatients.
We saw little carbapenem non-susceptibility in Enterobacteriaceae, precluding a robust temporal or inter-cohort analysis. Meropenem non-susceptibility was noted in fewer than 0.5% of all hospital Enterobacteriaceae, except for isolates from renal inpatients during 2009 -10, when an outbreak due to OXA-48-carbapenemase producing Klebsiella pneumoniae was detected. 17 Ertapenem nonsusceptibility also was noted in 1% -2% of Enterobacteriaceae, predominantly Enterobacter species, and was attributed to the breakpoint determining a 'tail' of AmpC-derepressed isolates to be non-susceptible.
Resistance in Pseudomonas
A total of 12 616 Pseudomonas spp. were identified, 10 226 of them confirmed as P. aeruginosa (Table 2). Across both hospitals and the renal cohorts, Pseudomonas spp. comprised 75.3% -88.9% of all non-fermenters, with 63.0% -77.1% identified as P. aeruginosa. Non-susceptibility to ciprofloxacin (Figure 2a There was a significant variation in ciprofloxacin nonsusceptibility rates between the two hospitals in critical care in Table 1. Sample types for isolates from the renal inpatient cohort comprised 11% blood cultures, 7% respiratory tract, 8% invasive tissue or fluid, 48% non-invasive wound swab and 26% urine. Sample types for isolates from Hospital 1 were 9% blood cultures, 13% respiratory tract, 10% invasive tissue or fluid, 37% non-invasive wound swab and 31% urine. Sample types for isolates from Hospital 2 were 5% blood cultures, 14% respiratory tract, 8% invasive tissue or fluid, 38% non-invasive wound swab and 35% urine. Heterogeneity of antimicrobial resistance in London 3411 JAC only one year, and at ward level in two years. Within-hospital comparisons demonstrated significant differences in all four years in Hospital 2, with the non-susceptibility rate in critical care almost double that in the general wards (Table 3). Temporal analysis found no significant variation in ciprofloxacin non-susceptibility in critical care, but showed significant falls at ward level in Hospital 2 between 2009 -10 and 2010 -11 (P ¼ 0.002) and in Hospital 1 between 2010 -11 and 2012 -13 (P ¼ 0.001). The renal inpatient cohort showed a significant fall (almost 50%) in ciprofloxacin non-susceptibility between 2010 -11 and 2011 -12 (P, 0.001). In the community, ciprofloxacin non-susceptibility among Pseudomonas spp. remained between 11.1% and 14.3% across the four years. Meropenem non-susceptibility was more prevalent than piperacillin/tazobactam non-susceptibility in all years and cohorts ( Table 2 and Figure 2b and c). Non-susceptibility to meropenem was significantly more prevalent (typically 2-to 3-fold) in critical care than in the general wards in all years at both hospitals Table 3). Instances of significant between-hospital variation in meropenem non-susceptibility were noted between corresponding levels of care, with less variation for piperacillin/tazobactam non-susceptibility.
There was no statistically significant temporal variation in nonsusceptibility to meropenem or piperacillin/tazobactam in the ward or critical care cohorts, in renal outpatients or in the community. In the renal inpatient cohort, by contrast, there were significant falls in the prevalence of non-susceptibility to both meropenem (33.3% to 8.9%; P,0.001) and piperacillin/tazobactam (23.2% to 3.7%; P,0.001) between 2010 -11 and 2011-12.
Resistance in enterococci
A total of 13643 enterococci were identified ( Table 2). Significant differences in GRE rates between ward and critical care areas were seen for all years in both hospitals (Table 3 and Figure 3a). A comparison between the hospitals demonstrated significant differences in the prevalence of GRE at ward level in all years but little significant variation between the critical care areas (with the exception of 2009 -10). GRE were consistently 2 -4 times more frequent among enterococci from renal inpatients than renal outpatients, and this was significant in all years. There was no significant year-on-year variation in the proportion of GRE in any cohort.
An analysis of amoxicillin-non-susceptible enterococci (i.e. presumptive Enterococcus faecium) demonstrated a significant variation between critical care and the general ward areas in both hospitals in all years (Table 3 and Figure 3b); specifically, the proportions of amoxicillin-non-susceptible enterococci in critical care were typically twice those in general wards in Hospital 1, and 3-6 times higher in Hospital 2. A significant variation was also demonstrated between renal inpatients and outpatients, with the former having amoxicillin-non-susceptible rates 4 times those for the latter. A comparison between the two hospitals demonstrated little significant variation in the proportion of amoxicillin-non-susceptible enterococci between the critical care cohorts (with the exception of 2012-13), but for general ward isolates Hospital 1 consistently had 1.5 -4 times higher rates than Hospital 2. Amoxicillin nonsusceptibility among community isolates was consistent and 10-fold below that of the other cohorts, at 1.0%-2.6%. Figure 4). MRSA rates were significantly higher in critical care than in the general wards at Hospital 1 only in 2009 -10 (Table 3). In Hospital 2, MRSA was more prevalent in critical care areas in two years, with its proportion peaking at almost twice that at ward level in 2011 -12. A comparison between the hospitals at ward level demonstrated an alternating trend as to which had the higher MRSA rate; these differences were significant until 2012-13. In critical care, Hospital 2 had persistently higher MRSA rates than Hospital 1 across all years, and this was significant in two years. Among renal patients, the proportion of MRSA from the inpatient cohort was significantly higher than from the outpatient cohort in three of the four years, peaking at almost double in 2009-10. An analysis over time showed a significant decrease in the proportion of MRSA at ward level in Hospital 1 between 2010-11 and 2011 -12, from 38.2% to 19.9% (P, 0.001), with no significant subsequent rebound. A similar decrease at ward level was observed in Hospital 2 over a longer period, from 38.
Antimicrobial usage
Biannual point prevalence studies consistently indicated that 33.3% -41.9% of patients were on antimicrobials, with this proportion rising to 62.9% -71.4% in critical care and to 71.8% -80.4% among renal inpatients. Analysis as DDDs/1000 OBDs, for the four most commonly prescribed antimicrobials (ciprofloxacin, amoxicillin/clavulanate, piperacillin/tazobactam and meropenem) among all inpatient groups is shown in Table 4, demonstrating persistently higher use in Hospital 1 than Hospital 2. This differential was 23% -56% for ciprofloxacin, 26% -53% for amoxicillin/clavulanate, 53% -82% for piperacillin/tazobactam, 74% -117% for meropenem and 27% -40% for total antimicrobial use. Although the renal unit had a lower use of amoxicillin/clavulanate than either hospital, the use of ciprofloxacin was 159% -213% higher than for Hospital 2, piperacillin/tazobactam use 104% -147% higher and meropenem use 39% -264% higher. There was little variation over time in the proportion of prescribing accounted for by these top four antimicrobials, except for a spike in meropenem use in Hospital 1 in 2010-11, a reduction by half for meropenem consumption among renal inpatients between 2010-11 and 2011-12, and for ciprofloxacin, a fall of 33% during the study period in Hospital 1 and of 20% in the renal inpatient cohort. A review of antimicrobial policies revealed only two major changes in the study period, both promoting the use of narrower-spectrum antimicrobials. The first, in 2010, was the introduction across the hospital network of an antimicrobial policy for infection in over-65-year-olds. This stipulated the use of narrow-spectrum antimicrobials, avoiding amoxicillin/clavulanate for urinary tract infections, peritonitis and pneumonia (advocating aminoglycosides, amoxicillin/metronidazole/gentamicin and amoxicillin, respectively). It aimed to reduce Clostridium difficile infections. The second major change related to antimicrobial stewardship in the renal cohort from 2009 onwards (see below).
Discussion
We found significant differences in antimicrobial non-susceptibility within and between the two hospitals for Enterobacteriaceae, enterococci, S. aureus and Pseudomonas spp. Furthermore, we found a substantial year-on-year fluctuation in non-susceptibility among most 'drug -bug' combinations but with few persistent trends. There was much less fluctuation in results for the community cohort, refuting the hypothesis that the variation in the hospital isolates represented a testing quality issue.
The data suggest a few instances where the cohort at one hospital had persistently higher non-susceptibility rates than the corresponding cohort at the other hospital. Notable examples include MRSA in Hospital 2 critical care, meropenem-non-susceptible Pseudomonas spp. in Hospital 1 critical care and amoxicillin-non-susceptible enterococci (i.e. E. faecium) in Hospital 1 ward areas. Nevertheless, the wider lack of consistency in relative rates or trends between the two hospitals suggests that short-term factors were a greater contributor to influencing the year-on-year variation. These short-term factors could potentially represent transmission, with many 'minioutbreaks' among patients who were hospitalized or who had frequent healthcare contact. A concept of 'mini-outbreaks', particularly in the critical care areas, is additionally supported by the fluctuations in the frequency with which species were encountered within each cohort (Table 1). Further investigation with prospective large-scale typing is indicated and may be facilitated by the increasing availability of whole-genome sequencing.
One factor that may contribute to the variation seen between the two critical care units is the impact of the use of selective digestive decontamination (SDD) 18 in Hospital 2 critical care but not in Hospital 1. This may help to explain why isolation rates of Enterobacteriaceae were generally lower in Hospital 2 critical care than Hospital 1. However, the proportion of Enterobacteriaceae displaying an ESBL/AmpC phenotype peaked at 52.2% in 2010 -11 at Hospital 2 (twice that in Hospital 1 in the corresponding year). Over the succeeding 2 years, the rates of isolates with ESBL/AmpC phenotype converged despite there being no change in SDD practice. Elsewhere, data on the impact of SDD on multidrug-resistant organisms is conflicting, and long-term cluster-randomized controlled trials are needed. 19,20 In contrast to the general lack of consistency in the differences in rates of resistance between the two hospitals, the results do suggest reasonably consistent 'within-hospital' variation, with higher resistance rates and a greater frequency of isolates in critical care versus ward areas. A consistent excess of resistance was also seen in the renal cohorts, where resistance rates in renal inpatients resembled those in critical care rather than those found at Heterogeneity of antimicrobial resistance in London ward level; conversely, resistance rates in renal outpatients resembled those of ward inpatients rather than community patients. This differential between the proportions of resistant isolates in general versus specialist cohorts was demonstrated for most 'drug -bug' combinations, with the exception of methicillin resistance for S. aureus and ciprofloxacin non-susceptibility in Enterobacteriaceae, for which fairly uniform rates were noted across all cohorts. One of the most likely causes for these 'withinhospital' variations may be the greater frequency of antimicrobial use in critical care areas/renal inpatients than in general wards. In our 6-monthly point prevalence studies, antimicrobial usage in critical care was higher than the benchmarked national point prevalence finding of 60.8% for critical care patients, 21 and usage in the renal inpatient cohort was higher even than in critical care. An increased use of devices and central intravenous cannulae in critical care and among renal inpatients may be driving the frequent empirical co-prescription of glycopeptides, selecting for GRE. 22 Nevertheless, markedly higher antimicrobial prescribing in Hospital 1 than Hospital 2 was not reflected in higher overall resistance rates.
Beyond the levels of antimicrobial usage, one of the biggest drivers of variation both between and within hospitals may be the spectra of activity of the particular antimicrobials prescribed, and it is widely suggested that resistance itself is encouraging increasingly broad-spectrum empirical antimicrobial use, thereby driving the selection of further resistance. A possible example was seen with the spike in meropenem usage in Hospital 1 between 2009-10 and 2010-11, which was temporally associated with spikes in piperacillin/tazobactam resistance among Pseudomonas spp. at both ward and critical care levels. This rise in meropenem use was associated with a non-significant but possibly consequent rise in meropenem-non-susceptible Pseudomonas spp. in both Hospital 1 cohorts, persisting in critical care for the subsequent year. Addressing this feedback loop through antimicrobial stewardship is key, and advances in rapid microbiological diagnostics to facilitate de-escalation may help. 23,24 One example of the successful interruption of such a feedback loop is demonstrated in the renal cohort data. Until and including 2010 -11, meropenem use in this cohort was high. Following an outbreak of K. pneumoniae with an OXA-48-carbapenemase in 2008 -10, carbapenem use was almost halved on the advice of infection specialists between 2010 -11 and 2011-12. While causality cannot be directly attributed, a beneficial yet unintended impact over a relatively short time frame was a significant fall in the proportion of Pseudomonas spp. non-susceptible to meropenem. A marked and concordant fall in piperacillin/tazobactam resistance in Pseudomonas spp.-despite plateaued consumption in this cohort over this period-may reflect the fact that both meropenem and piperacillin/tazobactam are affected by, and putatively select for, the same efflux-based resistance mechanisms in P. aeruginosa.
A further facet of antimicrobial use may contribute to the high burden of antimicrobial non-susceptibility, specifically homogeneous use, concentrating the selection pressure on a fraction of the antimicrobial armamentarium. 25,26 In our study, just four agents consistently represented 34% -50% of all antimicrobials prescribed in the hospitals and the renal inpatients, and two of these-amoxicillin/clavulanate and piperacillin/tazobactamare closely related. While the damaging consequences of homogeneous antimicrobial policies are not proven for all antimicrobials, 27 an argument for heterogeneity exists and might be achieved through antimicrobial cycling 28,29 or mixing. 30,31 The latter option is likely to be preferable, through offering broader choices of antimicrobials within policies and prospective monitoring to preserve the diversity of prescribing within those choices offered.
Critics of antibiogram-based surveillance data often cite inaccuracies in the sampling of selected patient populations and restricted geographical sampling. 32 We largely evaded these problems by extracting all the data from a laboratory serving multiple cohorts across two hospitals and an associated community that shared an overarching antimicrobial policy and infection specialists. Antibiograms can also suffer in accuracy, and therefore utility, when there are marked changes in patient mix. There was some change in the patient population through this study period, with solid organ oncology being consolidated from both hospitals to Hospital 2 in 2010 -11, and with the formation of a new heart attack centre at Hospital 1 in 2009-10. Changes in primary care in one part of West London between 2010-11 and 2011 -12 led to in a decrease in the number of community samples. Changing hospital configurations such as these highlight the need for regular antibiogram review and relation to the patient cohorts. One limitation of this study is that the susceptibility rates were not stratified by sample type. When a comparison by sample type was attempted, meaningful analysis was precluded by low numbers. It is acknowledged that rates may potentially vary between sample types, not least because of sampling bias. However, using an all-sample approach to resistance surveillance, rather than restricting data to blood culture results, may better reflect the drivers towards antimicrobial prescribing in clinical practice. A further limitation is the inability to delineate whether clinical isolates from patients in one cohort were acquired in that cohort or elsewhere (e.g. community-acquired isolates detected during inpatient stays or hospital-acquired infections becoming manifest in the community). The impact of community antimicrobial use, over and above hospital use, on the variation in susceptibility rates seen between cohorts could not be assessed and would require longitudinal, linked, primary and secondary care patient-level data, not presently available.
Conclusions
The Annual Report of the Chief Medical Officer, 33 the UK Five-Year Antimicrobial Resistance Strategy, 34 the Chennai Declaration 35 and the G8 Science Ministers Statement 36 all highlight surveillance as key to addressing antimicrobial resistance. Aggregated regional or hospital level data are no longer adequate and analysis at a finer resolution, as here, is needed; however, challenges are posed by the need to retain sufficient statistical power to detect variation. A standardization of reporting parameters urgently needs to be agreed to enable useful surveillance and benchmarking.
Despite (i) overarching antimicrobial and infection control policies, (ii) standardized laboratory practice and (iii) integrated infection specialists with an active stewardship programme, this study found a significant variation in antimicrobial susceptibility in common organisms between different patient cohorts in the multisite hospital network. Variations in antimicrobial use and clinical practice may be responsible, but relationships are far from clear and random fluctuations, potentially due to numerous 'mini-outbreaks', may be short-term modulators. The marked heterogeneity in antimicrobial susceptibility moreover suggests that whole-hospital antimicrobial policies may not be appropriate in hospitals with multiple sites or where units have markedly different patient populations. Local policies, rapidly responsive to short-term fluctuations of both antimicrobial resistance and prescribing patterns, may be necessary and desirable. Policies should also be mindful of the potential unintended consequences of reactive prescribing. | 2017-04-14T23:00:27.997Z | 2014-08-12T00:00:00.000 | {
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230525913 | pes2o/s2orc | v3-fos-license | Estimation of Quaternion Motion for GPS-based Attitude Determination Using the Extended Kalman Filter
In this paper, the Global Positioning System (GPS) interferometer provides the preliminarily computed quaternions, which are then employed as the measurement of the extended Kalman filter (EKF) for the attitude determination system. The estimated quaternion elements from the EKF output with noticeably improved precision can be converted to the Euler angles for navigation applications. The aim of the study is twofold. Firstly, the GPS-based computed quaternion vector is utilized to avoid the singularity problem. Secondly, the quaternion estimator based on the EKF is adopted to improve the estimation accuracy. Determination of the unknown baseline vector between the antennas sits at the heart of GPS-based attitude determination. Although utilization of the carrier phase observables enables the relative positioning to achieve centimeter level accuracy, however, the quaternion elements derived from the GPS interferometer are inherently noisy. This is due to the fact that the baseline vectors estimated by the least-squares method are based on the raw double-differenced measurements. Construction of the transformation matrix is accessible according to the estimate of baseline vectors and thereafter the computed quaternion elements can be derived. Using the Euler angle method, the process becomes meaningless when the angles are at 90 where the singularity problem occurs. A good alternative is the quaternion approach, which possesses advantages over the equivalent Euler angle based transformation since they apply to all attitudes. Simulation results on the attitude estimation performance based on the proposed method will be presented and compared to the conventional method. The results presented in this paper elucidate the superiority of proposed algorithm.
Introduction
The Global Positioning System (GPS) [1][2][3] is a satellite-based navigation system that provides a user with the proper equipment access to useful and accurate positioning information anywhere on the globe. In addition to the code observable commonly used for position and velocity determination, the carrier phase is the other type of observable that can be extracted from the GPS signals. The carrier phase observables contain noise much smaller than that of code observables. Due to its higher accuracy and precision compared to code observations, carrier phase observations can be used for relative positioning in centimeter level and has been widely applied to surveying, attitude determination [4][5][6][7][8][9][10][11][12][13][14], precision approach and automatic landing.
Traditionally used in precise static relative positioning, and thereafter in kinematic positioning, the GPS offers the interferometer [5] for attitude determination by processing the carrier phase observables, which enables the relative positioning in centimeter level. The relative positioning techniques using the carrier phase differential GPS (DGPS) based on interferometer principles can be adopted to solve for the baseline vectors, defined as the vectors between the antenna, designated master and one of the slave antennas, shown as in Fig. 1. The attitude of a vehicle can be precisely determined using the GPS carrier phase observables received at two or more nearby antennas attached to the vehicle. In the beginning of 1990s, Van Grass and Braasch [4] conducted research on the GPS to the field of aircraft attitude determination using carrier phase. In their work, the receiver-satellite double differenced observable was employed. The solution of the baseline vector is the approximate interferometer coordinates, which directly influence the performance of the GPS-based attitude determination. Real-time integer ambiguity resolution techniques [5,8,9,11] and attitude determination are two main issues to be resolved for determining the vehicle attitude when applying GPS double-differenced carrier phase. Very accurate relative position estimate will be available once the integer cycle ambiguities are properly resolved. Attitude determination using GPS does not have the error accumulation, which usually happens in the inertial navigation system (INS) [2].
The rotation angles that relate a coordinate system fixed in the body (body frame) to a coordinate system fixed in space are referred to as the attitudes. The space coordinate system is typically defined to be a local level NED (north-east-down) frame, also referred to as the navigation frame. The purpose of attitude determination essentially involves calculation of the three Euler angles, namely roll, pitch, and yaw. The quaternion method [15][16][17][18][19] uses four parameters instead of nine as in the Euler angle method, by defining the generalized complex number. The quaternion method possesses advantages over the equivalent Euler angle based transformation since they apply to all attitudes with the error equations bounded by the constraint equation. Furthermore, the numerical value of each parameter always lies within the range of −1 to 1, so that the scaling problems in the computing mechanization can be easily handled.
The purpose of the Kalman filter (KF) [2,20,21] is to provide an optimal (minimum mean-square error) estimate of the system state vector. As the nonlinear version of KF, the extended Kalman filter (EKF) deals with the case governed by the nonlinear stochastic differential equations. The EKF linearizes about a trajectory that is continually updated with the state estimates. Both the KF and EKF have been widely applied to the field of navigation, such as GPS receiver position and velocity determination, attitude Figure 1: Interferometric configuration considering the plane wave approximation of a GPS interferometer [5] determination, integrated navigation system design, and the carrier-smoothed-code (CSC) processing. Utilization of the EKF as the estimator of attitude related parameters enhances the accuracy and reliability of the attitude solution.
The remainder of this paper is organized as follows. In Section 2, preliminary background on the GPS carrier phase observation model is reviewed; the computed quaternion vectors based on the GPS interferometer is introduced. Section 3 presents the modelling of quaternion dynamics for the extended Kalman filter. In Section 4, simulation experiments are carried out to evaluate the performance on estimation accuracy using the proposed method as compared to the conventional one. Two numerical examples are presented for illustration. Conclusions are given in Section 5.
GPS-Based Computed Quaternion Vectors Based on the GPS Interferometer
In a GPS interferometer, the receiver-satellite double-differenced carrier phase observable has been commonly utilized to solve for the antenna baseline vector. The GPS carrier phase observables representing sum of range, an unknown integer ambiguity and some ranging errors -can be represented by: where the parameters involved in Eq. (1) are defined as follows. r: True range between a satellite and receiver; c: Speed of light; dt: offset of the satellite clock from GPS time; dT : Offset of the receiver clock from GPS time; d ion : Ionospheric error; d trop : Tropospheric error; : Carrier phase wavelength; N : Carrier phase integer ambiguity; v q , v ' : Measurement noises of code and carrier phases, respectively.
Formulation of the Transformation Using the Baseline Vectors
The receiver-satellite double differencing operator is defined asrDðÁÞ ¼ DrðÁÞ ¼ rðÁÞ À DðÁÞ, where DðÁÞ ¼ ðÁÞ 1 À ðÁÞ 2 denotes the between receiver single differencing operator for receivers 1 and 2, and rðÁÞ ¼ ðÁÞ i À ðÁÞ j denotes the between satellites single differencing operator for satellites i and j. Referring to the configuration as in Fig. 2 [4,14], when using the carrier phase signal from satellite i, the between-receiver single-differenced observable is a linear combination of two phase observables received by two antennas where the effects of errors associated with the satellites are greatly reduced. Similarly, for satellite j, we have where b is the baseline vector formed by two antennas, and e represents the line-of-sight unit vector from antennas to a satellite. Taking two independent single-differenced observables leads to the receiversatellite double-differenced observable: which eliminates or greatly reduces the satellite and receiver timing errors.
The signals received from n satellites by one GPS interferometer provide n À 1 independent double differences. When the integer ambiguity parameter (rDN ij 12 ) is resolved, the range-based equivalent of Eq. (4) is depicted as follows: which can be expressed in matrix formrDr ¼ Gb. The baseline vector can be obtained by the least-squares approachb ¼ ðG T GÞ À1 G T rDr. The solution of the baseline vector b ¼ ½ b x b y b z T is the approximate interferometer coordinates, which directly influences the performance of the GPS-based attitude determination.
Referring to Fig. 3, there are two body-frame-mounted non-collinear baseline vectors formed: b 1 ¼ S A À M and b 2 ¼ S B À M, where the master antenna (M) position is located at ½ 0 0 0 T in the body frame, while the other two slave antennas S A and S B are at ½ 1 0 0 T ' and ½ cos c sin c 0 T ', respectively. The parameter ' is the baseline length parameter used to adjust the length, and c is the angles between two baseline vectors which are adjustable for design flexibility. The accuracy of the attitude measurement depends on the baseline to noise ratio, and is also a function of antenna placement and GPS satellite geometry. The coordinate transformation matrix from body to local frameR b2n can be formed once the baseline vector is determined through the following calculation.
Since the conventional least-squares approach for baseline vector estimation is inherently noisy, incorporation of the Kalman filter into the GPS interferometer for performance improvement is accessible.
Transformation Matrix Involving the Euler Angles
In certain applications, the body angular velocities: p, q, r(which can be measured, for example, by body mounted rate gyros) information is required. They can be determined from the Euler rates: _ f, _ h, _ w. The relationship between the body angular velocities and the Euler rates can be written as: Once the Euler angle rates are available with sufficiently good accuracy, the body angular velocity can be obtained. Taking the inverse transformation for Eq. (7), we have the Euler rates in terms of the body angular velocities: The direction cosine matrix (DCM) R b2n is employed as a transition matrix to describe the transformation of a vector quantity defined in the body frame (denoted by 'b') to the geodetic frame (denoted by 'n'), f n ¼ R b2n f b . There are two common types of transformation approaches available for solving vehicle attitudes, typically including Euler angle method and quaternion method. The transformation matrix related b frame relative to n frame can be constructed in terms of the Euler's angles or the quaternion parameters.
where the subscripts n and b represent the local and body frames, respectively. Since R b2n is an orthonormal matrix, its inverse can obtained through its transpose: In Eq. (9), the notations S ðÁÞ sinðÁÞ and C ðÁÞ cosðÁÞ are defined. Since the vehicle attitude is defined by the angles between the NED frame and body frame, therefore, the rotation transformation matrix that relates the body and NED frames provides the information for finding the vehicle attitude [2,4]. The vehicle attitude can be obtained through the calculation: Figure 3: Antenna differential position vector geometry Using the Euler angle method, the singularity problem occurs when the angles are at 90 , the process becomes meaningless.
Transformation Matrix Involving the Quaternion Elements
A quaternion is a four-dimensional extension to complex numbers, containing four real parameters. The first is considered a scalar and the other three vector components in three-dimensional space: where a constant equation exists of the form q 2 1 þ q 2 2 þ q 2 3 þ q 2 4 ¼ 1. The transformation matrix from body frame to navigation frame axes in terms of the unit quaternion parameters is given by where q 1~q4 are the quaternion vector components. The relations between the quaternion q and the two vectors f b and f n are nonlinear.
Using the transformation matrix to obtain quaternion parameters can be done through By comparing Eq. (13) with Eq. (9), conversion of the quaternion parameters to Euler angles can be implemented through the following relationships: See [2] for more details on the quaternion method. The proposed algorithm for implementing the computation of the quaternion vector derived from the baseline vectors based on the GPS interferometer to be employed as the measurement of the EKF is provided in Fig. 4.
The interferometer offers the preliminarily computed quaternion vector using the GPS doubledifferenced carrier phase observables. The implementation procedure is highlighted as the following steps: (1) Determining the baseline vector using the receiver-satellite double-differenced carrier phase observables:b e 1 andb e 2 from the GPS interferometer; (2) Construct the transformation matrix according to Eq. (6); (3) Compute the quaternion elements.
Modelling of the Quaternion Dynamics for the Extended Kalman Filter
Consider a dynamical system whose state is described by a nonlinear, vector differential equation. The process model and measurement model are represented as It is assumed that f and h are known functions, uðtÞ and vðtÞ are two white-noise processes mutually independent with zero means and: where dðt À sÞ is the Dirac delta function, E ½Á represents expectation, and superscript "T" denotes matrix transpose. The nonlinear filtering deals with the case governed by the nonlinear stochastic difference equations. Assuming the process to be estimated and the associated measurement relationship may be written in the form: where the state vector x k 2 < n , process noise vector w k 2 < n , measurement vector z k 2 < m , and measurement noise vector v k 2 < m . The vectors w k and v k are zero mean Gaussian white sequences having zero cross-correlation with each other: where Q k is the process noise covariance matrix, R k is the measurement noise covariance matrix. The symbol d ik stands for the Kronecker delta function:
The Extended Kalman Filter
The nonlinear process model can be linearized along the currently estimated trajectory where the influence of the perturbations on the trajectory can be represented by a Taylor series expansion about the nominal trajectory.
The actual trajectory is the sum of the estimated trajectory and the small increment: x ¼x þ dx. The corresponding difference equation by converting the continuous-time model into a discrete-time model is given by When working with incremental state variables, the linearized measurement equation presented to the EKF is dz k ¼ z k À hðx À k ; kÞ ¼ H k dx k þ v k rather than the total measurement (nonlinear) z k . Consider the incremental estimate update equation at step k in which the measurement residual is given by: z k À hðx À k ; kÞ À H k dx À k , and the predictive measurement is the sum of hðx À k ; kÞ and H k dx À k . The residual involves the noisy measurement minus the predictive measurement based on the corrected trajectory rather than the nominal one.
Addingx À k on both sides of Eq. (20)x À k þ dx k ¼x À k þ dx À k þ K k ½z k Àẑ À k , we have the update equation: which shows that in an EKF it is accessible to keep track of the total estimates rather than the incremental ones. Oncex À k is determined, the predictive measurementẑ À k can be formed as hðx À k ; kÞ, and the measurement residual at k þ 1 is formed as the difference ðz k Àẑ À k Þ. Projection ofx k tox À kþ1 can be done through the nonlinear dynamicsx À kþ1 ¼ fðx k ; kÞ. Without the external aiding such as an inertial sensor to provide a reference trajectory, the process dynamics represent the total observer dynamics, as shown in Fig. 5. Implementation algorithm for the discrete EKF equations is provided in Tab. 1.
Reference
Estimate of states Figure 5: Configuration of the EKF
The Extended Kalman Filter for Quaternion Estimation
It can be shown that the quaternion elements are propagated according to the differential equation: The differential equations for describing the propagation of quaternion elements are given by (1) Evaluate the predicted state vector through the process model x À kþ1 ¼ fðx k ; kÞ (2) Propagate the predicted error covariance matrix kÞ, and the linear approximation equations for system and measurement matrices are obtained through the relations where the product terms in the parentheses are introduced by quaternion product between the angular rate and the quaternion, which can be written in matrix form Therefore, the quaternion vector is propagated according to the differential equation where q ¼ ½ q 1 q 2 q 3 q 4 T denotes the quaternion vector, and x b nb ¼ ½ p q r T describing the vector of body angular velocities. The symbol Ωðx b nb Þ ¼ ðx b nb ÂÞ represents the skew symmetric matrix with components of x nb in the body frame: The differential equations for describing the quaternion vector can be represented by where Wðx b nb Þ ¼ 0 Àp Àq Àr p 0 r Àq q Àr 0 p r q Àp 0 and Eq. (26) may also be written as Augmented by the propagation of the body angular rates, described by the random walk models, i.e., _ p ¼ u p ; _ q ¼ u q ; _ r ¼ u r , the differential equation has the form: _ x b nb ¼ ½ _ p _ q _ r T ¼ ½ u p u q u r T . The resulting state vector consists of seven states, in which the first four components are the quaternion elements, and the other three components are the body angular velocities. The corresponding Jacobian matrix can be shown to be The corresponding discrete state transition matrix is given by Φ k ¼ £ À1 ½ðsI À FÞ À1 ¼ expðFDtÞ ffi I þ FDt, where Dt is the step size.
Improved accuracy is accessible for the attitude solutions based on the EKF using the preliminarily computed quaternion vector from the GPS interferometer as the measurement. The measurement equations in this case are linear and are much simpler, and they are the four quaternion components i.e., where v i is the white noise measurement. The measurement model written in matrix form is given by With the GPS-based computed quaternions available as the measurement, the measurement matrix H k and noise v k can be expressed as respectively. The measurement model H k is a 4 Â 7 matrix. Although the measurement equations are linear, an EKF is still required since the process model is nonlinear. The attitude estimation is implemented based on the block diagram shown as in Fig. 6. Alternative models for body angular velocities in the process model.
or the first-order Gauss-Markov process
Illustrative Examples and Performance Evaluation
Simulation study has been carried out to evaluate the performance of the proposed approach in comparison with the conventional method for GPS-based attitude determination. Two illustrative examples were implemented through numerical experiments. Computer codes were developed using the Matlab® software. The commercial software Satellite Navigation (SatNav) Toolbox by GPSoft LLC (2003) [22] was employed for generation of the GPS satellite orbits/positions and thereafter, the satellite pseudorange, and carrier phase observables, required for simulation. Furthermore, the Inertial Navigation System Toolbox (2007) [23] was employed for generation of the Euler attitude angles of the threedimensional flight.
The error sources corrupting the GPS carrier phase observables include ionospheric error, tropospheric delay, receiver thermal noise and multipath errors. The variances of the receiver noise are assumed to be 1 m 2 . Most of the receiver-independent common errors can be corrected through the differential correction while the multipath and receiver thermal noise cannot be eliminated. The antenna geometry is set up as in Fig. 2, with the baseline length variable ' equal to 1 meter and c 90 degrees. ; where m ¼ 4 is the number of measurements and equals the number of quaternion elements. After resolving the integer ambiguity, the least-squares approach is utilized for constructing the baseline vector. Fig. 7 presents the body angular rates p, q and r for Example 1. There were 8 visible satellites available in the clear open sky during the time period of simulation. Fig. 8 provides the skyplot at the final time.
The proposed method in which Kalman filtering is used provides estimate of the quaternion vector with noticeable accuracy improvement. It should be noticed that it is risky to set the process noise variance zero to avoid filter divergence. As a result, when the system reaches steady state, the steady-state gain will not approach zero and, subsequently, the filter is able to catch the time-varying attitude dynamics. One still needs to find the suitable values to meet the specific design/mission requirement. The estimation accuracy To confirm the correctness of the solutions, the estimated quaternion vectors were examined, as shown in Fig. 9. Comparison of the attitude solutions is shown in Fig. 10; Tab. 2 summarizes the error statistic of attitude solutions for both the conventional and proposed methods. It can be seen that estimation accuracy by the proposed method has been remarkably improved.
(b) Illustrative Example 2-the three-dimensional flight. The scenario for the second example is a threedimensional flight. The 3D flight path is generated with the Inertial Navigation System toolbox, shown as in Fig. 11. Tab. 3 provides the description of the motion for the flight path. The body angular velocities as time progresses for this example are depicted in Fig. 12. In this example, the duration for simulation is 110 seconds with 8 visible satellites. The covariance matrices for the process and noise models, respectively, are given by 1e À 5 1e À 5 1e À 3 0:1 0:1 10 The standard EKF is sensitive to the changes of the process and measurement models, thus yielding poor performance. Furthermore, the EKF framework does not possess capability to deal with the non-Gaussian measurement errors/outliers. The EKF associates the contaminated measurements with an increase in the measurement covariance, causing the reformulated error covariance. This modified covariance inflation is known to cause an increase of error in the state estimation. Performance based on the EKF will degrade when the noise strength is varying and/or the actual distribution deviates from the assumed Gaussian model. To further improve the performance of the EKF, an adaptive mechanism or a robust technique can be incorporated for performance improvement. Due to appropriate tuning, the adaptive EKF (AEKF) exhibits robust behavior and therefore outperforms the standard EKF when the time-varying dynamic process and measurement models are involved.
Conclusions
A novel GPS-based attitude determination method has been presented. The quaternion vector derived from GPS interferometer has been used as the measurement of the EKF in the attitude determination system. Although utilization of the carrier phase observables enables the relative positioning to achieve centimeter level accuracy, nevertheless, the quaternion elements derived from the GPS interferometer are inherently noisy. This is essentially due to the fact that the baseline vectors estimated by the least-squares method are based on the noisy double-differenced measurements.
The preliminarily computed quaternion elements from the GPS interferometer is employed as the measurement of the EKF based quaternion estimator. The model based approach using the EKF is adopted for estimating the quaternion elements, which can then be converted to the Euler angles. Results show that, by incorporating the EKF into the GPS interferometer, the errors in the solutions of the baseline vectors, and thereafter the quaternion elements have been remarkably mitigated and the estimation accuracy of the attitude solutions has been noticeably improved. The proposed method provides several advantages, such as accuracy improvement, reliability enhancement, and real-time characteristics.
Since the EKF is sensitive to the changes of the process and measurement models, when implementing the EKF approach, poor knowledge of the noise statistics may seriously degrade the estimation performance, and even provoke the filter divergence. For achieving improved estimation accuracy, the designers are required to possess the complete a priori knowledge on both the dynamic process and measurement models. In the two illustrated examples, both the process and measurement noise parameters remained unchanged based on the stationary noise assumption. In the cases of high dynamic or multipath contaminated environments, the noise parameters in the two models need to be -properly tuned. Further investigation can be carried out using the AEKF as the noise-adaptive filter for tuning the noise covariance matrices in the high dynamic or multipath environments. Conflicts of Interest: The author declares that he has no conflicts of interest to report regarding the present study. | 2020-12-10T09:06:26.784Z | 2021-01-01T00:00:00.000 | {
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233185476 | pes2o/s2orc | v3-fos-license | The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans
Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.
Author summary
MicroRNAs play an essential contribution among the many mechanisms used by cells to maintain a proper gene expression program. These endogenous small RNAs regulate gene expression by binding to target mRNAs and blocking their translation into functional proteins. To achieve this, microRNAs rely on Argonaute and other proteins that form a complex called the miRISC. While research on microRNAs has brought a lot of interest over the last years, it is still unclear what modulators affect the activity of the miRISC. Our study identified proteins implicated in intracellular trafficking as important factors for miRISC function. We showed that the intracellular location of the Argonaute protein Introduction MicroRNAs (miRNA) are endogenous small RNAs that regulate a wide variety of biological functions through post-transcriptional gene silencing. Upon binding to Argonaute protein to form a functional complex termed microRNA-Induced Silencing Complex (miRISC), miR-NAs guide the effector complex to a target mRNA, typically in the 3' untranslated region (3' UTR), through base pairing of its seed sequence (nt 2-8) [1,2]. Alternatively, some miRNAs bind mRNA targets in a non-canonical manner and rely on the 3' half of the miRNA for binding [3,4]. With the help of effector proteins such as GW182, the miRISC can induce different molecular mechanisms leading to the abrogation of protein expression from the targeted mRNA such as translational repression, deadenylation, decapping or decay. While it is still unclear which one of these repression mechanisms occurs first, some studies suggest that the choice of favoured mechanism may be context specific (for example, see [5][6][7][8][9][10][11]). The composition and repression mechanism of miRISC therefore vary between cell types and stages of development.
Gene silencing by miRNA is known to occur in the cytoplasm of cells. However, the specific subcellular localization of miRNAs and their effector complex miRISC remains elusive. Several reports have linked miRNA function to endomembrane trafficking. Some studies have shown that miRISC components' association with multivesicular bodies (MVBs) is important for miRNA function both in mammalian cells and in Drosophila [12,13]. Interestingly, some studies have linked small RNA function to the Endoplasmic Reticulum (ER) and the Golgi apparatus. Forward genetic screens in C. elegans revealed that proteins associated with the Golgi apparatus are involved in miRNA-mediated gene regulation. For example, cogc-4, a member of the oligomeric Golgi complex, was reported to be involved in the miRNA pathway [14]. In addition, components of the Golgi Associated Retrograde Protein (GARP) complex were also identified as important factors for miRNA function [15].
A study in mammalian cells has shown that the siRNA and miRNA loading onto Argonaute, as well as target repression, occur at the rough ER [16]. Furthermore, studies in Arabidopsis thaliana also showed that miRNAs associate with membrane-bound polysomes in an Argonaute-dependent manner and that target repression occurs at the ER [17,18]. Other studies in mammalian cells suggested that miRNA targets need to be sent to the rough ER before repression can occur [19], and that Argonaute proteins are transported to the ER to be loaded with miRNA [20]. Although some studies identified the ER as a site of target repression, they could not exclude that other targets may be repressed at different cellular locations than the ER. This is well represented in neurons, where specific pre-miRNAs are transported in the dendrites to be processed as miRNA and repress locally mRNA targets to regulate dendritic development [21][22][23]. Moreover, this transport of pre-miRNAs to dendrites is mediated by late endosomes and lysosomes [24]. Altogether, these studies highlight the importance of the transport of miRNAs and miRISC components within the cell. However, while many reports point towards an important role for endomembrane trafficking in miRNA function, it is still unclear how these two mechanisms are related and how miRISC components are transported between different compartments of the cell.
In this study, we identify tbc-11, a Rab GTPase activating protein, as a new important factor for proper miRISC function in C. elegans. We demonstrate that tbc-11 is involved in miRNA-mediated gene regulation through mainly rab-6, a Golgi-associated small GTPase. We observed that misregulation of TBC-11 affects the localization of the miRNA-specific Argonaute ALG-1 as well as its association with miRNAs. This leads to an accumulation of miRNAunloaded ALG-1 in a perinuclear, high molecular weight complex, inducing defects in miRISC target repression in vivo.
The Rab GTPase activating protein TBC-11 contributes to miRNA function
We performed a forward genetic screen in C. elegans to identify new factors important for miRISC silencing. To focus on factors involved in miRISC function at the mRNA target level, we took advantage of the λN/Box B tethering system to recruit a functional λN tagged-ALG-1 protein to a GFP transgene in which 6 Box B sequences were inserted in the 3' UTR (as described in [9]; S1A Fig). In this condition, the recruitment of the miRISC-specific Argonaute ALG-1 to the reporter mRNA causes the repression of the GFP expression and mutations that disrupt miRISC function would be expected to alleviate this repression, leading to stronger GFP expression. Following chemical mutagenesis, we identified a mutant allele (qbc24) in which GFP repression was altered (S1B Fig). After genetic mapping that located the causal mutation on chromosome X and high-throughput genome sequencing of extensively outcrossed mutant animals, we identified the only missense/nonsense mutation on the chromosome X of qbc24 animal in the coding sequence of the tbc-11 gene (Fig 1A). To test if the qbc24 allele represents a dominant allele of tbc-11, we monitored the alae structure of heterozygote animals. As qbc24/+ animals did not display any defects (S1C Fig), we conclude that the qbc24 allele is a recessive allele.
TBC-11 is a conserved Rab GTPase activating protein (RabGAP) that acts on the Rab family of small GTPases. Rab proteins are implicated in all steps of vesicular trafficking and alternate between an active GTP-bound and inactive GDP-bound state (reviewed in [25,26]). GAPs such as TBC-11 catalyze the hydrolysis of GTP to GDP, therefore inactivating their targeted Rab. In addition to our isolated mutant tbc-11(qbc24), in which a conserved serine residue within the catalytic domain is changed for a proline, a deletion allele tbc-11(ok2576) was also available and further used for characterization ( Fig 1A).
To test whether tbc-11 is involved in the miRNA pathway, we first looked at the animals' alae formation. This cuticular structure of the worm is formed by the asymmetric divisions of seam cells throughout larval development followed by their fusion at the L4 to adult transition to form the alae. These programmed divisions of seam cells are tightly controlled by miRNA lin-4 and the let-7 miRNA family [27][28][29]. Disruption of genes associated to the miRNA pathway, such as alg-1, induces extra divisions of seam cells that leads to discontinuities in the alae structure (termed breaks) [30]. These defects have also been observed for many modulators of miRNA function (for example, see [15,[31][32][33]). We, therefore, scored the number of seam cells in both alleles of tbc-11 to assess their importance in this miRNA-regulated process. While wild-type animals have an invariable number of 16 seam cells at the adult stage, we observed a range in the number of seam cells in both tbc-11 mutant animals (S1D Fig). When we surveyed the alae structures of tbc-11 mutants, we observed the presence of breaks in both alleles, indicating that tbc-11 is important for the normal seam cell division pattern controlled by miRNAs ( Fig 1B).
To determine if tbc-11 is involved more broadly in the regulation by the let-7 family miR-NAs, we tested its synthetic effect with a let-7 mutant allele. This sensitized genetic background allows to monitor the involvement of various factors in the miRNA pathway as it presents partial penetrance on its own. The complete loss of let-7 miRNA induces a distinctive phenotype of bursting through the vulva [29]. The temperature-sensitive let-7(n2853) allele is completely penetrant at the restrictive temperature of 25˚C but only partially penetrant at the permissive temperature of 15˚C [29]. We thus used this mutant allele to test the involvement of tbc-11(ok2576) mutation on the bursting of let-7(n2853) animals. We could not assess the the two studied alleles. ok2576 is a deletion allele (a 1421 base pairs deletion that span from the middle intron 9 to the middle exon 11) and qbc24 allele is a missense mutation in the catalytic domain of the protein. PID: phosphotyrosine interaction domain. Bottom: ClustalW alignment of human (Hs) and C. elegans (Ce) protein sequence shows conservation between species that share 41.1% identity and 71.3% similarity. The amino acid mutated in qbc24 allele is conserved in humans. (B) Alae breaks of tbc-11(qbc24) and tbc-11(ok2576) young adult animals were scored under DIC Nomarski microscopy. The number of animals scored (n) is indicated. P value were obtained by two tailed t-test ( ��� p value < 0.0001). (C) Percentage of animals bursting through the vulva were scored at 15˚C. The number of animals scored (n) is indicated. P value were obtained by one-way ANOVA ( ��� p value < 0.0001). (D) Western blot of LIN-41 in wild-type (WT), tbc-11(qbc24) and tbc-11(ok2576) young adult worm extracts (left). Actin is used as a loading control. Quantification of 4 independent Western blots is shown (right). LIN-41 level is normalized to Actin levels. P value were obtained by one-way ANOVA ( � p value < 0.05). (E) Top: DIC and fluorescent microscopy of miR-228 activity reporter in intestine cells of L2 staged animals. Scale bar: 50μm. Bottom: Quantification of GFP fluorescence intensity in three intestine cells per animal. Quantification was performed by measuring GFP intensity in a consistent-sized circle that was drawn around each intestine cell nucleus. Images were taken using the same settings and exposition time for each animal. The number of animals scored (n) is indicated. P value were obtained by two tailed t-test ( � p value < 0.05, �� p value < 0.001).
We next wanted to assess whether the defects observed in the regulation by let-7 would alter the levels of its well-established target, LIN-41. To test this, we first used a GFP reporter fused to the lin-41 3' UTR region and measured the repression by let-7 in L4-staged animals, as let-7 is only expressed in late larval stages [29,34]. We observed a significant derepression of GFP upon depletion of tbc-11 (RNAi), suggesting that tbc-11 is important for repression of a reporter gene under the control of lin-41 3'UTR (S1E Fig). To further confirm that tbc-11 contributes to the regulation of LIN-41 protein in animals, we monitored the levels of endogenous LIN-41 expression by western blot in tbc-11 animals. We observed that the alteration of TBC-11 found in both tbc-11(qbc24) and tbc-11(ok2576) adult animals leads to a significant increase of LIN-41 protein compared to wild-type animal extracts (Fig 1D), confirming the implication of tbc-11 in the regulation of the microRNA target LIN-41.
To determine if tbc-11 affects miRNAs other than the let-7 family, we tested its contribution on the repression of a miR-228-dependent reporter [7]. While miR-228 represses the GFP reporter expressed in the intestine cells in wild-type animals, the alteration of tbc-11 leads to a significant derepression in both alleles (Fig 1E), indicating that tbc-11 is required for proper repression by miR-228. To confirm that this derepression was miR-228 dependent, we monitored if tbc-11 alleles had an effect on a reporter in which miR-228 binding sites have been removed. We observe no significant differences in GFP expression with this reporter (S1F Fig), confirming that the effect observed is miRNA-dependent. We therefore conclude that tbc-11 is important for the function of miRNAs in different tissues of the animal.
Altogether, these results suggest that tbc-11 is involved in miRNA function and that its loss induces repression defects for various miRNAs.
TBC-11 acts mainly on RAB-6 to affect miRNA function
Rab proteins need to alternate between an active and inactive state to regulate vesicular trafficking (Reviewed in [25,26]). Guanine nucleotide exchange factors (GEF) are implicated in activation of the Rab by releasing the bound GDP to allow binding of a GTP molecule. GTPase activating proteins (GAP) are important for hydrolysis of GTP to GDP, therefore inactivating their targeted Rab (Fig 2A). This cycle can then be repeated at every round of vesicular transport. Since distinct Rabs are associated with different steps of vesicular transports, we wanted to know which Rab was targeted by tbc-11 to regulate miRNA function. As the absence of a GAP (such as TBC-11) will lead to a constitutive activation of the targeted Rab, we expect that reducing its expression will rescue the phenotypes observed in the GAP mutant animals. In C. elegans, TBC-11 had never been characterized as a GAP for any Rab protein. Its human ortholog, TBC1D11, was however known to be a GAP for human Rab2, Rab4, Rab6, Rab11 and Rab14 (Reviewed in [25]). While Rab2, Rab4 and Rab11 can be deactivated by more than one GAP, Rab6 seems like a better candidate to test since TBC1D11 was its only known GAP. Moreover, Rab6 is known to localize at the Golgi apparatus and interact with the GARP complex, which was previously shown to be important for miRNA function [15]. We therefore tested first if TBC-11 could be a GAP for the two Rab6 orthologs RAB-6.1 and RAB-6.2 in C. elegans. We scored alae breaks in tbc-11(ok2576) and observed around 40% of animals with breaks. When exposed to RNAi against rab-6.1 or rab-6.2, we observed that alae breaks of tbc-11(ok2576) animals were suppressed, indicating that these defects were most likely caused by a persistent activation of RAB-6 ( Fig 2B and 2C). We further tested if alae breaks in tbc-11 (ok2576) animals could be suppressed by RNAi against other predicted Rab targets of tbc-11. We exposed WT and tbc-11(ok2576) animals to bacteria expressing dsRNA targeting rab-2 and rab-14 (the C. elegans ortholog of Rab4 and Rab14) and scored alae breaks (of note: RNAi against rab-11.1 and rab-11.2, the C. elegans ortholog of Rab11, caused lethality in animals, and thus alae structure could not be assessed). While both RNAi induce alae breaks in WT animals (suggesting a potential contribution to the miRNA pathway), they did not suppress the alae breaks in tbc-11(ok2576) animals at the same level as observed for rab-6.1 and rab-6.2 (S2 Fig). Based on these results and on the conservation of RAB and GAP proteins across species, we can conclude that RabGAP TBC-11 acts mainly on or through the small GTPase RAB-6.1 and RAB-6.2 (and is likely a GAP for these proteins) to control miRNA function in animals.
The RabGAP TBC-11 modulates the cellular distribution of ALG-1
RAB-6 is known to be located at the Golgi apparatus and is involved in many steps of trafficking such as endosome to Golgi, Golgi to plasma membrane, intra-Golgi as well as Golgi to ER (reviewed in [35]). Since we have shown that tbc-11 affects miRNA function through rab-6, we next asked if the alteration of tbc-11 could affect the localization of miRISC components to endomembranes. We first monitored the association of ALG-1 to the endomembranes by changing the level of detergent agents used during protein extracts preparations as detergent facilitates the extraction of membrane-associated proteins. We observed that in standard extraction conditions containing a low concentration of Triton (0.5%), protein extracts prepared from tbc-11(qbc24) animals had lower ALG-1 levels compared to WT, whereas tbc-11 (ok2576) animals had higher ALG-1 levels in their extracts (S3A Fig). When the extracts were prepared with lysis buffer containing a higher concentration of detergent (1.5% Triton), which helps solubilize endomembrane-associated proteins (as well as aggregation-prone proteins), we observed a significant increase in ALG-1 levels in tbc-11(qbc24) extracts and a decrease in tbc-11(ok2576) (Fig 3A). This difference in ALG-1 solubility might be explained by the nature of the tbc-11 mutations found in those alleles. The ok2576 allele carries a large deletion of 1421 base pairs that most likely corresponds to a loss of function of tbc-11 while the qbc24 allele corresponds to a point mutation in the catalytic domain of the gene, which may act as a catalytic dead RabGAP and thus have a different molecular outcome on Rab activation. Based on those observations, we hypothesize that in tbc-11(qbc24) animals, ALG-1 may be more associated to endomembranes, whereas it would be less associated to endomembranes in tbc-11(ok2576) animals. We next wondered if this effect was widespread to the other miRISC components. We monitored the levels of the GW182 ortholog AIN-1 and observed that while the protein levels are affected in tbc-11 mutants, this was not dependent on detergent concentration (S3B Fig), indicating that tbc-11 acts specifically on ALG-1. To confirm that the differences observed in ALG-1 were not caused by changes in the total ALG-1 protein level, we performed western blots on fully solubilized extracts and observed no substantial differences in ALG-1 levels (S3C
Fig 2. TBC-11 is likely a GTPase activating protein for RAB-6.1 and RAB-6.2. (A) The relationship between Rab
GTPase, GAPs and GEFs. Rab proteins are active when bound to GTP and inactive when bound to GDP. Guanine nucleotide exchange factors (GEF) activate Rab by releasing the bound GDP molecule to allow the binding of a GTP molecule. GTPase activating proteins (GAP) catalyze the hydrolysis of GTP to GDP to inactivate Rab. (B-C) Alae breaks of tbc-11(ok2576) were scored under DIC Nomarski microscopy. Animals were fed with bacteria expressing RNAi against rab-6.1, rab-6.2 or control RNAi (no targeting gene) for 48 hours and observed as young adults. 50 animals were observed for each condition. Each circle represents the mean of one independent RNAi experiment. P value were obtained by one-way ANOVA ( ��� p value < 0.0001).
https://doi.org/10.1371/journal.pgen.1009511.g002 Fig). This result indicates that the differences observed in ALG-1 levels in the prepared extracts are likely caused by the ability to solubilize and recover membrane-associated or aggregated proteins.
TBC-11 affects the association of ALG-1 to microRNAs
We next wanted to determine if the possible changes in ALG-1 endomembrane association altered miRNA levels as well as the loading of miRNAs onto the Argonaute, as these two properties are tightly linked [36]. We first monitored the total levels of a subset of mature miRNAs, including some let-7 family members, by RT-qPCR. We observed a significant decrease in all of the tested miRNA levels in both tbc-11 mutant alleles (Fig 3B). We then performed highthroughput sequencing analysis of total small RNAs isolated from young adult animal population to determine whether the alteration of tbc-11 has a global effect on miRNA levels. Even if the changes in total miRNA levels between wild-type and tbc-11 animals monitored by this method are not statistically significant (S3D Fig), we can observe a decrease in most miRNA levels in tbc-11 animals inferring that the loss of tbc-11 could have a general impact on miRNA levels in animals.
We next wanted to determine if the association of ALG-1 to endomembranes or to an aggregated complex altered its ability to bind miRNAs. To answer this, we immunoprecipitated ALG-1 in extracts from wild-type and tbc-11(qbc24) animals, prepared with low or high detergent concentration, and monitored miRNAs associated to ALG-1 in both conditions. We observed that in wild-type animals, the concentration of detergent used in the extracts does not alter the level of miRNAs loaded into ALG-1 (Fig 3C; left). However, in tbc-11(qbc24) animals, the level of miRNAs loaded onto ALG-1 is significantly decreased when extracts are prepared with high detergent concentration (Fig 3C; right). This data suggests that the pool of ALG-1 that accumulates on endomembranes or in aggregated complexes in qbc24 animals is not functional for gene silencing as it is not loaded with miRNAs. Moreover, this decrease in miRNA loading induces a decrease in total miRNA levels (as observed in Fig 3B) as these two properties are tightly linked.
ALG-1 localizes at the perinuclear region and accumulates in a high molecular weight complex in tbc-11(qbc24) animals
As we observed an accumulation of unloaded ALG-1 in tbc-11(qbc24) animals, we wondered if the subcellular localization of ALG-1 was altered. To test this, we observed the subcellular localization of an endogenous GFP-tagged ALG-1 in the seam cells of the animals. In wildtype animals, GFP::ALG-1 is localized broadly across the cytoplasm of the cell. However, in tbc-11(qbc24) animals, we observed that the pool of ALG-1 expressed in seam cells is mostly located around the nucleus (Fig 4). To confirm that this mislocalization of ALG-1 was , tbc-11(qbc24) and tbc-11 (ok2576) young adult worm extracts prepared with low (0.5%) or high (1.5%) detergent (Triton) concentration. High detergent concentration allows better extraction of membrane associated proteins. Actin is used as a loading control. Quantification of 3 independent Western blots is shown. ALG-1 level is normalized to Actin levels and normalized to 0.5% detergent for each condition. P value were obtained by one-way ANOVA ( � p value < 0.05). (B) Quantification of microRNA expression. The levels of let-7, miR-48, miR-58 and miR-228 were measured by RT-qPCR in tbc-11 mutants and normalized on the levels in wild-type animals (n = 3). Small nucleolar RNA sn2841 was used as a reference. The error bars indicate the confidence interval (α = 0.05) and P values were calculated with a two-tailed Student t-test. ( � p value < 0.05). (C) Expression level of ALG-1 associated miRNAs measured by RT-qPCR. RNA was extracted following ALG-1 immunoprecipitation in wild-type (WT) and tbc-11(qbc24) young adult worm extracts prepared with low (0.5%) or high (1.5%) detergent concentration. Ct values obtained for low detergent extracts were subtracted from Ct values obtained for high detergent extracts and log2 values were calculated. The Ct ratio was then normalized to the levels of immunoprecipitated ALG-1 (see S3E Fig for representative western blot) for each individual microRNA. Error bars represent standard deviation for each microRNA. P values were obtained by two tailed t-test ( � p value < 0.05). Three independent immunoprecipitation experiments were performed.
We have attempted to identify this perinuclear structure by expressing different organelle markers and observing co-localization with ALG-1. Since RAB-6 localizes at the Golgi, we first wondered if ALG-1 was accumulating at the Golgi in tbc-11(qbc24) mutants. We expressed the Golgi marker mannosidase::mCherry (MANS::mCherry), under the control of alg-1 promoter in WT and tbc-11(qbc24) animals and monitored its localization pattern in the seam cells. While we observed a fraction of ALG-1 that co-localized with MANS::mCherry by microscopy, we did not observe an elevated amount of MANS around the nucleus as we observed for ALG-1 in tbc-11(qbc24) animals (S4 Fig). We next wondered if ALG-1 could be accumulating at the endoplasmic reticulum (ER) located at the periphery of the nucleus since RAB-6 is involved in Golgi to ER transport [35]. Many processes occur at the ER, including protein synthesis, protein folding, lipid synthesis and calcium storage. There are two distinct forms of ER: the rough ER defined by a high density of ribosomes involved mainly in protein synthesis and the smooth ER which has few ribosomes and is associated with lipid synthesis (reviewed in [37]). It was previously shown that Argonaute proteins associate with the rough ER for miRNA loading and mRNA target repression [16]. Moreover, many studies have shown that Argonaute associates with polysomes (for example, see [32,[38][39][40][41]). We therefore hypothesized that the association of ALG-1 with the ribosomes at the perinuclear rough ER might be affected in the seam cells of tbc-11(qbc24) animals.
To determine if ALG-1 was accumulating at the rough ER in tbc-11(qbc24) animals, we attempted to express the ER marker TRAM-1 in the animals' seam cells but could not detect any protein expression in these cells. We therefore monitored the association of ALG-1 with polysomes in WT and tbc-11 animals by polysome profiling. We fractionated worm extracts on a 15% to 55% sucrose gradient and then performed western blots for ALG-1 in the collected fractions. We did not observe any difference in ALG-1 distribution in tbc-11(ok2576) animals compared to wild-type (S5A Fig). However, we observed that in tbc-11(qbc24) animals, ALG-1 co-sediments more with the heavy polysomes fractions than in wild-type animals (Fig 5A; polysome profiles are shown in S5B Fig). To determine if ALG-1 was truly associating to polysomes, we repeated the fractionation after EDTA treatment to dissociate polysomes. The addition of EDTA successfully dissociated polysomes in both wild-type and tbc-11(qbc24) animals (S5C Fig). As expected in wild-type animals, ALG-1 shifted to fractions corresponding to low molecular weight complexes (Fig 5B). In contrast, we did not observe a shift as major for ALG-1 in tbc-11(qbc24) animal extracts treated with EDTA ( Fig 5B). These results suggest that ALG-1 is not bound to polysomes in tbc-11(qbc24) animals but rather associates with an uncharacterized high molecular weight complex that is likely perinuclear, as observed by microscopy (Fig 4).
Fig 4. tbc-11 affects ALG-1 intracellular localization. (A)
Fluorescent microscopy of intracellular localization of endogenously tagged GFP::ALG-1 in seam cells of wild-type (WT) and tbc-11(qbc24) young adults animals. Animals were fed with bacteria expressing RNAi against rab-6.1 or control RNAi (-;no targeting gene) for 48 hours. The nucleus of the seam cell is indicated by a yellow arrow. A zoomed in image of tbc-11(qbc24) seam cell is showed. Images were taken using the same settings and exposition time for each animal. Scale bar: 10μm. (B) Quantification of representative images of GFP::ALG-1 in seam cells of wild-type (WT) and tbc-11(qbc24) animals exposed to RNAi against rab-6.1 or control RNAi (no targeting gene) for 48 hours. Quantification was performed by drawing a straight line starting from the middle of the nucleus and passing through the cytoplasm of the seam cell. The fluorescence signal was measured along the line using ImageJ. The distance in μm represents the distance from the middle of the nucleus. Each line represents the GFP quantification of a separate image of a different animal.
https://doi.org/10.1371/journal.pgen.1009511.g004 Fig 5. ALG-1 associates with a high molecular weight complex in tbc-11(qbc24) animals. (A) Detection of ALG-1 in different fractions by Western blot following polysome profiling of wild-type (WT) and tbc-11(qbc24) young adult animals performed with extracts prepared with high detergent concentration (1.5% triton). Low and high exposure of the same Western membrane are shown. Fractions corresponding to ribosomal subunits, light polysomes and heavy polysomes are indicated. (B) Detection of ALG-1 in different fractions by Western blot following polysome profiling of wild-type (WT) and tbc-11(qbc24) young adult animals performed with extracts treated with 10mM EDTA. Fractions corresponding to ribosomal subunits, light polysomes and heavy polysomes are indicated. Low and high exposure of the same Western membrane are shown. For both panels, the quantification of ALG-1 associated to heavy polysomes (to obtain the relative level of ALG-1 associated to heavy polysomes, the sum of the signal detected in those fractions was normalized to the total ALG-1 signal in all fractions) from Western blots of 2 independent experiments are shown (right). P value were obtained by two tailed t-test ( �� p value < 0.001).
Altogether, our results show that the alteration of tbc-11 induces an accumulation of unloaded ALG-1 in a perinuclear, high molecular weight complex that likely contains endomembranes (S6 Fig). This would prevent ALG-1 from binding to its mRNA targets and therefore induce miRNA-related defects in animals.
Discussion
In this study, we uncovered a novel function for the RabGAP TBC-11 identified in an unbiased genetic screen that aimed to identify new genes involved in the microRNA pathway. Our work identified the small GTPase rab-6 and the RabGAP tbc-11 as important new actors in the gene regulation mediated by miRNAs in animals. We showed that tbc-11 is likely a GAP for rab-6 in C. elegans and that this small GTPase regulation is important for miRNA function. We demonstrated that in absence of tbc-11, the pool of miRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes increases, leading to a misregulation of miRNA targets. The alteration of TBC-11 function causes an accumulation of ALG-1 at the perinuclear region of the cell and forms an uncharacterized high molecular weight complex. We propose that this complex would sequester ALG-1, likely unbound to miRNA, and prevent its normally induced degradation. Further studies will be important to further characterize the nature of this complex.
Interestingly, this study represents our second forward genetic screens that identified new RAB-6-associated genes involved in the regulation of the miRNA pathway in C. elegans, highlighting the important role played by RAB-6 in the control of this gene regulatory pathway in animals [15]. Our work further supports previous reports suggesting that miRNA-mediated gene regulation is tightly linked to endomembrane trafficking. While we tend to limit our view of miRNA target repression to granules such as the P bodies, it is not so surprising to find ALG-1 associating to endomembranes such as the ER and Golgi. Indeed, the first report of Argonaute proteins described them as GERp95 (Golgi-endoplasmic reticulum protein 95 kDa) before knowing their precise function [42]. Several studies since then have linked the ER and Golgi to different steps of miRNA maturation and function. This physical sequestration could be important to allow coordination of miRISC loading, target repression, target decay and finally, Argonaute turnover. We also propose that intracellular trafficking could be important to repress mRNA targets at different subcellular locations within the cell. While it may seem surprising that a screen designed to be independent of miRNA loading allowed us to identify a gene implicated in intracellular trafficking, the proper localization of the λN tagged ALG-1 is likely still needed to allow repression of the targeted mRNA.
The activity of RAB-6 in the control of ALG-1 localization is likely reflected in our analysis of both mutant alleles of tbc-11. In animals where TBC-11 is deleted (ok2576), the activity of RAB-6 may be partially regulated by other GAPs with low affinity and thus the localization of ALG-1 is not as affected. While in tbc-11(qbc24) animals, a non-functional form of TBC-11 that likely retains its capacity to bind RAB-6 is expressed but unable to hydrolyze GTP, leading to a hyperactive form of RAB-6. While our results suggest that RAB-6 is important for the shuttling of ALG-1 to the endomembrane system, the nature of this interaction remains unknown. In fact, it is still to be determined if RAB-6 binds directly to ALG-1 or if another protein mediates this interaction. As RAB-6 interacts with the retrograde complex GARP, previously shown to be involved in the miRNA function in C. elegans by controlling the level of the GW182 protein AIN-1 and miRNAs [15], it is plausible that the GARP complex could be the mediator of the interaction between RAB-6 and ALG-1. However, despite several attempts, we have not been able to detect any proof of interaction between VPS-52 and ALG-1, even in tbc-11(qbc24) animals. Thus, we postulate that RAB-6 could affect the miRISC at numerous steps; its association with the GARP complex would affect the transport of AIN-1 and its association with a yet unknown effector protein would affect the transport of ALG-1.
While this study identified rab-6 and the GAP tbc-11 as factors controlling the localization of ALG-1 to a perinuclear complex, we propose that other Rabs are most likely involved in the proper localization of the Argonaute proteins. Indeed, different Rabs are responsible for precise steps of vesicular trafficking, and Argonaute proteins are likely transported within different compartments of the cell. We would therefore expect that other Rab proteins may affect the function of miRISC. Indeed, we observed that rab-2 and rab-14 RNAi induced alae breaks in wild-type animals, suggesting that they might also play a role in miRNA function. However, when RAB-6 is constitutively active, it excessively shuttles ALG-1 to the perinuclear region (see Model S6 Fig). This mis-shuttling leads to an accumulation of unloaded and unfunctional ALG-1.
These results highlight the importance of the regulation of Rab proteins for proper Argonaute function. Further studies will be essential to assess the role of other Rab proteins on the localization of Argonaute and other components of the miRNA pathway. Moreover, the processes occurring at different subcellular localization need to be clarified. Indeed, the subcellular localization at which miRISC loading, target repression and miRISC recycling occur remains poorly characterized, especially in vivo. Nevertheless, our results demonstrate that RAB-6 and TBC-11 are important in the proper localization of ALG-1 and that accumulation of unloaded ALG-1 at a perinuclear, high molecular weight complex impairs miRNA-mediated silencing in animals. Overall, this study emphasizes the importance of intracellular trafficking for miRNA function and proper miRISC localization.
Worm culture
C. elegans strains were cultured in standard conditions [43]. Strains were grown on Nematode Growth Media (NGM) plates and fed E. coli strain OP50. Worms were grown at 20˚C unless specified.
RNA interference
RNAi was performed by feeding worms with IPTG inducible HT115 bacteria expressing dsRNA against rab-6.1, rab-6.2, rab-2, rab-14, alg-1 or tbc-11 (List of oligonucleotides used to make RNAi constructs can be found in S1 Table and name of plasmids used in S2 Table). L1 staged animals were exposed to RNAi for 48 hours at 20˚C and L4 or young adults were scored for either alae defects, ALG-1 localization or GFP expression.
Preparation of protein extracts
Extracts were prepared from~150,000 synchronized young adult animals. Worms were washed and resuspended in cold lysis buffer (10mM Potassium Acetate, 25mM Hepes-Potassium Hydroxide pH 7.0, 2mM Magnesium Acetate, 1mM DTT, 0.5% OR 1.5% [v/v] Triton X-100 and protease inhibitors) before being extracted with a Dounce homogenizer. The extracts were centrifuged at 17,000g for 20min at 4˚C and the clarified supernatants were collected. For experiments comparing 0.5% vs 1.5% Triton, a single worm pellet was equally separated and extracted in parallel with different detergent concentrations.
Immunoprecipitation and quantitative real-time PCR
Extracts were quantified with Bio-Rad Protein Assay and immuprecipitation (IP) was performed on 1mg of protein extract per condition. Anti-ALG-1 antibody was coupled to magnetic Dynabeads Protein G (ThermoFisher). Extracts were incubated with anti-ALG-1-coupled beads for 2h at 4˚C. 10% of IP was loaded on polyacrylamide gel to assess ALG-1 levels. The remainder was submitted to RNA extraction by Trizol/Chloroform. The RNA was retrotranscribed with Multiscribe reverse transcriptase (ThermoFisher) and miRNA levels were measured with specific Taqman probes.
Imaging and microscopy
DIC Nomarski images of worms' alae, GFP fluorescence of miR-228 reporter [7] and hypodermal let-7 GFP microRNA reporter were taken with a Zeiss AxioCam HRm digital camera mounted on a Zeiss Axio Imager M1 microscope. Fluorescence intensity was measured with Zen software. Images of the GFP::ALG-1 intracellular location in the seam cells were taken with Zeiss LSM700 confocal microscope. Images were taken using the same settings and exposition time for each animal.
Polysome profiles
Synchronized young adult animals were collected and washed in M9 supplemented with 1mM cycloheximide then washed once with lysis buffer (20mM Tris pH 8.5, 140 mM KCl, 1.5mM MgCl 2 , 1.5% [v/v] Nonidet P40, 1 mM DTT, 1 mM cycloheximide) before flash freezing. Thawed pellets were washed 3 times with lysis buffer and homogenized in 1 volume of lysis buffer supplemented with 0.4U/uL RNase using a Dounce homogenizer. For EDTA controls, a final concentration of 10mM of EDTA was added to the extracts following extraction.
Concentration of RNA was measured with a spectrophotometer. 10 OD 260 units were loaded onto a 15% to 55% sucrose gradient and further fractionated and analyzed as described in [45]. For EDTA controls, MgCl 2 and cycloheximide in sucrose gradients were replaced by EDTA. Recovered fractions were precipitated with 2 volumes of ethanol 100% and 10% of each fraction was loaded on an 8% SDS-polyacrylamide gel for western blot analysis.
miRNA cloning, sequencing and analysis
The TruSeq small RNA libraries were prepared and sequenced as described in [46,47]. The small RNA sequencing data are available at Gene Expression Omnibus (GEO) with the accession number: GSE141719.
Sequencing reads were mapped to the genome and cDNA using custom PERL (5.10.1) scripts and Bowtie 0.12.7 [48]. Databases used include C. elegans genome (WormBase release WS215), Repbase 15.10 [49], and miRBase 16 [50]. The Generic Genome Browser [51] was used to visualize the alignments. Detailed PERL scripts and related database files and analyses in this study are available upon request.
The samples were normalized to the total small RNAs including miRNAs, 22G-RNAs and 21U-RNAs. We used the average of triplicates for each strain to calculate the ratio of each small RNA reads of tbc-11 to the sum of tbc-11 + wild-type reads.
Figure data
Data for main text Figs 1-3 and 5 and S1 and S2 Figs can be found in S1 File.
Supporting information S1 Fig. Characterization of tbc-11(qbc24) and tbc-11(ok2576) mutant alleles. (A) GFP reporter repressed by λN::mCherry tagged ALG-1. GFP was fused to alg-1 endogenous promoter (alg-1p) and cog-1 3' UTR in which the lsy-6 binding sites were replaced by 6 box B sequences (cog-1Δlsy-6 bs). (B) DIC and fluorescent microscopy of WT and tbc-11(qbc24) pharynx in young adults. GFP is derepressed in tbc-11(qbc24) animals. ALG-1 expression (represented by mCherry) is not affected in these animals. Images were taken using the same settings and exposition time for each animal. Scale bar: 50μm. (C) Percentage of alae breaks of wild-type (WT), tbc-11(qbc24) homozygous, and tbc-11(qbc24) heterozygous (tbc-11(qbc24)/+) young adult animals. tbc-11(qbc24) heterozygous animals were observed as F1 of a genetic cross between tbc-11(qbc24) and wild-type animals. P value were obtained by one-way ANOVA ( �� p value < 0.001) (D) Number of seam cells in tbc-11(qbc24) and (ok2576) alleles. Strains were crossed with a strain expressing GFP in the seam cells (scm::GFP) in order to score them. Animals were observed as young adults. Wild-type (WT) animals have an invariable number of 16 seam cells. P value were obtained by Fisher's exact test ( ��� p value < 0.0001) (E) Left: DIC and fluorescent microscopy of col-10::gfp::lin-41 3'UTR reporter in hypodermal cells of L4 staged animals. Animals were fed with bacteria expressing RNAi against tbc-11 or control RNAi (no targeting gene) for 48 hours. Scale bar: 50μm. Right: Quantification of GFP fluorescence intensity in four hypodermal cells per animal. Images were taken using the same settings and exposition time for each animal. The number of animals scored (n) is indicated. P value were obtained by two tailed t-test. ( � p value < 0.05). (F) Left: DIC and fluorescent microscopy of miR-228 mutated reporter in intestine cells of L2 staged animals. Scale bar: 50μm. Right: Quantification of GFP fluorescence intensity in four intestine cells per animal. Images were taken using the same settings and exposition time for each animal. The number of animals scored (n) is indicated. P value were obtained by two tailed t-test. ns: non-significant. (TIF) S2 Fig. Effect of rab-2 and rab-14 RNAi on alae structure. Alae breaks of tbc- 11(ok2576) were scored under DIC Nomarski microscopy. Animals were fed with bacteria expressing RNAi against rab-2 (upper panel), rab-14 (lower panel) or control RNAi (no targeting gene) for 48 hours and observed as young adults. 50 animals were observed for each condition. Each circle represents the mean of one independent RNAi experiment. P value were obtained by one-way ANOVA ( ��� p value < 0.0001). (TIF) S3 Fig. Protein levels of ALG-1 and AIN-1 in tbc-11 animals and quantification of micro-RNA levels. (A) Western blot of ALG-1 and the GW182 protein AIN-1 in wild-type (WT), tbc-11(qbc24) and tbc-11(ok2576) young adult animals. Extracts were prepared with standard conditions (0.5% triton). Actin is used as a loading control. (B) Western blot of the GW182 protein AIN-1 detected in wild-type (WT), tbc-11(qbc24) and tbc-11(ok2576) young adult worms extracts prepared with low (0.5% triton) or high (1.5% triton) detergent concentration. High detergent concentration allows better extraction of membrane associated proteins. Actin is used as a loading control. (C) Western blot of ALG-1 protein in wild-type (WT), tbc-11 (qbc24) and tbc-11(ok2576). Proteins were fully solubilized by boiling animals in Laemmli buffer for 10 minutes. Actin is used as a loading control. (D) Small RNA sequencing of tbc-11 (qbc24) (left) and tbc-11(ok2576) (right) young adult animals compared to wild-type animals. The dotted line represents a two-fold change. The number of miRNA analyzed is indicated (n). p<0.22 for let-7 in tbc-11(qbc24), p<0.50 for miR-228 in tbc-11(qbc24), p<0.13 for let-7 in tbc-11(ok2576), p<0.25 for miR-228 in tbc-11(ok2576). P value for individual miRNAs were obtained by two tailed unpaired t test. The samples were normalized to the total small RNAs including miRNAs, 22G-RNAs and 21U-RNAs. | 2021-04-09T06:19:09.385Z | 2021-04-01T00:00:00.000 | {
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15799634 | pes2o/s2orc | v3-fos-license | Scale-Dependent Growth from a Transition in Dark Energy Dynamics
We investigate the observational consequences of the quintessence field rolling to and oscillating near a minimum in its potential,"if"it happens close to the present epoch (z<0.2). We show that in a class of models, the oscillations lead to a rapid growth of the field fluctuations and the gravitational potential on subhorizon scales. The growth in the gravitational potential occurs on timescales<<H^(1). This effect is present even when the quintessence parameters are chosen to reproduce an expansion history consistent with observations. For linearized fluctuations, we find that although the gravitational potential power spectrum is enhanced in a scale-dependent manner, the shape of the dark matter/galaxy power spectrum is not significantly affected. We find that the best constraints on such a transition in the quintessence field is provided via the integrated Sachs-Wolfe (ISW) effect in the CMB temperature power spectrum. Going beyond the linearized regime, the quintessence field can fragment into large, localized, long lived excitations (oscillons) with sizes comparable to galaxy clusters; this fragmentation could provide additional observational constraints. Two quoted"signatures"of modified gravity are a scale-dependent growth of the gravitational potential and a difference between the matter power spectrum inferred from measurements of lensing and galaxy clustering. Here, both effects are achieved by a minimally coupled scalar field in general relativity with a canonical kinetic term.
Introduction
Scalar fields are used ubiquitously in cosmology to provide a mechanism for accelerated expansion during both the inflationary [1,2,3] and current dark energy dominated epochs (for example, [4,5,6]). In the inflationary case, accelerated expansion occurs when the inflaton field is rolling slowly and it ends once the field starts oscillating around the minimum of its potential. The oscillatory phase is well studied and gives rise to rapid growth of inhomogeneities because of couplings to other fields or self interactions (see, e.g. [7,8,9,10]). For a recent review, see [11]. Like the inflaton, the quintessence field provides a mechanism for accelerated expansion in the slow-roll regime. However, unlike inflation, the possibility of the accelerated expansion ending through quintessence oscillations is rarely discussed in the literature (however, see e.g. [12]). If quintessence was rolling slowly in the past, it is "natural" (though certainly not necessary) for it to enter an oscillatory regime as it finds its way towards a local minimum in its potential.
In this paper, we wish to understand the observational signatures of a transition to an oscillatory regime in the quintessence field, if such a transition occurs in our recent past (z 0.2). An obvious observational signature is a late-time change in expansion history. More importantly, as is the case with the inflaton, the coherent oscillations of quintessence near an anharmonic minimum are unstable and quickly fragment in a spatially inhomogeneous manner, giving rise to additional structure. This instability driven by self interactions of the field (the anharmonic terms) leads to a growth in structure that is much faster than the usual gravitational growth. For example, all potentials that have a quadratic minimum and are shallower than quadratic away from the minimum (see Fig. 1) suffer from this instability (in particular for wavenumbers k m, where m 2 ≈ U (ϕ)| ϕ→0 . See, for e.g [13,14,9,15]). We find that while the gravitational potential is influenced strongly by such growth in the quintessence fluctuations, the over density in baryons and dark matter does not change significantly. The scale dependent growth in the gravitational potential and the absence of similar growth in the matter power spectrum provides a signature to confirm or rule out such a transition in the dynamics of the quintessence field. In addition, if the quintessence field becomes nonlinear, it can fragment rapidly into long-lived, localized excitations called oscillons (see e.g. [16,17,18,19,20,21,22,23,24,25,26,27,28,9,29,30,31,32,15]) with size l ∼ few × m −1 ∼ few × Mpc * . Although we do not pursue this in detail in this paper, in Appendix B, we comment on how such rapid nonlinear fragmentation of the field, and to a lesser extent, the time-dependent evolution of oscillon configurations themselves could have interesting integrated Sachs-Wolfe (ISW) signatures.
While similar resonant phenomenon are commonly invoked in the early Universe, for example, during preheating (z 10 20 ) [11], few observational signatures exist (e.g. [33,34,35,36]). The reason for this lack of observational signatures on astrophysical scales is that resonance is effective only on subhorizon scales. Since high redshifts imply a small * We will frequently relate the mass of the scalar field m, to length and time scales, without explicitly writing and c. We set = c = 1 throughout the paper.
Hubble horizon, only small astrophysically inaccessible scales today (of order meters for grand unied theory scale inflation) become excited. Moreover, on these small scales, the thermal radiation dominated state of the Universe at the time of big bang nucleosynthesis smooths out inhomogeneities. Since in our scenario the transition in quintessence is happening today, the resonance now happens on astrophysically accessible scales. As a result these models can be strongly constrained.
The observationally interesting phenomenology of quintessence oscillations comes at a price. In addition to the usual problems with quintessence models (such as the smallness of the energy density, long range forces, etc.), for the quintessence potentials we work with, we require that the transition to an oscillatory regime happens close to today. This imposes an additional tuning for the initial conditions of the field.
Related to the present paper [14], Johnson and Kamionkowski discuss the dynamical instability arising from a "small" anharmonic term in oscillating dark energy models and conclude that such instabilities render oscillating dark energy models unsuitable for providing a sustained cosmic acceleration period. While we rely on these very instabilities to source the rapid growth of the field fluctuations and the gravitational potential, we envision the transition happening late enough so that this instability does not significantly affect the observable expansion history. We then calculate and discuss in detail the additional observables that can be used to probe the rapid growth in the gravitational potentials arising from the dynamical instability. We reiterate, that we are not interested in the oscillatory phase providing the accelerated expansion.
We briefly mention a few papers that explore clustering in quintessence-like fields, though through very different mechanisms. Observational effects of quintessence clustering caused by phenomenologically varying its speed of sound (not from resonant behavior discussed here) has been investigated by several authors (see, for example, [37,38,39,40,41,42,43]). Instabilities in coupled dark energy-dark matter models have also been considered in [44]. In addition, rapid transitions in dark energy, in the context of coupled/unified dark matter and dark energy models where dark energy "switches on" at late times have been explored by [45] (also see references therein). In our case, dark energy "turns off" at late times as the quintessence field starts oscillating, leading to a rapid growth in field fluctuations. With a somewhat different motivation, in [46] the authors discuss how rapid, extremely low redshift (z 0.02) transitions in dark energy can be hidden from expansion history measurements. However, they did not consider the effects of such transitions on perturbations, which is the focus of this work.
In this exploratory paper, we restrict ourselves to single field quintessence models, assume a minimal coupling to gravity and impose no nongravitational couplings to other fields. We include Weakly Interacting Massive Particle (WIMP) dark matter along with the quintessence field and assume a spatially flat universe. We limit ourselves to a linear treatment of the fluctuations, including scalar gravitational perturbations, except when we discuss the nonlinear fragmentation of the quintessence field and the formation of robust, localized quintessence excitations (oscillons). We focus on a case that is consistent with expansion history and galaxy clustering but could potentially be ruled out by measurements of the gravitational potential, in particular, via the cosmic microwave background (CMB) temperature power spectrum at large angular scales. Our intention is to stress the interesting phenomenology of such models and to point out that such transitions can be better constrained by going beyond the measurements of expansion history and growth of structure in galaxies alone.
The rest of the paper is organized as follows. In Sec. (2) we introduce and motivate the form of the quintessence potential used in this paper. In Sec. (3) we work through the evolution of the quintessence field in an Friedmann-Robertson-Walker universe. We highlight initial conditions and important regimes of evolution as well as constraints placed on the parameters from observations of the expansion history. In Sec. (4) we discuss the evolution of linearized fluctuations in the quintessence field, dark matter and the gravitational potential, with a special emphasis on their evolution during the oscillatory phase of the background quintessence field. In the same section we also discuss the domain of validity of our linearized treatment and comment on the nonlinear evolution of the field. In Sec. (5) we compute observables such as lensing power spectra and the ISW contribution to the CMB temperature anisotropy. We present our conclusions in Sec. (6). We also include two Appendixes. In Appendix A, we provide some details of Floquet analysis and an algorithm used in this paper for calculating Floquet exponents. In Appendix B, we provide an estimate of the ISW effect resulting from evolution of quasi/nonlinear quintessence fluctuations and oscillons.
The model
We consider a quintessence field governed by a potential of the form (see Fig. 1): where 0 < α < 1. † This choice was motivated by monodromy and supergravity models of inflation [47,48,49,50,51] and a recent model of axion quintessence [52] (α = 1/2). The potential has a quadratic minimum and for very large field values it asymptotes to a shallower than quadratic form, The scale M determines where the potential changes shape, whereas, the scale m determines the curvature U (0) at the bottom of the well. We will consider cases where the field rolls † We write ϕ instead of |ϕ| to avoid clutter. slow roll oscillatory ϕ ∼ M U (ϕ) ∼ m 2 Figure 1: Initially, the field rolls slowly causing accelerated expansion of space. As it enters the oscillatory regime, the acceleration stops and a rapid, exponential growth of field fluctuations begins.
slowly for ϕ M , behaving like dark energy and then enters an oscillatory regime with ϕ ∼ M after z ∼ 0.2. When the field oscillates around the minimum, the "opening up" of the potential, leads to rapid, scale dependent growth of scalar field fluctuations via parametric resonance (see Sec. (4)). In the nonlinear regimes, it leads to the formation of localized field excitations called oscillons [9,29].
Similar phenomenology can arise in models such as pseudo-Nambu-Goldston-Boson quintessence (e.g. [53]) For potentials (1) and (4), a limited range of parameters will reproduce the observed expansion history, allow for a few oscillations in the field close to today, and allow for rapid growth of structure. For the pseudo-Nambu-Goldston-Boson-like models, the slow roll dynamics of the field necessary for accelerated expansion, only occur if M ∼ m Pl (unless ϕ → πM ). However, as we discuss in Sec. (4), efficient resonance in an expanding universe requires M m Pl . As a result we do not get rapid growth of structure here, except in cases of extreme fine tuning of the initial conditions and do not pursue this model any further in this paper.
For the potential in (1), we find the requirement of a few oscillations close to today translates into 10 2 H 0 M, m 10 −2 m Pl and α 1.
We study these constraints in more detail in the next section.
Finally, we note that resonant growth of fluctuations also arise when scalar fields oscillate Figure 2: (a) Homogeneous field evolution: note that the field is relatively constant until a osc after which it starts oscillating with a decaying amplitude. (b) The Hubble parameter increases initially compared to its ΛCDM counterpart but once the oscillations in the field begin, it starts decreasing again. The small oscillations in the Hubble parameter reflect the oscillations in the quintessence field. (c) The equation of state parameter w ≈ −1 until a osc , reflecting the cosmological constant like behavior due to the slowly rolling field, but starts oscillating about w = 0 after a osc , reflecting the approximately nonrelativistic matter like behavior of the oscillating field. (d) The acceleration parameter q reveals a period of decelerated expansion in the matter dominated era, followed by a period of accelerated expansion as the slowly rolling quintessence field begins to dominate the energy density. This continues until a osc , after which q starts oscillating due to the oscillations in the quintessence field.
In practice, when numerically evolving Eq. (6) according to the above choices of initial conditions and potential parameters, we find that in order to get agreement with the ΛCDM expansion history in the past as well as produce oscillations close to today, we require additional fine tuning of the initial conditions and parameters. The procedure described above results in a value of H 0 that is smaller than the ΛCDM value. This is because by assumption, the Hubble parameter for both ΛCDM and quintessence are identical. At late times, however, the oscillations in the quintessence field cause it to behave like dark matter, giving rise to a steeper fall off than the ΛCDM Hubble parameter. For better agreement with H 0 , we increase the value of m until H 0 agrees to better than a percent.
Performing the above iteration, we find that α affects whether or not the field starts oscillating at low redshift. This should be expected since the asymptotic slope of the potential determines the time it takes to transition from slow-roll to oscillations. We can calculate this approximate time from the derivative of the quintessence field when slow roll ends. Using the slow roll condition described above, the value of the quintessence field when slow roll ends is, Assuming 3Hφ ∼ −U , taking this transition to happen close to today, and using the constraints in Eq. (10), we find the change in scale factor ( a) between the beginning of oscillations (ϕ ∼ 0) and the end of slow roll is given by, The larger the α, the longer it takes for oscillations to start. Hence, consistency with a ΛCDM expansion history and having late-time oscillations limits α 1 for the case of nonzero α and M m Pl for vanishing α. For the rest of this paper, we specialize to α = 0. In summary, we keep M and a * as free parameters and choose m, ϕ i andφ i so that the value of H 0 agrees with the measured value, the field behaves like dark energy initially, and the field starts oscillating at a osc ≈ 0.8.
For the above prescription, we calculate the evolution of the quintessence field, the Hubble parameter normalized by ΛCDM's Hubble parameter, the field's equation of state parameter w and the acceleration parameter q ≡äa/ȧ 2 as a function of scale factor. We show the results in Unless otherwise stated we will use these as fiducial parameter values throughout this paper and refer to them as fiducial parameters. As constructed, the field remains approximately constant for a a osc , rolling slowly towards the minimum. Notice, as assumed initially, that ϕ M at early times. At a osc ∼ 0.8, the field starts to oscillate with a slowly decaying amplitude, ϕ ≈ ϕ osc (a) sin(ωt + ∆). (14) Note that when ϕ M , ϕ osc (a) ∝ a −3/2 . Because of the anharmonic terms in the potential, the frequency of oscillation (ω) depends on the amplitude of the field and is given by, where E is the complete elliptic integral. As ϕ → 0, ω → m. Note that in deriving the above expression for the period, we have assumed ω H 0 so that energy is approximately conserved during an oscillation.
The Hubble parameter matches ΛCDM at early times since both models have the same amount of dark matter [see Fig. 2(b)]. The Hubble parameter must then increase relative to ΛCDM in order for H 0 to agree, since the quintessence model's Hubble parameter falls off more steeply than ΛCDM at late times when the field starts to behave like nonrelativistic matter. The deviation, for the fiducial parameters, is at most 5%, which is consistent with recent observational constraints [55]. The equation of state parameter behaves as expected in the slow roll regime (w = −1) and oscillates between 1 and -1 after a osc as energy swaps between its kinetic and potential parts [ Fig. 2(c)]. Note that other models that give rise to an oscillating equation of state have been explored in the literature (for example, [56,57,58,59,60,61,62]). Lastly, the acceleration parameter reveals a period of decelerated expansion deep in the matter dominated era, followed by a period of accelerated expansion as the slowly rolling quintessence field begins to dominate the energy density. After a osc , the acceleration parameter starts oscillating around q = −1/2 [ Fig. 2 Observational constraints on the expansion history at late times arise primarily from distance measurements. Given our above prescription, we expect good agreement between distances calculated in our quintessence model and ΛCDM, which we show explicitly below. The comoving distance is given by, In Fig. 3, we plot the percent difference in χ(a) between quintessence and ΛCDM. As expected, deviations are smaller close to today, where the Hubble parameter was tuned to be identical to the observed H 0 and at early times, where dark energy can be ignored. The largest deviations are ∼ 4%. Since all observed distances, like the luminosity distance and the angular diameter distance, differ from the comoving distance by factors of the scale factor, Fig. 3 implies that current observations cannot distinguish between the background evolution of quintessence and the background evolution in ΛCDM [63,64,65,66,67]. We have restricted our analysis to a osc ∼ 0.8 because choosing a osc 0.8 gives rise to unacceptably large deviations from the observed expansion history.
Perturbation evolution
In this section we investigate the dynamics of the fluctuations in the quintessence field (δϕ), the gravitational potentials, and the overdensity in WIMPs (δ dm ) assuming linearized equations of motion. We ignore radiation and neutrinos, since we are interested in late-time dynamics. We work in the Newtonian gauge, where the metric is and Φ and Ψ are the two scalar gravitational potentials. Tracking the evolution of the gravitational potentials and the two matter components (WIMPs and quintessence) requires two second order differential equations. Normally, they are taken to be the energy-momentum conservation equations for the matter components. The gravitational potentials can then be obtained from the Einstein equations (constraints). However, since the most interesting dynamics happen in the gravitational potentials and the field fluctuations, we work with them as the degrees of freedom and then obtain the overdensity in WIMPs from the constraints. The equations of motion for the quintessence field fluctuations and the gravitational potential are (in Fourier space), The second equation is the diagonal, space-space component of the Einstein equations, where the right hand side is the pressure perturbation provided by the scalar field. Since the WIMP dark matter is assumed to be pressureless, it does not contribute to this source term. The second gravitational potential Φ k does not appear in the above equations because there is no anisotropic stress at linear order for single, minimally coupled scalar fields. This makes the two gravitational potentials equal via the off diagonal space-space part of the Einstein equations. ¶ After evolving the equations for δϕ k and Ψ k , δ dm follows from the time-time components of the Einstein equations,
Initial conditions and evolution during matter domination
We are interested in the behavior of δϕ k , Ψ k and δ dm for a 0.8. To solve (18) we need to specify δϕ k , δφ k , Ψ k ,Ψ k on some initial time slice. This can be done self-consistently using the ΛCDM solutions for Ψ k and δ dm if the initial time slice is chosen deep into the matter dominated era. During matter domination, the quintessence field is a small fraction of the total energy density. As a result, the δϕ k do not contribute significantly to Ψ k , and we can use Ψ k from a ΛCDM cosmology at these early times. We take these initial conditions for Ψ k andΨ k directly from the output of CMBFAST [68] at a i ≈ 10 −2 since by this scalefactor the anisotrotropic stress from neutrinos is negligible and the contribution of dark energy is yet to become important. We are then left with specifying the initial conditions in δϕ k , which requires understanding its evolution in the matter dominated era.
The evolution of adiabatic modes on superhorizon k/aH 1 scales is given by (see [69]) The term in the square brackets is constant during matter domination On subhorizon scales (k aH), during the matter dominated era, the gravitational potential is determined by the fluctuations in the WIMP overdensity. Since Ψ k is constant during matter domination, the equations of motion (18) becomë where we have assumed U (ϕ)/H 2 (k/aH) 2 . Under the assumption that U (ϕ) is slowly varying, the complete solution is where k H ≡ k/aH and c k , θ k are constants of integration set by initial conditions at the beginning of the matter dominated era. For solutions that are deep inside the horizon, k H 1 and only one term grows with time: The initial (transient) oscillatory behavior due to the homogeneous part of the solution 24 as well as the growth ∝ a 2 for a i a a osc due to the particular solution 25 can be seen in Fig. 5(a). We have also verified that although from Eqs. (22) and (25) we see that δϕ k grows as a 3 on superhorizon scales and as a 2 on small, subhorizon scales, their amplitude during matter domination does not become large enough to change the behavior of the potential Ψ k .
With the Eqs. (22) and (25) at hand, we set the initial conditions for all k H using a piecewise interpolation between the subhorizon and superhorizon solutions (dashed curve in Fig. 6). Although we have taken care to faithfully characterize the asymptotic behavior of the initial conditions of δϕ, in practice we find that changing the initial conditions by orders of magnitude does not affect our results significantly. This is because at late times, the particular solution of δϕ k (determined by Ψ k ) dominates, significantly reducing the dependence on initial conditions.
Evolution during the quintessence dominated era
In this section, we investigate the dynamics of fluctuations during quintessence domination and find that the evolution differs before and after a osc . We analyze these two regimes separately.
Before resonance a < a osc
When 0.5 < a < a osc , the homogeneous quintessence field rolls slowly with an energy density larger than, but comparable, to the WIMP density. The behavior of δϕ k on superhorizon scales is still determined by (21), but H decreases more slowly than during the matter dominated era.
In the subhorizon regime, the quintessence perturbations grow faster as we approach a osc because the field starts rolling more rapidly (see right-hand side of Eq. (18)). However, these fluctuations are still not strong enough to prevent the decay of the gravitational potential, which is caused by a transition to a dark energy dominated epoch. During this epoch, the evolution of the Ψ k is determined by H and does not show any resonant behavior. The behavior of the δϕ k and Ψ k discussed here can be easily seen in Fig. 5(a) and (b) for 0.5 a a osc . The thin, black line represents the evolution of the same mode of Ψ k in ΛCDM.
Resonance a > a osc
We now come to the most interesting era (a > a osc ) with regards to the evolution of fluctuations. In this era, the homogeneous field begins to oscillate with an amplitude dependent frequency ω < m (see Eq. (15)). This leads to a rapid growth in the field fluctuations for certain characteristic ranges of wavenumbers. To understand this, we initially ignore expansion (H = 0, a = 1) and the gravitational perturbations (Ψ k = 0) in Eq. (18).
Resonance in Minkowski space
The equation of motion for δϕ k then becomes, Since the homogeneous field ϕ is periodic in time, U (ϕ) is periodic as well as long as U (ϕ) = constant, which occurs for potentials with anharmonic terms (as is the case with our potential in (1)). This yields an oscillator with a periodically varying frequency, whose solutions can be analyzed via standard Floquet methods (for example, see [70]). For the interested reader, we review the main aspects of Floquet analysis in the Appendix. Under certain conditions (see Appendix A), Floquet's theorem guarantees that the general solution to Eq. (26) can be written as where ±µ k are the Floquet exponents and P ± (t) are periodic functions with the same period as U (ϕ). * * The Floquet exponent µ k depends on the amplitude of the "pump" field ϕ osc , as Yellow corresponds to a larger value than orange, whereas dark red regions have (µ k ) = 0. As the Universe expands, the wavenumber of a given mode as well as the amplitude of the background field redshift. As a result, a given mode "sees" different Floquet exponents as it traverses the Floquet chart along the thin white lines. In Fig. 4(b) we show the Floquet exponent "seen" by a mode with a comoving wavenumber k ∼ 0.05m (along the left-most dashed white line in Fig. 4(a). Modes with k 0.5m pass through many resonance bands in a single oscillation of the homogeneous field and undergo stochastic resonance (see text).
well as the wavenumber k. There exists an unstable, exponentially growing solution if the real part of the Floquet exponent (µ k ) = 0. In Fig. 4(a), we show | (µ k )| as a function of the amplitude ϕ osc and wavenumber. The color represents the magnitude of the real part of the Floquet exponent. Yellow corresponds to a larger | (µ k )| than orange, whereas red corresponds to (µ k ) = 0. Without expansion, neither ϕ osc nor the momentum k redshift with time. As a result, the evolution of modes is determined by the Floquet exponent at single point in the (k, ϕ osc ) plane. This no longer holds true in an expanding universe.
Resonance in an expanding universe
The equation of motion for δϕ k in an expanding universe (still ignoring Ψ k ) is To understand parametric resonance in an expanding background, we make the following identifications: Here k p is the physical wavenumber and ϕ osc (a) is the decaying envelope of the oscillating field. This identification (29) defines a trajectory in the k p − ϕ osc plane (white lines in Fig. 4(a)). The approximate amount of amplification undergone by a given mode is obtained by integrating | (µ k )| along the corresponding trajectory in the k p − ϕ osc plane. The | [µ k (t)]| as seen along such a trajectory (left-most dashed, white line in Fig. 4(a)) is shown in Fig. 4(b). Then, schematically, the evolution envelope of the amplified modes is (ignoring the oscillatory piece P ± (t)), where in the second equality we use the scalefactor a as a time co-ordinate and γ = 3/2 when ϕ osc M . We assume that the frequency of oscillation ω H. This expression (30) is meant to give intuition about the resonant behavior in an expanding universe and should be used with care, especially when multiple bands are involved since the phase of the oscillations can play a role when different bands are traversed.
When a mode traverses only the first resonance band, the fluctuations get a boost every time the homogeneous field crosses zero. This can be thought of as a burst of particle production. When a large number of bands are traversed within a single oscillation of the homogeneous field, we enter the regime of stochastic resonance. The amplitude of the mode still changes dramatically at zero crossings of the homogeneous field, however we are no longer guaranteed growth at every such instant. Although over longer time scales the fluctuations grow, at some zero crossings they can also decrease. For our scenario stochastic resonance is seen for modes with k 0.5m. A more detailed discussion of these different regimes can be found in [71].
For our scenario, the modes that grow the fastest are the ones with k m (ones traversing the first band). To get a sense of what is required of the parameters for these modes to grow rapidly, let us now concentrate on the right most expression in (30). If the argument of the exponent is significantly larger than 1, then we will have rapid growth in fluctuations. Using a osc ≈ 0.8, resonance takes place in the logarithmic interval ∆ ln a ∼ 0.2. Hence, one gets rapid growth in fluctuations when | (µ k )|/H 10. During the oscillatory regime Fig. 4, the real part of the Floquet exponent seen by a mode in the first band has value of | (µ k )| ∼ 0.1m. Thus we need M/m Pl 10 −2 for efficient resonance. However as we saw in Sec. (3), in the class of models described by (1), consistency with the observed expansion history and the requirement of a few oscillations in the homogeneous field close to today automatically yields M/m Pl (∆a) 3/2 ≈ .03 [Eq. (12)]. Hence in such models, resonance is almost inevitable. Importantly, this is a nongravitationally driven growth, driven by the homogeneous, oscillating field ω H. Hence this growth can happen on a time scale which is significantly shorter than H −1 .
Resonance in an expanding universe including local gravity
Let us now include the gravitational potentials in the equations of motion and consider resonant phenomenon in the complete coupled system given by Eq. (18). When the field driven resonance is efficient, the gravitational potential Ψ k does not play a significant role in the field dynamics. The evolution of Ψ k , on the other hand, is affected significantly by the resonant growth in δϕ k since the quintessence field dominates the energy density. We stress that Ψ k can only be ignored in the evolution of δϕ k for modes undergoing strong resonance when the self interactions dominate over the gravitational one. In particular, as we have seen in the previous sections δϕ k just before resonance is determined by Ψ k and as a result ignoring Ψ k before resonance is not justified. In addition, the gravitational potential includes contributions from the WIMP dark matter overdensity via the constraint equations. These two considerations make the analysis of Eq. (18) in the resonance regime nontrivial and we have to rely on numerical solutions. A more detailed analytical analysis of resonance in an expanding universe including the effects of the gravitational potential will be pursued elsewhere. Below we discuss the numerical solutions during resonance in a bit more detail.
Typical, rapidly growing solutions for δϕ k and Ψ k with k ≈ 0.05m are shown in Fig. 5(a) and (b), respectively. Note the rapid, nearly exponential growth of Ψ k and δϕ k after a osc ≈ 0.8 (vertical, dashed line). The thin, black lines show the evolution of the same modes in ΛCDM. The growth in the gravitational potential is somewhat delayed compared to the field. This is because the energy density in the field has to first become comparable to that of dark matter. Before this happens, the gravitational potential does not experience scale dependent growth (though it still responds to the changing expansion history). Figure 5: (a) Evolution of δϕ k with k ∼ 0.05m ≈ 0.01Mpc −1 . During matter domination, δϕ k ∝ a 2 and grows relatively slowly. However, after a osc , the mode grows exponentially fast due to parametric resonance. At a nl the fluctuations become nonlinear and linear evolution is no longer applicable. (b) Evolution of the scalar gravitational perturbation (k ∼ 0.05m). Before resonance, the evolution is similar to that expected in usual slow roll quintessence (or ΛCDM, thin black line) model. It is constant deep in the matter dominated era and starts decaying as quintessence takes over. Unlike slow roll quintessence, after a osc the potential grows rapidly until a nl . This leads to a scale dependent signal in observations that are sensitive to the gravitational potential. (c) The evolution of the WIMP overdensity is not significantly affected by the resonant growth. The departure from ΛCDM in this case is almost entirely due to the slight deviations in the expansion history. The normalization is set to one for all the above modes at a = a i .
So far we have ignored discussing the evolution of the WIMP overdensity since it is determined from the constraint Eq. (20). Naively one might expect to see scale dependent departures in the behavior of δ dm . However, despite the rapid growth in the gravitational potential after a osc , δ dm does not deviate significantly from its ΛCDM counterpart (see 5(c)) in the linear regime. Although difficult to see from the constraint Eq. (20), this behavior can be understood by considering the conservation equation for the WIMPs, Heuristically, we see that on subhorizon scales, δ dm is obtained from a double time integral of the potential Ψ k . This delays the response of the δ dm to the Ψ k . The small departure of δ dm from its ΛCDM counterpart can be accounted for by the difference in expansion history between the quintessence and the ΛCDM models. In particular, since the Hubble parameter in our quintessence model is always slightly larger than its ΛCDM counterpart (see Sec. 3), the growth of δ dm in the quintessence model is slightly suppressed.
From the above discussion we see that the linearized fluctuations in the field and the gravitational potential grow rapidly. Eventually, the field fluctuations will become nonlinear and we cannot trust the linearized treatment. Hence it becomes important to understand, at least qualitatively, when the field becomes nonlinear as well as what happens thereafter. Although we discuss the nonlinearity of the scalar field fluctuations, we do not include the usual nonlinearity in δ dm at late times. This nonlinearity in δ dm is of course well studied, but to include it would take us too far beyond the scope of this paper.
Nonlinearity and oscillon formation
We cannot ignore the nonlinearity of the fluctuations when U (ϕ) ∼ U (ϕ)δϕ. In the oscillatory regime this happen at the scale factor a nl when ∆ δϕ (k, a nl ) ∼ ϕ osc (a nl ).
In Fig. 6(b) the orange curves show the evolution of the (absolute value of) oscillatory homogeneous field and its envelope. The black curves show the maximum value of ∆ δϕ (k, a) as a function of a and its envelope. The location where the two curves intersect a nl ≈ 0.95 is taken as the point beyond which the linearized equations cannot be trusted. In Fig. 6(a) we show ∆ δϕ (k, a nl ) (solid, orange curve) and ∆ δϕ (k, a i ). As mentioned in Sec. 4.1, the normalization of ∆ δϕ (k, a i ) is set by ∆ Ψ (k, a i ), which we take from WMAP 7 [63](∆ 2 R = (9/25)∆ 2 Ψ = 2.42 × 10 −9 at k = 0.002 Mpc −1 and n s = 0.966). We also note that although k ∼ 0.05m ≈ 0.01 Mpc −1 is where we see the maximum deviation, resonance significantly enhances fluctuations for larger k as well, albeit through stochastic resonance. In order to calculate the effect of the rapid growth of the gravitational potential on observables, we need its evolution until today, ie. a ≈ 1. However, as discussed above, the nonlinearities of the field do not allow us to compute it for a > a nl = 0.95. To remedy this, for the linearized calculation, we take a conservative approach and "freeze" the value of the potential at a nl , setting Ψ k (a) = Ψ k (a nl ) for a nl ≤ a ≤ 1. More realistically, the potential will evolve further in a scale-dependent manner as the scalar field perturbations undergo nonlinear evolution. Fragmentation of the scalar field [discussed below] will enhance the perturbations, at least on smaller scales [we provide some simple estimates of the ISW effect due to this nonlinear evolution in Appendix B]. Along with the break-down of linearized equations, we also note that after a nl , large gradients in the scale field fluctuations will source anisotropic stress, violating our assumption of Ψ = Φ.
Although in this paper we do not pursue nonlinear evolution of the scalar field fluctuations, below we digress slightly and point out some of the interesting phenomenology that results. As nonlinearity sets in, the evolution of different k modes becomes coupled, and back-reaction from the perturbations curtails the resonant growth of fluctuations. The homogeneous field then fragments rapidly. For the type of potentials considered here (those having a quadratic minimum and a shallower than quadratic form away from the minimum), most of the energy density can eventually end up in localized, oscillatory, long-lived configurations of the field called oscillons (e.g. [16,18,29]). The central density of these oscillons can be greater than the background energy density and their sizes are of order a few m −1 ≈ 10 −3 H −1 0 ≈ 4 Mpc. Although oscillons radiate energy through scalar radiation (e.g. [17,72,73,30]), our limited radial simulations of individual oscillons reveal a lifetime of ∼ 10 6 m −1 ∼ 10 3 H −1 0 for an oscillon with width of a few m −1 and field amplitude of order M . This makes them effectively stable compared to current cosmological time scales. Unlike the usual oscillons (e.g. [16,18,73,29]), which have a relatively stationary energy density profile, the oscillons in our model have an energy density that breathes in and out (also see [15]) at about twice the field oscillation frequency.
This phenomenon of oscillon production has been studied in the context of the early Universe, in particular, at the end of a similar scalar field driven inflationary period (e.g. [32,10,9,15]). Here, we point out that it will likely happen at the end of the current period of cosmic acceleration as well. As with the early Universe studies, we expect the oscillons to dominate the energy density. However, unlike the early Universe, these oscillons have sizes which make them astrophysically accessible with novel signatures such as the existence of dark, low redshift clusters with time dependent configurations made of the quintessence field. We stress that it is not guaranteed that the Universe will become oscillon dominated before today. Although for the fiducial parameters, the field fluctuations will become nonlinear before today, it can take some time for the nonlinear field to enter a state where it is dominated by oscillons. This time can be longer than the time between a nl and today. Quantifying this process, requires simulating the full nonlinear dynamics of the quintessence field, including nonlinearities of the WIMP overdensity, which is beyond the scope of this paper. Given the similarity of the potential with [15], we expect many of the qualitative results to carry over. However, to perform a reliable calculation to be compared with observations, one cannot completely ignore the gravitational perturbations, which makes the simulations somewhat more challenging.
Power spectra and observables
With our understanding of the evolution of individual modes and the validity of the linearized equations, we now compute the power spectra of the gravitational potential ∆ 2 Ψ (k, a) ≡ k 3 P Ψ (k, a)/2π 2 and the WIMP overdensity P δ dm (k, a) and their impact on observables such as galaxy clustering, lensing and the CMB.
The calculated power spectra ∆ 2 Ψ (k, a nl ) and P δ dm (k, a nl ) (with a nl = 0.95) are shown in Fig. 7 (thick, orange lines). The thin, black lines show the corresponding power spectra for the ΛCDM cosmology. The gray, dashed lines show the power spectra at a i = 10 −2 . Note The difference from the ΛCDM counterpart (black line) is primarily due to the difference in expansion history between the quintessence and ΛCDM models. The dashed line represents 10 3 × P dm (k, a i ). Note that since we are using δ dm in the Newtonian gauge, P δ dm (k, a i ) shows an "upward turn" at small k values.
that since we are using δ dm in the Newtonian gauge, P δ dm (k, a i ) shows an "upward turn" at small k values. By construction, the initial conditions in the power spectra ∆ Ψ (k, a i ) and P dm (k, a i ) in our quintessence model and ΛCDM agree. The scale dependent departures in ∆ Ψ (k, a) arise after a osc ≈ 0.8. As discussed previously, the enhancement can be trusted until a nl = 0.95, after which we freeze the potential until a = 1. The wavenumber where the maximum departures are seen is k ∼ 0.05m ≈ 0.01 Mpc −1 . For k H 0 and k m, the final spectrum closely resembles the ΛCDM case. For fixed a osc , we have checked that the magnitude of departure from ΛCDM increases with increasing m. This is consistent with the notion larger m corresponds to a larger m Pl /M [see Eq. (10)] leading to more efficient resonance. As discussed for single modes, the potential ∆ Ψ (k, a) show dramatic, scale dependent departures from ΛCDM, but the WIMP overdensity power spectrum does not.
Assuming a model for the bias, the WIMP power spectrum can be probed by measuring the two point correlation function of galaxies. Given the lack of scale dependence, and the small difference ( 6%) with ΛCDM, we expect the departures from ΛCDM to be difficult to detect. † † The fluctuations in quintessence are not directly observable except through their gravitational imprint. This leaves us with the gravitational potential as the key probe to look for departures from ΛCDM. The rapidly changing potential in the quintessence model and the large scale dependent departures at low redshifts could affect the low multipoles of the CMB temperature and weak lensing power spectrum. With this in mind, we now calculate the weak lensing (convergence) power spectra and the CMB temperature anisotropy. We remind the reader that the differences that arise here are in spite of having made the expansion history consistent with current observations. An expansion history and galaxy power spectrum consistent with ΛCDM but scale dependent deviations in the lensing and CMB power spectra provides a unique probe of a late time transition in the quintessence field.
Integrated Sachs-Wolfe effect
When CMB photons travel through an evolving gravitational potential their energy changes (the integrated Sachs-Wolfe (ISW) effect [74]). Since the rapid growth in the quintessence fluctuations induces large changes in the potential, we expect imprints of the late-time quintessence transition on the CMB power spectrum through the ISW effect.
The angular power spectrum of the CMB temperature can be written as [75] where ∆ 2 Ψ (k)| prim is the primordial power spectrum. In the above, D l (k) ≡ ∆ l (k)/Ψ prim k , where ∆ l (k) on large angular scales (under the assumptions of adiabatic initial conditions and instantaneous recombination) is given by, In the above expression, the subscript "e" stands for emission (ie. surface of last scattering), χ a = 1 a da/a 2 H(a) is the comoving distance, and j l (x) are spherical Bessel functions. The second term is the ISW term, which gets large if the potential evolves significantly between last scattering and today. In ΛCDM the ISW term gets a small contribution just after recombination since we are not quite matter dominated at that time. But, the main contribution on the scales of interest comes from late times as the universe starts becoming Λ dominated and the potentials start decaying. The same effect is also present in the quintessence model. However, there is an additional contribution from the late-time rapid growth of the potential. This growth occurs in spurts [see Fig. 5(b)] and we can approximate ∂ a Ψ k as a Dirac-delta function at the location of the jumps in the gravitational potential. As a result, the contribution of each jump to the ISW term can be easily evaluated: where a j is the scale factor where the potential jumps. For a jump of order a few Ψ k , the "jump" term is larger than the smooth ISW contribution in ΛCDM. Note that there can be multiple jumps, both positive and negative, depending on the k mode in question. Also note that apart from the magnitude of the jump, the location a j also plays a role by fixing the argument of the Bessel function.
We expect that this growth affects the low multipole moments of the CMB since the changes in the potential occur very recently. More specifically, the multipole range where we expect deviations is l kχ a j , where k ∼ 0.05m ≈ 0.01Mpc −1 . For the fiducial parameters chosen here, this yields l few. Note that along with k, it is the smallness of χ a j that limits the effect to large angular scales.
In order to quantitatively calculate the effect of the quintessence perturbations on the CMB power spectrum, we use the Ψ k (a) and Φ k (a) evaluated from CMBFAST [68] for a < a i = 0.01. This captures the contributions from early ISW as well as early anisotropic stress (which we ignore at late times). For a > a i we use Ψ k (a) computed from our own independent code for ΛCDM and the quintessence model. As discussed before, for the quintessence case we set ∂ a Ψ k = 0 for a > a nl . With this entire solution at hand (for a e < a < 1) we compute the ∆ l (k) and C l . The ratio of the angular CMB power spectra for the quintessence and ΛCDM models is shown in Fig. 8(a). Note that this is large enough to potentially rule out this choice of parameters. Thus, we see that in spite of the effect being on large angular scales, ISW provides an excellent probe for constraining the considered transition in quintessence. One can change parameters, for example a osc or m (equivalently M ) to get the ISW effect small enough so that it is consistent with observations. One has to first make sure that such changes are still consistent with the expansion history (see discussion in Sec. 3). Increasing m or a osc or varying m and a osc in opposite directions can lead to an expansion history consistent with observations. Let us now look at perturbations. In general, for a fixed a osc , as we increase m (equivalently decrease M ), the fluctuations grow more rapidly, shifting the time when the potentials grow rapidly to smaller a values. One might expect that this will make the ISW contribution even larger. However, one finds that the effect on ∆ l (k) is not quite as simple. First the change in growth of potential is quite sensitive to the number of zero crossing of the homogeneous field. In addition, changing parameters changes the wavenumbers, which grow the fastest and the time when there is a rapid growth in the potential. Heuristically, this affects the argument of the kχ of the nonmonotonic spherical Bessel function in ∆ l (k). Similar considerations apply to changing a osc . As a result, it is somewhat difficult to apriori predict which combination of parameters (consistent with the observed expansion history) will also yield an acceptable ISW term. Indeed we find that m ≈ 2 × 10 3 H 0 and a osc ≈ 0.82 is entirely consistent with observations of the expansion history as well as the CMB. We also find regions of parameter space with smaller values of m but larger values of a osc compared to the fiducial model consistent with Figure 8: (a) Ratio of CMB temperature power spectrum at low multipoles between our quintessence and ΛCDM models. The large difference is due to the rapidly evolving gravitational potential in the quintessence model after a osc (b) Ratio of the lensing (convergence) power spectrum at low multipoles between our quintessence and ΛCDM models. The departures are restricted to low l multipoles due to the proximity of the transition in the quintessence field responsible for the rapid growth in the gravitational potential. For lensing, the model does not agree with ΛCDM at large l due to the different expansion histories. observations. A full sweep of parameters (including α) to determine regions consistent with observations is beyond the scope of this paper, but is certainly worth pursuing.
Before we move on to the calculation of the lensing power spectrum, let us briefly comment on the additional ISW that can result from nonlinear field evolution after a nl , ignored so far in the linearized treatment. As noted in Sec. 4.2.3, the rapid growth of the linearized scalar field perturbations is curtailed once the field perturbations become nonlinear (around a nl ). However, there will be further nonlinear evolution and rapid fragmentation of the field. To get an estimate of the resulting ISW effect, consider a spherical perturbation with mass M and radius R, collapsing at speed v. Such a perturbation yields a temperature decrement of order ∼ GMv/R. For a sphere with an initial radius R nl ∼ k −1 nl (with k nl ∼ 0.05m), an initial density comparable to the background energy density and v ∼ 0.05 (the approximate group velocity of a perturbation with wavenumber k nl ), the decrement can be ∼ few × 10 −5 for our fiducial set of parameters (m ∼ 10 3 H 0 ). The signal is qualitatively similar to the Sunyaev-Zel'dovich temperature decrement from galaxy clusters [76]. However, unlike arcmin angular scale of the SZ decrement, here, the angular scale is ∼ 30 degrees. This simple estimate shows that it might be possible to get additional constraints on the quintessence transition from the ISW effect due to the nonlinear field evolution. Individual oscillons, modeled by density configurations with time dependent radii ∼ few × m −1 can also yield an additional ISW signal on smaller scales. We discuss a toy model for estimating the ISW signal from such nonlinear perturbations in Appendix B. However, we again caution the reader that these numbers should be checked with input from detailed simulations of the nonlinear field dynamics including gravity.
Weak lensing
Observational constraints from weak lensing observations are often presented in terms of the weak lensing convergence (angular) power spectrum [77,78], where: is the power spectrum of the gravitational potential and η(χ) is the radial distribution of sources, normalized to η(χ)dχ = 1. We use the source distribution, with z med = 1.26. This distribution approximates the galaxy redshift distribution of the COSMOS survey [79]. As with the ISW effect, because of the closeness of the transition to the present day, we expect the signal to be largest at low multipoles l kχ(a nl ) ∼ few. The proximity of the transition also picks out sources at approximately 2χ(a nl ). As a result as a nl → 1(χ → 0), we run out of sources to be lensed. Thus in general, for a fixed enhancement of the gravitational potential, we expect the largest lensing signal to arise from the smallest a nl still consistent with the expansion history.
The ratio between the weak lensing convergence power spectrum for the quintessence model and ΛCDM is shown in Fig. 8(b). The errors associated with measuring the convergence power spectrum is given by [77]: where f sky is the fraction of the sky covered by the survey,n is the measured galaxy number density, and γ 2 int 1/2 ≈ .4 is the galaxy intrinsic rms shear in one component. Using survey parameters characteristic of the Large Synoptic Survey Telescope (LSST) (f sky ≈ .48,n ≈ 5.9 × 10 8 sr −1 ) [80], we find C κ l /C κ l ≈ {.9, .26} for l = {2, 30}. Hence this particular quintessence model cannot be constrained using LSST measurements of the weak lensing power spectrum. The weak lensing convergence power spectrum for the quintessence model does not asymptote to the ΛCDM prediction at higher l because there is a difference in expansion history between the two models. Even this deviation would be difficult to see using LSST since the minimum C κ l /C κ l ≈ 0.1 at l ∼ 300. Before moving on to our conclusions, we note that there are qualitative degeneracies between observational signatures (in particular, ISW at low multipoles) predicted by our scenario and those of other models, for example, interacting dark energy models with significant clustering (see [60,81,82,83]).
Conclusions
For a slowly rolling quintessence field, it is natural (though not necessary) that the field will eventually start oscillating at it approaches a minimum in its potential. In this paper we have analyzed the consequences on the expansion history and structure formation of a quintessence field, which initially behaves like dark energy and starts oscillating around the minimum in its potential at late times (a 0.8). The potentials considered in detail here have a quadratic minimum and are shallower than quadratic away from the minimum. When a spatially homogeneous scalar field (ϕ) oscillates about the minimum of such an anharmonic potential, it can pump energy into its spatially inhomogeneous perturbations (δϕ) through parametric resonance. The amount of energy transfer depends on both the wavenumber of δϕ as well as the number of oscillations that take place in the background field. This leads to a rapid fragmentation of the homogeneous field and rapid resonant growth of the field fluctuations on time scales significantly shorter than H −1 0 . In order to avoid discrepancies with the measured expansion history, and to simultaneously produce oscillations in the field, we have given a prescription for setting the initial conditions of the quintessence field and parameters in the potential. We have also explicitly shown that the potentials must be close to constant during the phase where the field is slowly rolling. Potentials that do not satisfy this requirement have too much of a delay between the end of slow roll and the beginning of oscillations, thus avoiding the rapid resonant growth of structure. Note that current data is consistent with a cosmological constant. Our model is in no way more natural than a cosmological constant or other dark energy models. However, we believe that the model's interesting phenomenology and potentially observable consequences warrants its study. Given a model that produces a background expansion history in good agreement with the measured expansion history, we have shown how the gravitational potential and the overdensity in WIMPs is affected by the resonant growth of the field fluctuations. We found that the metric perturbations develop scale-dependent growth, with the scale set by the mass of the scalar field potential m ∼ U (ϕ → 0) ∼ 10 3 H 0 (k 0.1m). On the other hand, the dark matter overdensity remains featureless and very similar to the ΛCDM solution aside from an overall slight suppression because of the small difference in expansion history between ΛCDM and the quintessence model. This is because, the dark matter does not have time to respond to the changing potential. Note that scale dependent changes in the gravitational potential are normally attributed to modified gravity (e.g. [84,85,86]). Here, however, we have shown that such a change can occur in general relativity with a minimally coupled quintessence field with a canonical kinetic term. Thus, if future observations find evidence for scale-dependent growth, it cannot be attributed to modified gravity.
The rapid growth of the potential significantly affects the ISW contribution to the temperature angular power spectrum of the CMB. For a range of parameters this yields the strongest constraint on such quintessence transitions. Since the metric perturbation develops a scale dependent change, the weak lensing power spectrum also offers a possible way to constrain this quintessence scenario, where dark energy undergoes a late-time transition described above. Unfortunately, because of the proximity of the decay in dark energy, deviations from ΛCDM occur at low l multipole moments. A full treatment, which includes nonlinearities in the quintessence field, however, could give rise to deviations in the weak lensing and temperature power spectrum, at larger l values.
The nonlinear dynamics of the quintessence field would give rise to a wealth of new phenomenon including field fragmentation and possible formation of localized scalar field lumps which could provide additional observational constraints. The full nonlinear analysis, combing N-body simulations for the dark matter and lattice simulations for the scalar field, is beyond the scope of this paper, but provides a promising avenue to explore quintessence transitions and their consequences further. Recall that in spite of its strong clustering properties, our quintessence field is minimally coupled with a canonical kinetic term and does not have nongravitational couplings to WIMP dark matter. This could make such models easier to simulate than models where gravity is modified (e.g. [87,88,89,90,91]), interacting dark energy models (e.g. [92,93,94,83]), nonminimally coupled quintessence (e.g. [95,96]) or when nonstandard kinetic terms are present.
In summary, if dark energy changes its nature close enough to the present time, it is possible to miss it in the expansion history measurements. For the models considered here, we have demonstrated that such a transition can dramatically change the gravitational potential power spectrum in a scale dependent way but leave the galaxy clustering unaffected, thus providing a possibly unique signature of such a transition. The best constraints on such transitions likely come from the ISW effect, followed by lensing. We expect, a more involved analysis, will provide additional constraints on such a transition once we include: (i) the nonlinear collapse in WIMPs and in the quintessence field, which will increase power on smaller scales; (ii) couplings to other fields (ignored here). It would also be interesting to explore similar resonant growth in models with multiple ultralight scalar fields (not necessarily dark energy) motivated in [97] and studied in more detail in the context of structure formation by [98].
Acknowledgements
We would like to thank Alan Guth, David Shirokoff, Raphael Flauger, Richard Easther, Andrew Liddle, Volker Springel, Robert Crittenden, Marco Bruni, Ignacy Sawicki, Jonathan Pritchard, Navin Sivanandam and Charles Shapiro for useful conversations. We would especially like to thank Mark Hertzberg for comments on an early draft of this work and Antony Lewis for repeated and prompt help when we were trying to check our ISW calculation using CAMB. We thank the anonymous referee for a number of constructive comments.
Appendix A Floquet's theorem and calculating Floquet exponents
The linearized equations of motion for the fluctuations, neglecting the Hubble expansion and metric perturbation, are (in Fourier space) where k is the wavenumber. Since the homogeneous field ϕ is oscillating, U (ϕ) is periodic in time. This results in a linear system with periodic coefficients that can be analyzed with Floquet theory. Floquet's theorem is most elegantly written in matrix form. Converting our second order equation of motion into a first order matrix equation, we find, where and Before stating Floquet's theorem, we need one more definition. The fundamental matrix solution O(t, t 0 ), of Eq. (42) satisfies The fundamental matrix solution evolves the initial conditions x(t 0 ) in time: Explicitly, O(t, t 0 ) consists of two columns that represent two independent solutions x 1 , x 2 , which satisfy x 1 (t 0 ) = (1, 0) and x 2 (t 0 ) = (0, 1). Note that det O(t, t 0 ) is the Wronskian, det O(t 0 , t 0 ) = 1, and there are no "friction" terms. Hence by Abel's Identity we have det O(t, t 0 ) = 1.
We are now ready to state Floquet's theorem (without proof): where x is a column vector and E is a real, 2 × 2 matrix satisfying where P (t+T, t 0 ) = P (t, t 0 ) and M(t Since det O(t 0 + T, t 0 ) = 1, we have µ 1 + µ 2 = 0. Suppose, from now on, that µ 1 = −µ 2 = µ. If M(t 0 ) has two linearly independent eigenvectors e ± (t 0 ) corresponding to ±µ (including µ = 0), then the general solution can be written as where P ± (t, t 0 ) = P (t, t 0 )e ± (t 0 ) are periodic column vectors. If there exists only one eigenvector corresponding to the repeated eigenvalue µ = 0, then the general solution becomes where P g (t, t 0 ) = P (t, t 0 )e g (t 0 ) with e g (t 0 ) is a generalized eigenvector.
Calculating the Floquet exponents explicitly for the problem at hand reduces to the following steps: 1. First we calculate the period T of U . The period of U (t) depends on the initial amplitude of the homogeneous fieldφ(t 0 ) (assuming ∂ tφ (t 0 ) = 0) and is given by In practice, we specify eitherφ max orφ min . The other is found by solving V (φ min ) = V (φ max ). For V (ϕ) = V (−ϕ) we haveφ max =φ min .
2. Next we solve ∂ t O(t, t 0 ) = E(t)O(t, t 0 ) from t 0 to t 0 + T to obtain, k (t 0 ) = 0, ∂ t δϕ (2) k (t 0 ) = 1}. This is equivalent to solving ∂ 2 t ϕ k + [k 2 + V (φ)] δϕ k = 0 for the above two sets of initial conditions from t 0 to t 0 + T . We have suppressed the dependence of T onφ max to reduce clutter.
3. Last, we find the eigenvalues of O(t 0 + T, t 0 ). They are where all quantities are evaluated at t 0 + T . Note that since the δϕ k (t 0 + T ) depends on k, the eigenvalues also depend on k. The Floquet exponents ±µ are then given by 9 Appendix B ISW effect from nonlinear dynamics of the quintessence field In the main body of the text, we discussed how the quintessence field fluctuations grow rapidly during the oscillatory regime. This rapid growth leads to a scale dependent growth in the gravitational potential which in turn leads to large changes in the CMB temperature anisotropies via the integrated Sachs-Wolfe effect (see Sec. 5.1). However, after a nl ≈ 0.95 the field perturbations become nonlinear and the homogeneous field fragments rapidly, potentially forming long-lived, localized excitations of the field called oscillons. Our linear analysis did not include the ISW effect resulting from the nonlinear evolution of the field perturbations. In this Appendix, we remedy this by estimating the change in the CMB temperature due to the following: • The change in the gravitational potential from the initial quasi/nonlinear evolution of the field perturbations.
• The time varying gravitational potential due to an isolated oscillon after it is formed.
Let us first look at the ISW effect from the initial evolution of quasi/nonlinear perturbations. A realistic calculation requires input from detailed simulations of the nonlinear field dynamics (for example, [15]). Here we concentrate on a related toy problem of calculating the ISW effect due to the collapse of a single, spherical top-hat overdensity. This represents a crude approximation to the initial rapid quasi/nonlinear evolution of density perturbations associated with our model of quintessence. Later, we will evaluate the CMB temperature anisotropies from the ISW effect due to a stable oscillon like configuration. Since the Universe expands very little between a nl ≈ 0.95 and a = 1, we will work in a Minkowski background. Our main purpose is to obtain an order of magnitude estimate and understand how the effect depends on aspects of nonlinear evolution.
Consider a spherical mass M with uniform density and a time dependent radius R(t) located at x c . The gravitational potential due to this mass is given by The partial time derivative of this potential is where u = R 2 (t) − |x − x c | 2 and Θ is the Heaviside function. The change in the CMB temperature due to a time varying potential (ISW term) is where n is a unit vector that defines observer's line of sight, t 0 is the time since last scattering ≈ 0.99H −1 0 , and we replace x by (t 0 −t)n since light travels on null geodesics. We have ignored anisotropic stress here (Ψ = Φ). Note that the time of integration starts at t nl ≈ 0.94H −1 0 (corresponding to a nl ≈ 0.95) since nonlinear perturbations do not exist beforehand.
where 0 < v < 1. This yields where t c = t 0 − x c · n = t 0 − |x c | cos θ, R c = R(t c ), r c = R 2 c − (1 − v 2 )|x c | 2 sin 2 θ and θ is the angle between n and x c . Note that t − and t + denote the time a light ray enters and exist the spherical mass. With these definitions at hand, the integral over time in Eq. (53) can be evaluated analytically. In particular, if t nl < t − < t + < t 0 (and R c > 0) we find Recalling that r c and R c depend on the angle θ, the above expression shows that we should expect a cold-spot in the CMB, with an angular size ∼ R c /|x c |. The anisotropy is negative and maximal when θ = 0 when the light ray passes through the center of the mass distribution, ∆T (n) The result depends only on the size of the sphere R c when the light passes through its center, the speed of collapse v and the mass M. To obtain an order of magnitude estimate, we use our ducial set of parameters. We choose an initial perturbation of size R nl ∼ k −1 nl ≈ (0.05m) −1 ≈ 0.02H −1 0 (see section 4.2.2 and 4.2.3), with density of order the critical density. We put the perturbation a distance |x c | = 0.03H −1 0 from us (corresponding ∼ (t 0 − t nl )/2) and impose that it collapses with v ∼ 0.05 (the approximate group velocity of a perturbation with wavenumber k nl ). We get which could provide additional constraints on our models. This signal is qualitatively similar to the Sunyaev-Zel'dovich temperature decrement from galaxy clusters [76]. However unlike arcmin angular scale of the SZ decrement, here, the angular scale is ∼ 30 degrees. As can be checked, the conditions t nl < t − < t + < t 0 and R c > 0 are satisfied for the above mentioned parameters. We remind the reader that this is merely an estimate based on a spherical top-hat collapse. The actual nonlinear field evolution can be more complicated. In addition, the amplitude of the effect depends on the parameters in a nontrivial manner. Nevertheless, this exercise shows that it is indeed interesting to pursue the nonlinear evolution of the scalar field perturbations in detail.
Let us now consider the ISW effect resulting from the time varying potential associated with a single oscillon. Based on our numerical solutions (radial only), oscillons with a central field amplitude of order M ‡ ‡ and width of order few m −1 , have a peak energy density of order m 2 M 2 and an energy density profile that breathes in and out. This is in contrast with a fixed energy density profile often used in the oscillon literature. For an oscillon, crudely approximated by the a top-hat density configuration, we take where for our fiducial model m ∼ 10 3 H 0 . We stress that the actual energy density profile of oscillons in our model is more complicated. The energy density profile resembles a Gaussian when the central field amplitude is at its maximum, but becomes flatter when the field amplitude passes through zero. As a result, a calculation based on realistic oscillon profiles could be somewhat different from the estimates below.
Unlike the linear collapse case, we did not find a simple analytic expression for the ISW contribution from individual oscillons. Evaluating it numerically, we find that for our fiducial set of parameters and assuming that the energy density within oscillons ρ ∼ m 2 M 2 ∼ 3H 2 0 m 2 Pl , we get ∆T (n) T | max ∼ ±GM osc m × 10 −2 ∼ ±10 −7 .
We have assumed that the oscillon is located at |x c | ∼ 0.03H −1 0 . The shape of the anisotropy pattern is ringlike. The phase of the radial oscillation φ determines whether the center has a temperature decrement or increment. Note that the radius associated with an individual oscillon is small compared to the quasilinear perturbations considered earlier in this Appendix (by an order of magnitude). As a result, one expects a smaller effect if one assumes that their density is still comparable to the average cosmological density. In addition, since the light crossing time is comparable to the oscillatory time scale of the energy density configuration, the ISW term undergoes cancellations, leading to a somewhat smaller ISW effect than what would be expected on purely dimensional grounds. Thus we expect the ISW contribution from individual oscillons to be smaller (and more localized) than that from the rapid collapse of nonlinear perturbations.
Before we end this Appendix, we would like to comment on a caveat regarding the emergence of oscillons. Our linear analysis in the main body of the paper revealed that perturbations become nonlinear at a nl . While the nonlinear perturbations will form oscillons eventually, it is worth asking whether we can see them as large overdensities with individual identities by today. To unambiguously understand the timescales associated with emergence of oscillons would require a full lattice simulation which is beyond the scope of this current paper. Our analysis here indicates that it is certainly worth exploring this further. While here, we have concentrated on isolated inhomogeneities, it would be interesting to look at the combined effect of a collection of such inhomogeneities (with a number density of order (k nl /2π) 3 ) [9], which would be closer to the actual scenario. | 2012-08-23T23:38:44.000Z | 2011-08-08T00:00:00.000 | {
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17261945 | pes2o/s2orc | v3-fos-license | Association of Bone Loss with the Upregulation of Survival-Related Genes and Concomitant Downregulation of Mammalian Target of Rapamycin and Osteoblast Differentiation-Related Genes in the Peripheral Blood of Late Postmenopausal Osteoporotic Women
We aimed to identify bone related markers in the peripheral blood of osteoporotic (OP) patients that pointed toward molecular mechanisms underlying late postmenopausal bone loss. Whole blood from 22 late postmenopausal OP patients and 26 healthy subjects was examined. Bone mineral density (BMD) was measured by DXA. Protein levels of p70-S6K, p21, MMP-9, TGFβ1, and caspase-3 were quantified by ELISA. Gene expression was measured using real-time RT-PCR. OP registered by low BMD indices in late postmenopausal patients was associated with a significant upregulation of autophagy protein ULK1, cyclin-dependent kinase inhibitor p21, and metalloproteinase MMP-9 gene expression in the blood compared to the healthy controls and in a significant downregulation of mTOR (mammalian target of rapamycin), RUNX2, and ALPL gene expression, while expression of cathepsin K, caspase-3, transforming growth factor (TGF) β1, interleukin- (IL-) 1β, and tumor necrosis factor α (TNFα) was not significantly affected. We also observed a positive correlation between TGFβ1 and RUNX2 expression and BMD at femoral sites in these patients. Therefore, bone loss in late postmenopausal OP patients is associated with a significant upregulation of survival-related genes (ULK1 and p21) and MMP-9, as well as the downregulation of mTOR and osteoblast differentiation-related genes (RUNX2 and ALPL) in the peripheral blood compared to the healthy controls.
Introduction
Osteoporosis (OP) is characterized by a low bone mass, poor bone quality, and an increased propensity to fracture. Osteoporotic fractures, which are often associated with decreased bone mineral density (BMD), are a major public health problem in the aging population [1]. However, the molecular mechanisms underlying OP related bone destruction are not completely understood at the present time. Recently it has been suggested that these mechanisms involve altered osteoblast and osteoclast differentiation and function [2].
Excessive bone degradation in OP is thought to result from an increased activity of resorptive osteoclasts, which express proteases such as metalloproteinase (MMP-9) and cathepsin K. Cathepsin K, a papain-like cysteine protease, is considered a key player in the process of bone resorption [3,4]. Osteoclast differentiation and function are regulated by the tumor necrosis factor (TNF ), receptor activator of nuclear factor kappa B ligand (RANKL), and macrophage colony-stimulating factor (M-CSF). These three cytokines may promote osteoclast survival by signaling through mammalian target of rapamycin (mTOR) as a common target [5]. mTOR is recognized as an evolutionarily conserved central coordinator of fundamental biological processes involving cell cycle progression, translational control, ribosomal biogenesis, transcription control, and autophagy [6]. Indeed, recent animal studies have shown that mTOR is capable of regulating osteoclastogenesis, while the downregulation of mTOR reduced osteoclast production and activity [7,8].
Differentiation-related phenotypic alterations in osteoblasts include changes in specific gene expression and corresponding protein synthesis during the course of proliferation, extracellular matrix maturation, and mineralization [9,10]. Initially, actively proliferating cells express cell cycle and cell growth regulating genes such as Runt-related transcription factor (RUNX) 2, an osteoblast transcriptional regulator, which also controls the expression of major bone matrix protein genes [11]. Osteoblast maturation is associated with production of the bone matrix from regularly and densely packed collagen fibrils and high mineralization. At this stage, the mechanical properties and composition of the bone matrix are regulated by transforming growth factor (TGF) 1 [12]. A decline in proliferative activity is accompanied by the enhanced expression of the CDK inhibitor p21(CIP1/WAF1) (p21) and alkaline phosphatase [9,13]. As a caspase-3 substrate, physiological amounts of p21 are responsible for the apoptotic activity observed in many cell lines, including osteoblasts [14].
Alterations in mTOR signaling may also be involved in osteoblast phenotypic conversions [15,16]. It has been shown that upregulation of mTOR signaling was associated with an increase in BMP2, RUNX2, and TGF expression in preosteoblasts [17]. In contrast, mTOR downregulation by rapamycin inhibited osteoblast proliferation and differentiation and reduced RUNX2, sialoprotein, and osterix gene expression, alkaline phosphatase activity, and mineralization capacity [18].
Furthermore, the downregulation of mTOR is accompanied by increased autophagy in many cell types [19]. Autophagy is a physiological cellular mechanism that degrades and recycles proteins to maintain an adequate amino acid level for survival purposes. It involves the formation of cytosolic double membrane vesicles (autophagosomes) associated with the upregulation of ULK (hATG)1-15 gene expression. Hyperautophagic conditions are capable of promoting caspase-dependent apoptotic cell death [20]. Recently, the importance of autophagy in OP development and progression was described [21,22]. In particular, the regulation of autophagy (ROA) pathway was shown to be significantly associated with wrist and arm BMD [23]. Moreover, autophagic proteins are important for the generation of the osteoclast ruffled border, their secretory function, and bone resorption [24].
The interplay of anabolic and catabolic factors, which are involved in major metabolic pathways and are associated with osteoporotic bone loss, is currently unclear. Clinical studies might help clarify this issue. However, bone specimens from postmenopausal OP patients are largely unavailable. On the other hand, it is well established that the number of genes simultaneously expressed in various cell types is higher in developmentally related tissues. As immune and bone cells originate from the mesoderm and the formation of bone and adaptive immune systems are phylogenetically closely related, these systems might involve identical regulatory cytokines and growth factors [25]. Moreover, it was suggested recently that peripheral blood mononuclear cells (PBMCs) contain substantial numbers of T-lymphocytes, which are capable of producing the proinflammatory cytokines IL-1 and TNF , which could be involved in postmenopausal bone loss [26,27].
Here, we compared expression of the genes associated with bone cell differentiation and bone resorption (RUNX2, TGF 1, ALPL, TNF , IL-1 , MMP-9, and cathepsin K) and the genes responsible for global cell survival and functioning (mTOR, p21, caspase-3, and ULK1) in the peripheral blood of late postmenopausal OP patients and healthy subjects. We observed a significant upregulation of ULK1, p21, and MMP-9 gene expression in the blood of the OP women compared to the healthy controls. This was associated with a significant downregulation of mTOR, RUNX2, and alkaline phosphatase (ALPL) gene expression, while the expression of cathepsin K, caspase-3, IL-1 , and TNF was not significantly different than that of the healthy subjects. We also observed a positive correlation between TGF 1 and RUNX2 expression and the BMD at femoral sites in these patients. We concluded that late postmenopausal osteoporosis is associated with an upregulation of cell survival as well as a downregulation of cell growth/proliferation and osteoblast differentiation/function related gene expression as registered in the peripheral blood cells.
Ethics.
The study protocol was approved by the Local Committee on the Ethics of Human Research and informed consent was obtained from all subjects.
Patients.
The study included 22 consecutive, unrelated, late postmenopausal, Russian women with idiopathic osteoporosis who visited the outpatient clinic of the Nasonova Research Institute of Rheumatology. The average age of the OP patients was 66.1 ± 7.2 years, with a range of 53-76 years of age. The average menopause duration was 18.0 ± 4.8 years, with a range of 10-30 years. Individuals with disorders known to cause abnormalities in bone metabolism, including diabetes mellitus, renal diseases, rheumatoid arthritis, and thyroid, parathyroid, and other endocrinological diseases, were excluded from the study. Women that had taken drugs, such as estrogen, progesterone, glucocorticoids, bisphosphonates, and alfacalcidol, were also excluded.
Twenty-six age-matched postmenopausal healthy volunteers (average age 63.0 ± 12.2 years, with a range of 49-78 years of age) who did not have any serious diseases, including osteoarthritis, and had not taken drugs known to affect bone and calcium metabolism were also recruited in the Moscow area. The study protocol was approved by the Local Committee on the Ethics of Human Research and informed consent was obtained from all subjects. The study was conducted in full accordance with the current version (2008) of the Declaration of Helsinki.
Measurement of BMD.
BMD of the lumbar spine (L1-L4), femoral neck, femoral trochanter, femoral intertrochanter, Ward triangle, and total femur was measured by dual-energy X-ray absorptiometry (DXA) using a QDR-4500w instrument (Hologic, USA) at the Nasonova Institute of Rheumatology. The diagnosis of osteoporosis was based on the criteria recommended by the World Health Organization [28], which included a -score < −2.5 SD. According to this test, all the OP subjects examined in this study were diagnosed with osteoporosis (Table 1).
Peripheral Blood Mononuclear Cell (PBMC) Isolation.
Peripheral blood (10 mL) was collected in Vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (BDH, England). The blood samples were taken in a standardized manner in the morning (between 07:00 AM and 09:00 AM). Whole blood fractionation was performed using a Ficoll density gradient. Upon centrifugation the peripheral blood mononuclear cells (PBMCs) located in the interphase were collected and washed twice in phosphate-buffered saline (PBS) [29]. The obtained cell fractions were frozen and kept at −70 ∘ C prior to protein extraction. The quantification of gene expression was conducted using a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as described previously [33]. Briefly, 1 L of RT product was subjected to real-time PCR in a 15 L total reaction mixture containing 7.5 L of TaqMan Universal PCR Master Mix (Applied Biosystems), 900 nM sense and antisense primers, 50 nM probe, and template cDNA. After a single step of 50 ∘ C for 2 min and an initial activation at 95 ∘ C for 10 min, the reaction mixtures were subjected to 40 amplification cycles (15 s at 95 ∘ C for denaturation and 1 min of annealing and extension at 60 ∘ C).
Quantification
Relative mRNA expression was determined using the delta-delta C T method, as detailed by the manufacturer guidelines (Applied Biosystems) [34]. The delta C T value was calculated by subtracting the C T value for the housekeeping -actin gene from the C T value for each sample. A delta-delta C T value was then calculated by subtracting the delta C T value of the control (each healthy patient) from the delta C T value of each OP patient. Each PCR was performed in duplicate. Three "no template" controls were consistently negative for each reaction.
Statistical Analysis.
A Kolmogorov-Smirnov normality test showed that the data were distributed according to a Gaussian distribution curve. Therefore, for statistical evaluations, Pearson's rank correlations and unpaired Student'stest were used for comparisons between the control subjects and the OP patients. As we compared only a control sample with OP sample in case of each gene and did not perform any multiple testing, no correction for multiple testing was made. Quantitative data were expressed as the mean ± SD. The Statistica 6 Software (StatSoft, Tulsa, OK, USA) was used for all statistical analyses. P values less than 0.05 were considered significant.
Whole Blood Gene Expression.
Examination of the gene expression in the whole blood of OP patients revealed that mTOR, RUNX2, and ALPL genes were significantly downregulated compared to the healthy subjects ( Figure 1). In contrast the expression of the p21, autophagy-related ULK1, and MMP-9 genes was significantly upregulated in the same OP patients. No significant changes were observed in the expression of caspase-3, TNF , IL-1 , TGF 1, and cathepsin K.
Protein Levels of p70-S6K, p21, MMP-9, TGF 1, and Caspase-3 in Isolated PBMC.
To further investigate the clinical significance of mTOR, p21, MMP-9, TGF 1, and caspase-3 gene expression in the whole blood of OP patients, we analyzed the protein levels of total p70-S6K serine/threonine kinase (a direct target of mTOR phosphorylation [30,31]), p21, MMP-9, TGF 1, and active caspase-3 in the PBMC fraction. These studies showed that OP patients demonstrated significantly lower p70-S6K protein concentrations in PBMCs compared to the healthy subjects (Figure 2). At the same time, p21 and MMP-9 protein levels were significantly increased, while no changes were observed in the amounts of active caspase-3 and TGF 1 in PBMCs of the OP patients, as compared to the healthy patients.
Association of Gene Expression with BMD.
The analysis of bivariate correlations using Pearson's correlation coefficient for the expression of the examined genes showed that mTOR, TGF 1, RUNX2, TNF , IL1 , cathepsin K, caspase-3, and p21 positively ( < 0.05) correlated with each other (Table 2). However, no correlation was observed between ULK and ALPL gene expression and the other genes. The expression of some of the examined genes also significantly correlated with BMD in the OP patients (Table 2). A positive correlation was noted between TGF 1 and RUNX2 gene expression with BMD of the total femur and its compartments (trochanter and Ward triangle) in the examined OP patients.
Discussion
Recently, the skeletal and immune systems have been thought to interact more than previously believed, and this interaction primarily involves regulatory aspects [35]. Moreover, T-and B-lymphocytes are thought to be key regulators of osteoclast and osteoblast formation, lifespan, and activity [26]. Therefore, we suggested that changes in gene and protein expression in the peripheral blood cells of osteoporotic patients might also be associated with the disease related metabolic alterations observed in the bone cells. Here, we used the PBMCs of late postmenopausal OP patients and healthy controls to investigate the differences in the expression of genes related to the regulation of bone cell differentiation and bone resorption. We show that the expression of osteoclast differentiation and function related genes was either unaltered (cathepsin K, TNF , and IL-1 ) or upregulated (MMP-9) in the peripheral blood of the examined OP women compared to the healthy subjects. Previous studies have also noted that osteoporotic women had serum IL-1 levels similar to those of normal controls [36]. In addition, no difference in the frequency of TNF expression was observed in the bone tissue of OP versus healthy women [37]. Upregulation of MMP-9 gene and protein expression in the PBMCs of the examined OP women is supported by the in situ hybridization results of others, which showed an increase in MMP-9 mRNA in osteoporotic bone tissue versus the normal controls [3]. However, some studies also reported decreased MMP-9 expression in the bone of the postmenopausal OP patients [38]. Although MMP-9 expression was upregulated in the PBMCs of the examined OP women, its proteolytic activity in osteoclasts might be limited by the unaltered expression of cathepsin K, as both proteinases are required to be equally expressed for tissue proteolysis [39]. At the same time, the osteoclast proteolytic activity might not be directly pursued in the peripheral blood as we did not observe any correlation of the examined osteoclast differentiation and function related genes with the BMD indices.
In contrast, significant downregulation of osteoblast differentiation-related RUNX2 and ALPL gene expression in the PBMCs of OP women compared to the healthy age-matched controls, as well as a positive association of RUNX2 gene expression with the BMD at femoral sites, might indicate a significant reduction in bone formation during late postmenopausal OP. Previously, a decrease in RUNX2 and 6 Journal of Osteoporosis ALPL expression was also observed in the bone tissue of OP patients compared to healthy subjects [38,40]. In addition, a lower RUNX2 expression and a subsequently increased bone loss were observed after treating of rodents with bone resorptive agents, such as rosiglitazone [41]. Therefore, bone loss during late postmenopausal OP might be associated with decreased bone formation rather than with increased bone resorption activity.
Our study also shows that late postmenopausal bone loss is associated with alterations in the non-tissue-specific gene and protein expression in the peripheral blood. It is well established that many human diseases occur when the precise regulation of cell growth (cell mass/size) and proliferation (rates of cell division) is compromised [42]. Therefore, the significant downregulation of mTOR gene expression that was associated with the decreased expression of osteoblast differentiation and function related genes (RUNX2 and ALPL) in the PBMCs of late postmenopausal OP women compared to healthy subjects was not surprising. Our observation is supported by the previous observations that the upregulation of mTOR signaling is associated with an increase in bone forming activity in rats and cultured human osteoblastlike cells [43,44]. In contrast, systemic administration of the mTOR inhibitor FK506 caused dramatic OP in animals [45,46], which was accompanied by the downregulation of RUNX2 expression [47]. Moreover, immunosuppressant FK506 therapy resulted in severe bone loss and an increase in fracture incidence in 65% of the patients [48]. Furthermore, a positive correlation between mTOR gene expression and the genes responsible for osteoblast (RUNX2 and TGF 1) and osteoclast (cathepsin K, TNF , and IL-1 ) related differentiation and activity indicates the association of bone loss during OP with a general declination of cell growth and proliferation activity in the examined women.
Significant upregulation of autophagy-related ULK1 gene expression in the examined OP women might involve an increase in cell maintenance, as autophagy has been shown to be associated with increased survival in many cell types [49]. Indeed, increased osteoclast survival was previously observed in association with rapid bone loss in glucocorticoid-induced OP in animal studies [50]. Other animal studies have shown that the downregulation of autophagy resulted in the inhibition of MCPIP (zinc finger CCCH-type containing 12A) induced expression of osteoclastic markers in monocytic osteoclast precursors [51]. Therefore, increased osteoclast survival in the examined OP patients may explain the excessive bone loss in the absence of the upregulation of the genes related to osteoclast differentiation and activity, such as cathepsin K and TNF .
The upregulation of p21, in association with the mTOR inhibition, which was observed in the PBMCs of the examined OP women, was also noted in cultured cells from various lineages following treatment with the mTOR inhibitor, rapamycin [52]. On the other hand, the upregulation of mTOR signaling in response to oscillatory shear stress was associated with a decreased expression of p21 in human osteoblast-like cells [44]. In addition, our observation of decreased RUNX2 gene expression associated with p21 upregulation in the blood of OP patients is supported by a previous study that showed p21 promoter repression by RUNX2 in osteoblast lineage cells [53]. Several other studies have also demonstrated an association between p21 upregulation and the inhibition of osteoblast proliferation and differentiation and an increase in the mononuclear precursor cell differentiation into osteoclasts [54][55][56].
Conclusion
Here, we show that peripheral blood cells of postmenopausal OP women exhibit significant upregulation of the ULK1, p21, and MMP-9 genes. This is associated with a significant downregulation of mTOR, RUNX2, and ALPL gene expression. Although the peripheral levels of cytokines and other factors regulating bone turnover do not always reflect the levels observed within the bone, the correlation of TGF 1 and RUNX2 gene expression with BMD suggests that the expression of these gene counterparts in the bone tissue might be similarly affected. Therefore, bone resorption in late postmenopausal OP might be associated with decreased bone formation and an increased survival of the bone degrading cells. Further experiments should be performed to confirm our observations. | 2018-04-03T05:56:16.078Z | 2015-02-10T00:00:00.000 | {
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211472785 | pes2o/s2orc | v3-fos-license | The Anti-Cancer Mechanisms of Berberine: A Review
Abstract Berberine (BBR) has been extensively studied in vivo and vitro experiments. BBR inhibits cell proliferation by regulating cell cycle and cell autophagy, and promoting cell apoptosis. BBR also inhibits cell invasion and metastasis by suppressing EMT and down-regulating the expression of metastasis-related proteins and signaling pathways. In addition, BBR inhibits cell proliferation by interacting with microRNAs and suppressing telomerase activity. BBR exerts its anti-inflammation and antioxidant properties, and also regulates tumor microenvironment. This review emphasized that BBR as a potential anti-inflammation and antioxidant agent, also as an effective immunomodulator, is expected to be widely used in clinic for cancer therapy.
Introduction
Cancer is a major cluster of diseases that seriously affects human health. Therefore, development of strategies to prevent and treat cancer is critical. 1 Berberine (BBR), a small molecule isoquinoline alkaloid extracted from the rhizomes of coptis chinensis and hydrastis canadensis, is traditionally used to treat bacterial diarrhea. 2 Recent studies showed that BBR reduced lipid levels and glycemic index, and exerted anti-tumor effects. [3][4][5][6][7] BBR lowered lipid levels via competitive inhibition of HMG-CoA reductase, and by interacting with the 3ʹ-UTR of the LDL receptor (LDLR) to improve the stability of LDLR mRNA. 8 In vivo experiment showed that BBR alleviated nonalcoholic fatty liver by activating SIRT3. 9 In foam cells, BBR promoted cholesterol efflux by increasing ROS production, and induced autophagy by inhibiting mTOR and Akt phosphorylation. 10 The mechanisms of the hypoglycemic effects of BBR have also been studied extensively. Studies showed that BBR improved insulin action through inhibition of mitochondrial and activation of AMPK. 11,12 In liver and muscle cells, BBR restored insulin sensitivity by up-regulating InsR expression. 13 In vitro experiments showed that BBR affected glucose uptake by down-regulating miR29-b and increasing Akt expression. 14 Recent studies have shown that BBR exerted anti-tumor effects against lung cancer, cervical cancer, liver cancer, leukemia, and other malignancies. [15][16][17][18] BBR inhibits cancer cell proliferation through various mechanisms. Here, in this review, we discussed the effects of BBR on cell cycle, cell apoptosis, cell autophagy, ability of inhibiting cell invasion and proliferation, expression of microRNA, telomerase activity, and tumor microenvironment. Currently, BBR is widely used in basic researches and clinical trials. This review clarified the potential of BBR as an anti-cancer drug, which may speed up its clinical application and eventually benefit cancer patients.
BBR Inhibits Cell Proliferation BBR Regulates Cell Cycle
Alterations in the cell cycle promote the development of cancer. 19,20 Studies showed that BBR regulated cell cycle and inhibited cell proliferation in multiple cancers. 6,21,22 BBR induced G1 phase cycle arrest in A549 lung cancer cells through inhibition of the expression of Cyclin D1 and Cyclin E1. 21 In addition, a combination of an Hsp90 inhibitor and BBR inhibited cell growth via inhibition of CDK4 expression and modulation of cyclin D1 in colorectal cancer cells. 22 In HepG2 human hepatoma cells, BBR suppressed cyclin D1 expression in vitro and in vivo. 6 Furthermore, BBR arrested the cell cycle at G1 via reduced expression of cyclin B1 and indirect inhibition of CDC2 kinase in several cancer cells. 23 In HBT-94 chondrosarcoma cells, BBR upregulated the expression of p53 and p21 by modulating activation of the PI3K/Akt and p38 signaling pathways, which resulted in G2/M phase arrest. 24 In MDA-MB-231 breast cancer cells, BBR arrested cells in S phase, which contributed to high sensitivity of cancer cells to chemotherapy. 25 BBR has also been shown to influence cell cycle through regulation of Rb. Specifically, BBR acted on the 3ʹ-UTR of Rb, which resulted in inhibition of Rb mRNA degradation, stabilization of Rb translation, and inhibition of cell cycle progression. 26 BBR also inhibited phosphorylation of Rb protein, which prevented dissociation of the transcriptional activator E2F from Rb, and resulted in inhibition of the transition from G1 to S phase. 27 Together, BBR inhibits cancer cell proliferation by affecting cell cycle progression.
BBR Regulates Cell Apoptosis
Apoptosis is a gene-controlled form of cell death that plays an important role in health and disease. 28 BBR has been shown to promote apoptosis by activating caspases. In leukemia, BBR contributed to cell apoptosis by increasing the expression of caspase-8 and caspase-9, and inhibiting the expression of bcl-2 through activation of caspase-3. 29 BBR activated caspases through increased levels of cytochrome C, 30 activation of AMPK, and increased ROS production. 31,32 Mitochondria are central to regulation of apoptosis. 33 A study showed that external stimulation increased the permeability of the mitochondrial membrane, which activated the caspase cascade, and resulted in apoptosis. 33 This signaling cascade was also involved in BBR-induced apoptosis in hepatoma cells. 34 BBR increased phosphorylation of p53 through activation of JNK/p38, which promoted the entry of the apoptotic proteins Bax and Bim into mitochondria. 35 In addition, studies showed that BBR promoted apoptosis through increased acetylation of foxo1/3a and increased expression of Bim and Bax. 36,37 In colon cancer cells, BBR promoted cell apoptosis by inducing the expression of ATF3 protein through increased p53 transcription activity. 38 In MDM2-overexpressing tumor cells, BBR treatment led to the degradation of MDM2, which induced cell apoptosis in acute lymphoblastic leukemia. 39 In conclusion, BBR contributes to cell death by inducing cell apoptosis through different mechanisms.
BBR Regulates Cell Autophagy
Autophagy is a form of programmed cell death that plays an important role in maintaining cellular homeostasis. 40 In glioblastoma, BBR targeted the AMPK/mTOR/ULK1 pathway and resulted in activation of autophagy. 41 In breast cancer, BBR induced autophagic death by modulating phosphorylation of JNK and contributing to dissociation of the bcl-2/ beclin-1 complex. 42 In hepatoma cells, BBR induced autophagy by promoting the release of beclin-1 from the bcl-2/ beclin-1 complex. 43 In addition, BBR has been shown to induce autophagy by increasing the binding capacity of GRP78 and VPS34 in cancer cells. 44 BBR also indirectly inhibited the expression of SREBP-1 through activation of AMPK, which activated the ULK1/mTOR1 signaling pathway to promote autophagy. 45 Studies have shown that autophagy-mediated drug resistance plays an important role in cancer development and that tumor cells evade apoptosis through regulation of autophagy. 46 BBR may be able to reverse drug resistance by regulating autophagy through activation of AMPK. A previous study showed that BBR promoted binding of the miR30 family with the beclin1 3ʹ-UTR region, which resulted in the inhibition of autophagy in adipocytes. 47 From the above findings, we found that the effects of BBR on autophagy are complicated, BBR either inhibited autophagy or induced autophagy to exert its antitumor effects. Regulation of BBR on autophagy has been widely studied; however, additional studies are needed to further characterize the mechanisms by which BBR may regulate autophagy.
BBR Inhibits Cell Invasion and Metastasis
Patients with cancer die due to destruction of tissues and organs resulting from uncontrolled cell proliferation, and tumor cell invasion and metastasis. In breast cancer cells, BBR inhibited cell proliferation and metastasis by targeting ephrin-B2 and inhibiting the expression of MMP-2 and MMP-9. 48 In addition, BBR inhibited the expression of MMP-2 and MMP-9 via downregulation of TGF-β1. 49 In melanoma, BBR inhibited the EMT through downregulation of RARα and upregulation of RARβ, which resulted in inhibition of the PI3K/Akt signaling pathway. 50 In triplenegative breast cancer cells, BBR inhibited cell proliferation by down-regulating IL8 expression through inhibiting the EGFR/MEK/ERK signaling pathway. 51 In addition, BBR has been shown to inhibit the COX-2/PGE2-JAK2/STAT3 signaling pathway, which resulted in reduced expression of MMP2 and MMP9. 52 Angiogenesis plays a key role in cancer progression and metastasis. 53 In vitro and in vivo studies showed that BBR inhibited the expression of VEGF mRNA in tumor cells, 54 induced phosphorylation of eEF2, 55 down-regulated the activity of HIF-1α, which resulted in reduced expression of VEGF, 56 and inhibited the PI3K/ AKT signaling pathway. 57 In SW480 colorectal cancer cells, BBR inhibited proliferation and migration by downregulating expression of GRP78. 58 In addition, BBR inhibited tumor metastasis by reducing the expression of the transcription factor snail-1. 16 In all, BBR inhibits cell invasion and metastasis by affecting the expression of tumorrelated signaling pathways and proteins.
BBR Regulates Tumor Microenvironment
Tumor cells secrete cytokines to alter the surrounding tumor microenvironment, which promotes tumor cell proliferation and metastasis. [59][60][61] In osteosarcoma, BBR altered the inflammatory microenvironment by downregulating the caspase-1/IL-1β signaling pathway, which resulted in cell apoptosis. 62 In autoimmune diseases, BBR suppressed the Th17 response through direct interaction with T cells and DC cells. 63 BBR has been shown to improve osteoarthritis through inhibition of IL-1β signaling, and inhibition of cartilage damage. 64 In all, BBR regulates tumor microenvironment by affecting inflammatory response and immune molecules.
BBR's Antiinflammatory Properties
In inflammatory macrophages, BBR induced Nrf2 activation in an AMPK-dependent manner and inhibited inflammation. 65 BBR reduced oxidized low-density lipoprotein (ox-LDL)-induced inflammation by regulating the AMPK/mTOR signaling pathway. 66 In hepatic fibrosis, BBR inhibited the inflammation induced by thioacetamide treatment. 67 In skeletal progenitor cells, BBR exerted its anti-inflammatory activities by activating the AMPKα-SIRT-1-PGC-1α signaling pathway and inhibiting the mitogen-activated protein kinase 4 (MKK4)-SAPK/JNK-C-JUN. 68 A study showed that BBR treatment downregulated TNFα, IL-1β and IL-6, which suppressed the seizure-like behavior in Zebrafish. 69 In adjuvant arthritis in mice, BBR significantly alleviated joint destruction and inflammatory cell infiltration by regulating the AMPK/NF-кB pathway. 70 Together, BBR inhibits inflammation by regulating different signaling pathways and cytokines.
BBR's Antioxidant Activities
In thioacetamide-induced liver fibrosis, BBR suppressed hepatic fibrosis by elevating hepatic antioxidant enzymes. 67 In spiral ganglion cells, BBR exerted antioxidant effects via mediating the generation of reactive oxygen species. 71 In experimental varicocele, BBR as an antioxidant agent, promoted testicular antioxidant potential, promoted spermatogenesis, and upregulated the sperm quality. 72 A study showed that BBR protected PC-12 cells from oxidative injury by inhibiting ROS via PI3K/AKT/ mTOR signaling pathways. 73 In the pentylenetetrazoleinduced kindling model of epilepsy in rats, BBR exerted antioxidant properties and anti-epileptogenic effects by upregulating superoxide dismutase levels. 74 In the intestinal tissue of mice, BBR treatment enhanced the antioxidant status by increasing the activities of catalase and glutathione peroxidase enzymes. 75 These findings demonstrated that BBR exerts antioxidant properties through various mechanisms, and BBR might be the potential antioxidant agent.
BBR Acts as an Effective Candidate for Tumor Immunotherapy
Immunotherapy for the treatment of tumors has received increased attention. BBR has been shown to exert positive effects on tumor immunotherapy. A study showed that BBR acted as a dopamine D1-and D2-like receptor antagonist to inhibit secretion of IFN-γ, TNF-α, IL-6, and IL-1β from LPSstimulated lymphocytes. 76 BBR also improved autoimmune neuropathy by down-regulating TNF-α and IL-1 levels, and by inhibiting proliferation of CD4+ T cells. 77 Moreover, BBR inhibited the phosphorylation of STAT1, which resulted in inhibition of IFN-γ-induced IDO1 expression. 78 These results indicated that BBR is a potential therapeutic candidate for tumor immunotherapy.
BBR's Other Functions Effects of BBR on microRNA
MicroRNA is a class of single-stranded RNA involved in post-transcriptional regulation. 79 More than 2500 microRNAs have been identified, and over-expression of many microRNAs has been shown to be closely related to onset and development of tumors. [80][81][82] In endometrial cancer cells and multiple myeloma cells, BBR inhibited cancer invasion and metastasis by interacting with microRNAs. [83][84][85] In hepatoma cells, BBR mediated the transcriptional activation of p21 and GADD45α by upregulating the expression of miR-23a. 86 In colon cancer cells, BBR down-regulated miR-429 and inhibited E-cadherin expression. 87 Together, BBR inhibits cancer invasion and metastasis by regulating the expression of microRNAs or interacting with them. Although studies have shown that BBR inhibited cancer development by interacting with microRNAs, the interaction sites and mechanisms associated with these inhibitory effects need to be further explored.
BBR Regulates Telomerase Activity
Telomerase activity is inhibited in normal cells, and abnormal activation of telomerase results in immortalization in tumor cells. 88 BBR inhibited binding of AP-2 to the hTERT promoter, which resulted in reduced expression of hTERT and reversal of tumor cell immortalization. 89 Another study showed that BBR stabilized the structure of the endogenous telomere G-quadruplex, 90 which resulted in inhibition of binding of hTR to this complex and inhibition of telomerase activity. 91 Together, BBR affects cell proliferation by inhibiting telomerase activity.
Discussion and Conclusion
The traditional Chinese medicine BBR has been shown to affect cell cycle, cell apoptosis, cell autophagy, and the tumor microenvironment. BBR has also been shown to exert anti-inflammatory and antioxidant effects. Tumor immunotherapy is a hotspot for tumor therapy in recent years, immune-suppressants such as PD-1/PD-L1 suppressants have emerged one after another. However, it is difficult to be widely used in clinic due to their high prices. BBR as an effective immunomodulator and a kind of cheap Chinese traditional drug, is expected to be widely used in clinical practice as an ideal drug for immunotherapy. As studies showed, BBR exerted its role on autophagy through different mechanisms. In several cancer cells, BBR inhibited cell proliferation by inducing autophagy and also reversed drug resistance by regulating cell autophagy. 45,46 However, in mature adipocytes, BBR maintained the cellular homeostasis by inhibiting autophagy. 47 Studies showed that autophagy plays an important role in maintaining a stable intracellular environment. 92,93 We inferred that autophagy plays different roles in cells. On the one hand, tumor cells evaded apoptosis through decreasing autophagy level; therefore, BBR treatment up-regulated autophagy and led to cancer cell death. On the other hand, BBR treatment lowered the original high level of autophagy in mature adipocytes to contribute to maintenance of a stable intracellular environment. Regulation of BBR on autophagy is complicated; therefore, studies are needed to make further progress on regulation of BBR on autophagy.
Although BBR exerts beneficial effects that may aid in the treatment of tumors, the efficacy of BBR is limited by poor solubility in water, rapid metabolism, and low absorption rate in intestines. Therefore, development of formulations that improve absorption of BBR in the intestines may have great potential for treatment of cancer. Xiao et al successfully designed nanoparticles that significantly increased the bioavailability of BBR, which demonstrated that nanotechnology may be a promising strategy for improving the pharmacokinetics of BBR. 94 Clinical trials mainly focused on the function of BBR on lowering lipid levels and regulating blood sugar. However, nowadays, anti-tumor effects of BBR are being investigated in a number of clinical trials.
In this review, we discussed the anti-tumor mechanisms of BBR (Figure 1), and its potential as an anti-cancer drug. BBR has been shown to inhibit cell proliferation and angiogenesis through modulating cell cycle, cell apoptosis, cell autophagy, and the tumor microenvironment. This review emphasized that BBR as a potential anti-inflammation and antioxidant agent, also as an effective immunomodulator, is expected to achieve clinical application for cancer therapy. | 2020-02-06T09:02:56.331Z | 2020-01-30T00:00:00.000 | {
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204788568 | pes2o/s2orc | v3-fos-license | Constraints on the String T-Duality Propagator from the Hydrogen Atom
We investigate the implications of a string-theory modified propagator in the high-precision regime of quantum mechanics. In particular, we examine the situation in which string theory is compactified at the T-duality self-dual radius. The corresponding propagator is closely related to the one derived from the path integral duality. Our focus is on the hydrogen ground state energy and the $1\text{S}_{1/2}-2\text{S}_{1/2}$ transition frequency as they are the most precisely explored properties of the hydrogen atom. In our analysis, the T-duality propagator affects the photon field leading to a modified Coulomb potential. Thus, our study is complementary to investigations where the electron evolution is modified as in studies of a minimal length in the context of the generalized uncertainty principle. The first manifestation of the T-duality propagator arises at fourth order in the fine-structure constant, including a logarithmic term. The constraints on the underlying parameter, the zero-point length, reach down to $3.9 \times 10^{-19}\, \text{m}$ and are in full agreement with previous studies on black holes.
Introduction
Symmetries lie at the heart of almost any theory in physics and imply far-reaching consequences: symmetries powerfully constrain the structure of terms that are allowed in a given action; e.g., of the electroweak theory [1]. They allow one to clearly extract the fundamental degrees of freedom of a theory by choosing an appropriate gauge (cf. gravitational waves; e.g., [2,3]). In the context of string theory, there are even symmetries which show the equivalence of whole theories. Among those is T-duality, which acts on the moduli space and relates string theories compactified on different backgrounds.
As a special case, T-duality relates toroidally compactified theories which emerge from each other under inversion of the compactification radius, R, and exchange of the Kaluza-Klein mode numbers, n, with the winding mode numbers, w. In the case of one extra dimension, the relation reads R → R 2 /R and n ↔ w. The self-dual radius R is mapped onto itself while compactification radii smaller than R are identified with larger ones. Thus, a notion of a smallest sensible length scale arises. The self-dual radius is located at the string scale, R = √ α , where α denotes the Regge slope. Starting from toroidally compactified bosonic string theory with compactification radius R , the authors of [4] derived an effective 4-dimensional propagator for the center-of-mass of closed strings (cf. also [5,6]). Compared to standard quantum field theory propagators, the presence of compactified extra dimensions implies a UV finite behavior. There are contributions from a tower of momentum and winding modes, out of which the massless mode is the most relevant for low-energy physics. It gives rise to the T-duality propagator. The T-duality propagator found its first, and so far, only application in the recent derivation of a quantum-gravity corrected black hole description [7]. Here, the self-dual radius plays a crucial role: It leads to a resolution of the curvature singularity at the black hole center and to a non-divergent black hole evaporation process without a final explosion. The latter difference to the Schwarzschild black hole solution offers an observable to test the hypothesis of the T-duality propagator.
A concept related to the T-duality ansatz which also tries to capture effects of the quantum nature of spacetime is the path integral duality. While the former is derived from string theory, the latter is an ad hoc ansatz in quantum field theory which introduces scale-inversion symmetric weights in the Schwinger representation of the propagator-at a scale known as zero-point length, l 0 [8,9]. Both the propagators, from the first order T-duality approach and from the path integral duality, agree. This allows one to relate the zero-point length to the self-dual radius, l 0 = 2πR = 2π √ α . Using effective actions, several quantum field theoretical aspects of the path integral duality have been examined; e.g., the Casimir effect and the thermal particle spectra of Rindler vacua [10]. In the context of gravity, investigations in the curved spacetime of cosmology [11,12] and of black holes [13] have been conducted. Recently, the regularization of path integrals has been generalized to the concept of so-called path densities [14]. But there are, to our knowledge, no studies that derive bounds on the zero-point length from experimental data.
For our work, modifications of electromagnetic interactions are relevant. In the literature, the path integral duality has been used to calculate deviations in the radiative corrections in quantum electrodynamics [15]. Radiative corrections for a whole family of propagators, which share a similar analytical structure and are referred to as smeared propagators, have been addressed earlier [16][17][18]. The aim of the present study is to analyze the concept of the T-duality induced zero-point length from a different, low-energy perspective. We chose a system in quantum physics which has been investigated to high precision theoretically and experimentally-the hydrogen atom [19]-and derive constraints on l 0 . The hydrogen atom has been used by several authors to test various high-energy concepts; e.g., [20][21][22][23][24].
We used two characteristics of the hydrogen atom: firstly, the ground state energy, and secondly, the transition frequency between the two lowest energy levels with vanishing orbital angular momentum, 1S 1/2 and 2S 1/2 -the spectral line which is experimentally known to highest precision [25]. The potential shift in these observables due to the T-duality concept strongly depends on the size of l 0 . We calculated those in Rayleigh-Schrödinger perturbation theory. From comparison with discrepancies between experimental and theoretical values and from their uncertainties, we obtained upper limits of l 0 .
In Section 2, we review the theoretical description of the hydrogen atom. We obtain the energy and frequency corrections from the T-duality propagator. The constraints from both observables are derived in Section 3 and are discussed in Section 4. Section 5 offers a summary. Useful mathematical identities are presented in the Appendix A. In this article we use natural units; in particular, c ≡h ≡ 1. For electromagnetic quantities we apply the Lorentz-Heaviside convention which additionally implies 0 ≡ µ 0 ≡ 1.
Hydrogen Atom Energy Levels
The hydrogen atom is a prime object in quantum mechanics. This section focuses on the energy spectrum in the conventional description and on the corrections arising from a T-self-dual spacetime. We start with the Schrödinger equation with fine-structure terms because we are considering a low-energy quantum system. Then, we introduce the T-duality induced modifications of the Coulomb potential. Finally, we derive shifts in energy levels and transition frequencies.
Conventional Description
The stationary Schrödinger equation, H 0 |ψ = E |ψ , with the eigenvectors |ψ and eigenvalues E, is the starting point of our recapitulation which follows the references [26,27]. In position space, the Hamiltonian for spherically symmetric systems is given by Here, µ is the reduced mass of the electron-proton system, ∆ is the Laplace operator, and L the angular momentum operator. The potential term follows directly from the Coulomb interaction, where α = e 2 /4π denotes Sommerfeld's fine-structure constant. The well-known separation ansatz in spherical coordinates reads with the principle, the orbital angular momentum, and the magnetic quantum numbers n, l, and m. The spherical harmonics Y lm (ϑ, ϕ) solve the angular part while the radial equation simplifies to − 1 2µ with the bound-state solutions Herein, we define κ ≡ µα/n and apply the associated Laguerre polynomials The spectrum of the energy eigenstates is discrete and depends on n only, The ground state, denoted by 1S 1/2 , shows the energy E S th ≡ E 1 = −µα 2 /2. Below, we will also employ the first excited state of spherical symmetry, 2S 1/2 . The associated wave functions read and the transition frequency between both states follows to be ν S th ≡ (E 2 − E 1 ) /2π. Next, we include relativistic corrections which lead to the so-called fine structure in the spectrum. These are encoded in the relativistically adjusted Hamiltonian H = H 0 + H fs . The terms for relativistic momentum correction, spin-orbit coupling, and zitterbewegung (Darwin term) constitute the Hamiltonian contribution which involves the Pauli matrices, σ.
The correction in the energy spectrum can be calculated by means of the time-independent Rayleigh-Schrödinger perturbation theory. The first order corrections partially break the degeneracy and explicitly depend on the total angular momentum quantum number j = l + s = l ± 1/2. They read In particular, the corrections to the 1S 1/2 and 2S 1/2 levels are We define the improved value of the ground state energy as E fs th ≡ E 1 + ∆E fs 1,1/2 and the corrected transition frequency as ν fs . The fine-structure corrections naturally arise in the Dirac treatment of the hydrogen atom and agree with Equation (12) to order α 4 . The state-of-the-art description of the hydrogen atom goes beyond idealizations like that of a point-like nucleus or vanishing nuclear polarizability, and uses methods of quantum field theory to include, e.g., multiple photon interactions in quantum electrodynamics or hadronic contributions to the proton self-energy from quantum chromodynamics. Those corrections appear at order α 5 or higher. For an overview of the contributions, we refer the reader to [19].
Contribution from T-Duality Propagator
The considerations up to now originate from quantum mechanics and quantum field theory. The standard model of particle physics, however, is generally being assumed to be incomplete. In contrast, superstring theory is a possible candidate for a unified theory also valid at high energies which reduces to the standard model and to general relativity as limiting cases [28]. In the following we consider closed bosonic string theory on a manifold with toroidal compactification where the compactification radius equals the self-dual radius under T-duality. Regarding the 4-dimensional propagation, the string center of mass deviates from excitations of quantum fields. The Euclidean propagator of a massless scalar field inherited from bosonic string theory reads [7] where K ν (x) are modified Bessel functions of the second kind. In the low-momentum limit, one obtains the standard scalar propagator, while there is an exponential suppression for momenta large compared to 1/l 0 . We regard such a kinetic modification to apply to all quantum fields, especially to bosons. Virtual-particle exchange then leads to modified interaction potentials which contain the zero-point length as a UV cutoff [7]. In electrodynamics, the potential energy reads The difference to the conventional Coulomb interaction can be used to identify manifestations of T-duality from the hydrogen energy spectrum. From that we can derive constraints on l 0 . Similar to the inclusion of fine-structure corrections, we apply the Rayleigh-Schrödinger perturbation theory to the amended Hamiltonian H = H 0 + H fs + H Td . The additional term comprises the modification of the Coulomb energy, which is presented in Figure 1.
dy y 2+2l e −2y/n L 2l+1 n+l (2y/n) Here we introduce y ≡ αµr and define x ≡ λ 0 α where λ 0 ≡ µl 0 . For the ground state, we find the following expression at first order in perturbation theory: We used the Euler-Mascheroni constant, γ ≈ 0.577; the Bessel functions of the second kind, Y ν (x); and the Struve functions, H ν (x). The level shift of 2S 1/2 reads: = µα 2 2 We provide the identities crucial in deriving this result in the Appendix A. Note that the corrections start at order α 4 . Note also that they are of the form ∆E ∝ const. + ln 1
Constraints on the Zero-Point Length
In the previous section, we derived the shifts in the energy levels 1S 1/2 and 2S 1/2 , and the shift in the associated transition frequency as a function of the zero-point length. Now we can contrast the shifts with experimental data in order to obtain constraints on the value of l 0 .
Ground State Energy
The reference values and uncertainties of the hydrogen ground state energy from theory and experiment are displayed in Table 1. Taking into account the respective standard deviations, we take the maximum difference between the fine-structure improved Schrödinger value, E fs th , and the experimental one, E exp , and that between the current theoretical value, E QED th , and the experimental one. In this context, the experimental precision ∆E exp by itself defines the smallest upper bound on l 0 . [19]. When expressed in eV, the actual precision of the current theoretical and experimental value is masked by the less-precisely known Planck constant [19]. For this reason, h is factored out and the values are given also in terms of MHz · h. Figure 2 shows the relative T-duality contribution ∆E Td 1,0 as a function of the zero-point length, l 0 . The reference values are included as well. The zero-point length has to be smaller than the intersection point value to comply with the corresponding bound. We found the upper bounds for l 0 to be 15.7 fm (from comparison of the fine-structure improved Schrödinger description with the experiment), 0.136 fm (from comparison of the state-of-the-art theoretical value with the experiment), and 0.112 fm (from the experimental precision), respectively.
Transition Frequency
Among all transitions of states in the hydrogen atom, the transition frequency between the 1S 1/2 and the 2S 1/2 level is experimentally known with the highest precision. The relative precision ∆ν exp /ν exp of the experimental value surpasses the relative precision of the absolute ground state energy, ∆E exp /E exp , by five orders of magnitude. Therefore, we obtained more stringent upper bounds from the transition data than from the absolute energy data discussed above. The theoretical and experimental values of the transition frequencies associated with their uncertainties are presented in Table 2. Our approach is analogous to the case of the ground state energy. In Figure 3 one finds the l 0 -dependent T-duality contribution to the transition frequency and the reference values. The corresponding upper bounds on l 0 are 15.7 fm (from comparison of the Schrödinger description including fine-structure corrections with the experiment), 1.45 × 10 −3 fm (from comparison of the current theoretical value with the experiment), and 3.90 × 10 −4 fm (from the experimental precision). For the sake of clarity and for contrasting with the other approach, the values are summarized in Table 3. Table 3. Bounds of l 0 . This table summarizes the findings from Section 3.
Discussion
Below, we discuss the validity and self-consistency of the results of Section 2 first. Then, we comment on the bounds on the zero-point length.
One can classify the different contributions to the hydrogen energy levels in terms of powers of the fine-structure constant. Generally, the higher is the order of a term, the smaller is its absolute contribution. The Schrödinger value sets the scale at α 2 and the Schrödinger fine-structure correction occurs at α 4 . The Dirac treatment reproduces the terms at order 4 and yields additional terms at order 6 and above. According to the standard theoretical description, further corrections set in at α 5 [19].
As presented in Equations (19) and (25), we obtained the corrections induced by the T-duality propagator in terms of Bessel and Struve functions. When expanding these results in powers of the fine-structure constant, Equations (21) and (27), we found the first manifestations at order α 4 and α 4 ln (1/α). At second order in perturbation theory, we expect terms starting with α 5 . Similarly, mutual interactions with the fine-structure corrections can only appear at order 5 and above. Therefore, we ensure describing a proper observable since we have taken all contributions to order 4 into consideration.
The other parameter which determines the amplitude of the T-duality induced correction is the zero-point length. For small l 0 , we found an approximately quadratic dependency, as evident in the series expansions Equations (20) and (26). Furthermore, the logarithmic term is dominating and is responsible for the overall sign of the corrections. Both these features are evident in Figures 2 and 3.
We stress that the fine-structure corrected Schrödinger value for the ground state energy lies below the experimental one. Additionally, the current theoretical value hints at stronger binding than the current experimental one (cf . Table 1). Similarly, the analogous theoretical transition frequencies are found above the experimental counterpart (cf. Table 2). Thus, at qualitative level, a shift towards weaker binding energy and smaller transition frequency improves the match between the theoretical and experimental results. Indeed, both hold true for the T-duality corrections.
At a quantitative level, there are two ways of comparing potential theoretical contributions with experimental data. One can compare the conventional theoretical value with the experimentally measured one. Under the assumption that the discrepancy is generated by the novel effect only, one can derive a bound on the underlying parameter. Alternatively, one can focus on uncertainties and require the extra contribution to be smaller than the experimental accuracy. Then, the experimental standard deviation is regarded as the reference scale. The latter approach usually results in stronger constraints and is used commonly in literature, e.g., [20,22]. We followed both approaches. However, we refined the first way in a conservative manner: we did not just take into account the discrepancy between the theoretical and experimental value; we also took into consideration the respective standard deviations. That way, we obtained a less strict, but more reliable bound.
In this paper we address the ground state energy and the energy difference between the 1S 1/2 and 2S 1/2 levels. The latter observable turns out most suitable: Firstly, the corresponding transition frequency is known to a better precision, experimentally and theoretically. For instance, the experimental relative error exceeds the one of the ground state energy by five orders of magnitude. Indeed, this transition frequency is the most accurately known hydrogen spectral line [25]. Secondly, the difference between two levels is relative by definition: It is insensitive to global energy shifts.
Overall, we found constraints in the range 1.6 × 10 −14 m down to 3.9 × 10 −19 m. Table 3 shows a compilation of the bounds obtained. Limits in the range of 10 −17 m were also found in related minimal length considerations [20,23] even though they arose from a different context: they rely on a generalization of the Heisenberg uncertainty relation while the limits presented here are the direct consequence of the T-duality propagator. There is a further sense of complementarity: they focus on a modification of the electron evolution and alter the Schrödinger or Dirac equation. In contrast, we applied the modified massless propagator to the electromagnetic field and studied the modified Coulomb potential while keeping the ordinary Schrödinger description. Although in both ways the energy contributions set in at α 4 , we found an additional logarithmic contribution.
More precise experimental and theoretical results would be helpful to further restrict the size of minimal lengths and to explore the viable range of quantum gravity modifications. Based on a consistency condition for the smallest sensible size of black holes, the authors of [7] expected the value of the zero-point length at l 0 = (2/3) 3/4 l P ≈ 0.8 l P , where l P is the Planck length. Testing this length scale with the hydrogen atom would correspond to a relative precision of 10 −47 for the ground state as well as for the transition frequency. While this seems out of reach with the techniques present today, astrophysical observations, e.g., of the maximum temperature of microscopic black holes, could provide stronger constraints and further insights.
Summary
String theory is a high-energy completion of quantum field theory and gravitation. Regarding quantum field excitations as strings instead of point-like particles, the authors of [4] derived the modified 4-dimensional propagator. It introduces a parameter called zero-point length, l 0 , which is closely related to the self-dual radius of T-duality. This approach was applied to the context of black holes; differences to the general relativistic counterparts were identified [7].
In this paper, we investigated how the modified propagator manifests itself in the hydrogen atom. The hydrogen atom is a well suited system since it has been investigated to high precision in theory and experiment. We derived the corrections to the hydrogen ground state energy and the 1S 1/2 − 2S 1/2 transition frequency by first order Rayleigh-Schrödinger perturbation theory. By comparison with experimental data, we could derive constraints on the zero-point length ranging down to l 0 < 3.9 × 10 −19 m.
Conflicts of Interest:
The authors declare no conflict of interest. | 2019-10-18T00:02:58.000Z | 2019-10-18T00:00:00.000 | {
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259263813 | pes2o/s2orc | v3-fos-license | Network Learning-Enabled Sensor Association for Massive Internet of Things
,
Introduction
Recently, newly emerged internet of things (IoT) applications such as smart agriculture, smart industries, and smart transportation systems have introduced massive growth in terms of connected devices and sensors, which increases wireless bandwidth consumption [1]. In an IoT environment, sensor device association plays a vital role in IoT applications like localization [2] and indoor positioning [3]. One of the well-known wireless communication technologies, the Institute of Electrical and Electronics Engineers (IEEE) 802.11 wireless local area networks (WLANs) has become a communication service provider to overcome the increasing demands for bandwidth consumption [4]. A WLAN for massive IoT is composed of several access points (APs) as gateways (GWs) for sensory data collection. In such a scenario, multiple GWs coexist with the same service set identifier (SSID) and create an extended service set (ESS) as a single WLAN environment. Such an example can be found in IoT applications such as smart industries or smart agriculture, where several smart devices (SDs) with attached sensors are required to connect to a single internet access GW. However, a WLAN environment suffers from network performance degradation as the number of connected devices grows. This occurs due to the limited number of available channels and bandwidth. Hence, channel resource scarcity has been one of the challenges for WLAN environments.
A newly arrived IEEE 802.11 WLAN, IEEE 802.11ax has introduced many new features such as spatial reuse (SR) and 5 gigahertz (GHz) bands [5]. One of the purposes of including these competitive features is to improve the channel access mechanisms that may have unsatisfactory efficiency in highly dense scenarios. In a WLAN, the channel scarcity problem is resolved using the carrier sense multiple access with collision avoidance (CSMA/CA) mechanism with the distributed coordination function (DCF) at the medium access control (MAC) communication layer. Currently implemented CSMA/CA is simple and effective and performs well for a small WLAN network with a limited number of users and low throughput requirements. With the rise in user density and higher throughput requirements, CSMA/CA fails to tackle the collision problem. In IoT network systems, such a situation can be handled by associating SDs with a stronger received signal strength indicator (RSSI) strategy. However, this may introduce an unbalanced use of different GWs for the sensor data. The issue with an RSSIbased strategy is the use of the physical layer RSSI as a metric for the strongest signal for an association, which does not consider the traffic situation on the GW or in the network. Fig. 1 depicts an example IoT scenario, where the two highlighted regions, blue and red, contain unbalanced associated SDs. As shown in the blue highlighted region of the figure, GW 1 and GW 2 may be associated with three SDs each; however, due to the RSSI-based strategy, GW 1 has a higher load than GW 2. Similarly, in the red highlighted region, GW 6 has four associated SDs, while GW 5 contains only one associated device. In each of the regions, the associated devices could be distributed more intelligently to enhance the overall network performance. Therefore, this work suggests considering the network situations as part of the network configuration as a suitable GW selection for connected SDs in an IoT network. Today, machine learning (ML) techniques have a proven ability to provide solutions to such optimization problems. In this paper a multi-armed bandits (MAB) [6], an ML technique, is used to address the GW association problems in an IoT network based on IEEE 802.11 WLANs. In MAB techniques, actions are performed by an agent within a defined environment, intending to maximize the accumulated reward. To do so, it considers SDs as agents that expect to learn the optimal action based on the environment to associate themselves with the GWs. However, this work addresses this problem in a decentralized multiagent context, where GWs and SDs compete for the common set of spectrum resources using multiple actions. This research work uses the MAB technique to perform the SD association with a GW for an IoT network. Our proposed MAB-based algorithm allows SDs to intelligently associate themselves with a suitable GW by learning from their experience (accumulated reward) through a learning-byinteraction approach to improve their future performance. In particular, it considers the use of MAB to study the case of the suitable GW association problem in a large WLAN-based IoT network. To do so, it utilizes the effectiveness of the ε-greedy algorithm as an optimal action exploitation strategy. The following are the key contributions of this paper, • This paper identifies the challenges posed by IoT sensor device associated with IoT gateways.
• A learning-based multi-armed bandit algorithm is proposed for the sensor's device association to an IoT GW. • In this study, several simulations are performed to evaluate our learning-based algorithm.
The remainder of the paper proceeds as follows; Section 2 describes the related research work. Section 3 describes the system model of the proposed ML-enabled algorithm. Section 4 describes the assessment of the problem statement using the MAB-based mechanism. Section 5 evaluates the performance of the proposed ML-enabled algorithm with the help of simulation results. Finally, Section 6 provides conclusions from this work.
Related Research Work
There are several research surveys and articles published on the IoT network systems. These studies mainly summarized the topics like age of information in massive IoT [7] and wireless sensor networks in massive IoT systems [8]. Most of their work targets the application design perspective of IoT systems. The authors discuss the influences of the buffers, queueing modeling, traffic scheduling, and MAC layer channel access mechanism to better design applications for IoT that require frequent updates from the network. In [9], the authors propose a data reduction algorithm to reduce the burden of the IoT gateway. There have been related research in this area, especially in the IEEE 802.11 WLAN context, which has addressed the challenge of the number of devices associated with a GW (or access point in WLAN terminology). In [10], the authors proposed an algorithm for the device's association for optimal throughput achievement. Their proposed mechanism estimated AP utilization based on the required throughput of the device to carry out the association. The authors in [11] proposed two different algorithms; the first was based on the quality of the up/down link channel, and the second used the airtime of each WLAN. In [12], the authors used the average payload of the WLAN to associate the network devices. Their work had the limitation of requiring changes to the standard messaging frames. In [13], the devices were moved (re-associated) to the AP with the lowest network load. However, in their proposed mechanism, the link quality was not considered, which significantly reduces the throughput of a WLAN. In [14], the authors explored a centralized approach for device association by introducing an online GW. Similarly, in [15], the authors introduced a software-defined network (SDN) solution for the unbalanced association of the devices with the GWs. In their proposed solution, the GW with congested associations was requested to re-associate by adjusting the transmission power.
Several authors have contributed to the use of ML-enabled techniques for device association purposes, for example, [16] utilized a reward-based algorithm for a GW AP selection. In [17], the authors proposed a MAB algorithm for the GW selection procedure by extending the ε-greedy exploration/exploitation mechanism. Moreover, a deep neural network-based wireless user association mechanism with six hidden layers was proposed in [18]. In [19], the authors proposed an association rule based on a mean-field game solver using MAB games. In [20], an optimization problem has been formulated to maximize the total gain of the network in different IoT requirements. The authors of this work proposed a stable matching mechanism algorithm to solve this high-complexity problem of IoT slices. An advocate RL algorithm is proposed to enable practical implementations and employs a learning framework to learn different IoT network dynamics [20]. The research in [21,22] studied IEEE 802.11 WLANs in a dense network for WLAN device association with deterministic (fixed) APs. In [23], the authors proposed to turn off as many GWs as possible based on low network load. In [24], the authors proposed a centralized device association with GWs based on an RL. Their proposed mechanism used minimized subscriber dissatisfaction in their RL mechanism.
Critical analysis: In light of this related research work, the current study sees that device association, especially for IoT network systems was either conducted in a traditional way (high signal strength strategy) or a heuristic way (retaining the best association as always). However, such strategies may lead to less efficient situations due to relying on high signal strengths only, which overburden GWs with the nearest sensors devices. Moreover, even in a heuristic mechanism case, an optimal association strategy cannot be reached due to the use of the highest experienced data rate as an instantaneous performance metric. Therefore, it is suggested to utilize ML-enabled solutions for association mechanisms.
The state-of-the-art mechanism for SD association in IoT networks is to examine all nearby available GWs and select the one with the highest RSSI. However, choosing the nearby GW (g) based on the highest RSSI may not be the best action at a particular moment, as it may lead to an unevenly connected device distribution, where network resources remain unused. Therefore, this work proposes an RL-based learning algorithm for IoT devices (SDs) as an agent. In RL-based ML techniques, the MAB addresses the problem where an interacting agent selects an action among a given number of actions (referred to as arms in a MAB algorithm) [6]. An agent chooses one action from the available set of actions alternatively, one at a time. Every time an arm is pulled as an action, a reward (feedback) is generated, which allows the agent to evaluate the performance of that specific action/arm. The objective of the agent is to learn the action with the optimal accumulated reward value. One of the challenges faced by MAB-based mechanisms is to consider the exploration and exploitation. In exploration and exploitation, an agent must balance the tradeoff between learning faster or more slowly. A learning rate parameter is introduced for this purpose. The tradeoff between slower learning and faster learning is crucial, as slow learning may waste too much time, while a faster learning rate may lead to less exploration. The ultimate goal of a MAB algorithm is to search for the optimal action that produces the maximum reward. Once an agent finds a converged maximum value action, say the agent has learned the environment. One of the ways to calculate this is to carefully measure a regret function for the actions performed by the agent. A regret function L i,t of an agent device (d i ) at time t after T total number of time steps can be given by [6], where R * i,t defines the reward of an optimal action at time t. The objective of the agent is to minimize the expected regret with the passage of time; that is, One of the challenges with MAB implementation is to choose a fair tradeoff between exploration and exploitation. In this study, an ε-greedy method is used for exploration with a probability (ε) and exploitation with the probability (1 − ε). Each IoT device acts as an agent and implements the ε-greedy method to explore/exploit to choose the available arms (GWs) in its sensing range. An agent receives a reward for each GW selected and accumulates these for future exploitation. At each iteration of time t, the devices perform reassociation with the nearby GWs. The experienced average throughput τ i of a device from a specific arm (GW) is used as the reward for the arm. Thus, an instant reward R i,t for a GW g i at time t is calculated as the average of the rewards received by an agent device from the associated GW g i as follows, where τ i is the achieved throughput of the device and is measured as the number of successfully transmitted data per unit of time. As mentioned earlier, ε-greedy is implemented at each sensor device (SD). We consider each of the GWs in the range as an arm for the sensor device. Thus, every SD as an agent keeps track of the reward collected against a specific GW selection, that is, the achieved performance. In Fig. 2, it shows the flowchart of the proposed MAB-based GW association algorithm, where the exploration and exploitation play a key role in selecting the GW either using the state-of-theart (random selection, which is exploration) or the GW with the maximum accumulated reward (which is exploitation). Each time an exploration/exploitation leads to a reassociation with the GW. The εgreedy explained in Fig. 2 works in the iteration of time t, which represents the association rounds. In addition, this work uses the achieved average throughput as a reward for each of the GWs. Thus, a sensor device accumulates the average throughput received as the result of any GW selection, and based on this accumulated throughput, the reassociation for the GW is performed using the ε-greedy mechanism.
One of the challenges for an ML-enabled algorithm is to extract the learning features from the data set and effectively tune its hyperparameters. Since this study is proposing an RL-based association mechanism for device association in IoT networks, which concerns how an intelligent agent (device equipped with RL capabilities) takes actions in an environment to maximize the notion of the accumulated reward. Our proposed ML-powered association mechanism differs from supervised or unsupervised learning in that it does not require explicit correction of extracted features with suboptimal actions. Since supervised learning and unsupervised learning are both used for problems where the desired output is known and provided in the training data, it is inconvenient to use such models for a dynamic environment like IoT. However, the benefit of RL is that it can handle problems with sparse or delayed rewards, which may not be well-suited for supervised and unsupervised learning. Additionally, RL can handle problems with long-term dependencies, such as sequential decisionmaking tasks, which can be challenging for other learning methods. It finds a balance between the exploration of the environment and the exploitation of the learned knowledge of the environment (learned in terms of the actions with the highest accumulated reward). One of the key parameters required for exploration/exploitation strategies such as the ε-greedy strategy is the ε. The tuning of this key parameter requires an educated guess depending on the level of exploration and exploitation. For example, a low value of ε allows the agent to exploit (1 − ε) more and explore less; on the other hand, a high value of ε allows the agent to exploit less and explore more. In this study, a 0.2 value of ε is used, so that an agent could exploit 80% of the time and explore 20%.
Simulation Setup
A simulation setup using MATLAB environment is developed, where a grid of 100 m 2 is created to randomly place IoT GWs. Table 1 shows some of the important simulation parameters used for the performance evaluation. To evaluate our proposed association mechanism, this study performed several simulations in a 100 m 2 area with eight IoT GWs and 32 IoT devices placed randomly in the area. A wireless network with a channel bandwidth of 20 megahertz (MHz) was used with a transmission power of 20-decibel milliwatts (dBm) and an interference signal level, clear channel assessment (CCA) of −82 dBm, and −72 dBm for the RSSI level. Fig. 3a shows an IoT environment with eight GWs randomly placed and 32 IoT devices using the standard RSSI-based mechanism to associate with the GWs in their range. We see from the figure that a few of the GWs were unnecessarily overloaded, such as GW 8, 6, and 1. This happens as every device tries to associate itself with the nearest GW with the highest received signal strength. This also affects the overall network performance in terms of the waste of resources and an increase in a collision due to congestion. In addition, it indicates that two of the GWs have no association with the sensor devices due to the comparatively longer distance between the SDs and GWs. However, these GWs may have access to the nearest SDs to lessen the burden from other overloaded GWs. More simulations are performed on a similar network to create an SD-GW association with the use of our proposed MABbased mechanism. Fig. 3b shows that GWs 5 and 4 were also associated with nearby sensors, which lessened the overload from GW 1 and GW 3. GW 6 is not highlighted as GWs 4-5 are; however, one can see that it has also contributed to the efficient association procedure. In this paper, the proposed association mechanism is evaluated in terms of the enhanced average (mean) throughput (that is, the successfully received data rate). Fig. 4 shows the comparison of the normalized throughput of an IoT network system with a fixed (deterministic association), RSSIbased (standard), heuristic (best from the past/history), and our proposed MAB-based mechanism. The results showed that in the case of the deterministic association mechanism (fixed), the system achieved the lowest normalized throughput due to the fixed number of GWs and SDs. Our proposed mechanism allows an IoT network system to explore and exploit the GWs with less load and associate the SDs with less loaded GWs. However, the heuristic algorithm reached closer to the proposed MABbased mechanism, due to its nature of utilizing the best experience from the past, which is somewhat similar to a MAB-based algorithm. In the case of an ML-enabled GW association, the network may also suffer from low throughput, which was mainly due to the exploration period of the algorithm. During the exploration, our proposed MAB-based algorithm searches for the best available GWs and may associate SDs with already overloaded GWs, resulting in lower network throughput. This study further evaluates the proposed MAB-based algorithm in terms of network satisfaction. The network satisfaction ( ) was calculated as follows,
Results and Discussion
where T * is the number of times a network is satisfied. In the above network satisfaction calculation, w determines whether the network is satisfied or not satisfied and is given by The above equation explains that the network is satisfied if the received data rate τ i at i th iteration is greater than or equal to the required data rate, that is, τ r . Fig. 5 shows a better representation of the performance of our proposed mechanism. This figure shows how the proposed MAB-based mechanism achieves network satisfaction over some time (simulation duration). As shown in the figure, an RSSI-based mechanism remains constant due to the one-time association based on the strongest signal strength strategy. Similarly, a deterministic approach (fixed association between SDs and GWs) may perform better than the RSSI-based approach due to prefixing the problem of device association. However, since wireless networks are usually dynamic and changing rapidly, this limits the performance of a fixed network association algorithm. On the other hand, our proposed MABbased algorithm allows the network to continue to be increasingly satisfied, even more so than the heuristic algorithm. The proposed MAB-based mechanism provides a higher average received data rate than the heuristic mechanism. One of the reasons for this performance improvement is that a heuristic mechanism tries to use the association links with the highest experienced data rate, which is an instantaneous performance metric. However, our proposed MAB-based mechanism exploits the association links with the highest accumulated reward, which shows optimal links over a longer period. This is also justified by Fig. 5, where the agents using the heuristic mechanism converged earlier and at a level lower than our proposed mechanism. Fig. 6 further explains this by adding more box plots to our results showing how the network devices reached their satisfaction level (4 Mbps in our case) using different association mechanisms. In the figure, it can be seen that the average successfully received data from a fixed algorithm remained very low compared to the required data rate. However, with the heuristic and MAB-based algorithms, the network was more satisfied; that is, it reached its required data rate.
Conclusions
Today's IoT networks include a massive number of connected sensor devices (IoT). Each of the sensor devices (SD) must associate itself with a nearby IoT gateway (GW) to transfer the collected sensory data. For this purpose, SDs are permitted to associate with any GW in the network, choosing naturally the one from which higher power is received (RSSI), regardless of whether or not it is the most ideal choice for the network's execution. Recently, a rise in the use of ML techniques for wireless networks as a viable way to determine the effect of various models in the system, and reinforcement learning (RL) is one of those ML models. In this research work, we used an RL-based model for the association of SDs with the nearby GWs in an IoT network. A multi-armed bandit solution was used to find the best GW to associate with a sensor device. The mathematical convergence of a greedy MAB-based mechanism helped achieve this purpose to evaluate our proposed mechanism alongside the standard and related mechanisms. The evaluation results indicated that our intelligent MAB-based mechanism enhances the association as compared to other approaches. The normalized throughput results indicated that the proposed mechanism also enhanced the system performance.
Limitations and Future Works
One of the challenges arrive with MAB-based algorithms is the trade-off between exploration and exploitation. In a network environment like GW selections for IoT devices is always very dynamic and changing. Therefore, an instant limitation that arrives with our proposed mechanism is the selection of ε-greedy parameters wisely. A higher value of ε (more exploration) may lead to a delayed learning rate. Similarly, a low value of ε (more exploitation) leads to immediate exploitation of the environment with very little information. In future works, we aim to explore the educated guess for and ε value. Moreover, we also aim to work on the other possible algorithms for exploration and exploitation, such as SoftMAX and upper confidence bound (UCB) [6]. | 2023-06-28T13:03:58.669Z | 2023-01-01T00:00:00.000 | {
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259974349 | pes2o/s2orc | v3-fos-license | Practical recommendations for home-nebulized corticosteroid use in children aged ≤ 5 years with asthma: A review and advisory group consensus
Background: Despite nebulized budesonide being identified by the Global Initiative for Asthma report as a viable alternative to inhaled corticosteroids (ICS) delivered by pressurized metered-dose inhalers (pMDIs) with spacers, practical guidance on nebulized corticosteroid use in the pediatric population remains scarce. Objective: To review the current literature and provide practical recommendations for nebulized budesonide use in children aged ≤ 5 years with a diagnosis of asthma. Methods: A group of 15 expert pediatricians in the respiratory and allergy fields in Thailand developed Delphi consensus recommendations on nebulized budesonide use based on their clinical expertise and a review of the published literature. Studies that evaluated the efficacy (effectiveness) and/or safety of nebulized budesonide in children aged ≤ 5 years with asthma were assessed. Results: Overall, 24 clinical studies published between 1993 and 2020 met the inclusion criteria for review. Overall, results demonstrated that nebulized budesonide significantly improved symptom control and reduced exacerbations, asthma-related hospitalizations, and the requirement for oral corticosteroids compared with placebo or active controls. Nebulized budesonide was well tolerated, with no severe or drug-related adverse events reported. Following a review of the published evidence and group consensus, a treatment algorithm as per the Thai Pediatric Asthma 2020 Guidelines was proposed, based on the availability of medications in Thailand, to include nebulized budesonide as the initial treatment option alongside ICS delivered by pMDIs with spacers in children aged ≤ 5 years. Conclusion: Nebulized budesonide is an effective and well-tolerated treatment option in children aged ≤ 5 years with asthma.
children aged < 5 years had more severe asthma with higher exacerbation rates compared with adolescents and adults aged < 55 years. 4Moreover, the National Health Interview Survey conducted in the United States (US) between 2001 and 2010 revealed that children aged ≤ 4 years had the highest rate of asthma-related hospitalization (5.2 per 100 persons with asthma) of any age group. 5Consequently, the management of pediatric asthma, the goal of which is to achieve good symptom control, maintain normal activity levels, and minimize the future risk of exacerbations, requires particular attention. 6Importantly, relative risks and benefits of treatments require careful consideration, especially in young children, to ensure normal physical and psychosocial development. 6he "Global Strategy for Asthma Management and Prevention 2021" report developed by the Global Initiative for Asthma (GINA) recommends a daily inhaled corticosteroid (ICS) for maintenance treatment of asthma in children aged ≤ 5 years at GINA treatment steps 2-4 (mild to moderate-to-severe asthma). 6As young children are unable to use dry powder inhalers or pressurized metered-dose inhalers (pMDIs) without assistance, 7 ICS can be delivered via pMDIs with spacers or nebulizers. 6However, aerosol delivery using pMDIs in infants and young children can present significant challenges, including a lack of cooperation, inability to breathe through the mouth and hold one's breath, rejection of masks, small tidal volume, and crying.All these factors can affect the breathing patterns of infants and adequacy of the seal between the mask and the face and consequently the drug dosage delivered to the lungs. 8][12] Nebulizers provide more consistent and less error-prone drug delivery compared with pMDIs with spacers, 12
Introduction
Asthma is a chronic inflammatory respiratory disease that affects approximately 339 million patients worldwide across all age groups. 1 The global prevalence of childhood asthma has significantly increased over the past 40 years, although substantial variations in the prevalence of asthma symptoms in children have been reported worldwide, with up to 13 fold differences between countries. 2Notably, the prevalence of childhood asthma in Thailand stands at over 10%, which is comparable to the global prevalence at 11.2%. 1 Childhood asthma exerts a substantial financial burden on healthcare systems, especially from direct costs associated with exacerbations and subsequent hospitalizations. 3Indeed, a retrospective study in Thailand reported that annual direct medical costs were the highest in patients diagnosed with asthma before 5 years of age. 3 Furthermore, treatment costs increased significantly for patients who had at least one exacerbation compared with those without an exacerbation. 3 population-based cohort study investigating over 0.4 million patients in the United Kingdom (UK) showed that [title/abstract] OR suspension [title/abstract]) AND (pediatric [title/abstract] OR paediatric [title/abstract] OR children [title/abstract] OR infants [title/abstract]) NOT (review OR case series OR case report).Additional studies were identified via a Google scholar search.The PubMed search was updated prior to final submission of the manuscript to include publications up to 25 January, 2023.All 15 committee members participated in the inclusion/exclusion of retrieved articles from all literature search results.Studies were restricted to clinical studies, including RCTs, observational studies, and real-world studies, that were published in English and that assessed the efficacy (effectiveness) and/or safety of nebulized budesonide either as maintenance therapy or in the management of acute exacerbations in children aged ≤ 5 years with an asthma diagnosis.Case reports/series, review articles, systematic reviews, and meta-analyses were a priori excluded from the study: case reports, because they represent low quality evidence; 22 the latter three article types, because they are not primary data sources.Based on the review of published studies and the opinions of clinicians, an algorithm on asthma management in children aged ≤ 5 years was developed to provide health care providers (HCPs) with simplified practical recommendations to ensure seamless transition of evidence-based medicine into routine clinical practice.
In addition to the literature review, the Delphi technique 23 was used to gain consensus on a questionnaire comprising 11 questions related to asthma management in children aged ≤ 5 years, that was developed through verbal discussions prior to the steering committee meeting and completed in one round during the meeting.Statements from the Delphi technique that obtained a mean consensus score of at least four out of five were concluded to be expert recommendations.
require minimal patient cooperation, 11 are easy to use as they do not require breath-holding, 11 and offer adjustable doses 11 with the potential to mix with other compatible nebulized drugs, such as bronchodilators. 11,13Moreover, nebulizers cause less irritation because of their hydrating effect on the airways 10 and can be used with supplemental oxygen. 11udesonide, a second-generation ICS, provides fast onset of local action, dissolves rapidly in human bronchial fluid, 14 and has a strong bronchial vasoconstrictive effect. 15Additionally, the unique esterification of budesonide prolongs its anti-inflammatory action and improves airway selectivity, 16 resulting in low systemic exposure and side effects with favorable long-term tolerability. 17Notably, a systematic review in children aged ≤ 5 years, which included nine articles on asthma maintenance therapy, confirmed that nebulized budesonide significantly reduced the risk of further asthma exacerbations compared with placebo, cromolyn sodium, and montelukast in randomized controlled trials (RCTs). 18][21] Although pMDIs with spacers are generally used to deliver corticosteroids in pediatric patients with asthma, the latest GINA report 6 has identified nebulized budesonide as the only effective and viable alternative for patients who may be unwilling or unable to use a pMDI with the correct inhaler technique. 7However, although expert consensus recommendations on asthma management using nebulized corticosteroids in children and adolescents with acute asthma exacerbations in the hospital setting have been previously published, 7 practical guidance on the use of nebulized budesonide in young children remains scarce.Therefore, we present expert consensus recommendations on the use of nebulized corticosteroids in multiple settings specifically in children aged ≤ 5 years with a diagnosis of asthma based on published evidence and clinical experience.
Methods
A steering committee comprising 15 hospital-based pediatric pulmonologists and allergists treating asthma at public and private tertiary medical centers across Thailand was convened in Bangkok, Thailand, on November 27, 2020, to discuss the role of nebulized corticosteroids in asthma management in children aged ≤ 5 years.Committee members were jointly selected by AstraZeneca and a group of pediatric advisors who had worked collaboratively with the Pediatric Medical Association in Thailand (Pediatric Society of Thailand) as guideline committee members and were invited to participate in the current study based on a relevant publication history and their clinical experience and expertise in pediatric asthma.A narrative review of published studies on nebulized budesonide in children aged ≤ 5 years with asthma was undertaken to evaluate the evidence and develop expert recommendations through panel discussions for implementation in clinical practice.
Publication
Efficacy outcomes Safety outcomes Connett, 1993 Cough scores improved significantly with budesonide (p < 0.05) One child developed a facial skin rash, which resolved after parents were reminded to wipe their child's face after drug administration Ilangovan, 1993 Requirement for treatment with oral steroids reduced and overall health as scored on a visual analog scale improved with nebulized budesonide (p < 0.05 for both) Two children developed an eczematous rash in the area of the face mask.Treatment was not stopped and the rash was controlled by applying a barrier cream before nebulization and washing the face afterwards Wennergren, 1996 Although an overall minimal effective maintenance dose could not be demonstrated, 47% of patients achieved symptom control on 0.
Kemp, 1999
All budesonide inhalation suspension doses produced significant improvements in nighttime/daytime symptoms and decreases in reliever medication use, while improvements in FEV(1) were observed in the 0.5 mg and 1 mg budesonide inhalation suspension groups (p < 0.05).
Treatment was discontinued by 4 children because of AEs: bronchospasm (1 child each in the 0.25 mg and 0.5 mg budesonide groups), meningitis (1 in the 0.25 mg budesonide group), and cellulitis (1 in the 0.5 mg budesonide group).No significant differences between placebo and any budesonide group in basal and ACTH-stimulated cortisol levels were reported.
Baker, 1999
All budesonide inhalation suspension doses produced statistically significant improvements in lung function and daytime and nighttime symptoms compared with placebo (p ≤ 0.05).
The incidence of AEs deemed possibly or probably related to treatment was similar between placebo and budesonide groups.Mean height and weight changes were similar between the treatment groups.No clinically relevant changes in basal cortisol levels were found in any treatment group.
Mellon, 2000
Budesonide inhalation suspension via a face mask or mouthpiece resulted in clinical improvements in nighttime and daytime asthma symptoms compared with placebo (p < 0.05).
The overall incidence, type, and severity of non-asthma-related AEs were similar in the placebo and budesonide groups.The overall incidence of any AEs among budesonide-treated children was slightly higher in those who received treatment with face masks (85%) than mouthpieces (78%).
Leflein, 2002
The budesonide group had a mean (median) asthma exacerbation rate of 1.23 (0.99) per year compared with 2.41 (1.85) for the cromolyn group, significantly longer times to the first exacerbation and first use of additional long-term asthma medication, greater improvements in asthma symptom scores, reduced use of reliever medication, and fewer urgent care visits.
There were no clinically relevant differences in the incidence or type of AEs between groups.Levels of basal and ACTH-stimulated plasma cortisol were similar between groups.
Murphy, 2003
Improvements from baseline in domain-specific (activities and emotional function), and total PACQLQ scores were greater at each time point (weeks 8, 28, and 52) for caregivers of patients treated with budesonide compared with caregivers of patients receiving cromolyn sodium.
In the budesonide group no discontinuations were attributable to AEs or disease deterioration.Other safety parameters were not assessed or reported.
Delacourt, 2003
Overall, 51.7% and 40.4% of patients on budesonide and beclometasone dipropionate treatments, respectively, did not experience major exacerbations (p = 0.28).Both treatments were also associated with marked reductions in the number of nights with wheezing and the number of days of oral steroid use.
The incidence and AE profile were equivalent between the groups.Urinary cortisol and urinary cortisol/creatinine ratios were not significantly affected.
Berger, 2005
Compared with patients receiving placebo, the mean percentage of symptom-free days was numerically greater for patients receiving 0.5 and 1.0 mg budesonide (37.5%, 48.8%, and 43.4%, respectively).
Treatment-related differences in physicians' global assessments of patient health status did not reach statistical significance, but physicians rated 90% and 85% of patients in the 0.5-mg budesonide and 1.0-mg BIS groups, respectively, a ''great deal better'' or ''somewhat better'' compared with 67% of patients in the placebo group.Budesonide treatment resulted in a greater reduction in daytime and nighttime symptoms compared with placebo.
The safety profile of budesonide was similar to that of placebo, with no suppressive effect on adrenal function.
Adverse events were mild and of similar frequency in both groups.
Camargo, 2007
Patients who had a claim for budesonide inhalation suspension had a lower risk of a subsequent hospitalization or an ED visit (HR, 0.55; 95% CI, 0.41-0.76;p < 0.001) than patients who did not have budesonide inhalation suspension claims. N/R
Decimo, 2009
Airway resistances were significantly reduced at day 7 (p < 0.01 flunisolide; p < 0.05 budesonide) and day 21 (p < 0.05 flunisolide; p < 0.05 budesonide) versus baseline in both groups, although at day 7 the reduction occurred faster in the flunisolide group than in the budesonide group (p < 0.01).During the first 7 days of treatment, symptom scores decreased in both groups; however, the decrease was greater in the flunisolide group (p < 0.05).
Morning serum cortisol level after 21 days of treatment did not differ versus baseline (p = 0.5) (Table IV).The other blood parameters evaluated were within normal limits in both groups.There were no cases of oral Candida infection or dysphony.
Zeiger, 2011
The daily regimen of budesonide did not differ significantly from the intermittent regimen with respect to the frequency of exacerbations, with a relative rate per patient-year of 0.99 (95%CI, 0.71-1.35;p = 0.60).
There were no significant differences in the proportions of children with serious AEs (including all hospitalizations) and nonserious AEs between groups.Five children in the intermittent-regimen group and four in the daily-regimen group were hospitalized for asthma exacerbations.
Nagakura, 2012
N/R No significant differences in salivary cortisol levels were seen from baseline for budesonide and cromolyn sodium inhalation suspensions, suggesting that they are safe and without any inhibitory effects on adrenocortical function.There were no drug-related adverse reactions recorded in any group.
Szefler, 2013
No difference was observed between the budesonide inhalation suspension and montelukast groups in median time to the first additional asthma medication over 52 weeks (183 vs 86 days).Statistically significant differences were observed in favor of budesonide inhalation suspension over montelukast in the percentage of patients requiring oral steroids at 52 weeks (21.9% vs 37.1%; p = 0.022), the rate (number/patient/year) of additional courses of medication (1.35 vs 2.30; p = 0.003), the rate of additional oral steroid therapy (0.44 vs 0.88; p = 0.008), and caregivers' ability to manage patients' symptoms (p = 0.026) The incidence of most commonly reported adverse events was similar in both groups and mostly mild.No discontinuations due to adverse events were reported with budesonide, versus 4 discontinuations with montelukast (asthma [n = 2], upper respiratory tract infection, and pneumonia).Other safety parameters were not assessed or recorded.
Yanagida, 2015
There were no significant differences between the two groups in terms of the severity of attacks and duration of management or in terms of therapeutic efficacy, duration of wheezing, or period of hospitalization.The frequency of inhalations on days 3 to 6 of hospitalization was lower in the budesonide inhalation suspension group than in the methylprednisolone group, and the cortisol level at discharge was significantly higher in the budesonide inhalation suspension group (13.9 ± 6.1 μg/dL) than in the methylprednisolone group (8.0 ± 2.1 μg/dL) (p = 0.008) A significantly higher number of children in the methylprednisolone group (5 of 15) versus budesonide group (0 of 18; Fisher exact test, p = 0.013) had cortisol levels below the reference value of 4 mg/dL.Other safety parameters were not assessed or recorded.
Table 2. (Continued) Publication Efficacy outcomes Safety outcomes
Zhou, 2016 Symptom scores in the severe disease group were higher than those in the mild group at weeks 1, 3, and 5 (p < 0.05), but not at week 7 (p > 0.05).Further, more patients in the mild group achieved disease control at any time point (98.6% at 3 weeks and 99.7% at 7 weeks), compared with the patients in the severe group (p < 0.001).The proportion of patients requiring bronchodilators differed between the groups until week 5 (p < 0.001).
No severe or drug-related adverse events were reported.
Bian, 2017
The overall effective rate of treatment in the control group was 73.33% (40% with marked improvement, 33.33% with some improvement and 26.67% with no improvement) and that in the treatment group was 96.67% (73.73% with marked improvement, 23.33% with some improvement and only 3.33% with no improvement); p < 0.05.Overall improvement in FEV(1), FVC, FEV(1)/FVC and PEF was higher in the treatment group than the control group (p < 0.05).ESR and CRP levels in the treatment group were improved to a greater degree than in the control group (p < 0.05).
There were only minor adverse reactions in two patients in the treatment group, and the overall rate of adverse reactions was not significantly different between the two groups.
Saito, 2017
Wheezing disappeared after an average of 5 days, with no significant difference in days of oxygen use.
Serum cortisol levels remained unchanged in the budesonide inhalation suspension group and were significantly decreased in the prednisolone group compared with the budesonide inhalation suspension group (p = 0.0036).On the 4 th day of hospitalization serum cortisol levels were 17.0 μg/dL and 10.9 μg/dL in the budesonide and prednisolone groups, respectively, with significant suppression in the prednisolone group.No adverse events were reported in either group.
Razi, 2017
The discharge rate in the budesonide inhalation suspension group was significantly higher than that in the placebo group (p < 0.001).
N/R 3. ICS are recommended as the preferred maintenance medication for recurrent wheezing in children aged ≤ 5 years, especially in those experiencing frequent symptoms, using relievers more than twice a month, or with a history of ≥ 3 exacerbations per year or ≥ 2 severe exacerbations in 6 months.4. As reported by GINA, 6 adequate efficacy and safety data in children are available for nebulized budesonide (≥ 1 year) and fluticasone propionate pMDI (≥ 4 years).
Nebulization requires minimal patient cooperation
and results in fewer errors in drug delivery in young children with asthma when compared with other inhalation methods.
Summary of expert consensus and clinical recommendations included in the proposed treatment algorithm Summary of expert consensus
The following statements received an average score of at least four out of five using the Delphi technique and were therefore considered to be expert recommendations: 1.The goal of asthma treatment in children is to achieve symptom control; therefore, patients should be categorized by their level of control (controlled vs uncontrolled).
Education of children's families and healthcare
workers is a key factor in achieving asthma control in children.
6. Nebulized budesonide is a viable ICS treatment option along with budesonide delivered by pMDIs with spacers in children with asthma aged ≤ 5 years.7. Should a nebulized mode of delivery be selected, jet nebulizers may be the preferred delivery devices for nebulizing ICS. 8.The use of nebulizers should be recommended for pediatric patients who are unwilling or unable to use a pMDI with a spacer properly.
Unmet needs in asthma management in children aged ≤ 5 years
In recent years, the prevalence of asthma has increased globally in children and adolescents, particularly in low-and middle-income countries. 48oreover, asthma-related hospitalizations are particularly common in children aged < 5 years, with a rising prevalence over the past two decades. 48There are several unique challenges pertaining to pediatric asthma management that require urgent attention.Notably, adherence to inhaled medication is generally poor among children with asthma; indeed, a systematic review conducted in the US, Canada, and the UK reported adherence rates ranging from 28% to 67%, 49 emphasizing the need for educational initiatives to improve adherence to asthma medications.Additionally, in non-specialist practices in rural areas of Thailand, most of the burden of pediatric asthma results from underdiagnosis or misdiagnosis (cross-diagnosis with pneumonia, virus-induced wheezing, and bronchiolitis) and underuse of and limited adherence to maintenance medications.This indicates the need for accurate and timely diagnosis and management aligned with evidence-based recommendations.Notably, adherence to maintenance medications remains problematic across practice types, particularly in non-specialist practices.Therefore, while several guidelines recommend the referral of pediatric patients with asthma to a specialist to minimize the burden on healthcare systems, 50 simplified treatment algorithms that focus on inhaled ICS treatment with the correct inhaler technique are warranted.Thus, an expert consensus on the optimal use of nebulized budesonide in young children was urgently required.
Implementation of budesonide nebulization in maintenance therapy in pediatric asthma
Since the goal of asthma treatment in children is to achieve symptom control, pharmacological management of children should focus on age-specific treatments according to clinical severity and the level of asthma control; this is determined by the interaction between a patient's ongoing treatment, environment, and psychosocial factors. 51ebulized budesonide is the only nebulized ICS recommended for children aged ≤ 5 years in the 2021 GINA report 6 due to its broad evidence base in childhood asthma.Several trials in young children have shown that nebulized budesonide significantly improves asthma symptoms, including exacerbations, when compared with placebo [24][25][26]39 and disodium cromoglycate 32,40,42 in RCTs. In double blind, placebo-controlled study of 100 children aged 7-72 months hospitalized for asthma exacerbations, Razi et al. 52 showed that 2 mg/day of nebulized budesonide added to standard treatment significantly reduced the length of stay in hospital.Another study by Razi and colleagues, 43 which included 100 preschool children aged ≤ 6 years who presented to an ED with acute wheezing attacks, reported that addition of nebulized budesonide significantly decreased hospital readmission rates and increased discharge rates. Althougha 52-week, open-label, randomized, active-controlled, multicenter study in 203 children aged 2−4 years showed no significant differences between nebulized budesonide and montelukast in median time to first additional asthma medication (183 vs 86 days), statistically significant differences were observed in favor of nebulized budesonide over montelukast in the proportion of patients requiring oral steroids at 52 weeks (21.9% vs 37.1%; p = 0.022) and caregivers' ability to manage patients' symptoms (p = 0.026).20 Moreover, in two separate RCTs in young children (aged ≤ 5 years) with asthma exacerbations, nebulized budesonide was therapeutically equivalent to the systemic corticosteroids prednisolone and methylprednisolone for symptom control 21,28 and period of hospitalization.21 However, in contrast to systemic corticosteroid treatments, nebulized budesonide did not suppress adrenocortical function.21,28 Based on the review of 24 published clinical studies and the consensus reached by the expert panel, a treatment algorithm was proposed (Figure 3).This algorithm was adapted from the Thai Pediatric Asthma 2020 Guidelines 47 as per the availability of drugs in Thailand and included nebulized budesonide as the initial treatment option alongside ICS delivered by pMDIs with spacers in children aged ≤ 5 years.The algorithm proposes that young children who have frequent symptoms, experience a severe exacerbation, or require the use of reliever medications more than twice a month should be prescribed low-dose ICS (0.5 mg nebulized budesonide/day, 0.1-0.2mg budesonide/day delivered via pMDI with a spacer, or 0.125 fluticasone/day delivered via pMDI with a spacer) with as-needed reliever medications. Alernatively, a regular leukotriene receptor antagonist (LTRA; 4 mg/day) or intermittent high-dose nebulized budesonide (1-2 mg/day for 7 days after the first sign of acute respiratory tract infection) can be prescribed.The initial recommended dosage for nebulized budesonide during periods of severe asthma exacerbations or while tapering off OCS is 0.5-1 mg twice daily, whereas the dosage for maintenance treatment is 0.25-0.5 mg twice daily.The recommended oxygen flow for nebulization is 6-8 liters.Home nebulization can also be used to administer multiple drugs simultaneously, i.e., budesonide with albuterol or ipratropium bromide together in one dose. Asneeded reliever medication is usually sufficient for children with non-severe, infrequent symptoms.
As recommended by GINA, 6 symptoms should be reassessed at intervals of 1-3 months.Patients with well-controlled symptoms can continue treatment and step down their ICS doses when there is no risk of future exacerbations in the next 6-12 months.Once full asthma control is achieved, HCPs can consider stopping ICS treatment altogether.However, in patients whose symptoms are not fully controlled, HCPs should either increase the ICS dose or add another maintenance medication, such as an LTRA.Upon periodic reassessment at 1-3 months, HCPs should consider further increasing the ICS dose, adding an LTRA, or prescribing intermittent high-dose ICS.Finally, HCPs should consider referring patients with uncontrolled asthma to specialists to ensure optimal asthma management.
A real-world analysis of claims data from > 10,000 patients aged ≤ 8 years with ≥ 1 asthma exacerbation requiring hospitalization or ED visit from the Florida Medicaid database revealed that patients treated with nebulized budesonide had a 71% lower risk of repeat asthma exacerbations than those receiving other ICS medications delivered through other modes (hazard ratio [HR], 0.29; 95% confidence intervals [CI], 0.18-0.48;p < 0.001). 37oreover, treatment with nebulized budesonide in the first 30 days after hospitalization or an ED visit for asthma was associated with a 45% reduction in the risk of subsequent asthma-related hospitalizations or ED visits (HR, 0.55; 95%CI, 0.41-0.76;p < 0.001). 37 retrospective analysis of three randomized, double-blind, placebo-controlled, 12-week studies 36,39,53 showed that all nebulized budesonide dosage regimens (0.25-1 mg once daily; 0.25-1 mg twice daily) were effective in improving asthma control days, symptom-free days, and rescue medication-free days in pediatric patients aged 6 months to 9 years with mild to moderate persistent asthma. 54Two meta-analyses have also confirmed the efficacy of nebulized budesonide in children with asthma.A meta-analysis of nine studies (n = 1,473) reported that addition of nebulized budesonide to systemic corticosteroids decreased the length of hospital stay by more than 1 day and significantly improved the acute asthma score among children (birth to 18 years) with acute asthma in ED settings. 55Another meta-analysis of 21 RCTs reported that nebulized budesonide reduced the hospitalization rate (random effects-odds ratio [RE OR], 0.34; p = 0.003) and worsening of symptoms (RE-OR, 0.38; p = 0.001) compared with conventional treatments in 12,787 patients, including pediatric patients (6 months to 18 years) with asthma who were admitted to an ED. 56verall, nebulized budesonide is well tolerated; an analysis of three pooled randomized, double-blind, placebo-controlled, multicenter, short-term (12 weeks) studies 36,39,53 revealed that the incidence, type, and severity of nonasthma-related adverse events were similar between nebulized budesonide and placebo in infants and young children (6 months to 8 years of age) with persistent asthma. 19Furthermore, long-term treatment with nebulized budesonide, as assessed in 52-week extension studies of the 12-week double-blind trials, was well tolerated, with the incidence of reported adverse events comparable between nebulized budesonide and other conventional asthma therapies. 19oreover, the OCS-sparing effects observed with nebulized budesonide 20,21,25,26,28 may improve the overall safety of asthma therapy in patients with persistent asthma and during acute exacerbations. 57However, to date, no study has directly compared the efficacy and safety of nebulized budesonide with those of budesonide delivered using pMDIs with spacers.Therefore, additional clinical studies are warranted to examine the comparative efficacy or effectiveness of these two modes of budesonide delivery in children aged ≤ 5 years.
Based on the results of published studies, daily low-dose ICS is recommended for optimal clinical benefits in children with asthma.][60] Higher doses of ICS have been associated with an increased risk of local and systemic adverse effects, including the risk of adrenal suppression in some children; 61 therefore, ICS must be carefully titrated and considered for their benefits and risks. 6owever, poorly controlled asthma itself may affect patients' growth; 62 consequently, it is essential that ICS therapy be gradually tapered to the lowest effective dose once the desired symptom control is achieved. 51
Role of home nebulization and practical recommendations
In instances where HCPs, patients' families, or caregivers deem nebulized therapy to be more effective than inhalers, home nebulization may be continued for a short period following discharge from a healthcare facility and after a demonstrable improvement in clinical status following an acute asthma exacerbation. 7Moreover, children may be more compliant with treatment when at home in a familiar environment.The nebulizer should be used in a room that is isolated from other household members to minimize the risk of transmission of respiratory viral infections.Crucially, parents and caregivers should be educated that failure to protect the skin or eyes during ICS delivery using a nebulizer may result in local side effects, such as steroid rashes; 6 therefore, the skin on the nose and around the mouth should be cleaned shortly after inhalation.Home nebulization for ICS maintenance treatment can be implemented under three potential scenarios: 1) ICS maintenance treatment for 1-3 months to prevent an acute exacerbation; 6 2) intermittent high-dose treatment (1-2 mg once daily for a week) at the onset of a respiratory infection; and 3) management of an acute exacerbation. 7,63This guidance should be followed especially when access to healthcare facilities is limited, and to reduce the risk of viral infections and severity of asthma exacerbations during the COVID-19 pandemic.Patients with mild or moderate asthma exacerbations can be effectively managed with home nebulization, instead of receiving treatment at a healthcare facility.Notably, high-dose (0.5-1 mg/dose) nebulized ICS can be added to a systemic corticosteroid in the first hour of treatment to ensure a rapid onset of action. 7Home nebulization also facilitates the administration of multiple asthma medications, including budesonide and bronchodilators, in one dose, especially during asthma exacerbations.However, it is essential that all patients, including those perceived to be without future risk of exacerbations, are monitored carefully.Although both jet nebulizers and ultrasonic nebulizers produce the desired particle size in an aerosol drug output, require only tidal breathing, and allow dose modification, 64 jet nebulizers are sturdier and less expensive and nebulize suspensions more effectively than ultrasonic nebulizers, which may cause drug degradation in suspension formulations. 64However, all nebulizers have inherent limitations, including the length of treatment time and ambient contamination by escaped aerosols. 64
Asthma education
The education of children's families, caregivers, and healthcare workers is an essential component of asthma management in children.Indeed, a combination of health programs at school and home improved asthma control in children aged 2-6 years from low-income families in the US. 65However, results of a global survey of national Member Societies of the World Allergy Organization from 31 countries highlighted disparities in the availability of adequate educational material among responding countries, with many reporting a lack of suitable material locally. 66hese findings emphasize the need to develop clear, age appropriate information that can be easily translated and delivered in a culturally and educationally effective format. 66dditionally, it has been determined that education of patients and caregivers should focus on the identification and avoidance of triggers, increase in understanding of prescribed treatments and the need to adhere to maintenance medications, and the need for appropriate choice and use of delivery devices. 67
Conclusion
Nebulized inhalation therapy provides multiple advantages that overcome common challenges in the delivery of inhaled medications for asthma, making it an effective treatment strategy for asthma management in pediatric patients who cannot use pMDIs with spacers effectively.While home nebulization should be widely recommended for pediatric patients in specific clinical scenarios, all patients should be monitored carefully including those perceived to be without future risk of exacerbations.Based on the literature review and expert opinion, nebulized budesonide is an effective and well-tolerated treatment option in children aged ≤ 5 years with asthma and should therefore be considered as initial therapy in this patient population.We hope that this algorithm is integrated into routine clinical practice for pediatric asthma management in Thailand and across other countries.
Figure 3 .
Figure 3. Practical treatment algorithm for children aged ≤ 5 years with a diagnosis of asthma.This treatment algorithm is adapted from the Thai Pediatric Asthma 2020 Guideline 47 and based on available medications in Thailand.
Re-assessment at 1-3 months Partly/uncontrolled
*Based on the drugs available in Thailand † Future risk of exacerbations is characterized by uncontrolled symptoms, > 1 severe exacerbation in the past 12 months, exposure to tobacco smoke, pollution, and aeroallergens especially in those with respiratory tract infections, low adherence, and inaccurate inhaler technique.ICS, inhaled corticosteroids; LTRA, leukotriene receptor antagonist; OCS, oral corticosteroids; pMDI, pressurized metered-dose inhaler; SABA, short-acting β 2 -agonist. | 2023-07-20T06:17:46.170Z | 2023-07-16T00:00:00.000 | {
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4988980 | pes2o/s2orc | v3-fos-license | Global evidence directing regional preventive strategies in Southeast Asia for fighting TB / HIV
Tuberculosis (TB) and human immunodeficiency virus (HIV) co-epidemics form a huge burden of disease in the Southeast Asia region. Five out of eleven nations in this region are high TB/HIV burden countries: Myanmar, Thailand, India, Indonesia and Nepal. The trends of TB incidence in these countries have been rising in recent years, in contrast to a falling global trend. Experts in the field of TB control and health service providers have been perplexed by the association of TB and HIV infections which causes a mosaic clinical presentation, a unique course with poor treatment outcomes including death. We conducted a review of contemporary evidence relating to TB/HIV control with the aims of assisting integrated health system responses in Southeast Asia and demystifying current evidence to facilitate translating it into practice.
Introduction
Tuberculosis (TB), a centuries-old disease, causes more than a million deaths every year.After World War II, TB was well-controlled globally with TB mortality declining dramatically [1].However, it reemerged after the 1980s following the global outbreak of the human immunodeficiency virus (HIV) infection [1].A large number of HIV-infected individuals were rapidly killed by TB in high TB burden settings such as Africa and Thailand, as well as its neighboring countries in Southeast Asia [1][2][3].The World Health Organization (WHO) declared TB a global health emergency in 1993.Afterward, global and national responses were launched progressively but heterogeneously across the countries with differing resources and health systems.
Today, TB remains a disease which is highly infectious, difficult to diagnose, and slow to treat.Its treatment and curability by multiple-combination chemotherapy is being challenged by an increase in drug resistance worldwide [2].The technical knowledge of TB control has been hindered by the association of TB and HIV infections which causes a mosaic clinical presentation, a unique course and poor treatment outcomes including death.
In this review, both conclusive and inconclusive evidence was appraised in terms of epidemiology, diagnosis, pathophysiology, treatment, and prevention of TB/HIV co-infection.It was aimed to identify the gaps in research and integrated health system responses in Southeast Asia, catalyzing translation of contemporary evidence to practice.
TB/HIV regional epidemiology in Southeast Asia
Globally the trend of TB burden has been falling in the last few years, but this scenario is being threatened by TB/HIV co-epidemics.The World Health Organization (WHO) estimated that in 2010 the global burden of TB cases stood at 8.8 million, while 34 million people were living with HIV and 1.1 million were TB/HIV co-infected patients [2,4].TB/HIV coinfection caused 500,000 deaths worldwide in 2008, 380,000 deaths in 2009 and 350,000 deaths in 2010 [5].The burden of these two diseases is highest in Africa followed by Southeast Asia.Southeast Asia is home to 3.5 million people living with HIV (PLWH) and 5 million TB patients, which represents 41% of global TB patients.Five out of eleven nations in the Southeast Asia region are high TB/HIV burden countries: India, Indonesia, Myanmar, Nepal and Thailand [6].Neighboring countries such as Cambodia and Vietnam are also relatively high TB/HIV burden countries.The WHO estimated that HIV prevalence in Southeast Asia was 0.3%, while HIV prevalence among TB patients was 5.7% [4].However; the burden of TB/HIV co-infection in the Southeast Asia region is heterogeneous across the 11 countries (Figure 1).PLWH residing in high TB prevalent countries experience infection and reinfection of Mycobacterium tuberculosis (M.tuberculosis) resulting in a large number of TB/HIV co-infected patients and high consequent mortality.Such incidence of HIV-associated TB is highest in Myanmar, followed by Thailand, India, and Indonesia [4].Moreover, the TB incidence in those countries has been rising in recent years, compared to a falling global trend [3,7].
Immunopathology of TB/HIV co-infection
Insight into the immunopathological interrelationship between these two severe diseases has been explained by many observational and interventional studies over the last three decades [6,8,9].HIV-infected individuals are more susceptible to TB infection from a new source than HIV-negative individuals.Persons co-infected with TB and HIV are 21 to 34 times more likely to develop active TB disease than persons without HIV [5].Moreover, the incidence of severe and extra-pulmonary TB, such as TB meningitis, and death rate are higher in HIVinfected patients.The natural course of TB has been exacerbated by the HIV manipulated immunological reaction against TB, affecting macrophage function, cytokine production, and failure to contain initial or latent M. tuberculosis infection and disruption of granuloma.Several hypotheses have been proposed to explain how HIV increases the risk of TB infection and how M. tuberculosis infection may exacerbate HIV infection, but the exact mechanism is still poorly understood [9].
Granuloma, the major hallmark of the human immune response to TB, is a structure composed of macrophages, lymphocytes, dendritic cells, neutrophils, and sometimes fibroblasts, often with a necrotic center.It serves optimal immunologic functions to contain the M. tuberculosis bacilli and acts as an immune microenvironment for cellular interactions that limit M. tuberculosis replication.HIV co-infection disrupts the granuloma structurally and functionally [9].The exacerbated pathology of TB in patients with HIV infection is probably due to qualitative (functional) and quantitative changes in immune response against M. tuberculosis, especially inside the granulomas [9].
Quantitative changes
HIV replication is increased at sites of M. tuberculosis infection leading to an exacerbated pathological process.Several studies that measured HIV levels in lungs, pleural cavities, and the associated macrophages observed an increased HIV replication at sites of M. tuberculosis infection.Nakata et al. (1997) showed that there are greater HIV p24 levels and viral loads in bronco alveolar lavage fluid (BAL) from TB-involved lungs than in BAL fluid from TB uninvolved lungs [8].
HIV induces primary or reactivated TB through the killing of CD4 + T cells within granulomas.CD4 + T cells play a major role in controlling the virulence of M. tuberculosis inside and outside the granulomas.Lawn et al. showed that the HIV-infected individuals with fewer peripheral CD4 + T cells are more prone to TB than HIV-infected individuals with relatively higher CD4 + T cells [10].Furthermore, the study of Diedrich et al. reported that the reactivation of latent M. tuberculosis in cynomolgus macaques infected with simian immunodeficiency virus (SIV) is associated with early peripheral T cell depletion even before the rise in SIV viral load [11].
Qualitative changes
The killing mechanisms of macrophage containing M. tuberculosis are manipulated by HIV co-infection.Macrophages are major compartments of human innate immunity in containing M. tuberculosis infection.HIV and M. tuberculosis co-infected macrophages induce less tumor necrosis factor (TNF)dependent apoptosis than macrophages infected with only M. tuberculosis [12].Moreover, co-infected macrophages release less TNF than macrophages infected with only M. tuberculosis [12].Revealing another aspect of functional changes in macrophages, Deretic et al. showed that HIV further decreases the ability of M. tuberculosis-infected macrophages to acidify vesicles [13].
HIV induces functional changes in M. tuberculosis-specific T cells.Apart from killing M. tuberculosis-specific T cells, HIV infection induces some functional changes in those cells decreasing their ability to contain M. tuberculosis.Geldmacher et al., in their studies, observed fewer interferon-gamma (IFN-γ + )-producing M. tuberculosis-specific memory CD4 + T cells after HIV infection in individuals with latent TB [14].
Additionally, HIV exerts its adverse effects by interfering with the cell cycle [15].The virus lowers IFN-γ mRNA production and therefore cellular proliferation in airways of patients with AIDS and TB as opposed to individuals with TB alone [15].These findings were further enhanced by the observations of lower IFN-γ, TNF, and interleukin 2 (IL-2) production and cellular proliferation in M. tuberculosis-specific peripheral T cells in HIV-positive individuals as opposed to HIV-negative individuals with active TB [16].Overall, the immune pathological understanding of TB/HIV co-infection has improved to explain the more severe and silent course of the two diseases in one host, but is still in the evolution phase in terms of applying improved diagnosis and treatment.
Diagnosis of active TB disease in HIV-infected persons
TB diagnosis has never been easy and straightforward, especially in HIV-infected patients, and it cannot be made by a single diagnosis test.A physician's decision, based on a clinical work-up of symptoms, chest X-ray (CXR), and sputum smear microscopic examination, triggers the TB treatment at district level hospitals within the developing setting of Southeast Asia, where culture is usually not included in the routine diagnosis algorithm [17].Recently, the WHO-recommended TB diagnosis models were compared in HIV-infected TB suspects, in terms of cost effectiveness, to reduce the mortality within 6 months of antiretroviral therapy (ART) initiation [18].The GeneXpert algorithm was found to be less costly than either smear-CXR or smear-CXR-culture algorithms.Culture and GeneXpert algorithms were more cost effective in reducing mortality than the current practice [18] (Table ).This report represented a new diagnostic outline, skipping traditional diagnosis methods, which is likely to save time and resources.
Algorithm for TB diagnosis in people living with HIV
In recent years, TB diagnosis research has been conducted worldwide.However, studies done in Southeast Asia followed the diagnostic algorithm approach, which reinvented the traditional clinical parameters of ruling out active TB in order to start ART or to provide isoniazid preventive therapy (IPT) in a program approach.The WHO 2007 algorithm for TB diagnosis in HIV patients was evaluated in Cambodia during 2008-2009 [19].The median time to TB treatment initiation was 5 days (interquartile range, IQR: 2-13 days), ranging from 2 days (IQR: 1-11.5 days) for extra-pulmonary TB, over 2.5 days (IQR: 1-4 days) for smear-positive pulmonary TB, to 9 days (IQR: 3-17 days) for smear-negative pulmonary TB [19].However, the sensitivity of the algorithm was very low (58.8%)despite its inclusion of no cough or cough less than two weeks as a criterion in suspects with constitutional symptoms or abnormal chest Xray.In 2010, Cain et al. evaluated another algorithm, applying cough for any duration and other symptoms such as fever, night sweats and loss of appetite, as a way of identifying TB in PLWH (median CD4 count of 242 cells/mm 3 ) in three countries: Thailand, Vietnam and Cambodia.The sensitivity of the algorithm was improved to 88% [20].The authors suggested that patients lacking these symptoms could be candidates for IPT, although a small proportion, about 3%, might have active TB and be undertreated [20].These studies changed the paradigm of cough "for more than two weeks" to be cough "for any duration" when screening TB among PLWH.
Current gold standard diagnostic tests
The sputum smear microscopic examination of acid fast bacilli (AFB), discovered more than 125 years ago, still remains the mainstay bacteriological TB diagnosis test in Southeast Asia, where more than half of the reported TB incident cases were smear AFB negative [7].Mycobacterial culture is the current gold standard diagnostic test for TB diagnosis.However, the solid culture testing system is timeconsuming, while the liquid culture testing system is expensive and not feasible in the Southeast Asia setting, except in tertiary care hospitals [21].Adding these tests to symptoms screening and chest X-ray increases the level of TB case detection.However, smear-negative TB is highly prevalent among PLWH and atypical presentation of chest radiography and silent clinical features are not uncommon in immunecompromised patients with a very low CD4 count.
Nucleic acid amplification tests (NAATs)
NAATs are rapid TB diagnostic tests, which are faster than both smear microscopy and culture.Former generation NAATs were not sensitive enough for the effective TB screening.However, newer NAATs have increased the case detection rate of smear-negative TB among HIV-positive patients within high prevalent HIV settings.Establishment costs and human resource needs formed significant barriers, meaning that the diagnosis of TB among HIV-infected people remained a challenge in the resource-poor Southeast Asian setting.Recently, the WHO started to implement application of the GeneXpert machine and or the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA).It is a cartridge-based fully automated NAAT for TB diagnosis and rifampicin resistance testing [22].It purifies concentrates, amplifies by rapid real-time PCR assay, and identifies targeted nucleic acid sequences in the TB genome from unprocessed sputum samples and offers TB diagnosis within two hours, with minimal hands-on technical time.The sensitivity in detecting smear-negative culture-positive TB patients is 72.5% [22].It is suitable for use in TB and HIV disease-endemic countries.All eleven countries in Southeast Asia have ordered at least one GeneXpert instrument according to the WHO monitoring of GeneXpert implementation as of 2012 [23].
Microscopic observation drug susceptibility assay (MODS)
The microscopic observation drug susceptibility assay (MODS), first evaluated in Peru, is a wellknown, low-cost and low-technical direct culture method, providing TB diagnosis and drug susceptibility results in 7 to 14 days [24].It has been evaluated in Asian countries such as Thailand and Vietnam with variable results [17,25].The TB diagnosis performance of MODS in HIV-positive patients was promising, but the sensitivity for smearnegative cases was weak at 38% [26].However, MODS identified TB meningitis in cerebrospinal fluid samples with a very high accuracy (positive predictive value 100% and negative predictive value 78.7%) as well as at a low cost of 0.53 US$ per sample [25].
Serological tests
The commercial serological tests can mislead diagnosis and increase costs.After twelve months of rigorous analysis by global experts involving evaluation of evidence from 67 studies for pulmonary TB and 27 studies for extra-pulmonary TB, the WHO issued an explicit "negative" policy recommendation to stop the use of serological tests for TB [27].
Urine tests
Detection of TB-specific biomarkers, namely Lipoarabinomannan (LAM), in the urine is an alternative diagnostic option.A newer invention, the LAM strip test offers an easy-to-perform bedside TB diagnosis for PLWH with a very low CD4 count (< 200 cells/m 3 ) and most at risk of mortality [28].Active TB cases were diagnosed at bedside within 25 minutes and at a cost of 3.5 US$ per test.The sensitivity and specificity were 66% and 96%, respectively.It could serve as a "point of care" TB diagnosis test in advanced immunocompromised patients.These performance characteristics indicated the possibility to rule in TB among PLWH with low immune status in the Southeast Asia setting.A newer sandwich immunoassay, to detect the LAM and other biomarkers in complex patients' samples, is being developed but has yet to be evaluated.Many other new tests are still in the TB diagnostic research pipeline, in the form of single tests or parts of algorithms, in the ongoing challenge of TB diagnosis.
Treatment of active TB disease in HIV-infected persons
There are three main options for the treatment of HIV-associated TB in the developing setting of Southeast Asia: 1. TB therapy, involving a combination of 4 to 6 anti-TB drugs; 2. antiretroviral therapy, with at least 3 antivirals; and 3. therapy using other drugs for prevention of opportunistic infections, such as fluconazole and co-trimoxazole preventive therapies in cases of immunodeficiency.
Timing of ART and TB treatment
The optimal timing for initiation of ART antiretroviral drugs, in relation to TB therapy, remained unclear until 2010.The pros and cons were the survival benefit and the risk of immune reconstitution inflammatory syndrome (IRIS), respectively.Afterward, three clinical trials, namely SAPiT, STRIDE and CAMELIA, conclusively confirmed the substantial survival benefit in early initiated ART [29][30][31].Mortality was highest in sequential ART, i.e., starting ART after the completion of TB treatment [29].ART initiated during TB therapy reduced the mortality rate in all three trials [29][30][31].How early to begin ART during TB therapy should be judged by the level of immune deficiency according to the CD4 + T cell count of the patients.
Among patients with CD4 + T-cell counts less than 50 cells/mm 3 , ART initiated two weeks after the commencement of TB treatment is beneficial in reducing AIDS-defining illnesses and death (hazard ratio 0.62, 95% CI 0.44 to 0.86.P = 0.006), and outweighs the risk for IRIS [30,31].In a clinical trial in Cambodia, the same survival benefit of early ART was reported among patients with CD4 + T cell counts less than 50 cells/mm 3 and CD4 + T cell counts between 51-200 cells/mm 3 [31].The goal of early ART initiation is to shorten the time that the patient has profound immunodeficiency.
ART initiated in patients after two weeks of TB treatment reduced mortality rate by 41.7%, compared to those who started ART after 8 to 12 weeks.Even a brief delay made a huge difference.Recent trial results in Thailand could not find any significant difference when comparing ART initiated in patients after four weeks compared with ART started in patients at 12 weeks [32].However, a brief delay in starting ART 4 to 8 weeks after the initiation of TB therapy in patients with CD4 + T cell counts more than 200 cells/mm 3 did not convey any increase in the risk of a new AIDSdefining illness or death, with a reduced risk of IRIS [29].Moreover, immunological studies revealed that delaying the initiation of ART for up to 12 months during complete treatment of pulmonary TB among PLWH with preserved CD4 + T cells greater than 350 cells/mm 3 may not accelerate a decline in immunologic function [33].However, the current WHO 2010 ART guideline recommends initiating ART in TB/HIV co-infected patients irrespectively of the CD4 count.
Underpinning the implementation of this consolidated evidence was an integration of healthcare management of TB and HIV clinics which can provide a CD4 test and TB diagnosis to TB-suspected PLWH within two weeks concomitantly.Another point of view is to consider the influence of the TB site on mortality.All the studies described above recruited pulmonary TB patients.The incidence of extra-pulmonary TB was increasingly common among the Asian TB/HIV cohort and was associated with higher mortality [34].A randomized control trial of HIV-associated TB meningitis in Vietnam reported a very high mortality rate (59.8% and 55.6 %) regardless of whether ART started within seven days or after two months following the start of TB treatment.The treatment outcome was not significantly different in relation to the timing of ART initiation [35].Better therapeutic options are needed for HIV-associated extra-pulmonary TB patients, especially those with meningitis.
Despite the clinical trials results showing that survival benefit outweighed the adverse effect, the practicality of those regimens could be weakened by other factors such as common drug-drug interactions, side effects, pill burden, and the patient's compliance [6].Anti-TB drug-induced hepatotoxicity is common.Interaction between anti-TB and highly active antiretroviral therapy (HAART) can cause failure in either treatment.Still, it is difficult to predict hepatotoxicity during TB treatment.Moreover, directly observed treatment, short-course (DOTS) strategy, the well-known compliance enhancer in a TB control program, has not been evaluated in relation to integrated TB/HIV treatment.
Treatment of latent infection for preventing TB in HIV-infected persons
Before the HIV epidemic, the Bacille Calmette-Guérin (BCG) vaccine could prevent the fatal forms of TB, namely meningitis and disseminated TB.However, there is no currently effective vaccine to prevent all forms of TB in PLWH.A newer TB vaccine is still in trial.Meanwhile, preventing active TB among PLWH, in other words, treating latent TB infection (TLTI) to prevent active TB, is an attractive alternative strategy.Currently, it is one of the constituents in the WHO's TB preventive strategies, namely the 3Is: 1. intensive case finding; 2. infection control; and 3, Isoniazid preventive therapy (IPT).IPT could make a difference in reducing the incidence of TB among PLWH and consequently offer a survival benefit to PLWH.
TLTI or efficacy of IPT reducing TB incidence among PLWH
IPT reduced the TB incidence among PLWH before and in the ART era.According to Cochrane's review of 12 randomized clinical trials (RCTs) in the pre-ART era [36], TB incidence in IPT-treated PLWH was 3% compared to 6% in the placebo group.Overall, IPT reduced the risk of active TB by 33% (relative risk 0.67, 95% CI 0.54 to 0.85).Among tuberculin skin test (TST) positive PLWH, TB incidence after receiving IPT was 2% compared to 7% in the placebo group, and IPT reduced the risk of active TB by 64% (relative risk 0.36, 95% CI 0.25 to 0.57).TB incidence among TST-negative HIV patients after receiving IPT was 3.8%, compared to 4.5% in the placebo group.IPT reduced the risk of active TB by 14% (relative risk 0.86, 95% CI 0.59 to 1.26) in TST-negative PLWH.The effect of IPT in the ART era was also reported by a prospective cohort study in a high TB burden setting of Africa: in that study, the risk of TB was drastically reduced (89%) by IPT in PLWH receiving ART, and TB incidence was lowest among those who received IPT preceding ART when compared to those who received only ART or those received only IPT [37].
Efficacy of IPT offering survival benefit among PLWH
IPT might also benefit the survival of PLWH as treating latent infection can prevent the drastic immune-deficiency triggered by active TB in HIVinfected hosts.In 2010, a study in South Africa compared the reduction of death in the ART cohorts in 12 months of follow-up and revealed a significantly longer survival time in IPT-treated than non IPTtreated groups [38].IPT reduced the mortality of PLWH when provided with or before ART initiation [38].Recently, a non-randomized comparative study in Thailand reported the impact of TST guided IPT on four-year follow up.TB incidence was significantly lower among PLWH attending the hospital with TSTguided IPT provision than among patients attending the hospital without it [39].In contrast to the African report, Khawcharoenporn et al. reported that the mortality in IPT-treated and non IPT-treated cohorts were similar [39].Those two studies were different in terms of IPT provision protocol, sample size, and the higher TB prevalence in Africa.A randomized trial in a Southeast Asian setting may give a conclusive answer.
Despite the convincing evidence cited above, IPT reached only 12% of eligible HIV patients globally in 2010 [2].In the Southeast Asia region, only 4 of 11 countries implemented and reported the provision of IPT in 2009 [40].Most of the countries in Southeast Asia did not include IPT in their national guidelines, according to WHO TB/HIV collaborative activities, although IPT was provided in Bhutan and Thailand [7].One of the challenges underlying this sluggish roll-out was the difficulty in diagnosing patients with latent tuberculosis among PLWH as candidates for IPT [40].
Identification of latent infection in HIVinfected persons
To identify the HIV-infected individual who could benefit from IPT, tests to diagnose latent tuberculosis infection (LTBI) and their performance characteristics in high TB burden settings are to be determined.Since there is no gold standard for the diagnosis of LTBI, it is challenging to diagnose LTBI.Until recently, the tuberculin skin test (TST) has been the sole diagnostic for LTBI.As the TST is based on delayed-type hypersensitivity response mediated by lymphocytes, especially T cells after injection of a purified protein derivative (PPD), the sensitivity of the TST in the HIV-infected population is highly reduced due to immune suppression [41].In addition, TST is also known to be confounded by prior BCG vaccination or infection of non-tuberculous mycobacterium (NTM) due to cross-reactivity of antigens, which are common in HIV-endemic areas [42].
In the last decade, interferon-gamma (IFN-γ) release assays (IGRAs) were developed for diagnosing LTBI.IGRAs detect the release of IFN-γ in response to highly M. tuberculosis-specific antigens, such as early-secreted antigen 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), which are absent from all BCG vaccine strains and most of the non-tuberculous mycobacteria (NTM) [43].Currently, two types of IGRAs are commercially available.One of the IGRAs, QuantiFERON-TB Gold In-Tube (QFT, Cellestis Ltd., Victoria, Australia) is based on the ELISA assay and uses whole blood for antigen stimulation.QFT contains an additional M. tuberculosis-specific antigen, TB7.7 in addition to ESAT-6 and CFP-10.Another IGRA, T-SPOT.TB (T-SPOT, Oxford Immunotec Ltd., Abingdon, UK), is based on the Enzyme-Linked ImmunoSpot (ELISPOT) Assay and uses purified peripheral blood mononuclear cells (PBMCs) for antigen stimulation.As IGRAs stimulate antigen-specific T cells with M. tuberculosis specific antigens, it has been demonstrated that IGRAs have higher specificity than TST among BCG-vaccinated populations [44].IGRAs have also been shown to have higher sensitivity than TST [44].Since their development, IGRAs have been well accepted in many developed countries and incorporated into their TB programs.The advantages of IGRAs over TST include not only higher specificity, but avoidance of a second visit, objective test results, and booster effect.However, it would be important to note that IGRAs do have some limitations [45].Specifically, IGRAs cannot distinguish between active TB and LTBI, or recent infection and remote infection [43].As IGRAs measure immune responses against M. tuberculosis infection, as the TST does, the sensitivity of IGRAs in HIV-infected populations also decreases [46].Although the count of CD4 + T cells decreases with the progression of HIV infection, which could affect the sensitivity of IGRAs, it has been reported that IGRAs generally perform better among HIV-infected populations compared to TST [47].Because QFT uses whole blood regardless of lymphocyte count, and T-SPOT uses a certain count of PBMCs, the sensitivity of QFT is supposed to be lower than that of T-SPOT in the HIV-infected population.Furthermore, IFN-γ response to mitogen (the positive control) in QFT was proportionate to the CD4 + T cell counts in PLWH with CD4 counts less than 50 cells/ul.Thus fewer IFN-γ +producing M. tuberculosis-specific memory CD4 + T cells in PLWH with latent TB lead to poor sensitivity of QFT [14,48].In fact, several studies have shown that the responses of T-SPOT are well retained even in HIV-infected populations, whereas those of QFT, as well as TST, decrease as the count of CD4 + T cells declines [49].Therefore, the best available diagnostic test for LTBI in HIV-infected populations, at present, appears to be T-SPOT; however, it is important to improve the diagnostic accuracy.More recently, it has been reported that combining TST and T-SPOT increases the diagnostic sensitivity in HIV-infected individuals [50].Another approach to improve the sensitivity of LTBI diagnosis is to search for other potential biomarkers, such as IFN-γ inducible protein 10 (IP-10).
Numerous studies have been undertaken since the development of IGRAs, showing them to be superior to TST in diagnosing LTBI.So far, it is not conclusive whether the use of IGRAs improves the identification of PLWH who could benefit from IPT.Further studies are needed to investigate how to optimize the performance of IGRAs in PLWH, especially in Southeast Asian settings where most of the PLWH have been BCG vaccinated and IGRAs were underutilized.
The current WHO-recommended approach for resource-limited settings is to screen PLWH for active TB using a clinical algorithm of four symptoms (current fever, cough, weight loss, and night sweats) and to provide IPT to those who are negative on screening [51].The negative predictive value of this type of algorithm is 97.7% (95% CI 97.4-98.0) in places with 5% TB prevalence among PLWH to rule out active TB [51].The risk of active TB misdiagnosed as LTBI is minimal.Further studies are necessary to evaluate the implementation and impact of this guideline and its barriers in Southeast Asia.
Evidence to implementation: health service delivery models
The WHO advocated collaborative TB/HIV control activities years ago.Integrated collaboration is a challenge in most of the Southeast Asian nations.Early provision of ART in TB/HIV co-infected patients is evidence-based and supported by consistent guidelines.To translate these evidence-based guidelines into practice, countries need an adequately funded health system with a highly integrative healthcare delivery service model [52].To initiate ART services for HIV-infected active TB patients, a onestop service that provides the basic diagnostic needs to confirm TB diagnosis, HIV tests, and CD4 counts is required.It demands a close collaboration between TB and HIV programs and health service providers.The best model for a TB/HIV health-care delivery service provides services from the national level to catchment area community hospitals.A study of three health-care service models in Malawi is worth reiterating (Figure 2) [52]: 1. in the referral based model, TB and HIV clinics refer the patients for each service; 2. in the partially integrated models, a synchronized service of TB and HIV clinics for TB/HIV patients is offered; and 3. in the integrated models, all TB and HIV/AIDS services (HIV counselling as well as ART and TB treatment) are provided at the same clinic or the same, one-stop shop.
Current clinics in Thailand are mostly based on either model 1 or 2. The experience in Malawi has shown model 3 as the best cost-effective model that improves the patients' outcome [52].Barriers to patient care such as a long distance to travel between HIV and TB clinics, a limited budget for ART, health care providers' delay, and a lack of a universal coverage system may possibly result in a delay in implementing the recommended guidelines for treatment.Moreover, a politically committed health system, well-funded for diagnostic and treatment services, is essential.It requires facilities for testing HIV and the CD4 count, as well as TB diagnostics and facilities for DOTS.Practically, indecisive clinical presentations, such as coughless TB, smear-negative TB, and high CD4 counts require a skillful physician to assess the patient and come to a clinical decision; however, this is hardly possible in catchment area clinics at the community level of developing countries.
Where the diagnostics of TB such as culture systems are lacking and where a physician to start TB treatment in query cases is not feasible, delayed referrals, under-diagnosis and consequent further transmission of TB, resulting in poor outcome for patients, will still occur in the resource-poor setting.
To resolve these realities in developing nations in Southeast Asia, health-care providers at the community level must be trained to proceed with a high index of TB suspicion among PLWH, to enable appropriate urgent referral for treatment with sensitive and easily applicable TB diagnostic testing, such as GeneXpert or LAM strip tests.
Recently, a multinational analysis showed that every US$100 per capita government health expenditure was associated with a 33% (95% CI: 24%-42%) decrease in TB/HIV mortality rates [53].Implementing wider access to ART in a TB/HIV burden country needs huge financial resources.Access to ART is very difficult for PLWH in countries with the "out of the pocket" payment system such as the one in Myanmar.
Therefore, a mechanism for mobilizing the national budget, as well as effective external support, through faster and proper entry points, is required.
Research gaps in awareness studies
TB was a well-known disease in the past and it is re-emerging as a common epidemic disease, globally.The awareness and knowledge of TB among the persons at most risk, such as PLWH, is very important in preventing the transmission of infection and needs immediate attention today [54] There have not been many studies assessing such awareness.A decline in TB incidence and mortality before World War I preceded the discovery of drugs and vaccines [1].This was a historically evident example of health promotion via public awareness, which uplifted general wellbeing and reduced TB incidence.It would be beneficial to assess the awareness and knowledge of TB among PLWH and their family members, in order to deliver health education that can promote awareness of TB infection prevention.
Conclusion
We reviewed the evidence available on a global scale, with the aim of formulating a strategy for counteracting the TB/HIV disease burden in Southeast Asia.Contemporary evidence points out the benefit of early treatment and prevention of TB among PLWH.A diagnosis test for effective TB screening in PLWH is still a gap demanding further exploration in basic science and operational research.The two diseases, TB and HIV, can form an alliance in one patient, thereby challenging human existence and well-being.In response to this challenge, a completely integrated TB/HIV health care delivery model and local health system adjustment, allowing an integrated program of health services, is an urgent necessity.
Figure 1 .
Figure 1.HIV prevalence (deep red line) and HIV prevalence among TB cases (blue bars) in WHO Southeast Asia Region
Figure 2 .
Figure 2. Three health care delivery service models adapted from the study of Kachiza C et al., 2010 [52]
Table .
[18]cost of three TB diagnostic algorithms for newly initiated ART among PLWH who manifest either current cough, fever, night sweats, or weight loss at the initiation of ART, South Africa, 2011 †[18] † ART, antiretroviral therapy; PLWH, people living with HIV; RIF, rifampicin.* Sputum smear using 2 specimens.α 1 sputum Mycobacterial culture using the automated Mycobacteria Growth Indicator Tube (MGIT).# Unit cost of TB diagnosis and treatment and ART costs. | 2017-04-02T03:25:43.234Z | 2013-03-14T00:00:00.000 | {
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220420811 | pes2o/s2orc | v3-fos-license | Tobacco smoking and smoking cessation in times of COVID-19
INTRODUCTION Tobacco smoking may increase susceptibility to and severity of coronavirus disease 2019 (COVID-19). This information may influence smoking cessation related beliefs in smokers. METHODS Online questionnaires were answered from 26 March to 3 April 2020 in the Netherlands by 340 smokers willing to quit smoking. Beliefs regarding (quitting) smoking and (consequences of) the coronavirus are described and associations with motivation to quit due to the coronavirus are reported. RESULTS While 67.7% of the smokers indicated that the coronavirus did not influence the number of cigarettes smoked per day, 18.5% smoked less cigarettes and 13.8% smoked more cigarettes per day due to the coronavirus. One-third of the smokers were more motivated to quit smoking due to the coronavirus. Motivation to quit due to the coronavirus was positively associated with beliefs about the coronavirus as a serious threat, being at high risk of catching the coronavirus and developing severe illness, smokers being at higher risk than non-smokers, quitting smoking to reduce complaints, the social environment endorsing quitting, and perceived stress. CONCLUSIONS Subgroups of smokers may be receptive to smoking cessation advice due to COVID-19. Because of the measures taken to reduce the spread of the virus (e.g. stay at home as much as possible), personalized digital health interventions may be particularly suitable to reach smokers at home.
INTRODUCTION
Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus. The COVID-19 pandemic has determined daily life in most countries in the past weeks/months and there is no end in sight.
The current situation may influence (beliefs about) smoking behavior. Smokers may smoke more tobacco because of higher stress levels due to the crisis situation 1 . Yet, smokers may also perceive increased susceptibility to and severity of COVID-19, potentially increasing motivation to quit.
The coronavirus may pose an additional threat to smokers. First, the hand-to-mouth movement while smoking can facilitate viral infection. Public health measures strongly advise to avoid touching eyes, nose and mouth, since hands can transfer the virus to the face and from there enter the body. Second, tobacco smoke damages the lungs of the smoker and compromises the immune system resulting in an increased risk for respiratory infections and negative disease progressions 2 .
There is distinct evidence that smoking status is associated with severity and mortality of COVID-19. A recent meta-analysis revealed that current smoking increases the risk of severe COVID-19 by around twofold (pooled OR=1.98; 95% CI: 1.29-3.05) 3 . Mehra et al. 4 Smokers may be influenced by (news) reports on these findings about the link between smoking and COVID-19. Therefore, this study describes Dutch smokers' perceptions regarding susceptibility to and severity of COVID-19 and its effect on smoking behavior (i.e. number of cigarettes smoked) and difficulty to quit smoking. Associations between beliefs and motivation to quit due to the (consequences of) coronavirus are reported. As there may be gender differences in psychological responses to crises, differences in beliefs between men and women are also described 5 .
METHODS
From 26 March to 3 April 2020, a questionnaire was distributed to smokers willing to quit smoking. Inclusion criteria were that participants were aged ≥18 years, have smoked cigarettes in the past 7 days, and were motivated to quit smoking within 5 years. The sample was provided by Flycatcher, a Dutch ISO certified internet research agency with more than 10000 voluntary members. In return for full participation, panel members received a lottery ticket and points, which can be redeemed for gift vouchers. Informed consent was obtained online. The questionnaire was part of an overarching project on digital smoking cessation interventions for which ethical approval was granted by the institutional ethical review committee (FHML-REC/2019/072). Potential participants were selected for invitation based on the inclusion criteria. Flycatcher sent an invitation e-mail to 463 members of their panel, of which N=340 completed the questionnaire (response rate: 73%).
The I-Change model 6 , aimed at explaining motivational and behavioral change by integrating various social-cognitive theories, served as a theoretical framework for the beliefs about smoking and the coronavirus and covered risk perception, attitude, social norms, self-efficacy, and action planning ( Figure 1). All items were measured on a 5-point Likert scale ranging from 1='Strongly disagree' to 5='Strongly agree'. Means and 99.99% confidence intervals for the beliefs were calculated and are reported as diamonds in the left-hand panel of Figure 1.
Correlations between the individual items and motivation to quit (MTQ) due to (consequences of) the coronavirus were calculated. MTQ due to the coronavirus (i.e. 'Because of the coronavirus, I'm now more motivated to quit smoking') was measured on a 5-point Likert scale ranging from 1='Strongly disagree' to 5='Strongly agree'. The association strength is reported in the right-hand panel of Figure 1. The diamonds visualize the correlation coefficients with 95% confidence intervals.
The confidence interval-based estimation of relevance (CIBER) approach was employed to visualize the results 7 . The tool is part of the 'userfriendlyscience' 8 package for the statistical computing environment R.
Furthermore, changes in smoking behavior due to the coronavirus (i.e. 'Because of the coronavirus, I now smoke more/less a day') were measured with response options being 1='Less cigarettes', 2='Unchanged', and 3='More cigarettes'. For response options 1 and 3, the exact number of cigarettes smoked less or more was measured as well.
Demographics were assessed by age, gender, and education level (low, intermediate, high). Cigarette dependence was assessed by the Fagerström Test 9 . The six items of the scale were summed into an overall score ranging from 0 to 10. Willingness to quit smoking was assessed by asking participants when are they planning to quit smoking (within 1 month, within 6 months, within 1 year, within 5 years).
Smoking and the coronavirus
Of the participants, 33.8% were more motivated to quit smoking because of the coronavirus and 66.2% were not more motivated to quit due to the coronavirus. Of our sample, 18.5% indicated that they smoke less cigarettes due to the coronavirus (M=4.1 cigarettes/day, SD=3.3), 13.8% indicated that they smoke more cigarettes (M=7.3 cigarettes/ day, SD=8.2), and 67.7% did not alter the number of cigarettes.
The CIBER analyses (Figure 1) reveal that MTQ due to the coronavirus was positively associated with beliefs about the coronavirus as a serious threat, being at high risk of catching the coronavirus and developing severe illness, smokers being at higher risk than non-smokers, quitting smoking to reduce complaints, the social environment endorsing quitting, and perceived stress. All beliefs reported in Figure 1, except for self-efficacy ('Because of the coronavirus, I find it hard to quit smoking'), were significantly (p<0.01) positively associated with MTQ due to the coronavirus. These items together explained 66-77% of the variance in MTQ due to coronavirus.
Analysis of variance also confirmed that smokers motivated to quit were significantly (p<0.01) more convinced of all these items than smokers not motivated to quit.
DISCUSSION
One-third of our smokers were more motivated to quit smoking due to the coronavirus. Our results reveal which beliefs may be especially important to incorporate in smoking cessation interventions in times of COVID-19, such as the risks of being a smoker, and support from their social environment.
Since people are advised to stay at home in many countries to reduce the spread of the coronavirus, digital health smoking cessation interventions form an effective way to reach smokers 10 . In order to reach as many smokers as possible and reduce the spread of the coronavirus, we advocate evidencebased (digital health) interventions that are easily accessible for all citizens and include the latest evidence about the relation between smoking and COVID-19.
Limitations
A limitation of this study is that we included all smokers willing to quit smoking within 5 years. Thus, the sample consists of smokers highly motivated to quit and smokers less motivated to quit. Inclusion criteria of only smokers who are contemplating or preparing to quit may lead to different results.
CONCLUSIONS
The results of the present study provide evidence that perceiving the coronavirus as a serious threat and acknowledging that smokers are at higher risk than non-smokers is associated with motivation to quit. Believing that quitting decreases that risk and that friends and family endorse quitting is also associated with motivation to quit. | 2020-07-02T10:31:19.589Z | 2020-07-01T00:00:00.000 | {
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1379342 | pes2o/s2orc | v3-fos-license | Jejunojejunostomy intussusception after gastric bypass: Case report of a rare but serious complication
Obesity, and the comorbidities associated with it, have become endemic within society. Roux-en-Y gastric bypass (RYGB) surgery is an increasingly common procedure with medical and cosmetic benefits (Li et al., 2014) [1]. However, as the case volume increases so do the rate of uncommon complications and it is imperative for surgeons to be aware of management guidelines of these complications. We present a case of Retrograde intussusception (RI) which is a rare complication status post RYGB. It is most commonly reported at the jejunojejunostomy (JJ) site, and it is hypothesized to be secondary to an antiperistaltic (retrograde) telescoping of the common limb going into the jejunal anastomosis (Varban et al., 2013) [2,3]. We present another case study as well as some points to consider in clinical management.
Case background and diagnosis
A thirty four year old female, with no significant past medical history, who had undergone gastric bypass one and half year ago presented to the ED with excruciating diffuse abdominal pain. She reported a one hundred pound weight loss (change in BMI of 17.7, % Excess BMI lost of 60% and% Total Body Weight Loss of 43.4%.) On day of admission, she presented to the emergency room in the afternoon with acute onset of severe generalized abdominal pain after her last meal earlier that morning. She endorsed nausea with no complaints of vomiting, constipation or diarrhea. Her abdominal pain was minimally responsive to dilaudid and morphine.
Patient underwent CT of the abdomen that showed 'anastomosis to the jejunum and intussusception at the more distal jejunal anastomosis; no bowel obstruction is manifest [ed] at this time and no evidence of extravasation at either anastomosis'. Patient was taken emergently to the operating room for an exploratory laparotomy with the possibility of bowel resection.
Treatment
The procedure was intended to be started laparoscopically. However, upon placement of a nasogastric tube by the anesthesia team, there was a moderate amount of bleeding which raised higher suspicion for ischemic bowel. The operative approach at * Corresponding author.
E-mail addresses: akohli23@gmail.com (A. Kohli), lgutnik@montefiore.org (L. Gutnik), anil.narula@nbhn.net (A. Narula). this point was switched from laparoscopic to open. Upon entering the peritoneal cavity a retrograde intussusception was found: the distal bowel had intussuscepted retrogradely into the jejunojejunostomy. The segment of bowel that had intussuscepted was almost 30-40 cm long. Additionally, there was a 20 cm segment of bowel that was found to be necrotic and nonviable (not showing any evidence of peristalsis) and was resected.
Outcome and follow up
The patient had an uncomplicated course. Her pain was well controlled initially with a hydromorphone PCA and later with IV morphine and IV tylenol. Foley catheter was removed on post-operative day 1 and nasogastric tube was removed on postoperative day 2. Her diet was advanced to clear liquids on post-operative day 2 and subsequently to regular foods on postoperative day 3 and she tolerated both well. Bowel function returned on post-operative day 3. She remained hemodynamically stable throughout the admission. She was discharged home on post-operative day 3. She was seen in the clinic 2 weeks later, had regular bowel movements and was tolerating a diet with no more episodes of bowel movements.
Discussion
Most cases of intussusception within adults are anterograde but Retrograde Intussusception (RI) is most commonly seen in patient status post Roux En Y gastric surgery [1]. One believed explanation may be related to small bowel motility disturbances [2,4]. Oradand aborad-propagated migrating motor complexes were found in the Roux limb of patients having undergone bariatric surgery which likely would be secondary to minimal phase 2 motor activity and also the failure of motility conversion to a 'fed pattern' after a liquid meal. Normally, small bowel motility is initiated within the duodenum, where the smooth muscle cells have the fastest frequency of the pacesetter potential. Intestinal motility is enhanced after a meal with contractions which mix digested food with enzymatic juices in an ab-orad direction. The most important factors determining this mechanism are the integrity of the vagal nerve, the presence of the duodenum as well as the type of ingested food [5,6]. When a patient undergoes Roux En Y, with transection of the jejunum to construct a Roux limb (bowel distal to the transection) the distal jejunum is separated from the duodenal pacemaker. This disruption causes a drop in the pacesetter potential in the Roux limb which subsequently creates ectopic pacemaker potentials to arise within the Roux limb. It is postulated that these ectopic pacemakers create potentials that migrate not just distally, but sometimes also in an orad direction, which can cause delaying emptying or stasis of the Roux limb. This hypothesis is considered to be the underlying phenomenon behind RI although the exact mechanism is still unknown. Another theory behind this type of intussusception is the anastomotic staple line acting as a lead point [6,7].
From a clinical standpoint, our operative approach was in line with suggested management as indicated within the limited publications in literature. All patient with a diagnosis of RI underwent surgical exploration [8]. Studies have shown that the management of RI involved either resection with revision of the jejojeunal anastomosis or simple reduction with or without enteropexy. Additionally, all patients with either an obstructed or a nonreducible RI with ischemia underwent resection. Although our patient's RI was reduced, there was a section of ischemic bowel that necessitated resection. Without presence of ischemic bowel, the decision to resect would have been a more challenging and not straightforward.
Limitations
Care was taken to ensure a comprehensive presentation of the case presentation. However, the study had a few limitations. Firstly, this was a single occurrence of such an acute complication within a large inner city hospital setting that required operative intervention. Future presentations could help determine the clinical application of an altered operative course and whether or not bowel resection, as performed in our patient, could be avoided. Secondly, our report presents the treatment of the patient in the short termwithin the hospital setting as well as close clinical follow up -and doesn't highlight patient observation for future visits. We, however, will continue to monitor the patient through outpatient visits into the future for any further complications.
Conclusion
As Roux-en-Y gastric bypass (RYGB) becomes a more widely in the management of obesity, it is important to understand possible complications that may present, especially in the long term period. RI is an uncommon, but potentially very serious, complication that requires expedient surgical management. Clinically, it can present with significant abdominal pain with or without symptoms of bowel occlusion and internal hernia should be included in the differential. While no operative method can guarantee the absence of recurrence, simple reduction with possible resection of bowel segment is the preferred route in non-ischemic intussusception [3].
State any conflicts of interest
None.
Sources of funding for your research
None.
Ethical approval
This case report was anonymized without any disclosure of identifying patient information. There was no indication for ethics approval.
Consent
All identifying information has been removed.
Author contribution
Ajay Kohli-case analysis, write up, peri-operative patient care. Lily Gutnik-case analysis, peri-operative patient care. Danielle Berman-case write up, peri-operative patient care. Anil Narula-chief surgeon, peri-operative patient care, case analysis.
Guarantor
Author accepts full responsibility. | 2018-04-03T03:21:45.941Z | 2016-12-07T00:00:00.000 | {
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11640632 | pes2o/s2orc | v3-fos-license | The Left Atrial Appendage in Health and Disease
Atrial fibrillation (AF) is the commonest sustained cardiac arrhythmia and results in significant morbidity and mortality. The left atrial appendage (LAA), a small embryonic remnant of the left atrium (LA), has been shown to play a key role in the pathophysiology of AF-related stroke and thromboembolism. As a consequence the LAA, in spite of its meagre size, has been described as ‘our most lethal human attachment’. Despite being a recognised harbinger of disease, the LAA has also been shown to play an important role in health. This review seeks to address our current understanding of this vital structure in both health and disease states.
Introduction
Atrial fibrillation (AF) affects 2% of the general population rising with age to affect over 10% of octogenarians [1].Importantly AF has been predicted to increase in prevalence by three-fold by 2050 [2].This arrhythmia is by no means benign since it confers a five-fold risk of stroke and thromboembolism and independently results in a two-fold risk of excess mortality [3].Although formerly a somewhat neglected structure, the left atrial appendage (LAA) has been found to be a key player in stroke secondary to AF. Accordingly the LAA has in recent times been proposed as a potential therapeutic target.
LAA anatomy
The LAA is a remnant of the embryonic left atrium (LA), which is formed during the third week of gestation.The LA itself develops later from the pulmonary veins [4].The LAA, an outpunching of the LA, is a long, tubular structure lying in the left atrioventricular groove between the left upper pulmonary vein and the left ventricle.It comprises a single layer of endothelium and is trabeculated with underlying pectinate muscles lining the cavity.The LAA varies considerably in size from 16-51mm in length, 10-40mm in diameter and 0.77-19.27cm 3in volume [5,6].The LAA also appears to differ in morphology with distinct variants including the 'chicken wing', 'cactus', 'windsock' and 'cauliflower' being described in 48%, 30%, 19% and 3% of cases respectively [7].
LAA Physiology
It has become increasingly apparent that the LAA has not only specific anatomical, but also, physiological properties.Like the LA, the LAA is a dynamic structure and plays a number of mechanical roles throughout the cardiac cycle.It serves as a reservoir during left ventricular systole, a conduit during early diastole, provides an active pump function in late diastole and its elasticity enforces backward flow to refill in early systole [8].The LAA also been shown to maintain intravascular volume status through activation of stretch receptors located within its body.These afferent signals play a role in fluid haemostasis and control of heart rate in response to changes in LA pressure.In support of this, one study demonstrated that 30% of all cardiac atrial natriuretic peptide (ANP) was found within the LAA [9].Furthermore in the healthy human heart, ANP concentration in the LAA is present in 40-times the concentration of the rest of the LA [10].The significance of the LAA in fluid balance has been reinforced in humans after clamping of the LAA during cardiac surgery yielded increased LA and left ventricular filling pressures [8].Animal studies have also shown that by removing the LAA, reduction of both LA compliance and LA function occurs [11].Notably, a dramatic reduction in cardiac output, of nearly 50%, was witnessed in guinea pigs following ligation of the LAA.This finding was attributed to the contractile function of the LAA [12].Conversely, distension of the LAA in a dog model was found to increase diuresis as well as sodium excretion and heart rate [13].
The LAA in disease
The most notable association between the LAA and disease is in the context of AF.In this setting, reduced contractility and stasis of the LAA occur, resulting in thrombus formation and thereafter the potentially catastrophic consequences of embolisation.In individuals with non-valvular AF, 90% of thrombi have been identified within the LAA; (Figure 1) [14].Additionally it has been observed that up to 14% of patients have thrombus identifiable in the LAA within 3 days of AF onset [15].It is these observations which have led to the LAA being termed 'our most lethal human attachment [16].Remodelling of the LAA in subjects with AF has been observed with chamber enlargement and decreased pectinate muscle volume [17].Typical histological appearances include endocardial thickening, fibrosis and myocyte hypertrophy [18].A reduced LAA peak flow velocity studied during transoesophageal echocardiography (TOE) is established to be an independent and powerful predictor of thromboembolic risk [19].Likewise LAA morphology has been proposed as another marker of thromboembolism with the 'cauliflower' LAA conferring the highest risk of thrombus.In contrast patients with a 'chickenwing' appearance LAA have lowest risk of embolism [7].
Mini Review
The Left Atrial Appendage in Health and Disease Similar to the LA, the LAA has been shown to increase in size in patients with a history of hypertension when compared controls.was also associated with a reduction in emptying velocities demonstrated during TOE [20].
Likewise it appears that the LAA also plays a dynamic role in the setting of left ventricular dysfunction.Firstly left ventricular impairment results in a 10-fold increase in LAA ANP concentration [10].Furthermore, another study indicated that after successful heart failure therapy, LAA function improved significantly.It was also noted that, after treatment, LAA size reduced markedly more than LA size, demonstrating relatively increased compliance [21].
The LAA as a therapeutic target
Since the LAA has been documented to be a major culprit of thromboembolism in AF it has also been proposed as a potential therapeutic target through LAA occlusion or ligation.This treatment option is of considerable interest given the welldocumented bleeding risk of anticoagulants and the significant proportion of patients who are intolerant of anticoagulation therapy [22].To date both surgical and transcatheter LAA exclusion have been investigated with encouraging results, although trials have been criticised both for their small sample size and lack of randomisation in the majority.Recently published long-term data from the PROTECT-AF trial, randomising patients to transcatheter LAA ligation or warfarin, suggests in fact that LAA ligation may be superior to warfarin for prevention of stroke, systemic embolisation and all-cause mortality [23].
Conclusion
In summary the LAA plays a vital role in the pathogenesis of stroke secondary to AF. Furthermore from the available evidence it is clear that the LAA is not a redundant structure in the absence of disease.Despite outcomes suggesting that surgical and transcatheter LAA exclusion may be an alternative to conventional stroke prevention therapy, interference with the mechanical and neurohumoral functions of the LAA may result in, as yet unaccounted for, clinical sequelae.These potential consequences merit further evaluation in future trials.
Figure 1 :
Figure 1: Thrombus identified with in the left atrial appendage during TOE. | 2017-07-28T23:20:55.290Z | 2015-07-23T00:00:00.000 | {
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105478063 | pes2o/s2orc | v3-fos-license | Physical Model of Nernst Element
Generation of electric power by the Nernst effect is a new application of a semiconductor. A key point of this proposal is to find materials with a high thermomagnetic figure-of-merit, which are called Nernst elements. In order to find candidates of the Nernst element, a physical model to describe its transport phenomena is needed. As the first model, we began with a parabolic two-band model in classical statistics. According to this model, we selected InSb as candidates of the Nernst element and measured their transport coefficients in magnetic fields up to 4 Tesla within a temperature region from 270K to 330K. In this region, we calculated transport coefficients numerically by our physical model. For InSb, experimental data are coincident with theoretical values in strong magnetic field.
Introduction
One of the authors, S. Y., proposed [1] the direct electric energy conversion of the heat from plasma by the Nernst effect in a fusion reactor, where a strong magnetic field is used to confine a high temperature fusion plasma. He called [1,2] the element which induces the electric field in the presence of temperature gradient and magnetic field, as Nernst element. In his papers [1,2], he also estimated the figure of merit of the Nernst element in a semiconductor model. In his results [1,2], the Nernst element has high performance in low temperature region, that is, 300 -500 K. Before his works, the Nernst element was studied in the 1960's [3]. In those days, induction of the magnetic field had a lot of loss of energy. This is the reason why the Nernst element cannot be used. Nowadays an improvement on superconducting magnet gives us higher efficiency of the induction of the strong magnetic field. We started a measuring system of transport coefficients in the strong magnetic field to estimate efficiency of the Nernst element on a few years ago [4]. We need criteria to find materials with high efficiency. The first model is one-band model which was proposed by S. Y. However is model cannot explain the temperature dependence of the Nernst coefficient above the room temperature for intrinsic indium antimonide, InSb X [4,5]. We improved the one-band model to the two-band model. In this paper, we measured InSb B which is doped Te heavier than InSb X. Near room temperature, the sample InSb B transits from the extrinsic region to the intrinsic region. To calculate transport coefficients of InSb B in a magnetic field, we use the two-band model, In this paper, we report the calculations by the two-band model. (In Ref. [6], we also measured and calculated transport coefficients of Ge in a magnetic field near room temperature.)
Theoretical calculations
As the physical model to describe transport phenomena of the material in the Nernst element, we use a parabolic two-band model in the classical statics. We have the following parameters of this model; • µ n (µ p ) : mobility of an electron (a hole), • ε G : energy gap, ε F : fermi energy.
Using these parameters, we obtain concentrations of carriers as follows: where n(p) is the concentration of free electron (hole). Here N C (N V ), the effective density of state in the conduction (valence) band is given by We also obtain the concentration of electrons (holes) in the donor (acceptor) level, n D (p A ) as follows: We suppose the charge neutrality as Substituting the concentrations of carriers with eqs. (1)-(6) in eq. (7), we obtain the following algebraic equation in value where Using the fermi energy which is given from eqs. (8) and (9) , we can solve the Boltzmann equation of this model in a magnetic field with a perturbation theory and the relaxation time approximation. See Ref. [1] for details. Here we define the following parameters to simplify formulation as We also define the following integrals as Using the above eqs. (10) -(12), we obtain transport coefficients in a magnetic field B, as follows: R H (B) = 3π 2 zen nJ 1 α(B) = k ze where is the conductivity, R H the Hall coefficient , α the thermoelectric power, and N the Nernst coefficient for electron (z = −1) . For hole (z = 1), we must use p, η + ε G , and µ p instead of n, µ n and η. Relations between these one-band transport coefficients and the two-band ones are written as [7] where the subscripts 1 and 2 denote the contribustion from conduction and balence bands, respectively. The parameter D is described as By the above algorithm, we calculate the transport coefficients in a magnetic field. In this calculations, we must prepare physical quantities i.e. effective masses, energy levels, concentrations of impurities, mobilities, energy gap. From the previous works [8], we can get the following parameters: where m 0 is the bare electron mass. Using eq. (22), we calculate transport coefficients.
Comparison between experimental and theoretical results
We measured transport coefficients of indium antimonide in a magnetic field. The sample X has the electron carrier concentration n = 6.6 × 10 20 m −3 and mobility µ n = 21m 2 /V/s at 77K. The sample B has n = 2.1 × 10 22 m −3 at 77K.
The conductivity and the Hall coefficient are measured by the van der Pauw method. The thermoelectric power and the Nernst coefficient are also measured for the bridge shaped sample [8]. In Fig. 1, we plot the thermoelectric power of InSb X as a function of magnetic field. The Nernst coefficient of InSb X is plotted in Fig. 2. These figures show that these transport coefficients can be calculated by the two-band model. For InSb B, we also measured the conductivity, the Hall coefficient the thermoelectric power and the Nernst coefficient. These results are plotted in Figs. 3-6. These transport coefficients given by the theoretical calculations are coincident with the experimental values.
Discussion and conclusions
From comparison the experimental and the theoretical values, we conclude that the two-band model is enough good model to estimate the transport coefficient. We need to measure thermal conductivity to estimate the thermomagnetic (i.e. Nernst ) figure-of-merit Z N = σ(N B) 2 /κ. The thermal conductivity has phonon scattering mechanism. We, therefore, improve the physical model to include the phonon scattering phenomena. This is a future problem. | 2019-04-10T13:12:22.673Z | 1998-05-24T00:00:00.000 | {
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15399887 | pes2o/s2orc | v3-fos-license | Rocking ratchet induced by pure magnetic potentials with broken reflection symmetry
A ratchet effect (the rectification of an ac injected current) which is purely magnetic in origin has been observed in a superconducting-magnetic nanostructure hybrid. The hybrid consists of a superconducting Nb film in contact with an array of nanoscale magnetic triangles, circular rings or elliptical rings. The arrays were placed into well-defined remanent magnetic states by application of different magnetic field cycles. The stray fields from these remanent states provide a magnetic landscape which influences the motion of superconducting vortices. We examined both randomly varying landscapes from demagnetized samples, and ordered landscapes from samples at remanence after saturation in which the magnetic rings form parallel onion states containing two domain walls. The ratchet effect is absent if the rings are in the demagnetized state or if the vortices propagate parallel to the magnetic reflection symmetry axis (perpendicular to the magnetic domain walls) in the ordered onion state. On the other hand, when the vortices move perpendicular to the magnetic reflection symmetry axis in the ordered onion state (parallel to the domain walls) a clear ratchet effect is observed. This behavior differs qualitatively from that observed in samples containing arrays of triangular Ni nanostructures, which show a ratchet of structural origin.
INTRODUCTION
Ratchets are present in a wide variety of natural and artificial systems, ranging from biological motors to electronic rectifiers. They are characterized by the directional motion of out-of-equilibrium particles induced by a periodic asymmetric potential (e.g. a saw-tooth potential), without a net average driving force or temperature gradient. There are two main types of ratchets: i) flashing ratchets, in which the ratchet potential is time-dependent, as in molecular motors [1]; and ii) rocking ratchets when the ratchet potential is time-independent and the zero average driving force is periodic, as in superconducting rectifiers [2,3]. The preparation and study of artificially prepared ratchets may shed light on the basic physics issues related to these nonequilibrium systems and may give rise to new and unusual applications [4][5][6].
A superconducting-magnetic hybrid is a well-defined artificial system in which a ratchet effect can be induced. These types of hybrid systems consist of a superconducting (e.g. Nb) film in contact with an array of magnetic (e.g. Ni) nanostructures. The ratchet effect manifests itself as a rectification of an a.c. current along specific geometric directions of the array. The underlying physical origin of this effect is the motion of superconducting vortices in the asymmetric periodic potential produced by the nanostructured array. In addition to the ratchet effect, these superconducting-magnetic hybrids exhibit a variety of other interesting effects such as quantum matching phenomena in which periodic matching is observed in the flux flow resistance, or bistable superconductivity which depends on the magnetic history [7][8][9][10][11]. A widely investigated ratchet of this type consists of a superconducting film in proximity to a square array of triangular magnetic nanostructures. For triangles arranged with one edge parallel to the x direction of the array, a ratchet effect is observed for an a.c. current along the x direction leading to vortex motion along the y direction, along which the potential is asymmetric. There is no ratchet for vortex motion along the x direction [2]. In this case the ratchet is caused by the intrinsic structural asymmetry of the triangular array. In addition, an interesting sign reversal of the superconducting ratchet is observed with the amplitude of the a.c. current, i.e., the rectified d.c. voltage changes sign with increasing amplitude of the a.c. driving force.
In the present work, ratchets of magnetic origin are investigated in a Nb/Ni system with structural symmetry. The superconducting vortices in the Nb film play the role of the nonequilibrium particle ensemble, the rocking ratchet potential is induced by an array of Ni rings, and the zero average driving forces are induced by a.c. currents injected into the hybrid samples. We show that the ratchet produced this way is purely magnetic in origin and has a different symmetry from the one produced by an array of triangular nanostructures with structural asymmetry [2]. We first present the fabrication and magnetic characterization of the arrays and the magnetotransport measurement of the hybrid. We then show that the rings provide very effective pinning sites for superconducting vortices and that they modify substantially the vortex lattice dynamics, especially the ratchet effect. We show that, for this system, the ratchet has a purely magnetic (i.e. non-structural) origin and has a different symmetry from a structurallyinduced ratchet.
EXPERIMENTAL METHODS
The samples studied here consist of a superconducting Nb film in contact with arrays of magnetic Ni triangles, circular rings or elliptical rings. The Ni nanostructures were fabricated on Si (100) substrates, and then coated with the Nb layer. The preparation of the Ni triangles has been described previously [2]. The equilateral triangles were arranged in a square array with x-axis period 770 nm, triangle base length 620 nm, y-axis period 746 nm, triangle height 477 nm, and thickness 40 nm. The circular and elliptical Ni rings were prepared by electron-beam lithography in a Raith 150 electron-beam writer using polymethyl methacrylate resist and liftoff processing. Fabrication procedures for similar samples have been described elsewhere [12]. The 20 nm thick Ni film was deposited by sputtering in a system with a base pressure of 10 -8 Torr. The geometries of the ring samples are listed in Table I. Two samples (A: elliptical rings, B: circular rings) were made without Nb overlayers, for MFM and magnetometry study, while two samples (C: elliptical rings, D: circular rings) were made with Nb overlayers. The dimensions of A and C, and of B and D, differed slightly. The 100 nm thick Nb films were deposited using magnetron sputtering on top of the nanostructured Ni arrays. Electrical leads were patterned using photolithography and etching techniques to form a 40µm × 40µm bridge, as shown in Figure 1.
The uncoated Ni rings were characterized magnetically by magneto-optical Kerr effect (MOKE) magnetometry on a Durham Magneto Optics NanoMOKE2 system [13] for inplane magnetic fields. The beam was focused to a 30 µm diameter spot size, capturing the average reversal behavior of ~10 3 rings. With a repetition rate of 11 Hz, typically ~10 3 loops were averaged to obtain a single hysteresis curve. In addition to conventional major hysteresis loops, a first-order reversal curve (FORC) method [14][15] was also employed to help determine the magnetic state of the Ni nanostructures. After positively saturating the sample, the applied in-plane field was reduced to a given reversal field H R and the magnetization M was then measured back to positive saturation thereby tracing out a FORC. This process was repeated for increasing negative H R until negative saturation is reached and the major hysteresis loop is filled with FORCs (Fig. 2). The FORC distribution gives details of the switching process of the Ni rings.
Magnetic imaging was performed on samples made without the Nb overcoat by magnetic force microscopy (MFM, Digital Instruments Nanoscope), with a low-moment magnetic tip. Micromagnetic modeling of rings was performed using the two dimensional OOMMF code [16] with 2 nm × 2 nm cell size and parameters appropriate for pure Ni, with the cubic anisotropy oriented randomly in each cell.
The electrical resistivity of the hybrid system was measured using the standard 4-point probe method, in the geometry shown in Fig. 1, with a magnetic field applied perpendicular to the sample plane. In this geometry we are able to induce vortex motion in the x and y in-plane directions of the array. The relevant transport properties of all hybrid samples are similar, i.e. the critical temperatures are 8.10 K (elliptical ring sample), 8.15 K (circular rings) and 8.30 K (triangles), and the mean free paths are 8 × 10 -10 m (elliptical rings), 12 × 10 -10 m (circular rings) and 10 × 10 -10 m (triangles).
RESULTS AND DISCUSSION
The magnetic state of the ring arrays was examined for three cases: circular rings with the in-plane field along one axis of the square array, elliptical rings with the field along the long axis (LA) of the ellipses, and elliptical rings with the field along the short axis (SA) of the ellipses. In all three cases the FORC magnetometry data (Fig. 2) and the MFM data ( Fig. 3) indicate that the rings form onion states at remanence after saturation at fields > 600 Oe. Onion states consist of two domains separated by domain walls at opposite sides of the ring, aligned parallel to the direction of the saturating field. For rings of these dimensions, thickness, and material, the simulation shows that the domain walls have transverse character with in-plane magnetization. These results are in agreement with previous results on thin film magnetic rings [18,19]. For the circular rings and the elliptical rings magnetized along their long axis, practically all of the rings form onion states of identical orientation, and the dark or bright contrast of the domain walls is evident in Figs. 3g and 3h respectively. For the elliptical rings magnetized along the short axis, some of the rings (<10%) form 'vortex' states with no domain walls and no magnetic contrast, as seen in Fig. 3i, but the majority form parallel onion states with domain walls located along the minor axis of the ellipse.
In contrast, demagnetization of the ring arrays is expected to lead to a magnetic configuration consisting of onion states oriented in different directions, in addition to 'vortex' states. In this case the stray magnetic field fluctuates aperiodically across the sample providing a random magnetic potential landscape.
The magnetic properties of the array of Ni triangles have been reported previously [17]. At remanence after in-plane saturation the triangles form magnetic vortex states exhibiting both clockwise and counterclockwise chiralities and 'up' and 'down' polarities of the vortex core.
In general, structural defects are very effective vortex pinning centers [20]. In these types of systems close to the superconducting critical temperature, the magnetoresistance exhibits pronounced periodic minima when the vortex density is an integer multiple of the pinning site density [7]. Hence, the number of vortices n per Ni nanostructure is controlled by the external magnetic field H z perpendicular to the sample, and can be characterized using magnetoresistance measurements. Since the perpendicular applied magnetic fields are less than 400 Oe, we expect that the remanent states of the rings are not substantially modified during the experiment due to the high demagnetizing factors of the thin film structures. Fig 4 shows magnetoresistance vs. H z of three hybrid samples that were initially in the demagnetized state, i. e. samples as deposited. The triangular, elliptical rings and circular rings have similar geometric sizes and periodicities, and the three samples show similar periodicity in the magnetoresistance fluctuations. However, the samples containing rings exhibit a significantly larger number of minima than the ones containing triangular nanomagnets. Thus the rings in these hybrids are very effective pinning centers and are able to pin a larger number of vortices than hybrid samples containing magnetic triangles. Fig. 5 shows a comparison between the ratchet effect in samples containing triangular and elliptical rings measured at a perpendicular field H z corresponding to n=3, i.e. three vortices per pinning site. The a.c. injected current is parallel to the triangular base or the ellipse short axis. Therefore, the vortex motion is from triangle base to tip, or parallel to the ellipse major axis, respectively. The motion is caused by the Lorentz force, (where φ 0 is the quantum fluxoid, z r is a unit vector parallel to the applied magnetic field and J is the a.c. current density) acting on the vortices. Although the time-averaged force on the vortices 0 = L F , a non-zero d.c. voltage drop is observed for the triangles, as seen previously [2], and for the rings measured at remanence after saturation, where they are in parallel onion states. Since the electric field ( v v B E r r r × = r and B r being the vortex-lattice velocity and the applied magnetic induction, respectively), the voltage drop (V dc ) along the direction of the injected current probes the vortex motion along the perpendicular direction. Therefore, the timeaveraged vortex velocity is , where d is the distance between the voltage contacts.
As noted above, the Ni triangles in the demagnetized state develop a magnetic vortex state, with random chirality (clockwise or counter clockwise) and polarity (up or down) [13,17]. Thus, in the absence of a periodic magnetic potential, the ratchet effect in this case must originate from the structural shape asymmetry of the array of triangles [2]. It is interesting to note that for the purely geometric structural asymmetry exhibited by these triangles the ratchet effect is odd with the direction of the applied magnetic field, i.e. reversing the magnetic field reverses the sign of V dc .
On the other hand, the samples with elliptical rings are structurally symmetrical and therefore show no ratchet effect in the demagnetized state (empty circles in Fig. 5), but an unambiguous ratchet effect (solid black circles in Fig. 5) occurs when the elliptical rings are magnetized in parallel onion states. The latter ratchet is attributed to a pure magnetic asymmetry. This magnetic origin is further confirmed by the fact that the sign of V dc does not change when the applied magnetic field direction is reversed, i.e., the ratchet effect is even. The magnetic ratchet effect (in the rings) and the shape-induced ratchet effect (triangles) share certain similarities: i) both are adiabatic, i.e., independent of frequency [2] up to the highest attainable frequency in our experiments (10 kHz), and ii) both show the same experimental trends, i.e., the magnitude of the output d.c. voltage increases (at constant applied field) with decreasing temperature and (at constant temperature) with increasing applied magnetic field [21]. There is also an important difference between the two ratchet effects: for the ring samples the sign of V dc (the ratchet polarity) does not change with increasing number of vortices or with the a.c. input driving currents, i.e., there is no vortex ratchet reversal for a purely magnetically-induced ratchet. In contrast, structurally asymmetric ratchets do exhibit a sign reversal [2,3,21] in which the d.c. output voltage polarity can be tuned by external parameters. For instance, at n = 4 the sign of the d.c. voltage changes with increasing amplitude of the a.c. drive current as shown in the inset of Fig. 5. Fig. 6 shows the ratchet effect for both circular and elliptical rings magnetized in parallel onion states for different temperatures and vortex motion directions (along or perpendicular to the magnetization axis, i.e. parallel or perpendicular to the domain walls). When the vortex motion is perpendicular to the domain walls (and to the direction of the initial saturating in-plane field) the ratchet is absent, as expected from a purely magnetic effect, because the magnetic potential is symmetrical. The driving force threshold and the magnitude of the ratchet effect increase with decreasing temperature as found earlier [21].The ratchet in circular rings shows similar trends to the elliptical rings. The magnitude of V dc for the circular sample is similar to that measured for the elliptical samples for vortex motion along the minor axis, but much smaller than the V dc measured for elliptical rings when the vortex motion is along the major axis. This result suggests that the separation between the magnetic potential wells plays an important role in the strength of the ratchet effect and therefore in the amplitude of the d.c. output voltage.
As we have seen, the magnetization direction in the ring dictates the direction along which the vortices have to move to produce a ratchet effect. This is analogous to the triangular elements, in which the structural asymmetry defines the directions along which a ratchet effect can be observed [2,21]. However in the case of the rings, the ratchet is tuneable according to the magnetic state of the pinning sites, and can be 'switched off' by demagnetizing the array. The results shown in Fig. 6 indicate that an asymmetric magnetic potential is only found when the vortices move along the magnetization direction parallel to the direction of the initial saturating field and to the remanent domain walls. Experimentally, the asymmetric ratchet potential is therefore clearly related to the differences in the interaction between the vortex motion and the magnetization direction in the rings. The variation of the amplitude of the ratchet effect for different directions and types of ring arrays implies that the interaction of the vortex motion with the magnetic fields produced by the onion states plays an important role in the magnitude of the effect.
Recently, a pure magnetic ratchet potential has been discussed theoretically [22,23]. This ratchet is based on pinning by arrays of magnetic dipoles, and this model has been applied in the case of superconducting vortices. In this theory, the origin of this pure magnetic ratchet effect is the periodic vortex pinning potential with broken reflection symmetry created by the magnetic dipoles. This pinning of the vortex lattice by dipole arrays occurs if the vortex lattice periodicity is pinned by and commensurate with the dipole array periodicity. This theoretical model is applicable here, since the remanent onion state induces periodic dipolar magnetic potentials, and the applied field H z was chosen such that the vortex lattice is commensurate with the periodicity of the magnetic rings, based on the magnetotransport measurements.
Superficially similar results [24] in hybrids of Pb films with array of triangular Co microrings and Al films with Co microbars have been reported. These results were analysed with the aforementioned model [22,21] invoking the appearance of vortexantivortex pairs. However, contrary to our results, several polarity reversals were observed with increasing number of vortices. Furthermore, a non-zero ratchet effect was found even in zero applied fields. Since some of these experiments rely on arrays which are structurally asymmetric, the reported ratchet effects may not be purely magnetic in origin, and the relationship to the results reported here is not yet clear.
CONCLUSIONS
Superconducting-magnetic hybrid systems based on periodic arrays of Ni rings in contact with Nb films show rocking ratchet effects when the superconducting vortex lattice moves in an asymmetrical magnetic potential landscape induced by the periodic array. The most relevant results can be summarized as follows: i) The ratchet effect is observed only when the ring array is magnetized at remanence into parallel onion states, and not when the array is demagnetized. ii) The magnetic ratchet observed here resembles the ratchet produced by structurally asymmetric pinning sites in that both are adiabatic, and the amplitude and threshold of the driving force increases with decreasing temperature. However, they differ qualitatively in that the magnetic ratchet does not change its polarity when the applied magnetic fields are reversed or the driving force strength (a.c. current) is increased. iii) The origin of the ratchet effect lies in the interaction between the superconducting vortex screening currents and the periodic asymmetric stray magnetic fields produced by the rings when placed in parallel remanent onion states. This interaction establishes a net direction for the vortex motion when it is driven by a zero average a.c. current. While the detailed mechanism for this interaction has not been established, our experiments show that this effect must be purely magnetic in origin. Table I) where the applied field is (a) parallel and (b) perpendicular to the long axis (major axis) of the ellipses. (c) FORC's measured on an array of circular Ni rings (sample B in Table I). The insets show a sketch of the onion magnetic states and the asymmetric magnetic reflection axis (double arrows). Fig. 3. (a,b,c) OOMMF simulation of the remanent state of (a) a circular ring after 5000 Oe saturation, (b) an elliptical ring after 5000 Oe saturation along the long axis, (c) an elliptical ring after 5000 Oe saturation along the short axis; (d,e,f) topographic and (g,h,i) magnetic remanent images of (d,g) the circular ring array at 65nm lift height; (e,h) the elliptical ring array at 50 nm lift height, long axis field; (f,i) the elliptical array at 65 nm lift height, short axis field. Images are 6 µm square. The white dotted lines indicate the locations of some of the rings in the MFM images. Table I) and circular rings (open dots, sample D) measured with the magnetic nanostructure arrays initially in the demagnetized state. The temperature T/T c was 0.99. Current densities: J = 8.0 x 10 2 Acm -2 (triangle sample); J = 2.5 x10 3 Acm -2 (elliptical rings); J = 6.0 x 10 3 Acm -2 (circular rings). The data for the triangles has been scaled by a factor of two for clarity. The Ni rings were measured in both the demagnetized state (open circles) and in parallel onion states after saturation along the long axis (solid circles). The vortex lattice motion corresponds to translation from the triangle base to the apex in the array of triangles and along the major axis in the case of the array of elliptical rings. The frequency of the injected a.c. current is 10 kHz and T/T c = 0.98. The inset shows ratchet reversal in the array of Ni triangles obtained with increasing a.c. drive current. Fig. 6. Experimental data showing the ratchet effect for samples with elliptical and circular Ni rings for different vortex motion directions and temperatures. In all cases the rings were magnetized into parallel onion states. The vortex motion is along either the major axis (LA) or minor axis (SA) in the case of the elliptical rings. All the measurements were made with the vortex motion along the direction of the domain walls (DW), parallel to the initial in-plane saturating field, except for the data shown with solid circles in which there is no ratchet effect. The measurement temperatures were 0.98 T c (solid triangles and solid diamonds) and 0.97 T c (open triangles and open diamonds). F L is the Lorentz force on the vortices and <v> their average velocity. Table I Dimensions of the circular and elliptical ring arrays in the Ni/Nb hybrid samples. | 2016-03-14T22:51:50.573Z | 2009-12-01T00:00:00.000 | {
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78088621 | pes2o/s2orc | v3-fos-license | Peak cement-related CO 2 emissions and the changes in drivers in China
In order to fight against the climate change, China has set a series of emission reduction policies for super-emitting sectors. The cement industry is the major source of process-related emissions, and more attention should be paid to this industry. This study calculates the process-related, direct fossil fuel- and indirect electricity-related emissions from China’s cement industry. The study finds that China’s cement-related emissions peaked in 2014. The emissions are, for the first time, divided into seven parts based on the cement used in different new-building types. The provincial emission analysis finds that developed provinces outsourced their cement capacities to less-developed regions. This study then employs index decomposition analysis to explore the drivers of changes in China’s cement-related emissions. The results show that economic growth was the primary driver of emission growth, while emission intensity and efficiency were two offsetting factors. The changes in the construction industry’s structure and improvement in efficiency were the two major drivers that contributed to the decreased emissions since 2014.
Introduction
Being the largest CO2 emitter and energy consumer (Guan et al. 2009), China is taking increasing responsibility for global climate actions. To reduce greenhouse gas (GHG) emissions in more effective and efficient ways, China has set a series of relevant strategies and specific policies targeting super-emitting sectors (NDRC 2016c), including power generation , coal mining (NDRC 2016b), and the iron and steel industry (MIIT 2016). Cement is one of the largest key sources of process-related emissions in China and worldwide. According to China's official GHG emission inventories from National Communications on Climate Change (as shown in Figure S1), process-related GHG emissions from the cement lime industry reached 157.78 million tons (Mt) (or 57% of the total process-related emissions) in 1994 (NDRC 2004), 411.57 Mt (or 72%) in 2005(NDRC 2013, and 834.03 Mt (or 70%) in 2012 (NDRC 2016a). With such a large amount and rapid growth of GHG emissions, the cement industry has become a key sector in GHG emissions mitigation. Indeed, several policies have been proposed to reduce the energy consumption and emission intensity of the cement industry in China. In the latest 13th five-year plan for the cement industry, the government aims to achieve a 30% reduction of the air pollutant emissions of the cement industry in 2020 compared to the 2015 level (China Cement Association, CCA, (2017). The energy consumption per ton of clinker production should be kept under 105 kilograms (kg) coal equivalent in 2020, while those of 2015 amounted to 112 kg. With the efforts of the government, the process-related CO2 emissions from China's cement industry peaked in 2014 (Shan et al. 2018a), maintaining the same trend as China's total emissions .
Accurately accounts of the cement-related emissions and understanding the driving forces of the changes in emissions is of considerable value, especially has practical significance for further emission reduction policymaking. Oda et al. (2018) developed a global high-spatial-resolution gridded CO2 emissions data inventory including cement production as a part of nonpoint emission sources. Şanal (2018) evaluated the CO2 emissions of different types' cement and discussed emission reduction capacities of cement replacement in concrete production. Andrew (2018) presented a new analysis of global process emissions from cement production, which were 30 % lower than those reported by the Global Carbon Project (Le Quéré et al. 2015). Apart from the carbon source function, Xi et al. (2016) found that carbonation of cement materials has offset 43% of CO2 emissions from cement production from 1930 to 2013.
Previous studies have employed the Index Decomposition Analysis (IDA) -logarithmic mean Divisia index (LMDI) model to analyze the variations of carbon emissions. Branger and Quirion (2015) investigated the changes of CO2 emissions in the European cement industry from 1990 to 2012 and found that most of the emission change (activity, clinker trade, thermal and electrical energy efficiency, and electricity decarbonization) could be attributed to the activity effect. When it comes to China, Xu et al. (2012) got a similar result. They found that the activity effect (calcining process and electricity consumption) was the main driver of cement-related emissions increase while clinker share, structural shift, and kiln efficiency were main negative drivers. Wang et al. (2013) identified the main drivers that influence China's cementrelated greenhouse gas emissions. Boqiang Lin (2016) found that the labor productivity was the major driving force to increase the cement-related CO2 emissions from 1991 to 2010.
Despite that nearly all previous studies in emission accounts and analysis involved the cement process in the emission inventories, the emissions are usually calculated with the cement production (Shan et al. 2016b;Liu et al. 2015;Shan et al. 2018b). As the process-related CO2 emissions are majorly produced alongside the clinker production, such a cement production-based calculation method is not accurate enough. It may overlook the regional diversity in the cement manufacturing technique and cementclinker ratio. Therefore, some recent studies calculated the emissions with clinker's production (Cai et al. 2016). Considering the regional diversity in China's cement manufacturing, this study first investigates the emissions from China and its regions' cement industry. This study examines the process-related CO2 emissions (calculated based on the clinker productions), direct emissions from fossil fuel combustion, and indirect emissions induced by purchased electricity in the cement industry. The cement-related CO2 emissions are divided into seven parts according to the cement demands of different new-building types for the first time. This study also describes the regional diversity in cement production and the cement production capacity shifts among the provinces.
This study then employs the LMDI method to break down the changes in China's cement-related CO2 emissions into four drivers, including the construction industry's structure, emission intensity, efficiency, and economic growth. To the best of our knowledge, our study is the first to examine the factor of "construction industry's structure", which is measured by the cement used for different new-building types, in the analysis of the drivers of China's cement-related emissions. We particularly compared the changes in drivers before and after these emissions peaked in 2014.
Our study provides robust and transparent data support for further environmental evaluations and emission reduction/sustainable production policy making for the cement industry in China.
Cement production process and related emissions
Cement is normally produced in three steps, as shown in Figure 1 (Worrell et al. 2001). First, the limestone (primarily CaCO3) is crushed and ground into raw meal. Then, the raw meal is calcined in kilns to clinker. Finally, the clinker is ground together with additives (fly ash, pozzolana, gypsum, anhydrite, etc.) to form cement.
Figure 1 Cement production process and emission calculation
The crusher and grinder are physical reactions that do not emit any CO2 during the process. In contrast, calcination is a chemical reaction, during which the processrelated CO2 may be emitted. The process-related emissions are the CO2 emitted as a result of chemical reactions in the production process rather than the energy combusted by industry (Shan et al. 2016a). During the calcination of raw meal, the limestone is heated to lime (CaO) and CO2; see Equation 1. The CO2 emissions are process-related emissions during the cement production process.
Although the crushing and grinding processes do not emit process-related emissions, they consume abundant energy for power, such as coal and electricity. The CO2 emitted during coal combustion and electricity generation is counted as direct emissions and indirect emissions, respectively (Liu 2016b). The indirect emissions are normally produced in power plants rather than the cement plants.
Overall, this study considers the process-related CO2 emissions, direct CO2 emissions from coal combustion, and indirect CO2 emissions induced by electricity consumption in China's cement industry. We adopt the mass balance method recommended by the Intergovernmental Panel on Climate Change to account for the emissions (IPCC 2006).
Process-related CO2 emissions
The process-related CO2 emissions during cement production can be estimated as the cement or clinker production timed by the related emission factors. As discussed above, most of the previous studies use the cement production to calculate the cement process-related emissions. These cement production-based emission accounts may overlook the regional diversity in the cement manufacturing process and cement-clinker ratio. Therefore, the present study calculates the process-related emissions based on clinker production to achieve more accurate emission accounts of the cement industry; see Equation 2 (IPCC 2006).
= × Equation 2
In the above equation, refers to the clinker production, while is the emission factor for clinker production, i.e., the CO2 emitted during per unit production of clinker.
The emission factor for cement is collected from Liu et al. (2015), which is 0.4964 tons CO2 per ton of clinker production.
Direct CO2 emissions from coal combustion (coal-related CO2 emissions)
The direct coal-related CO2 emissions are estimated using Equation 3 (IPCC 2006).
In the above equation, is the direct coal-related CO2 emissions in the cement production, (activity data) refers to the coal consumption, and is the emission factor of coal, which is made up of three components: (net caloric value), (carbon content), and (oxygenation efficiency). The parameters are collected from our previous study on China's coal quality based on an extensive investigation of 4243 coal mines (Liu et al. 2015). The of coal is 20.60 PJ/mt, the is 26.32 tC/TJ (Shan et al. 2018a), and the is 92%. The overall emission factor of coal ( ) is 0.499 ton CO2 emissions per ton of coal consumption.
Indirect CO2 emissions from electricity consumption (electricity-related CO2 emissions)
The indirect CO2 emissions induced by purchased electricity consumption are calculated using Equation 2 (IPCC 2006).
= × Equation 4
In the equation, is the electricity-related CO2 emissions in the cement production and (activity data) refers to the electricity consumption, while refers to the emission factor. This study uses the regional average electricity emission factors (NDRC 2011) for each province (see Table 1).
CO2 emissions induced by domestic construction and exports
Cement is mainly used in the construction industry to build new-buildings. Therefore, the cement-related CO2 emissions are closely associated with the new investment in the construction industry. In order to better analyze the CO2 emissions, we split the total cement-related emissions into seven parts according to the cement demands of different domestic new-building types and export.
We firstly exclude the export-related emissions from the total amount. The exportrelated emissions are calculated based on the cement clinker export amount, which is collected from the CCA (2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014). The remaining CO2 emissions are associated with domestic new-building's construction, which can be categorized into six types: residential, manufacturing, infrastructural, commercial, science-education-culturehealth, and other buildings. We collect each new-building types' construction outputs from CCA (2005CCA ( -2014 and calculate the respectively proportion of each type. We then divide the domestic emissions into six parts according to the proportion of each newbuilding type.
Index decomposition analysis (IDA)
Understanding the drivers leading to the cement-related CO2 emission peak has practical importance for further emission reduction policymaking. Techniques available for conducting such analyses include structural decomposition analysis (SDA) (Rose and S. Casler 1996) and index decomposition analysis (IDA) (Ang 2004;Liu et al. 2012b), both of which have been extensively applied to quantify the socioeconomic driving factors of a dependent variations, such as energy consumption or CO2 emissions (Dhakal 2009;Mi et al. 2017;Baležentis et al. 2011;Guan et al. 2018). SDAs enable us to capture both direct and indirect effects along the entire supply chain on the basis of input-output tables ). This study focuses on the emissions from 1996-2016 and uses IDA because of two reasons. First, all the cement-related CO2 emissions are from the sector of non-metallic products, only a small part of sectors in China's economy is involved in the supply chain. Second, the time-series input-output tables in China are not available. Thus, we use IDA to decompose the cement-related CO2 emissions.
A variety of methods for IDA have been developed, most of which are versions of the Laspeyres index or Divisia index methods. Ang (2004) proposed a log mean Divisia index (LMDI) method based on the Divisia index. The LMDI method is the most preferred method, as it passes a number of basic tests for a good index number. The decomposition is perfect, which means that there is no residual term that other methods might produce. The LMDI method can also deal with zero values better than other methods (Ang and Liu 2007a;Ang and Liu 2007b). The LMDI method has the advantages of "path independence, consistency in aggregation and easily interpreted results" (Liu et al. 2012a;Meng et al. 2016). By using the LMDI method, previous studies have explored the drivers of regional emission growth Liu 2016a;Zhang et al. 2016;Chong et al. 2012;Wang et al. 2014;Liu et al. 2007). This study, therefore, employs LMID to quantify the drivers of CO2 emissions' changes in China's cement industry. Detailed methods are presented in the Supporting Information.
Four driving factors are defined in Equation 5 to explain the total changes in the cement-related CO2 emissions: the construction industry's structure, emission intensity, efficiency, and fixed capital formation. The changes in each factor help quantify the change in CO2 emissions from the cement's different usages, environmental effects, technological advancement, and economic growth aspects.
In the above equation, is the total cement-related CO2 emissions. are emissions induced by different new-building types, which reflects the construction industry's structure in China. We merge the seven types defined in the section 2.2.4 to three for a clear and concise decomposition analysis: residential and commercial buildings; infrastructural buildings, manufacturing buildings, science-educationculture-health buildings, exports and others. The building types merged together have similar characters. is the cement production, and represents the fixed capital formation in respective years.
The four different factors are: 1) S = ⁄ (proportion of CO2 emissions, in %) measures the share of CO2 emitted from cement usage , representing the construction industry's structural effect; 2) I = CE/P (emission intensity, in ton/ton) measures the CO2 emissions per unit of cement production, representing the environmental impacts in the cement production; 3) E = P/F (input efficiency, in ton/Chinese yuan) measures the cement production per unit of fixed capital formation, representing the technological advancements in the cement production; 4) F (fixed capital formation, in Chinese yuan) stands for the economic growth.
Cement and clinker production
National Bureau of Statistics NBS (2018) provides the national and provincial cement production from 1996 to 2016. The CCA (2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) publishes the cement production of the country from 1996 to 2013, clinker productions for the country from 2002 to 2013, and the cement/clinker for the provinces from 2005 to 2012. We compare the national and provincial aggregated cement production of NBS and CCA. We find that the difference between the two sources is within ±1% (as shown in Table S1). This demonstrates that the quality of China's cement statistics is relatively high. In order to achieve more accurate CO2 emission accounts for China's cement industry, we integrate both NBS and CCA's data and obtain the cement and clinker production data.
At the national level: 1) The cement production from 1996 to 2013 are collected from CCA, and the data from 2014 to 2016 are collected from NBS. The clinker production from 2002 to 2013 are collected from CCA.
2) We then calculate China's clinker-to-cement ratios from 2002 to 2013. The clinker-to-cement ratio is calculated as the clinker production divided by the cement production. 2) The clinker-to-cement ratios of every province from 2005 to 2012 are calculated based on the provinces' cement and clinker production volume.
3) The clinker-to-cement ratios of every province from 2002 to 2004 and 2013 are estimated with the country's overall ratios and the provinces' ratios. We assume the provinces have the same change rates in clinker-to-cement ratio as those at national level. 5) The clinker production of every province from 1996 to 2004 and 2013 to 2016 are estimated as the product of clinker-to-cement ratios and corresponding cement production volume.
The cement and clinker productions, and the clinker-to-cement ratios of China (for both the nation and provinces) are presented in Table S2, Table S3, and Table S4, respectively.
Coal and electricity consumptions
The CCA (2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) published the total energy consumption and coal consumption of each province from 2005 to 2012. We estimate the provinces' energy consumption for the remaining years assuming that the energy intensities (per unit of cement production energy consumption) remain the same. The electricity consumption is estimated as the total energy consumption minus the coal consumption, as coal and electricity are the primary energies used in China's cement industry (CCA 2005(CCA -2014.
The coal and electricity consumption in China and each province's cement industry are presented in Table S5 and Table S6, respectively.
Other indexes used in IDA
The Table S7.
All the data and results can be download freely from the "China Emission Accounts and Datasets (CEADs)" at http://www.ceads.net for re-use. The growth of cement-related CO2 emissions was coupled with the trends of China's GDP growth (NBS 2018). This correlation can be explained by the relationships among the emissions, production/demand, and economic inputs in the cement industry: 1) the CO2 emissions are calculated based on the clinker/cement production; 2) the cement is a building material that is not easy to preserve, and the plants normally produce cement based on the market demand or purchasing orders; and 3) cement demand is closely associated with the number of new-buildings, which is primarily stimulated by the country's fixed capital formation. In this way, it could be inferred that the cement-related CO2 emissions were highly driven by the country's economic gains.
Emissions from China's cement industry
After the three increasing phases, China's cement-related emissions peaked in 2014.
In 2015, the overall cement-related emissions appeared to decline for the first time. The emissions decreased by 76 Mt (or 7.5%) in one year. This decline was mainly caused by a decrease in cement production. The total cement production decreased from 2,492 Mt in 2014 to 2,359 in 2015.
By investigating the detailed emission mix, we find that the process-related CO2 emissions were the primary source of cement-related emissions in China, accounting for 63.1% in 2016. The coal-and electricity-related emissions accounted for the remaining 12.3% and 21.6%, respectively. The shares of emissions caused by different sources have changed slightly over the past 20 years. The process-related emissions decreased from 67.0% in 1996 to 63.1% in 2016 (with an average value of 66.1%), while the emissions induced by purchased electricity consumption increased from 20.2% in 1996 to 24.4% in 2016 (with an average value of 21.6%). The coal-related emissions remained stable at approximately 12.3% over the 20-year study period. As we use the same emission factor for emission accounts over years, the unchanged emission mix implies that the energy intensity (energy consumption per units of cement production) has not changed greatly, i.e., the energy efficiency and utilization technology in China's cement production remained at the same level during the past 20 years.
Figure 3 Cement-related CO2 emissions by new-building types
Note: SECHG is short for science-education-culture-health-government. Figure 3 presents the CO2 emissions induced by the cement consumption in each newbuilding type in selected study years. The results show that the emissions induced by cement produced for different new-building types remain stable over time, implying a relatively stable structure in China's construction industry. Residential buildings are the major contributors to the cement-related emissions, accounting for approximately 40%, followed by infrastructure (approximately 25%) and the manufacturing plants (approximately 10%). Science-education-culture-health-government (SECHG) and commercial buildings emitted approximately 7% and 4% of total emissions, respectively. The emissions induced by the cement export accounted for only 1% of the total emissions due to the small export volume. Cement is expensive for longdistance transport due to its short shelf life and high density. It is much more costeffective to self-produce cement. China exports less than 10 Mt of cement and clinker to Africa and the USA/EU every year, a very small proportion of the 2,410 Mt cement and 1,295 Mt clinker produced.
This study also calculates the emission intensity of cement production. The emission intensity is defined as CO2 emissions per unit of cement production. We find that the emission intensity of the cement production slightly decreased during the past two decades from 0.54 (in 1996) to 0.42 ton CO2 per ton of cement production (in 2016).
The process-related, direct coal-related, and indirect electricity-related CO2 emissions of cement industries at both the national and individual province levels are presented in Table S8, Table S9, and Table S10, respectively. The total cement-related CO2 emissions are summarized in Table S11.
Regional differences in cement production
Despite the overall cement-related emissions having peaked in China, considering the regional differences in China, not all provinces have peaked their cement-related emissions yet. Certain provinces are still in the rapidly increasing stage. We classify the 31 provinces (excluding Taiwan, Hong Kong, and Macao due to a lack of data) into four groups at different peak stages, as shown in Figure 4.
Figure 4 Provincial CO2 emissions from the cement industry
Note: the number following the province name presents the emission intensity of the province in 2016. The early-peaked, mid-peaked, late-peaked, and fast-growing provinces are shown in green, blue, red, and yellow, respectively.
The early-peaked group includes the four most developed provinces in China: Beijing, Shanghai, Tianjin, and Zhejiang. These four provinces peaked their cement-related emissions around 2006. The early-peaked provinces have the lowest emission intensity (0.35 ton CO2 per ton cement production) among the four province groups. The mid-peaked group includes 11 provinces, most of them located in northern and northeast China. These provinces peaked their cement-related emissions around 2012. The average emission intensity of these 11 mid-peaked provinces is 0.43 ton CO2 per ton cement production. Another 10 provinces belong to the late-peaked group, which peaked their cement-related emissions around 2014. The fast-growing provinces are still continuously increasing their cement-related emissions. Yunnan, Guangxi, Hainan, Guizhou, Chongqing, and Tibet belong to this group. The fast-growing group, therefore, has the highest average emission intensity of 0.47 ton CO2 per ton cement production.
Production capacity shifts among provinces
The provinces' different technical levels could affect their emission peak stages and emission intensities. The early-peaked provinces have advanced technologies, which consume less energy during the cement production. However, the discrepancies can be largely explained by the clinker trade among provinces, which can be shown from the clinker-to-cement ratios of the provinces.
Figure 5 Clinker-to-cement ratio changes in provinces
We present the provinces' clinker-to-cement ratios in Figure 5. The overall national ratio decreased uniformly from 72.4% in 2002 to 53.7% in 2016 with an average value of 65.0%. A higher ratio indicates a larger clinker production and a smaller cement production, whereas a smaller ratio indicates a smaller clinker production but a larger cement production.
Figure 5-a shows that the early-peaked provinces' clinker-to-cement ratios decreased markedly since 2006, especially Shanghai, Tianjin, and Zhejiang. The decline of the clinker-to-cement ratios implies that these provinces reduced their clinker production and imported clinker from other provinces to produce cement. As most of the CO2 is emitted during the clinker production process (Worrell et al. 2001), a clinkeroutsourcing policy effectively reduced the provinces' overall CO2 emissions. Taking Shanghai as an example, its clinker-to-cement ratio decreased considerably from 33.9% (in 2005) to 16.6% (in 2006) and further to 8.5% (in 2010). Shanghai massively reduced its clinker production two times, in 2006 and 2010. Its clinker production reduced by 1.7 Mt (or 46.9%) and 0.8 Mt (or 57.5%), respectively, in the two years. However, the cement production of Shanghai remained stable at the same time (10.5 Mt in 2005, 11.3 Mt in 2006, and 6.7 Mt in 2010, which illustrates that Shanghai reduced its clinker production and imported the clinker from other regions. As a result, the emission intensity of Shanghai is the lowest among all the provinces, with 0.09 ton CO2 per ton cement production. Similarly, Figure 5-b shows that most of the mid-peaked provinces' clinker-to-cement ratios decreased from 2007 to 2010, such as Heilongjiang and Jilin. As for the latepeaked provinces (shown in Figure 5-c), there is no remarkable sudden drop in the clinker-to-cement ratio. Despite the ratio of Anhui decreasing continuously since 2003, this decrease is mainly caused by the increase in Anhui's cement production rather than the decrease in its clinker production. Anhui's clinker production kept increasing until 2014, which caused Anhui's cement-related emissions to peak at 9,145 Mt in 2014. Anhui is the largest clinker production base in China. In 2016, the clinker production of Anhui was 121 Mt, accounting for 9.3% of the national production. However, Anhui's cement production accounted for only 5.6% (or 136 Mt) of the national total production, illustrating that Anhui exports large amounts of clinker to other regions. The average clinker-to-cement ratio of Anhui was as high as 134.5% over the past 20 years, which is more than twice the national average level of 61.7%. Figure 5-d presents the clinker-to-cement ratios of the six fast-growing provinces. We find that most of the provinces had a relatively stable ratio during the past 20 years. It is worth noting that the clinker-to-cement ratio of Tibet increased suddenly from 75.7% (in 2008) to 91.0% (in 2009), mainly due to the increase of Tibet's clinker production. Tibet enlarged its cement production capacity since 2000 and formed the integrated company "Huaxin Cement (Tibet)" in 2009 (Liu 2017;Shan et al. 2017).
To summarize, certain developed provinces have closed or outsourced their clinker production to other less-developed regions in the past ten years. Such outsourcing may be effective in reducing the developed provinces' emissions in the short term, but it may not be beneficial to the country's overall emissions reduction (Shan et al.).
The developed provinces usually have more advanced technologies than those in lessdeveloped regions, i.e., low emission intensities. Moving the production capacities from regions with advanced technologies to less-developed regions will, therefore, increase the country's overall emissions. Thus, greater attention should be paid to these key cement production provinces, such as Tibet, which will continue developing its cement production in the future (Tibet autonomous region government 2011), and Anhui, which is the largest cement production base in China. Figure 2 shows that the total cement-related CO2 emissions started to grow rapidly since 2002 after China joining the WTO and then peaked in 2014. Understanding the changes in the drivers of the cement-related CO2 emissions since 2002, especially before and after the peak point (2014) has great policy implications for emissions control in the cement industry. By employing the LMDI method, this study decomposes the changes in China' cement-related CO2 emissions from 2002 to 2016 into four factors: the construction industry's structure, emission intensity, efficiency, and economic growth. The first factor, "construction industry's structure", is measured by three indicators: the emission proportion induced by cement produced for new residential buildings, for new infrastructural buildings, and for a new proportion of export and others. The results are shown in Table S7 and Figure 6. Emission intensity and efficiency were two major factors offsetting the growth of cement-related CO2 emissions. Efficiency offset 14.5% of the emission growth from 2014 to 2016. The efficiency decreased by 14.3% (from 1.19 to 1.02 tons cement production per 10 thousand yuan fixed capital formation). This change implies a technical improvement in cement production, as the plants produced more cement with less economic inputs. As for the intensity factor, the emission intensity of cement production decreased slightly during the past two decades from 0.54 (in 1996) to 0.42 ton CO2 per ton cement production (in 2016), as discussed above. As a result, the emission intensity consistently offset 3.5% to 4.5% of the emissions' growth since 2008.
Changes in the drivers of the cement-related CO2 emissions in China
The changes in the construction industry's structure had significant influences on the related emissions changes since 2010. The proportion of newly constructed residential buildings first increased the cement-related emissions by 6.9% and 2. Comparing the contributions of each factor between 2012 to 2014 and 2014 to 2016, this study finds that the improvement in efficiency and the reduction in residential building construction were the two major reasons causing the cement-related CO2 emissions' decrease since 2014.
Conclusions and policy implications
The cement industry is the primary source of process-related CO2 emissions in China and worldwide. This industry contributed 11% of the total emissions in China. As the cement industry is regarded as one of the key energy-intensive manufacturing sectors, greater attention should be paid to its sustainable production and emission control. Understanding the emission characteristics of China's cement industry and the drivers of the emission changes is an essential foundation for related policy making.
Our study first calculates the CO2 emissions from the cement industries in China. The emissions include process-related emissions, direct emissions from fossil fuel combustion, and indirect emissions from purchased electricity. This study then calculates the cement-related emissions of 31 provinces in China and cluster the provinces into four groups based on different peak stages. Finally, the study calculates the drivers of the changes in cement-related CO2 emissions with the LMDI decomposition analysis.
The emission accounts of China's cement industry finds that: 1) the total cementrelated CO2 emissions in China peaked in 2014 at 1,093 Mt; 2) the emissions growth was coupled with the trend in China's GDP growth, which implied that the emissions were highly driven by the country's economic gains; 3) the process-related emissions were the primary source of the cement-related emissions in China, accounting for 63.1% in 2016, and the emission mix of the process-related, direct, and indirect emissions has changed slightly over the past 20 years; and 4) the emissions induced by different new-building types also remained stable over the time, implying a relatively stable structure in China's construction industry. Residential buildings are the major contributor to the overall emissions, followed by infrastructure and manufacturing plants.
Then, our regional analysis of China's cement-related CO2 emissions have significant implications to manage the cement industry and production capacities of every province; and optimize the whole country's cement production and utilization. Four developed provinces belong to the early-peaked group with the lowest emission intensities, whose cement-related emissions peaked around 2006, and 11 provinces are classified as the mid-peaked group, which peaked their emissions around 2012. Another 10 provinces that peaked their emissions around 2014 are grouped as the late-peaked provinces. The remaining six provinces are still increasing their cementrelated emissions due to their increasing production capacities. The analysis of provincial clinker-to-cement ratio finds that the above discrepancies in provincial peak stages can be explained by the capacity outsourcing among provinces. The developed provinces have closed or outsourced their clinker production capacities to other lessdeveloped regions. Despite the similar outsourcing can be found in other industries (Shan et al. 2018b), this is certainly not a sustainable development pathway. Such outsourcing may effectively reduce the developed provinces' emissions in the short term, but it may not be beneficial to the country's overall emissions reduction. Moving the production capacities from regions with advanced technologies to less-developed regions will increase the country's overall emissions. Stimulated by the "Economic Stimulus Plans" and other development strategies, China is still in the stage of expanding infrastructure construction, especially in the currently backward cities. The central and local governments should co-ordinate the management of cement production in various provinces and cities, avoid overcapacity in the region, and largescale unnecessary cement production outsourcing and shift among provinces in China. Also, backward areas should learn from developed regions to improve their cement production efficiency and reduce unit output emissions.
The decomposition analysis of the cement-related CO2 emissions illustrates the driving forces hidden behind the emission changes. Economic growth was the major source of the growth in cement-related emissions for a long time, especially in the period 2008-2010. The emissions intensity and efficiency were two major factors offsetting the growth of cement-related CO2 emissions. Since 2014, the efficiency played a more importation role in reducing the emissions, which lead to the decoupling of economic growth and cement-related CO2 emissions (Wu et al. 2018). As more and more studies have found that China is entering a phase of decoupling economic growth from carbon emissions, efficiency gains can further reduce the emissions in the future. For example, the cement plants may consider using a cleaner energy mix to reduce the emission intensity or use low-cost energies (non-recycled plastics and paper as alternative fuels) to improve the efficiency (Huh et al. 2017;Bourtsalas et al. 2018). These low-cost energies should be used in a clean way. Cleaner production techniques should also be developed and applied to the cement plants in China, such as the "calcium looping CO2 capture" (Schakel et al. 2018).
Apart from the economic driver, the changes in the structure of the construction industry also had significant influences on the related emissions changes since 2010. The proportion of new residential buildings became a negative contributor to emissions growth since 2014, mainly influenced by the housing price fluctuation in China. However, the change in house prices is highly sensitive. The governments should plan the construction industry and new-building construction to eliminate the erratic effects of housing prices on cement production. In this way, the overcapacity and waste of cement production can be avoided.
In the future, we will conduct a more detailed investigation of each province' cement industry to analyze the impact of the cement-related CO2 emissions. Also, further study could use life-cycle assessment and carbon footprint analysis to provide a more detailed evaluation of the emission performance of cement production and consumption. The LCA and carbon footprint analysis can allocate the cement-related environmental performance to the end use in the construction industry (Fořt and Černý 2018). Then, the emissions from the cement industry can be controlled from both the production and demand perspectives.
Supplemental Figures and Tables
Index Tables Table S1 Comparisons of cement production of NBS and CCA | 2019-03-11T19:44:21.550Z | 2019-02-08T00:00:00.000 | {
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271223302 | pes2o/s2orc | v3-fos-license | High-Performance Supercapacitor Electrodes from Fully Biomass-Based Polybenzoxazine Aerogels with Porous Carbon Structure
In recent years, polybenzoxazine aerogels have emerged as promising materials for various applications. However, their full potential has been hindered by the prevalent use of hazardous solvents during the preparation process, which poses significant environmental and safety concerns. In light of this, there is a pressing need to explore alternative methods that can mitigate these issues and propel the practical utilization of polybenzoxazine aerogels. To address this challenge, a novel approach involving the synthesis of heteroatom self-doped mesoporous carbon from polybenzoxazine has been devised. This process utilizes eugenol, stearyl amine, and formaldehyde to create the polybenzoxazine precursor, which is subsequently treated with ethanol as a safer solvent. Notably, the incorporation of boric acid in this method serves a dual purpose: it not only facilitates microstructural regulation but also reinforces the backbone strength of the material through the formation of intermolecular bridged structures between polybenzoxazine chains. Moreover, this approach allows ambient pressure drying, further enhancing its practicability and environmental friendliness. The resultant carbon materials, designated as ESC-N and ESC-G, exhibit distinct characteristics. ESC-N, derived from calcination, possesses a surface area of 289 m2 g−1, while ESC-G, derived from the aerogel, boasts a significantly higher surface area of 673 m2 g−1. Furthermore, ESC-G features a pore size distribution ranging from 5 to 25 nm, rendering it well suited for electrochemical applications such as supercapacitors. In terms of electrochemical performance, ESC-G demonstrates exceptional potential. With a specific capacitance of 151 F g−1 at a current density of 0.5 A g−1, it exhibits superior energy storage capabilities compared with ESC-N. Additionally, ESC-G displayed a more pronounced rectangular shape in its cyclic voltammogram at a low voltage scanning rate of 20 mV s−1, indicative of enhanced electrochemical reversibility. The impedance spectra of both carbon types corroborated these findings, further validating the superior performance of ESC-G. Furthermore, ESC-G exhibits excellent cycling stability, retaining its electrochemical properties even after 5000 continuous charge–discharge cycles. This robustness underscores its suitability for long-term applications in supercapacitors, reaffirming the viability of heteroatom-doped polybenzoxazine aerogels as a sustainable alternative to traditional carbon materials.
Introduction
Supercapacitors, also called electrochemical capacitors, represent a novel class of energy storage devices acclaimed for their distinctive attributes.Combining the virtues of dielectric capacitors and rechargeable batteries, they excel in swiftly delivering substantial power while surpassing conventional capacitors in energy retention.Their superiority is underscored by a markedly higher power density relative to batteries, primarily attributed to the utilization of carbon materials [1][2][3][4][5].Carbon's ascendancy is fueled by its superior performance metrics, ecological compatibility, and cost-effectiveness, with Gels 2024, 10, 462 2 of 15 porous carbons emerging as frontrunners in energy storage endeavors.Their expansive surface area, commendable electrical conductivity, and inherent stability render them coveted candidates for electrode material in supercapacitors.Yet, the efficacy of porous carbons is impeded by sluggish kinetics, chiefly stemming from in-pore ion transport hindrance and elongated ion diffusion pathways within the electrode matrix.Consequently, when pore dimensions diminish, the electrode's potential experiences a notable decline, impeding ion conveyance particularly at elevated current densities, thus severely curbing overall electrode efficiency [6][7][8][9][10].Addressing these challenges remains imperative for advancing supercapacitor technology towards broader applications and heightened performance thresholds.
Among the myriad materials available, carbon aerogels emerge as a highly promising option, as these materials possess dense, interconnected networks of pores that are both continuous and open.This unique structure endows them with exceptional characteristics, including a large surface area, outstanding electrical conductivity, and high porosity.These attributes make carbon aerogels particularly suitable for supercapacitor applications.However, despite their impressive properties, the production of carbon aerogels remains relatively expensive due to the complexity involved in their synthesis process.Thus, developing cost-effective and straightforward methods for producing multifunctional polymer aerogels remains a crucial area of research [11][12][13][14][15][16].
Polybenzoxazine (PBz), representing an aromatic thermosetting polymer, stands out as a promising material due to its unique properties, such as low water absorption, minimal curing shrinkage, and superior thermal and mechanical characteristics.The synthesis of PBz involves ring-opening polymerization of the benzoxazine (Bz) monomer, which can be initiated through heating or the use of chemical initiators.The Bz monomer itself is derived from a phenolic compound, a primary amine, and formaldehyde, enabling the design of aerogel materials through precise molecular selection and chemical modification.Despite these advancements, the production of PBz aerogels often requires the use of environmentally harmful solvents such as DMF (dimethylformamide) or NMP (N-methyl-2-pyrrolidone) to achieve homogeneous monomer dissolution.The reliance on these solvents raises significant environmental and health concerns, highlighting the need for the development of more sustainable and safer processing methods [17][18][19][20][21].
The ongoing challenge is to optimize these materials and reduce production costs, thereby making supercapacitors a more viable and widespread solution for energy storage needs.Expanding upon prior discussions, this research introduces an innovative method for producing nitrogen-doped carbon aerogel known as ESC-G.This approach, characterized by its simplicity, cost-effectiveness, and efficiency, involves polymerizing a multifunctional benzoxazine monomer.The resulting aerogel exhibits high specific surface area and porosity, making it ideal for various applications.The advantages of both hetero-atom doped carbon and increasing the surface area through gel formation have been utilized in synthesizing ESC-G.Specifically, ESC-G was investigated as an electroactive material for constructing working electrodes tailored for supercapacitors.This study meticulously assessed the electrochemical properties of supercapacitors employing ESC-G compared with those utilizing ESC-N, elucidating the advantages and potential enhancements in performance.
Characterizations of the Synthesized Benzoxazine Monomer
Schematic illustration of the preparation processes of E-St-Bz and Pbz-based porous carbons are shown in Figure 1a and Scheme 1.The chemical structure of the E-St-Bz benzoxazine monomer was meticulously confirmed using Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy techniques.In the FT-IR analysis (Figure 1b), distinct peaks revealing the benzoxazine ring structure were identified.Notably, peaks at 1236 and 1028 cm −1 represented the asymmetric and symmetric stretching of the C-O-C bond, respectively, while the peak at 936 cm −1 indicated the Gels 2024, 10, 462 3 of 15 fusion of a benzene ring with an oxazine ring [22].Further examination unveiled peaks at 1147 and 1224 cm −1 , corresponding to C-N-C stretching and methoxycarbonyl stretching, respectively.Additionally, intense peaks at 2924 and 2854 cm −1 , denoting C-H stretching vibrations of the alkyl side chain of stearylamine were notable [23,24].
Schematic illustration of the preparation processes of E-St-Bz and Pbz-based porous carbons are shown in Figure 1a and Scheme 1.The chemical structure of the E-St-Bz benzoxazine monomer was meticulously confirmed using Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy techniques.In the FT-IR analysis (Figure 1b), distinct peaks revealing the benzoxazine ring structure were identified.Notably, peaks at 1236 and 1028 cm⁻ 1 represented the asymmetric and symmetric stretching of the C-O-C bond, respectively, while the peak at 936 cm⁻ 1 indicated the fusion of a benzene ring with an oxazine ring [22].Further examination unveiled peaks at 1147 and 1224 cm⁻ 1 , corresponding to C-N-C stretching and methoxycarbonyl stretching, respectively.Additionally, intense peaks at 2924 and 2854 cm⁻ 1 , denoting C-H stretching vibrations of the alkyl side chain of stearylamine were notable [23,24].In concurrence with the FT-IR findings, 1 H-NMR analysis also provided further structural insight, represented in Figure 1c.Peaks at 4.0 and 4.9 ppm were associated with Ar-CH₂-N and O-CH₂-N protons of the oxazine ring, while a singlet at 3.8 ppm confirmed the presence of -OCH₃ protons.Doublets at 3.2, 5.9, and 5.1 ppm were attributed to allyl protons, and multiplets at 2.67, 1.2 and 0.8 ppm indicated the long aliphatic chain attached to the oxazine ring.Furthermore, the presence of the solvent (CDCl₃) was indicated by a peak at 7.2 ppm [25,26].Subsequent 13 C-NMR spectra, as shown in Figure 1d, supported these findings, exhibiting characteristic carbon resonances relevant to the oxazine ring, -OCH₃ group, and allyl carbons.In summary, both the FT-IR and NMR spectroscopy techniques provided robust confirmation of the E-St-Bz monomer's chemical structure, elucidating crucial molecular intricacies essential for understanding and application.The polymerization behavior of E-St-Bz was analyzed using differential scanning calorimetry (DSC) under nitrogen, with a heating rate of 10 °C/min from 30 to 350 °C.As observed via the DSC thermogram (Figure 2a), the benzoxazine monomer (E-St-Bz) started to In concurrence with the FT-IR findings, 1 H-NMR analysis also provided further structural insight, represented in Figure 1c.Peaks at 4.0 and 4.9 ppm were associated with Ar-CH 2 -N and O-CH 2 -N protons of the oxazine ring, while a singlet at 3.8 ppm confirmed the presence of -OCH 3 protons.Doublets at 3.2, 5.9, and 5.1 ppm were attributed to allyl protons, and multiplets at 2.67, 1.2 and 0.8 ppm indicated the long aliphatic chain attached to the oxazine ring.Furthermore, the presence of the solvent (CDCl 3 ) was indicated by a peak at 7.2 ppm [25,26].Subsequent 13 C-NMR spectra, as shown in Figure 1d, supported these findings, exhibiting characteristic carbon resonances relevant to the oxazine ring, -OCH 3 group, and allyl carbons.In summary, both the FT-IR and NMR spectroscopy techniques provided robust confirmation of the E-St-Bz monomer's chemical structure, elucidating crucial molecular intricacies essential for understanding and application.
Thermal Behavior of E-St-Bz and Poly(E-St-Bz)
The polymerization behavior of E-St-Bz was analyzed using differential scanning calorimetry (DSC) under nitrogen, with a heating rate of 10 • C/min from 30 to 350 • C. As observed via the DSC thermogram (Figure 2a), the benzoxazine monomer (E-St-Bz) started to melt at 49 • C, marked by a distinct endothermic peak.The curing process began around 218 • C, with an exothermic peak maximum at 230 • C, indicating the maximum curing process.This curing process between 200-250 • C signifies ring-opening polymerization, a trend that can generally be observed in all sorts of benzoxazine monomers [27,28] Thermogravimetric analysis (TGA) was utilized to evaluate the thermal stability of poly(E-St-Bz), and the results are depicted in Figure 2c,d.This provided essential parameters including initial degradation temperature (T i ), temperatures at 5% (T 5 ) and 10% (T 10 ) weight loss, and char yield (CY) at 800 • C. Notably, poly(E-St-Bz) exhibited superior thermal stability, displaying T i = 284 • C, T 5 = 324 • C, and T 10 = 353 • C. Furthermore, a char yield of approximately 23.9% was obtained at 800 • C.This thorough analysis sheds light on the complex polymerization behavior and thermal properties of E-St-Bz, hinting at its potential applications across various fields requiring resilient and thermally enduring polymers [32,33].
Structural Analysis of the Prepared Carbon Samples
The benzoxazine monomer underwent two different processes: (i) curing, carbonization, and activation to produce ESC-N; and (ii) gelation, carbonization, and activation to produce ESC-G.To gain a deeper understanding of the structural characteristics and graphitic properties of the synthesized carbon samples, ESC-N and ESC-G, Raman spectroscopy and wide-angle X-ray diffraction (XRD) techniques were employed.The Raman spectra presented in Figure 3a offer valuable insights into the chemical composition and degree of graphitization in the carbon materials.Both ESC-N and ESC-G exhibited two prominent peaks: The D band at 1351 cm −1 signified the presence of disordered carbon structures, while the G band at 1594 cm −1 denoted the more ordered, graphitic carbon domains.The intensity ratio of these bands (I D /I G ) served as an indicator of the graphitization level.For ESC-N, a pronounced D band was observed, suggesting a higher concentration of defects within the carbon structure [34].This could be attributed to the incorporation of nitrogen and oxygen atoms during processing.The calculated I D /I G value for ESC-N was found to be 0.96, which is higher compared with the I D /I G value of ESC-G (0.85).This signified that the ESC-N possessed a more disordered structure with a higher prevalence of defects compared with the ESC-G.Interestingly, despite the difference in I D /I G values for both the samples, they had relatively similar chemical composition and structure, being produced via either the aerogel method or chemical activation.Further details regarding the graphitic domains within the carbon aerogels were obtained through XRD analysis (Figure 3b).Both ESC-N and ESC-G exhibited two broad diffraction peaks at approximately 2θ = 24 • and 45 • .These peaks corresponded to the (002) and (100) planes of hexagonal graphitic carbon, confirming the presence of graphitic domains within both samples [35].
To quantify the interlayer spacing within these graphitic domains, Bragg's equation was employed.This equation relates the wavelength of the incident X-rays (λ = 1.5418Å), the diffraction order (n = 1), the measured angle (θ), and the interlayer spacing (d) within the crystal lattice.Applying this equation revealed a d-spacing (d 002 ) of 0.40 nm for each graphite lamella in both the carbons.This value was slightly higher than that observed in conventional graphite (0.33 to 0.34 nm).This enlarged interlayer spacing within the carbon framework could offer a significant advantage for supercapacitor applications [36][37][38][39][40].
Nitrogen sorption measurements were performed on the synthesized samples, ESC-N and ESC-G, to thoroughly investigate their porosity and textural characteristics.These analyses provided crucial insights into specific surface area, pore size distribution (PSD), and pore volume, all of which are key factors influencing the electrochemical performance of supercapacitor electrodes.The N 2 physisorption isotherms, shown in Figure 3c, included a combined pattern of type I and type IV isotherms for both ESC-N and ESC-G.The type I isotherm, indicative of microporous materials, displayed a sharp rise in N 2 adsorption at low relative pressures (P/P 0 ), which in ESC-G suggested a significant presence of micropores, providing numerous adsorption sites for electrolyte ions.Additionally, both samples showed hysteresis loops at high relative pressures (P/P 0 close to 1.0), indicating the presence of mesopores with relatively uniform sizes.These mesopores are essential pathways for electrolyte transport within the electrode structure [41][42][43].
Figure 3d further reveals the pore size distribution (PSD) of ESC-N and ESC-G.Both samples exhibited pore diameters less than 2 nm and between 2 to 50 nm, indicating the presence of microporous and mesoporous materials.However, a distinct difference was observed between the two: ESC-N obtained via calcination had a broad mesopore distribution, whereas ESC-G derived from the aerogel featured a narrower mesopore distribution along with a significantly higher pore volume.This specific mesopore structure in ESC-G was attributed to the unique properties obtained through the aerogel process.Moreover, the specific surface area obtained from BET was found to be 289 m 2 g −1 for ESC-N and 673 m 2 g −1 for ESC-G.This substantial difference underscores the effectiveness of the aerogel approach in creating a highly porous material, along with increased surface area [44].
Gels 2024, 10, x FOR PEER REVIEW 6 of 16 being produced via either the aerogel method or chemical activation.Further details regarding the graphitic domains within the carbon aerogels were obtained through XRD analysis (Figure 3b).Both ESC-N and ESC-G exhibited two broad diffraction peaks at approximately 2θ = 24° and 45°.These peaks corresponded to the (002) and (100) planes of hexagonal graphitic carbon, confirming the presence of graphitic domains within both samples [35].To quantify the interlayer spacing within these graphitic domains, Bragg's equation was employed.This equation relates the wavelength of the incident X-rays (λ = 1.5418Å), the diffraction order (n = 1), the measured angle (θ), and the interlayer spacing (d) within the crystal lattice.Applying this equation revealed a d-spacing (d₀₀₂) of 0.40 nm for each graphite lamella in both the carbons.This value was slightly higher than that observed in conventional graphite (0.33 to 0.34 nm).This enlarged interlayer spacing within the carbon framework could offer a significant advantage for supercapacitor applications [36][37][38][39][40].
Nitrogen sorption measurements were performed on the synthesized samples, ESC-N and ESC-G, to thoroughly investigate their porosity and textural characteristics.These analyses provided crucial insights into specific surface area, pore size distribution (PSD), and pore volume, all of which are key factors influencing the electrochemical performance of supercapacitor electrodes.The N2 physisorption isotherms, shown in Figure 3c, included a combined pattern of type I and type IV isotherms for both ESC-N and ESC-G.The type I isotherm, indicative of microporous materials, displayed a sharp rise in N2 adsorption at low relative pressures (P/P0), which in ESC-G suggested a significant presence of micropores, providing numerous adsorption sites for electrolyte ions.Additionally, both samples showed hysteresis loops at high relative pressures (P/P0 close to 1.0), Scanning electron microscopy (SEM) revealed intricate details about the morphology of the synthesized carbon materials, ESC-N and ESC-G.Unlike an ideal supercapacitor electrode, ESC-N features an irregular microparticle structure.These particles, which vary in size from several hundred nanometers to a few micrometers, aggregate to form bulky, porous clusters.Interestingly, these clusters displayed a dual nature, exhibiting both sheet-like and three-dimensional bulk characteristics.The SEM images in Figure 4a-c highlight the disparity between the largest and smallest particles, emphasizing this nonuniformity.Notably, ESC-N exhibited a complete absence of pores, presenting a rough, unbroken surface without visible cavities or macropores.This lack of porosity suggests a limited surface area, which could potentially hinder its performance in supercapacitor applications.
sheet-like and three-dimensional bulk characteristics.The SEM images in Figure 4a-c highlight the disparity between the largest and smallest particles, emphasizing this nonuniformity.Notably, ESC-N exhibited a complete absence of pores, presenting a rough, unbroken surface without visible cavities or macropores.This lack of porosity suggests a limited surface area, which could potentially hinder its performance in supercapacitor applications.In contrast, the surface morphology of ESC-G, derived from the activation of carbon aerogel, underwent a remarkable transformation.SEM images revealed numerous micropores and mesopores embedded on the surface of the carbon, along with very few voids in the macropore range with a pore diameter of 0.5 mm (Figure 4d-f).These macropores are likely to have been formed by the expulsion of residual solvent during the gelation process.Such extensive porosity is highly advantageous for supercapacitors, as In contrast, the surface morphology of ESC-G, derived from the activation of carbon aerogel, underwent a remarkable transformation.SEM images revealed numerous micropores and mesopores embedded on the surface of the carbon, along with very few voids in the macropore range with a pore diameter of 0.5 mm (Figure 4d-f).These macropores are likely to have been formed by the expulsion of residual solvent during the gelation process.Such extensive porosity is highly advantageous for supercapacitors, as it significantly increases the surface area, offering more sites for electrolyte ion interaction and enhancing capacitance.
Transmission electron microscopy (TEM) provided a deeper, high-resolution view of the internal morphology of ESC-G.The TEM images of ESC-G (Figure 5a-d) revealed an open-pore network, a crucial feature for efficient supercapacitor performance.This nanoarchitecture offers several benefits: it shortens the diffusion pathways for ions, enabling their rapid movement within the electrode, and provides a continuous electron pathway, improving electrical conductivity.Additionally, ESC-G displayed a higher abundance of pores arranged in a more orderly manner.This suggests the presence of sp 2 -bonded carbon in ESC-G, which is highly desirable for its superior electrical conductivity.
X-ray photoelectron spectroscopy (XPS) was employed as a potent analytical method to delve into the chemical composition and bonding environment of nitrogen and oxygen species present on the surface of the synthesized carbon materials, ESC-N and ESC-G.As shown in Figure 6, the XPS spectra displayed distinct peaks for C 1 s, N 1 s, and O 1 s for both the samples.This confirmed the incorporation of nitrogen and oxygen atoms into the carbon framework, attributable to the use of a benzoxazine monomer as the initial carbon precursor.Additionally, the XPS results verified the absence of impurities in the nitrogen self-doped carbon materials.The XPS survey scan for ESC-N and ESC-G highlighted the characteristic photoelectron peaks for carbon (C), nitrogen (N), and oxygen (O) at binding energies around 281, 401, and 533 eV, respectively [45][46][47].To gain a deeper understanding of the surface chemistry, deconvolution analyses were performed on the individual C 1 s, N 1 s, and O 1 s peaks, and the results are presented in Figure 6b-d and Table 1.
open-pore network, a crucial feature for efficient supercapacitor performance.This nanoarchitecture offers several benefits: it shortens the diffusion pathways for ions, enabling their rapid movement within the electrode, and provides a continuous electron pathway, improving electrical conductivity.Additionally, ESC-G displayed a higher abundance of pores arranged in a more orderly manner.This suggests the presence of sp 2 -bonded carbon in ESC-G, which is highly desirable for its superior electrical conductivity.X-ray photoelectron spectroscopy (XPS) was employed as a potent analytical method to delve into the chemical composition and bonding environment of nitrogen and oxygen species present on the surface of the synthesized carbon materials, ESC-N and ESC-G.As shown in Figure 6, the XPS spectra displayed distinct peaks for C 1 s, N 1 s, and O 1 s for both the samples.This confirmed the incorporation of nitrogen and oxygen atoms into the carbon framework, attributable to the use of a benzoxazine monomer as the initial carbon precursor.Additionally, the XPS results verified the absence of impurities in the nitrogen self-doped carbon materials.The XPS survey scan for ESC-N and ESC-G highlighted the characteristic photoelectron peaks for carbon (C), nitrogen (N), and oxygen (O) at binding energies around 281, 401, and 533 eV, respectively [45][46][47].To gain a deeper understanding of the surface chemistry, deconvolution analyses were performed on the individual C 1 s, N 1 s, and O 1 s peaks, and the results are presented in Figure 6b-d and Table 1.The deconvoluted C 1 s spectrum shown in Figure 6b revealed four distinct peaks.The first peak, located at 284.7 eV, is attributable to hydrocarbon chains involving C=C and C-C bonds.The second peak at 285.6 eV corresponds to carbon atoms bonded in C-N configurations.The third peak at 286.5 eV signifies the presence of O-C=O, C=N, and C-OH functionalities.The fourth peak at 288.9 eV is indicative of carbon atoms bonded with both oxygen and nitrogen groups (HN-C=O).
Examining the nitrogen environment, the deconvoluted N 1 s spectrum shown in Figure 6c exhibited three distinct peaks, each representing different nitrogen functional groups.The peak at 398.6 eV corresponds to pyrrolic nitrogen, while the peak at 400.8 eV indicates graphitic nitrogen.The third peak at 406.2 eV is associated with pyridine N-oxide, suggesting possible interactions with oxygen-containing species on the surface.The O 1 s spectrum shown in Figure 6d provided additional insights into the oxygen functionalities present on the carbon surface.Deconvolution revealed four peaks at binding energies 531.3, 533.1, 533.6, and 537.1 eV, corresponding to hydroxyl (C-OH), epoxy (C-O-C), carbonyl (C=O)/carboxyl (COO-), and chemisorbed oxygen or water functional groups, respectively [48][49][50].This confirmed the presence of oxygen-containing functionalities and possibly entrapped water molecules within the carbon matrix.The XPS analysis unequivocally confirmed the presence of nitrogen and oxygen functionalities within the structure of the synthesized carbon materials.The deconvoluted C 1 s spectrum shown in Figure 6b revealed four distinct peaks.The first peak, located at 284.7 eV, is attributable to hydrocarbon chains involving C=C and C-C bonds.The second peak at 285.6 eV corresponds to carbon atoms bonded in C-N configurations.The third peak at 286.5 eV signifies the presence of O-C=O, C=N, and C-OH functionalities.The fourth peak at 288.9 eV is indicative of carbon atoms bonded with both oxygen and nitrogen groups (HN-C=O).
Examining the nitrogen environment, the deconvoluted N 1 s spectrum shown in Figure 6c exhibited three distinct peaks, each representing different nitrogen functional groups.The peak at 398.6 eV corresponds to pyrrolic nitrogen, while the peak at 400.8 eV indicates graphitic nitrogen.The third peak at 406.2 eV is associated with pyridine Noxide, suggesting possible interactions with oxygen-containing species on the surface.The O 1 s spectrum shown in Figure 6d provided additional insights into the oxygen functionalities present on the carbon surface.Deconvolution revealed four peaks at binding
Electrochemical Characterizations of ESC-N and ESC-G Electrodes
ESC-N and ESC-G are nitrogen-enriched porous carbons derived from biomass, exhibiting distinct microstructures that hint at their potential for use as supercapacitor electrode materials.To validate this potential, a comprehensive electrochemical evaluation was conducted using a three-electrode system with 1 M H 2 SO 4 aqueous electrolyte.A pivotal technique in this assessment was cyclic voltammetry (CV), which gauged the capacitive properties of the electrode materials.Figures 7 and 8 display the electrochemical properties of the ESC-N and ESC-G electrodes.Figures 7a and 8a present the CV curves for ESC-N and ESC-G electrodes across a range of scan rates (5 to 100 mV s −1 ) within a potential window of 0-1 V. Remarkably, even at the highest scan rate of 100 mV s −1 , the CV curves retained a near-rectangular shape.Moreover, the CV curve area of ESC-G was larger compared with ESC-N.This characteristic suggests an impressive capacitive performance for the ESC-G electrode, indicative of efficient charge storage and release processes.
ESC-N and ESC-G electrodes across a range of scan rates (5 to 100 mV s -1 ) within a potential window of 0-1 V. Remarkably, even at the highest scan rate of 100 mV s -1 , the CV curves retained a near-rectangular shape.Moreover, the CV curve area of ESC-G was larger compared with ESC-N.This characteristic suggests an impressive capacitive performance for the ESC-G electrode, indicative of efficient charge storage and release processes.Galvanostatic charge-discharge (GCD) measurements further elucidated the charge storage and delivery capabilities of supercapacitors.Figures 7b and 8b illustrate the GCD curves for ESC-N and ESC-G electrodes, revealing nearly perfect triangular shapes with minimal IR drop.The slight deviations from perfect triangularity point to some degree of pseudocapacitive behavior.This phenomenon is linked to the presence of nitrogen atoms Galvanostatic charge-discharge (GCD) measurements further elucidated the charge storage and delivery capabilities of supercapacitors.Figures 7b and 8b illustrate the GCD curves for ESC-N and ESC-G electrodes, revealing nearly perfect triangular shapes with minimal IR drop.The slight deviations from perfect triangularity point to some degree of pseudocapacitive behavior.This phenomenon is linked to the presence of nitrogen atoms on the carbon surface, as confirmed by X-ray photoelectron spectroscopy (XPS) analysis [51][52][53].The presence of nitrogen functionalities enhances pseudocapacitance by creating an electrochemically active interface between the electrolyte ions and the electrode surface.An important performance metric for supercapacitors is specific capacitance, which varies with current density.As depicted in Figures 7c and 8d, the specific capacitance values calculated from the GCD data showed a decline with increasing current density for both the electrodes.Specifically, at current densities ranging from 0.5 to 10 A g −1 , the specific capacitance decreased from 82 to 52 F g −1 for the ESC-N electrode and from 151 to 63 F g −1 for the ESC-G electrode.This trend highlights the rate limitations of the electrode material, as higher charging/discharging rates reduce its efficiency.
Electrochemical impedance spectroscopy (EIS) offered additional insights into the interfacial dynamics, diffusion processes, electronic conductivity, and charge transfer resistance within the electrode-electrolyte system.Figures 7d and 8d show the Nyquist plots for the ESC-N and ESC-G electrodes.The plotted intercept on the real axis denotes the solution resistance (R s ), and the diameter of the semicircle denotes the charge transfer resistance (R ct ).Notably, ESC-G exhibited a very low R ct value of 0.95 Ω, compared with the ESC-N electrode (R ct = 1.45 Ω), signifying excellent electronic conductivity-a vital trait for high-performance supercapacitor electrodes.This low R ct value significantly contributed to ESC-G's impressive rate capability.
Obviously, the reduced performance of the ESC-N electrode was attributed to its poorly developed porosity, leading to a lower surface area and uneven nitrogen atom distribution throughout the material.In summary, this study underscores the exceptional potential of ESC-G electrode, a biomass-derived nitrogen-containing porous carbon, as a supercapacitor electrode material.The near-rectangular CV curves, triangular GCD profiles, and low R s and R ct underscore its outstanding capacitive behavior and robust rate capability [54].This research advances the development of high-performance supercapacitors using sustainable, biomass-derived materials, promoting environmental sustainability while achieving high efficiency in energy storage applications.
Comparison of the Electrochemical Performance of ESC-N and ESC-G Electrodes
Figure 9a-d provide a comparative visualization of the two electrodes.The CV curve for the ESC-G electrode, evaluated at a scan rate of 20 mV s −1 , enclosed a noticeably larger area than that of the ESC-N.This indicated a higher capacitance for ESC-G.Similarly, the GCD curves at a current density of 0.5 A g −1 revealed a longer discharge time for ESC-G, implying its superior ability to store and release charge.The enhanced performance of ESC-G can be attributed to its unique microporous structure, further complemented by additional micro-, meso-, and macropores.This hierarchical porosity offers numerous advantages.Micropores provide a large surface area essential for efficient ion adsorption, contributing to pseudocapacitance.Meanwhile, meso-and macropores facilitate the smooth infiltration of the electrolyte throughout the electrode, thus maximizing the accessible surface area for ionic interactions.The synergy between pore size and distribution is pivotal in achieving high capacitance [55,56].
Figure 9c quantifies the significant improvement in specific capacitance (C s ) achieved by incorporating a gel network into the ESC-G electrode.At a current density of 0.5 A g −1 , the ESC-N electrode exhibited a C s of 82 F g −1 .Remarkably, this value escalated to 151 F g −1 for the ESC-G electrode.This substantial enhancement was directly linked to the optimized porous structure.The interconnected network of pores in ESC-G allows efficient electrolyte penetration, maximizing the electrode surface area available for ion interaction.This effectively meets a key requirement for high capacitance, positioning ESC-G as a promising candidate for supercapacitor applications [57][58][59][60][61][62].
To further explore the practical viability of these electrodes, their cycling stability was evaluated through a GCD study over 5000 charge-discharge cycles (Figure 9d).Notably, the C s of the ESC-G electrode exhibited exceptional stability throughout the test at a current density of 0.5 A g −1 , although a slight decrease from 151 to 117 F g −1 was observed after 5000 cycles and the capacitance retention ratio maintained at 89%.This outstanding stability underscores the long-term durability of ESC-G electrodes, rendering them highly suitable for real-world supercapacitor devices.
ity while achieving high efficiency in energy storage applications.
Comparison of the Electrochemical Performance of ESC-N and ESC-G Electrodes
Figure 9a-d provide a comparative visualization of the two electrodes.The CV curve for the ESC-G electrode, evaluated at a scan rate of 20 mV s -1 , enclosed a noticeably larger area than that of the ESC-N.This indicated a higher capacitance for ESC-G.Similarly, the GCD curves at a current density of 0.5 A g -1 revealed a longer discharge time for ESC-G, implying its superior ability to store and release charge.The enhanced performance of ESC-G can be attributed to its unique microporous structure, further complemented by additional micro-, meso-, and macropores.This hierarchical porosity offers numerous advantages.Micropores provide a large surface area essential for efficient ion adsorption, contributing to pseudocapacitance.Meanwhile, meso-and macropores facilitate the smooth infiltration of the electrolyte throughout the electrode, thus maximizing the accessible surface area for ionic interactions.The synergy between pore size and distribution is pivotal in achieving high capacitance [55,56].
Conclusions
An efficient, adaptable, cost-effective, and highly time-efficient approach was utilized to synthesize heteroatoms containing activated porous carbons, ESC-N and ESC-G, from fully bio-based benzoxazine.This method employed a non-templating technique combined with KOH activation.By utilizing polybenzoxazines as organic precursors, we successfully created a carbon aerogel using ethanol (an ecofriendly solvent), featuring an optimal pore size, making it ideal for electrochemical applications, particularly as an electrode material.This process not only simplifies the synthesis but also enhances the functional properties of the resulting carbon aerogel, broadening its potential applications in various electrochemical devices.The findings from this study clearly demonstrate that the biomass-derived ESC-G electrode, characterized by its hierarchical pore structure and the presence of heteroatoms, offers superior electrochemical performance compared with the ESC-N electrode.The efficient electrolyte penetration and maximized accessible surface area of 673 m 2 g −1 for ESC-G translate into a significant improvement in specific capacitance and cycling stability.These characteristics make ESC-G a promising candidate for the development of high-performance supercapacitors.The advanced structural design of ESC-G, with its
Scheme 1 .
Scheme 1. Schematic illustration showing the preparation process of E-St-Bz monomer and PBzbased carbon aerogel.
Figure 4 .
Figure 4. SEM images of (a-c) ESC-N and (d-f) ESC-G at different magnifications.
Figure 4 .
Figure 4. SEM images of (a-c) ESC-N and (d-f) ESC-G at different magnifications.
Figure 7 .
Figure 7. (a) CV curves at different scan rates; (b) GCD curves at various current densities; (c) specific capacitances with changing current densities; and (d) an EIS Nyquist plot of the prepared ESC-N electrode.
Figure 7 . 16 Figure 8 .
Figure 7. (a) CV curves at different scan rates; (b) GCD curves at various current densities; (c) specific capacitances with changing current densities; and (d) an EIS Nyquist plot of the prepared ESC-N electrode.Gels 2024, 10, x FOR PEER REVIEW 11 of 16
Figure 8 .
Figure 8.(a) CV curves at different scan rates; (b) GCD curves at various current densities; (c) specific capacitances with changing current densities; and (d) an EIS Nyquist plot of the prepared ESC-G material.
FigureFigure 9 .
Figure 9c quantifies the significant improvement in specific capacitance (Cs) achieved by incorporating a gel network into the ESC-G electrode.At a current density of 0.5 A g -1 , Figure 9. (a) CV; (b) GCD; (c) specific capacitance; and (d) cyclic stability of the ESC-N and ESC-G electrodes.
Table 1 .
Elemental composition of ESC-N and ESC-G.
Table 1 .
Elemental composition of ESC-N and ESC-G. | 2024-07-17T15:18:41.883Z | 2024-07-01T00:00:00.000 | {
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17101903 | pes2o/s2orc | v3-fos-license | Monomerization of Viral Entry Inhibitor Griffithsin Elucidates the Relationship between Multivalent Binding to Carbohydrates and anti-HIV Activity
Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.
INTRODUCTION
A variety of infections caused by enveloped viruses are currently the subject of extensive research aimed at discovery and development of preventive and therapeutic antiviral agents. In particular, infections caused by viruses such as HIV, SARS-associated corona virus, ebola hemorrhagic fever virus, and hepatitis virus are in need of novel ways of prevention and therapy. One of the promising, although not well-developed strategies for combating these viral infections is inhibiting viral entry with lectins such as cyanovirin-N (CV-N), scytovirin (SVN), or griffithsin (GRFT) (Balzarini, 2007). GRFT, originally isolated from extracts of red alga Griffithsia sp., has demonstrated antiviral activity against both HIV (Mori et al., 2005) and SARS (O'Keefe et al., 2010;Zeitlin et al., 2009). It has the most potent activity of all known lectins against various HIV-1 isolates, with EC 50 in the midpicomolar range. Furthermore, GRFT has been shown to be amenable to large-scale, agricultural manufacturing (O'Keefe et al., 2009). The stability and low immunogenicity of GRFT make it a candidate for formulation as a component of vaginally or rectally applicable microbicides (Zeitlin et al., 2009).
Earlier work in our laboratories has resulted in elucidation of the structural, biophysical, and biochemical properties of GRFT, along with its mode of binding to several mono-and disaccharides Mori et al., 2005;Zió lkowska et al., , 2007b. However, we were not able to prepare soluble complexes of GRFT with branched carbohydrates corresponding to those present on viral envelopes, such as a synthetic nonamannoside or a slightly larger Man9 (Figure 1), since such complexes precipitated from solution even at very low concentration. For that reason, previously we used molecular modeling to derive the putative structure of the GRFT-Man9 complex (Zió lkowska et al., 2007a). GRFT is a domain-swapped homodimer with the first 16 amino acids of one monomer completing the b-prism-I fold of the other monomer . Each monomer comprises one binding surface at opposite ends of a double-prism homodimer, and each binding surface is composed of three carbohydrate-binding pockets, for a total of six binding pockets per GRFT dimer. Still to be answered are questions about the exact mechanism for the antiviral activity of GRFT and its mode of binding to high-mannose, branched carbohydrates. Logical models for the mechanism of GRFT's activity include (i) coating of viral particles and blocking interaction with cell surface receptors, (ii) inhibiting viral entry postattachment, (iii) aggregation/agglutination of viral envelope glycoproteins or particles, and (iv) enhancement of the cellular immune response. One approach to finding answers to these questions appeared to be through engineering, expression, and purification of a monomeric form of GRFT (here called mGRFT). The availability of such a protein was expected to allow us to directly address the role of multivalency and crosslinking in GRFT binding to carbohydrates and to elucidate how these binding interactions relate to antiviral activity. It was also anticipated that an antiviral protein of reduced size might exhibit potentially more advantageous physiological attributes.
We have engineered several variants of mGRFT and studied their physical properties using ultracentrifugation, isothermal titration calorimetry, and X-ray crystallography, as well as investigated their biological activity using anti-HIV infectivity assays. We found that the monomers were fully capable of binding carbohydrates, but were less stable thermodynamically than GRFT and had greatly diminished antiviral activity. The latter result provides a clue to the mechanism responsible for the antiviral activity of GRFT.
Attempts to Create mGRFT by Mutation of Amino Acids
Analysis of the previously solved X-ray crystal structures of GRFT (Zió lkowska et al., 2006, 2007b pointed to several possible approaches to engineering of mGRFT. Our initial efforts at creating monomeric GRFT were aimed at stabilizing its hinge region (residues 15-19) so as to allow the monomeric form to predominate, and on removing the hydrogen bonded interactions with the N-terminal peptide. We have thus created a series of mutants, starting from the replacement of Ser65 (which makes a hydrogen bond with the amide group of Gly8) by an aromatic residue, such as tyrosine or tryptophan. Additional mutations were made at positions 2 (L2V), 6 (K6V), 106 (S106E), and 119 (E119I), both singly and in combination with the mutation S65Y. None of these initial attempts resulted in the formation of monomeric GRFT and the resulting proteins retained their dimeric character.
Creation of mGRFT by Insertion of Residues into the Hinge Region (1GS and 2GS)
Another approach to creation of monomeric GRFT relied on the detailed analysis of the region responsible for domain swap- The three branches are labeled D1-D3, and the individual mannose moieties are numbered in a manner consistent with the nomenclature used for Man9 (Man 9 GlcNAc 2 ) in the earlier studies of GRFT binding to branched carbohydrates (Zió lkowska et al., 2007a). Man9 differs from the nonamannoside by replacement of the pentyl chain by two N-acetyl glucosamines connected to mannose 3 by a b1-4 linkage. Carbohydrate residues that bind to one molecule of mGRFT are red, the residue bound to its symmetry mate is blue, and the atoms not visible in the electron density map of the complex of the nonamannoside with 1GS-S are gray.
ping. The dimerization interface of GRFT consists of a ''hinge'' formed by the swapped strands (containing Ser16 and Gly17), as well as a ''lock'' created by hydrophobic interactions of Leu2, Ile101, and Tyr117 ( Figure 2A). Furthermore, structures of GRFT in complex with maltose and N-acetylglucosamine (Zió lkowska et al., 2007b) contain an ethylene glycol molecule spanning the 7.4 Å gap between the swapped strands at the dimerization interface. We hypothesized that the gap between the swapped strands can be bridged by insertion of two or more residues between Ser16 and Gly17, at least one being a serine (to provide polar interactions similar to those of ethylene glycol). Additionally, sequences containing GS are commonly found in natural linkers (Lubkowski et al., 1999); thus, we selected either one or two of these dipeptides in designing mGRFT.
Both constructs, here called 1GS and 2GS, were successfully expressed, purified, and crystallized. Their predominantly monomeric nature at lower concentrations was initially confirmed with analytical ultracentrifugation. The dissociation constants of 1GS and 2GS calculated by sedimentation equilibrium ultracentrifugation (SEU) were 0.12 and 1.17 mM, respectively. The weight-averaged molecular mass obtained using a singlespecies model was 15.96 ± 0.05 kDa and 15.11 ± 0.08 kDa for the 1GS and 2GS mutants, respectively. These data can be interpreted as showing limited self-association of monomers that does not involve domain swapping.
The 1GS protein crystallized in the space group P6 1 with a monomer in the asymmetric unit and its structure was solved at the resolution of 1.1 Å ( Table 1). Each of the three carbohydrate binding pockets of this form of mGRFT was occupied by molecules present in the crystallization and cryoprotection solutions. Pocket 1 , adjacent to Asp112, contained a glycerol molecule. Pocket 2, near Asp30, was filled by the terminal carboxylate group belonging to a symmetryrelated molecule ( Figure 2B), whereas pocket 3, near Asp70, contained two water molecules. The 2GS mGRFT crystallized in the space group P6 5 22 and the structure was solved at 1.7 Å ( Table 1). Each of the potential carbohydrate-binding pockets contained a glycerol molecule. Electron density for the inserted sequences on both of these mGRFT molecules were clearly seen in the jFoj-jFcj maps ( Figure 3). As predicted, these insertions led to formation of loops, allowing the first 16 amino acids to complete the b-prism fold of the same molecule.
Increasing Monomerization and Sensitivity to TEV Protease (1GS-S) To facilitate monomerization we also reduced the interactions at the hydrophobic patch opposite to the strand swap region (the lock) by creating the L2S variant of the 1GS mGRFT (hereon called 1GS-S). An unexpected result of the L2S mutation was that the N terminus of mGRFT was dramatically more susceptible to cleavage by TEV protease (Phan et al., 2002) for the removal of the N-terminal hexahistadine affinity tag (Parks et al., 1994) (not shown). Because of the ease of its purification, this mutant was used for crystallization of the Man9 complex and for titration isothermal calorimetry studies. This mutant was uniformly monomeric with a weight-averaged molecular mass obtained for this mutant by SEU analysis of 11.58 ± 0.11 kDa.
Elimination of Residual Monomer-Monomer
Interactions (1GS-SDNDY) Crystal packing of the 1GS variant of mGRFT was such that the C terminus of one molecule of mGRFT was bound into the carbohydrate binding pocket 2 of an adjacent mGRFT molecule ( Figure 2B). In order to eliminate this interaction we mutated the C terminus of 1GS-S, changing its sequence from EQY to DN (hereon referred to as 1GS-SDNDY). The stability of this mutant was reduced (see Table 2 available online) and its weightaveraged molecular mass appeared to be the lowest of all mGRFT mutants (11.03 ± 0.99 kDa). 1GS-SDNDY crystallized in the space group P3 1 21 and, although the crystals still diffracted to atomic resolution, showed a high degree of disorder in the carbohydrate-binding surface, with two binding pockets containing only water molecules and the third one incorporating the guanidinium moiety from Arg80 of a symmetry-related molecule, along with a chloride ion. The chloride ion mediates most of the interactions between the symmetry-related Arg80 and the binding pocket.
Structure of 1GS-S in Complex with Nanomannoside (1GS-Sm9) Crystallization trials of 1GS-S in the presence of nonamannoside resulted in the growth of crystals under a wide range of conditions. However, regardless of the crystallization conditions, all tested crystals were isomorphous in the tetragonal space group I4 1 . Diffraction data were obtained at a resolution of 0.97 Å ( Table 1). The electron density maps show very clearly that the complex was formed, delineating unambiguously the position of the D1 and D2 branches of the nonamannoside ( Figure 4A) and of the two connecting b-mannoses. The density was partial and weak in the region where mannose 5 of the D3 branch would be expected. The binding mode of nonamannoside to 1GS-S is different in some important respects from our previous computational model (Zió lkowska et al., 2007a), or from the recently proposed mode of binding of Man9 to actinohivin, a lectin isolated from Longispora albida which is also presumed to contain three carbohydrate-binding pockets creating an equilateral triangle (Tanaka et al., 2009). The terminal mannose 11 of the D1 branch and mannose 8 of the D2 branch of the nonamannoside bind into two pockets of 1GS-S (pockets 3 and 1 according to the previously introduced nomenclature; Zió lkowska et al., 2006), with the rest of the nonamannoside molecule spanning an arch between these two binding pockets. In addition, binding pocket 2 is occupied by mannose 7 of the D2 branch of a symmetry-related nonamannoside molecule, and hence the overall ratio between polysaccharide and lectin is still one to one. The weight-averaged molecular mass of the nonamannoside-saturated complex was 16.73 ± 0.13 kDa, suggesting a two-to-one ratio of nonamannoside to 1GS-S in solution. This apparent stoichiometric ambiguity can be explained by the difference in the primary structure of the three binding The hinge region of the swapped strands is shown in the dashed rectangle with the glycerol molecule seen in two structures colored in cyan. The lock region is shown in the dashed oval with the relevant hydrophobic residues (L2, I101, and Y117) from both chains in stick configuration. All mutations other than the insertions at the hinge region relevant to this study are mapped onto the gray monomer. Successful and unsuccessful mutants are colored green and red, respectively, on the chain and labels. Positions that yielded mixed results are colored orange on the chain. (B) Carbohydrate binding surface of mGRFT (1GS). The relative orientation and interaction of two symmetry related molecules (green and yellow) are shown with the C-terminal residues of one monomer in stick configuration. Hydrogen bonds that stabilize Y121 are shown in black dashed lines. Water (red sphere) and glycerol (gray) molecules in adjacent carbohydrate binding pockets are shown for the yellow molecule.
pockets. Tyrosines 27, 68, and 110 form the internal walls of the mannose-binding pockets and create an almost perfectly symmetric triangle at the center of the binding surface. The preceding residues are Asp67 and Asp109 in pockets 3 and 1, respectively, whereas Ser26 forms part of pocket 2. This difference in primary structure leads to significant alteration of the electrostatic landscape of the carbohydrate-binding surface ( Figure 4B). Whereas the surface potential is highly negative in pockets 1 and 3, it is much less negative in pocket 2. In addition, Asp67 and Asp109 are both within hydrogen bond distance from the nonamannoside, while Ser26 in pocket 2 is too far to contribute to binding. It is thus likely that pockets 1 and 3 offer better binding to the terminal mannoses of two branches of nonamannoside, while pocket 2 is occupied more transiently. It can be postulated that when this site becomes temporarily occupied by b-mannose 7 of a nonamannoside bound to another protein molecule, the resulting symmetric interactions lead to creation of a stable crystal lattice, which ultimately changes the apparent stoichiometry of the complexes. These results point out that the originally proposed model in which only the terminal mannoses of each branch bound to a single molecule of GRFT (Zió lkowska et al., 2007a) was most likely oversimplified, with the observed mode of crosslinking being only one of many that lead to prevention of the viral entry by this class of lectins.
Differential Scanning Calorimetry
To further quantitate the GRFT monomerization process, the thermal stabilities of the GRFT monomer variants were studied using differential scanning calorimetry (DSC). Here, the heatcapacity change upon unfolding was monitored with respect to temperature, and the extrapolated midpoint of the transition, the melting temperature (T m ), and integrated area under the curve, the enthalpy of unfolding (DH unf ), were obtained (Table 3).
Dimeric GRFT melted at a T m of 78.82 C, with an enthalpy of unfolding (DH unf ) corresponding to 248 kcal/mol (Table 3). The 1GS mGRFT construct, where a GS sequence was introduced between Ser16 and Gly17, melted at a dramatically decreased T m of 67.33 C and DH unf of 91 kcal/mol. Insertion of two GS sequences in the 2GS GRFT construct resulted in a similar unfolding as observed in the 1GS construct. The 2GS GRFT appeared to have greater conformational stability (DH unf = 125 kcal/mol), although it melted at a temperature about a degree less than its 1GS counterpart. The 1GS-S GRFT melted at an even further reduced T m of 63.48 C and appeared to have an almost 2-fold greater structural stability (DH unf = 163 kcal/mol) compared with 1GS, suggesting that the L2S mutation increased the monomerization potential while stabilizing the overall domain structure of the protein (Table 3). Last, and in complete agreement with the analytical ultracentrifugation experiments, the elimination of residual monomer-monomer interactions in the 1GS-SDNDY was corroborated in the DSC experiments, this protein melting at the lowest temperature (Tm = 51.34 C) of all the tested variants of mGRFT. The enthalpy of unfolding of 1GS-SDNDY GRFT was integrated as 117 kcal/mol, and the DDH unf = 46 kcal/mol, between this monomer variant and the 1GS-S mGRFT, pointed to possible intermolecular monomermonomer contacts that stabilized the 1GS-S but were missing in the 1GS-SDNDY construct.
Isothermal Titration Calorimetry
We previously used isothermal titration calorimetry (ITC) to characterize the thermodynamics of binding of GRFT to monoand disaccharides and showed that the protein preferentially binds to mannose and mannose-containing sugars (Zió lkowska et al., , 2007b. We have extended this analysis here to study larger branched oligomannose polysaccharides to derive a more biologically relevant model for the antiviral activity of griffithsin. A panel of linear and branched oligomannose sugars was assembled and their binding to dimeric GRFT and to the 1GS-S mutant was examined and compared using isothermal titration calorimetry (ITC). The derived thermodynamic binding parameters are shown in Table 2.
In terms of energetics, all interactions observed between the sugars and the proteins were exothermic, indicating favorable binding contacts. In addition, entropically favored bindings (positive TDS values) were mediated by all carbohydrates except for the nonamannoside, suggesting that the conformational entropy of the sugars (mannose, mannobioses, and mannopentaoses) and/or of the protein regions at the binding interface were not compromised during complex formation. Among the dimannoses, 1/6a mannobiose was the weakest GRFT binding partner with a K d value almost twice that of the other disaccharides and with very slight enthalpy contributions. Unlike the 1/6a dimannose (Zió lkowska et al., 2007a), the 1/2a dimannose and 1/3a dimannose bound GRFT with a 2-fold better binding affinity as compared with mannose alone, pointing to the importance of proper presentation of the terminal mannoses in the tested sugars. The enthalpy values among the mannobioses also pointed to possible differences in complex formation between monomeric and dimeric forms of GRFT, the sugars appearing to mediate more contacts with the mGRFT protein (negative DH), although crystal structures of dGRFT and mGRFT showed nearly identical integration of the sugar hydroxyl and carbon backbones within the protein's binding sites. Since in ITC experiments the binding interactions were studied in solution, naturally the results would report all global and dynamic effects of complex formation, the proteins
Structure
Monomerization of Griffithsin and sugars freely tumbling and accessing varying conformational states of their binding partner. Thus, according to the calorimetric data with mGRFT, the sugars appeared to access more surface area contacts that were probably precluded in the dimer structure due to tumbling or steric effects. However, these slight differences in binding enthalpy did not amount to a corresponding difference in binding affinity, suggesting that the increased surface area contacts with mGRFT were nonspecific in nature, and indeed the monomeric and dimeric forms of the Figure 1. (B) Computed surface electrostatic potential of 1GS-S monomer (space filling representation) colored by charge from negative (red) to positive (blue), with bound nonamannosides shown as sticks. One nonamannoside molecule (yellow) binds to two carbohydrate-binding pockets, with negative electrostatic potential due principally to Asp67 and Asp109. The second nonamannoside molecule (gray) binds to the third binding pocket formed in part by Ser26 instead of Asp in the equivalent position and carrying a more positive electrostatic potential.
protein exhibited marked similarities in binding to any particular sugar ( Table 2).
As seen in Table 2, a significant shift in affinity (and enthalpy) occurred when the GRFT proteins were titrated with branched oligomannoses (mannopentaose and nonamannoside). Compared with the dimannose titration experiments, titrations of GRFT and mGRFT with mannopentaose resulted in a 3-fold increase in affinity, presumably due to the three terminal nonreducing mannoses of the mannopentaose. However, the 6-fold increase in enthalpy could not be accounted for by a simple additive enthalpic contribution of the exposed terminal mannoses in the mannopentaose (DH mannopentaose > > 2DH a1,3 dimannose + DH a1,6 dimannose ), suggesting that chemically equivalent nonreducing mannoses in the context of a branched sugar structure (mannopentaose) yielded better binding contacts and better binding affinity with GRFT as compared with the linear dimannoses. In the absence of a crystal structure of this branched sugar with GRFT, it is unclear how these terminal mannoses occupy the sugar binding sites. However, from examining the entropy of binding it was apparent that crosslinking of proteins by the mannopentaose did not occur, as this thermodynamic parameter remained favorable (Table 2, positive TDS). Similar to the dimannoses, the mannopentaose appeared to mediate more contacts with the mGRFT protein (negative DH) than with dGRFT.
In the nonamannoside titrations of GRFT and mGRFT, more than a 100-fold increase in affinity over the dimannoses, and more than a 30-fold increase in affinity over the mannopentaose Structure Monomerization of Griffithsin was measured (Table 2). In terms of energetics, the bindings were largely enthalpically driven (negative DH; DG = DH -TDS), with 50-fold increase in enthalpy over the dimannoses, and an approximate 10-fold increase in enthalpy over the mannopentaose. Such an increase in enthalpy indicated the presence of numerous favorable contacts between the sugar and the protein, in addition to those mediated by the sugar-binding pockets. This was suggested by the observed crystallographic results (Figure 4) that showed how a single nonamannoside molecule spanned an arch of 15 Å on 1GS-S mGRFT. The expanse of protein surface area bridged by the sugar could have allowed the two binding partners to come close enough together to mediate multiple, short-distance contacts to account for the large increase in enthalpy observed in the calorimetry experiments. Moreover, the observed calorimetric stoichiometry of two nonamannosides bound per mGRFT (data not shown) indicated multisite binding and additionally supported analytical ultracentrifugation experiments which showed a weighted average of 16.73 kDa for the nonamannoside-mGRFT complex and which suggested that, on average and in solution, two molecules of nonamannoside were bound to one molecule of 1GS-S mGRFT. Where the binding entropy of the smaller sugars was favorable, the unfavorable binding entropy (negative TDS) of nonamannoside-GRFT complex strongly suggested that nonamannoside acted as a multivalent ligand, binding separate protein molecules and greatly limiting the conformational freedoms of both binding partners as well as potentially trapping more water molecules and further reducing the entropy of the system. The calorimetry data, taken together with the crystallographic structures and the analytical ultracentrifugation data, indicated that the multisite binding by GRFT and the multivalent binding mediated by the nonamannoside contributed to the tight affinity/avidity of the binding system. The avidity of the multisite, multivalent interaction between GRFT and nonamannoside was also seen in dynamic light scattering experiments (data not shown) where the percent polydispersity indicated the presence of multiple species ranging from monomer to higher combinations of the protein and oligosaccharide.
The ability of the mGRFT and dGRFT molecules to mediate multisite, multivalent interactions with oligomannoses was next tested in experiments with HIV-1 glycoprotein gp120, which contains multiple high-mannose oligosaccharides believed to be the recognition elements of antiviral proteins such as griffithsin and cyanovirin-N. Previously, the antiviral lectin cyanovirin-N (CV-N), which has two carbohydrate-binding domains, has been shown to be able to simultaneously bind to both HIV gp120 and gp41 (O'Keefe et al., 2000). This interaction was later shown to be due to specific multivalent interactions between CV-N and high mannose oligosaccharides on the surface of HIV envelope glycoproteins (Shenoy et al., 2001). Thus, it was reasonable to investigate the effect that multivalency might play in the carbohydrate-binding and biological activity of both mGRFT (with three unidirectional binding sites) and dGRFT (with a bidirectional composition of six binding sites) with gp120. As can be seen from the results of the ITC experiments (Table 2), dGRFT shows approximately 15-fold greater affinity for gp120 than mGRFT. This difference in measured affinity is likely more related to a change in avidity resulting from the decreased off-rate of dGRFT (which caused significant precipitation of gp120 in solution) as compared with mGRFT (which did not cause similar precipitates). This result was additionally supported by dynamic light scattering experiments (data not shown) which showed the dGRFT-gp120 complex to be fully precipitated, whereas the mGRFT-gp120 complex to be in a fluid, transitory, and fully soluble system composed of a number of differently sized species. Hence, the reduction in crosslinking of HIV envelope glycoproteins by the more limited carbohydratebinding interface of mGRFT, as evidenced by the lack of precipitates, resulted in a significant reduction in the measured affinity/ avidity of this molecule for gp120. The functional relevance of this lack of crosslinking on antiviral activity was further investigated via assessment of the anti-HIV activity of the various GRFT mutants.
Cell-Based Anti-HIV Activity Assay
To assess the antiviral activity of the various mutant forms of GRFT, we utilized a previously described assay that measures the cytopathic effect of HIV infection on T-lymphoblastic cells (Gulakowski et al., 1991). Nine different mutant forms of griffithsin were tested for their activity in protecting CEM-SS cells from HIV. As can be seen in Table 4, dGRFT was potently active against HIV with and EC 50 < 0.2 nM. Several additional mutants made in the effort to design an obligate monomeric from of GRFT were also tested. The mutant forms of GRFT which did not form monomers (S65W, S65W/S106E, E119I/ S65Y, K6V/S65Y, L2V/S65Y) retained potent anti-HIV activity with EC 50 values below 1 nM. However, all monomeric forms of GRFT exhibited severely diminished anti-HIV activity. Interestingly, such activity seems to be directly correlated to the thermal stability of the mutants and inversely correlated to their propensity to self-aggregate (Tables 3 and 4). HIV-1 gp120 has been estimated to bear 24 N-linked oligosaccharides of which 11 are high mannose (Zhang et al., 2004). As our ITC results have shown that the binding affinity of GRFT and mGRFT for branched high-mannose oligosaccharides are essentially identical, the antiviral results argue for the conclusion that it is the crosslinking of these oligosaccharides on individual or close neighbor gp120 molecules that results in the striking antiviral activity displayed by GRFT.
DISCUSSION
Analysis of the previously published crystal structures of GRFT (Zió lkowska et al., , 2007a(Zió lkowska et al., , 2007b has led us to a conclusion that this protein should be considered as ''a candidate for domain swapping,'' as defined by Schlunegger et al. (1997). Unlike in the case of true domain-swapped proteins, there was no previous indication of the existence of a monomeric form of this lectin, although the general fold of GRFT is the same as of the members of jacalin family (Bourne et al., 2002). Whereas jacalin and related proteins have been shown to form oligomers, none of them make domain-swapped dimers . However, the reported dimeric nature of banana lectin (Meagher et al., 2005;Singh et al., 2005) may be related to its comparatively high antiviral activity (Swanson et al., 2010). It has been also pointed out that other known antiviral lectins, such as CV-N (Bewley et al., 1998) and SVN (Moulaei et al., 2007), form at least quasidimers (Botos and Wlodawer, 2005). Domain-swapped dimers and a variety of multimeric forms have been reported for the members of the structurally unrelated b-prism-II family (Chandra et al., 1999) and served as a basis of evaluating multivalency of binding to carbohydrates, using molecular modeling (Ramachandraiah et al., 2003). Creation of a monomeric form of GRFT was considered by us as a step in the direction of correlating the quaternary structure of the antiviral lectins with their biological activity, but the successful engineering of mGRFT also yielded an unexpected benefit of allowing a crystallographic study of a complex of this protein with the branched carbohydrate nonamannoside. The latter results form the basis of a proposed model that explains the role of multivalency of carbohydrate binding on the antiviral activity of b-prism-I family. Initial attempts at forming stable monomers of GRFT centered on stabilizing the hinge region in GRFT that provides the flexibility necessary for domain swapping, as well as on preventing hydrogen bonding between the N-terminal peptide and the body of the protein. These mutants, involving aromatic substitutions of Ser65, and sometimes additional 1-2 amino acids, did not have the desired effect and thus an alternative approach was necessary. A clue to the best way of designing mGRFT was provided by the presence of a glycol molecule in the vicinity of residues 16 and 17 0 (Zió lkowska et al., 2007b), suggesting a path for their connection. Similarly to the case of an engineered Cro monomer (Albright et al., 1996), it appeared that insertion of two or more residues into the hinge region might prevent domain swapping and result in a monomeric form of GRFT. Whereas utilization of a string consisting of five different residues (DGEVK) led to engineered monomeric Cro, it was sufficient for GRFT to insert only one or two repeats of the sequence GS, frequently found in linking regions of proteins due to its flexibility (Lubkowski et al., 1999).
Both the 1GS and 2GS constructs yielded stable lectins, with almost identical thermal stability as judged by their melting temperatures of 67 C (Table 3). Interestingly, both constructs were less stable than the dimeric GRFT, and several additional mutations destabilized them even further. The most telling of these additional mutations were the 1GS-S and 1GS-SDNDY forms that resulted in melting temperature of 63 and 51 C, respectively, a loss of up to 27 of thermal stability compared with GRFT. The results of the DSC studies suggest that the mutations necessary to form true monomeric GRFT result, in this instance, in a less stable protein. These studies were followed up with experiments designed to assess the effect of nonamannoside binding on the stability of both GRFT and various mGRFT mutants. As shown in Table 3, although GRFT shows no change in stability coincident with carbohydrate binding, mGRFT showed a definite increase in thermal stability upon binding to nonamannoside. With the overall reduced thermal stability of mGRFT compared with GRFT, the increase in melting temperature upon binding nonamannoside is suggestive of conformational changes leading to such enhanced stability.
Not surprisingly, in isothermal titration calorimetry experiments both the monomeric and dimeric forms of GRFT exhibited similar affinity to disaccharides, pentamannoside, and virtually identical avidity of binding to the branched oligosaccharide nonamannoside. The progression of both binding enthalpies and dissociation constants indicates that both GRFT and mGRFT share similar binding interfaces to carbohydrates. What is telling, however, is the significant increase in the strength of the binding interaction when one moves from likely single-site binding interactions (i.e., mannobioses) to those that potentially involve multiple binding sites on individual GRFT monomers (e.g., pentamannoside and, to a greater degree, nonamannoside). Despite the similarity of their interactions with isolated oligosaccharides, the binding of GRFT and mGRFT to the viral envelope protein gp120 differed significantly, with the avidity of mGRFT being approximately 15-fold lower. Another major difference was observed in their antiviral properties, with GRFT exhibiting EC 50 of <0.2 nM, whereas the activity of mGRFT was reduced by three orders of magnitude under similar conditions (EC 50 of 323 nM). This observation is in agreement with the results reported for the monomeric and dimeric forms of CV-N, where the presence of at least two binding sites, whether located on a single molecule or on two molecules, was shown to be necessary to for their antiviral properties (Liu et al., 2009). Furthermore, the activity of CV-N mutants that were inactive as monomers was restored by their dimerization (Matei et al., 2010). However, another quasidimeric lectin, SVN, behaves in a different way, since even just its N-terminal domain (SD1) exhibits significant antiviral properties (Xiong et al., 2006). It should be noted that several other mutants of GRFT created for this study did not show either decreased antiviral activity or true monomeric character. The antiviral assay and thermodynamic results described here detail the close association of multivalent interactions between dGRFT and gp120 with its antiviral activity. The significant reduction in the affinity of mGRFT for gp120 is mirrored by its drastic reduction in antiviral activity. The results showing that both dGRFT and mGRFT bind with similar enthalpies and dissociation constants to the Man9 analog nonamannoside indicate that the interactions between individual high mannose oligosaccharide moieties and individual monomeric units of GRFT are not sufficient to engender significant antiviral activity. It is only in the crosslinking of multiple high-mannose oligosaccharides present on gp120 when the carbohydrate-binding capabilities of lectins such as GRFT confer an antiviral effect. These results further explain our previous results which detailed the large difference in anti-HIV EC 50 values for a number of monovalent and multivalent monosaccharide-specific lectins (Zió lkowska et al., 2007b).
The ability to grow crystals of the complex of mGRFT with nonamannoside provides an indirect evidence of the most likely mode of the antiviral activity of GRFT. All previous attempts to grow crystals of the complexes of GRFT with Man9 or nonamannoside failed due to precipitation of the material from solution (T.M. and A.W., unpublished data). This was the case even when both components were mixed at very low concentration and then concentrated. These results indicate that the activity of GRFT might be compared with that of an antibody, which requires the presence of dimers in order to crosslink the antigens. Whereas the absence of such crosslinking would not influence carbohydrate binding, it would abolish the ability to interfere with viral infection. A similar explanation of the basis of antiviral activity was also recently proposed for CV-N (Matei et al., 2010).
The mode of binding of nonamannoside to mGRFT seen in the crystals was quite unexpected and did not correspond fully to the previously published model of their interactions (Zió lkowska et al., 2007a). The model structure assumed that the three terminal mannoses of branches D1-D3 of the nonamannoside would each occupy sites 1-3 on a GRFT monomer (as defined by , whereas the remaining mannoses would participate in only limited or no interactions. In agreement with that model, we found that in the crystals of mGRFT one nonamannoside molecule inserts its mannose 11 from the D1 branch into site 3 on the protein, whereas mannose 8, terminal in branch D2, is located in site 1. However, it is mannose 7, the second unit on the D2 branch from a symmetry-related nonamannoside molecule, that is located in site 2 of mGRFT. It is very likely that this mode of binding is a crystallization artifact, which, incidentally, must have helped in the growth of that particular crystal form of the complex. However, this fortuitous mode of binding may actually be used to explain why GRFT, at least in its dimeric form, possesses strong antiviral activity, whereas jacalin (with one binding site per monomer), or b-prism II lectins such as garlic lectin, despite their three sites, do not. A major difference between GRFT and garlic lectin is the distance between carbohydrate-binding sites on a monomer, 15 Å for the former and a minimum of 22 Å for the latter. Thus, a single molecule of nonamannoside cannot reach more than one site on the garlic lectin (Ramachandraiah et al., 2003) but can easily reach two or three on GRFT (Zió lkowska et al., 2007a). If all three GRFT sites were occupied by the terminal residues of a single nonamannoside molecule, there would not be any more branches left to provide crosslinking. However, with two sites that are bound to terminal mannoses in the D1 and D2 branch offering different electrostatic properties than the third one ( Figure 4B), the latter (and presumably weaker) site may provide an anchoring point for other nonamannosides and thus initiate crosslinking. The presence of two, almost identical, sites that can be reached by a single molecule vastly increases the avidity of binding, whereas the third site enhances crosslinking. Such organization is unique to GRFT (other lectins mentioned above either have fewer sites, or the existing sites cannot be spanned by a single complex carbohydrate molecule). With dimers allowing much more elaborate crosslinking patterns than monomers, their antiviral activity is enhanced. While highly likely, this interpretation will still require further verification through mutagenesis studies that would involve modification of the individual binding sites.
Mutagenesis and Protein Expression
All restriction enzymes where purchased from New England Biolabs. All chemicals were obtained from American Bioanalytical, unless otherwise stated. An expression vector containing the gene for GRFT downstream of a hexahistidine tag and thrombin cleavage site was used as a PCR template. The amplified cassette was cloned into pET-15b expression vector in between the NcoI and XhoI sites. Standard site-directed mutagenesis technique was used to mutate the thrombin cleavage sequence to TEV cleavage sequence and to create all mutant forms of GRFT using mutagenic primers (IDT), PfuUltra DNA polymerase (Stratagene), and DpnI (NEB). All mutants were expressed in BL21(DE3) pLysS cells (Novagen) at 37 C and 226 rpm. Induction was initiated at optical density of 0.7 measured at 600 nm by addition of IPTG to a final concentration of 0.5 mM and continued for 10 hr.
Protein Purification
Cell pellets were lysed in 50 mM Tris (pH 8.0), 500 mM NaCl, and 5% v/v BugBuster 103 Protein Extraction Reagent (Merck), and the lysate was centrifuged at 17,000 rpm in a SS-34 rotor for 30 min. The supernatant was loaded onto a Ni-NTA Superflow column (QIAGEN), equilibrated with five column volumes of buffer A (20 mM Tris [pH 8.0], 100 mM NaCl). The column was washed with buffer A and eluted with buffer A containing 250 mM imidazole. TEV protease was added in 1:100 molar ratio of protease to eluted protein and the sample was incubated at room temperature for 1 hr. At this stage, two different strategies were used to complete the purification. For 1GS and 2GS proteins, the sample was loaded onto an amylose column equilibrated with buffer A. The column was washed with buffer A and eluted with buffer A containing 250 mM maltose. For 1GS-S and 1GS-SDNDY proteins, the sample was heated at 50 C for 10 min and then centrifuged at 12,000 rpm in a SS-34 rotor for 10 min. The supernatant was loaded onto a Ni-NTA column (QIAGEN) equilibrated with buffer A and the flowthrough was collected. For all versions of mGRFT, the purified protein was dialyzed against deionized, double-distilled water overnight at 4 C, and then concentrated.
Crystallization, Data Collection, and Refinement
All proteins were concentrated to 25 mg/ml. To create the complex with nonamanoside, the carbohydrate was dissolved in water to a concentration of 10 mM and then titrated into a 1 mg/ml sample of 1GS-S, 2 ml at a time, followed by gentle mixing. The final molar ratio of nonamanoside to 1GS-S was 1.5:1. The complex was subsequently concentrated to 25 mg/ml. Initial screens for crystallization conditions were performed using a Phoenix liquid handling robot (Art Robbins Instruments) in 96-well Intelli-Plates (Hampton Research), with protein to well solution volumetric ratios of 1:2, 1:1, and, 2:1.
Initial crystallization conditions for the 1GS mutant were obtained from EasyXtal Ammonium Sulfate crystallization suite (QIAGEN). These conditions were optimized and diffraction quality crystals were grown in 0.1 M sodium acetate (pH 4.0), 0.15 M ammonium sulfate, and 25% w/v PEG 3350 at 20 C by hanging drop vapor diffusion method. The protein and well solution were mixed in 1:1 volumetric ratio. All other crystals of mGRFT mutants were obtained directly from crystallization suites, with 1:1 volumetric ratio of protein to well solution.
The 1GS-Sm9 crystallized under a variety of conditions from a number of crystallization suites. Nevertheless, all crystals were isomorphous in the space group I4 1 . The highest resolution diffracting crystal was grown in 0.1 M mixture of imidazole, sodium cacodylate, MES, bis-tris (pH 6.5), 0.1 M mixture of L-Na-glutamate, alanine, glycine, lysine HCl and serine, and 30% w/v mixture of PEG MME 550 and PEG 20000 (Molecular Dimensions Morpheus, #85).
All crystals were soaked for 3 min in a cryoprotectant solution consisting of 4:1 volumetric ration of mother liquor to 80% v/v glycerol and rapidly cooled in a nitrogen stream (100 K). Data for the 2GS protein were collected with a MAR345 dtb image plate detector mounted on a Rigaku MicroMax-007HF generator operated at 40 kV and 30 mA. All other data were measured at the SER-CAT beamline 22-ID, at the Advanced Photon Source, on a MAR 300CCD detector. The atomic resolution data for 1GS-Sm9 were measured in two passes, with a low-resolution (30-3.0 Å ) pass and a high-resolution (5.0-1.0 Å ) pass. The measured intensities for both passes were scaled and merged together. All data were processed and scaled using the HKL3000 package (Minor et al., 2006). Initial phases were calculated by molecular replacement method using the program Phaser (McCoy et al., 2007) and a model derived from the structure of GRFT (PDB ID 2HYR), using residues 17-121 of chain A and 1-16 of chain B. The structures were refined with the program Refmac5 (Murshudov et al., 1997) and rebuilt with Coot (Emsley and Cowtan, 2004). Statistics of data processing and refinement are given in Table 1.
Isothermal Titration Calorimetry
ITC experiments were performed using a MicroCal VP-ITC microcalorimeter (MicroCal, Northampton, MA). The oligomannose sugars were diluted in the same exact buffer (20 mM Tris, 150 mM NaCl [pH 7.5]) used to dialyze the GRFT protein variants as well as gp120. The concentrations of the oligomannose sugars were determined by oligosaccharide composition analysis by HPAEC at the Complex Carbohydrate Research Center (CCRC, University of Georgia, Athens, GA). The concentrations of all proteins were assessed by amino acid analysis. In titrations with the oligomannose sugars, the protein solutions were in the range of 0.1-0.3 mM, and the sugars in the concentration range of 1-15 mM. In a typical titration experiment, the oligomannose sugar was placed in the syringe injector and the GRFT protein solution was placed in the calorimeter cell. In the experiments with HIV-1 glycoproteins, a protein solution of gp120 was placed in the calorimeter cell at a concentration of 1.0 mM, and the GRFT proteins were loaded into the syringe injector at a concentration of 0.175 mM. In all experiments, a total of 55 injections (5 ml per injection) were made into a rapidly mixing solution (300 rpm), with 600 s spacing between injections. The temperature was set at 25 C and the reference power to 7 mcal/s.
The heats of dilution of the oligomannoses were accounted for by blank titrations, where the oligomannoses were titrated into a buffer solution. After subtracting these baseline thermograms from experimental thermograms, the data were fit using Origin 5.0 nonlinear least-squares program supplied by the manufacturer, and values for the binding enthalpy (DH) and the dissociation constant (K d ) were derived.
Differential Scanning Calorimetry DSC experiments were performed using a MicroCal VP-DSC microcalorimeter (MicroCal, Northampton, MA). The oligomannose sugars were diluted in the same exact buffer (20 mM Tris, 150 mM NaCl [pH 7.5]) used to dialyze the GRFT monomer variants. The concentrations of all proteins were assessed by amino acid analysis. In a typical experimental format, buffer (20 mM Tris, 150 mM NaCl [pH 7.5]) was placed in both the reference and sample cells and the calorimeter was allowed to cycle at a heating rate of 60 C/hr, scanning from 10 C to 90 C, with data recordings every 15 s. The calorimeter cells were self-pressurized to approximately 30 psi of positive pressure to prevent evaporation of solutions at the higher temperatures. During the down-scan at 25 C, the buffer solution in the sample cell was quickly and efficiently replaced by a GRFT protein solution (60 mM), positive pressure was reapplied, and the calo-rimetric cells were allowed to cycle between a total of six alternating up-and down-scans from 10 C to 90 C to measure reversibility of the unfolding process. Using Origin DSC Analysis software provided by the manufacturer, the melting profiles of all samples were corrected for buffer effects by subtracting the first up-scan of each experiment. To take into account the heatcapacity changes between the native and denatured states of the protein, a cubic baseline was extrapolated from pretransition and posttransition baselines and was subtracted from the melting profile. The resulting thermogram was fit to a two-state melting model and the calorimetric transition enthalpy (DH unf ) was obtained from the numerical integration of the area under the excess heat-capacity peak, and the midpoint of the transition was extrapolated as the melting temperature.
Sedimentation Equilibrium Ultracentrifugation
Sedimentation equilibrium ultracentrifugation experiments were performed in a Beckman Optima XL-A ultracentrifuge at 20 C with absorbance optics at 280 nm. All protein samples were in a buffer containing 20 mM Tris (pH 8.0) and 100 mM NaCl. Samples were spun in a Beckman An-60 Ti rotor using 1.2 cm two-sector charcoal-fillled Epon centerpieces with quartz windows at speeds of 21,000, 25,000, and 30,000 rpm. Equilibrium was reached after 24 hr for each speed and verified by comparison with a second scan taken 2 hr later. All data were analyzed using the programs Sedfit and Sedphat (Schuck, 2000) with a buffer density of 1.00293 g/cm 3 and a buffer viscosity of 0.01023 Poise as calculated using the program Sedenterp. Partial specific volumes were also calculated with Sednterp to be 0.7164 (1GS), 0.7155 (2GS), 0.7142 (1GS-S), and 0.7134 (1GS-SDNDY) in units of g/cm 3 . All data were fit using the simplex algorithm.
Whole-Cell anti-HIV Assays
An XTT-tetrazolium based assay was used to determine the anti-HIV activity of GRFT against a T-tropic laboratory strain (HIV-1 RF ) in CEM-SS cells as previously described (Gulakowski et al., 1991). In brief, CEM-SS cells were maintained in RPMI 1640 media without phenol red and supplemented with 10% fetal bovine serum (BioWhittaker), 2 mM L-glutamine (BioWhittaker), and 50 mg/mL gentamicin (BioWhittaker). Exponentially growing cells were washed and resuspended in medium, and a 50 ml aliquot containing 5 3 10 3 cells was added to individual wells of a 96-well round-bottom microtiter plate containing serial dilutions of GRFT, GRFT mutants, or AZT in a volume of 100 ml medium. Stock supernatants of HIV-1 RF were diluted in medium to yield sufficient cytopathicity (80%-90% cell kill in 6 days), and a 50 ml aliquot was added to appropriate wells. Plates were incubated for 6 days at 37 C, then stained for cellular viability using 2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT). | 2014-10-01T00:00:00.000Z | 2010-09-07T00:00:00.000 | {
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55758535 | pes2o/s2orc | v3-fos-license | Gastric Mucosal Changes and Ghrelin Expression and Their Relation to Weight Reduction After Sleeve Gastrectomy
Background: Weight loss after laparoscopic sleeve gastrectomy (LSG) is usually attributed to the volume restrictive effect of the procedure in addition to specific hormonal changes. Objective: The present study aimed to investigate the changes in plasma ghrelin levels, the number of ghrelin producing cells and histopathological changes in the remaining pouch after LSG. Methods: The present study included 27 patients with morbid obesity. The plasma ghrelin levels were measured before and six months after LSG and the change in their levels was assessed in relation to body mass index (BMI) after LSG. Immunohistochemical staining of cellular ghrelin was used to evaluate the number and distribution of ghrelin producing cells in the resected specimen and the mucosal changes in the remaining gastric pouch after LSG were assessed at 6 months postoperatively. Results: The mean age of patients was 33.9± 21.9 years. At six months after LSG, BMI decreased from 52.6± 12.8 to 40.8± 7.2 Kg/m 2 (p= 0.0001). The plasma ghrelin level decreased significantly from 564.1± 15 to 434.7± 22.6 at six months after LSG. There was strong positive correlation between BMI after LSG and plasma ghrelin level (R= 0.906, p= <0.0001). A significant improvement in the inflammatory parameters was noticed by histopathologic examination. The mean ghrelin positive cell per specimen decreased significantly from 25.37± 3.5 to 13.7 ± 2.12. Conclusion: There was good positive correlation between weight loss and lowering of plasma ghrelin level, also complete removal of the fundus was associated with more weight loss.
Introduction
Obesity is a pan-endemic health problem in both developed and developing countries that can be associated with a high incidence of complications and decrease in life expectancy, especially among younger adults. Obesity is classified into classes according to the body mass index (BMI) with morbid obesity defined as BMI ≥ 40 kg/m 2 or a BMI ≥ 35 kg/m 2 in patients with at least one associated comorbidity [1].
While conservative measures may succeed in treatment of obesity in some patients, bariatric surgery remains a viable option for the treatment of morbid obesity, resulting longlasting weight loss, improved quality of life, and resolution of obesity-related comorbidities [2]. With the application of laparoscopy to bariatric surgery a major expansion in the number of laparoscopic bariatric procedures has been noted worldwide [3].
Laparoscopic sleeve gastrectomy (LSG) is one of the most popular bariatric procedures performed for morbid obesity [4]. The mechanism of weight loss after sleeve gastrectomy is attributed to a combination of volume restriction, creation of a high pressure system, and the induction of a favorable hormonal change [5].
A decline in appetite after LSG has been observed, presumably due, in part, to ghrelin cell removal. Ghrelin is an orexigenic hormone produced primarily by cells in the oxyntic glands of the stomach mainly in the gastric fundus as it has been demonstrated that the expression of Ghrelin mRNA expression in the fundus is three fold higher than in the pre-pyloric area and the same pattern was also seen in ghrelin cell distribution [6].
Ghrelin hormone plays a physiologic role in the regulation of food intake as its plasma levels rise during food deprivation and decline after food ingestion [7]. In addition, Ghrelin is involved in many biological processes ranging from appetite regulation and growth hormone release to gut motility [8]. The changes in plasma ghrelin levels after different bariatric procedures vary according to the procedure. While plasma ghrelin levels tend to decrease after gastric bypass and after LSG, they tend to rise after gastric banding in a way similar to dieting [9].
The present study aimed to evaluate the changes in plasma ghrelin hormone at six months after LSG, correlating these changes with weight loss. The study also aimed to investigate the distribution of ghrelin producing cells in different regions of the stomach and the change in their number after LSG and to evaluate the histopathological changes after sleeve gastrectomy since the evaluation of these changes can improve the understanding of the local mechanism and the outcome of LSG as Vrabie et al. implied [10].
Patients and Methods
This is a prospective cohort study on patients with morbid obesity who underwent LSG in the General Surgery Department of Mansoura University Hospitals in the period of May 2015 to May 2017. Ethical approval of the study was obtained from the Institutional Review Board (IRB) of Mansoura Faculty of Medicine.
All patients with morbid obesity with BMI >40 kg/m 2 or BMI >35 kg/m 2 with at least one obesity-related comorbidity who failed conservative treatments were included to the study.
We excluded patients who underwent previous bariatric surgery, patients unfit for anesthesia as patients, patients with gastric pathologies as peptic ulcer or neoplasm, patients with secondary obesity due to endocrine disorders, patients with psychological problems or patients unwilling to comply with postoperative diet regimen.
Patients were asked about the onset and duration of obesity and any obesity-related comorbidities such as diabetes mellitus, hypertension, sleep apnea, and joint pain. Patients were also asked about previous treatments for morbid obesity including diet regimens, weight loss medications, exercise programs, interventional therapies as intragastric ballon, and any previous surgical procedures for morbid obesity or other abdominal conditions. Detailed dietary history was taken from all patients with regards to type and frequency of meals, eating snacks in between meals, drinking of water or beverages, level of appetite, and feeling of satiety after each meal.
General examination was conducted to all patients. Patients' weight and height were recorded and BMI was then calculated using the following equation: BMI= Weight in kilograms/ (Height in meters) 2 . Abdominal examination was performed to exclude presence of any abdominal masses, ascites, and ventral or groin hernias.
Routine preoperative laboratory investigations including complete blood count, liver and kidney functions tests, prothrombin time, and random blood glucose were done to confirm anesthetic fitness. Endocrine and metabolic panels including serum lipid profile, thyroid function tests, and fasting and postprandial blood sugar levels were done to exclude secondary causes of obesity. Plasma ghrelin levels were measured in all patients within 48 hours before the procedure.
Abdominal ultrasonography was performed in all patients to exclude any associated abdominal pathologies. The cardiac function was evaluated by electrocardiography and echocardiography in select patients and pulmonary function tests were done to assess respiratory functions and chest condition.
Informed consent for the study was taken from all patients. Liquid diet was prescribed for 24 hours before the procedure with fasting for at least six hours before the onset of anesthesia.
Preoperative measures against thromboembolism were taken in the form of: wearing elastic stocking 24 hours before the operation, subcutaneous administration of low molecular weight heparin (Enoxaprin, 40 IU) at the night of surgery. Prophylactic antibiotics (cefotaxime 1 gm) were administered intravenously on induction of anesthesia.
All procedures were done under general anesthesia with end tracheal intubation. Patients were placed in the supine position being appropriately secured to the operating table with padding of all pressure points. The operating surgeon stood between the legs of the patient and the two assistants stood on each side of the patient.
After scrubbing of the abdomen with povidone iodine, peumoperitoneum (15)(16)(17) was established by insertion of veress needle above the umbilicus and insufflation of CO2. Trocars were inserted under direct vision as follows: a 5-mm trocar is inserted in the subxiphoid area for the liver retractor, another 5-mm trocar is placed in the left upper quadrant at the anterior axillary line just below the 12 th rib for the assistant, additional 12-mm trocars were placed in the right upper quadrant, epigastrium, left upper quadrant, and right paramedian regions for inserting staplers.
Upon identification of the pylorus by visualizing the prepyloric vein of Mayo, the greater curvature of the stomach was devascularized using an advanced vessel-sealing device (Harmonic scalpel TM or Ligasure TM ) starting at 6 cm away from the pylorus. Devascularization continued proximally onto the fundus until the left crus of the diaphragmatic hiatus was clearly identified. All the short gastric vessels were divided along the way. After devascularizaion was completed, the hiatus was assessed for hernias and if large hiatal hernia was encountered, it was repaired using standard laparoscopic techniques.
A 36 French bougie was then inserted transorally by the anesthesiologist, advanced under direct vision to the pylorus, and then positioned against the lesser curvature. The gastric transection was then performed using sequential applications of 60-mm linear staplers beginning at 6 cm proximal to the pylorus. As the gastric transection proceeds, the height of the staples may need to be adjusted according to the thickness of the tissue. We used green stapler at pylorus, then gold staplers at middle of stomach, then blue staplers at fundus.
After the stomach was transected, an intraoperative leak test was performed by injection of methylene blue in the gastric sleeve. The resected portion of the stomach was extracted through the right upper quadrant trocar then the trocar sites were closed and an intra-abdominal drain was inserted.
Patients were monitored in the regular ward with regards vital signs, output of abdominal drain, and postoperative complications. Intravenous fluids were administered for 24 hours postoperatively and one gram of cefotaxime was administered intravenously every 12 hours. Analgesia was achieved using non-steroidal anti-inflammatory drugs (NSAIDs) or Nalufin amp. A second dose of low molecular weight heparin was given subcutaneously 8 hours after the operation then was repeated every 24 hours. Early ambulation was advised in all patients. Resumption of oral feeding was scheduled in every patient. Once assured to be stable, started oral fluids, with no complaints, patients were discharged home within 48 hours of the procedure.
Ghrelin immunoreactivity was determined on sections after microwave pretreatment (10 min in 0.01 M citric acid solution) and overnight incubation at 4°C with human polyclonal antibody (Chongqing biopsies Co., Ltd., Chongqing, China) diluted 1:100. Diaminobenzidinehydrogen peroxide was used as chromogen, and the sections were counterstained with diluted hematoxylin.
Ghrelin positive cells were counted in three (hot spots) in areas containing mucosa in full thickness. The total number was divided by 3 to obtain mean Ghrelin positive cell per specimen.
Patients were asked to visit the outpatient clinic at one and two weeks after sleeve gastrectomy for assessment of general condition, wound status, and for removal of stitches, then every month for six months.
At Six months after the procedure, body weight was recorded and the amount and percentage of weight loss was calculated and compared with the preoperative value. BMI was calculated to estimate the reduction in BMI after surgery. A blood sample was withdrawn and plasma ghrelin levels were measured and compared to the preoperative value. In addition, all patients underwent gastroscopy to evaluate the condition of mucosa of the remaining pouch and take three tissue samples from the remaining part of the stomach.
Tissue samples were examined pathologically with the same techniques described earlier to assess postoperative changes in ghrelin gene expression, mucosal cells distribution and to demonstrate any histopathological changes.
Data were analyzed using SPSS program version 23 (IBM corp., Bristol, UK). Continuous variables were expressed as mean and standard deviation (SD) or median and normal range. Categorical variables were expressed as number and percentages. Student t-test was used for processing of quantitative data whereas Fisher's exact test was used for processing of qualitative data. The correlation between BMI and plasma ghrelin levels was measured using Pearson correlation test. P value less than 0.05 was considered significant.
Results
The present study included 27 patients with morbid obesity who underwent LSG. Patients were all females except one male. The mean age of patients was 33.9± 21.9 years, ranging from 21 to 63 years.
Twenty-four patients had obesity-related comorbidities: nine patients had type II DM, five had arterial hypertension, ten had dyslipidemia, and four had joint pain. In addition, 12 patients had fatty liver and two had gall bladder stones in abdominal ultrasonography.
Figure 1. Correlation between excess weight loss and ghrelin level at 6 months after LSG.
Histopathological examination of the postoperative specimen and random endoscopic biopsies from the remaining gastric pouch revealed the following (table 3): 1. Superficial gastritis was detected in six patients (4 moderate and 2 mild) and superficial congestion in two patients.
2. Regarding the presence of lymphoid follicles, the number of patients with grade 0 increased from 15 to 22, the number of patients with grade I decreased from 9 to 5, and the number of patients with grade II decreased from 3 to 0 (p= 0.059) (figure 2).
Figure 2. Gastric mucosa with lymphoid follicle (arrow) (H&E stain, 100x).
3. Regarding the presence of interstitial inflammation, the number of patients with grade 0 increased from 12 to 20, the number of patients with grade I decreased from 12 to 7, and the number of patients with grade II decreased from 3 to 0 (p= 0.03) (figure 3). 4. The mean ghrelin positive cell per specimen decreased from 25.37± 3.5 to 13.7 ± 2.12 (p< 0.0001).
5. The mean number of ghrelin producing cells in the fundus of the resected specimen was significantly higher than the body and the antrum as shown in table 4 and figure 4.
Discussion
Twenty-seven patients with morbid obesity were admitted and underwent LSG in this prospective observational study. Almost all patients were females reflecting the female predominance of obesity as documented in the literature. The majority of patients were of middle age in concordance with the average age of patients undergoing bariatric surgery for morbid obesity [4].
Significant weight loss at 6 months after LSG was recorded with a significant decline in the preoperative BMI. The average percentage of excess weight loss was around 40%. Improvement in associated co-morbidities was observed yet was not statistically significant, perhaps due to the short duration of follow-up.
We studied plasma ghrelin levels before and after LSG, correlating the changes in these levels with weight loss after the procedure. A significant decrease in plasma ghrelin levels at 6 months after LSG was recorded which was associated with significant weight loss as demonstrated by decline in BMI. In concordance, other researchers found total plasma ghrelin levels decrease significantly at 3 months after LSG, with simultaneous significant weight loss [11]. Plasma ghrelin levels have been shown to decrease after sleeve gastrectomy and its low levels can be maintained up to five years after surgery [12,13]. Since ghrelin is mainly produced by oxyntic cells in the stomach in addition to other parts of the gastrointestinal tract, it is logical that plasma ghrelin levels decline after resecting around 75% of the stomach volume in LSG [14].
We identified a weak correlation between plasma ghrelin levels and BMI before surgery, nevertheless this positive correlation became stronger at 6 months after LSG and this correlation was statistically significant. Similarly, the percentage of excess weight loss at 6 months had moderately strong negative correlation with plasma ghrelin implying that the drop in plasma ghrelin levels was associated with higher percentage of weight loss. In discordance, Goitein et al. [11] found no distinct correlation between plasma ghrelin levels and weight loss which they explained that plasma ghrelin is only one of multiple factors contributing to the success of LSG and that weight loss per se is not responsible for ghrelin level change [15].
The significant strong correlation we observed between plasma ghrelin and BMI after surgery can be attributed to different patient characteristics compared to the study of Goitein et al. [11] where the BMI of our patients was obviously higher than their study. In addition, we made the correlation at six months after LSG, whereas the other investigators made the same correlation at shorter period of follow-up (three months postoperatively).
Ghrelin hormone has been recognized to contribute to the incidence of morbid obesity through different mechanisms. Ghrelin is one of the orexigenic peptide transmitters know to stimulate appetite and induce a positive energy balance leading to weight gain. It is implicated in regulating mealtime hunger and meal initiation. Since ghrelin hormone is considered a signal of starvation, it can induce sustained adiposity as long as its levels are elevated [16].
Histopathological examination of the resected part of the stomach after LSG revealed abnormal pathologic changes in 55.5% of patients in the present study. This implies that around 45% of patients had normal histopathology of the stomach preoperatively which is lower than other studies [17][18][19] that reported normal histopathology in 50-54% of patients. However, similar to our findings other investigators reported normal histopathology of the stomach in 35.2 and 46.3% of patients [20,21].
Abnormal histopathological findings in the present study involved chronic gastritis, either active or inactive which is in line with Safaan et al. [17] who reported chronic gastritis to be the most common abnormal histopathological findings in the resected part of the stomach after LSG. Nonetheless, the study by Safaan and colleagues [17] documented other important pathological findings as intestinal metaplasia, GIST, leiomyoma, lipoma, and pancreatic heterotropia, however, these abnormal findings were found in a very low incidence of patients. In fact, the incidence of GIST ranged between 0.2 and 1% and the incidence of intestinal metaplasia ranged between 0.2 and 1.4% across several studies [17][18][19][20][21][22][23].
No incidental gastric malignancy in the postoperative specimen was detected in our study, in concordance with largest study [17] of histopathological diagnoses in LSG patients. The observation of lack of malignancies further raises the question of any added value of routine histopathological examination of the resected part of the stomach after LSG as AbdullGaffar et al. [18] implied. In light of financial restrictions that warrants evidence-based decision-making based on the cost-effectiveness concept, routine histopathological examination of surgical specimens has been previously questioned [24,25].
We used two markers to measure inflammatory activity in gastric specimen after LSG and in the remaining pouch on follow-up. Presence of lymphoid follicles and interstitial inflammation were graded from 0 to 3 according to their severity. Presence of lymphoid follicles was observed in around 45% of patients in the resected specimen and in less than 20% of patients in the remaining gastric pouch in agreement with Onzi et al. [26]. who also reported decrease in inflammatory activity from 33% to less than 10% of patients in the remaining pouch after LSG Similarly, 55.5% of patients showed evidence of interstitial inflammation of varying degree in the postoperative specimen which decreased significantly to around 25% in the remaining pouch at 6 months of follow-up. The improvement of the inflammatory parameters may be due to less exposure of patients to aggressive dietary factors owing to the restrictive effect of LSG as Onzi and coworkers [26] stated.
Additionally, a relation between chronic gastritis and Helicobacter pylori infection has been described in approximately 75% of asymptomatic patients, indicating that these bacteria could be an initiating factor of the process of gastritis [27]. Onzi et al. [26] verified the relation between Helicobacter pylori and chronic gastritis in obese patients because all patients with chronic gastritis in their study also had Helicobacter pylori according to positive urease test. Since we did not examine for the presence of Helicobacter pylori infection in the patients studied, we cannot conclude similar findings.
The number of ghrelin positive cells showed a significant decrease in the remaining pouch after LSG. This marked decrease can be explained that ghrelin positive cells are mainly located in the gastric fundus, which is removed in LSG, and tend to decrease in number towards the antrum. This decrease of ghrelin cell count along the stomach is consistent with similar findings demonstrating higher levels of expression in endoscopically obtained samples from the fundus and antrum of healthy individuals [28].
Regarding the prevalence of ghrelin-secreting cells detected by immunohistochemical staining, the number of ghrelin positive cells in the gastric fundus was significantly higher than the antrum. In contrast, Goitein et al. [11] found the difference in ghrelin cell count between fundus, body, and antrum of the stomach to be statistically insignificant, perhaps due to the small sample size included in their study.
The presence of ghrelin-secreting cells in the antrum, though small in number, is of special interest. The removal of antrum was correlated with sustained weight loss on the long term, perhaps due to removal of additional ghrelin-producing cells in the antrum. Leaving intact antrum may lead to weight regain partly due to preservation of ghrelin producing cells in the antrum and partly due to sleeve dilatation which minimizes the restrictive effect of LSG. On the other hand, preservation of the pumping mechanism of the antrum may serve to facilitate gastric empting [29,30].
The significant decrease in ghrelin producing cells after LSG was paralleled by a similar decrease in plasma ghrelin level and significant weight loss at 6 months postoperatively. Weight regain in some patients after LSG was linked to the formation of neo-fundus as demonstrated in follow-up contrast studies [31]. Neo-fundus is formed by stretching and enlargement of the upper part of the remaining gastric sleeve and is associated with slow increase in plasma ghrelin level, though it remains lower than the preoperative levels [32].
Limitations of the current study include the small number of patients included and short follow-up. Furthermore, the presence of Helicobacter pylori in the resected specimen and the remaining gastric pouch was not investigated which may have a relation with the improvement in inflammatory activity postoperatively.
Conclusion
There was good positive correlation between weight loss and lowering of plasma ghrelin level, also complete removal of the fundus was associated with more weight loss. Similarly, a significant decrease in the number of ghrelin-producing cells was noted after LSG with evidence of higher number of ghrelin positive cells in the gastric fundus compared to the body and pre-antral regions. Improvement in inflammatory parameters as demonstrated by resolution of lymphoid follicles and interstitial inflammation was also observed. | 2019-03-17T13:11:49.561Z | 2018-03-19T00:00:00.000 | {
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225687544 | pes2o/s2orc | v3-fos-license | Intensification of the East Asian summer monsoon lifecycle based on observation and CMIP6
Long-term changes in the East Asian summer monsoon (EASM) lifecycle since 1979 are analyzed based on observational datasets and historical simulations of the Coupled Model Intercomparison Project Phase 6 (CMIP6). According to the observation, the active and break phases of EASM have intensified resulting in a shorter but stronger rainy season, followed by a longer dry spell. This intensification in the active-phase precipitation is accompanied by increased lower tropospheric southwesterly wind and subsequent convergence of water vapor flux. These changes are accompanied by the widely reported westward extension of the North Pacific Subtropical High, which has been associated with the warming climate. CMIP6 models generally underestimated the observed intensification of the EASM lifecycle and the monsoon precipitation. However, some of the models did simulate the intensified EASM lifecycle similar to that observed. The result highlights the reasonable performance on EASM shown in some CMIP6 models and those simulations lend support to a dynamically-driven intensification of the EASM lifecycle in the warmer climate.
Introduction
Throughout East Asia, summer precipitation variability in intraseasonal timescales has been increasing, resulting in more frequent floods and intense dry spells seemingly coexisted during the same season. Summer rainfall in northeast Asia is driven by the distinct lifecycle of the East Asian Summer Monsoon (EASM). The EASM lifecycle includes a stepwise northward and northeastward advance, derived from successive passages through (i) its rainbands (Changma, Meiyu, or Baiu), (ii) the western Pacific subtropical high, and (iii) tropical cyclone activity (Chen et al 2004, Yihui andChan 2005). As a result, the EASM features active, break, and revival phases, with intraseasonal timing of these phases dependent upon location. The intensification of this lifecycle was manifest in the 2018 Japan flood-heat wave succession event, in which more than 1000 people were killed in one month (Imada et al 2019, Wang et al 2019. The temporal and spatial variability associated with the EASM lifecycle makes trend analysis difficult and produces geographically inconsistent results. Several studies have indicated that summer precipitation over East Asia has increased (Kim et al 2005, Kornhuber et al 2019, but rainfall trends also vary by month. In Korea, Kim et al (2005) found an overall increase in summer precipitation, while In et al (2014) found that only July and August saw increased precipitation.
The impact of global warming on the EASM is an ongoing dispute. Several studies have asserted that global warming has increased EASM precipitation (Yun et al 2008, Seo et These seemingly conflicting claims arguably result from the limitation of climate models in simulating EASM features (Liu et al 2018, Tian et al 2019. Additionally, the coexistence of various weather systems in East Asia (Utsumi et al 2017) and their nonlinear changes with global warming have further complicated the matter (Utsumi et al 2016). Meanwhile, Li et al (2010) have suggested the chance of both drought and flood over East Asia would increase together due to global warming, a proposition that may reconcile these notions.
In this paper, we analyzed the variability of the EASM lifecycle and subsequently evaluated the performance of coupled climate models in CMIP6. In section 2, we described the observational and modeling datasets as well as the analysis methods. Section 3 shows the observed intensification to the EASM lifecycle and the variability in the CMIP6 models. A conclusion is provided in section 4.
Data
Daily precipitation data is obtained from the Climate Prediction Center Global Unified Precipitation dataset provided by the NOAA Earth System Research Laboratory's Physical Sciences Division at https://www.esrl.noaa.gov/psd/. Spatial coverage is the global terrestrial area on a half degree grid. Daily outgoing longwave radiation (OLR), daily precipitation data from Asian Precipitation Highly Resolved Observational Data Integration Towards Evaluation (APHRODITE), and from GPCP Version 1.2 are used to check the robustness of the change in unified CPC (table 1). Global 6-hourly meteorological data (850mb geopotential height, u-wind, and v-wind, and column integral of horizontal water vapor flux) at the 1.25 • × 1.25 • resolution is obtained from JRA-55 (Kobayashi et al 2015, Harada et al 2016. We also used the ERA-interim dataset for comparison (Dee et al 2011).
To assess the CMIP6 model performance, we adopted a single ensemble member from the historical simulations of 32 different models retrieved from the PCMDI database; these models are summarized in table 2. Following the sampling approach of Knutti et al (2010), only one member from each model experiment was selected to avoid potential systematic bias when calculating linear trends from the multi-model ensemble mean. CMIP6 outputs are linearly interpolated to 0.5 × 0.5 • , same as the unified CPC rainfall dataset and ocean masking is applied. The analysis period is from 1979 to 2010 for the CMIP6 historical runs and from 1979 to 2017 for the reanalysis data. In case of precipitation from APH-RODITE and GPCP, the analysis period is from 1979 to 2015 and from 1997 to 2015 respectively, due to data accessibility.
Methods
This study categorizes East Asia into three subregions, including the Korean peninsula (125 • -130 • E, 33 • -38 • N), the central-eastern part of China (115 • -120 • E, 27 • -33 • N), and the south-western part of Japan (130 • -139 • E, 31 • -38 • N) as shown in figure 1 inset. The EASM lifecycle has three-phases (active, break, and revival) but the variability of revival is mixed with tropical cyclone activity. Thus, this study focuses on active and break phases only. Various meteorological variables have been used to define the onset and withdrawal of the monsoon rainband (Ellis et al 2004, Yihui and Chan 2005, Seo et al 2011, Pradhan et al 2017; however, for simplicity, we used daily climatology of precipitation to define the active and break phases. Researchers have previously defined the Changma onset as the first day of more than 3 d of at least 5 mm d −1 rainfall (Seo et al 2011), but a defined active period based on this criteria is not always aligned with the operational active period. For example, the active period in Korea under this criteria is defined from 17 June to 5 September, but operationally the Korean Meteorological Agency defined the active phase from 19 June to 24 July (Seo et al 2011) and the Japan Meteorological Agency defined the active phase in Kyushu from 5 June to 19 July. 1 Here, the active phase is defined by 3 consecutive days of rainfall greater than 6 mm d −1 and the break phase is defined as the 14 d after the active phase. Figures 1(a)-(c) display the rainfall pattern and timing of the active phase and subsequent break over Korea, centraleastern China, and southwestern Japan. The dates for active, peak and break phases are listed in table 3, which is comparable to those used in the regional operational centers. In order to examine the extreme precipitation, 'peak phase' is defined as the days in which rainfall exceeds 8 mm d −1 over 1 standard deviation in all three regions. To estimate the dry spell associated with the break phase, we have introduced the Dry Severity Index (DSI), which is defined as the product of the dry spell length and precipitation deficit. Dry spell length (DSL) reflects the duration of the dry event and is the length of the successive dry days (P = 0). Precipitation deficit (PD) reflects the magnitude of a dry event (Tallaksen et al 1997) and is defined as three times the long-term mean precipitation for break periods over time (P) minus annual precipitation (P(t)) divided by mean precipitation. (Multiplying the long-term mean precipitation by three ensures PD is always positive and thus simplifies the interpretation.) In the atmosphere, the amount of water vapor (E-P) is the sum of the water vapor mass change depending on time and water vapor flux transfer (Barnes 1965) (equation (3)). The sum of the water vapor changes ( ∂w ∂t ) is small and negligible. At the surface, the convergence of water vapor flux (-∇ · Q) infers the amount of moisture pooling (equation (4)).
Observational analysis
Linear trends of rainfall in the active and break phases were examined in figure 2 for the three target regions. During the active phase, positive precipitation trends prevail in most areas (figure 2(a)). Active phase precipitation over East Asia has increased (figure 2(c)) and the trend is more pronounced in the peak rainfall of the active phase (figure 2(e)). In Japan, a strong positive trend in the southwest (Kyushu) dominates weak negative trends in the Osaka and Nagoya regions. For both active and peak phases, the precipitation trend over Japan is consistently significant (p<0.05) but in Korea, the increasing trend is only significant in the peak in active phase (p<0.1). This contrasts with precipitation trend over China, which is only significant in the active phase (p<0.1) but not in its peak phase. Nonetheless, the active phase precipitation has increased and this observation suggests enhanced EASM rainfall in active over time. This result is in agreement with a recent study on Taiwan's EASM active rainfall (Tung et al 2020).
During the monsoon break phase, figure 2(b) shows a negative trend in precipitation in Korea and central-eastern China, while a weak but positive trend is exhibited in southwest Japan. In terms of the break phases over each region, the linear trend is weak and statistically insignificant (figure 2(d)). To examine the reduced precipitation amount during the break phase, we calculated the dry severity index (DSI) described above to examine the intensity and duration of precipitation reduction together (figure S1 (available online at stacks.iop.org/ERL/15/0940b9/mmedia)). Although the trend is not statistically significant, dry spells have become drier and have lasted longer, with the exception of central-eastern China. The severity of dry periods in Korea and southwest Japan has increased. Combined, these findings indicate that the overall EASM lifecycle has enhanced, leading to a higher chance of both wet and dry extremes within a single summer season. This is consistent with the results of Singh et al (2014) and Li et al (2010) for the long-term trends, as well as the consecutive floodheat wave events over Japan during the summer of 2018 (Wang et al 2019). Temporal and latitudinal evolution of rainfall is investigated using a latitude-time Hovmöller diagram ( figure 3(a)). Climatologically, the rainband propagates northward and passes East Asia from early June to mid-July. As the rainband passes East Asia, it causes the Meiyu-Baiu-Changma season from central eastern China to southwest Japan to Korea, respectively, with increased precipitation observed along this migrating rainband in the Meiyu stage (~25˚N). A slight drying trend is observed in the periods before and after this rainband passage, suggesting the EASM rainband has intensified and this intensification is associated with adjunct drying. There is dry signal over East Asia from mid-July, proposing enhanced dry signal in break phase. The result is robust regardless of the other datasets used (see figure S2 for the validation analysis). ∆OLR, which is defined as 235-OLR, values larger than zero is used in this analysis as an indicator of deep convection. The trend of precipitation from GPCP is not calculated because the analysis period is too short. APHRODITE, GPCP, and OLR all show consistent result with that from the unified CPC precipitation. Since the characteristics of each precipitation data are unique, their consistent trends lend support to the robustness of the life-cycle change of the EASM precipitation.
The rainfall migration in summer over East Asia is closely related with the low-level southwesterlies (Seo et al 2013), so we defined the temporal evolution of the 850 hPa south westerlies as SW = u * sin (45 • ) + v * cos (45 • ) and the convergence of water vapor. As shown in the Hovmöller diagram (figures 3(b)), the rainband's evolution coincides with that of the southwesterly wind and their trends are in phase, suggesting that the increased precipitation along the EASM rainband may be dynamically driven. Next, we plotted the migration of convergence of water vapor in figure 3(c) and found that the migrating pattern of − ∇ · Q agrees well with that of the precipitation and southwesterly wind, both in the climatology and the respective trends. Noteworthy is the similar but opposite trends in − ∇ · Q suggesting that the drying trends adjacent to the EASM rainband are accompanied by increased divergence of water vapor flux. The climatology and trend of the south westerlies and − ∇ · Q were crosschecked with the ERA-interim dataset (figure S3), and the result is robust regardless of which dataset used. The cause of these systematic changes may be related to the documented westward expansion of the Western North Pacific Subtropical High (WNPSH) in the warmer world. As shown in figure S4, the WNPSH ] from JRA-55 and averaged from 115E to 139E covering target area. Contour is climatology and distinct value is plotted. (a. 6~10mm day -1 ; b. 3~5m s -1 ; c. -8~-4 *10 -5 kg m -1 s -1 ). The trend is shaded ranging (a) from -0.2mm day -1 yr -1 to 0.2mm day -1 yr -1 , (b) from -0.15m s -1 yr -1 to 0.15m s -1 yr -1 and (c) -0.3*10 -5 kg m -1 s -1 yr -1 to 0.3*10 -5 kg m -1 s -1 yr -1 , respectively. Dotted area is significant at 95% confidence level. has expanded westward in recent decades causing the high pressure to arrive earlier and reach farther in East Asia, hence increasing the south-westerlies. This finding echoes the observation by Woo et al (2017) that the Changma rainfall has increased due to the expansion of WNPSH modulating the jet-stream over Korea and inducing anomalous anti-cyclones south of Korea. Intensified southerly and southwesterly winds also cause moist convection to increase, eventually enhancing the EASM precipitation (Seo et al 2013).
Simulation by CMIP6 models
Next, we examined the historical simulations of CMIP6 for evaluation. The similarity of the observations and the simulated climatological features of EASM lifecycle is quantitatively evaluated using the pattern correlation that is illustrated on a Hovmöller diagram of precipitation (here, x-and y-axis are for time and latitude, respectively) of precipitation. For active and break phases over East Asia (latitude of 27˚N-38˚N, and time period from 7 June to 2 August in figure 4), the pattern correlation between observations and CMIP6 models was calculated to examine how the models catch the migrating pattern. However, it does not consider the intensity of the precipitation, so we included that averaged precipitation amount as an additional measure. CMIP6 models with R 2 larger than 0.5 and the total averaged precipitation for active and break phases over East Asia within ± 20% of observations (7.54) are categorized as 'group A' and the others as 'group B' . In the end, 8 models and 24 models are in group A and B, respectively (table 4). Interestingly, strong correlation between climatological rainfall and its long-term change measured by pattern correlation (figure S5) strongly infers importance of better climatological rainfall in the EASM. Table 4. Pattern correlation coefficient in CMIP6 model. Correlation coefficient larger than 0.70 (r 2 > 0.5) and total precipitation for active and break phase over East Asia is within ± 20% of observation (6.03-9.05) is classified as group A, and the others are in group B. The climatology of the spatiotemporal evolution of precipitation (solid line) is reasonably simulated (figure 4), though simulated precipitation in models both in group A and B tends to be underestimated. Models in group A depict the climatological rainfall onset and its migration to the north well (figure 4(a)). However, the intensity of precipitation is weak. There is a slightly positive trend at the beginning of the rainband, but a negative trend exists throughout the monsoon period, implying underestimation of the EASM precipitation. Precipitation time-series for the overlapping active, break and peak phases in three regions are shown in figure S6. Observations show a distinct increasing trend in the active and peak phases and decreasing trend in break phase, supporting the observed intensification of the EASM lifecycle. Precipitation of group A models shows a distinct decreasing trend in the break phase and slightly increasing trend in active and peak phases. Despite the underestimated precipitation in group A models, northward propagation of the rainband, intraseasonal characteristic, is realistically simulated. Furthermore, the overall pattern of wetting trend (drying trend) in the earlier (later) part of the rainy season is well simulated. All models in group A do simulate climatological propagation of the rainband but the timing and the intensity of the monsoon onset are different (figure S7). Nevertheless, some models in group A (CESM2, CESM2-WACCM, CESM2-WACCM-FV2) reproduce the intensification in both the active and break phases (figure S9). When it comes to models in group B, the rainfall peak in summer is captured but the amount of precipitation is underestimated and they mostly fail to simulate the migrating pattern of EASM ( figure 4(b)). Nevertheless, some models depict the climatological propagation of the rainband, but the rainband reaches to the East Asia lately or its intensity is weak (CNRM-CM6-1, CNRM-CM6-1-HR, CNRM-ESM2-1, EC-Earth3, EC-Earth3-Veg, INM-CM4-8, IPSL-CM6A-LR, MIROC-ES2L, MIROC6, NorCPM1) (figure S8). Also, most models fail to simulate the precipitation trend in either phase except for EC-Earth3, MIROC6, CNRM-CM6-1 (figure S8). These models catch the wetting trend (drying trend) in the earlier (later) part of the rainy season, but the timing of the rainy season is wrong. The climatological propagation of the rainband simulated by EC-Earth3 and MIROC6 is too fast and the rainband reproduced by CNRM-CM6-1 is stagnated at the lower latitude. Time-series of precipitation for overlapped active, break, and peak of MIROC6 phases show similar trend with that of observation (figure S10).
According to previous studies of Park et al (2020) and Li et al (2020) using CMIP5 models, it was found that certain features of the EASM were simulated but its more detailed characteristics were not properly captured, such as the second peak and northward propagation of precipitation. On the other hand, some of CMIP6 models, those in group A simulate the EASM lifecycles and its long-term change, which is a positive sign.
Concluding remarks
In this paper, the long-term change in the EASM lifecycle was analyzed based on observational datasets, and the capacity of CMIP6 models to reproduce the mechanisms of the long-term trends. It was found that precipitation in the active phase has increased and the increasing trend is more significant in its peak, which means that the monsoon peak or extremes is getting stronger. This precipitation trend coincides with the increasing trends of southwesterly wind and the convergence of water vapor flux accompanying the rainband. The WNPSH and its westward extension help intensify the southwesterly wind. It was also found that the break phase tends to be enhanced after the rainband passage, and this tendency is further illustrated by the DSI index, showing the combined intensity and duration of the dry spell. Thus, it can be concluded that the EASM lifecycle has intensified, making it more likely for southwest Japan and Korea to experience heavy rainfall followed by enhanced drying. This result is in agreement with previous studies (Kim et al 2005, Seo et al 2013 in terms of a generally intensified EASM system, but adds further details in that both the rainy season and dry spell within the EASM lifecycle have intensified. Apparently, the observed EASM variability is more complex than just the summer mean value.
The performance of CMIP6 models in simulating the EASM climatological evolution and its variability is still limited, but some models are showing sign of an improved performance. In general, the intensity of monsoon precipitation tends to be underestimated, but group A models realistically simulate the latitude-time propagation of the rainband and the general pattern of the long-term intensification of EASM. Not only that, group A models also depict the precipitation time-series at the active and break phase that is consistent with the observational dataset. While models in group B did not accurately simulate the progression of the summer rainband and they underestimated the EASM rainfall, they still depicted the rainfall peak in summer. This evaluation highlights some of CMIP6 models that exhibit a marked performance in simulating intraseasonal characteristic of the EASM as well as its long-term change. | 2020-06-18T09:03:11.591Z | 2020-06-10T00:00:00.000 | {
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218513393 | pes2o/s2orc | v3-fos-license | A specialised delivery system for stratospheric sulphate aerosols (part 2): financial cost and equivalent CO2 emission
Temporary stratospheric aerosol injection (SAI) using sulphate compounds could help avoid some of the adverse and irreversible impacts of global warming, but comprises many risks and uncertainties. Among these, the direct financial cost and carbon emissions of potential SAI delivery systems have hitherto received only modest attention. Therefore, this paper quantifies the initial and operating financial costs and initial and operating equivalent CO2 (CO2eq) emissions of the specialised aircraft-based SAI delivery system developed with relatively high-fidelity tools in part 1 of this series. We analyse an interval of operating conditions, within which we devote special attention to four injection scenarios outlined in part 1: Three scenarios where H2SO4 vapour is directly injected at several dispersion rates and one SO2 injection scenario. We estimate financial cost through Raymer’s adjustment of Rand Corporation’s Development and Production Costs for Aircraft (DAPCA) model, augmented by additional data. CO2eq emission is computed from existing data and the computed fuel consumption for each of the scenarios. The latter estimates include an emission weighting factor to account for non-CO2 aircraft combustion products at altitude. For direct H2SO4 injection, both financial cost and CO2eq emission are sensitive to the design dispersion rate. For scenarios where higher dispersion rates are achieved, the delivery system’s cost and CO2eq are relatively small compared with the presumed benefits of SAI. The most optimistic H2SO4 scenario is found to have a financial cost and CO2eq emission similar to that of SO2 injection, while potentially allowing for reductions in the annual mass of sulphur injected to achieve a target negative radiative forcing. The estimates of financial cost and CO2eq emission were subjected to sensitivity analyses in several key parameters, including aircraft operational empty weight, engine specific fuel consumption, fuel price and aerosol price. The results indicate that the feasibility of the considered scenarios is robust.
Introduction
If emission of greenhouse gases and associated global warming proceed along a high Representative Concentration Pathway (RCP) (IPCC 2018), one may consider enacting temporary measures to limit further increases in the probability and severity of adverse and irreversible impacts of continued climate change, until emission reductions and potential carbon capture efforts have returned the greenhouse gas concentration in the atmosphere to safe levels. Although they are by no means suggested to be a solution to the climate change problem, such temporary measures could include solar radiation management (SRM) techniques such as cirrus cloud thinning (Storelvmo et al. 2013;Lohmann and Gasparini 2017) (CCT), marine cloud brightening (Latham et al. 2012) (MCB) and stratospheric aerosol injection (Crutzen 2006) (SAI). While SAI is generally regarded as the most feasible option (Shepherd 2009), it comes with a long list of risks and uncertainties (Robock 2014), spanning i.a. the environmental (Kravitz et al. 2013), social (Irvine et al. 2016), ethical (Lin 2013), policy (Reynolds 2019) and technical domains. Here, we focus on the financial and environmental implications of developing and operating a delivery system capable of lifting very large payloads to unconventionally high altitudes. McClellan et al. (2010) and Smith and Wagner (2018) propose specially developed aircraft for delivering aerosol precursors to the stratosphere. They estimate that these likely form realistic delivery platforms at a manageable financial cost per injected unit mass. Part 1 of this series corroborated the technical viability of specialised aircraft delivery systems by designing a fleet of aircraft and its operation at a higher degree of technical fidelity than these studies. However, it did not yet quantify the delivery system's financial cost. In this second part of the series, we therefore perform a detailed analysis of the financial costs for developing and operating this delivery system 1 .
Furthermore, neither McClellan et al. nor Smith and Wagner address the contribution to the atmospheric greenhouse gas concentration of developing, producing and operating a large fleet of aircraft for SAI (McClellan et al. 2010;Smith and Wagner 2018). However, in order to make a full assessment of the risks and benefits of SAI, it is imperative to establish the carbon footprint of proposed delivery systems. Therefore, this paper will also quantify the equivalent CO 2 (CO 2 eq) emissions incurred by initially conceiving and operating the delivery system proposed in part 1.
Part 1 addressed four operating scenarios: Three featuring direct injection of H 2 SO 4 vapour along with one SO 2 injection scenario. The H 2 SO 4 scenarios are advantageous in that they offer the potential of maximising the effectiveness of SAI per unit mass of injected S (Vattioni et al. 2019). However, they might also require greater technical resources. As a result, it is unclear whether their deployment-related financial costs and CO 2 eq emissions are competitive with respect to SO 2 injection. Therefore, we analyse and compare the financial cost and CO 2 eq emission of each of the SAI scenarios developed in part 1.
The paper first briefly describes the SAI system and injection scenarios developed in part 1. This is followed by analyses of the financial cost and CO 2 eq emission over an interval of dispersion rates that encompass the injection scenarios. These analyses are accompanied by descriptions of their respective estimation methods. Finally, the sensitivity of financial cost and CO 2 eq emission to several important input parameters subject to uncertainty is examined.
Summary of the delivery system
The following subsections summarise the features of the SAI delivery system developed in part 1 salient for the analysis of financial cost and CO 2 eq. The design choices and factors concerning aerosol material and delivery are first outlined, followed by the candidate injection scenarios. Then, the most important aspects of the aircraft and flight profiles that emerge from these scenarios are highlighted.
Aerosol and delivery requirements
In order to facilitate realistic estimates of financial cost and CO 2 eq emission, the delivery system considered here is based on the use of sulphate aerosols. These grow naturally in the stratosphere after volcanic eruptions, leading to relative certainty concerning their impacts on temperature and the climate system (Pope et al. 2012;Keith et al. 2016;Dykema et al. 2016). Sunlight-scattering sulphate aerosols in the stratosphere consist of H 2 SO 4 particles. These can be introduced into the atmosphere either by injection of precursor compounds, such as SO 2 , or by directly injecting condensable H 2 SO 4 . Part 1 of this study discusses how direct H 2 SO 4 injection appears promising with respect to SO 2 injection, as it facilitates targeting the growth of optimally sized aerosols. While direct comparisons between SO 2 and H 2 SO 4 injection are relatively sparse and significant uncertainties in their long-term, large-scale application remain (Pope et al. 2012;Pierce et al. 2010;Benduhn et al. 2016;Vattioni et al. 2019), the potential benefits of direct H 2 SO 4 injection make this the main focus of our aircraft-based delivery system.
To provide conservative measures of the costs and emissions of an SAI delivery system in a fully operational phase, we target a constant, rather large annual delivery rate of 15 Mt H 2 SO 4 yr −1 (5 Mt S yr −1 ). This corresponds to contemporary estimates of the rates needed to achieve 2.5-3 Wm − 2 negative radiative forcing (IPCC 2018;Pierce et al. 2010;Vattioni et al. 2019). If desired, the target rate can be adapted by changing the fleet size of the delivery system. We estimate the sensitivity of our cost and emission figures to such modifications in Section 5. Here, we choose to focus on a single delivery rate for the sake of clarity. For effective, global radiation reduction, delivery will take place near altitudes of 20 km at latitudes centred around 15 • north and south (Rasch et al. 2008;Pierce et al. 2010;Tilmes et al. 2017;Vattioni et al. 2019).
Two parameters drive the growth of optimally sized particles (here we target initial particle sizes of approximately 0.1 μm (Pierce et al. 2010)) from direct H 2 SO 4 injection: the initial H 2 SO 4 concentration in molecules cm −3 and background flow diffusivity (Benduhn et al. 2016). These two parameters can be related to financial cost and CO 2 eq emission through dispersion rate DR-the mass of aerosol dispersed per flown unit length-in kg H 2 SO 4 m −1 , which additionally depends on the volume flow rate of injection and aircraft speed. High DR allows short flights with high payloads, which in turn is beneficial for minimising the operation's financial cost and emissions. The high initial H 2 SO 4 concentrations associated with high DRs require a high diffusivity for favourable initial particle growth (Benduhn et al. 2016). To achieve this, we propose dispersing the H 2 SO 4 in a single engine's exhaust plume. However, the diffusivity in aircraft engine plumes is neither constant, uniform nor well-established (Yu and Turco 1998;Schumann et al. 1998). To capture the resulting uncertainty, part 1 of this study considered three direct H 2 SO 4 injection scenarios with different combinations of diffusivity and initial concentration. We briefly introduce these below and refer the reader to part 1 for a more elaborate motivation on their choice.
The lowest DR considered and upper bound to estimates of financial cost and CO 2 eq emission is set by the conservative core injection (CI) scenario. This entails that the diffusivity is assumed to be low (100 m 2 s −1 ) and that H 2 SO 4 is only injected into the uniform, high-velocity core flow of the specialised turbofan engine of the aircraft. Targeting an initial particle radius of ≈ 0.1 μm at the assumed diffusivity requires an initial H 2 SO 4 concentration of approximately 10 16 cm −3 (Benduhn et al. 2016), leading to a relatively low DR of 0.002 kgm −1 .
The second full injection (FI) scenario considers injection into the full engine flow to help increase DR. This is incommensurate with the plume simulations upon which we base our results, which assume a uniform, well-mixed initial flow (Pierce et al. 2010;Benduhn et al. 2016). Therefore, the viability of this scenario is somewhat less certain. However, owing to the higher total volume flow, it should achieve a DR that is an order of magnitude higher (0.02 kgm −1 ) than the CI scenario at the same assumed diffusivity and initial concentration. Therefore, we still consider it highly relevant to analyse the potential of such a scenario.
Improved engine flow mixing technology or more accurate measurements of plume growth might show that diffusivity values higher than those considered in the first two scenarios can be attained. This is likely, given that detailed engineering optimisations have yet to be performed for this purpose. Thus, the third, optimised full injection (OFI) scenario considers a relatively high value of diffusivity, 3.16 · 10 2 m 2 s −1 , in combination with both core and bypass injection. This corresponds to a much higher initial H 2 SO 4 concentration and DR of 3.16 · 10 17 cm −3 and 0.5 kgm −1 , respectively.
When precursors such as SO 2 are injected, aerosol formation does not occur until long after delivery and is thus virtually independent of DR. Hence, cost-efficient, short, highpayload flights can be employed. However, aerosol particles formed from SO 2 injection have a lower radiative forcing efficiency, which we account for by assuming that approximately twice the amount of S is required when injecting SO 2 instead of H 2 SO 4 (Pierce et al. 2010;Vattioni et al. 2019). The final SO 2 scenario thus assumes the delivery of 20 Mt SO 2 yr −1 (10 Mt S) at the same altitudes and latitudes as the H 2 SO 4 scenarios at a DR that is as large as technically possible. A summary of the setup of all scenarios is reported in the first part of Table 1.
Coupled aircraft/flight profile design
The simultaneous optimisation of the aircraft configuration and flight profiles described in part 1 resulted in an unmanned aircraft featuring a large, slender, strut-braced wing and four custom-designed turbofan engines. For the direct injection scenarios, H 2 SO 4 is kept at high temperature onboard and evaporated upon injection into a single outboard engine plume. The main characteristics of the operation that results for each of our scenarios are summarised in the second portion of Table 1. The flight profiles of the CI, FI and OFI scenarios have a similar main structure, consisting of an initial climb to the outgoing dispersion leg at 20 km, followed by a second climb to the return dispersion leg, located 500 m above its outgoing counterpart. Operations are constrained by a critical flight condition encountered during climb. High lift and high thrust requirements can both become critical here (C L,crit , T crit ) and constrain the payload the aircraft can carry; in practice, the T crit limit is reached first in these scenarios (see Section 4.2.2 of part 1 for a more detailed description of these limits). The SO 2 scenario is simpler, in that it only consists of a climb to 20 km altitude, the quick release of all payload and return to the airport. However, even in this scenario, the climb is constrained by the T crit condition. Correspondingly, in the following analyses of financial cost and CO 2 eq emission, the high-performance limit is set by the T crit condition in the flight profile.
Cost model description
The estimation of financial cost is based on Raymer's 1999 adjustment of Rand Corporation's Development and Production Costs of Aircraft (DAPCA) model. This method uses statistical cost estimation relationships (CERs) to separately determine initial cost (consisting of research, development, testing and evaluation -RDT&E -and production cost) and annual operating cost and is commonly used at the conceptual design stage of transport aircraft (Raymer 1999). The relations comprising the model are detailed in McClellan et al. (2010).
The CERs provide either cost for a particular development phase or labour hours for different professions involved (e.g. production materials CERs have cost as an output, whereas RDT&E engineering CERs provide engineer labour hours). In order to convert labour hours to cost, we use labour rates for the corresponding profession obtained from the U.S. Bureau of Labour statistics, May 2017 National Occupational Employment and Wage Estimates United States (Bureau of Labor Statistics U.D.o.L. 2017b). These are multiplied by a factor 2.5 (Raymer 1999) to give a conservative estimate of the hourly wrap rates, which are tabulated in the supplementary material. The wrap rates are multiplied by 1.03 to convert FY2017 to FY2018 USD (Bureau of Labor Statistics U.D.o.L. 2017a). To estimate pilot labour costs, which deviate from standard practice due to the unmanned concept, one operator is assumed to control four unmanned aircraft. Direct cost outputs are multiplied with 1.51 to convert FY1999 to FY2018 USD (Bureau of Labor Statistics U.D.o.L. 2017a). Moderate changes in labour rates do not have a large impact on total costs. Raymer's RDT&E and production equations were scaled by a technological implementation factor (TIF) of 1.5 to incorporate expected cost increases due to difficulties resulting from the extremity of the design and material costs were scaled by a factor of 1.5 to account for the use of the relatively expensive composite material in the aircraft's wings.
Furthermore, the custom-developed, high-performance turbofan engine's RDT&E and production cost incorporates a difficulty factor of 2 and scales with the high maximum thrust; the engine's maintenance cost is also affected by these factors (McClellan et al. 2010). For the initial cost of adjustment of airports to SAI operations, a fixed investment of 0.6 B USD per airport was assumed. This was based on the total investment as of 2017 of Deutsche Post DHL group for their recent Central Asia cargo hub at Hong Kong International Airport (DHL press 2017). Although the annual mass of cargo to be handled per airport in the scenarios featuring injection of 15 Mt aerosol yr −1 is larger than the DHL hub capacity, it is assumed that the simple nature of the cargo and infrastructure required as well as standardisation will keep the costs comparable.
Operating cost was estimated using Raymer's method for commercial aircraft (Raymer 1999), motivated by the similarly high flight frequency in airline operations and in our scenarios. The fuel price and the aerosol payload price input parameters were set to 90 USD bbl −1 and 350 USD ton −1 , respectively (International Air Transport Association 2018; Boyd 2014). Although commercial prices for H 2 SO 4 and SO 2 differ, we use the same aerosol price for all four scenarios for the sake of comparison. Commercial market SO 2 prices are higher than H 2 SO 4 prices, implying that the reported financial cost of the SO 2 scenario might be an underestimation. However, it is also likely that commercial prices do not hold for operations at this scale.
In addition to the parameters mentioned, the annual delivery requirement of 15 Mt yr −1 and the payload per flight govern the number of flights, fleet size and number of airports needed for the delivery scenario in question, which make up the scenario-dependent inputs to the cost model.
Financial cost over an interval of DR
Since the financial cost is a strong function of the chosen payload-delivery range combination, cost estimates for an annual delivery rate of 15 Mt H 2 SO 4 yr −1 were made for a matrix of payload-delivery range combinations, obtained using repeated aircraft-flight optimisations as described in part 1. The matrix spanned an interval which included the CI, FI and OFI H 2 SO 4 delivery scenarios. The results are shown in Fig. 1 a and b, respectively, where the initial and operating costs are indicated by colour bands. We also include contour lines along which DR-and thus the ratio of payload to delivery range-is constant.
(a) Initial financial cost (b) Operating financial cost Modifying payload and delivery range affects the aircraft's take-off weight, which impacts the thrust requiredat the end of the climb segment. Since this cannot exceed T crit , excessive combinations of payload and delivery range are technically unfeasible. In Fig. 1 a and b, possible mission scenarios are therefore confined by the broken red line. Changing the take-off weight also impacts take-off C L , storage volume, early-flight load cases and margins to aeroelastically unstable modes. However, as none of the these aspects approached their min-or maximum allowed values in the baseline aircraft design, these values were allowed to vary without reexamining their influence on aircraft feasibility.
The cost values for the four scenarios, marked in Fig. 1 a and b, are given in Fig. 2. The figure indicates how initial costs primarily depend on aircraft production related costs, with relatively small airport adjustment and RDT&E costs, even in the presence of a customdeveloped engine. This is simply due to the high material and labour cost associated with production of a considerable number of aircraft, each having four powerful engines. At large fleet size, the engine production cost constitutes the largest proportion, but this is overtaken by labour costs when the fleet size comes down. Operating cost was found to be primarily determined by maintenance materials cost, which in turn comprises the cost of materials, replacement components and other maintenance supplies. These costs are relatively high for this particular aircraft, due to its extreme design and the specialised high-performance engines. They additionally scale with flight hours and number of sorties, which are also high for SAI aircraft. As the model depends heavily these factors, the operating cost estimate is thought to be relatively conservative. These observations suggest that scenario changes that allow fleet size reductions should be the primary target to bring down initial and operating cost. Figure 1 a and b confirm that the most cost-effective payload-range combination for a given prescribed DR lies on the T crit boundary. For both the initial and operating costs, changes in the prescribed DR are therefore most economically dealt with by changing the delivery range and payload along the T crit line. It is possible to operate below T crit , but this increases cost through inefficient use of the aircraft's capacity. The three scenarios CI, FI and OFI are therefore positioned on this line. The FI and OFI scenarios achieve substantially lower initial and operating costs than the CI scenario. This is primarily caused Fig. 2 Comparison and breakdown of initial and operating financial cost for the delivery scenarios by the increased DR in the latter two scenarios, allowing for faster aerosol delivery. This results in both lower initial costs due to the reduced size of the fleet of aircraft required and lower operating costs due to lower fleet maintenance requirements and fuel consumption. Consequently, for direct H 2 SO 4 injection scenarios, the most cost-effective solution is to operate at the point defined by T crit and maximum achievable DR. Figure 2 directly compares the initial and operating cost of the four scenarios. It shows that if the FI scenario can be realised, the initial costs reduce by 70% and operating costs reduce by over 75% with respect to the CI scenario. If the OFI scenario can be achieved, the cost decreases are 80% and 85%, respectively. This variation spans an order of magnitude in operating cost, and results directly from the uncertainties in achievable plume diffusivity and, consequently, DR that underpin the different scenarios. Therefore, even if the CI scenario may still be considered manageable when compared with e.g. global annual military (Stockholm international peace research institute 2019) or health care (World Health Organization 2019) expenses, there is a strong motivation to research the achievable plume diffusivity for H 2 SO 4 scenarios in particular and engineering solutions that maximise DR in general. Figure 2 also includes results for the SO 2 scenario. This scenario has no prescribed DR and can therefore operate at the optimum point with minimum delivery range and maximum payload. While cheaper than the CI and FI scenarios, its higher annual aerosol delivery requirement renders it more expensive than the H 2 SO 4 OFI scenario. As the aerosol payload price was kept the same for the SO 2 scenario, whereas commercial prices are higher for SO 2 than for H 2 SO 4 , the financial cost advantage of the OFI over the SO 2 scenario might be larger in reality, as aerosol price makes up almost 30% of the operating cost in the SO 2 scenario. As there is currently limited knowledge on the relative effectiveness in practice of SO 2 and H 2 SO 4 injection, however, the ultimate ratio of required annual injection rates to achieve a certain effect might differ from the one used here. These factors, along with the ultimately achieved DR in the OFI scenario, will determine their relative costs. As the SO 2 scenario currently has less uncertainties due to its analog with volcanic emissions, this highlights the need for further research of the OFI scenario, in order to validate its feasibility and the potential benefits of direct H 2 SO 4 injection in the form of e.g. lower costs and sulphur injection.
In situ production of SO 2 has also been proposed Smith and Wagner 2018), which has the advantage of having to carry only S to stratospheric altitudes. This has the potential to reduce the required annual mass delivery rate. However, the benefits of such a reduction may be somewhat offset by a possible increase in the required aircraft residence time at delivery altitude while S combustion takes place (Smith and Wagner 2018). Still, as this approach has the potential to reduce financial cost, further research on in situ SO 2 (or other) aerosol production and delivery should also be pursued.
The observations above pertain to operation variations for the fixed aircraft configuration described in part 1 of this series. Any scenario with a maximum thrust requirement less than the current T crit value during its critical climb phase would likely benefit from a redesign. This could yield a smaller wing, lower structural and fuel weight, potentially yielding a more conventional design with lower financial cost and CO 2 eq emission than the baseline configuration considered here.
Such a more conventional design is presented in Smith and Wagner (2018), which proposes 25 tons of payload can be lofted to 20 km by an aircraft with a 100 ton take-off weight. With less aggressive climbs and 1 hour of station keeping, the aircraft proposed in Smith and Wagner (2018) would likely achieve this mission at delivery range values which are similar to those of the aircraft presented here. However, our design requires a 150 ton take-off weight to do so, which follows directly from the detailed analysis procedures described in part 1. While the altitude profiles, flight profiles, aerosol species, operating concept, annual delivery target and cost models also differ between the two aircraft, the difference in estimated take-off weight likely plays a major role in the disparity in predicted operating costs for the delivery of 25 tons of payload per flight (approximately 1800 with USD ton −1 here, compared with approximately 1400 with USD ton −1 in Smith and Wagner (2018)). It will be necessary to fully understand and reduce this disparity to further advance the discussion on the financial implications of developing and operating SAI delivery systems.
CO 2 eq emission analysis
Given that SAI's main objective is to curb the detrimental effects brought about by excessive anthropogenic greenhouse gas emissions, it is imperative to quantify the carbon footprint of the SAI delivery system itself. While many aerosol-related aspects of SAI that lead to environmental risks have been studied (Robock 2014), emission figures for specialised aircraft-based SAI delivery systems are to our knowledge missing from the contemporary literature. Therefore, we compute these figures in this section.
As was the case for the financial cost analysis, we distinguish between initial and operating emissions. The following subsections provide quantitative estimates for these two components for the different delivery scenarios. The quantification is made in terms of CO 2 eq in order to provide an indication of their relative impact on global warming. The reported CO 2 eq values also account for the global warming potential of non-CO 2 emissions by using an emission weighting factor (EWF). This approach has some limitations in the current context, as described in Section 4.2.
Initial CO 2 eq emission
Estimates of initial CO 2 eq emission were made based on emissions due to aircraft development and production, as well as those due to airport construction works.
Emissions due to aircraft development and production were computed using the 2017 average CO 2 eq estimate for large airliners described in Airbus (2017), multiplied by the required fleet size. Emissions due to airport modification are based on the conservative assumption that the operation will use existing airports that are complemented with an additional runway and with extended ground facilities to accommodate the added number of flights and logistics required for SAI. The associated CO 2 eq estimates were then obtained from the analysis of airport expansions performed in Sturgis Carbon Profiling LLP (2013) (note that there are also local health effects resulting from NO x emissions due to construction work, but these are not included here). The resulting initial CO 2 eq emission estimates for all four scenarios appear in the first part of Fig. 4.
Operating CO 2 eq emission
Estimates of operating CO 2 eq emission were obtained by adding the emissions of running the required airport facilities to those associated with aircraft operation. Airport facility CO 2 eq emissions were estimated using reference data for existing airports which handle around 400,000 aircraft movements per year, which is on the order of magnitude of the annual number of flights per airport in the CI scenario and is thus conservative for all other scenarios which feature lower flight numbers (Federal Aviation Administration 2017).
The annual CO 2 eq emissions associated with in-flight aircraft operation result from estimates of CO 2 emissions, which are scaled by an EWF. CO 2 emissions are computed by multiplying the total fuel consumption of a scenario (obtained from our aircraft model) with the amount of CO 2 released per kg fuel burned: a constant value of 3. 16 (Penner et al. 1999). In contrast, determining an adequate EWF is not trivial, due to the complex interaction of aircraft engine combustion products with the environment.
In addition to CO 2 , aircraft engine combustion produces H 2 O and NO x , as well as aerosol particles consisting of e.g. soot, sulphate and hydrocarbons. The impact of these emissions depends on the altitude at which they are emitted as well as local conditions. Emitted H 2 O can produce contrails in the troposphere, which both reflect solar radiation and trap thermal radiation. Generally, their net effect on the earth's surface is warming (Azar and Johansson 2012). Stratospheric warming due to heat absorption by emitted water vapour and particulate matter can further increase stratospheric water vapour content and lead to ozone depletion, intensifying the earth's surface warming due to increased solar radiation passing through the atmosphere (Azar and Johansson 2012;Heckendorn et al. 2009). In contrast, NO x emitted at high tropospheric altitudes typically results in increased ozone formation. Another effect of high altitude NO x is methane depletion, which, on longer time scales, results in decreased ozone concentrations, in contrast to the initial NO x effect. The initial NO x ozone/methane response thus has a net cooling effect, but this effect reverses as time progresses (Azar and Johansson 2012). The aerosols produced by combustion can have a reflection-induced cooling effect, although this depends on the balance of their specific radiative properties and their tendency to promote contrail formation. Generally, their effect on warming is uncertain and dependent on local conditions (IPCC 2013).
Due to i.a. these complexities and the different economic and physical metrics available (see Azar and Johansson 2012), the EWF values for aviation span a large range. They typically vary between 1.3 and 2.9 for operations as considered here, with a best estimate of 1.7 when using global warming potential as the relevant metric (Azar and Johansson 2012). Therefore, we use an EWF of 1.7 in our computations.
The resulting operating CO 2 eq emission estimates for all four scenarios appear in the second part of Fig. 4. As before, local health effects due to NO x emissions from ground operations are not accounted for.
CO 2 eq emission over an interval of DR
Similar to the estimates of financial cost, the estimates of CO 2 eq emission are highly sensitive to payload and range, which in turn are a function of DR. Thus, plots of initial and operating CO 2 eq emission for the same range of conditions considered in Section 3.2 are given in Fig. 3 a and b. The CO 2 eq emission follows trends similar to those of the financial cost: high DR scenarios have smaller initial and operating CO 2 eq emission due to fleet size reductions and lower fuel consumption. A sharp increase in initial CO 2 eq emission is seen for payload values below ≈ 6 tons, which is due to an increase in the number of airports required to accommodate the increasing number of flights per day for lower flight payloads. A difference between CO 2 eq emission and financial cost behaviour is seen for very low DRs. Here, possible delivery scenarios below the T crit line have moderately lower operating CO 2 eq emission at constant DR, as fuel savings per flight for smaller payload and delivery range combinations slightly outweigh the extra fuel required by the increased flight numbers of such combinations. However, this difference is small.
The CO 2 eq emission breakdown in Fig. 4 shows how both initial and operating CO 2 eq emissions are largely determined by a single component. Initial CO 2 eq is dominated by airport construction related emissions, which scales with the number of airports required to support the required fleet size. The number of airports is constant; four airports can accommodate each of the four scenarios. However, since fleet size decreases as DR increases across the scenarios, the relative contribution of aircraft development and production to initial CO 2 eq emission decreases. The airport modification emission estimates are based on conservative values from literature and could turn out to be lower in reality. However, this is unlikely to significantly affect the total CO 2 eq emission, since for a mission spanning several years, it would clearly be dominated by the operating CO 2 eq emission.
Operating CO 2 eq is essentially determined by fuel combustion. The percentage contribution of this parameter decreases as DR increases, but only very slightly. Given the large fuel-related contributions to both operating financial cost and CO 2 eq emission (> 17% and > 99% respectively in OFI), it is once again important to attain high DRs to reduce range and flight numbers. This is also clearly demonstrated in Fig. 4, which shows large reductions in CO 2 eq for the FI and OFI scenarios. The figure also shows that, like the financial cost, the CO 2 eq emission of OFI is similar to that of SO 2 . Fig. 4 Breakdown of initial and operating CO 2 eq emission for the delivery scenarios An indication of the relative impact of the proposed delivery method is given on the rightmost y-axis in Fig. 4, which shows the operating CO 2 eq emission as a percentage of the global annual 2016 CO 2 eq emission (Olivier et al. 2017). The values are appreciable for a single aeronautical operation, especially for scenarios that deviate far from OFI. While the corresponding effect is still rather small relative to the beneficial anticipated cooling effects of the delivered aerosol, this again indicates the importance of development of high DR technologies.
Sensitivity analysis
Aside from the uncertainties in the achievable DR, the analyses presented in the previous sections depend on a number of input parameters to our model that are also subject to uncertainty. In the following, the most important of these are listed and the sensitivity of financial cost and CO 2 eq emission to their variation is quantified and briefly discussed. In the supplementary material, the complete, detailed version of this sensitivity analysis is provided. The sensitivity of financial cost and CO 2 eq emission to design choices, i.e. propulsion system and operating altitude, as described in part 1, is also presented in the supplementary material.
For the sensitivity analysis, DR and critical climb segments are kept constant; less critical flight phases are allowed to change. Hence, perturbations in design parameters drive changes to the payload-range combinations (along a DR-isoline) and the coupled procedure is iterated until either the thrust constraint imposed by T crit or the lift constraint imposed by C L,crit is encountered. The resulting payload-range combination can be unambiguously translated into changes in operating parameters and financial cost and CO 2 eq emission.
The results of the sensitivity analysis are only presented for the CI scenario. The general conclusions remain similar for the other scenarios, although the specific magnitudes resulting from the analysis differ.
Input parameters
The following six parameters and perturbations have been considered.
Annual aerosol delivery
Range 50-150% of baseline value 2. Operational empty weight (OEW) Range 90-110% of baseline value 3. Specific fuel consumption (SFC) Range 80-120% of baseline value 4. Technological implementation factor (TIF) Range 1-2 5. Fuel price Range 60-120 USD bbl −1 (International Air Transport Association 2018) 6. Payload price Range 250-450 USD ton −1 (Boyd 2014) Several other aircraft and cost-model related inputs were also examined for their effects on financial cost and CO 2 eq emission, but were found to be of minor importance in comparison with those listed.
Sensitivity of financial cost
The changes in financial cost of the CI scenario due to individual variations of the input parameters are summarised in Fig. 5.
Annual aerosol delivery impacts both initial and operating cost by increasing the fleet size and number of flights required. In total, a 50% increase and decrease in annual aerosol delivery lead to a 39.9% increase and 42.1% decrease in initial cost, respectively, and a 48.8% increase and 48.9% decrease in operating cost, respectively.
OEW variations also notably affect the financial cost of the system, primarily through large changes in the fleet size and number of flights. This is a result of the extreme operating conditions leading to low payloads that quickly decrease if aircraft efficiency is reduced. Around the CI scenario, 10% increases and decreases in OEW lead to 29.5% and − 13.2% changes to initial costs and 28.3% and − 11.1% changes in operating costs. During the aircraft development cycle, minimising the weight of the aircraft components should thus be assigned appropriate importance, especially given the higher sensitivity to cost-increasing OEW increases.
The sensitivity of operating cost to a 20% SFC decrease is − 11.8%, while a 20% increase leads to a 15.2% increase. These values are remarkably moderate for a 20% variation in input parameter value. This is a positive finding, as the robustness of cost to SFC increases the viability of the development and use of custom engines with comparatively high SFC uncertainties.
Despite a relatively large TIF interval, its effect on initial cost is moderate, amounting to a ± 9.16% variation over the interval considered. This again increases the economic viability of developing custom technology for the system.
The last parameters considered, fuel price and payload price, do not significantly affect operating cost, as operating cost is dominated by the maintenance materials cost. In the more optimised direct H 2 SO 4 injection scenarios and the SO 2 scenario, the contribution of payload cost to the operating financial cost increases substantially (up to 26% for OFI). In such scenarios, payload cost might be an important factor, but is not expected to fatally affect feasibility.
(a) Financial cost sensitivity (b) CO 2 eq sensitivity
Sensitivity of CO 2 eq emission
The changes in CO 2 eq emission of the CI scenario due to individual variations of the input parameters are summarised in Fig. 5a. Increases in the annual aerosol delivery rate affect the initial CO 2 eq emission due to the increased number of airports required for the CI scenario. Increases in OEW and SFC also increase the number of required flights and thus the number of airports and initial CO 2 eq emission. As the CI scenario already employs the minimum number of airports (four), decreases in annual aerosol delivery rate, OEW or SFC have very little impact.
Changes in operating CO 2 eq emission due to changes in annual aerosol delivery, OEW and SFC are more substantial. The first of these scales the overall mission, while OEW and SFC affect the amount of payload which can be carried per flight. Increases in any of these inputs thus increase the number of flights required and hereby fuel consumption, the main factor determining operating CO 2 eq emission.
Increasing or decreasing the annual aerosol delivery by 50% changes the operating CO 2 eq emission by 50.0% and − 50.0%. The operating CO 2 eq emission is more sensitive to increases than to decreases in OEW, amounting to + 28.0% and − 12.7% changes, respectively. Increases in SFC result in more fuel burned per flight and consequently more emissions, but also flight number increases occur, as the fuel weight increases at the expense of payload weight per flight. A 20% increase or decrease in SFC thus produces changes of + 28.2% and − 23.8% in operating CO 2 eq emission, respectively. For both OEW and SFC, the larger upward sensitivity is due to the low CI payload, as was the case for financial cost sensitivity.
As the CO 2 eq emission of CI is significant, achieving a low OEW and fuel consumption is highly desirable for this scenario. This is also important for the FI and OFI scenarios, for which fuel makes up a larger fraction of the operating CO 2 eq emission. However, in FI and OFI, the relative impact is small compared with other greenhouse gas sources.
Sensitivity to combined uncertainties
The sensitivities presented in the previous section were evaluated by considering uncertainties in each input parameter separately. This is appropriate for most cases, as the effects of uncertainties in the majority of the parameters are independent of each other (e.g. those due to fuel price and aerosol Mt yr −1 ). However, determining the sensitivities to combined uncertainties in OEW and SFC is more complex, as their effects are strongly coupled via the flight profile optimisation procedure. Therefore, Fig. 5 also includes estimates of the sensitivities to simultaneous 10% and 20% changes to OEW and SFC, respectively. The plotted bar shows the extremes, when both changes have the same sign.
Lower OEW and SFC lead to financial cost and CO 2 eq reductions that do not exceed the sum of their individual effects. This is because this scenario simultaneously exchanges aircraft empty weight for payload and allows longer ranges to be flown at the same fuel weight. Yet, the increasing range required for increasing payload at constant DR limits cost and emission decreases by increasing fuel required. Hence, the effect of the combined variation is moderate.
Combined increases in OEW and SFC are more problematic, as the payload per flight becomes so low that flight numbers and fleet size become very high. Fleet size and flight number increases are causative of the major part of cost variations and operating CO 2 eq variations, whereas the increase in initial CO 2 eq is primarily due to the higher number of airports required. The amplification effects are considerable, leading to > 100% increases in initial and operation costs and CO 2 eq. These are approximately double the summed effects of individual OEW and SFC variations. As the aircraft configuration in part 1 was designed using validated models, it is unlikely that the actual variations in OEW and SFC will exceed a few percent. However, it will be highly important that the final aircraft design process monitors these variations closely.
Conclusions and recommendations
This paper has presented the financial cost and CO 2 eq emission analyses for the relatively detailed SAI delivery system developed in part 1 of this series. These have been performed for scenarios considering the direct injection of H 2 SO 4 , as well as SO 2 injection. The SO 2 injection scenario is relatively simple, as its financial cost and CO 2 eq emission are primarily determined by the number of flights and aircraft required and are thus minimised when payload per flight is maximised. The situation is more complex for direct H 2 SO 4 injection, which requires specific combinations of initial H 2 SO 4 concentration and plume diffusivity to produce favourably sized aerosol particles. The results demonstrate that the financial cost and CO 2 eq emission of direct H 2 SO 4 injection decrease strongly with increasing dispersion rate (DR).
High DR allows shorter flights that carry more payload, reducing the required fleet size and fuel consumption to achieve a given delivery target. As fleet size is the single largest contributor to financial cost, and fleet size and fuel consumption drive CO 2 eq emission, maximising DR minimises both. This is illustrated by three H 2 SO 4 injection scenarios with varying assumptions on engine exhaust injection volume and diffusivity. For the very conservative CI scenario, cost and emissions are relatively high, 412 and 153 B USD for initial and annual operating financial costs, and 13.3 and 366 Mt CO 2 eq for initial and annual operating CO 2 eq emissions. For the likely achievable FI scenario, this drops to 124 and 35.5 B USD, with 10.7 and 64.1 Mt CO 2 eq. For the more optimistic OFI scenario, the numbers become quite low: 80.1 and 20.3 B USD with 10.3 and 25.2 Mt CO 2 eq. The values for this latter H 2 SO 4 scenario appear comparable with those associated with SO 2 injection, under the assumption that SO 2 injection requires twice as much annual S delivery. An analysis of realistic value ranges for uncertain input parameters indicates that the above conclusions are robust.
The strong sensitivity of financial cost and CO 2 eq emission to DR motivates a more detailed treatment of the assumptions underpinning our direct H 2 SO 4 injection scenarios. In particular, a more detailed understanding of aerosol growth in realistic aircraft plumes, what levels of diffusivity can be attained in such plumes by engineering and the resulting impact on achievable DR, could narrow the uncertainty margins in these numbers considerably. This may in turn better inform trade-offs between direct H 2 SO 4 injection and SO 2 injection.
Although only part of the foreseen and unforeseen effects of SAI are covered in this study, the anticipated financial cost and CO 2 eq emissions of developing and operating a fleet of specialised aircraft for direct H 2 SO 4 injection can be considered to be manageable, and will likely be substantially outweighed by SAI's intended economic and environmental effects.
in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommonshorg/licenses/by/4.0/. | 2020-05-06T14:49:38.758Z | 2020-05-06T00:00:00.000 | {
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260753717 | pes2o/s2orc | v3-fos-license | P1018: UPDATED RESULTS FROM MANIFEST ARM 2: EFFICACY AND SAFETY OF PELABRESIB (CPI-0610) AS ADD-ON TO RUXOLITINIB IN MYELOFIBROSIS
Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis, splenomegaly and cytopenias. Progressive BM fibrosis results from aberrant megakaryopoiesis and proinflammatory cytokine expression, processes that are regulated by BET protein-mediated expression of genes. The current standard of care for MF is
Background:
Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis, splenomegaly and cytopenias. Progressive BM fibrosis results from aberrant megakaryopoiesis and proinflammatory cytokine expression, processes that are regulated by BET protein-mediated expression of genes. The current standard of care for MF is treatment with Janus kinase inhibitors (JAKis) such as ruxolitinib (RUX). However, unmet need persists due to the limited depth and durability of responses with JAKi monotherapy, high rates of discontinuation and toxicities (Tefferi A. Am J Hematol 2021;96(1):145-162). Pelabresib (CPI-0610; PELA) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κ B signaling and other genes involved in MF disease pathways. PELA is under investigation as monotherapy and in combination with RUX in pts with MF in the ongoing, open-label Phase 2 MANIFEST study (NCT02158858).
Aims:
To present updated results from Arm 2 of the MANIFEST study on the efficacy and safety of PELA as 'add-on' to ongoing RUX in pts with suboptimal/lost response to RUX.
Methods:
Pts received oral PELA 125 mg QD in 21-day cycles combined with continuous oral RUX (at dose taken at time of screening). Pts in Arm 2 are stratified as transfusion dependent (TD) and nontransfusion dependent (non-TD). The primary endpoints are conversion from TD to transfusion independence (TI) in the TD cohort and ≥35% reduction in spleen volume from baseline (BL; SVR35) at Wk 24 in the non-TD cohort. Secondary endpoints include ≥50% total symptom score reduction from BL (TSS50) per MF Symptom Assessment Form v4.0 at Wk 24. Durability of responses, Hb changes, BM biopsies and safety were evaluated.
TD to TI conversion rate was 36.8% (14/38). Median TI duration was 37 wks (range 15-192). In the non-TD cohort, 24% (7/29) of pts had a Hb response (mean Hb increase of ≥1.5 g/dL from baseline without transfusions over 12 wks). TSS50 at Wk 24 was 38% (32/85) overall. A total of 55% (47/85) of pts had TSS50 at any time. Median TSS reduction was 48% at Wk 24. BM fibrosis improvement by ≥1 grade was observed in 26% of pts after 24 wks by central pathology review; 54% of pts maintained the improvement at the next available assessment or longer and 35% had ≥1 grade improvement at any time (best response).
Conclusion:
Conclusion: Treatment with PELA as add-on to RUX in pts with a suboptimal/lost response to RUX monotherapy resulted in durable and deepening splenic and symptom responses, and was generally well tolerated. Transfusion independence was achieved in 36.8% of pts, and improvement in BM fibrosis, as a marker of disease modification, was observed in 35% of pts. | 2023-08-10T15:02:18.354Z | 2023-08-01T00:00:00.000 | {
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237956847 | pes2o/s2orc | v3-fos-license | Economic Sustainability of Small-Scale Hydroelectric Plants on a National Scale—The Italian Case Study
: The feasibility of hydroelectric plants depends on a variety of factors: water resource regime, geographical, geological and environmental context, available technology, construction cost, and economic value of the energy produced. Choices for the building or renewal of hydroelectric plants should be based on a forecast of the future trend of these factors at least during the projected lifespan of the system. In focusing on the economic value of the energy produced, this paper examines its influence on the feasibility of hydroelectric plants. This analysis, referred to as the Italian case, is based on three different phases: (i) the economic sustainability of small-scale hydroelectric plants under a minimum price guaranteed to the hydroelectric operator; (ii) an estimate of the incentives for reaching the thresholds of “acceptability” and “bankability” of the investment; (iii) an analysis of the results obtained in the previous phases using a model of the evolution of the electricity price over the 2014–2100 period. With reference to the Italian case, the analysis suggests that, to maintain the attractiveness of the sector, it is necessary to safeguard the access to a minimum guaranteed price. With the current tariff plan, complete sustainability is only achieved for plants with p ≤ 100 kW. For the remaining sizes, investments under current conditions would not be profitable. The extension of minimum guaranteed prices could make new medium-large plants (500–1000 kW) more attractive. The current incentive policy is not effective for the development of plants larger than 250 kW, as systems with lower capital expenditures are preferred. Uncertainty about the evolution of the price of energy over time is a concern for the sector; the use of evolutionary models of technical economic analysis tried to reduce these criticalities, and it was shown that they can be transformed into opportunities. It was also found that profitability due to the growing trend expected for the price of energy cannot be highlighted by a traditional analysis.
Introduction
Hydropower accounts for about 20% of worldwide electrical power production, with a higher percentage in mountain regions [1]. It is a clean source of energy as well as an economic resource for regions rich in usable water. Hydroelectric production is managed mainly according to water availability and the selling price of electricity [2]. Electricity demand and price generally depend on societal and economic development, but they are also subject to changes related to weather variability, of both a seasonal and long-term type (e.g., variation due to climate change [3,4]). Water availability depends largely on climatic and hydrological conditions and, therefore, can have significant variations in both space and time [5].
While seasonal and year-to-year variability of river runoff has always been taken into account in the past, the recent concern is related to the effect of climate change on the productivity of hydroelectric plants. This effect is related not only to precipitation volumes and time patterns, but also to evapotranspiration and consequently to average paid, it produces more energy than other sources, since its useful life is much longer. Hydroelectric plants have a much higher investment cost than other energy production technologies, but operating costs are a lot lower since no type of fuel is required, which is often the most important cost component. The relationship between the energy produced during the useful life and the energy consumed to build, install, and dispose of it at the end of its life is of an order of magnitude higher than that for other types of renewable sources. Moreover, this relationship ensures the security of the energy supply since hydroelectricity is the only programmable renewable source; reservoir plants produce only when necessary, thus stabilizing the National Transport Network and allowing the Italian electricity system to adapt to our consumption hour by hour. Even run-of-the river plants have a predictable production in the short term, and therefore they have a qualitatively better role in the energy system than wind power or photovoltaic plants. In any case, there are two main critical issues that undermine the economy of an investment in hydroelectric plants: the cost of water and the lack of economies of scale since the plants are tailor-made for their respective sites. The design of small-scale hydroelectric plants is a challenge involving several factors: hydrological, technological, environmental, and social. Moreover, each plant must undergo to a strict and selective authorization process, facing the regulatory uncertainties regarding possible incentives.
The objective of the following study is to analyze the economic sustainability of small-scale river hydroelectric plants with a power concession of up to 1 MW in the current regulatory context, in order to provide planners, legislators, and stakeholders with reflections that are useful for the transition to a new configuration of incentive mechanisms. The choice of focusing on small-scale hydroelectric plants is due to the peculiarity of the territory. The Alps and the Apennines are completely saturated, and it is impossible to build large systems.
Materials and Methods
The study is divided into three phases. The first is an analysis of the economic sustainability of hydroelectric plants with power concession up to 1000 kW in the absence of incentives for the first 1,500,000 kWh produced. Particular attention is paid to the incidence of water concession fees on the economic evaluation of the investment. The second phase is an estimate of the value of the incentive needed to achieve economic sustainability for hydroelectric plants, compared to the investment "acceptability" and "bankability" thresholds typical for these types of plants. The last phase consists of an evaluation of the sustainability of the plants in the complicated context of climate change, with reference to the most influential factors that govern the phenomenon in such a way as to offer the most reliable and truthful forecast possible. The aim is to understand whether the incentives can be seen to be a shock absorber capable of effectively meeting the economic needs of the hydroelectric sector, in order to ensure that the latter remain strategic in the Italian production system.
To evaluate the economic suitability of small-scale hydroelectric plants, two levels of feasibility were adopted: (a) Economic feasibility: This is determined by the profitability rate, that is, the internal rate of return (IRR), between 7% and 9%. This represents a profitability range typically considered acceptable by the entity that promotes the investment. (b) Banking feasibility: Rates higher than 9% on average are considered acceptable by credit institutions to guarantee the bankability of a hydroelectric project.
An economic analysis of the suitability of small hydroelectric plants can be conducted using different methods. The simplest is to compare the relationship between the total investment and the installed power or the ratio between total investment and annual energy yield. These criteria do not identify the value for money of the systems since the revenues are not considered; they can only be used to obtain general indications on the investment. In this study, the net present value (NPV) methodology was used. This makes it possible to obtain a faithful estimate of the profitability of the project by estimating the IRR. The NPV is simply the difference between cash inflows and outflows, throughout the duration of the investment, both discounted at a rate called the discount rate [19][20][21]. The formula to calculate the NPV [22], given the condition that the cash flows occur at regular time intervals, is: where I i is capital expenditures in period i, R i is cash inflow in period i, O i is operating cash costs in period i, M i is maintenance and repair cash costs in period i, and V r is current residual value of the investment at the end of its lifetime, r is the discount rate or opportunity cost of capital, and n is the number of periods considered.
Only projects with positive NPVs can be considered acceptable. The IRR indicates the rate of return expected from an investment: the higher the IRR is, the better value-formoney the investment represents. The limit condition is as follows:
Case Study
In Italy there are 3700 hydroelectric plants, and they achieve a total production of 42.4 TWh, which is about 14% of the country's production [19]. Most such plants are small and have marginal influence. In 2015, 77% of Italian hydropower was produced by plants with power >10 MW, covering merely 17% of the total electricity production. The distribution of hydroelectric plants is quite heterogeneous, as well as the density of national installed power. Figure 1 shows a map of Italian maximum annual hydroelectric production. The largest number of plants in Italy is in the northern regions with very high percentages in Piedmont, Lombardy, and Trentino Alto-Adige. According to data provided by the GSE (year 2016), over 55% of all the plants are installed in these three regions alone, with a considerable density in Piedmont and Lombardy where there are 36% of all the plants installed, which generate a total of 42% of the national installed hydroelectric power [19]. Lombardy was chosen as a case study, given the high presence of hydroelectric plants and because it is a virtuous example in terms of the canons of concession for small plants. Consequently, if the results were not advantageous for these types of plants and in this context where the ferment and private initiative are masters, they would not be advantageous in other regions either, where the overall rents are higher. In the calculation, the following standard sizes were used: 100, 250, 500, and 1000 kW, all of which are in the category of microhydroelectric plants.
The Italian Tariff System
One of the main concerns regarding the economic sustainability of hydroelectric plants in Italy is that while the average energy price is constantly decreasing (i.e., about 20% less than that in 2012), the sum of the water concession fees/surcharges continues to rise; in some regions there was an increase in taxes related to the use of water resources by local authorities by almost 160% [23]. Figure 2 shows the growing trend of water concession fees in recent years for the two regions "at the extreme" as regards the values for small plants in comparison to the national single price (PUN). After the peak of 2012, the PUN went down (−17% overall from 2009 to 2018), while the water concession fees, especially in Piedmont, increased significantly (+159% from 2009 to 2018). The increase in Lombardy is less marked and more gradual (+12% in the period 2009-2019). The trend is rather disconcerting, considering that the fees are fixed costs for the hydroelectric operator and are not related to either the economic value of production or actual water availability [23]. The water concession fee varies according to the region, and at above a predefined threshold (220 kW), it is necessary to pay surcharges as defined by a national standard. Table 1 shows an example of annual water fees for Lombardy. Table 1. Annual water fees for Lombardy [23].
Annual Water Fees In Lombardy
Water concession fee 16 The investment in a small hydroelectric plant involves several payments distributed over the life of the project and provides incomes, also distributed over time. Outputs include a fixed component such as the cost of capital, insurance, taxes other than income taxes, etc., and a variable component represented by operating expenses and maintenance, which are costs that absolutely cannot be ignored for a correct assessment of economic profitability and above all with a view to efficient operation throughout its useful life. At the end of the project, which generally coincides with the duration of the concession, the residual value should be positive. The sale price of the energy produced is defined through the so-called PMGs (minimum guaranteed prices) or according to a simplified tariff mechanism that allows producers to sell the electricity fed into the grid, transferring it directly to the GSE who remunerate them for it based on precise and variable rates every year, paying a price for each kilowatt hour drawn. In this economic model, it is assumed that the PMGs are constant for the entire duration of the plant s concession. The choice is dictated firstly by the awareness that in the last ten years these rates have remained virtually unchanged and secondly by the desire to recognize them as being of more and more strategic importance to support the sector; in this way, the study can constitute a wellfounded alternative for a possible future proposal of legislation devoted to environmental protection. Tables 2 and 3 show PMGs until 2013 and current PMGs (2019) respectively. Table 2. Minimum guaranteed prices in 2013 [23].
Guaranteed Minimum Prices (EUR/MWh) 2019
0-250,000 (kWh) 156.1 250,000-500,000 (kWh) 107.2 500,000-1,000,000 (kWh) 67.7 1,000,000-1,500,000 (kWh) 58.5 Table 4 shows the incentive plans proposed in recent years for flowing water systems; in the last decade, the incentive has dropped by about 30%. The incentive tariffs provided for medium-large sized plants underwent a percentage decrease in the incentive tariffs higher than that for all the other sizes.
The Economic Value of Energy Produced
Several models aimed at estimating the electricity prices were recently introduced in Europe in the wake of the liberalization of the energy market at the end of the 1990s. Such liberalization led to the country-wise definition of the free market [27]. In Italy, Legislative Decree 79/1999 allowed for such liberalization. Energy prices set after the energy liberalization are made available continuously by the Energy Markets Operator (GME), an Italian authority with the mission of promoting the development of a national competitive electricity system, according to the criteria of neutrality, transparency, and objectivity. Competition in the electricity market is guaranteed by the Borsa Elettrica, an electricity stock market. It promotes the application of efficient equilibrium prices, allowing for the sale and purchase of electricity based on greater economic convenience. It is organized as a real physical market, with the definition of sales and purchases through hourly charts, according to the criterion of economic merit. This consists of considering the prices in increasing order for sales and the prices in decreasing order for purchases. Price definition takes place as in a physical market, by matching supply and demand. Electricity offers are accepted in order of economic merit, i.e., in order of increasing price, until their sum in terms of kWh completely meets the demand. The kWh price of the last accepted bidder, i.e., the one with the highest price, is attributed to all offers, and according to European Directive 2009/28, renewable energies, such as hydropower, have priority in terms of access to the market. In so doing, in each zone of the Italian territory with given technical constraints, the equilibrium prices are defined, i.e., those that are found at the intersection of the supply and demand curves. Subsequently, the PUN is established by GME. The economic value of electricity is difficult to express by means of a relationship between the independent variables, even only at a national level. These can be physical, economic, social, and political variables and are therefore all specific to a sociopolitical context, generally referable to a national scale. In literature, some models were proposed for the economic value of electricity linked to more general quantities that are useful if future projections are to be made [28,29]; among them there are multi-agent models [30], parametric models [31], stochastic models [32][33][34], and computational models [35]. In addition, hybrid, or mixed, models have also been developed [3,4,[36][37][38][39]. The availability of water resources in the coming years, strongly influenced by climatic changes [28], is expected to greatly influence hydroelectric production and, consequently, the economic value of energy produced. Moreover, hydroelectric energy is greatly affected by weather conditions; its productivity can be subject to significant seasonal and annual variations. The climatic conditions affect the hydrological cycle, the energy demand, and the price of electricity. Bombelli et al. [3,4] investigated how hydroelectric production is influenced by the climate, the fluctuations in demand, and price constraints, and in doing so they extrapolated a hypothetical trend of electricity price up to the end of the 21st century, from three global climate models of the IPCC AR5, RCP2.6, RCP4.5, and RCP8.5 ( Figure 3). The method was applied to the case study of the Italian electricity market, showing acceptable capacity for modelling recently observed price fluctuations. The average annual price of energy is expected to undergo a significant increase over the 87 years analyzed, going from around 64 EUR/MWh to around 169 EUR/MWh. The immediate outcome of such a projection is the increase in expectations of the entire hydroelectric sector. If currently the economic sustainability of a hydroelectric project cannot be separated from incentive policies, such a scenario may instead reserve the possibility of investing in the sector without the need to rely on subsidized tariffs.
Phase 1
In phase 1 the economic sustainability of hydroelectric plants with concession power up to 1000 kW in the absence of incentives and access to the PMG for the first 1,500,000 kWh produced was analyzed. The energy exceeding the PMG threshold is sold at the market price, that is, the average value of the last 5 years of the PUN on the day before market, was assumed. Moreover, particular attention was paid to the incidence of water concession fees on the economic evaluation of the investment. In calculating, energy was weighted by a reduction coefficient equal to 0.85 to consider that a plant is not always at its maximum potential due to periods of inactivity or other external factors that the operator cannot exclude. These could be periods of drought (in which the plant runs at reduced power) or periods of full extremes (in which it runs at maximum power or is stopped). From this point of view, the resulting economic simulation of the profitability of a plant is certainly more reliable and representative of reality because it considers the unpredictability. Furthermore, according to this logic, the average hours of operation considered are effective (nonoperating hours for maintenance excluded). The calculation is usually carried out over 30 years because due to the discounting, both expenses and income weigh shortly after many years. This aspect, which could be considered a "limit" of this economic model, is not, however, influential in this analysis since it is customary to consider a duration equal to the period of concession of the plants, which in the greatest number of cases is around 20-30 years. Figure 4 shows the trend of the IRR varying the average annual hours of operation between 3000 and 8000 h and the CAPEX (capital expenditure) between 4000 and 7000 EUR/kW for sizes of 100, 250, and 500 kW, and between 3000 and 6000 EUR/kW for size 1000 kW. Average OPEX (operating expenses) of 125 EUR/kW for the size of 100 kW and of 115 EUR/kW for the others and an annual inflation rate of 1% were assumed. In each graph, two areas were highlighted to mark the investment acceptability threshold in orange (for profitability rates between 7% and 9%) and the investment convenience threshold in green (for rates higher than 9%). Although the average operation of the entire national hydroelectric park, including the reservoir plants, is equal to 3370 h/year, the plants analyzed are generally characterized by greater hours of operation; this is because little run-of-river systems, which guarantee a more persistent functioning throughout the year that easily reaches 6000-7000 h/year, were considered. The IRR of the investment is less than 7% for almost all cases, except the first. The rate worsening as the size increases is substantially due to the lesser relevance of the PMG provided for plants with a concession power less than 1000 kW, within the first 1,500,000 kWh produced. Only the smaller plants can take advantage of these subsidized prices for all the produced energy, while the larger ones must operate mainly on the market price. As shown in Figure 4, a 100-kW system is the only interesting one: it achieves a threshold of acceptability for a CAPEX of 4000 EUR/kW, 5000 EUR/kW and 6000 EUR/kW respectively after 4500, 5500 and 6500 h. For plants with an installed power of 250 kW, the area of acceptability is only slightly crossed with a CAPEX of 4000 EUR/kW. Systems with the most expensive installation costs (CAPEX equal to 6000 and 7000 EUR/kW) achieve a very low IRR. When a plant exceeds 5,000,000 kWh of energy production, according to PMG 2019, the price of energy undergoes a sharp decrease of approximately 37%, from 107.2 to 67.6 EUR/MWh. The cash flow is therefore gradually reduced as the production and consequently also the IRR increase. As shown in Figure 4c,d, the situation worsens for plants of 500 and 1000 kW, as these can reach very high productions, of about 3,000,000 and 7,000,000 kWh, respectively. In such cases, the effort to use substantial resources for development is not rewarded at all by the high productions because the subsidized tariff plan does not reward them. The simulation concerning the 1000 kW system has the lowest CAPEX, i.e., between 3000 and 6000 EUR/kW, in order to consider the scale factor; unit installation costs that are too high would lead to a hugely unreliable investment. The rents (in which royalties are also considered) have a strong impact on the profitability of the plant: these represent an average annual cost equal to approximately 6% of the revenues for a 100 kW plant, also exempt from the payment of mountain watershed and local authorities, between 9% and 12% for a 250 kW plant, between 15% and 17% for the 500 kW case, and between 19% and 22% for a 1000 kW system. If incentive tariffs on the same cases were applied, any plant size would become economically sustainable, even hitting profitability peaks of over 25%. Figure 5 shows results for the following incentives: 0.219 EUR/kWh for 250 kW plants (Figure 5a), 0.179 EUR/kWh for 500 kW plants (Figure 5b), and 0.1561 EUR/kWh for 1000 kW plants (Figure 5c). To consider the scale factor, the rate was discounted as the size increases. Despite this, all lines intersect the areas of acceptability and convenience.
In Figure 5, only the minimum and maximum CAPEX were represented. The IRR achieves the profitability threshold for all combinations even for the highest CAPEXs. This proves that, in a regulatory context that supports the sector with advantageous tariffs, there could be significant sustainable development rates for all plant sizes without further concerns about investment attractiveness. Newly built plants to which the PMGs are applied do not appear to be economically sustainable, according to the current thresholds. They were extended to up to 2,000,000 kWh to highlight the positive influence produced by GMPs, especially for high hours of operation. Given the low price guaranteed for the last bracket, i.e., between 1,000,000 kWh and 1,500,000 kWh, instead of increasing the latter, it was decided to add the 500,000 additional kWh that is missing to reach 2,000,000 kWh in equal measure (i.e., 250,000 kWh each) in the two central brackets. For all plant sizes, the curves at CAPEX 4000 EUR/kW were compared with two alternative tariff plans: the tariff plans of PMGs 2013, which show more favorable prices, and PMGs 2019, with an extended PMG to 2,000,000 kWh ( Figure 6). For all plant sizes, both alternatives involve advantages, except for the minimum size of 100 kW, which is characterized by low energy production that does not benefit from the extension of the PMGs or from the increase in prices. With reference to the PMG 2013, there is an increase of profitability of approximately 21%, 40%, and 46% in plants with installed power equal to 250 kW, 500 kW, and 1000 kW, respectively, while with the PMG 2019, slightly lower percentages were recorded, i.e., about 17%, 18%, and 23% for the same plant sizes.
In conclusion, the PMGs that are enforced only ensure complete profitability for the smallest plants size, i.e., with an installed power of less than or equal to 100 kW, whose OPEX is not compensated by the energy sale revenue at market price. For the other sizes, there is a deterioration in the profitability of investments as the installed power increases; this is due to the lower impact of the PMG. To make the development of new medium-large plants more attractive, an extension of the PMGs is needed.
Phase 2-Remodulation of the Tariff Plan
The aim of this phase is to identify a so-called feed in tariff (FIT) to economically support the microhydroelectric sector in order to guarantee the maintenance of minimum plant profitability. Feed-in tariffs (FIT) are fixed electricity prices that are paid to renewable energy producers for each unit of energy produced and injected into the electricity grid. Several countries introduced this kind of energy policy [40][41][42][43][44][45][46]. The FIT provided by the DM 4 July 2019 was 0.080 EUR/kWh, FIT provided by DM 6 July 2012 and DM 23 June 2016 were equal to 0.155 EUR/kWh and 0.150 EUR/kWh, respectively. The same thresholds of investment acceptability and bankability as phase 1 were adopted. In this case, the cash flows were also discounted, assuming that the lifespan of the hydroelectric project is 30 years and that the energy produced was sold to the PUN. Figures 7-10 show the value of FIT to reach an IRR equal to the two pre-established thresholds (7% and 9%) for a plant size of 100 kW, 250 kW, 500 kW, and 1000 kW, with the hours of operation and the CAPEX varied. The current FIT (DM 4th July 2019) is suitable for guaranteeing a net economic return of 7% but not to reach 9%, while the FIT of DM 6 July 2012 and DM 23 June 2016 would be sufficient for both profitability thresholds. The FIT to reach an IRR of 7% and 9%, when averaging the values associated with the various CAPEX values and assuming 6000 h of operation, would be 0.129 EUR/kWh and 0.208 EUR/kWh, respectively. Figure 8 shows some improvement for the bankability threshold: the FIT of DM 4th July 2019 (0.155 EUR/kWh) makes it possible to reach a profitability for all systems of this size with an installation cost of between 4000 and 5000 EUR/kW. Higher CAPEXs are unsustainable for this incentive rate. The FIT that would be necessary to reach an IRR of 7% and 9%, when averaging values of different CAPEX values and assuming 6000 h of operation, would be 0.125 EUR/kWh and 0.168 EUR/kWh, respectively.
With the increase in installed power, the remuneration conferred by the DM 4th July 2019 fell to 0.110 EUR/kWh with significant repercussions on the economic sustainability of a hydroelectric project (Figure 9). The minimum profitability limit is not guaranteed for an IRR of 7% (for a CAPEX of 6000 and 7000 EUR/kW) or for an IRR of 9%. The FIT to reach these profitability thresholds, when averaging values of different CAPEX values and assuming 6000 h of operation, would be 0.124 EUR/kWh and 0.155 EUR/kWh respectively.
For 1000 kW systems, the FIT provided by the DM 4 July 2019 is insufficient to guarantee an economic sustainability of 7% and 9% ( Figure 10). On the contrary, the FIT established by the previous ministerial decrees, i.e., DM 6 July 2012 and DM 23 June 2016, would be sufficient for both profitability thresholds. The FIT to reach these profitability thresholds, when averaging of the values of different CAPEX and assuming 6000 h of operation, would be 0.107 EUR/kWh and 0.127 EUR/kWh, respectively. With the FIT guaranteed by the DM 23 June 2016, almost all plants reach acceptable yields at around 6000 h of operation, unlike that with the last tariff plan (DM 4 July 2019). Only systems with installed power less than or equal to 250 kW (for any CAPEX) and those of 500 kW for small CAPEX (about 4000 EUR/kW) guarantee the minimum profitability of 7% when calculating for the same number of operating hours. All other combinations do not guarantee the achievement of minimum economic sustainability. Table 5 summarizes the values of the FIT resulting from the analysis. The current FIT (DM 4 July 2019) is insufficient for the development of plants larger than 250 kW. While in the past the incentive policies allowed for an across-the-board development of a microhydroelectric plant that affects all sizes, today they are excessively restrictive, and they risk paralyzing not only the increase in total installed power but also any technological development.
Phase 3-Economic Sustainability of Microhydroelectric Plants in the Period 2014-2100
The evaluation of the effect of the price of energy on production is important when studying the profitability and benefits associated with energy systems. As previously discussed, the demand and the price of electricity depend not only on economic and social developments, but they may also be subject to seasonal variability and other medium-long term variations due to climate change. With reference to the model in Figure 3, the benefit that an increase in electricity price would bring to the economic profitability of small-scale hydroelectric plants was explored. Simulations were performed by applying the NPV methodology; the cash flows to discount over 30 years of the investment life were updated from year to year according to the kilowatt hours of energy produced and the average annual price. Two different analyses were carried out: the first compares the performance of hydroelectric projects undertaken in two opposing scenarios, the second investigates the evolution of the IRR from 2020 to 2070.
Analysis 1: Comparison of Performance for Two Opposite Scenarios
For each plant size, the trend of the IRR for two opposite scenarios, characterized by regimes of electricity prices at the extremes of the pre-established temporal projection (periods 2020-2049 and 2070-2099, respectively) were compared. Consistently with previous phases, CAPEX was varied from 4000 to 7000 EUR/kW for plants with an installed power of 100 kW, 250 kW, 500 kW, and from 3000 to 6000 EUR/kW for 1000 kW systems. To consider the uncertainty linked to future changes in water concession fees from now up to 2100 in calculations, two distinct criteria were adopted: Approach 1: Relying on the trend in the average annual energy price according to the projection in Figure 3, the NPV was estimated by calculating the updated cash flows from year to year according to the electricity price for the current year.
Approach 2: The calculation of the NPV was developed by assuming the price of electricity to be constant over time, so that all cash flows calculated for the entire useful life of the investment are equal. Figures 11 and 12 compare the results of the two approaches. With reference to Approach 1, The IRR at 6000 h of operation for the period 2020-2049 never reaches profitability values equal to or greater than 7% for any CAPEX. Considering the scenario 2070-2099, the IRR always exceed the 7% threshold, up to reaching values that exceed the minimum investment convenience limit (9%) for a CAPEX below 6000 EUR/kW. The different profitability between the two stages is underlined if the situation is analyzed at 7000 average hours of operation: while in the first scenario the IRR reaches acceptable values only for installation costs equal to 4000 EUR/kW, in the second scenario the IRR exceeds 9% for all types of investments (CAPEX of 4000, 5000, 6000, and 7000 EUR/kW). Considering a system characterized by an installed power of 1000 kW, both scenarios benefit from a more favorable IRR. The average annual production of 6000 h in the 2020-2049 scenario exceeds the minimum limit of 7% only with the minimum CAPEX, while in the 2070-2099 scenario all the IRRs record a profitability of over 9%, with values even close to 18%. The same trend occurs for 7000 average hours of production: while in Scenario 1 acceptable investments are achieved only for a CAPEX of 3000 EUR/kW and 4000 EUR/kW, in Scenario 2 all the CAPEXs exceed the convenience threshold of 9%. The positive effect on IRR of the increase in electricity prices over the course of the century is significant and reflects the expectations of the sector. On average, the increase recorded between Scenario 1 and Scenario 2, at the threshold of 6000 average hours of operation, even exceeds 100%; in other words, the profitability is doubled. Comparing results from Approach 1 and Approach 2, in all simulations the difference of IRR is appreciable. Over the useful life of a hydroelectric plant operating in the free market, the factor with the most relevant specific weight for its profitability is the price of energy. To consider the future, the evolution of the price of electricity is important for evaluating the suitability and reliability of an investment.
Analysis 2: IRR Evolution over the Period 2020-2070
The aim is to trace the trend of the investment profitability over the years, calculating the IRR by discounting the cash flows envisioned by the NPV methodology over the entire useful life of the system and associating the IRR to the year in which the investment is undertaken. For each year, the resulting curve indicates the percentage of the IRR of the investment, calculated by discounting the cash flows over the next 30 years (i.e., the value of profitability associated with the year 2032 is the result of the economic analysis carried out using the NPV method considering the cash flows in the years 2032-2061 and the corresponding energy prices). The simulations were carried out sequentially from 2020 to 2070. For each case study (i.e., 100 kW, 250 kW, 500 kW, 1000 kW), the trends of the IRR corresponding to the minimum and maximum CAPEX were calculated in order to highlight the range within which the investment profitability of similar projects can fluctuate ( Figure 13). Between one unit's installation cost and another, there is a difference ranging from 3 to 5 percentage points at the beginning of the projection (2020) and from 8 to 10 percentage points at the end of the projection (2070). The IRR increases with an almost linear trend, as expected from the trend in the price of electricity. Considering the maximum CAPEX of 7000 EUR/kW, starting from an IRR of approximately 1%, within 50 years an IRR of almost 8% is reached; considering the minimum CAPEX of 4000 EUR/kW, the initial IRR settles at around 5% and the final one at around 12.5%. The influence of the CAPEX on the marginal growth of the IRR was confirmed. Even in the case of a 1000 kW system, the IRR trend continues to increase. However, the marginal growth of this parameter is different for the two CAPEX cases considered. For the minimum CAPEX (3000 EUR/kW), the IRR recorded in 2020 is about 7.5%, while in 2070 it settles at around 18%. For the maximum CAPEX (EUR 6000/kW), the IRR starts from a value of 1% and increases to just over 8%. In the period 2020-2070, the increase of the IRR for the maximum CAPEX is approximately 7%, and for the minimum CAPEX it is greater (about 11%). This trend can be traced back to the economic investment costs associated with the size of the plant: for the same installed power and for the maximum sizes of the microhydroelectric plant, the CAPEX assumes a greater specific weight in the projection of the IRR. For the smaller CAPEX, there is a marginal increase in the IRR, higher than the value for the same unit installation cost, for the smaller sizes. Relating the feasibility analysis of a hydroelectric project to an accurate model of future projections of the value of electricity is certainly a more truthful approach, despite the uncertainties connected with this kind of model.
Conclusions
In this study, the evolution of the profitability of an investment, according to the plant sizes and the economic context, was analyzed for the Italian scenario, as a function of the tariffs recognized by the hydroelectric energy market, the unit cost of installing the plants, and their average hours of operation. Three phases were distinguished: the first, was characterized by the analysis of the economic sustainability of the microhydroelectric plants under the PMG; in the second, the value of the incentive to reach the thresholds of "acceptability" and "bankability" of the investment, for the same hydroelectric plants as the phase 1, was estimated; in the third, an analysis of the results obtained in the previous phases was conducted using a model of the evolution of the price of electricity for the period 2014-2100. The results obtained suggest that, to maintain the attractiveness of the sector, it is necessary to safeguard access to the PMG. With PMG 2019, complete sustainability is only achieved for plants with P ≤ 100 kW. For the remaining sizes, investments under current conditions would not be profitable. The extension of PMGs could make new medium-large plants (500-1000 kW) more attractive. The current incentive policy (DM 4 July 2019) is not effective for the development of plants larger than 250 kW; systems with lower CAPEX should be preferred. Uncertainty about the evolution of the price of energy over time is a concern for the sector; the use of evolutionary models of technical economic analysis tries to reduce these criticalities and shows that they can be transformed into opportunities. Profitability due to the growing trend expected for the price of energy cannot be highlighted by a traditional analysis.
Conflicts of Interest:
The authors declare no conflict of interest.
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236504132 | pes2o/s2orc | v3-fos-license | The Archaeal Na+/Ca2+ Exchanger (NCX_Mj) as a Model of Ion Transport for the Superfamily of Ca2+/CA Antiporters
The superfamily of Calcium/Cation (Ca2+/CA) antiporters extrude Ca2+ from the cytosol or subcellular compartments in exchange with Na+, K+, H+, Li+, or Mg2+ and thereby provide a key mechanism for Ca2+ signaling and ion homeostasis in biological systems ranging from bacteria to humans. The structure-dynamic determinants of ion selectivity and transport rates remain unclear, although this is of primary physiological significance. Despite wide variances in the ion selectivity and transport rates, the Ca2+/CA proteins share structural motifs, although it remains unclear how the ion recognition/binding is coupled to the ion translocation events. Here, the archaeal Na+/Ca2+ exchanger (NCX_Mj) is considered as a structure-based model that can help to resolve the ion transport mechanisms by using X-ray, HDX-MS, ATR-FTIR, and computational approaches in conjunction with functional analyses of mutants. Accumulating data reveal that the local backbone dynamics at ion-coordinating residues is characteristically constrained in apo NCX_Mj, which may predefine the affinity and stability of ion-bound species in the ground and transition states. The 3Na+ or 1Ca2+ binding to respective sites of NCX_Mj rigidify the backbone dynamics at specific segments, where the ion-dependent compression of the ion-permeating four-helix bundle (TM2, TM3, TM7, and TM8) induces the sliding of the two-helix cluster (TM1/TM6) on the protein surface to switch the OF (outward-facing) and IF (inward-facing) conformations. Taking into account the common structural elements shared by Ca2+/CAs, NCX_Mj may serve as a model for studying the structure-dynamic and functional determinants of ion-coupled alternating access, transport catalysis, and ion selectivity in Ca2+/CA proteins.
INTRODUCTION
Membrane-bound ion transport proteins (channels, transporters, and pumps) can selectively recognize and transport physiologically important cations such as H + , Na + , K + , Ca 2+ , and Mg 2+ in accordance with the physiological requirements of a given cell type and thereby control the vast majority of biochemical reactions in nearly every living cell (Carafoli, 1987;Berridge et al., 2003;Bers 2008;Forrest et al., 2011;Keller et al., 2014). Ion transporters couple the free energy of the transmembrane electrochemical potential of one solute/ion to the transmembrane movement of another (Bahar et al., 1997;Roux et al., 2010;Forrest et al., 2011;Cheng and Bahar, 2019). The basic paradigm underlying the transporters' function was put forward more than 5 decades ago by Jardetzky (1966), who described the alternative access of the substrate (ion) binding sites to either one side of the membrane or the other during the transport cycle (Forrest et al., 2011;Drew and Boudker, 2016). According to this phenomenological model, the membrane-bound transporter protein must undergo at least two major conformational states during the transport cycle, designated as the inward-facing (IF) and outward-facing (OF) conformation states.
Today it is clear that transporters undergo numerous conformational changes, although the structure-dynamic segregation and the characterization of functionally important intermediates remain challenging, even for transporters with known structural details (Roux et al., 2010;Lockless, 2015;Deng and Yan, 2016;Drew and Boudker, 2016;Cheng and Bahar, 2019). Structure-dynamic resolution of the underlying ion transport mechanisms may provide new opportunities for selective and effective pharmacological targeting. The relevant information can provide "game-changing" opportunities to advance the most demanding applications in modern biomedicine and technology, although this requires the development of sophisticated multidisciplinary approaches that combine both advanced experimental and computational approaches (Bahar et al., 2010;Roux et al., 2010;Deng and Yan, 2016;Drew and Boudker, 2016;Cheng and Bahar, 2019;Garibsingh and Schlessinger, 2019).
The aim of the present review article is to summarize our current understanding of the structure-dynamic mechanisms underlying the ion transport mechanisms shared by a huge superfamily of Ca 2+ /Cation (Ca 2+ /CA) exchangers (antiporters). This is achieved by using the structure-based functional model of the archaeal Na + /Ca 2+ exchanger protein derived from Methanococcus jannaschii (NCX_Mj) (Figure 1). Even though the structural model of NCX_Mj provides a basis for investigating the general mechanisms underlying ion transport in NCX and similar proteins, certain limitations exist. For example, it is well known that NCX_Mj (like many other archaeal proteins) possesses a very rigid protein structure; therefore, the structuredynamic features of NCX_Mj should be carefully considered before advocating the general mechanisms of ion transport shared by Ca 2+ /CA proteins. Nevertheless, the structural template of NCX_Mj (Liao et al., 2012;Liao et al., 2016) is instrumental for a structure-based analysis of the ion transport mechanisms in NCX and structurally related proteins (Nishizawa et al., 2013;Waight et al., 2013;Wu et al., 2013;Giladi et al., 2016a;Giladi et al. 2016b;Giladi et al. 2016c;Giladi et al., 2017;Khananshvili 2017).
In general, the membrane-bound Ca 2+ /CA antiporters extrude Ca 2+ in exchange with distinct ions (Na + , K + , H + , Li + , and Mg 2+ , among others) by utilizing the downhill gradient of counter-cation species such as H + , K + , or Na + (Blaustein and Lederer, 1999;Philipson and Nicoll, 2000;Lytton, 2007;Emery et al., 2012). Thus, the structuredynamic model of the archaeal Na + /Ca 2+ exchanger (NCX_Mj) represents a feasible model for studying the iontransport mechanisms, since twelve ion-coordinating residues (forming four binding sites) are highly conserved among Ca 2+ / CAs, while sharing some common structural motifs (Philipson and Nicoll, 2000;Lytton, 2007;Liao et al., 2012;Schnetkamp, 2013;Marinelli, et al., 2014;Khananshvili, 2020) ( Figures 1A,B). Typically, the Ca 2+ /CA proteins contain ten transmembrane helices (TM1-TM10), where two inversely swapped hubs (TM1-TM5 and TM6-TM10) generate an inverted twofold topology with "pseudo-symmetry" (Figures 1A-C). Four transmembrane helices (TM2, TM3, TM7, and TM8) contain highly conserved repeats (α 1 and α 2 ), where the assembly of the inversely banded TM2/TM3 (α 1 ) and TM7/ TM8 (α 2 ) segments form an ion passageway with the ioncoordinating residues (Figures 2B,C,D). The Na + /Ca 2+ exchange cycle involves a separate translocation of the 1Ca 2+ -and 3Na +bound species. Green and red spheres denote the Na + and Ca 2+ ions, respectively, whereas a dashed line denotes the sliding cluster (TM1/TM6). According to this model, the transition state is situated between the two occluded states, where the sliding event of the TM1/TM6 cluster takes place during the transition state and it is associated with the OF/IF swapping in either direction. (B) The backbone dynamics of the four ion-coordinating helices of NCX_Mj are represented by the overlay of the HDX-MS hit maps (Giladi et al., 2017) on the crystal structure of 3Na + -bound NCX_Mj (5HXE). The arrow direction indicates the increased rigidity of the backbone dynamics (the color key shows the percentage of deuterium incorporation). Notably, the backbone dynamics of TM2B is much more constrained than the other ioncoordinating segments (TM2C, TM7B, TM7C, and TM8A). Thus, the helix-breaking residues (G49, P53, G208, and P212) belonging to the GTSLPE repeats (allocated at the interface of the TM2B/TM2C and TM7B/TM7C segments) may specifically shape the local backbone dynamics at "catalytic" ion-coordinating residues. (C) Singlepoint mutations of residues allocated at TM2B, TM2C, TM3A, TM7B, TM7C, and TM8A differentially affect the rate-equilibrium relationships of bidirectional ion movements (Giladi et al., 2016a;Giladi et al., 2016b;Giladi et al., 2016c;van Dijk et al., 2018). Inversely matched pair residues (shown in the same colors) differentially affect the ion transport activities, thereby suggesting that the functional asymmetry of inversely located pair residues occurs at the single residue level. (Reeves and Hale, 1984;Bers and Ginsburg, 2007;Shlosman et al., 2018). The Na + -or Ca 2+ -bound species are separately translocated across the membrane at different stages of the transport cycle (Khananshvili, 1990;Niggli and Lederer, 1991;Hilgemann et al., 1991). High-resolution crystal structures of NCX_Mj capture the outward-facing (OF) conformation with four putative sites, namely, S ext , S mid , S int , and S Ca , which are separated from each other by ∼4Å, thereby forming a diamondshaped configuration (Liao et al., 2012) (Figures 1E,F). Notably, according to the crystal structure, the S int and S ext sites have high selectivity to Na + , whereas the S Ca and S mid sites show no preferential selectivity to Na + or Ca 2+ . It was initially proposed that the 3Na + ions occupy the S ext , S mid , and S int sites, whereas 1Ca 2+ binds to the S Ca site (Liao et al., 2012). According to this model, NCX_Mj can bind to either the 1Ca 2+ or 3Na + ions at mutually exclusive sites, meaning that the Na + and Ca 2+ ions occupy completely different sites at separate steps of the transport cycle.
The follow-up studies with MD simulations and mutational analyses of ion fluxes put forward a revised model for assigning four binding sites in NCX_Mj (Marinelli et al., 2014;Almagor et al., 2014;Giladi et al., 2016a, Giladi et al., 2016b, Giladi et al., 2016c, Giladi et al., 2017, van Dijk et al., 2018Khananshvil, 2020). According to this model, the 3Na + ions occupy the S int , S ext , and S Ca sites, whereas the 1Ca 2+ ion occupies the S Ca site8 ( Figures 1E,F). Recent crystallographic studies of NCX_Mj further underscored a unique feature of the S Ca site, which is alternatively occupied by the Na + or Ca 2+ ion (Liao et al., 2016). The mutational analyses of the ion-exchange activities are in agreement with the revised model of the binding site assignment Marinelli et al., 2014;van Dijk et al., 2018), HDX-MS (Giladi et al., 2016a;Giladi et al., 2016b;Giladi et al., 2016c;Giladi et al., 2017). Despite this progress, the functional status of the S mid site remains unclear. Interestingly, the MD simulations and X-ray data suggest that the S mid site may become occupied by a water molecule through the protonated D240 in either the ion-bound (3Na + , 2Na + , 2H + , or 1Ca 2+ ) or apo states (Marinelli et al., 2014;Liao et al., 2016).
High-resolution crystal structures of three H + /Ca 2+ exchanger proteins (all belonging to the CAX family) depict the inwardfacing (IF) conformation in the open, semi-open, and occluded states (Nishizawa et al., 2013;Waight et al., 2013;Wu et al., 2013;Lu et al., 2020). According to these structural assignments of the ion-binding sites, the CAX proteins contain the mutually exclusive sites for 1Ca 2+ (S Ca ) or 2H + (S ext and S int ) binding. According to this model, the stoichiometry of the H + /Ca 2+ exchange must be 2H + :1Ca 2+ , although experimental evidence for this stoichiometry is absent. Even though the CAXs and NCX share common structural features, the ion selectivity of S Ca might differ in the CAX and NCX_Mj proteins. Namely, according to the crystal structures of CAXs, the S Ca site binds Ca 2+ (but not H + ), whereas the S Ca site of NCX_Mj can alternatively bind either Ca 2+ or Na + Marinelli et al., 2014;Giladi et al., 2016a;Giladi et al., 2016b;Giladi et al., 2016c;van Dijk et al., 2018).
Since the crystal structures of the NCKX and mitochondrial NCLX remain unavailable, the structural template of the NCX_Mj and CAX proteins could be useful for resolving the underlying mechanisms of ion selectivity and transport. Notably, MD simulations have shown that the occupation of four binding sites by 4Na + is thermodynamically forbidden in NCX_Mj (Marinelli et al., 2014;Liao et al., 2016), but not in NCKX (the K + -dependent Na + /Ca 2+ exchanger that transports 1Ca 2+ plus 1K + in exchange with 4Na + ) (Zhekova et al., 2016). This is very intriguing, since ten (out of twelve) ion-coordinating residues (besides T50 and E213) are identical in NCX_Mj and NCKXs ( Figure 1B). Thus, NCKX can alternatively bind either 4Na + ions (at S int , S mid , S ext , and S Ca ) or 1Ca 2+ (at S Ca ) plus 1K + (at S mid ).
Strikingly, nine (out of twelve) ion-coordinating residues differ between the NCLX and NCX_Mj ( Figure 1B), where NCX possesses high selectivity for the Na + /Ca 2+ exchange, whereas NCLX can mediate either the Na + /Ca 2+ or Li + /Ca 2+ exchange (Palty et al., 2010;Refaeli et al., 2016;Khananshvili, 2020). The replacement of different ioncoordinating residues in NCX_Mj generates a new NCLX_Mj chimera that can generate either Na + /Ca 2+ or Li + /Ca 2+ exchange activities, as a native NCLX does (Refaeli et al., 2016;. However, the stoichiometry of the Na + /Ca 2+ and Li + /Ca 2+ exchange remains highly uncertain. The inside-positive potential accelerates the NCX_Mj-mediated Na + /Ca 2+ exchange with an electrogenic stoichiometry of 3Na + :1Ca 2+ , whereas the NCLX_Mj-mediated Na + /Ca 2+ or Li + /Ca 2+ exchange rates are insensitive to voltage clamp, consistent with the electroneutral (e.g., 2Na + :1Ca or 2Li + :1Ca 2+ ) ion-exchange mode Assali and Sackler, 2021). The HDX-MS and mutational analyses of NCLX_Mj revealed that S Ca binds to Na + , Li + , or Ca 2+ , whereas the binding of 3Na + (or 3Li + ) to NCLX_Mj is highly improbable (Refaeli et al., 2016;. Interestingly, the residues coordinating Ca 2+ at the S Ca of NCX_Mj (T50, E54, T209, and E213) completely differ from the Ca 2+ -ligating residues FIGURE 2 | and transition states are schematically represented in the case of Ca2+. In the Ca2+ occluded states the hydrophilic gap between the TM2C (P53) and TM7B (P212) segments becomes closer to each other for a hydrophobic patch, although the HDX-MS data reveal that TM2C, TM7B, TM7C, and TM8A, nearby the hydrophobic patch, remain quite flexible upon ion occlusion (Giladi et al., 2017). After the ion occlusion, the hydrophobic patch may undergo further conformational adjustments through the interactions of "catalytic" residues (E54, E213, D240, S51, S77, and T209) with Ca 2+ . As a result, the sliding of the TM1/TM6 cluster can take place to accomplish the OF/IF swapping (the movement of the TM1/TM6 cluster during the transition state is denoted by an arrow). The present considerations may represent the basis for future MD simulations.
Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 722336 of NCLX_Mj (N50, D54, N209, and D213). Namely, two negatively charged residues (E) at the S Ca of NCLX_Mj are replaced by shorter side chain residues (D) and two carbonyl coordinating residues (T) are replaced by rather bulky (N) side chain residues.
Structure-Dynamic Basis of Ion-Coupled Alternating Access and Transport
In general, the swapping of the IF and OF conformations may occur either in the presence or absence of ligand (ion), whereas an alternating access mechanism takes place differently in distinct groups of "secondary" transporters (Jardetzky, 1966;Forrest et al., 2011;Keller et al., 2014). For example, the action of the antiporter proteins mainly differs from the other groups of secondary transporters (e.g., cotransporters) in that that the ligand (ion) binding to respective sites is required to perform the OF/IF swapping (Forrest et al., 2011;Drew and Boudker, 2016). However, it is unclear how the selective recognition of ions at multiple sites of antiporter proteins is coupled to the ion-induced conformational changes associated with OF/IF swapping. The underlying mechanisms are not trivial to resolve (even for proteins with a known crystal structure), since subtle conformational changes might be involved in the dynamic recognition and coupling of ion transport. High-resolution crystal structures in the OF state, possessed by NCX_Mj (Liao et al., 2012;Liao et al., 2016), provided the primary clue regarding the major conformational changes underlying the ion-coupled alternating access in the Ca 2+ /CA proteins, otherwise known as the sliding mechanism. According to the originally proposed model, the binding of 3Na + or 1Ca 2+ to respective sites of NCX_Mj induces the sliding of the tilted twohelix (TM1/TM6) cluster (on the protein surface) in front of the remaining part of the protein (TM2-TM5 and TM7-TM10 hubs). Follow-up discoveries of the CAX structures in the IF state provided further support for the major structural elements involved in the sliding mechanism, thereby suggesting that the Ca 2+ /CA proteins share a common mechanism of ion-coupled alternating access (Nishizawa et al., 2013;Waight et al., 2013;Wu et al., 2013). Despite this progress, the structure-dynamic details underlying the ion-induced movement of the TM1/TM6 bundle remain incompletely understood (Giladi et al., 2016b;Giladi et al., 2017;van Dijk et al., 2018;Khananshvili, 2020).
In the absence of a crystal structure of NCX_Mj in the IF state, the HDX-MS studies provided important complementary information on the structure-dynamic mechanisms underlying the ion-coupled sliding mechanism of NCX_Mj (Giladi et al., 2016b;Giladi et al., 2017). The striking finding is that in both the OF and IF states the TM2B segment is much more constrained (less flexible) than the neighboring TM2C, TM7B, and TM7C segments ( Figure 2B). The HDX-MS observations are especially interesting from the perspective of structural evidence indicating that the ion binding/occlusion closes a hydrophilic gap between P53 (TM2C) and P212 (TM7B), thereby yielding a hydrophobic patch (Nishizawa et al., 2013;Giladi et al., 2016b). Even though the occlusion of Na+ or Ca2+ can promote the formation of a hydrophobic patch, the HDX-MS analysis shows that TM2C, TM7B, and TM7C segments remain quite flexible in the ion bound occluded state, though the hydrophobic patch is not stable enough to accomplish the TM1/TM6 sliding.
Extended ion flux analyses of single-point mutations revealed that the side chains of six residues (S51, E54, S77, E213, D240, and T209) are essential for ion transport activities, thereby suggesting that these "catalytic" residues interact with ions in the transition state van Dijk et al., 2018;Iwaki et al., 2020). These data are consistent with the proposal that the TM7B, TM7C, TM2C, and TM8A segments become more stable in front of the "ever-rigid" TM2B upon ion binding/occlusion, where the subsequent interactions of ions with "catalytic" residues in the transition state might drive the TM1/TM6 sliding toward OF/IF swapping (Figure 2). Computer-aided GNM (Gaussian Network Model) analysis of NCX_Mj identified key mechanical sites that potentially act as hinges or anchors for supporting the collective dynamics of the exchanger (van Dijk et al., 2018). These GNM results correlate well with the observed mutational effects on the Na + /Ca 2+ and Ca 2+ /Ca 2+ exchange rates, thereby identifying functionally important segments, namely, TM2 (G42-S51), TM3 (G76-C80), TM7 (P212-S217), and TM8 (G231-D240), which actively contribute to ion transport and thus, can limit the transport rates. Notably, the singlepoint mutations of ion-coordinating and non-coordinating residues within these segments have a devastating effect on the transport rates, thus implying that relevant regions play a mechanochemical role in controlling the ion transport rates. Thus, this analysis suggests that single-point mutations within functionally important hinge sites impact the ion-exchange kinetics in NCX_Mj.
In conjunction with GNM analyses, a close examination of collective motions by use of ANM (Anisotropic Network Model) approaches (Bahar et al., 1997;Eyal et al., 2015) have identified the correlated motions of TM1, TM6, and TM7 (block 1) and the strong coupling between TM2, TM3, TM4, TM5, and TM8 (block 2). In conjunction with the HDX-MS and mutational analyses of ion fluxes, the GNM and ANM analyses support the notion that block1 and block2 undergo anticorrelated motions with respect to each other. Thus, they might represent the dynamic features of hinge sites that may control the ion-coupled alternating access in NCX_Mj (van Dijk et al., 2018).
According to the sliding mechanism (Liao et al., 2012), the eight-helix core (TM2-TM5 and TM7-TM10) constitutively remains static (unmoved) during the multiple conformational transitions that take place during the transport cycle. According to this model, the static segments do not undergo any significant conformational changes in the OF/IF swapping, although they provide a protein surface for the TM1/TM6 sliding (Liao et al., 2012;Liao et al., 2016;Nishizawa et al., 2013;Waight et al., 2013;Wu et al., 2013;Lu et al., 2021). In contrast with this assumption, the GNM and ANM analyses revealed that TM4, TM5, TM9, and TM10 of NCX_Mj are quite flexible (van Dijk et al., 2018) and thus, may or may not affect the ion passageway. The structuredynamic features of these "static" segments as well as their effects on the ion transport activities, must be carefully addressed in future research.
Structure-Dynamic Hallmarks Associated With Ion Occlusion
The high-resolution crystal structures of NCX_Mj, in conjunction with highly dedicated MD simulations, provided useful information regarding the mechanisms underlying ion recognition and occlusion that take place at the extracellular vestibule in the OF conformation (Marinelli et al., 2014;Liao et al., 2016). These studies have demonstrated that at low concentrations of Na + , NCX_Mj adopts the "semi-open" OF conformation, in which two Na + ions occupy the S int and S Ca sites, thereby exhibiting a high affinity occupancy of these sites. At high Na + concentrations, the low affinity binding of the third Na + to the S ext site is associated with a "subtle" backbone bending at the interface of the TM7A and TM7B segments, thereby resulting in the occluded state of 3Na + -bound species in the OF conformation. Notably, when Na + binds to the S ext site at high Na + concentrations, the N-terminal part of TM7 bends into two short helices (TM7A and TM7B). According to this mechanism, TM7B occludes all ion-binding sites from the external bulk phase, since the Na + coordination through the backbone carbonyl of A206 and the bulk aromatic ring of F202 prevents ion dissociation from the extracellular vestibule (Liao et al., 2016;Giladi et al., 2017). However, when the S ext site is empty (at low Na + concentrations), the TM7A/TM7B segment is straight (thus, the carbonyl group of A206 and the aromatic ring of F202 are in a pullback position). Moreover, an open gap exists between the bent TM7A/TM7B and the C-terminal half of TM6 when Na + does not occupy the S ext site, whereas the occupation of S ext by Na + generates a hydrophobic patch between two helixes. These ion-induced conformational changes, associated with ion occlusion, are interesting in the context of the sliding mechanism, according to which the ion-coupled sliding of TM1/TM6 on the protein surface promotes the OF/IF switch (Liao et al., 2012, Liao et al., 2016Marinelli et al., 2014;Giladi et al., 2016b;Giladi et al., 2017). The ATR-FTIR analysis identified multiple signals for Na + binding to NCX_Mj, which may represent specific steps of ion binding/occlusion at the extracellular vestibule of NCX_Mj (Iwaki et al., 2020). The Na + interactions with respective sites result in Amide I and multi-dentate carboxylate ν s stretches, which represent distinct signals for the high-and low-affinity K d values observed for Na + binding (Iwaki et al., 2020). These observations are consistent with the stepwise binding/occlusion of 3Na + ions at respective sites. Notably, the Amide I signal refers to Na + -dependent changes in the secondary structure upon the high-affinity binding of 2Na + ions to the S int and S Ca sites, whereas the changes in the carboxylate IR signal represent the low-affinity binding of the third Na + ion to the S ext site through the ligation of the E213 carboxylate. Collectively, the IR analyses of the apo-and Na + -bound species of NCX_Mj reveal the underlying details of 3Na + ion binding/occlusion at the extracellular vestibule in the OF conformation.
HDX-MS (hydrogen-deuterium exchange massspectrometry) has been explored to analyze the apo-and ionbound states of NCX_Mj, with the goal of mapping the backbone dynamic patterns in the OF and IF states (Giladi et al., 2016b;Giladi et al., 2017;. In general, HDX-MS quantifies the exchange rates of backbone amide hydrogen with deuterium in solvent; therefore, more flexible or solvent-exposed regions take up more deuterium than do the rigid segments or the regions that are less exposed to solvent (Demmers et al., 2000;Englander et al., 2003;Chalmers et al., 2011;Konermann et al., 2011;. Thus, HDX-MS is the method of choice for analyzing the local backbone dynamics of ion-occluded states, since relatively small and slow conformational changes in the backbone dynamics can be detected in apo and ligand (ion) bound states (Englander et al., 2003;Chalmers et al., 2011;Konermann et al., 2011).
Notably, the Na + or Ca 2+ binding to NCX_Mj slightly (but specifically) modifies the backbone dynamics at respective ionbinding sites, although the incremental conformational changes in the OF and IF states are largely predefined by signature landscapes exemplified in the apo-OF and apo-IF protein structures (Giladi et al., 2017). Consistent with the X-ray crystallographic data, the HDX-MS analysis reveals that the Na + -dependent bending occurs at TM7AB (202-FTLV-205), where the 3Na + occlusion at the extracellular vestibule may remotely couple the interactions between TM7AB and TM6 (Giladi et al., 2016b;Giladi et al., 2017;. According to the HDX-MS analysis, the bending of TM7AB, upon the occlusion of the 3Na + ions, may affect the hydrophobic interactions between TM6 and TM7 (involving L204), which is consistent with the higher solvent exposure (higher deuterium uptake) associated with the S ext occupation by the third Na + ion, thereby achieving the 3Na + occlusion (Giladi et al., 2017).
STRUCTURE-DYNAMIC ASYMMETRY OF BIDIRECTIONAL ACCESS/PERMEATION IN NCX_MJ
Biochemical and kinetic tests revealed that access of ions to the extracellular and cytosolic vestibules of eukaryotic and prokaryotic NCXs is highly asymmetric (Khananshvili et al., 1995(Khananshvili et al., , 1996Almagor et al., 2014;Giladi et al., 2017;van Dijk et al., 2018). This functional asymmetry is especially interesting in the context of α1 and α2 repeats, since they form an inverted two fold (pseudo)-symmetry for ion passageway in the Ca 2+ /CA proteins. Notably, the repetitive structural elements were generated through the duplication and fusion of genes during evolution; consequently, the functional outcomes of a given protein can be affected in many different ways (Duran and Meiler, 2013;Forrest, 2015;Drew and Boudker, 2016;van Dijk et al., 2018).
In agreement with biochemical studies, the ATR-FTIR analysis revealed the high-and low-affinity K d values for Na + or Ca 2+ binding to purified NCX_Mj, which may represent the different affinities of ion binding in the OF and IF states (Iwaki et al., 2020). Moreover, the ion-flux assays have shown that the K m Cyt values are at least 7-10-times lower than the K m Ext values for either Na + or Ca 2+ . Finally, the different K d values measured by ATR-FITR are comparable with the K m Cyt and K m Ext values measured by the ion-exchange assays Iwaki et al., 2020). Collectively, the available data support the notion that ions have an asymmetric access to the ion-binding pocket at the opposite sides of the membrane under steady-state conditions. Extended mutational analysis of inversely matched pair residues in NCX_Mj revealed that the structure-functional asymmetry occurs at the level of single amino acid residues; this suggests that specific structural elements within the ion passageway differentially control the rate-equilibrium relationships of bidirectional ion movements in NCX_Mj (Giladi et al., 2016a;Giladi et al., 2016b;Giladi et al., 2016c;Giladi et al., 2017;van Dijk et al., 2018).
The HDX-MS analysis of NCX_Mj revealed that ionpassageway entities (involving the TM2, TM3, TM7, and TM8 helices) exhibit characteristic profiles in the local backbone dynamics when they adopt the OF and IF states (Giladi et al., 2016b;Giladi et al., 2017). For example, TM2A and TM8A are more flexible in the IF than in the OF state, whereas the opposite is true for TM7A ( Figures 2E-G). Moreover, the OF state is characterized by higher water accessibility at the extracellular than at the cytosolic entry, whereas the opposite is true for the IF state. Thus, pseudo-symmetry-related structural entities at the cytosolic and extracellular entries exhibit reciprocal levels of deuterium uptake (as revealed by HDX-MS) in the IF and OF states. Therefore, they represent hallmark differences in the structure-dynamic preorganization of the OF and IF states prior to ion binding/occlusion. Interestingly, the Na + or Ca 2+ ion binding to NCX_Mj results in relatively small (but specific) changes in the local backbone dynamics, although the overall conformational asymmetry is largely retained (Giladi et al., 2017). Thus, structure-dynamic preorganization of NCX_Mj underscores the importance of the apo-protein conformational state, which is asymmetric in nature.
The present considerations raise the following question: how can the "minute" differences in the signature sequences shape the structure-dynamic features of ion ligation geometry and how can this affect the ion binding affinity, selectivity, and transport rates. Notably, all the relevant structure-dynamic determinants may predefine the physiologically important parameters, such as the ion-binding affinity, the intrinsic asymmetry of bidirectional ion movements, the ion-exchange stoichiometry, and electrogenicity, among many others. Moreover, the relevant structural variations must somehow reconcile with a general mechanism of ioncoupled alternating access shared by Ca (Liao et al., 2012(Liao et al., , 2016Nishizawa et al., 2013;Khananshvili, 2020).
The 49-GTSLPE-54 and 208-GTSLPE-213 repeats contain six ion-coordinating residues (T50, S51, E54, T209, S210, and E213) and four helix-breaking residues (G49, P53, G208, and P212) ( Figures 2B-D). The HDX-MS and ion flux analyses of mutants revealed that the folding/unfolding features of the local backbone segments (at the pore center) control the transport rates Giladi et al., 2016a, Giladi et al., 2016bGiladi et al., 2016c;Giladi et al. 2017;van Dijk et al., 2018). Moreover, the HDX-MS analyses of NCX_Mj revealed that in the apo state the 49-GTSLPE-54 segment is more rigid than its counterpart segment (208-GTSLPE-213); thus, it represents an asymmetrically rigidified structural template for relevant ion-coordinating residues. Interestingly, the binding of the Na + or Ca 2+ ions specifically (and incrementally) rigidify the backbone dynamics at the respective ion binding sites ( Figures 2F,G) (Giladi et al., 2017). Notably, the mutation of the non-coordinating residues (G49, P53, G208, or P212) within 49-GTSLPE-54 and 208-GTSLPE-213 (next to the ion-coordinating carboxylates) have a devastating effect on the transport rates. At this end, it is unclear how the local backbone dynamics at signature sequences control ion ligation in the ground and transition states. The emerging working hypothesis is that characteristic disparities in the local backbone dynamics at signature sequences may control the helix folding/unfolding energies at ion-coordinating residues, consequently, shaping the ion selectivity and transport rates.
PROTONATION/DEPROTONATION STATUS OF ION-COORDINATING CARBOXYLATES IN DISTINCT ORTHOLOGS
Notably, the ion-binding pocket of NCX_Mj contains three carboxylates (E54, E213, and D240), whereas the eukaryotic NCXs contain two carboxylates (E54 and D213) at matching positions ( Figure 1B). Previous studies with MD simulations and mutational analysis of ion fluxes revealed that D240 of NCX_Mj (exclusively belonging to the S mid site) is protonated, although the deprotonation of D240 is not essential for ion transport activities (Marinelli et al., 2014). In agreement with this, the ATR-FTIR tests have shown that mono-dentate D240 cannot bind to either Na + or Ca 2+ (Iwaki et al., 2020), thereby suggesting that the S mid site lacks the capacity for Na + or Ca 2+ binding (at least in the ground state). Accumulating data support a model according to which only two deprotonated carboxylates take part in the 3Na + or 1Ca 2+ ligation, either in the prokaryotic (E54 and E213) or eukaryotic (E54 and D213) NCX prototypes. According to this model, the 3Na + -bound species might carry a positive charge (Z + 1), whereas the 1Ca + -bound species are electroneutral (Z 0) either in the prokaryotic or eukaryotic NCXs. An important outcome of this model is that the voltage-sensitive translocation of positively charged 3Na + -bound species might be a rate-limiting step during the action potential swings in excitable tissues. For example, in cardiomyocytes, the membrane-potential oscillations (from −90 mV to +50 mV and back) might affect the translocation of 3Na + -bound species.
Recently performed MD simulations and extended mutational analysis of ion fluxes have shown that the carboxylate residue at the S mid site of NCKX (equivalent to D240 of NCX_Mj) could be in a deprotonated state, thereby allowing the binding of either Na + or K + ions at the S mid site of NCKX (Zhekova et al., 2016;Jalloul et al., 2018). These results seem to be very interesting, since the eukaryotic NCKXs (E54, D213, and D240) and prokaryotic NCX_Mj (E54, E213, and D240) contain three carboxylates within the ion-binding pocket ( Figure 1B). However, the striking difference between the eukaryotic NCXs and NCKXs could be that the mono-dentate carboxylate at the S mid site adopts a deprotonated state in NCKX, but not in NCX. This may underscore a fundamental difference between the NCX and NCKX prototypes, although some common electrostatic features can be shared by NCXs and NCKXs as well. More specifically, the 4Na + -bound species of NCKX may carry a positive charge (Z + 1), whereas the 1Ca + +1K + -bound species might be electroneutral (Z 0). According to this proposal, the voltage-sensitive translocation of positively charged 4Na + -bound species could to be rate limiting during the membrane potential changes in health and disease, thereby showing a certain similarity to the eukaryotic NCXs.
ALLOSTERIC EFFECTS CANNOT EXPLAIN THE HUGE KINETIC VARIANCES AMONG THE PROTOTYPE PROTEINS
Even though the prokaryotic and eukaryotic NCXs share a common stoichiometry of ion-exchange (3Na + :1Ca 2+ ), the turnover rates of the ion-exchange cycle dramatically differ in the eukaryotic NCXs (∼5,000 s −1 ) and NCX_Mj (∼0.5 s −1 ) (Niggli and Lederer, 1991;Hilgemann et al., 1991;Baazov et al., 1999;Almagor et al., 2014). These huge differences in the transport kinetics definitely have a physiological significance for a proper handling of NCX-mediated extrusion of Ca 2+ from a given cell type, although the structure-dynamic determinants of this fascinating phenomenon remain unresolved (Blaustein and Lederer, 1999;Bers 2008;Khananshvili, 2013;Khananshvili, 2014). The structure-dependent control of kinetic capacities is especially interesting in light of the fact that "only" three (out of twelve) ion-coordinating residues differ among NCXs (T50S, E213D, and D240N) ( Figure 1B)-therefore, one may posit that these structural differences may account for kinetic variances (at least partially) among NCXs. However, a systematic replacement of differing ion-coordinating residues in NCX_Mj cannot recapitulate the high turnover rates of those possessed by prokaryotic NCXs van Dijk et al., 2018;Iwaki et al., 2020). These findings are consistent with the notion that the rigid structure of NCX_Mj prohibits an effective ligation of ions in the occlusion and/or transition states, which in turn, may limit the transport rates.
The striking difference in the protein structure of prokaryotic and eukaryotic NCXs is that the cytosolic 5L6 loop (between TM5 and TM6) of eukaryotic NCXs is very long (∼520 residues) and contains the Ca 2+ binding regulatory domains (CBD1 and CBD2). In contrast, the prokaryotic NCXs have a very short 5L6 loop (12-32 residues) in the absence of regulatory CBD domains (Philipson and Nicolle, 2000;Hilge et al., 2006;Liao et al., 2012). The eukaryotic NCXs undergo an extensive splicing at CBD2 and are expressed in a tissue-specific manner in order to fulfill the functional requirements of a given cell type (Lytton, 2007;Khananshvili, 2016;Khananshvili, 2017;Khananshvili, 2020). Notably, the Ca 2+ binding to the regulatory CBD domains of eukaryotic NCXs can activate the NCX-mediated transport activities up to 20-fold (Boyman et al., 2011). Thus, at resting levels of cytosolic Ca 2+ , the transport rates of eukaryotic NCX might be at least 10 3 -times faster than those of prokaryotic NCX_Mj. Moreover, the proteolytic shaving of the regulatory CBD domains of the cardiac NCX results in a maximal activation of the transport rate, although the regulatory responses are completely lost (Doering et al., 1996). Interestingly, the elongation of the 5L6 loop of NCX_Mj (by 8-16 residues) results in a 5-10-fold activation of the transport rates , thereby suggesting that the 5L6 elongation may facilitate the dynamic features of TM1/TM6 toward OF/IF swapping. Thus, it would be interesting to determine what are the specific structure-dynamic determinants that predefine so high rates in eukaryotic NCXs.
CONCLUDING REMARKS AND PERSPECTIVE
A systematic application of multidisciplinary approaches, which included structural (X-ray crystallography), computational, biophysical (HDX-MS, ATR-FITR), and biochemical techniques, shed light on the ion transport mechanisms operating in the Ca 2+ / CA proteins. High-resolution crystal structures of NCX_Mj (in the OF orientation) and of CAX proteins (in the IF orientation), in the open, semi-open, and occluded conformation states provided a fundamental basis for considering the "sliding mechanism" as a common mechanism for ion-coupled alternating access in Ca 2+ / CA proteins. Follow-up studies with MD simulations and mutational analysis of ion fluxes have established the functional features of individual ion-binding sites and have identified the key residues controlling the ion transport activities in the NCX and NCKX proteins. HDX-MS has identified the ion-induced changes in the local backbone dynamics and thereby provided specific clues for "subtle" conformational changes that accompany the ioncoupled alternating access. ATR-FTIR elucidated the protonation/deprotonation states of ion-coordinating carboxylates, thereby underscoring the charge-carrying features of ion-bound species with different numbers of carboxylates within the ion-binding pocket. Collectively, the structural and functional data gained during the last years have provided a conceptual framework for studying the dynamic mechanisms by using the template model system of the archaeal NCX_Mj protein. An increasing body of evidence has revealed that the "sliding mechanism" can be considered as a framework model (with specific modifications) for the ion transport mechanisms in the Ca 2+ /CA proteins, although a considerable amount of work is required to resolve subtle conformational changes that seem to be essential for performing the OF/IF swapping during the transport cycle. This is especially true for eukaryotic NCX, NCKX, and NCLX proteins, whose the structural details remain unavailable. This is important since numerous isoform/splice variants of these proteins are expressed in a tissue-specific manner to fulfill cell-specific physiological requirements in a given cell type; From the structure-functional standpoint, the challenge is to determine how the allosteric signals are transferred from distantly located regulatory domains to transport sites and how the output of allosteric signals is modified in distinct variants.
Given the physiological importance of NCX, NCKX, and NCLX proteins in health and disease, a future discovery of mammalian protein structures may provide new opportunities for long-wanted pharmacological targeting of tissue-specific isoform/splice variants. In the longterm, this may have a clinical significance. Taking into account the accumulating data discussed here, the structural template of NCX_Mj can be further explored to better understand the ion transport mechanisms in NCX and similar proteins.
AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and has approved it for publication.
FUNDING
This work was supported by the Israel Science Foundation to DK (#1351/18). | 2021-07-30T13:06:24.254Z | 2021-07-30T00:00:00.000 | {
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119415242 | pes2o/s2orc | v3-fos-license | Lorentz invariance violation in top-down scenarios of ultrahigh energy cosmic ray creation
The violation of Lorentz invariance (LI) has been invoked in a number of ways to explain issues dealing with ultrahigh energy cosmic ray (UHECR) production and propagation. These treatments, however, have mostly been limited to examples in the proton-neutron system and photon-electron system. In this paper we show how a broader violation of Lorentz invariance would allow for a series of previously forbidden decays to occur, and how that could lead to UHECR primaries being heavy baryonic states or Higgs bosons.
I. INTRODUCTION
UHECRs are an enduring mystery. Without the introduction of new particles or interactions, evading the Greisen-Zatsepin-Kuz'min (GZK) cutoff [1,2] requires unidentified nearby sources. Even without the GZK cutoff, the "bottom-up" approach faces the challenge of finding in nature an accelerator capable of energies in excess of 10 20 eV.
"Top-down" scenarios assume that the UHECRs result from the fragmentation of a ultra-high energy hadronic jet produced by cosmic strings [3] or by the decay of a supermassive particle [4]. In the supermassive particle (wimpzilla) scenario, the UHECRs are of galactic origin, resulting from the decay of relic supermassive (M > ∼ 10 13 GeV) particles. Wimpzillas can be produced copiously in the early universe [5], thus solving the energy problem. Since they would cluster in the dark matter halo of our galaxy [6,7,8], they also solve the distance problem. Detailed analysis of these decays, however, show that at high energy top-down scenarios produce more photons than protons [9,10,11] in the UHECR spectrum seen at Earth.
The top-down prediction of photon preponderance in UHECRs is the one major problem in an otherwise simple explanation. Results from the Fly's Eye [12], Haverah Park [13], and AGASA [14] cosmic ray experiments all indicate that at energies above around 10 19 to 10 20 eV, protons are more abundant than photons in UHECRs. While photons are disfavored, it is not possible to be sure that the primary is indeed a proton.
The idea of violating LI (see Ref. [15] for a broad overview) has recently been studied in the context of UHECRs for proton decay [16,17], atmospheric shower development [18], and modifications to the cutoffs of proton and photon spectra due to cosmic background fields [19,20,21,22,23,24]. As shown in Ref. [25], LI violation can also lead to vacuum photon decay, and in general the decay of particles to more massive species. We exploit this fact to show how particles produced in top-down decays can undergo an "inverse cascade" to produce superheavy UHECR primaries.
A. Modified Dispersion Relations
There are two approaches to breaking LI commonly used in the literature. The first [16,20,21,25,26] is generically to modify the dispersion relations for a particle of mass m and 3-momentum p = |p|.
The second method, which we will not utilize here, is instead to write down a particle Lagrangian [27] which includes Lorentz and CPT violating terms. A mapping of the Lagrangian terms to a dispersion relation is nontrivial (see [23] for a simple case); however, to first order the changes to the photon and electron propagators induce shifts to c γ and c e [17]. Similarly, it was shown by [28] that loop quantum gravity effects produce modified dispersion relations similar to those considered elsewhere and here.
The simplest way to break LI, as shown in [17], is to write down a dispersion relation for a particle species i as This is changing the "speed of light" or Maximum Attainable Velocity (MAV) for each particle to something slightly different than c. 1 The MAVs for different particle species are assumed a priori to be different in that and similar treatments [26].
In such a case, it is possible for previously unallowable reactions to occur, such as p → ne + ν e [16,17], γ → e + e − [25,26] and γ → νν [25]. A conclusion reached, using the first reaction, is that neutrons may make up the dominant baryonic component of the UHECR primaries. This, strictly, would be true if one was only tuning the proton and neutron MAVs and leaving all others equivalent (= c). On the other hand, keeping every MAV as individually tunable gives an overwhelming number of free parameters.
In order to examine the consequences of varying all particle MAVs using the fewest number of free parameters, we extend the method used in Ref. [21] and write the dispersion relation as Here, f ( In this section we consider the tree-level photon decay process γ → e + e − , which is kinematically forbidden for c e = c. Following Ref. [17], we define the parameter δ γe as 3 Using astrophysical constraints [29,30], δ γe can be limited to the range 10 −16 > δ γe > −10 −17 . For δ γe > 0, photon decay occurs above a photon energy threshold given by This decay rate of was computed in Coleman & Glashow (1997) as For decay product energy above about 2×10 20 eV, photons will outnumber protons, so we set E th to this value and suggestively rewrite the decay rate above threshold as: This would rule out all but a terrestrial source of UHE photons. The value δ γe = 3 × 10 −29 is used is a lower limit; if δ γe would be any lower the energy threshold would become too high. This means that only δ γe ∈ (3 × 10 −29 , 10 −16 ) will correct for the photon overabundance in top-down scenarios.
Now we see what is required to have δ γe in the necessary range to allow for this photon decay. Using Eqs. (6) and (9), we can write Since the functions g(x) and h(x) are a priori arbitrary, so are the values and signs of their derivatives. It is argued in Ref. [21] that too strongly varying functions would be unphysical, and that the derivatives should be of order unity. 4 Adopting this, we can ignore the g ′′ (0) term in the above equation. In this case, in order to have photon decay we need to require h ′ (0) negative. Then δ γe ∼ mc 2 /2M ∼ 3 × 10 −23 and E th = 2m e c 2 e c 2M/mc 2 = 2 √ 2m e M c 2 ≈ 2 × 10 17 eV. This value of δ γe is (logarithmically) in the middle of our allowable range.
Returning to our original impetus of addressing the feasibility of "Top Down" scenarios, we see that requiring h ′ (0) ∼ −1 is sufficient to eliminate any photons among the UHECR primaries. This model, however, eliminates any protons as well, which we will now address.
C. Making the impossible possible
Unlike previous investigations [17,29] where the MAV for each particle could be chosen independently, this is not so in our case. Since Eq. 2 holds for all types of particles, it is straightforward to see other effects of this level of Lorentz invariance violation. Using Eq. 6, we can in general write for two particles A and X where we have used h ′ (0) ∼ −1.
The consequences of this for particle decays are as follows, as first noted by [17], and we reproduce their argument here.
Consider the decay A → {X}, where {X} is a collection of massive particles. We want to find the minimum possible energy E min where this can occur. We can always lower final state energy by removing transverse components of momenta, so we're working in one dimension only. We want to minimize subject to the constraint The {p X } are variables, and p A , E A > E min are constants. Using the method of Langrange multipliers, E is minimized when which becomes Thus all products move with the same velocity λ. Note that λ < c X for all X. For the example we consider, in the event of massless particles among the decay products, they would minimize final state energy by carrying exactly zero momenta [17]. Since the {c X } are the maximum attainable velocies for the decay products, that implies that λ < min{c X }.
It is this last point we focus on: min{c X } in our case corresponds to max{m X }. In the ultrarelativistic limit, E A ≈ p A c A and so on, and the particle with the lowest speed of light will carry most of the momentum (in the minimum energy case). Further, in this threshold case, λ is also the velocity of the incident particle [17]. Since λ ≈ c A in the regime we are considering, that means that the decay can only occur if c A > min{c X }. From Eq. 14, that implies that m A < max{m X }.
In other words, above a certain threshold, a decay may only be allowed if at least one decay product is more massive than the decaying particle. This can have serious consequences for the particle content of UHECRs, as we will now address.
III. THE INVERSE CASCADE
In this model, for each individual reaction there is a threshold energy E T above which particles can only decay if at least one of the decay products is more massive. For the decay A → B + ..., where B is the most massive decay product, m B > m A , and m ... ≪ m B the threshold energy goes like Using equation 14, this reduces to Due to the large value of the Planck mass, this threshold is around the same order of magnitude for a wide range of reactions involving particles of mass much less than M .
Consider an UHECR of mass m 0 produced with energy E 0 ≫ E T . If normally (at energies E ≪ E T ), particle 0 can be a decay product of a more massive particle 1, then here (E ≫ E T ), 0 can decay into 1. We ignore the lighter products that will result as well; the substantial fraction of the incident energy will be carried by the most massive decay product. Now, we have a particle of energy E 1 < E 0 and mass m 1 > m 0 . If it can, it will also decay into a particle 2 with m 2 > m 1 and energy E 2 < E 1 . As the decays continue into more and more massive final states, the final energy continues to decrease, until at some point we reach a particle N with energy E N < E T and mass m N > m 0 . Since we are now below threshold, the N particle can decay as usual into N-1 and so on into less massive particles.
So, to summarize, this inverse cascade decay process occurs in two stages.
1. Decay up: Decays into more and more massive, yet less energetic particles, when E > E T , and 2. Decay down: Regular decay schemes once E < E T .
IV. PANDORA'S BOX (NEWLY ALLOWED REACTIONS)
We saw in Section II that at sufficiently high energies particles can only decay if one of the decay products is more massive. In Section III we saw how this can produce an "inverse cascade" to more and more massive particle species. In this section we address the specifics of how this cascade occurs, by examining which previously forbidden decays can now occur. All of the following are assumed to be occuring well above threshold, and we list them in several classes.
A. Tree-level QED
Here the bare QED vertex γ → Q + Q − where Q is any charged particle is allowed as a decay. This also includes final states with extra photons and bound states of Q + Q − , like γ → π 0 γ.
B. Tree-level weak
It is in the weak sector that most of the interesting decays in the inverse cascade occur, due to the flavorchanging W vertex.
In this Lorentz-violating scheme, weak decays (such as that of the neutron) can happen in reverse, allowing proton decay via p → ne + ν and flavor changing decays such as n → Λπ 0 .
Not only that, decays to weak bosons become permissible, such as ν l → l ± W ∓ and l ± → ν l W ± . Included in this are decays of leptons to heavier leptons due to virtual W 's. In the quark sector, this allows decays such as p → nW + and so on. Generally, the flavor changing weak decay q → q ′ W will convert all quarks to t quarks. In the absence of a fourth generation, a bare t quark is stable.
For the bosons; W + → tb is allowed, and Z-Cerenkov is possible for the W as well (this is a bare weak vertex, as is W → W ZZ and W → W Zγ). The Z is unstable to the decay Z → tt. Now consider the Higgs: indeed we might be above the energy of weak symmetry breaking. Including a single massive Higgs H would allow; {f, W, Z} → {f, W, Z}H ("Higgs-Cerenkov" for fermions and weak bosons) and {W, Z} → {W, Z}HH. In the absence of new physics beyond the Standard Model, the H is stable if it is heavier than the top quark. If it is lighter than the top, the Higgs is unstable to H → tt.
C. Tree-level Strong
Since the bare QCD vertex preserves quark flavor and charge, not much happens in the bare quark sector, apart from the reversings of strong decays such as N → ∆π and π → ρπ. Just as for the massless photon, free gluon decay is allowed via g → qq.
D. One-loop processes
One could formulate many more decays at the 1-loop level, but the rates for these for them are suppressed compared to the tree level decays. For this reason, we do not consider them further here, except to note two interesting decays of the photon: γ → ν lνl (QED and weak process). Note that since neutrinos are so much lighter than their heavy partners and the quarks, the threshold for photon to neutrino decay could be very low depending on how light the neutrinos are.
γ → Zγ (weak process). This photon Z-Cerenkov decay occurs due to a W -loop.
E. Spin up
For bound states (and now restricting ourselves to bound states of three valence quarks) there exist higher mass resonances that only differ in the mass and total angular momentum (e.g., the ∆ and higher resonances for the proton and neutron). Since the state with higher angular momentum is more energetic, it is more massive and thus processes like N → N * π, N * → N * * π and so on can act to "spin up" the particle to more and more massive states.
Thus, even the all top quark baryon ttt isn't completely stable, but can decay via spin up.
V. ESTIMATES OF DECAY RATES
For the photon decay γ → e + e − , the decay rate Γ well above threshold was We adapt this form of the decay rate to other processes to make estimates, and argue for it as follows. At ultrarelativistic energies, the only energy scale is the incident energy E, so Γ ∝ E. It is proportional also to the effective coupling constant involved in the decay, and to kinematic factors. We schematically write this as For the photon decay, the coupling is α and the kinematic factor is δ γe . We generalize these factors as follows. We (first) consider decays of the form A → B + C where m A < m B , m B ≫ m C . At threshold and beyond, most of the momentum of the final state is carried by the particle with the lowest speed of light, thus highest mass (particle B). The relevant kinematical factor will then be δ AB . This will continue to be true when final states with more than 2 particles are allowed, as long as B continues to be the most massive particle.
The coupling factor is determined by looking at the tree level Feynman diagrams and counting vertices involved in the reaction.
Consider then the decay of the proton; in addition to the inverse of the neutron decay p → ne + ν e ("leptonic" decay), the decay p → nW + ("bosonic" decay) is also allowed.
For the leptonic decay; E T ∼ M (2m p ) ∼ 4.5 × 10 18 eV, and as most of the energy goes into the n, δ pn ∼ (m n − m p )/2M ∼ 5 × 10 −23 . This reaction has two weak vertices with coupling α 2 W ∼ 10 −3 . Thus, at Note that the distance travelled before decay is ∼ 1/Γ, and givenhc = 6.57 × 10 −24 eV pc, the 1/Γ for these reactions is incredibly small: 10 −24 pc for the bosonic and 10 −17 pc for the leptonic. In the context of a UHECR, assuming comparable rates (within a few orders of magnitude) to the above, this inverse cascade happens essentially immediately, so that by the time any decay products reach the Earth, they have gone through both stages of the cascade and are now sub-threshold particles.
VI. CONSEQUENCES FOR UHECR COMPOSITION
Given the rapid rate of the inverse cascade process, there can be two outcomes for the UHECR population at earth.
First, if both the Decay Up and Decay Down sequences occur, then the consituent particles could, depending on the exact decay chains and nature of initial UHE primary, consist of protons, electrons, photons, and their antiparticles. Each particle spectrum would exhibit a cutoff at their prospective threshold energies, which are all related by the h ′ (0) parameter.
Second, and more interesting, if the initial UHE primary is of high enough energy, the decay up sequence may occur and leave particles that can no longer decay into more massive particles, but are still above threshold for decay into less massive particles. These Most Massive Particles (MMPs) would then be the most energetic component of the UHECR population. What exactly the MMPs are depends on the model of particle physics used. Of particles known to exist know, the MMPs would consist of top quarks and excited bound states of top (tt mesons and ttt baryons) quarks. If the Higgs is more massive than the top, then the MMPs would consist of single Higgs bosons. Including Supersymmetry and its expected spectrum of more massive superpartners, if the decay rate is still rapid enough the MMP would be the most massive superparticle (MMSP).
Of course, in the first outcome, our proposed solution of using LI violation to solve the photon abundance issues in top-down models is not obviously successful. For both outcomes, in fact, one would have to re-examine the decay process starting with the initial decay of the supermassive relic (which is assumed to be sub-threshold, so it decays "normally"), and follow the decay products to see what results.
Simulations of MMP-induced shower development would have to be performed to see if they are consistent with actual events. It was shown in Ref. [32] that stable UHE primaries of mass greater than about 50 GeV should be distinguishable from proton primaries by their atmospheric shower profiles. As the lightest conceivable MMP (a tt meson) would be much heavier than this bound, the result of [32] might be used to exclude the MMP as the primary for UHE showers. However, a key assumption is that the primary is stable. For a MMP just above the threshold energy, it may take only a few collisions before the MMP energy is lowered below threshold, where it will decay "normally" (e.g., tt → W + W − ) into a shower of particles, perhaps mimicking the shower of a nucleus UHECR. A MMP with incident energy farther above threshold would then penetrate deeper. In any event, further investigation into MMP UHECR shower profiles is warranted.
Also of concern is that, while energy and momentum are conserved in a certain preferred frame, they are necessarily not conserved in all other frames. As such, due to the motion of the earth and the solar system, small departures from energy conservation might be observed as well in the UHECR atmospheric showers.
This work was supported in part by the Department of Energy and by NASA (NAG5-10842) and by NSF grant PHY 00-79251. | 2019-04-14T02:35:36.515Z | 2003-06-29T00:00:00.000 | {
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1734533 | pes2o/s2orc | v3-fos-license | Social Pharmacy and Clinical Pharmacy—Joining Forces
This commentary seeks to define the areas of social pharmacy and clinical pharmacy to uncover what they have in common and what still sets them apart. Common threats and challenges of the two areas are reviewed in order to understand the forces in play. Forces that still keep clinical and social pharmacy apart are university structures, research traditions, and the management of pharmacy services. There are key (but shrinking) differences between clinical and social pharmacy which entail the levels of study within pharmaceutical sciences, the location in which the research is carried out, the choice of research designs and methods, and the theoretical foundations. Common strengths and opportunities are important to know in order to join forces. Finding common ground can be developed in two areas: participating together in multi-disciplinary research, and uniting in a dialogue with internal and external key players in putting forth what is needed for the profession of pharmacy. At the end the question is posed, “What’s in a name?” and we argue that it is important to emphasize what unifies the families of clinical pharmacy and social pharmacy for the benefit of both fields, pharmacy in general, and society at large.
Introduction
"What's in a name? That which we call a rose. By any other name would smell as sweet." This reference to William Shakespeare's play Romeo and Juliet is used to imply that the names of things do not affect what they really are. Could this question not just as easily be posed about the disciplines of clinical pharmacy and social pharmacy?
We have experience in working within both clinical and social pharmacy as educators and researchers and, through this, observed the commonalities and differences between these two sister disciplines. The differences have been allowed to dominate the discourse within pharmacy as a profession. We argue that this focus is illogical and detrimental to furthering pharmacy science and practice development.
A simplistic differentiation is that those who entitle themselves clinical pharmacists are practitioners, with little emphasis on research and teaching. Conversely, social pharmacy is an academic university discipline which does not really have a practitioner group with this as a specialization, but has often worked with or supervised community pharmacists. We will argue that this is not a fruitful distinction based on developments within the field of health care.
Our work as researchers and educators has mostly been carried out in the Nordic countries, but we have also sought to incorporate our knowledge of the Anglo-Saxon (North America, UK, and Australia) and European countries outside the Nordic region.
Social Pharmacy Defined
The remit of social pharmacy has been defined by authors from Denmark and the USA [1] as studying . . . " . . . the drug/medicine sector . . . from the social scientific and humanistic perspectives. Topics relevant to Social Pharmacy consist of all the social factors that influence medicine use, such as medicine-and health-related beliefs, attitudes, rules, relationships, and processes." Concepts and nomenclature other than social pharmacy are used within Anglo-Saxon literature such as pharmacy practice and pharmaceutical policy. In North America, pharmacy practice as a research discipline has primarily been carried out by clinical and/or hospital pharmacists. British literature refers to pharmacy practice both as a discipline for primary and secondary care pharmacists. A notable exception is also that researchers in England have written a textbook with the title Social Pharmacy [2] and define the concept as: " . . . social pharmacy is a relatively recent concept with Britain, often subsumed under the generic term "Pharmacy Practice". Initially it was synonymous with the demography of medicines use and "pharmacoepidemiology" but its remit now extends well beyond drug-use surveillance." The same authors, in a paper from 1993 [3], state that social pharmacy cannot be equated to the sociology of pharmacy but that it explores the . . . " . . . hitherto neglected social domain in which pharmacy is practised from the practitioner's social perspective." This differs considerably from the definition put forth by Sørensen et al. [1] who see it more broadly from a societal perspective rather than pharmacy as a profession.
The term social pharmacy has been used in Britain and occasionally in North America; however, it has been most widely used in the Nordic context and in other (mostly Eastern) European countries [4]. In the USA, pharmacoepidemiology, pharmacoeconomics, and pharmaceutical policy-all considered part of social pharmacy in Europe-are seen as fields separate from pharmacy practice. The UK tends to include pharmaceutical policy and social pharmacy within the term pharmacy practice.
In addition to the lack of unified terminology, the discipline of social pharmacy has been found lacking consensus and a common understanding of what constitutes the research area [5]. This is quite obvious when studying the definitions put forth and the lack of literature about what constitutes the field of social pharmacy.
Clinical Pharmacy Defined
Clinical pharmacy has been defined by professional bodies on both sides of the Atlantic. The European Society of Clinical Pharmacy (ESCP) [6] has defined it as: " . . . a health specialty, which describes the activities and services of the clinical pharmacist to develop and promote the rational and appropriate use of medicinal products and devices.
Clinical Pharmacy includes all the services performed by pharmacists practicing in hospitals, community pharmacies, nursing homes, home-based care services, clinics and any other setting where medicines are prescribed and used." The American College of Clinical Pharmacy (AACP) [7] put forth both a long and an abridged definition. The latter states that clinical pharmacy is: " . . . defined as that area of pharmacy concerned with the science and practice of rational medication use." Additionally, the AACP in their unabridged definition emphasize that clinical pharmacy must be engaged in research to contribute to the generation of new knowledge that advances human health and quality of life and that clinical pharmacists should include research as part of their work [7].
Sørensen et al. [1], who discussed the definition of social pharmacy, said that "Clinical Pharmacy serves as a bridge that overlaps with and connects the natural sciences and Social Pharmacy."
Aims
In this article, we will explicitly dig into what defines these two areas of clinical and social pharmacy, what they have in common and what still sets them apart. We aim to challenge researchers, educators, and practitioners relating to either of these fields to find a common ground and thereby strengthen their impact in society.
Other Healthcare Professions
Some healthcare providers feel that they have a strong knowledge about medicines, yet this level of knowledge does not match the level of pharmacists. For example, in Norway, nurses specializing in geriatric nursing take over many tasks that clinical pharmacists currently perform or could perform due to their expert knowledge of pharmaceuticals. In Denmark, some of the regions have decided to put the responsibility on general practitioners to do medicine reviews for their patients where pharmacists play only a minor administrative support role. The rise of the related medical specialty of clinical pharmacology [8] has both been a help and a hindrance as the medical counterparts have either been very willing to cooperate or have worked openly to oppose clinical pharmacy from advancing in the healthcare system [9].
A Wide Range of Knowledge
Increased focus on medicines management for nurses and medical doctors can be seen as a natural progression since they increasingly deal with a heavy drug burden, particularly in the elderly [10]. Such a natural progression to fulfill the needs of patients does not follow for pharmacists. The pharmacy profession as a whole is not sufficiently patient-focused to break new ground, being more occupied with the intricacies of systems of drug distribution (i.e., product-focused) [11,12].
Another important challenge lies in the fact that clinical and social pharmacy span a wider range than other disciplines within pharmacy, such as pharmaceutics and medicinal chemistry. This leads them to be more fragmented and have a harder time covering what is needed to teach within these areas [13]. Researchers addressing the topics within social pharmacy have major challenges due to the many different areas of investigation and application [1].
Weak Profession
During the last decades, legal and structural pharmacy ownership reforms in three of the Nordic countries have shown that the pharmacy profession is somewhat weak and divided [14]. Consequently, attempts to increase professionalism within community pharmacies were easily foiled by new commercial owners. Clinical pharmacy was mostly absent from these legal and structural reforms and discussions. One can only wonder how the clinical pharmacy discipline could have contributed to the discourse by arguing for a more distinct role of the pharmacist in safe and effective medicine use in the primary community pharmacy sector.
Favorable Job Market
One important common threat to both clinical and social pharmacy in many countries is a very favorable job market for pharmacists. In Norway, for instance, community pharmacy chains are in great need of labor. Practically every pharmacist can get employment without making efforts either to update their knowledge or to work with patients on their medicine use, i.e., practicing pharmaceutical care and taking responsibility for the patient's drug-related needs [15].
The challenge an abundant and fragmented job market poses is two-fold. On the one hand, there is no push to develop the field of clinical pharmacy in the primary care sector. On the other hand, pharmacists lose even more ground to de-professionalization in community pharmacies due to increasing proportions of the revenue being linked to beauty and well-being products. In countries with a strong pharmacy industry employing many pharmacists, the competitive environment can shift quickly, leaving pharmacists with the challenge of job loss.
Understanding the Environment
It is therefore imperative to understand the forces in play. To counteract powers shifting the professional services over to commercial well-being products, clinical and social pharmacy should collaborate on increasing understanding and preparing to meet new challenges. Some of the most important new challenges are: the aging of the population and increased complexity in medicine use and polypharmacy; shortened hospital stays coupled with an aging population, which make a smoother sector transition more imperative than ever before; increased cultural diversity challenging the health information and education of patients; increasing use and focus on high-cost drugs (biologicals) in society and the rational use of these by prescribers and patients. These new challenges increase the need for more clinical pharmacy skills in primary care and the increased collaboration of pharmacists across sectors with each other and with other health care professionals.
Pharmacy Schools
University structures are traditionally divided into faculties, institutes and departments. Internal politics and a lack of understanding for the common ground sometimes limit or even counteract the collaboration of clinical and social pharmacy, thus weakening both fields. The disciplines should be aware of how the healthcare system perceives pharmacists' abilities to contribute to patient care. Political and organizational aspects, pharmacist-patient communication, and inter-professional communication can be studied using social pharmacy approaches within the "territory" of clinical pharmacy. If this understanding is strong, the clinical pharmacy projects have a greater chance of being accepted. Thus, research in social pharmacy can help to study the important opportunities and challenges facing clinical pharmacy within healthcare. If this understanding is lacking, it makes research of clinical pharmacy services within healthcare difficult.
The Push to Do Research
Social pharmacy is first and foremost a research discipline without a practitioner group with this as a specialization. Social pharmacists, however, have often worked with or supervised community pharmacists. Clinical pharmacy is primarily constituted by practitioners with less emphasis on research and teaching. For the medical profession, however, it has become vital, as clinicians in the hospital environment must publish research results in peer-reviewed journals to be recognized as a strong specialty inside the profession. This trend is also visible within primary care and general practice medicine. It is therefore a weakness that most clinical pharmacists do not do research as a regular part of their practice or only carry out in-house development projects. Even though projects may result in posters presented at practitioner conferences, they do not get published for the wider health professional research arena. Many pharmacists take post-graduate degrees in clinical pharmacy. These degrees focus primarily on becoming better practitioners with less emphasis on research. Combining a PhD and clinical work is increasingly becoming more common, but still not to the extent seen in the profession of medicine.
Priorities in Hospital Pharmacy
Hospital pharmacy in most countries still prioritizes logistic distribution of drugs above clinical pharmacy services. This is quite natural, as the income of most hospital pharmacies depends solely on this area and they are not necessarily remunerated specifically for providing clinical services. Notably, American clinical pharmacists have often been paid specifically for services by the "users" themselves (i.e., the hospital wards), which has made for growth in the USA [16]. Another important and related leap was taken by US hospital pharmacists at the Hilton Head Consensus Conference in 1985 where hospital pharmacy made the unified decision that hospital pharmacies should function as clinical departments with a mission of fostering the appropriate use of medicines. This was a very important idea because most hospital pharmacists thought in terms of adding discrete clinical services rather than conceptualizing the totality of the department's work as a clinical enterprise [17].
Cross-financing of clinical services with revenues from drug sales and drug distribution naturally slows down the expansion of clinical services to hospital wards [18]. As the hospital pharmacy managers have this focus, there is no impetus to prioritize research-let alone encourage clinical pharmacists on staff to enter PhD programs or to seek adjunct faculty positions at universities. In contrast, medical doctors neither have to excuse themselves for performing research nor have to provide evidence that they are cost-effective in their professional role.
Key Differences between Clinical and Social Pharmacy
Historically there are clear differences with respect to at least four facets of these two disciplines: the levels of study within pharmaceutical sciences, the location where the research has been conducted, the choice of designs and methods, and the use of theories.
Levels of study within pharmaceutical sciences: The research questions in clinical pharmacy are often at the pharmacological organ-level or study particular pharmacokinetic and pharmacodynamic questions. In social pharmacy, the focus has been more on groups and society at large. Location: As clinical pharmacy had its infancy in hospitals, its research was also primarily conducted within this realm. Traditionally, Nordic research within these two fields has been divided so that social pharmacy researchers study community pharmacy with a stronger academic profile, whereas clinical pharmacists study hospital pharmacy and are less inclined to connect directly with faculties of pharmacy. Choice of research designs and methods: There have been some differences in preferred research designs between clinical and social pharmacy research. Clinical pharmacy primarily focused on the efficacy and safety of selected medicines or pharmacology-related research questions. Researchers have therefore been prone to using randomized trials and quantitative methodology in general. Social pharmacy research, on the other hand, has used a broader palette of designs and methods from the social sciences. Theoretical and methodological foundations: Lastly, clinical pharmacy research has, to a very small extent, been based on theoretical foundations other than biological or epidemiological models. Social pharmacy, in contrast, has historically leaned more towards a social science theoretical approach and a range of qualitative research methods.
Common Strengths and Opportunities
As academics and researchers, we currently observe that the traditional differences between clinical pharmacy and social pharmacy are shrinking. Gradually, clinical pharmacists recognize the need to expand their repertoire of research designs, methods, and theories in order to understand the organizations and the patients they work with. Social pharmacy needs to broaden the research activities to studying pharmacy as it is practiced in a wider context-including institutional pharmacy. Increasingly, hospitals and communities are no longer two separate worlds. It is currently vital to study the patients as they move across the healthcare system, be it in hospitals, home care or primary care.
With the emphasis on medicine use by patients, a clinical mindset does not preclude pharmacists from understanding medicine use at higher levels than the individual patient. There is a need for an evidence base for the use which comes through knowledge about randomized controlled trials (RCT) and pharmacoepidemiology. To increase effective and safe patient care, pharmacoepidemiology and pharmacovigilance can define the problems clinical pharmacy should tackle. In order to operate in times of cost-cutting, clinical pharmacists also need to have a strong understanding of the organization and society they operate within. It is therefore unnatural to make the distinction between social pharmacy and clinical pharmacy when studying how pharmacy makes inroads into new clinical territories.
A prime example of the importance of understanding the environment comes from the hospital pharmacy enterprise in the Central Norway Regional Health Authority that has worked strategically to include clinical pharmacists in hospitals [19]. The increased role of pharmacy in patient care can be attributed to those pharmacists who were in charge of forging ahead, armed with a thorough understanding of the system they worked within. They were able to use this knowledge to influence the Regional Health Authority to understand how pharmacists could be used as a drug knowledge resource in hospital services, securing the seamless transfer of medicine information throughout the hospital stay and back into the community.
Pharmacoepidemiology-historically mostly dealt with in social pharmacy research-has recently become relevant also in clinical pharmacy. For instance, many abstracts and a plenary session at the 30th International Society for Pharmacoepidemiology (ISPE) conference addressed clinical pharmacy and patient perspectives in pharmacoepidemiologial research. Similarly, at the latest conference of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR), abstracts regarding clinical pharmacy research had a sizeable presence.
The Nordic countries all have individual level prescription databases generated from prescriptions dispensed in community pharmacies [20]. Others also generate data on multi-dose dispensed drugs to assess prescribing quality [21]. These have been used extensively by social pharmacy researchers to gain insights into the rational use of medicines and clinical pharmacists are starting to utilize the possibilities of performing research using these data [22]. Social pharmacy researchers have performed research on databases and identified problems in the quality and safety of medicine use. Clinical pharmacists are well situated to identify patients who have a need for tailored interventions. Increased collaboration between these two can be instrumental in furthering outcomes research and making it more patient-focused and relevant for healthcare. Clinical pharmacists are seen as patient safety experts when it comes to medications. Their research collaboration with social pharmacy could thus result in the design of "off the rack" solutions for increasing safe medication use in hospitals and in primary care. These types of solutions, when well defined and described, can then be applied to other healthcare professions (technicians or nurses) for routine implementation.
How to Join Forces
We argue that the reconciliation of forces and strengthening of common ground in clinical and social pharmacy can be developed in two main areas: (1) Research within these two sister areas increasingly requires multi-disciplinary input. The disciplines can combine the strong methodological and theoretical foundation (in social pharmacy) and the strong foothold in practice (evident in clinical pharmacy) to work with medicine, nursing, social sciences, and health services researchers. (2) The political aspects of the work going on to enhance rational medicine use require both internal and external key actors. Internally they must unite at the institution level (university, hospital, pharmacy, pharmaceutical societies) and put forth what is needed in education, practice, and research. By not having a united front towards external actors deters them from lobbying and speaking in one voice to policy-makers and other professions.
It is imperative that the message about the ability of the profession to take on more responsibility in patient care is not diluted by focusing on what is "different". We rephrase Harding and Taylor's [3] view on social pharmacy: that it is not the sociology of pharmacy, but rather pharmacy's use of the social sciences to further its usefulness to society.
Conclusions
What's in a Name?
It is important to emphasize what unifies the disciplines of social and clinical pharmacy. No longer can it be stated that clinical pharmacy serves as a bridge between social pharmacy and the rest of the pharmaceutical sciences. It increasingly incorporates many of the same research questions, designs, methods, healthcare system interests and professional autonomy issues as social pharmacy. Social pharmacy, on the other hand, has a need to get closer to practice in order to understand and study the important research questions of today.
So, what's in a name? Juliet argues that it does not matter that Romeo is from her family's rival house of Montague-that he is named "Montague". We argue that it is indeed time to unite the families of clinical pharmacy and social pharmacy for the benefit of both fields, pharmacy in general, and society at large. | 2016-04-04T08:54:49.290Z | 2015-12-22T00:00:00.000 | {
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3185621 | pes2o/s2orc | v3-fos-license | Edinburgh Research Explorer Associations between Season and Gametocyte Dynamics in Chronic Plasmodium falciparum Infections
Abstract
Introduction
In a markedly seasonal malaria setting, the transition from the transmission-free dry season to the transmission season depends on the resurgence of the mosquito population following the start of annual rains.The sudden onset of malaria outbreaks at the start of the transmission season suggests that parasites persist during the dry season and respond to either the reappearance of vectors, or correlated events, by increasing the production of transmission stages.Here, we investigate whether Plasmodium falciparum gametocyte density and the correlation between gametocyte density and parasite density show seasonal variation in chronic (largely asymptomatic) carriers in eastern Sudan.
Materials and Methods
We recruited and treated 123 malaria patients in the transmission season 2001.We then followed them monthly during four distinct consecutive epidemiological seasons: transmission season 1, transmission-free season, pre-clinical period, and transmission season 2. In samples collected from 25 participants who fulfilled the selection criteria of the current analysis, we used quantitative PCR (qPCR) and RT-qPCR to quantify parasite and gametocyte densities, respectively.
Results and Discussion
We observed a significant increase in gametocyte density and a significantly steeper positive correlation between gametocyte density and total parasite density during the pre-clinical
Introduction
In many areas, malaria transmission is seasonal and limited to a short window of time, often a few months, depending on availability of mosquito vector breeding habitats [1][2][3], temperature and relative humidity [4][5][6].During the transmission-free dry season, Anopheles mosquitoes are often undetectable or at extremely low density and confined to a few refugia.This occurs in endemic areas across the Sahel [7], Sudan [2,8], Senegal [1], Burkina Faso [9] and The Gambia [10].Following the start of annual rains, vector densities increase rapidly [10], suggesting that they persist locally, possibly via aestivation [8], or appear after long-distance migration [11,12].However, it takes at least 2 months after the start of rains for the appearance of new malaria cases [2,13].The gap between the revival of mosquitoes and appearance of clinical cases of P. falciparum-here denoted the pre-clinical period-is observed in areas with a short transmission season, such as eastern Sudan [1,2,10].
Whether P. falciparum modulates gametocyte investment in response to the appearance of vectors at the start of a new transmission season is unknown.Studies on rodent models have yielded contradictory results for whether malaria parasites respond to probing by mosquitoes by increasing gametocyte investment [34,35].A more recent experiment using an avian malaria parasite species revealed that parasites responded to mosquito biting by enhancing transmission, but whether this involved an increase in gametocyte investment is unknown [36].If P. falciparum parasites adjust their transmission strategy in response to the availability of vectors, this could significantly impact malaria transmission dynamics, particularly in seasonal malaria settings, by enabling parasites to maximise transmission opportunities.
Here, we study naturally occurring human infections of P. falciparum in an area of distinct seasonal transmission in eastern Sudan.Our aim is to assess whether gametocyte production shows seasonal variation coinciding with the limited duration for transmission.Specifically, we hypothesise that if parasites respond to, or prepare for, the start of the transmission season, then gametocyte density and the slope of the correlation between gametocyte density and parasite density (i.e. the proportion of parasites that are gametocytes) will increase during the preclinical period compared to the preceding transmission-free season.
Study area
The study was carried out in Asar village (longitude 358.300E and latitude 138.300N), Gedaref state, eastern Sudan.P. falciparum is the predominant malaria parasite, accounting for more than 90% of all malaria infections.Anopheles arabiensis is the main mosquito vector.The entomological inoculation rate is less than one bite per person during the transmission season [37].
Characterisation of seasons
We defined four distinct seasons based on a combination of records for temperature, rainfall, humidity, Anopheline abundance and incidence of malaria cases as described by previous studies at the study site [2,37].Anopheles mosquitoes generally appear in late July to early August reaching peak densities in late September/October.Malaria cases are usually reported between late September and November with a peak in October [2,37].By January, the number of malaria cases drops substantially.Previous entomological surveys have shown no evidence of transmission during the long dry season [2,37].The pattern of rainfall during the study period was as the following: in November 2001 (the start of study recruitment), the monthly average rainfall was 1. to increase humidity, creating optimum conditions for mosquito breeding and larval development [5].Therefore, for our study period we defined four seasons of epidemiological importance as: (a) transmission season 1 (November and December 2001) with mosquitoes present and malaria cases reported; (b) transmission-free season (January to late July 2002) without Anopheles mosquitoes or malaria cases [2]; (c) pre-clinical period (August to late September 2002) with the presence of Anopheles mosquitoes but before clinical cases are reported [2]; and (d) transmission season 2 (October to December 2002), with the presence of Anopheles mosquitoes and clinical cases [2] (Fig 1B).
Study design, subjects and samples
The details of enrolment of subjects, sample collection and processing are reported in [26].Briefly, during November 2001 (start of transmission season 1), a cohort of 123 symptomatic malaria patients, aged ! 10 years, with positive P. falciparum blood films was recruited to the study and treated with chloroquine and/or sulfadoxine/pyrimethamine according to malaria treatment guidelines at the time of the study [26].Venous blood (2 ml) was collected in heparinised tubes from each participant before treatment and during the monthly follow up visits until December 2002 (except November 2002) [26].Blood samples were centrifuged, plasma was separated from the blood cells at the study site and samples were immediately preserved in liquid nitrogen and transported to University of Edinburgh, where they were kept at -80˚C.Ethical clearance for sample collection in 2001 was obtained from the Ethical Committee of the Ministry of Health, Sudan.Blood samples were collected with written or oral informed consent of all patients or their parent/ gradient.For the current analysis no ethical approval was sought, as samples were archive samples [26].
Exclusion/inclusion criteria
Of the 123 recruited participants, 25 individuals with the following infection characteristics were included in the present study: (1) Sustained chronic sub-microscopic parasite densities, determined as at least 3 time points during the study that were parasite-positive by qPCR.Fourteen (56%) participants had 7 to 12 positive time points, 5 (20%) participants had 5 to 6 positive time points and 6 (24%) participants had 3 to 4 positive time points.(2) No evidence of new infections during the pre-clinical period (August and September); in terms of either sudden appearance of high parasite density or appearance of new parasite clone.(3) No self-treatment with anti-malarials during the period between June and September, to rule out any possible effect of drug on gametocyte density during the pre-clinical period.(4) Participants who received treatment (n = 2) or who developed new infections (identified by presence of new alleles and high parasitaemia) during transmission season 2 were not excluded from the analysis because their gametocyte density during the transmission-free season and the pre-clinical period could not be affected by future drug treatment.
Over the follow up period we analysed 270 samples from the 25 eligible participants.There were 30 missing samples across 12 different time points.For the current analysis, only data from samples collected after drug treatment were described (i.e.samples collected at enrolment in November 2001 were excluded).
Quantification of parasite and gametocyte densities
The total number of parasites in the samples was quantified from DNA extracted in 2002 (using the Chelex method) from blood spotted onto filter paper [26].Only 64 time points had missing DNA samples; for those samples DNA was re-extracted in 2012 from stored frozen blood cells using the QIAamp DNA mini kit (Qiagen).P. falciparum 18S ribosomal RNA gene copy number was quantified by qPCR as described in [38], except using TaqMan Universal PCR Master Mix (ThermoFisher) and probe concentrations reduced to 100nM.18S copy numbers were converted to parasite numbers using a calibration curve generated from P. falciparum clone 3D7 parasite DNA, a range of 0.014-138938 parasites/μl of DNA.The 18S qPCR amplification efficiency was 97.7% (s.e.0.01%), the inter-assay variability (as a measure of qPCR reproducibility, but not uncertainty that may arise from blood sampling low level of parasitaemia [39]) between standard curves efficiency was <5% at all densities and the correlation between log 10 parasite numbers and Cq values was significant (adjusted R 2 >0.98 for all PCRs with P <0.001).The lower limit of detection was 0.139 parasites/μl of DNA corresponding to 0.1 parasites/μl of blood.Total parasite density was quantified in duplicate for each sample and the correlation between duplicate Cq values was significant (R 2 = 0.95, P <0.001).
To quantify gametocytes, RNA was extracted in 2002 using the High Pure RNA isolation kit (Roche) [26,40] and stored at -80˚C until used for the current study.RNA was extracted for a few missing RNA samples using the SV Total RNA Isolation System (Promega, UK) in 2012.Extracted RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher, UK).From the cDNA, expression of the pfs25 (female gametocyte specific) and pfs230p (male gametocyte specific) genes were quantified by PCR as described in [41], with the exception that a TaqMan probe (6FAM-ACTGGAATCGAACAACA-MGB, 250nM) and FastStart Taq dNTPack (Roche) were used to quantify male gametocytes.Levels of pfs25 and pfs230p in the cDNA samples were converted to female and male gametocyte counts, respectively, using calibration curves of in vitro DNAs (PCR amplified sequence of DNA spanning the region of the qPCR amplicons) and their relationship to P. falciparum female and male gametocyte counts from in vitro culture [41].The female and male gametocyte RT-qPCR assays performed with amplification efficiencies of 94.2% (s.e.0.03) and 89.6% (s.e.0.02) respectively.Inter-assay variability for both assays was <6% at all densities, and log 10 male or female gametocyte numbers and Cq values were significantly correlated (adjusted R 2 >0.98 for all PCRs with P <0.001).The correlation between duplicate quantification of the same samples was strong (R 2 = 0.950, P <0.001; 10% of samples randomly tested).Total gametocyte numbers were calculated by summing the male and female gametocytes, with a lower limit of detection of 0.5 gametocytes/μl of blood.
Because DNA was extracted from unknown amounts of blood spotted on filter paper and RNA was extracted from 50uL of blood cells (after plasma removal), the association to full blood volumes could not be simply determined.Therefore, absolute quantification of parasiteand gametocyte densities per μl of blood was not possible.Instead, parasite and gametocyte counts were normalised against human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).GAPDH was quantified as described in [42], with a modified reverse primer (SCTGGCGACG CAAAAGA; S = G or C).GAPDH qPCR and RT-qPCR reaction volumes, mastermixes and probe concentrations were the same as those for the 18S PCR and pfs25 RT-PCR, respectively.Between-individual potential differences in GAPHD, were controlled for by using patient identification (ID) (categorical variable) as a random effect in the statistical analysis.
Statistical analysis
Generalized linear mixed models (GLMMs) [43] were used to estimate the changes in parasite prevalence, total parasite density, gametocyte prevalence and gametocyte density across seasons.These parameters were used as response variables in different models and allowed to vary between participants by including participant ID as a random effect.This, both increases the ability to detect changes over time as there is substantial inter-participant variability, and also accounts for the missing time points by supplying a predicted value.Total parasite density and gametocyte density were analysed on the same scale in which they were estimated (i.e.logarithm to base 10) to reduce biases caused by diagnostic measurement error.Sample processing differences and different WBC reference samples (healthy volunteers from Oman (n = 5) for DNA and from the UK (n = 10) for RNA) that were used for normalisation of gametocyte and parasite counts resulted in relative quantification differences between gametocytes (RNA) and total parasites (DNA) from the same blood sample.Whereas parasite and gametocyte dynamics for each participant can be followed over time, absolute densities of parasites and gametocytes of the same sample are not directly comparable.The relative difference would lead to the appearance that some samples had a greater number of gametocytes than total parasites.Therefore both gametocyte and total parasite densities are described as arbitrary units, denoted as parasites or gametocytes /μL blood DNA or /μL blood RNA to emphasize these are not absolute quantities and are not directly comparable between DNA and RNA samples.
A variety of error distributions (negative binomial, zero-inflated negative binomial, zeroinflated Poisson and zero-inflated normal) were tested to describe patterns for total parasite density and gametocyte density.A zero-inflated normal distribution gave the most parsimonious fit and was used for both of the continuous dependent variables.Parasite prevalence and gametocyte prevalence were fitted to binomial distribution models.Models were built using forward stepwise selection, and Log likelihood Ratio Tests (LRT) were used to determine the most parsimonious model.If more than one model fitted the data, the one with the lowest Akaike Information Criterion (AIC) was selected.R program version 3.0.1 and glmmADMB package [44] were used for the analysis.
For parasite prevalence and density models, fixed effect variables included season (categorical variable), and for gametocyte prevalence and density models fixed effect variables included season (categorical variable), total parasite density, and their interactions.The most parsimonious models included season (for parasite prevalence and parasite density); total parasite density (for gametocyte prevalence); and season, total parasite density and the interaction between them (for gametocyte density).
Seasonal variation in parasite prevalence and density
Parasite prevalence and density data are presented in Table 1, alongside the estimates from our statistical models, in which between-participant variation has been controlled for.The average estimated parasite prevalence among the participants (n = 25) was 81.3% (s.e.= 2.48) one month after drug treatment at the end of transmission season 1 (December 2001).Prevalence varied across the seasons (X 2 3 = 8.966, P = 0.029).The average estimated prevalence decreased significantly in the transmission-free season (X 2 1 = 7.71, P = 0.005) compared to December 2001, and stayed at a similar level during the pre-clinical period (X 2 1 = 0.64, P = 0.423) compared to the transmission-free season (X 2 1 = 0.318, P = 0. 0.573; Table 1).There was no significant difference in parasite prevalence between the pre-clinical period and transmission season 2 (X 2 1 = 0.186, P = 0.666; Table 1).The average estimated parasite density was 1.63 log 10 (s.e.= 0.04) parasites/μl blood DNA after drug treatment during transmission season 1, i.e.December 2001.Parasite density varied across the seasons (X 2 3 = 74.08,P < 0.001).Specifically, average estimated parasite density decreased during the transmission-free season (X 2 1 = 24.33,P < 0.001) compared to December 2001 (Table 1).The average estimated parasite density remained similar between the transmission-free season and the pre-clinical period (X 2 1 = 0.99, P = 0.320) and increased from the preclinical period to the transmission season 2 (X 2 1 = 5.08, P = 0.024) (Table 1).
Discussion
The unique epidemiological setting of eastern Sudan, where malaria transmission occurs over a period of 8 to 10 weeks, and then ceases for 9 to 10 months, provides an opportunity to examine the hypothesis that seasonal variation in gametocyte density coincides with temporal fluctuations in transmission opportunities.This epidemiological setting spans the poor savannah belt of Sub-Saharan Africa [1,2,[7][8][9][10], where annual rains are restricted to few months and the rest of the season remains hot and dry.Among the examined cohort, densities of P. Gametocyte density is presented in arbitrary units, denoted as μL blood RNA and is not directly comparable to total parasite density due to differences in sample processing and normalization to different WBC calibration curves.doi:10.1371/journal.pone.0166699.g002falciparum total parasites and gametocytes fluctuated at sub-microscopic levels (0.64 log 10 total parasites/μl blood DNA and 0.36 log 10 gametocytes/μl blood RNA , on average) in the transmission-free season between January and July 2002.However, following the start of the annual rains in the pre-clinical period (August to September 2002)-prior to the appearance of clinical cases-gametocyte densities increased while total parasite density remained at levels similar to the transmission-free season.This suggests an increase in gametocyte investment, which is further supported by the steeper correlation between gametocyte density and parasite density during the pre-clinical period compared to the transmission-free season.
The stability of parasite prevalence, density and gametocyte prevalence in the pre-clinical period compared to the transmission-free season suggests that the increase in gametocyte density is unlikely to be due to the acquisition of new infections.Instead, we suggest that P. Correlation between parasite and gametocyte densities across seasons.The correlation between gametocyte and parasite densities varied across the seasons (i.e.there is a season by parasite density interaction).During the pre-clinical period, a strong and positive correlation was observed.This suggests that a larger proportion of total parasites are gametocytes during the pre-clinical season, compared to the other 3 seasons.Points represent raw data; lines represent the best-fit between values of log 10 parasite and gametocytes densities as classified by season.Arbitrary numbers are used to present gametocyte and total parasite densities, denoted as blood RNA and blood DNA , to account for sample processing differences that might result in the appearance that some samples contain more gametocytes than total parasites.doi:10.1371/journal.pone.0166699.g003falciparum parasites may respond to, or prepare for, the appearance of transmission opportunities by up-regulating investment into gametocytes (i.e.increasing conversion rate).Data from animal models shows that P. relictum responds to mosquitoes biting their avian host by increasing transmission potential [36] but whether these parasites detect mosquitoes directly (e.g.salivary proteins) or indirectly via a host reaction to bites (e.g.immune responses to salivary proteins) is unknown.P. falciparum may also respond to mosquito biting or, alternatively, respond to seasonal changes in host physiology.Seasonal changes occur in many aspects of human physiology and behaviour, including immune responses [45,46].How parasites sense environmental change and translate this information into a change in phenotype is unclear but it has been suggested that Ca 2+ dependent protein kinases are involved in activation of stage-specific development [47,48].
Variation in gametocyte density during infections has been linked to many in-host environmental and parasite genetic factors, including anti-malarial drug treatment [32,49,50], anaemia [25,27,28], and the presence of multiple genotypes within an infection [26].Seasonal variation in these factors is likely to occur and could obscure or confound parasite responses to transmission opportunities.For example, new infections are more often symptomatic [2] and thus often result in drug treatment and/or anaemia [51].New infections can also increase the multiplicity of existing infections [52] which has been linked to prolonged carriage of gametocytes [26].We can probably rule out a direct impact of drug treatment as a confounding factor because participants who self-treated during June to September (prior and during the pre-clinical period) were excluded from analysis.All individuals received drug treatment at enrolment, and 20/25 participants' received no further treatment during the transmission-free or the preclinical periods.However 5 individuals received self-treatment at the start of the transmissionfree season, but none of them showed clinical symptoms beyond February 2002.The parasites in these individuals have apparently survived drug treatment, and could potentially be drug resistant.However, genotyping of drug resistance mutations (pfcrt codons 72-76 and pfmdr1 codon 86) linked to chloroquine resistance showed a significant decrease in the proportion of drug-resistant parasite genotypes and parallel significant increase in wildtype from transmission season to the pre-clinical period among participants treated with chloroquine (data not shown).In the same period, gametocyte production increased and therefore, the pattern we observed cannot be attributed to increased gametocyte production by chloroquine resistant parasites.Anaemia and haemoglobin levels were not measured, and we cannot exclude potential effects of seasonal variation in haemoglobin levels that may occur due to e.g.diseases or physiological changes.However, if only malaria infections were to be considered, it is unlikely that haemoglobin levels would vary significantly between the transmission-free season and pre-clinical period as a result of sub-microscopic asymptomatic malaria infections.With regard to parasite multiplicity, it has been demonstrated that multiplicity among asymptomatic carriers can decrease over time [26]; we expect this to have remained stable during the relatively short period between the transmission-free season and pre-clinical period in the absence of new blood-stage infections.Therefore, we predict that seasonal variation in other aspects of the in-host environment or biting by mosquitoes provides parasites with more accurate information on transmission opportunities.
In addition to uncovering the information parasites may use, it is also necessary to test whether they respond in a manner that maximises fitness [53].For example, does our observation of a decline in both total parasite and gametocyte densities from the pre-clinical period to transmission season 2 suggest that parasites prematurely increased gametocyte production?This is unlikely because a number of factors could reduce parasitaemia and gametocytaemia in the transmission season, including the acquisition of new infections that boost immune responses and the indiscriminate use of anti-malarials for any malaria-like symptoms [54,55].
Instead, we suggest that transmission of gametocytes occurs as soon as mosquitoes are available because the time frame required for gametocytes to be produced, mature, and undergo sporogony in mosquitoes, plus the exo-erythrocytic development of malaria in the host, corresponds to the lag between the appearance of mosquitoes and clinical cases [56].
Our main observations are supported by epidemiological surveys in other sites where annual rains and malaria transmission are short and highly seasonal [1,2,7-10], presenting circumstantial evidence for seasonality in the carriage of P. falciparum gametocytes [3,9,57,58].For example, a longitudinal study in Burkina Faso found that season is an independent determinant of gametocyte prevalence and density and that it is significantly higher in the wet season compared to the dry season [9].Furthermore, analysis of three historical data sets from different regions with seasonal malaria (Thailand, Tanzania and Nigeria) suggests that the intense seasonal pattern of uninfected mosquito bites during annual rains is associated with elevated gametocyte prevalence and can ignite transmission [59].More recently, it has been shown that gametocytes produced at the start of the transmission season in Burkina Faso are more infectious to mosquitoes than those during the peak of the transmission season or the dry season [56].
In our study we have observed similar gametocyte prevalence during the transmission-free and the pre-clinical periods.Indeed, gametocytes must be present to obtain any transmission, however increasing gametocyte densities generally improves transmission success particularly at very low gametocytaemia [60,61] and therefore also represents a fitness benefit to the parasite.The increase in gametocyte density but not prevalence can be attributed to three main factors, as the following: (a) the small cohort and the possibility that not all infections (isolates) are committed to gametocyte production (3 participants did not show gametocytes throughout the study period).Early in vitro studies suggested that P. falciparum isolates could vary significantly in their capacity to produce gametocytes [18,62].(b) Due to periodicity of gametocytogenesis we may not have picked up all gametocyte producer infections at all sampling times.(c) Owing to the highly sensitive methods used for detection, the proportion of samples with gametocytes below the detection limit is small in all seasons, and therefore gametocyte prevalence is unlikely to be statistically different between seasons.
Some limitations of our study include first, our sampling method precluded estimates of the gametocyte conversion rate.Estimating conversion rates remains inherently difficult in natural Plasmodium infections, because of overlapping cohorts of gametocytes circulating at the same time, which makes it difficult to establish which proportion of gametocytes originates from a specific asexual replication cycle 10-14 days earlier [63].Instead we have taken a commonly used approach and investigated the association between gametocyte and total parasite dynamics.The increase in gametocyte densities in absence of changes in asexual densities from the transmission-free to the pre-clinical season, and the observed steeper slope of the correlation between total parasite and gametocyte densities support the hypothesis of increased gametocyte production during the pre-clinical period.Second, entomological data were not collected during this study and the seasons have been defined based only on weather data (rains and temperature).However, the weather data were consistent with previous reports in the study village, in which a uniform pattern of annual rains and appearance of mosquitoes was shown [2].We therefore believe that our definition of season, based on weather data, is sufficient to link the pre-clinical period (August-September) to the expected appearance of mosquitoes.Third, sample sizes were limited by ethical constraints and the low prevalence of infection in the area.Limited sample sizes, combined with inter-assay variability in qPCR quantification of parasites, can make data analysis difficult.However, these issues are reflected in our error estimates, and did not prevent the detection of a significant pattern.
In summary, in eastern Sudan, an area of distinct seasonal transmission, P. falciparum sustains chronic asymptomatic infections throughout the transmission-free season, characterised by low parasite densities, but producing gametocytes.Despite, some limitations to our study, we have shown that gametocyte production increases after the transmission-free season.This suggests that in areas of seasonal malaria, the parasite may have evolved to recognise vector abundance, or a proxy for it, and responds in a manner that maximises transmission opportunities.Control measures aiming at reducing asymptomatic carriage, during the transmissionfree season, should therefore have a significant impact on control of cyclical malaria in such areas.
2 mm/month.There was no rain detected between December 2001 and February 2002.In March, April and May 2002 the average rainfall was 9.4, 4.6 and 2.2 mm/month, respectively.The main annual rains started in June 2002, with an average rainfall of 95.7 mm/ month, and increased to reach a peak of 290 mm/month in August 2002 (Fig 1A).The highest and lowest temperatures recorded during the study period were 43.3˚C (May 2002) and 21.0˚C (August 2002), with the latter coinciding with the peak of the rains (Fig 1A).The temperature increased again in September to a maximum of 37˚C, which combined with the rains
Fig 1 .
Fig 1. Seasonality of annual rains, mosquito abundance, and categorisation of seasons.(A) Monthly average rainfall in mm (blue line), maximum (red line) and minimum (green line) temperature in the study area between November 2001 and December 2002 (Meteorological Authority, Sudan).Mosquito symbols indicate the expected appearance of mosquitoes (July 2002) and peak mosquito densities (October 2002).(B) Distinct epidemiological phases of malaria transmission; transmission season 1 (November to December 2001), transmission-free season (January to July 2002), pre-clinical period (August and September 2002) and transmission season 2 (October and December 2002).doi:10.1371/journal.pone.0166699.g001
e
Model estimates are shown, which are more informative than raw data because of inter-participant variation and, for gametocyte estimates, also the effect of seasonal variation in parasite densities have been controlled for.f Significant difference compared to the previous season doi:10.1371/journal.pone.0166699.t001transmission-free season (X 2 2 = 11.03,P = 0.004), then increased during the pre-clinical period (X 2 2 = 9.18, P = 0.010), and decreased during transmission season 2 (X 2 2 = 11.75,P = 0.003) (Fig 2).
Fig 2 .
Fig 2. Association between gametocyte density and season.Gametocyte density, as predicted by the best-fit model, is significantly higher in the pre-clinical season compared to the transmission-free season and transmission season 2. Error bars represent the standard error of the mean.Gametocyte density is presented in arbitrary units, denoted as μL blood RNA and is not directly comparable to total parasite density due to differences in sample processing and normalization to different WBC calibration curves.
Fig 3 .
Fig 3. Correlation between parasite and gametocyte densities across seasons.The correlation between gametocyte and parasite densities varied across the seasons (i.e.there is a season by parasite density interaction).During the pre-clinical period, a strong and positive correlation was observed.This suggests that a larger proportion of total parasites are gametocytes during the pre-clinical season, compared to the other 3 seasons.Points represent raw data; lines represent the best-fit between values of log 10 parasite and gametocytes densities as classified by season.Arbitrary numbers are used to present gametocyte and total parasite densities, denoted as blood RNA and blood DNA , to account for sample processing differences that might result in the appearance that some samples contain more gametocytes than total parasites.
Table 1 . Prevalence and densities of total parasites and gametocytes in samples taken from 25 individuals selected during transmission 1. Season Transmission 1 Transmission-free Pre-clinical Transmission 2 (Only data from December 2001) January to July 2002 August and September 2002 October and December 2002 Presence of Anopheles mosquitoes [2,7]
a Total number of samples collected during each season from the 25 participants b Raw data are presented to describe the infection parameters as observed among the study cohort.c Number of participants with at least one positive sample during a season.d Mean (continuous variables) or percentage (categorical variables) and standard error (s.e.) are presented. | 2018-04-03T01:35:16.148Z | 0001-01-01T00:00:00.000 | {
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246775210 | pes2o/s2orc | v3-fos-license | Treatment of Graves' Disease Associated With Severe Neutropenia
Severe neutropenia in newly diagnosed hyperthyroidism is a diagnostic and therapeutic dilemma since antithyroid drugs (ATDs) cannot be started if the absolute neutrophil count (ANC) is <1 x 109/L. We report the case of a patient followed for hyperthyroidism associated with severe neutropenia treated with dexamethasone and ATD. The patient was 51 years old and was hospitalized for hyperthyroidism with a thyroid stimulating hormone (TSH) level <0.005 (0.4-4) mUI/L, T4 at 415 (9.3-17.1) ng/L and T3 at 148 (2-4.4) pg/mL on Graves' disease (GD) confirmed by the TSH receptor antibodies at 38 IU/mL and scintigraphy, associated with neutropenia, with ANC at 0.4 x 109/L. He was put on prednisolone 60 mg/day and propranolol 60 mg/day for three weeks without improvement. Faced with the association of hyperthyroidism and severe neutropenia, we could not start the ATD for fear of agranulocytosis; we put the patient on propranolol 60 mg and dexamethasone 6 mg with progressive degression resulting in a spectacular increase of ANC from 0.4 x 109/L to 7.1 x 109/L, which allowed us to start the ATD (carbimazole) at a dose of 30 mg, and then 50 mg, with monitoring of ANC and transaminases every 48 hours. Euthyroidism was achieved after 15 days. A curative treatment with radioactive iodine ablation was administered. Our patient did not respond to prednisolone but responded dramatically to dexamethasone; this leads us to consider using dexamethasone for the rapid preparation for radical treatment of patients with GD.
Introduction
Graves' disease (GD) was first described by the Irish physician Robert James Graves in 1835 [1]. It is a specific autoimmune disease of the thyroid gland affecting approximately 12% of the population, with a female preponderance. It is characterised by the presence of TSH receptor antibodies (TRAb) and T and B lymphocyte infiltration [2]. The mechanism that triggers the autoimmune response remains unclear, but a genetic predisposition interacting with environmental factors may be involved [3].
Treatment modalities for GD include prescription of antithyroid drugs (ATDs), surgery and radioactive iodine (RAI) therapy with iodine-131 (131I). The choice of treatment depends on several factors such as severity of thyrotoxicosis, size of goiter, response to treatment and presence of other comorbidities [4]. However, the treatment of first choice remains the ATDs, which have the effect of restoring euthyroidism [5][6].
ATDs are an effective and safe therapy for GD hyperthyroidism, but contraindications and adverse reactions may limit their use, including severe neutropenia, which is a well-known adverse effect of ATDs, and which may occur during treatment of hyperthyroidism [7]. This neutropenia may also be a sign of newly diagnosed hyperthyroidism, which is why the American Thyroid Association suggests that a complete blood count should be checked before starting treatment with ATDs and that they should not be started when the absolute neutrophil count (ANC) is <1 x 10 9 /L [4]. Therefore, the discovery of severe neutropenia in newly diagnosed hyperthyroidism is a major diagnostic and therapeutic dilemma.
We report the case of a patient admitted for severe hyperthyroidism associated with severe neutropenia treated successfully with dexamethasone and the ATD.
Case Presentation
The patient was 51 years old, admitted for hyperthyroidism, with a history dating back to nine months before admission, onset of a weight loss of 20 kg contrasting with polyphagia associated with palpitation, tremors of extremities and digestive disorders such as motor diarrhea. Clinical examination revealed a blood pressure of 150/70 mmHg, tachycardia at 100 bpm, temperature of 37°C, weight 65 kg with a weight loss of 20 kg, and bilateral exophthalmos with European Group on Graves' Orbitopathy (EUGOGO) stage 1 orbitopathy without pretibial myxedema. Cervical examination revealed a homogeneous, soft, pulsatile, grade 1 goiter with no irregularities and free lymph nodes.
In the etiological work-up of hyperthyroidism, TRAb were elevated at 38 UI/mL and scintigraphy showed a goiter under endogenous stimulation in favour of GD.
The patient was put on prednisolone 60 mg and propranolol 60 mg/day for three weeks before hospitalisation without improvement. We admitted him for the management of hyperthyroidism in the context of GD associated with severe neutropenia for which a myelogram was performed showing a rich marrow with numerous megakaryocytes. All lineages were represented with marked dysgranulopoiesis. A bone marrow biopsy also showed a normal rich marrow with non-specific maturation disorders.
Given the severity of the hyperthyroidism and the very high T4 levels, a paraneoplastic syndrome had to be ruled out, which is why the tumor marker assay (human chorionic gonadotropin, lactate dehydrogenase) was performed and was negative.
Given the combination of severe hyperthyroidism and severe neutropenia, we were unable to start ATDs for fear of agranulocytosis and scheduled plasmapheresis sessions in preparation for curative treatment.
While waiting for the response of the health care system with regard to plasmapheresis, we put the patient on propranolol 60 mg and dexamethasone 6 mg resulting in an increase of ANC from 0.4 x 10 9 /L to 7.1 x 10 9 /L, which allowed us to start the ATD (carbimazole) at a dose of 30 mg with monitoring of ANC and transaminases every 48 hours ( Table 1). We increased carbimazole to 50 mg while decreasing dexamethasone until it was stopped (
Evolution
After curative treatment with radioactive iodine ablation, thyroid balance was checked once a week for the first month and then once a month. After two months, the patient gained 18 kg and presented hypothyroidism for which a substitution with levothyroxine was started.
Discussion
The association between neutropenia and hyperthyroidism was first described by Caro in 1907 and confirmed one year later by Koche who proposed the triad of leukopenia, neutropenia and lymphocytosis as an early diagnosis of Graves' disease [8,9]. Subsequently, various studies were conducted to investigate the relationship between hyperthyroidism and neutropenia.
Coexistence of severe neutropenia (ANC ≤0.5 x 10 9 /L) with GD poses diagnostic and therapeutic challenges.
It has been shown that neutropenia associated with GD is most often mild [10]. Therefore, other etiologies of severe neutropenia should be ruled out before simply linking it to GD. Several studies have found an inverse correlation between thyroid hormone level and neutrophil count, so more severe hyperthyroidism appears to be a predictor of neutropenia [11,12].
In cases of GD with severe neutropenia, curative treatment with radioactive iodine or surgery can be considered but due to the high thyroid hormone levels, the risk of acute thyrotoxic crisis is not negligible.
Our patient had severe neutropenia prior to the initiation of treatment; the prescription of an ATD was not possible for fear of inducing agranulocytosis. And the introduction of a curative treatment was dangerous because the risk of acute thyrotoxic crisis was not negligible in view of the very high level of free T4.
Moreover, our patient did not respond to prednisolone but responded spectacularly to dexamethasone, which led us to think of using dexamethasone in Graves' ophthalmopathy and rapid preparation for the radical treatment of patients with GD, even if there exist no studies on this subject as per our knowledge.
Mechanism of neutropenia in hyperthyroidism in GD
The mechanism of neutropenia in GD remains unclear, but different theories could explain the close relationship between GD hyperthyroidism and neutropenia. Excessive thyroid hormones seem to significantly affect the proliferative potential of haematopoietic progenitor cells [13].
Some studies have suggested an autoimmune basis and shortened survival of peripheral neutrophils. The study by Weitzman et al. provided evidence that TRAb, and "classical" antineutrophil antibodies, may bind to the TSH receptor on neutrophils so that they can mediate neutrophils in some patients with GD [14]. The reduction in circulating neutrophils could be due to abnormal distribution and marginalisation, as shown in an animal study [15,16]. Adhesion molecules such as E-selectin promote the adhesion and aggregation of leukocytes to blood vessel walls. In one study, E-selectin levels were higher in patients with leukopenia than in GD patients with normal leukocytes. In addition, there was a negative correlation between leukocyte count and E-selectin levels. Interestingly, glucocorticoid (prednisone) treatment of patients with leukopenia resulted in a significant reduction in E-selectin levels [17].
Evolution of neutropenia
In a meta-analysis investigating the association between neutropenia and hyperthyroidism, in all patients with GD, treatment with ATDs and/or irradiation was also used to treat coexisting neutropenia [17]. The time to resolution of neutropenia in two studies was 14-55 days and occurred in parallel with the restoration of euthyroidism [10,18]. A similar time frame (60 days) was required to achieve the normalisation of severe neutropenia (ANC 0.2 × 10 9 /L) after treatment with carbimazole in a case study by Hegazi et al. [19].
Our patient did not respond to prednisolone but leukocyte levels increased dramatically after two days of dexamethasone that may have a more powerful effect on the demargination of neutrophils. The ANC continued to increase after the introduction of ATD and progressive normalisation of thyroid function.
Conclusions
The combination of hyperthyroidism in GD and severe neutropenia is a diagnostic and therapeutic challenge. The introduction of an ATD for an ANC < 1 x 10 9 /L is not recommended by current guidelines. In our patient, dexamethasone produced a spectacular increase in the ANC after 48 hours that allowed for the introduction of the ATD and euthyroidism within 15 days. This suggests that a dose of dexamethasone can be given as a test treatment in patients with hyperthyroidism associated with GD and severe neutropenia after the diagnosis of malignancy has been ruled out.
Additional Information 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. | 2022-01-09T16:10:33.157Z | 2022-01-01T00:00:00.000 | {
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219703705 | pes2o/s2orc | v3-fos-license | Diabetes Epidemiology in the COVID-19 Pandemic
Diabetes has been identified as an important risk factor for mortality and rates of progression to acute respiratory distress syndrome (ARDS) in hospitalized patients with coronavirus disease 2019 (COVID-19). However, many recent reports on this topic reflect hurried approaches and have lacked careful epidemiologic design, conduct, and analysis. Features of prior studies have posed problems for our understanding of the true contribution of diabetes and other underlying comorbidities to prognosis in COVID-19. In this Perspective, we discuss some of the challenges of interpreting the current literature on diabetes and COVID-19 and discuss opportunities for future epidemiologic studies. We contend that the COVID-19 pandemic is a defining moment for the field of epidemiology and that diabetes epidemiology should play a significant role.
Early reports from Wuhan noted a higher prevalence of diabetes among severe cases of COVID-19 (2)(3)(4). A high prevalence of diabetes was also noted in reports of COVID-19 hospitalizations in Italian populations (5,6).
Authors have responded to the pressing need for data in a short time frame. However, many studies, including those in published in major medical journals, reflect hurried approaches and have lacked careful epidemiologic design, conduct, and analysis. Features of these studies have posed problems for our understanding of the true contribution of diabetes and other underlying comorbidities to prognosis in COVID- 19. First, what are often being called retrospective cohort studies are actually large case series (7)(8)(9). These studies have consisted of convenience samples and selected patients for inclusion on the basis of their outcome statusdtypically death or survival. Analyses of these data are not truly prospective, as those patients who transferred out of the hospital or who were still in the hospital at the time of the analysis were not included. The reliance on hospitalized cases can also make the disease appear more severe, as more mild cases of COVID-19 are not captured in these samples.
Second, most studies to date have relied on data retrospectively obtained from electronic health records (EHR). This poses several challenges. EHR data are often inaccurate, rife with missing data, and reflective of selective testing. For example, a hospitalized COVID-19 patient with diabetes and a history of cardiovascular disease will be more likely to receive an echocardiogram or troponin testing than other patients. A patient with poorly controlled diabetes will have their glucose measured more often than a patient with good glycemic control who is otherwise similar. The lack of systematic laboratory testing or standardized assessment of risk factors can result in detection bias and other errors, producing spurious associations. Moreover, some studies have cherry-picked associations. For example, one prominent article examined the association of laboratory values on admission in COVID-19 patients with ARDS (2). In this study, the analytic population was restricted to COVID-19 patients with available laboratory measures (complete case analysis) and only those associations that met an arbitrary threshold for significance were reported in the article. Such an approach represents significant reporting bias.
Third, studies of comorbidities have lacked sufficient multivariable adjustment for characteristics of patients at baseline. There has been rampant use of stepwise regression in COVID-19 publications. Stepwise regression is inappropriate in this setting (10,11). To isolate the independent association of any single comorbidity, models need to include demographics and other confounding health conditions. Age and sex are known to be important risk factors for mortality in COVID-19. Yet some early and influential reports from Wuhan did not adjust for either age or sex (2). Also, some studies with age adjustment have modeled age as a simple linear term (4,7,9,12). Modeling age linearly may not fully account for its effect on mortality, as all evidence points to an exponential association (13)(14)(15)(16). Obesity and smoking are two important patient characteristics that are also typically poorly characterized in EHR data and have not been included as covariates in many studies.
Fourth, a number of studies have evaluated models that included measurements that change greatly during observation of an acute illness. The development and specification of some of these models have been problematic. For example, studies of risk factors for ARDS and death have adjusted for in-hospital laboratory measurements such as random glucose levels, white cell count, and troponin (17)(18)(19). These laboratory measurements are not risk factors in the usual sense, but they may be relevant to outcomes. In-hospital laboratory tests will reflect disease severity and progression, and their associations with outcomes may reflect reverse causality. Alternatively, some metabolic factors that are altered during the course of the disease may causally contribute to outcomes. Studies have identified baseline hyperglycemia, in-hospital glycemic control, and glycemic variability as risk factors for progression and death in patients with diabetes, yet careful measurements of glycemia have not generally been available (4,20). Elevated glucose and glycemic variability are common in acutely ill patients even when they were not known to have diabetes previously. It is not yet clear what these findings mean for the care of patients with COVID-19.
Fifth, some studies have been small with few clinical events, generating highly imprecise statistical estimates. Publication bias is a concern, as small studies with few events are only powered to detect large effects; this can result in false positive results.
Sixth, there have also been concerns about reporting results from the same patients in different articles or even duplicate publications (21). This is problematic for future meta-analyses, as investigators will not be able to determine the independence of study observations. Systematic reviews and meta-analyses will play an important role in synthesizing the literature and pooling data to provide more precise estimates of the effects of diabetes and other comorbidities on outcomes in COVID-19.
Finally, many reports have lacked longterm follow-up. Identifying the frequency of and risk factors for readmission is urgent. We also need surveillance for longer-term outcomes, especially possible pulmonary, neurological, and cardiac sequelae of COVID-19. Understanding morbidity in recovered patients is particularly important in adults with diabetes who are already at higher risk for neurological disorders and diseases of the lung and cardiovascular system.
DIABETES EPIDEMIOLOGY AND COVID-19: WHERE TO GO FROM HERE?
To understand the independent contribution of diabetes on risk of death and ARDS in COVID-19, exposures and covariates need to be measured rigorously and systematically in patients who are not selected with respect to the development of certain outcomes. Regression models need to be thoughtful and reflect our best understanding of the disease. Investigators need to distinguish between clinical characteristics of patients prior to admission (i.e., age, sex, diabetes, cardiovascular disease history, obesity, medication use)dwhich may be important independent risk factors for prognosisdand in-hospital measures (e.g., inflammation, glucose, troponin), which reflect disease progression. Studies need to be large and community-based, with sufficient followup and end points to generate precise estimates. The stakes are substantial. These studies are driving clinical and public health decision-making.
Many of the epidemiologic challenges facing the broader COVID-19 epidemic are applicable to patients with diabetes. Mild cases and deaths have been missed due to undertesting. As a result, it is challenging to estimate infection risk and outcomes for the majority of patients, including those with diabetes. Further, rapid changes in screening policies and approaches make estimating the burden of disease and epidemiologic associations a moving target.
To address these challenges, we need to ask the right questions. From our viewpoint, there are five diabetes-related questions that deserve our attention and should be a focus of future epidemiologic studies.
1) Is Diabetes a Risk Factor for Infection With SARS-CoV-2?
Press coverage of the novel coronavirus has conflated risk factors for infection with SARS-CoV-2 (susceptibility) with risk factors for mortality and disease progression in COVID-19 (prognosis). It is unknown whether diabetes or its complications might directly increase the risk of becoming infected with SARS-CoV-2. It is possible that features of diabetes make individuals physiologically more susceptible to infection with SARS-CoV-2. Indeed, individuals with diabetes are known to be more likely to acquire certain infections (22,23). More likely, however, is that the overlap of diabetes with drivers of health disparities and historical inequities including poverty, racism, housing and residential segregation, environmental risks, and employment and neighborhood factors are causing higher rates of infection (24).
SARS-CoV-2 is disproportionately spreading among our racial and ethnic minority communities and individuals of lower socioeconomic status. The highest rates of hospitalization have been in some of our most vulnerable populations. Concerns have also been raised about bias in COVID-19 treatment. These disparities are long-standing, but the pandemic has brought them to the forefront, including to the pages of the top medical journals and mainstream media reports. In combating this pandemic, it is critical that government and policymakers use this as an opportunity to address some of the fundamental inequities in health care and our social safety net. It is also imperative that, going forward, epidemiologic studies include information on race/ethnicity and rigorous measures of the social determinants of health to best inform policy.
2) Does Diabetes Independently Contribute to Risk of Progression to ARDS and Death in Hospitalized Patients With COVID-19?
Type 2 diabetes is much more prevalent in older age and is a risk factor for chronic kidney disease, cardiovascular disease, and premature mortality. COVID-19 has been described to contribute to cardiac strain and acute myocardial injury. It has not been established whether diabetes itself independently increases risk of progression from mild symptoms to life-threatening illness in COVID-19, or whether associated conditions including obesity, hypertension, and cardiovascular or kidney disease may be driving worse outcomes. These conditions are not likely to be modified after diagnosis of infection, but other aspects of diabetes may be modifiable. Glucose control is important in hospitalized patients and can be modified. Studies that rigorously address the independent associations of diabetes and glycemic control with outcomes of COVID-19 in the intensive care setting are needed.
3) Are There Medications That Are More or Less Effective in Adults With Diabetes in COVID-19?
It is not clear whether disease progression in adults with diabetes and COVID-19 can be modified by diabetes-specific interventions such as intravenous insulin, continuous glucose monitoring, or specific pharmacotherapies. One class of therapies common among adults with diabetes are angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs). These medications are recommended for adults with diabetes, hypertension, and proteinuria. SARS-CoV-2 uses the angiotensinconverting enzyme 2 (ACE2) receptor in host cell membranes to facilitate entry into cells in the lungs and other tissues (25). ACE inhibitors and ARBs increase the abundance of ACE2 receptors, affording more access points for the virus to bind and enter cells (26,27). It has been hypothesized that use of these agents may increase risk of COVID-19 infection and more severe lung injury. In contrast, others have thought these receptors might contribute to viral fixation and lower the risk of infection. This has been a major debate in the field (28). Clinical trials to address these questions are ongoing.
4) What Is the Role of Obesity in COVID-19?
Emerging evidence suggests that obesity is an important risk factor for progression and death in hospitalized adults with COVID-19 (29). The role of overweight and obesity in COVID-19 may relate to ventilation; obesity is a known risk factor for abnormal ventilation and can contribute to reduced functional residual lung capacity and chest wall elastance (30). It is also possible that adiposity may play a direct role, for example, via inflammatory pathways and/or local biological effects of epicardial adipose tissue (31) or other fat depots in the body.
Relying on EHR data will not provide the data we need to address the role of overweight and obesity in this pandemic. At the most basic level, we need studies with systematic measurements of height and weight to rigorously address this question.
5) What Are the Implications of the Pandemic for Diabetes Prevention and Management?
Stay-at-home policies during the pandemic and social distancing have discouraged many activities and changed lifestyles and behavioral patterns in large swaths of the population. There have been anecdotal reports of increased consumption of unhealthy foods and weight gain. Elective procedures, preventive outpatient visits, and ambulatory maintenance care are being delayed. Routine care including diabetes screening and recommended routine screening for complications and HbA 1c testing in patients with diabetes have been deferred for millions of patients. There have also been reductions in hospitalizations for myocardial infarction and other acute events, likely as a result of patients avoiding the health care system and forgoing care due to fear of SARS-CoV-2 transmission (32)(33)(34). Implications of this deferred care on short-and long-term complications of diabetes will be an important topic of subsequent research.
Recent reports have provided helpful guidance on management of mild COVID-19 disease (the majority of cases) in the setting of diabetes (35). An unfortunate challenge is that the pandemic has caused problems of access to medications for some patients. Whereas, a promising trend is that telemedicine has been widely adopted and represents a particular opportunity for diabetes medicine, as aspects of diabetes care may lend themselves well to telemedicine technology. The pandemic has also fueled interest in remote monitoring technologies; continuous glucose monitoring devices are poised for adoption for such purpose (36).
Social isolation and loneliness as a result of stay-at-home policies and social distancing are concerns. The psychological consequences of the pandemic are likely to disproportionately affect older adults and those with preexisting health conditions including diabetes. Older age and diabetes are known risk factors for depression (37). The psychosocial effects of the physical isolation that has accompanied the pandemic and measures of mental health and well-being during this global pandemic are important considerations for future studies.
CONCLUSIONS
The COVID-19 epidemic represents an unprecedented challenge to patients with chronic disease, especially diabetes. Epidemiology has never been more important. Epidemiologists are public health professionals who typically toil in the background. We have now been foisted to the forefront of the pandemic, to television news and the front covers of newspapers. When public health interventions work, they are seamless and invisible, preventing the spread of disease and improving health without any front-page headlines. The pandemic has made our "invisible profession" of public health highly visible. We are at the beginning of this pandemic. For many aspects, we don't even know what we don't know. But we do know that this is the moment for team sciencedfor interdisciplinary groups to come together to identify the most important questions and address them with rigor and responsible research.
Prospective studies with systematic data collection and extended follow-up, surveys with probability sampling of the general population, randomized clinical trials of therapeutic approaches, and collaborative studies in diverse populations are needed. Sound epidemiologic thinking is indispensable to the design of those studies that will answer questions central to the COVID-19 pandemic and inform clinical and public health policy. This pandemic has already shown us that individuals with diabetes are a vulnerable population who need particular consideration. Diabetes epidemiology should play a significant role in helping address this public health crisis.
Funding. E.S. was supported by National Institutes of Health National Heart, Lung, and Blood Institute grant K24 HL152440. Duality of Interest. No potential conflicts of interest relevant to this article were reported. | 2020-06-16T20:06:47.948Z | 2020-06-15T00:00:00.000 | {
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225379633 | pes2o/s2orc | v3-fos-license | The response of growth rate in different husk biochar application of Allium cepa l in sandy soil
Shallot (Allium cepa L) is typically cultivated in Coastal area of Yogyakarta with high input of water and organic fertilizer. This area has sandy soil type with low water holding capacity and low soil fertility. Husk biochar is a potential alternative soil amendment to improve soil quality and may increase crop productivity. The objective of this study is to characterize growth rate of Allium cepa L under different husk biochar application in sandy soil. Experiment was carried out in growth chamber with four different treatments. The treatments were (1) control; (2) Husk Biochar; (3) Husk biochar and compost (0.5:0.5); and (4) Husk biochar and compost (0.65:0.35). Plant growth and irrigation were monitored every day for 30 days experiment. Daily temperature, relative humidity inside and solar radiation growth chamber were around 30 – 34.5°C, 46.7 – 66.7% and 5.3 – 20.7 W/m2 respectively. The results showed that soil amendments treatment in various comparisons affected the growth of shallots (Allium cepa L). Husk biochar and compost treatment (0.5:0.5) significantly increased plant growth. Moreover, husk biochar treatment has better water use efficiency (WUE) than other treatment (0.09 g/mm).
Introduction
Sandy soil on the southern coast area in Yogyakarta, Indonesia has great potential to be developed into agricultural land. This area has some advantages, i.e. wide area, flat, huge amount of sunlight, shallow ground water, and neutral soil pH. However, in utilization as agriculture, coastal sand land has limiting factors related to the physical, chemical and biological properties of soil. These limiting factors include the ability to store low water, high infiltration and evaporation as well as low fertility and organic matter [1]. In addition, on dry land, water stress in plants can occur due to reduced water availability or due to the very high transpiration rate compared to normal conditions [2]. This condition affects the productivity of plants as well as the yields [2,3]. WUE (water use efficiency) of plants is one of indicator that show comparison between crop productivity and water use by different plants [4].
In addition, some plants species are adaptable to drought stress. Such tolerance in different plants may vary due to different adaptation mechanism [5]. Shallot (Allium cepa L) is typically cultivated in Coastal area of Yogyakarta. Shallot plants are considered suitable for planting in coastal sand because these plants are very responsive to changes in environmental conditions, especially soil.
There are some soil amendments that will increase plant tolerance to drought as well as WUE. Biochar is an example of amendments that contain about 80% of carbon and has ability to conserve water for plants [2]. Biochar was selected as raw material for soil amendments based on the production of untapped plant residues [6]. Biochar itself is charcoal from agricultural waste such as rice husks, corncobs, coconut shells and wood residues. These materials are difficult to decompose which requires incomplete combustion process (pyrolysis) in order to obtain charcoal with activated carbon content to be applied to the soil. Biochar is known as a material that can last long enough in the soil and is relatively resistant to microorganism attack, thus the decomposition process runs slowly [7].
Many studies have investigated the addition of soil amendments on the sandy soil. According to [8], farmer in coastal area Yogyakarta applied clay (0.75 -1 m 3 per 100 m 2 ) and organic fertilizer (20 t/ha) for shallot cultivation. However, there was lack information about biochar application ant its impact on plant productivity as well as water use efficiency in sandy soil. This study aims to determine the effect of the addition of husk biochar in sandy soil with a certain ratio to the plant growth rate and water use efficiency (WUE).
Site and experiment description
The experiment was conducted in growth chamber Laboratory of Farm Structure Environment Engineering, Faculty of Agricultural Technology, Universitas Gadjah Mada. Microclimatic conditions in the growth chamber during the study included temperatures between 30.1-34.5 o C, air humidity of 47.67-66.67% and solar radiation 5.3-20.7 W/m 2 .
The experiment was carried out for 30 days from July to August 2019. The plant was shallots (Allium cepa L) and planted in polybags with a 10 cm diameter and 13cm height. The planting media was sandy soil from Bantul coastal area, D.I. Yogyakarta Province, Indonesia. Each planting media has different treatment, by using different comparison of sand, biochar, and compost as mention in Table 1. All treatment followed complete randomized design with four replications. Shallot was planted by using tubers sliced at the ends to accelerate bud growth. All treatment was irrigated manually by adding water in accordance with shallot water requirement, about 123 mm/day.
Plant height.
The growth of shallot was measured every three days, started from 0 day during vegetative phase. Height was measured from the surface of growing media to the leaf using a ruler. On the 18 days after planting, the plants were collected in order to determine of fresh and dry mass. Dry mass was obtained after dying in an oven (65 °C) for 24 hours. Data in the form of plant height during the vegetative phase were analyzed to obtain plant growth rate by using monomolecular equation. The monomolecular function explains the progress of a simple first-order irreversible reaction. Assuming a constant and independent growth rate of plant height, the equation is obtained [9] : (1) (1), resulting in equation (2) (2) The integral form of equation (2) is equation (3): The integral form of equation (3) solved into equations (4) : : surface area (m 2 ) Water Use Efficiency (WUE) analysis for each treatment using the equation: Y : shallot bulb yield (g polybag -1 ) ET : seasonal evapotranspiration (mm)
Soil condition
Soil samples were mixed with different treatment and resulted different soil chemical properties (table 2). PBK2 treatment (biochar sand and compost) with a ratio of 1: 0.35: 0.65 resulted the highest pH value i.e. 7.07 of the four treatments, while PBK1 (sand, biochar and compost) 1: 0.5: 0.5 was 7.03. This result show that the provision of organic material (compost) and soil amendments (biochar / husk charcoal) significantly improved soil pH. C-organic in the four treatments also has different result, where PBK1 treatment has the highest C-Organic content (5.30 %) compared to other treatments. C-organic in planting media plays an important role in improving soil properties both physically, chemically and biologically. Moreover, total nitrogen of PBK1 also showed highest N-total (0.18%), followed by PBK2 (0.16%). Another parameter is the potassium content in the four treatments obtained the highest K value for the control treatment (sand) while the lowest value in the PBK1 treatment. In the PBK1 treatment the potassium value is quite low because the potassium levels are absorbed by the plants. Potassium nutrients play a role in vegetative growth to strengthen stem stands and increase carbohydrate levels [11]. Furthermore, Phosphorus availability also increase with addition of biochar. Soil used in this experiment was sandy soil from tropical condition with neutral pH, low organic carbon, and low total nitrogen availability. Addition of soil amendments (husk biochar and compost) have positive response to soil parameters (table 2). This results has similar trends in different application of biochar by [2,12].
Effect of Treatment on Plant Growth Rate 3.2.1. Plant growth.
Plant height was measured as one of the plant growth parameters. The height was assessed at 0 days after planting to 18 days after planting and plotted into a graph by following monomolecular function (Fig 1). Plants growth rate was higher in PBK1 treatment (sand, biochar and compost; 1 : 0.5 : 0.5) as compared to other treatments. PB (sand and biochar) treatment was lowest plant growth rate. Moreover, control (sand) treatment in this study was resulted higher growth rate than PB and PBK 2. Therefore, addition of husk biochar and compost was found to be more effective than other treatment to increase plant growth rate of shallot. Composition of a balanced planting media can support a good aeration process, so that the roots can grow well and optimally absorb nutrients so the plants grow well [13]. Growing media is one of important factor that must be considered in conducting cultivation for plant growth and development. Provision of soil amendments, such as biochar and compost can improve the physical and chemical properties of the soil by improving the structure, texture, water retention ability, aeration and soil nutrient content [14].
Effect of Treatment on Water Use Efficiency (WUE)
Efficient use of water is the ratio between plant dry weights and water requirements [15] and shows the ability of plants to convert available water into dry matter [16]. Comparison of WUE of control (sand) and other treatment is presented in table 3. Addition of husk biochar in sandy soil significantly increase WUE. Biochar treatment (PB) was resulted highest WUE (0.09 g/mm). Husk biochar together with compost (PBK2 and PBK1) has also improved the WUE (0.04 and 0.05 g/mm respectively). Therefore, husk biochar in sandy soil was proved to be more effective to increase WUE of shallot. Other studies related to the value of WUE in onion plants (Allium cepa L) at harvest time were 0.1169-0.289 g/mm [10]. One significant increase in the value of WUE is influenced by the addition of biochar and organic fertilizer [17].
In the other hand, dry weight of shallot was different result, where control treatment resulted higher dry weight than other treatment. It may be caused by data measurement. In this study, plant growth and dry weight of shallot were measured at the beginning of vegetative phase that would be have impact on the result. Moreover, evapotranspiration (ET) of shallot was also different between treatment. Control treatment (P) was significantly has highest ET (30.87 mm) with lowest WUE (0.02 g/mm). Husk biochar as well as husk biochar and compost were decreased ET. Previous study by [18] resulted that biochar added to sandy loam increases water-holding capacity and might increase water available for crop use. increasing of water holding capacity of sandy soil will improve water use efficiency of crop in agricultural system.
Conclusion
Based on the results of the study showed that the treatment of different growing media in various comparisons affected the growth of shallots (Allium cepa L). In PBK1 treatment, the ratio between sand, biochar (husk biochar) and compost (1:0.5:0.5) positively affected plant growth of shallot. As for the efficient use of water, PB treatment(sandy soil and husk biochar) with a ratio (1:1) was higher than other treatments (0.09 g/mm) as well as lowest value of evapotranspiration (6.38 mm). Therefore, addition of biochar (husk biochar) and compost fertilizer improved plant growth and increased Water Use Efficiency (WUE). | 2020-08-13T10:02:28.637Z | 2020-08-07T00:00:00.000 | {
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2477607 | pes2o/s2orc | v3-fos-license | A Model for the Induction of DNA Damages and their Evolution into Cell Clonogenic Inactivation
Model/Radiation Mechanism/Inactivation Cross Section/DSB. The dependence of the initial production of DNA damages on radiation quality was examined by using a proposed new model on the basis of target theory. For the estimation of DNA damage-production by different radiation qualities, five possible modes of radiation action, including both direct and indirect effects, were assumed inside a target the molecular structure of which was defined to consist of 10 basepairs of DNA surrounded by water molecules. The induction of DNA damage was modeled on the basis of comparisons between the primary ionization mean free path and the distance between pairs of ionized atoms, such distance being characteristic on the mode of radiation action. The OH radicals per average energy to produce an ion pair on the nanosecond time scale was estimated and used for indirect action. Assuming a relation between estimated yields of DNA damages and experimental inactivation cross sections for AT-cells, the present model enabled the quantitative reproduction of experimental results for ATcell killing under aerobic or hypoxic conditions. The results suggest a higher order organization of DNA in a way that there will be at least two types of water environment, one filling half the space surrounding DNA with a depth of 3.7–4.3 nm and the other filling all space with a depth 4.6–4.9 nm.
INTRODUCTION
To understand the mechanism of radiation action on living systems, many phenomenological and mechanistic models have been proposed (see reviews in Refs. 1 and 2).One general agreement is that the target for radiation action is DNA itself, with emphasis on the importance of DNA doublestrand breaks (DSB) acting as lethal lesions.It is also well known that non-homologous DNA end-rejoining (NHEJ) is an important repair mechanism for ionizing radiationinduced DSB in mammalian cells 3,4) .Reviewing the mechanism of radiation-induced death in mammalian cells, it is not certain yet that the dependence of radio-sensitivity upon radiation quality can be explained in terms of un-repaired DSB production, although some past work has demonstrated that the repair of DSB in cellular DNA can be a major cause of radiation-quality dependence of cellular radiosensitivity [5][6][7][8] . T explore the mechanism of radiation action in mammalian cells, the processes of radiation action for cell killing must be separated into at least two classes, damageinduction and damage-repair and this was done in the previous work 9) .This need arises from the current status of theoretical radiation biology in which no studies have yet shown a single or unique set of descriptive variables to be sufficient for understanding of radio-sensitivity as a whole in mammalian cells and its dependence on radiation-quality.
An inherent difficulty with mammalian cells regarding the present subject is the lack of experimental data concerning the initial induction of DNA damage immediately after exposure to ionizing radiation.As far as DSB induction is concerned, the yields of DSB are measured by various techniques including pulsed field gel electrophoresis (PFGE) for particle radiations of differing linear energy transfer (LET) 10) .Observed difference in relative biological effectiveness (RBE) values between DSB induction and for other cellular effects such as cell killing, mutation and cell transformation, are seen according to the differing repair ability for DSB of the cells and variations related to the techniques used 10) .Initial DSB induction among normal cells with 90 kV X-rays has recently measured using a technique called immunofluorescence 11) .This kind of data is important but it is still lacking especially for information concerning radiation quality dependences.As an alternative, data concerning the initial production of DSB that has been observed experimentally in repair-deficient mutant lines such as NHEJ-defi-cient cells, which are considered to be useful because they will involve the failure of such functions as DNA end-binding hetero-dimer of Ku70/Ku80, catalytic subunit of DNA-PK, XRCC4 gene product and DNA ligase IV [3][4] . Frther, the cellular mutations related to the above functions are known to make cells sensitive to ionizing radiations due to these marked deficiencies 12,13) .One useful finding is that increased radio-sensitivity is accompanied with increased production of un-repaired DSB in NHEJ-deficient cells 14,15) .
From this finding the higher radio-sensitivity observed in Ataxia telangiectasia (AT) cells is deduced to be due to an increased rate of production of un-repaired DNA damages including DSB.The experimental data [14][15][16][17][18][19] on AT-cell inactivation for various radiation qualities, therefore, are useful for estimation of un-repaired DNA damages or approximated initial DNA damages.
Using these data the present investigation on induction of the initial DNA damage proposes a mathematical model developed on the basis of target theory 20) .Although many different ideas of modeling have been developed so far, the concept of a hit in an amorphous volume (volume-like) or segment (linear-like) target has been developed and allow the use of such descriptive variables as radiation absorbed dose 21) , linear energy transfer (LET) 22) , ionizations in a unit of track segment [23][24][25] , and primary ionization mean free path 26) .None of these descriptors separates the processes into induction and repair for modeling of radiation action.Using the primary ionization mean free path ( l ) and a molecular structure consisting of DNA and surrounding water, the present study focuses on the relationship between initial DNA damages and cell lethality.
The present model is based on the following notions: 1. Various DNA damages are formed by a subset of single tracks traversing the cell nucleus.
2. Un-repaired DNA damage has a probability of causing cell lethality.Some of the mis-repaired DNA damages will be responsible for mutagenesis or oncogenesis in surviving cells.
The importance of a single track to induce DNA damages including DSB is now well studied both experimentally and theoretically 27,28) .And it is further suggested that various DNA damages produced by single tracks, as classified in Fig. 1, are potentially lethal if they are un-repaired, though the exact mechanisms involved are as yet unclear 29,30) .The initial damage is defined here more generally as the remaining DNA damage uncommitted to a repair mechanisms 31) , and both DNA damages uncommitted to a repair system and un-repaired or mis-repaired DNA damages may eventually be responsible to cellular effects including cell lethality 31) .
MATERIALS AND METODS
In this section we describe the techniques for (a) setup of the structure of the molecular target in which the initial DNA damages are defined, (b) specification of the modes of radiation action for DNA damage-production within the target, and (c) a definition in which cell lethality (inactivation cross section) is related to the initial DNA damages.
We have used for the model data on the inactivation of ATcells by X rays 14,-19) and heavy charged particles 15,16) .
Methods of the model consist of several steps as described in detail below.
1.The molecular entity for DNA damage is assumed to be any of those shown in the Fig. 1 being caused within a distance of 10 base-pairs 32,33) .No specification for the complexity of the entity has been taken into account in the definitions, though it can be a matter for repair processes, which are tentatively out of consideration in the present study.
2. A molecular target is defined as a 10 base-pairs unit (denoted here as dna, and DBS and SSB in this target denoted here dsb and ssb respectively) at the central part of a 15 mer B-DNA of d(CGCGCCATTACTCGC) 2 and water surrounding DNA with an appropriate depth of c nm (Fig. 2).The molecular target is defined in a box (16.77 ¥ 16.98 ¥ 27.06 nm 3 ) large enough to contain the DNA produced by a molecular dynamics computer code 34) and protocol 35) .The (x, y, z) coordinates of all atoms of DNA (948 atoms) and water (189,000 water molecules) stabilized at 300 K are used.
3. A single track of an ionizing particle traversing the target interacts with any atoms along the track.It is assumed that the track is linear within the target and that interactions are caused successively along the track in an interval of distance simply characterized by the primary ionization mean free path l in liquid water 26,41) .The interaction at either end of a single unit of l along the track is classified as either direct or indirect action depending on the atom involved.If it is any atom of the dna or any atom of a water molecule, it is defined as an origin of a direct action (D) or an indirect action (W), respectively.
4. It is assumed that water relevant to the indirect action is limited inside the depth c nm (Fig. 2) and that only OH radicals produced by water radiolysis are relevant to cause any of or a part of each dna damage (Fig. 1).Taking diffusion distances equivalent to water of depth c nm and experimental evidences 36,37) into consideration, we assume that the yields of OH radical at the time of nanoseconds is relevant to the indirect action.The yield of OH radicals at nanoseconds of time by a ionizing particle of l is estimated 38) per W-value [energy required to produce an ion pair, denoted here as g ( l ) (see Appendix)].
5. As potential processes of dna damage-production five modes of radiation action from (a) to (e) are assumed, as described below.As an ionizing track is specified by the distance l and orientation between tracks and the target (Fig. 2) may be random over a large number of similar targets, possible distances of any of two atoms among different molecules in the target are a clue to classify the reaction modes.The average distance over the classified distances according to the mode is used as the characteristic distance (t 0j ) of the j-th mode (j=1, ..., 5) .The number of potential distances occurring in a mode, over which the average distance is calculated, determines the relative importance of the mode among the five modes.
(a) Mode D-D: To cause either type of damage (i)-(iv) (Fig. 1) both strands of dna within 10 base-pairs are hit by a single track at the same time as a direct action.The average distance (t 01 ) is calculated from a sample of distances of number of occurrence of n 1 (=[nd 1 ¥ nd 2 ]), where nd 1 and nd 2 are the numbers of atoms of each strand, respectively.It is noted that atoms not Fig. 2. A molecular target was defined in a molecular system that consisted of a 15-mer B-DNA and 189,000 water molecules in a rectangular box (16.77 ¥ 16.98 ¥ 27.09 nm 3 ).The molecular target of the present model was a central 10 base-pair DNA (denoted as dna in the text) and waters inside a water depth c that was cut out from the molecular system.Coordinates of all atoms stabilized a temperature of 300 K were produced by the computer software AMBER 4.1 34) with a protocol 35) .One of the results was that 635 atoms of the central dna and a water layer of depth c = 4.6 nm, therefore 15,505 water molecules were involved to form the molecular target of the present model.This mode and mode D are independent of grouping of the water molecules.7. The probability of the interaction by an ionizing particle traversing linearly in the molecular target is calculated as follows.Along the track a distance x between any successive occurrences of ionization is probabilistically fluctuated as (1/ l )exp(-x/ l ) , where l is the primary ionization mean free path.From this the probability of occurrence of more than one distance l within a distance t along the track is [1 -exp(-t/ l )] .The distances relevant to the reaction modes (a) -(e) have their own frequency distributions (not shown here).The probability of occurrence of ionization in each mode is calculated by an integration of [1 -exp(t/ l )] folded by the normalized frequency distribution of distance.This integration can be well approximated by [1 -exp(t 0j / l )] , where t 0j is the average distance of the j-th mode calculated with the frequency distribution.
8. To relate the probability of coincidence of structure between a track and the probability of occurrence of a lethal damage, the efficiency of each mode is considered.We assume that the efficiency is unity for any type of mode causing damages in both strands of dna as types (i) -(iv) in the Fig. 1, and that the efficiency is p (<1) for the mode causing a damage in either strand of dna as types (v) or (vi) in the Fig. 1.The efficiency q of an OH radical produced around DNA eventually causing SSB is obtained from experiments and found to be 0.11 39) .We assumed also this value of efficiency for occurrence of a base damage.9. Another type of reduced efficiency is assumed for the damages such (i) -(iv) in the Fig. 1 that are induced by either mode D-W or mode W-W, compared to those induced by mode D-D.The time involved in single action D and single action W is the order of 10 -15 sec and 10 -9 sec, respectively and the time involved in subsequent molecular rearrangement for ionized or excited dna due to intra-molecular interactions is the order of 10 -12 sec 40) .Considering the possibility of interaction of two independent sub-lethal hits within 10 -9 sec to form a lethal damage in the mode D-W or mode W-W, we assume the probability of two hit [1 -exp (-t0j/l)] 2 for these modes for a single track traversal.
10.The following equations are mathematical statements of Steps 7-10 described above.The inactivation cross sec-Fig.3. Five modes to cause DNA damage.l is the primary ionization mean free path 41) in liquid water.The symbols of ★ and • show lesion on a DNA strand and OH radical production, respectively.Mode D-D produces lethal damage consisting of two lesions in each strand by direct action from a single track.Mode D-W or mode W-W produce lethal damage through a direct action on one strand and an attack by an OH radical on the opposite strand or attacks by two OH radicals on opposite strands from a single track with an efficiency of q (= 0.11) 39) for an OH radical induce lesion.Mode D or mode W produces lethal damage with an efficiency of p through a single direct action or a single attack by an OH radical from a single track.
tion for the initial production of radiation-induced lethal dna damage consists of five components as a function of the primary ionization mean free path l as the following, (2) where s g is the geometrical cross section of the cell nucleus, t 0j (j = 1, 2, 3, 4 and 5) the average distance and n j (j = 1, 2, 3, 4 and 5) the frequencies of the modes of D-D, D-W, W-W, D, and W, respectively.The factor q is the efficiency of dna damage-production by diffusion of OH radicals and p is the probability of lethal damage-production through the mode D or the mode W. The yield of OH radicals at the nanosecond scale of time per energy deposition of W-value per ion pair is expressed by g( l ) 38) (see Appendix).The quantity N is a normalization constant such that s ( l )/ s g =1 for the ionizing particle of a small l (here l = 0.1 nm).
11.The primary ionization mean free path l in liquid water is defined as the mean path length of successive ionizations along the track of particles 41) .(8) where W is the energy to produce an ion pair in liquid water, T av is the average energy of the secondary electrons and L is the collision stopping power in liquid water.The published data 41) were used for heavy ions and were basis of estimation of l for photons.
12. The number of parameters for the present model is three.These are s g for geometrical cross section on cell inactivation, c for the depth of water, and p for the probability of lethal damage production in the modes D and W. The factor q was not considered a free parameter, but set as q = 0.11 from Step 8.By estimating these three parameters the obtained inactivation cross section, s i ( l ), according to eq.( 1) of the present model is subjected to a comparison with experimental data published on AT cells [14][15][16][17][18][19] . Eperimental inactivation cross section, s exp [ m m 2 ] can be obtained by equation 42) , s exp = 0.16 ¥ L ¥ a / r , where L is LET, a [Gy - 1 ] is the linear coefficient obtained from the linear-fitting of actual experimental data on cell survivals and r is the density of liquid water.The comparison is judged by following the least square fit among possible choice of the three parameters as following eq.( 9), (9) where s i ( l ) was the inactivation cross section estimated by eq. ( 1) and s i-exp ( l ) the i-th experimental data.
RESULTS
The present model is developed to estimate the initial induction of dna damage, which is potential for cell death.The dna damage, whatever its origin, direct or indirect effect, is assumed to be induced by two paths, an interaction of two molecular alterations on each strand of dna within the 9) as a function the water depth c for survival data on AT cells under aerobic [14][15][16][17][18][19] (----), hypoxic 16) (--+--) conditions and their sum c 2 (solid curve).Each point was the minimum c 2 with optimized values of s g and p. Twopparent minimum c 2 = 26.2around 2.2 nm < c < 2.8 nm (denoted here as structure A) and c 2 = 24.7 around 4.6 nm < c < 4.9 nm (structure B) were observed.The total number of data was 35 (24 aerobic and 11 hypoxic).The water depth c is ranged in an interval of 0.3 nm from 0.4 nm to 6.1 nm. cs l s l s l range of 10 base-pairs, or a single molecular alteration on either strand of dna, induced by a single track.These conditions occur when the characteristic distance for the five different modes of action match the primary ionization mean free path.OH radicals produced by the average energy deposition for an ion pair are considered to be the agents of indirect action. Bsed on the notions described above, the present model for initial dna damage was used to study the change in inactivation cross sections for AT-cells as a function of radiation qualities.For this purpose, the optimization of the model parameters was required for determination of the geometrical cross sections (sg) of AT cells, the depth (c) of water around dna and the efficiency (p) for lethality of a single molecular alteration in either strand of dna.The efficiency (q) of OH radicals for a dna damage in either strand of dna was assumed to be constant as q = 0.11 within the water depth of c (Step 8).The experimental data on the inactivation for AT cells used for the present study were obtained from previously published reports [14][15][16][17][18][19] .
First, the optimization was explored with c 2 values using eq.( 9) for s (l) vs. depth (c) between the range of 0.4 nm to 6.1 nm.In Fig. 4 the changes of c 2 values were obtained for AT cells under aerobic (----) and hypoxic (--+--) conditions together with that of their total values (solid line).The curves show the existence of minima with slight difference in atmospheric conditions of the irradiations in the following two ranges; one for c 2 =26.2 lies between 2.2 nm < c < 2.8 nm and the other for c 2 =24.7 between 4.6 nm < c < 4.9 nm.Taking account of the fact that two different values of c 2 were smaller than the sample size of 35 (24 for aerobic and 11 for hypoxic), two of these optimum minima were statistically accepted as a possible depth for the water (Fig. 4).Tentatively, these two different sizes of water layer were denoted as structure A for the thinner one and structure B for thicker one.The existence of two different structures for water was thought to be an important subject for discussion particularly concerning whether these structures are due to the higher order organization of DNA and its surrounding water.
The optimal parameters obtained for the present model for structure A and B are summarized separately in Table 1.The results indicate that the efficiency p for production of a lethal damage by mode D or mode W is higher by 5.4 or 5.75 times for the aerobic group than for the hypoxic cells with either of structures A or B, respectively.The values can be taken as an expression of oxygen enhancement for dna damage induction.The difference of sg between aerobic and hypoxic groups may be another expression of oxygen enhancement, so that the ratio of aerobic sg to hypoxic sg was 1.06 ± 0.09 with structure A and 1.04 ± 0.04 with structure B, respectively.
For further detailed analysis, the changes in fraction of the modes of radiation action was explored as a function of l under aerobic and hypoxic conditions with structures A and B. The results are shown in Figs. 5 to 8.Each of the Figs.consist of two panels (a) and (b).Later, a comparison of the data was made between the present study (Figs. 5 -8) and other studies with repair-proficient human T1 cells 43,44) .The data for repair-proficient cells were used for calculating a ratio of the inactivation cross section for T1 cells vs. that estimated for AT cells as a measure of repair.
Figures 5 and 6 show the results of estimation with structure A. Good agreement with the experimental data [14][15][16][17][18][19] is seen in Fig. 5 (a).One can see that the change of inactivation cross section with radiation quality is dependent on the probability for dna damage induction.This probability can be estimated as a sum of the contributions from the different modes.The decrease in s (l) in the modes associated with OH radicals as l decreased was found due to the decrease in the yields g(l) of OH radicals at nanoseconds level (see Appendix).The induction of lethality was dominant for such indirect modes as W and W-W for low LET radiations as l increases (Fig. 5 (b)).This is apparently due to the dominance in the frequency of occurrence in indirect modes, and additionally with a larger value of g(l).On the other hand for high LET particles as l decreases, the induction of lethal damge was dominant through such direct modes as D-D, D and (D-W).This is apparently due to a higher probability for the induction of lethal damage in direct modes, even though both their frequency of occurrence and the yield g(l) of OH are not higher than those of the indirect modes.The result in Fig. 5 (b) shows that the change in contribution fraction of the modes reflects that of un-repaired dna damages in T1cells.One possibility would be that repair works quite effectively in the modes W and W-W and optionally in the modes D-W or D but less effectively in the modes D-D.
The results in Fig. 6 show some difference between hypoxic and aerobic conditions.The estimation of the inactivation cross section using the present model shows quite good agreement with data obtained by the experiments [14][15][16][17][18][19] (Fig. 6 (a)).The dependence of the change in fraction of modes as a function of l was attributed to a small contribution of water and a smaller value of p for hypoxic cells, which appeared to reduce the contribution to dna damage production with modes D and W (Fig. 6 (b)).Under such conditions a repair mechanism may be able to work effectively for the produced dna damages in almost all of modes W, W-W, D-W and D. However, it does so optionally for such dna damages produced with mode D-D.Upon this somewhat surprising result the results with structure A were again examined in a different manner as shown blow.
The results in Figs.7 and 8 show that there is some difference between structure A and structure B. One finding is that the indirect modes of W and W-W were distinctively dominant in all radiation qualities because of the strong involvement of surrounding water for dna damage induction.The other finding was that the results of calculation for inac-tivation cross sections of AT-cells under aerobic (Fig. 7 (a)) and hypoxic (Fig. 8 (a)) were in good agreement with those of experimental data [14][15][16][17][18][19] . Tirdly as a remark, the difference in the probability p between aerobic and hypoxic groups was considered to include some bias due to a possible difference in g(l) for OH radicals between the two groups, since it has been reported 45) that the production of OH radicals under aerobic condition is less than under hypoxic conditions.Nevertheless it can be said that a repair function may work effectively only for the dna damages produced with mode W but optionally for those with mode W-W or doubtfully for those with mode D, and further probably not for those with mode D-D even under hypoxic conditions.16) , Heions 15) , 95 kV X 14) , 200 kV X 18,19) , 225 kV X 16) , 250 kV X 15) , 137 Cs g 17) and 60 Co g 15) , are plotted against estimated l 41) .The solid curve shows the inactivation cross section estimated by the present model.44) , 225 kV X 44) and 250 kV X 43) , to the present estimates for AT-cells.The experimental error of data of T1 cells and estimated uncertainty of the inactivation cross section calculated for AT cells were taken into account and combined in a single estimated error bar.
(a) (b)
Fig. 6.(a) Inactivation cross section of AT-cells in hypoxic condition for structure A as a function of the primary ionization mean free path l.Experimental data 16) are plotted against estimated l 41) .(b) The fraction of the five reaction modes and the ratio of the inactivation cross sections under hypoxic conditions for T1-kidney cells 16) to the present estimates for AT-cells are shown as a function of l.
(a) (b)
As for the higher order organization of DNA and structural protein, nucleosomes have been recognized to be a subunit of chromatin, which is composed of two full turns of DNA wrapping around a core of histone proteins (an octamer).This fundamental packing unit is the most elementary framework of chromatin conformation known as "beads-ona-string" 46) .The beads as core part of the nucleosome are linked to each other by a short length of DNA, called linker DNA 46) .From the "beads-on-a-string" framework of chromatin two different situations of DNA exposure to water can be assumed.DNA strings wrapping cores may be exposed only on one side to water molecules but linker-strings may be exposed on both sides.In this situation DNA wrapped around histone cores may correspond to structure A because of less water or more probably because of half the exposure to water and the linker DNA to structure B because of more water or because of full exposure.The calculations of inactivation cross sections for AT-cells according to the present model were made by following the approximation for wrapped DNA the average distance of mode W-W should be half of that of full water exposure, as well as for modes D-W, W-W, D and W. With this approximation an optimization was examined for the depth of water (Fig. 9).Interestingly a single minimum of c 2 around c = 3.7-4.3nm was observed, which we call structure C. The optimal values estimated for the parameters required are given in Table 1.Good agreement was obtained similar to the optimization of inac- 11 (a)) conditions.The fractional changes of modes of action in AT-cells and of un-repaired dna damages in T1cells were shown for aerobic conditions (Fig. 10 (b)) and for hypoxic conditions (Fig. 11 (b)).Both patterns of modefractions in aerobic and hypoxic conditions were similar to their respective pattern for the structure B, and the optimally obtained parameters (sg and p) for structure C show no statistical difference from those for structure B either for aerobic or hypoxic conditions (Table 1).The parameters obtained may be useful for identifying some molecular structures of the target.Moreover, the oxygen enhancement is suggested to be a slightly higher for structure C than for structure B, 9.4 times in terms of p and 1.085 ± 0.09 in terms of sg.
So far as the initial yield of dna damages is concerned, the primary ionization mean free path l was shown to be a good descriptive variable of radiation quality as shown in panel (a) in Figs.7-8 and 10-11 but not so good for modeling the repair mechanism especially under hypoxic conditions because the same l in the fraction of un-repaired dna damages showed differences in T1-cells depending on the incident particles (Figs.In summary, the present model has made it possible to estimate the initial induction of dna damages as the source of radiation-induced cell death under aerobic or hypoxic conditions.The results of estimation in terms of l have disclosed that the total inactivation cross section is the sum of l-dependent processes defined in five different actions, which are attributed to both direct and indirect effects of radiation.Consequently, the primary ionization free path l was found to be a useful descriptive variable to describe the difference of dna damage-induction by different radiation qualities.The dependence of un-repaired dna damages on radiation quality may correlate preferentially with those induction modes relevant to direct actions.Finally, the present results of parameter estimation suggest that at least a compound of structures B and C is quite possible as a practical description of target structure, where B can be related to the linker DNA and C to the DNA wrapping histone beads (of about 73% of chromatin subunit consisting of 146 base pairs / 200 base pairs) 46) .12) as a function the water depth c for survival data on AT cells under aerobic [14][15][16][17][18][19] (----) , hypoxic 16) (--+--) conditions and their sum c 2 (solid curve) for the calculations with the assumption that DNA was surrounded by water for half of the surface like that for wrapped DNA around a nucleosome bead of chromatin structure.A single minimum c 2 = 26.9 (tol) around 3.7 nm < c < 4.3 nm (denoted here as structure C) was observed.The total data was 35 (24 aerobic and 11 hypoxic).
Relation between the molecular target and the geometrical cross sections (s g)
The present model suggests two types of molecular targets, a cylindrical volume which was calculated as V1 = pr 2 h for the structure B and V2 = pr 2 h/2 for the structure C, where r = t01 + c, h = 3.4 + 2c (nm) and c is the depth of the water.Introducing a chord length of each volume lj (= 4Vj/Sj; Sj is the area of surface of the volume Vj including the lateral surface and the bases of the cylinder), an effective cross section seff of the molecular target were weighted by two cross sections sj for fractions of linker and wrapped DNA as seff = 0.27 ¥ s1 + 0.73 ¥ s2, where sj was the geometrical cross section sj = Vj/lj (j=1,2 for the structure B and C respectively).From Table 1 we obtained seff as 126.8 nm 2 and 140.8 nm 2 for aerobic and hypoxic conditions.For high LET radiation where l = 1 nm for instance, the macroscopic effective inactivation cross section sgeff was 151.2 mm 2 , 140.8 mm 2 (= 0.27 ¥ 143 + 0.73 ¥ 140) for aerobic and hypoxic conditions respectively from Table 1.The ratio sgeff /seff was 1.19 ¥ 10 6 and 1.0 ¥ 10 6 for aerobic and hypoxic respectively and suggests about a million of the molecular targets were involved in a single traversal of the ionizing particle with l = 1 nm.On the other hand the total length of human DNA is almost 2 m long and contains about 2.94 ¥ 10 7 chromatins (of 200 b.p.) or 588 ¥ 10 6 present molecular targets (of 10 b.p.).It may be estimated for the aerobic condition that 1/494 of the total number of the molecular targets, or 1/49.4 of the total number of the molecular targets corresponding to the part of the exon (here called the total exon molecular targets) may be attributed to the cell death of AT cells.For the same radiation, around 50-80 % of the initial damages will be repaired in T1-cells (as shown in the panel (b) of the Figs.6, 7, 9, and 10).For the case of 80% repair the molecular targets of 1/247 of the total exon molecular targets, or 1/ 2470 of the total number of molecular targets will still have un-repaired dna damages but the whole DNA will enable the cell to survive.
These estimations are modified if the range of an ionizing particle is so short that it is not enough to cross the cell nucleus.This modification is not included in all present calculations with the eq.(1).
Relation between the lethal damage and the dsb
The initial yields of DSB is estimated to be 35 DSBs per cell per Gy for both normal and radiosensitive cells irradiated by 90 kV X-rays over the absorbed dose range of 1m Gy -80 Gy in aerobic conditions 11) , the cross section sdsb which becomes 4 mm 2 per track of the photoelectron of LET = 0.713 keV/mm and l =114 nm from the 90 kV X-rays 41) ) meaning ndsb = 3.15 ¥ 10 4 (= 4 mm 2 /126.8nm 2 ) molecular targets per track each of which contains at least measurable dsb with immunofluorescence.For this radiation the inactivation cross section was 1.40 mm 2 (= 1.258 ¥ 0.27+1.459¥ 0.73, where s (l) = 1.258 mm 2 and 1.459 mm 2 for structure B and C respectively using eq.( 1) or from Figs. 7(a are likely associated with DSBs induced only in the part of bead 11) and therefore for each track 582.8 nucleosomes having un-measured DSB of Linker DNA are estimated.From an agreement between these estimates it is suggested that the entity of lethal damages are most likely the DBS induced in Linker DNA and that almost all DSB on the beads on the other hand may be restored structurally due to the binding situation of DNA with a nucleosome core of 8 histones.It may be also suggested that the structure B is relevant to this context and that the entity of lethal damage induced by any of the five modes of the present model is most likely a dsb.These interpretations, however, are only preliminary, because measured yields of DSB may well depend on various conditions including the method itself of the measurement 10) .Nevertheless, the present model suggests that the entity of the dsb, however, may be different depending on induction-mode, and may affect repair processes as seen in Figs.7(b) and 10(b).Un-repaired dsbís are induced almost only by direct modes, which results from the setup that all atoms of dna are involved.The same analysis is possible with a setup that involves only atoms of the sugar-phosphate backbone to estimate the contribution of modes relevant to direct action (D), though it may result in more reduced contribution from these direct modes than the present results.A study of repair processes, however, may help judge which setup is better.The efficiencies for lethality relevant to modes assumed in Step 8 are also subjects for study through the modeling of repair processes.
Amount of water involved in indirect action
The real depth of the water around DNA relevant to indirect action is not yet exactly known.The potential of ethyl alcohol as a scavenger of OH radicals has suggested the depth of water around DNA to be 4 ~ 6 nm 36) , while the scavenging potentials of other small molecules around DNA has suggested a smaller range of depth 37) .Three different sizes of possible depth (range) of water proposed here.The first is between 2.2~2.8nm for structure A, the second between 4.6~4.9nm for structure B and the third between 3.7-4.3nm for structure C. The possible existence of structure A will depend on repair mechanisms if they work to support the result of Fig. 6 (b).The result in Fig. 6 (b), which was not expected, is apparently different from those in Figs.8(b) and 11(b).At present, therefore, it would be reasonable to conceive that a compound structure of B (linker-DNA) and C (histone-DNA) is possible as an entity, so that the depth of the water should be 4.6-4.9nm.However, the water around the histone-beads can be expected to be thicker than 3.7-4.3nm.The present result for structure C would be a thinner entity in terms of depth of water, because experimental inactivation data may include the possible effect of protection by the histone octamer from the induction of dna damages.Track structure simulations may make the present subject clearer with an application to a higher order organi-zation of DNA molecule 47) .
The amount of the water affects the estimation for the relative contributions of direct and indirect effect.From the present model for electrons of l = 1000 nm (about 2 MeV) 15% of the DNA damage was estimated to be due to direct effects and 85% due to indirect effects under aerobic conditions in both structures B and C.This result is different from the literature 48) for the same radiation, in which the values for direct and indirect effect were 45% and 55%, respectively estimated by the reactivity of active species including OH radicals to DNA.
Relevant time of hit kinetics and intra-molecular interaction
The mode D-D of radiation action may occur as a .singlehit event within a time duration of about t1 = 6.3 ¥ 10 -16 sec.This was estimated from the example that argon ions of 30 MeV/amu can traverse such a range of diameter (11.4 nm ~1.58 + 4.9 ¥ 2 nm) inside the water depth.The duration of the diffusing time for OH radicals through a water of depth 0.3-6.1 nm was estimated as t2 = 3.3 ¥ 10 -13 -1.3 ¥ 10 -10 sec, where 0.3 nm is the average distance of bulk water molecules 49) , and the minimum distance between DNA and water molecules is 0.3 nm except those in hydrated situations.The difference between t1 and t2 in mode D-W and the magnitude of time order of 10 -12 sec for intra-molecular interaction 42) will suggest some different pathways of DNA damage -induction through different chemical consequences from that of the mode D-D.A similar possibility may be reserved for the DNA damage -induction by mode W-W when compared with mode D-D or mode D-W.It is the rationale of the present model that mode D-W and mode W-W could be expressed by two hits kinetics, while mode D-D definitely by one hit kinetics.Further evidence, however, will be needed on whether the dna damage induced from mode D-W or mode W-W has as lower efficiency in lethality than those from mode D-D as the difference in hit kinetics suggests.
Complexity of hits and their consequences
When mode D is due to a single ionization, i.e., ejecting a single electron at most and mode W is simply due to a single OH adduct, their intermediate chemical consequence (ssb) leading to a lethal damage can be considered to be more or less the same 31) .Situations, however, will be more complex if the cases are of multiple ionizations, i.e., multiple electrons ejected (after an inner-shell ionization), or multiple hits to dna due to OH radicals in close proximity with all modes of radiation action.The present model does not take account of such complexity that it may result in much more complex consequences of chemical intermediates, chemical dynamics of intra-molecular reactions and its involvement of dissolved oxygen and counter ions.A more sever case of these complexity must be a gap, a loss of nucleotids of either strand or a deletion, a loss of a few basepairs nucleotides in both strands of DNA.It is not clear now how to analyze these complexities in terms of the present model, though these complexities would be expressed anyhow by numbers of affected molecular targets of the present model.These complexities may provoke another type of complexity of repair process though it is far beyond the application of the present model.Hole migration 50) may induce other diverse complexity such as dependence of DNA damage induction on base sequence.And the hole migration could be a mechanism to induce the damage (iv) in Fig. 1 from the damage (v) or (vi) in Fig. 1, where the second damage is migrated from outside the target.From these points of view it may well be the case that chemical entities of clustered damage 24,25,32,33) or MDS (multiply damaged site) 27,37) are more complicated phenomenon than those situations currently proposed by track structure simulations 25,26) , in which the spatial pattern of ionization or excitation events in liquid water and/or induced and diffused OH radicals with their time profile is simply superposed on DNA structure.
APPENDIX
The yields of OH radicals of the present work were calculated using a prescribed diffusion model (here it is referred to as the radical model) 38) .The radical model solves deterministically a set of differential equations for the yield of radical species induced by radiolysis of neutral water as a function of time after an irradiation.Study on a descriptive variable to explain the dependence of yields of water radicals on radiation quality was elaborated in terms of "spur distance" 38) , in which the primary ionization mean free path l was found to be unsuccessful as a descriptive variable for the estimation of radical yields.Optimization of the relevant parameters of the radical model was made by comprehensive experimental Fricke G-values of different radiation qualities (as the yields of microsecond time scale), and as well by data of the decay of OH radical as a function of time measured by pulse radiolysis experiments for the time of range of 200 picoseconds -microseconds using electrons of 20 MeV or He ions.The optimized radical model enables the estimate the yields of OH radicals G(l) expressed as a function of l for neutral water at the time of nanosecond with an estimated uncertainty of a factor of three (Fig. 12).The solid curves with symbol (○ ) and (• ) were estimates for electrons or photons, and He ions respectively.The electron of energy of 0.14 keV has a minimum l at 4.3 nm.Electrons of energies lower than 0.14 keV having a longer l than the minimum l induce slightly higher yields of OH radicals than that of the minimum l .For electrons of larger l than 1112 nm (or higher energy than 2.27 MeV) it was assumed that G( l ) = 5 per 100 eV.For heavy ions of lighter than Ar ions the yields of He ions were assumed in the present study.For heavy ions of longer l than 1454 nm (or higher energy than 1 GeV/amu), it was assumed that G( l ) = 4 per 100 eV.The yields g( l ) of OH used in eq.( 1) was calculated by that g( l ) = G( l ) ¥ W/100 , where it is simply assumed W = 34 eV.
Fig. 5 .
Fig. 5.(a) Inactivation cross section of AT-cells under aerobic condition for the structure A as a function of the primary ionization mean free path l.Experimental data: Ar-and Ne-ions16) , Heions15) , 95 kV X14) , 200 kV X18,19) , 225 kV X16) , 250 kV X15) , 137 Cs g 17) and 60 Co g15) , are plotted against estimated l41) .The solid curve shows the inactivation cross section estimated by the present model.Letters D-D, D-W, W-W, D and W indicate the curves for the five components or modes of the estimated inactivation cross section, each of which shows specific dependence on radiation quality l.The same letters for the curves are used with the same meaning throughout panel (a) of the following Figs.for the aerobic case.(b) The fractions of five reaction modes are shown for the inactivation cross section of AT-cells as a function of l.The data are plotted as a ratio of inactivation cross sections under aerobic conditions for T1-kidney cells: Ar-, Ne-and C-ions44) , 225 kV X44) and 250 kV X43) , to the present estimates for AT-cells.The experimental error of data of T1 cells and estimated uncertainty of the inactivation cross section calculated for AT cells were taken into account and combined in a single estimated error bar.
Fig. 5.(a) Inactivation cross section of AT-cells under aerobic condition for the structure A as a function of the primary ionization mean free path l.Experimental data: Ar-and Ne-ions16) , Heions15) , 95 kV X14) , 200 kV X18,19) , 225 kV X16) , 250 kV X15) , 137 Cs g 17) and 60 Co g15) , are plotted against estimated l41) .The solid curve shows the inactivation cross section estimated by the present model.Letters D-D, D-W, W-W, D and W indicate the curves for the five components or modes of the estimated inactivation cross section, each of which shows specific dependence on radiation quality l.The same letters for the curves are used with the same meaning throughout panel (a) of the following Figs.for the aerobic case.(b) The fractions of five reaction modes are shown for the inactivation cross section of AT-cells as a function of l.The data are plotted as a ratio of inactivation cross sections under aerobic conditions for T1-kidney cells: Ar-, Ne-and C-ions44) , 225 kV X44) and 250 kV X43) , to the present estimates for AT-cells.The experimental error of data of T1 cells and estimated uncertainty of the inactivation cross section calculated for AT cells were taken into account and combined in a single estimated error bar.
Fig. 7 .
Fig. 7. (a) Inactivation cross section of AT-cells under aerobic conditions for structure B as a function of the primary ionization mean free path l.(b) The fraction of the five reaction modes and the ratio of the inactivation cross sections under aerobic conditions for T1-kidney cells to the present estimates for AT-cells are shown as a function of l.
Fig. 8 .
Fig. 8. (a) Inactivation cross section of AT-cells under hypoxic conditions for structure B as a function of the primary ionization mean free path l.(b) The fraction of the five reaction modes and the ratio of the inactivation cross sections under hypoxic conditions for T1-kidney cells to the present estimates for AT-cells are shown as a function of l.
Fig. 9 .
Fig. 9.The results of c 2 of eq.(12) as a function the water depth c for survival data on AT cells under aerobic[14][15][16][17][18][19] (----) , hypoxic16) (--+--) conditions and their sum c 2 (solid curve) for the calculations with the assumption that DNA was surrounded by water for half of the surface like that for wrapped DNA around a nucleosome bead of chromatin structure.A single minimum c 2 = 26.9 (tol) around 3.7 nm < c < 4.3 nm (denoted here as structure C) was observed.The total data was 35 (24 aerobic and 11 hypoxic).
Fig. 10 .
Fig. 10.(a) Inactivation cross section of AT-cells under aerobic conditions for structure C as a function of the primary ionization mean free path l.(b) The fraction of the five reaction modes and the ratio of the inactivation cross sections under aerobic conditions for T1-kidney cells to the present estimates for AT-cells are shown as a function of l.
) and 10(a)) meaning n lethal = 1.11 ¥ 10 4 (= 1.40 mm 2 /126.8nm 2 ) molecular targets per track each of which contains at least a lethal damage.A nucleosome is associated with approximately 200 bp of DNA and consists of 146 bp of wrapped DNA around a nucleosome core (bead) and 54 bp of Linker DNA (string).In terms of the number of nucleosomes per track ndsb = 1,575 (= 3.15 ¥ 10 4 /20) nucleosomes which contain at least a dsb and nlethal = 555 (= 1.11 ¥ 10 4 /20) nucleosomes which contain at least a lethal damage.The total number of nucleosomes having a DSB per track is estimated to be 2,175 (= 1575/0.73),because measured DSBs
Fig. 11 .
Fig. 11.(a) Inactivation cross section of AT-cells under hypoxic conditions for structure C as a function of the primary ionization mean free path l.(b) The fraction of the five reaction modes and the ratio of the inactivation cross sections under hypoxic conditions for T1-kidney cells to the present estimates for AT-cells are shown as a function of l.
Fig. 12 .
Fig. 12.Yields of OH radicals per 100 eV on the nanosecond time scale as a function of the primary ionization mean free path l .The solid curves with the symbol (○ ) and with the symbol (• ) show the estimates for electrons (photons) and for He ions respectively, details of calculations are given in the ref.38.For heavy ions the values of He ions were assumed as an approximation in the present study.
Table 1 .
Optimum values for parameters of the present model | 2017-04-01T01:31:11.046Z | 2006-06-01T00:00:00.000 | {
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244212299 | pes2o/s2orc | v3-fos-license | Intelligent and Data-Driven Fault Detection of Photovoltaic Plants
: Most photovoltaic (PV) plants conduct operation and maintenance (O&M) by periodical inspection and cleaning. Such O&M is costly and inefficient. It fails to detect system faults in time, thus causing heavy loss. To ensure their operations are at an ideal state, this work proposes an unsupervised method for intelligent performance evaluation and data-driven fault detection, which enables engineers to check PV panels in time and implement timely maintenance. It classifies monitoring data into three subsets: ideal period A, transition period S, and downturn period B. Based on A and B datasets, we build two non-continuous regression prediction models, which are based on a tree ensemble algorithm and then modified to fit the non-continuous characteristic of PV data. We compare real-time measured power with both upper and lower reference baselines derived from two predictive models. By calculating their threshold ranges, the proposed method achieves the instantaneous performance monitoring of PV power generation and provides failure identification and O&M suggestions to engineers. It has been assessed on a 6.95 MW PV plant. Its evaluation results indicate that it is able to accurately determine different functioning states and detect both direct and indirect faults in a PV system, thereby achieving intelligent data-driven maintenance.
Introduction
Nowadays, PV energy represents the third-largest source of renewable energy after wind and hydro [1,2]. Many countries are developing PV projects to utilize such renewable sources. For example, a massive solar park with 7.2 million PV panels has been built in Egypt to increase its generation capacity [3], and an Iowa farm [4] in the USA uses solar power to generate fuel and fertilizer on-site. In order to increase the efficiency of generating power, PV power plants have shifted their focus from large-scale development to large-scale operation and maintenance (O&M) [5,6]. Under this circumstance, intelligent O&M methods are being widely researched. By employing them, PV power stations are capable of analyzing their operation process automatically, coping with faulty situations timely, thus greatly improving the overall efficiency of maintenance and management.
Most intelligent O&M methods are based on a video/image analysis or monitoring database. Video/image-based methods [6][7][8] utilize UAVs, satellite or 24 h cameras to get videos or images of PV stations and then train deep learning models to detect potential anomalies. However, to obtain a reliable and accurate model requires a large number of labelled samples (anomalies in the PV panels, e.g., short circuit and cell cracking). Yet, quisition. Huang et al. [25] utilizes the AdaBoost algorithm to establish a fault diagnostic model. Momeni et al. [26] uses a graph-based semi-supervised learning (GBSSL) algorithm to identify, classify, locate, and correct faults. Ma et al. [27] focuses on a partial shading scenario, and apply a multiple-output support vector regression (M-SVR) to estimate the shading strength. Chen at al. [28] proposes a random forest (RF) based fault diagnosis model and takes the real-time operating voltage and string currents of the PV arrays as fault features. Compared to the above-mentioned PR, I-V and statistical methods, the prediction methods are data-driven, which learn the diagnosis knowledge based on historical data and is free of the expertise domain background. Moreover, such machine learning-based models can detect faults in real-time and classify their specific type with high prediction accuracy. However, these methods require data to be collected from both normal and faulty conditions. Our proposed method is an unsupervised prediction method, and we do not directly predict which fault occurs. Instead, we predict the expected ideal and worst power generation and then make two comparisons between them, and real-time ones. Hence, we are able to evaluate its real-time performance and identify faults. Our work is novel and advances the area of intelligent O&M in the following aspects: (1) Applying unsupervised detection method. PV panels' performance depends on meteorological conditions, and a large number of faults may appear. It is difficult to get a dataset covering all possible fault scenarios. Thus, some methods must artificially produce labeled anomalous data by intentionally making some open circuit or short circuit to PV panels [29,30]. This undermines the total power generation of PV stations and declines their operation efficiency. On the contrary, our method is unsupervised without relying on the labelled faulty samples, and simply makes use of the existing monitoring data to evaluate operating status and detect anomalies.
(2) Building non-continuous regression models. Considering the special characteristic of non-continuity in PV generation (Non-continuity is caused by the current-limiting nature in photoelectric conversion [1,29,31], where data values are not continuous in the whole scope and some ranges are meaningless and thus no data values are found there in practice.), we build non-continuous regression models. First, we deploy the ensemble treebased regression algorithm that adjusts a tree structure according to the data characteristics and thus handles non-linear functions better [32]. Moreover, we implement clusteringbased modification to the regression predictions so as to ensure that there is no inexistent value in such non-continuous regression tasks. To the best of our knowledge, our method is the first to deal with such non-continuous issues in PV predictive models.
(3) Detecting indirect faults sensitively. Unlike direct faults that lead to conspicuous performance loss and can be identified easily, indirect faults (caused by dust, module degradation and so on) result in such a gradual PV generation loss that many methods fail to detect it [33,34]. Instead, by comparing the real-time measured value with both the upper and lower references, our method can accurately distinguish different states of PV panels, and hence detect indirect faults sensitively and also provide instantaneous alarm of degradation.
Proposed Framework
The main objective of the proposed O&M framework is to enable PV system production to reach its expected level of efficiency intelligently [19]. Therefore, the proposed approach aims at PV system failure detection, performance evaluation, and O&M planning. The notations frequently used in this paper and their descriptions are summarized in Table 1. The concrete steps of the proposed method are detailed in the following sections.
The maximum value of cluster C i Min i , i = 1, 2, . . . , k The minimum value of cluster C i l i {1, 2, 3, . . . , k}, i = 1, 2, . . . , m Class label of the i-th sample in D f A , f B Upper and lower regression models Predictions from the upper and lower model p A ,p B Modified upper and lower predictions, also simplified as a and b α 1 , α 2 , β 1 , β 2 Coefficients that divide up a baseline range w Weather scale factor A general framework of the proposed method is presented in Figure 1. First, we apply data preprocessing (including outlier detection, feature analysis and data pre-classification) to both historical meteorological and PV power datasets. Then, historical data are preclassified into three subsets that represent different operational statuses, namely, ideal period dataset D A , transition period dataset D S and downturn period dataset D B . We apply an XGBoost-based regression algorithm to datasets D A and D B , so as to train upper and lower baseline models of a PV plant's power output. Moreover, since PV power data are noncontinuous, we deploy k-means [35] to cluster hierarchical PV power data and use the statistical results of every cluster to modify the prediction values. Furthermore, due to very low PV generation in bad weather (e.g., rainstorm, blizzard, hail, and sandstorm), we consider its corresponding weather scale factors to revise both references. Thus, by integrating the results of upper and lower reference models, clustering results and weather scale factors, we acquire the final upper and lower reference curves. Comparing the measured power with two reference curves, we can evaluate their performance, detect faults, and carry out intelligent data-driven O&M. It is noted that our method does not use the information related to a PV system's components like inverters, which means that it is a generic data-based method and not limited to a certain type of PV systems. We intend to evaluate the different operating statuses of real-time power generation so as to better implement O&M. Thus, we propose the five stages (determined by two references) and present corresponding definitions, which are detailed in Section III.C. If only using a simple threshold to identify how close the actual power value is to the expected value, there is only one reference indicating the expected generation, so it would be easy and clear to identify whether the PV panels are in the expected state or not. However, it is not qualified to answer the following important questions: which operating situation (ideal or malfunction period) the PV panels are in when the actual generation values are higher than the expected ones, and how to distinguish the downturn period when covered by light-barriers and the malfunction periods when suffering from short-circuit (their power outputs in such cases are far below the expected reference values)? Using only one threshold makes it difficult to make more fine-grained performance evaluations. Therefore, we propose to use two references indicating the expected best and worst generation. With two references, the above questions can be easily answered. Moreover, it is more accurate to determine the operating status and evaluate real-time generation efficiency.
Data Preprocessing
Before a prediction model is applied, the first step is to conduct data preprocessing, including outlier detection, feature analysis, and data pre-classification.
Outlier Detection
Due to the error or failure of sensor data transmission, there are various anomalies in raw PV monitoring data, such as missing, negative, and duplicated values. Apart from conducting basic preprocessing towards such obvious outliers, we pay attention to detecting others, e.g., extreme and unmatched values in the original dataset so as to thoroughly clean the data.
First, we apply classical statistical methods, i.e., box-plot and 3σ criterion, on each single feature and try to detect outliers that deviate far away from most data. Note that, such statistical methods achieve local detection that only identify extreme values in a single feature. As for global detection, we concentrate on unmatched values. For exam- We intend to evaluate the different operating statuses of real-time power generation so as to better implement O&M. Thus, we propose the five stages (determined by two references) and present corresponding definitions, which are detailed in Section 3.3. If only using a simple threshold to identify how close the actual power value is to the expected value, there is only one reference indicating the expected generation, so it would be easy and clear to identify whether the PV panels are in the expected state or not. However, it is not qualified to answer the following important questions: which operating situation (ideal or malfunction period) the PV panels are in when the actual generation values are higher than the expected ones, and how to distinguish the downturn period when covered by lightbarriers and the malfunction periods when suffering from short-circuit (their power outputs in such cases are far below the expected reference values)? Using only one threshold makes it difficult to make more fine-grained performance evaluations. Therefore, we propose to use two references indicating the expected best and worst generation. With two references, the above questions can be easily answered. Moreover, it is more accurate to determine the operating status and evaluate real-time generation efficiency.
Data Preprocessing
Before a prediction model is applied, the first step is to conduct data preprocessing, including outlier detection, feature analysis, and data pre-classification.
Outlier Detection
Due to the error or failure of sensor data transmission, there are various anomalies in raw PV monitoring data, such as missing, negative, and duplicated values. Apart from conducting basic preprocessing towards such obvious outliers, we pay attention to detecting others, e.g., extreme and unmatched values in the original dataset so as to thoroughly clean the data.
First, we apply classical statistical methods, i.e., box-plot and 3σ criterion, on each single feature and try to detect outliers that deviate far away from most data. Note that, such statistical methods achieve local detection that only identify extreme values in a single feature. As for global detection, we concentrate on unmatched values. For example, in one PV plant, when irradiation is more than 1000 W/m 2 , the corresponding PV generation should also be quite large, e.g., 1000 KWh. However, there is a record with 1000 W/m 2 irradiation but very low generated power, e.g., 20 KWh. In the proposed framework, such unmatched structural outliers are removed by an unsupervised machine learning algorithm, i.e., DBSCAN (Density-Based Spatial Clustering of Applications with Noise) [36], which is able to identify hidden outliers from the global perspective. DBSCAN has two major parameters: radius ε determines the scope of a cluster, and minimum number of points N means the minimum number of members in a cluster. It can be regarded as a simple binary classification (normal data vs. outliers) method. Although it is an excellent anomaly detection method, it cannot be directly used in a monitoring system for classifying different operating status and different faults. Due to its design, it is sensitive to data distribution and depends heavily on the manual off-line setting of parameters, which is not suitable for online detection. Especially, under a situation where data are recorded every minute, DBSCAN would gradually turns to be unstable and inaccurate without timely human supervision. The biggest motivation of intelligent PV fault detection is to identify faults instantly and warn engineers of anomalous situations in time, so that they don't have to keep their eyes on these monitoring data but still can notice anomalies at the first time. Using only DBSCAN is hard to perform such goal, so we propose the methods depicted next. After detecting and removing outliers in raw data, we obtain our dataset D.
Feature Analysis
Since we try to predict PV generation, it is necessary to carry out detailed analysis of PV power data (p). First, it is greatly affected by the fluctuation and uncertainty of meteorological factors, and hence exhibits variability and volatility. Particularly, under the nonstationary and low illumination intensity in cloudy and rainy days, PV power data are prone to fluctuating violently [2,26,31,37]. Second, due to the current-limiting nature, a PV system has non-continuous output characteristics of power generation [2,29,37]. However, traditional methods, e.g., linear regression and support vector regression, fit continuous data and output continuous value. The special characteristics of PV power data increase difficulties and challenges for the accurate prediction of PV output. We deal with such non-continuous regression tasks by clustering-based modification which is presented in detail in the next sections.
Besides analyzing PV power data, we pay attention to the factors that affect or contribute to PV output. The most directly related factors are meteorological one. Commonly used factors include solar irradiance (r), temperature of PV panel (τ), relative humidity (h), wind velocity (v), and wind direction (d). In order to capture the non-linear relationship between meteorological factors and PV power, as many feature engineering methods do, we construct two additional features: r L and h L , which are the logarithmic values of r and h. To capture the changing trend (increasing or decreasing) of solar irradiance, we add feature r which denotes the differential irradiance between two adjacent r values.
Data Pre-Classification
In the proposed method, a critical part is to build two models: upper and lower reference models. It is of great importance to select suitable valid data from the original data and use them to train two models. We dig out the original data D and manually select the ideal period dataset D A and the downturn period dataset D B for the upper and lower model, respectively.
As shown in Figure 2, we propose to pre-classify the original data into three periods. For most PV plants, as time goes by, if there are no faults during running, the PV panels degrade due to dust or module deterioration. Thus, the power generation presents a declining tendency as shown in Figure 2. The states of PV panels are divided into the following three periods: 1) Ideal period A: The first time when the panels are brought into operation maintenance (e.g., cleaning and washing), the PV panel is in a healthy and clea without any light barriers. At this time, the efficiency of photoelectric conve comparatively high. The power generation in a PV plant is also at an ideal state, n relatively high and stable.
2) Transition period S: Under a natural state and without any interference, P els gradually accumulate dust and some light barriers (e.g., bird dropping, leave and plastic bags). Under this circumstance, the conversion efficiency slows dow power generation gradually declines too. The total PV power generation makes a al transition from ideal state to a lower state.
3) Downturn period B: When there is visible dust or too much light barriers panels, PV cells receive little solar irradiation, or when they are aging, the photo conversion efficiency reaches its lowest limit, and the generated power continue sluggish.
Among these operating periods, we pay special attention to A and B perio collect data from these two periods to construct the ideal period dataset and turn period dataset . Note that in this paper we manually classify the datase select suitable data for and . According to the definitions of operating perio our preliminary investigation, we are able to select data for and based on w and maintenance records. Based on experience, these two factors are the most ones in regard to generation efficiency. Besides, it is convenient to get access to historical operating records and the weather-related data online. Our method can ily applied in other similar tasks. In the future, we consider labeling historical with different period labels and then train a classification model, thus avoiding selection of data.
Non-Continuous Regression Models
The proposed method builds the upper and lower baseline models with respectively. The training procedure of these two models are similar, and the o ference lies in a different PV dataset we input for training.
As mentioned above, PV power data have special characteristics of variabil non-continuity, which motivates us to deploy an ensemble trees-based regression od called extreme gradient boosting (XGBoost) [38]. It assembles a number of (Classification and Regression Tree) as base learners, which can deliver more a (1) Ideal period A: The first time when the panels are brought into operation or after maintenance (e.g., cleaning and washing), the PV panel is in a healthy and clean state without any light barriers. At this time, the efficiency of photoelectric conversion is comparatively high. The power generation in a PV plant is also at an ideal state, namely, relatively high and stable.
(2) Transition period S: Under a natural state and without any interference, PV panels gradually accumulate dust and some light barriers (e.g., bird dropping, leaves, snow and plastic bags). Under this circumstance, the conversion efficiency slows down, and power generation gradually declines too. The total PV power generation makes a gradual transition from ideal state to a lower state.
(3) Downturn period B: When there is visible dust or too much light barriers on PV panels, PV cells receive little solar irradiation, or when they are aging, the photoelectric conversion efficiency reaches its lowest limit, and the generated power continues to be sluggish.
Among these operating periods, we pay special attention to A and B periods. We collect data from these two periods to construct the ideal period dataset D A and downturn period dataset D B . Note that in this paper we manually classify the dataset D and select suitable data for D A and D B . According to the definitions of operating periods and our preliminary investigation, we are able to select data for D A and D B based on weather and maintenance records. Based on experience, these two factors are the most related ones in regard to generation efficiency. Besides, it is convenient to get access to its own historical operating records and the weather-related data online. Our method can be easily applied in other similar tasks. In the future, we consider labeling historical data D with different period labels and then train a classification model, thus avoiding manual selection of data.
Non-Continuous Regression Models
The proposed method builds the upper and lower baseline models with D A and D B , respectively. The training procedure of these two models are similar, and the only difference lies in a different PV dataset we input for training.
As mentioned above, PV power data have special characteristics of variability and non-continuity, which motivates us to deploy an ensemble trees-based regression method called extreme gradient boosting (XGBoost) [38]. It assembles a number of CART (Classification and Regression Tree) as base learners, which can deliver more accurate prediction. It inherits the advantages of a decision tree algorithm and handles well non-continuous functions, which exactly suits the prediction task on non-continuous PV power data. Hence, we deploy it as our regression algorithm. The power generation p is the output of our prediction model whose inputs are the combination of meteorological features (r, τ, h, v, d), time-related features (T), and additional features ( r, r L , h L ). Then, our regression prediction model is denoted as: where f is an XGBoost-based prediction function. Note that we cannot obtain explicit expressions in a tree-based regression method. Hence, f is a simplified notation of a tree structure and corresponding parameters. Using D A and D B as training datasets, we can obtain two prediction models f A and f B . By inputting a real-time feature vector: into f A and f B , we can conduct PV generation prediction and acquire the upper and lower references, i.e., Although the proposed XGBoost-based regression model is suitable for fitting noncontinuous PV data, it is still a regression method and sometimes obtain outputs that do not exist in a real PV system. Considering the non-continuous characteristic of PV generation, it is necessary to implement further modification to refine (3) and (4), i.e., modifying with the weather scale factors and power data clustering results, which is detailed as follows. Due to the above mentioned current-limiting principle, PV power data are of obviously hierarchical discreteness. Power values belong to several particular groups where they are continuous. Between two adjacent groups, there is a blank gap with no data. We propose to cluster the original power data by using k-means algorithm [35]. In k-means, there is only one key parameter: the number of clusters denoted as k. After clustering, we calculate the minimum and maximum values for each cluster. Hence, we can know the ranges to which actual values belong. For upper or lower predictions located outside existing ranges, we propose to modify them with the maximum or minimum values of their closest cluster.
For the upper prediction value p A , if it does not belong to any cluster, then the principle of proximity is adopted to correct it. We replace it with the maximum value of the closest cluster. The modified prediction value iŝ where Max j is the maximum value of the j-th cluster. Similarly, for p B located beyond any existing cluster, we modify it with the closest minimum value, as follows: where Min j is the minimum value of the j-th cluster.
Considering the variability of PV generation under different weather conditions, we propose the weather scale factors so as to make our prediction more robust. When predicting expected PV generation in bad weather (here, bad weather refers to the case of greatly unstable irradiance or extremely low irradiance, e.g., rainstorms, blizzards, hail, and sandstorms), we multiply them by weather scale factors. In the proposed method, a weather scale factor w is defined as the percentage of reduction of power generation in bad weather. It can be computed as the ratio of average power output from a normal day to that of a bad weather day, which can be derived from the historical monitoring data. Then, (5) and (6) are modified byp A = wp A andp B = wp B . Prediction modification is realized in Algorithm 1. classify it into the closest category: 7. Let Moving each cluster centroid µ j to the mean of the points assigned to it: Repeat above for-loop until the change of centroids is less than a certain threshold 10. Obtain the clustered data (5)
Performance Evaluation, Fault Detection, and O&M Planning
Generally, a PV system can be affected by different types of faults that result in the significant loss of power [20,39,40]. According to the factors causing PV faults, two types of faults can be distinguished: direct and indirect faults. Some direct faults such as cell cracking, nonconnected module, open circuit and short circuit in a PV system, and broken fuse or cable, cause conspicuous performance loss. Indirect factors, such as shading due to dust or light barriers, encapsulation degradation due to ultraviolet and yellowing EVA (ethylene vinyl acetate), module degradation due to light or heat, and rust due to water infiltration, lead to the gradual deterioration of PV panels, and hence result in the gradual power loss [34]. Using the monitored data, a PV monitoring system has to decide whether there is degradation in its generation performance [41].
In the proposed approach, apart from the real-time PV power-versus-time curve displayed in the monitoring system, there are also two reference curves (A and B) from regression models exhibited in the same figure. For each real-time record (including a feature vector x and its corresponding power generation p), we obtain its expected ideal and worst PV generationsp A andp B by inputting its feature vector x into Algorithm 1. To simplify, we set a =p A and b =p B . Our method evaluates the PV panels' real-time status by comparing real-time PV power p with a and b. As in Table 2 BetweenB andB: Fluctuating nearB: Far belowB baseline : If real-time power exhibits more than a given percentage of generation losses, they are classified into a downturn or malfunction period. In our method, we set α 1 , α 2 , β 1 , and β 2 (α 1 > α 2 , β 1 > β 2 and α 1 , α 2 , β 1 , β 2 ∈ [0, 1]) to divide up the warning ranges. Users receive an alarm if a PV panel produces more than α 1 of the expected ideal baselineB, i.e., p > (1 + α 1 )a, or less than β 1 of the expected worst baselineB, i.e., p < (1 − β 1 )b, which means that the sensors or PV panels may break down or the data transmission is incorrect. If p is fluctuating nearB, i.e., (1 − β 2 )b < p < (1 + β 2 )b, the PV panels are of low efficiency, and they need timely maintenance, such as manual cleaning and equipment repair. For α 1 and β 1 , smaller values mean stricter alert and more sensitive detection; larger values mean looser limitation, but help reduce false alarms. On the contrary, a larger β 2 means larger range of Stage 4, which may result in more observations classified into the downturn period, hence bring in more false alarms.
If p is nearB, i.e., (1 − α 2 )a < p < (1 + α 2 )a, it indicates that the PV power generation is running in an ideal state. There is no need to implement any maintenance. Furthermore, there is no warning or alarm when p is betweenB andB, i.e., (1 it is in the transition period, and we consider it as a normal life cycle of PV panels. Note that, if α 2 is too large, there are more data classified into the ideal period, leading to the risk of misclassification of potential faults.
Data Description
We have conducted experiments based on the proposed method and used it in a 6.95 MW PV plant. Apart from zero-records (at night or missing), available effective monitoring data consist of 5936 records (In our experiments, the monitoring system records sensor data every 15 min). Although the time range of the collected dataset covers less than one year, our data included all kinds of weather situations, especially some extreme weather, e.g., snow, high temperature and rainstorms. Examples of the collected data are shown in in Supplementary File. As mentioned in Section 3, we collect power data p and features x = [r, τ, h, v, d, T, r, r L , h L ] in (2).
Results of Data Preprocessing
We use DBSCAN [36] to detect outliers and the parameters are set as ε = 46 and n = 25. To explore raw data intuitively, we plot PV data of a week in Figure 3. Only daytime hours (from 6:00 to 18:00) are considered in our PV forecasting application. As in Figure 3, PV power experiences violent fluctuation within a day as well as drastic variation among days. Meanwhile, we plot all measured PV power data versus time, as shown in Figure 4. It is noticeable that PV power data is non-continuous. Looking at the picture from the bottom to top, there are many blank gaps between two adjacent data dot clusters. Obviously, power data belong to different groups. In sum, the characteristics of raw data: intensive fluctuation and variability, and hierarchical non-continuity, motivate us to apply an XGBoost algorithm that suits most for non-continuous PV power regression task.
from the bottom to top, there are many blank gaps between two adjacent data dot clusters. Obviously, power data belong to different groups. In sum, the characteristics of raw data: intensive fluctuation and variability, and hierarchical non-continuity, motivate us to apply an XGBoost algorithm that suits most for non-continuous PV power regression task. We aim to select appropriate data for two datasets, and , reflecting the ideal and downturn periods, respectively. First, we get by considering maintenance records of a studied PV plant. The scheduled maintenance plan for year 2018 is to do the cleaning work every month, and each last for nearly half a month (from 16th to the end of that month). Obviously, the cleaning work is quite frequent. Under this circumstance, PV panels always stay in a comparatively clean status, namely, the ideal period. (Since we need the downturn period data, we stop the monthly cleaning from May.) Moreover, we consider that PV panels are perfectly clean two days after cleaning, so we take data from 18th to the end of the month from January to April as an ideal period dataset. For example, in January, data are collected into . In order to obtain valid , we suspend the monthly cleaning from May. Without cleaning, PV panels depend on the rain to wash away dust or other light barriers. So, it is important to find data originated from to apply an XGBoost algorithm that suits most for non-continuous PV power regression task. We aim to select appropriate data for two datasets, and , reflecting the ideal and downturn periods, respectively. First, we get by considering maintenance records of a studied PV plant. The scheduled maintenance plan for year 2018 is to do the cleaning work every month, and each last for nearly half a month (from 16th to the end of that month). Obviously, the cleaning work is quite frequent. Under this circumstance, PV panels always stay in a comparatively clean status, namely, the ideal period. (Since we need the downturn period data, we stop the monthly cleaning from May.) Moreover, we consider that PV panels are perfectly clean two days after cleaning, so we take data from 18th to the end of the month from January to April as an ideal period dataset. For example, in January, data are collected into . In order to obtain valid , we suspend the monthly cleaning from May. Without cleaning, PV panels depend on the rain to wash away dust or other light barriers. So, it is important to find data originated from continuous sunny days, where PV panels may be covered with dust or light barriers, and hence in the downturn period. By checking the historical weather, which is freely available online, we are able to select suitable valid data for from May to July. Take We aim to select appropriate data for two datasets, D A and D B , reflecting the ideal and downturn periods, respectively. First, we get D A by considering maintenance records of a studied PV plant. The scheduled maintenance plan for year 2018 is to do the cleaning work every month, and each last for nearly half a month (from 16th to the end of that month). Obviously, the cleaning work is quite frequent. Under this circumstance, PV panels always stay in a comparatively clean status, namely, the ideal period. (Since we need the downturn period data, we stop the monthly cleaning from May.) Moreover, we consider that PV panels are perfectly clean two days after cleaning, so we take data from 18th to the end of the month from January to April as an ideal period dataset. For example, in January, data are collected into D A . In order to obtain valid D B , we suspend the monthly cleaning from May. Without cleaning, PV panels depend on the rain to wash away dust or other light barriers. So, it is important to find data originated from continuous sunny days, where PV panels may be covered with dust or light barriers, and hence in the downturn period. By checking the historical weather, which is freely available online, we are able to select suitable valid data for D B from May to July. Take July as an example, it is rainy in the first week. Thus the data are not appropriate for dataset D B . But since 8 July 2018, it has been cloudy, overcast or sunny, which suits our selection principle of dataset D B . So, we take data from 9 July 2018 to 15 July 2018 into dataset D B .
To conclude, by data preprocessing, we determine the XGBoost-based regression algorithm according to the special characteristics of PV power data. Moreover, we present how to construct datasets D A and D B . More detailed analyses about data preprocessing can be viewed in Supplementary File.
Results of Non-Continuous Regression Models
We compare XGBoost with seven universal regression methods, and each of them has achieved good performance in existing research. This includes multivariable linear regression (MLR) attempts to model the relationship between two or more explanatory variables and a response variable by fitting a linear equation to observed data. ElaticNet [42] is a regularized regression method that linearly combines the L1 and L2 penalties of the lasso and ridge methods. Support vector regression [27] is a version of support vector machine (SVM) for regression. Here, we apply kernel-based SVM. Support vector regression with linear kernel is denoted as SVR, and the one with radial basis function kernel is denoted as SVR-RBF. Decision tree regression (DTR) [43] uses tree structure to predict the continuous output on the basis of input or situation described by a set of properties. Random forest regression (RFR) [28] is an ensemble learning method, which constructs a multitude of decision trees at training time and outputs the mean prediction of the individual trees. Gradient boosting decision tree (GBDT) [44] builds the ensemble model in a stage-wise fashion like other boosting methods do, and it generalizes them by allowing optimization of an arbitrary differentiable loss function. Note that we compare their basic regression performance and do not implement the proposed modifications (clustering-based one and weather scale factors-based one). The compared algorithms are listed: (1) Multivariable linear regression (MLR) (2) ElasticNet (3) Support vector regression with linear kernel (SVR) (4) Support vector regression with radial basis function kernel (SVR-RBF) (5) Decision tree regression (DTR) (6) Random forest regression (RFR) (7) Grmadient boosting decision tree (GBDT) The above algorithms are available in scikit-learn [45] which is a free software machine learning library for the Python programming language. Among them, RFR and GBDT are similar to XGBoost. They are tree-based ensemble methods but others use a single model.
To validate the generalization performance, we extract four datasets from the dataset D: April, May, June, and July. They are under different meteorological conditions. Then, for each dataset, we split 50% for training regression models and the rest for testing. We apply five-fold cross validation to search the optimal parameters that show the highest accuracy.
We use three performance metrics on test data. They are the ratio of root mean squared error to the mean value denoted as E, the mean absolute error denoted as E, and the goodness of fit denoted as R 2 . where y i is the ground truth of p,ŷ i is the prediction value, y i is the mean of all y i , and n is the number of test samples. All experiments are carried out in Python 3.7 with an Intel Core i5-8250 CPU and 8G memory. Table 3 shows the performance results on each dataset, and the best one is highlighted in bold. From Table 3, compared to other methods, XGBoost generally achieves much better performance. Even if it does not rank the best on dataset May, it achieves the second-best with a small gap to the best one, i.e., RFR. Table 4 presents the average evaluation metrics on the four datasets. XGBoost outperforms its peers with the best average performance metrics. In particular, XGBoost achieves better performance than RFR and GBDT, both of which are the state-of-the-art ensemble regression methods. In sum, compared with its seven peers, XGBoost achieves higher average accuracy and more generalized performance under different meteorological conditions. Table 3. Accuracy of algorithms.
Algorithms
April May June July Then, we apply XGBoost to train upper and lower reference models. We randomly split 50% of selected D A and D B to train regression models. The rest of D A and D B are for testing. The optimal XGBoost parameters for upper models are as follows: maximum depth is 3, learning rate is 0.1 and the number of estimators is 125. As for the lower model, maximum depth is 3, learning rate is 0.06, and the number of estimators is 300. For both upper and lower models, other not-mentioned parameters are set as default values. As discussed in Section 3, we deploy a k-means clustering algorithm to classify original PV power data and then take the clustering results to modify the prediction values. In the k-means algorithm, we set parameter k = 16 to ensure that our data are exactly classified into 16 classes (As in Figure 4, there are 16 classes). The clustering results are presented in the Supplementary File section. We calculate the maximum value (Max) and the minimum value (Min) of every cluster. Then, it is necessary to take the weather scale factors into consideration to avoid incorrect near-zero PV prediction. The near-zero values usually are regarded as data from a malfunction period, but for extreme weather, it is reasonable to get zero PV output. Some methods may produce false alarm under this situation. The weather scale factors are used to modify the predictions under the case of greatly unstable irradiance or extremely low irradiance, thus effectively avoiding the abovementioned false alarm. The weather scale factors are computed as the ratio of average power output from a normal day to that of a bad-weather day. The studied PV plant is operated under normal weather conditions, i.e., the collected datasets does not include data under extreme weather conditions (e.g., blizzards, hail, and sandstorms), and we thus set w = 1. Our future work plans to find more datasets that cover all types of weather conditions and conduct related analysis to show how weather scale factors can be well-used to improve model performance. Following Algorithm 1, we obtain the final predictions for the test samples. Table 5 presents model performance on training and test datasets. E and E are low on two test datasets, R 2 is very close to 1 on both models, indicating that our two prediction models are highly accurate and reliable. Note that, the performance in Table 5 is not as good as those in Tables 3 and 4. This is because our selected datasets D A and D B consist of records from different months, and hence the training and test datasets are less stable than those in above experiments. To better present the superiority of our proposed modification methods, i.e., the clustering-based modification and weather scale factor-based one as shown in Algorithm 1, Figure 5a,b show an example of the monitoring system before and after modifica-tion, respectively. Two trained models are applied to the monitoring data of a day in May which are presented as black hollow circles. The blue curve is the upper reference from f A , and the purple one is the lower reference deriving from f B . In Figure 5, it is noticeable that purple one is sometimes above the blue one, e.g., curves during 5:28-6:43, 8:28-9:28, and 16:28-17:58. Moreover, there are many observations below or above the reference lines. Instead, in Figure 5, there is no overlap, and the purple reference line is below the blue one. Furthermore, the modifies references are more consistent with the changing trend of actual PV power values, and there are also less observations located outside two references.
Processes 2021, 9, x FOR PEER REVIEW 14 of 21 the ratio of average power output from a normal day to that of a bad-weather day. The studied PV plant is operated under normal weather conditions, i.e., the collected datasets does not include data under extreme weather conditions (e.g., blizzards, hail, and sandstorms), and we thus set = 1. Our future work plans to find more datasets that cover all types of weather conditions and conduct related analysis to show how weather scale factors can be well-used to improve model performance. Following Algorithm 1, we obtain the final predictions for the test samples. Table 5 presents model performance on training and test datasets. � and � are low on two test datasets, R 2 is very close to 1 on both models, indicating that our two prediction models are highly accurate and reliable. Note that, the performance in Table 5 is not as good as those in Tables 3 and 4. This is because our selected datasets and consist of records from different months, and hence the training and test datasets are less stable than those in above experiments. To better present the superiority of our proposed modification methods, i.e., the clustering-based modification and weather scale factor-based one as shown in Algorithm 1, Figure 5a,b show an example of the monitoring system before and after modifica-tion, respectively. Two trained models are applied to the monitoring data of a day in May which are presented as black hollow circles. The blue curve is the upper reference from , and the purple one is the lower reference deriving from . In Figure 5, it is noticeable that purple one is sometimes above the blue one, e.g., curves during 5:28-6:43, 8:28-9:28, and 16:28-17:58. Moreover, there are many observations below or above the reference lines. Instead, in Figure 5, there is no overlap, and the purple reference line is below the blue one. Furthermore, the modifies references are more consistent with the changing trend of actual PV power values, and there are also less observations located outside two references.
Results of Performance Evaluation, Fault Detection, and O&M Planning
Based on two modified prediction models, we obtain the corresponding upper and lower references of power generation. We compare them with the measured power value, and assess PV panel status according to their distributions in the reference range. After different tests about selecting suitable parameters, we recommend to set 1 = 1 = 0.5 and 2 = 2 = 0.1, which show satisfactory results in most experiments. To make a
Results of Performance Evaluation, Fault Detection, and O&M Planning
Based on two modified prediction models, we obtain the corresponding upper and lower references of power generation. We compare them with the measured power value, and assess PV panel status according to their distributions in the reference range. After different tests about selecting suitable parameters, we recommend to set α 1 = β 1 = 0.5 and α 2 = β 2 = 0.1, which show satisfactory results in most experiments. To make a comprehensive comparison, we show pictures of different weather conditions, i.e., a sunny day in Figure 6a, and a cloudy dayin Figure 6b. There are five kinds of typical distributions. As discussed before, data in Stages 1, 4, and 5 can provide early warning for the engineers in a PV station. In Stages 1 and 5, actual values deviate far from the references. This is the malfunction period, where the sensors may break down and the data transmission may be incorrect. Specially for Stage 5 where the power is relatively low, chances are high that there are open circuits or short circuits in PV panels. In Stage 4, the generated power is comparatively low. PV panels are in a downturn period, and may be covered by dust and need cleaning. It is worth noting that some disturbance in the power grid also leads to the decrease of output power. It may fall below the lower reference curve, but it is not due to a failure in the PV plant. After such warning, on-site engineers need to conduct further inspection and corresponding O&M plans. Stages 2 and 3 do not trigger warning, because they correspond to an ideal period and transition period. According to our previous data analysis and definitions of stages, the transition period (Stage 3) and the downturn period (Stage 4) are the most common ones. Generally, a normal operating PV station does not frequently break down with severe faults or always yield an ideal power generation with high operational efficiency. Hence, a malfunction period (Stages 1 and 5) and ideal period (Stage 2) are the relatively less common ones. comprehensive comparison, we show pictures of different weather conditions, i.e., a sunny day in Figure 6a, and a cloudy dayin Figure 6b. There are five kinds of typical distributions. As discussed before, data in Stages 1, 4, and 5 can provide early warning for the engineers in a PV station. In Stages 1 and 5, actual values deviate far from the references. This is the malfunction period, where the sensors may break down and the data transmission may be incorrect. Specially for Stage 5 where the power is relatively low, chances are high that there are open circuits or short circuits in PV panels. In Stage 4, the generated power is comparatively low. PV panels are in a downturn period, and may be covered by dust and need cleaning. It is worth noting that some disturbance in the power grid also leads to the decrease of output power. It may fall below the lower reference curve, but it is not due to a failure in the PV plant. After such warning, on-site engineers need to conduct further inspection and corresponding O&M plans. Stages 2 and 3 do not trigger warning, because they correspond to an ideal period and transition period. According to our previous data analysis and definitions of stages, the transition period (Stage 3) and the downturn period (Stage 4) are the most common ones. Generally, a normal operating PV station does not frequently break down with severe faults or always yield an ideal power generation with high operational efficiency. Hence, a malfunction period (Stages 1 and 5) and ideal period (Stage 2) are the relatively less common ones.
(a) (b) Figure 6. Five kinds of distribution on (a) a sunny day and (b) a cloudy day.
In order to explicitly present the performance evaluation, we plot the warning boundary lines in Figure 7a,b. In the monitoring system, the engineers are capable of directly distinguishing the PV panel statuses and getting suggestions about how to carry out proper O&M plans. As in Figure 7a, the PV power generation experiences an abrupt decline and drops greatly at 14:28 in 2018/5/11, which indicates that the PV station is in a malfunction period and maintenance is required. According to the abrupt decline and long-lasting Stage 5, we consider that there is direct fault in the PV plant, e.g., nonconnected modules and short/open circuits. Such direct faults are relatively easy to notice in a monitoring system. They usually happen in Stage 5, accompanied by an obvious and long-term decline of PV generation. In this case, further O&M plans lie in checking detailed PV records about each panel and then locating the faulty one(s). As in Figure 7b, a cloudy day in summer, the solar irradiance is strong, so the curves of p, a, and b are nearly sinusoids shape. At 10:37, both A and B baselines fall greatly, whereas the actual p stays in the original trend. There is a high chance that meteorological sensor errors or transmission mistakes appear. The wrong data are input to our prediction models, so we get wrong results. We suggest that further O&M plans attach importance to check the original database and repair or replace faulty sensors. In order to explicitly present the performance evaluation, we plot the warning boundary lines in Figure 7a,b. In the monitoring system, the engineers are capable of directly distinguishing the PV panel statuses and getting suggestions about how to carry out proper O&M plans. As in Figure 7a, the PV power generation experiences an abrupt decline and drops greatly at 14:28 in 2018/5/11, which indicates that the PV station is in a malfunction period and maintenance is required. According to the abrupt decline and long-lasting Stage 5, we consider that there is direct fault in the PV plant, e.g., nonconnected modules and short/open circuits. Such direct faults are relatively easy to notice in a monitoring system. They usually happen in Stage 5, accompanied by an obvious and long-term decline of PV generation. In this case, further O&M plans lie in checking detailed PV records about each panel and then locating the faulty one(s). As in Figure 7b, a cloudy day in summer, the solar irradiance is strong, so the curves of p, a, and b are nearly sinusoids shape. At 10:37, both A and B baselines fall greatly, whereas the actual p stays in the original trend. There is a high chance that meteorological sensor errors or transmission mistakes appear. The wrong data are input to our prediction models, so we get wrong results. We suggest that further O&M plans attach importance to check the original database and repair or replace faulty sensors. As for the validation, as there is no label about which performance stage the PV system is in and which fault it suffers (which makes it difficult to conduct detailed verification and give specific classification metrics), we have manually labelled the data and conducted classification experiments to verify the performance of our method compared with other advanced machine learning classification algorithms. The input of classification models are the monitoring records that include both meteorological data as listed in (2) and corresponding generated PV power data. The output of classification models indicates which performance period the PV system is in, i.e., malfunction, ideal, transition, or downturn periods. We compare our method with several widely-used and powerful algorithms under their classification implementations, i.e., support vector machine classification [46][47][48] with linear kernel (SVC-Linear), support vector machine classification with RBF (SVC-RBF), decision tree classification (DTC) [49], random forest classification (RTC) [50], gradient boosting decision trees classification (GBDTC) [51] and extreme gradient boosting trees classification (XGBC) [52]. The above algorithms are available in scikit-learn [45]. The classification performance metrics are shown in Figure 8a-g and Tables 6-12. As for the validation, as there is no label about which performance stage the PV system is in and which fault it suffers (which makes it difficult to conduct detailed verification and give specific classification metrics), we have manually labelled the data and conducted classification experiments to verify the performance of our method compared with other advanced machine learning classification algorithms. The input of classification models are the monitoring records that include both meteorological data as listed in (2) and corresponding generated PV power data. The output of classification models indicates which performance period the PV system is in, i.e., malfunction, ideal, transition, or downturn periods. We compare our method with several widely-used and powerful algorithms under their classification implementations, i.e., support vector machine classification [46][47][48] with linear kernel (SVC-Linear), support vector machine classification with RBF (SVC-RBF), decision tree classification (DTC) [49], random forest classification (RTC) [50], gradient boosting decision trees classification (GBDTC) [51] and extreme gradient boosting trees classification (XGBC) [52]. The above algorithms are available in scikit-learn [45]. The classification performance metrics are shown in Figure 8a-g and Tables 6-12. with RBF (SVC-RBF), decision tree classification (DTC) [49], random forest classification (RTC) [50], gradient boosting decision trees classification (GBDTC) [51] and extreme gradient boosting trees classification (XGBC) [52]. The above algorithms are available in scikit-learn [45]. The classification performance metrics are shown in Figure 8a-g and Tables 6-12. Based on whether PV generation is matched with real-time meteorological records, we manually classify the original data into 4 classes, i.e., malfunction, ideal, transition and downturn periods. With meteorological records, it is possible to calculate the nominal power generation by formulas of photoelectric conversions. Specifically, the generated PV power in an ideal period is supposed to be close to the nominal power generation; the generation in a transition period is slightly lower than the nominal one; the generation in a downturn period is relatively low but reasonable (due to too-much light barriers or aging panels); and the generation values in a malfunction period are extremely larger or lower than the nominal values. Such manual divisions are conducted based on expert knowledge and prior experience. We label the malfunction, ideal, transition and downturn periods with the class indices 0, 1, 2, and 3, respectively. Then, we split 75% for training classification models and the rest for testing. We apply five-fold cross validation to search the optimal parameters that show the highest performance. Figure 8 shows the confusion matrix (CM) of all classes in test dataset. Tables 6-12 details the performance metrics (precision, recall, f1-score) of each compared algorithm and our method. From Figure 8a-g and Tables 6-12, we can conclude that our method achieves the best classification performance with the highest averaged precision 0.94, recall 0.93 and f1-score 0.93. Moreover, the other compared methods are far behind, which validates the superiority of our method.
In addition, we assess model performance by consulting engineers and judge whether the proposed method gives right performance evaluation and accurate fault alarm. We apply the proposed method to the monitoring system of our studied PV plant. According to the feedback from their on-site engineers, our method achieves accurate performance evaluation and fast fault detection. First, our method is able to present instantaneous evaluation for each real-time observation. With the assistance of our method, the O&M engineers do not have to keep their eyes on the curves, and they only check the database when Stages 1 and 4 appear. Second, our method is able to detect both direct and indirect faults in a PV system. It presents an accurate classification and seldom misses potential anomalous situation, which greatly enhances the operation safety and maintenance efficiency. More results and analyses are presented in the Supplementary File section.
Although our proposed method runs well in a practical PV plant, from Figure 7a,b we can tell that there are still a few unusual observations in early morning and late afternoon when illumination intensity is quite weak. We plan to improve the robustness of our prediction models as future research, so that they can make more accurate prediction even when the power output is pretty low.
Discussion
In practical scenarios of PV stations, direct faults, like open circuit and transmission errors, are comparatively easy to notice in the monitoring system. There is an abrupt shift from previous trend. Among indirect factors, encapsulation or module degradation is common in the life cycle of PV panels, which is unavoidable. Therefore, the difficult task of O&M in a PV plant is to intelligently implement panel cleaning, including dust removal and anti-blocking. Compared to direct faults, shade reduces a small amount of output power, which is hard to be detected. In the past, the cleaning O&M of PV panels was mainly periodically manual or robotic cleaning, such as once a month or once a week. Now with the proposed method, which evaluates the state of PV panels and provides instantaneous alarm of degradation, the cleaning maintenance is triggered only when needed. Furthermore, the proposed framework can easily detect direct PV faults and offer timely O&M suggestions.
Conclusions
This paper presents an O&M framework consisting of an intelligent detection structure which can enhance the O&M efficiency in the PV monitoring system and reduce the burden on monitoring staff. Our method evaluates operating performance and identifies anomalies by comparing to two reference baselines, which is an unsupervised way and exerts no dependence on labeled faulty data. Moreover, considering the special characteristic of non-continuity in PV generation, we build corresponding non-continuous regression models, which are based on XGBoost algorithm and refined by the results of k-means clustering. Last, by comparing the real-time measured value with both the upper and lower references, our method is sensitive to indirect faults and can provide instantaneous alarm of degradation. Results on a 6.95 MW PV plant indicate that the proposed method is able to evaluate different operating statuses and provide faults identification and O&M suggestions to engineers.
Our work focuses on performance monitoring, fault detection and diagnosis, and O&M optimization in large complex systems. With proper data of ideal and downturn periods, the proposed method can be easily applied to other similar engineering scenarios, such as the assessment of workshop equipment and fault detection in wind power plants. Moreover, our method can be transferred to the application of RUL (remaining useful life) [53] prediction and equipment's PHM (prognostic and health management) [54]. In future studies, we plan to concentrate on the classification refinement of detected faults and predictive maintenance based on the proposed method. | 2021-10-18T18:26:45.393Z | 2021-09-24T00:00:00.000 | {
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12252334 | pes2o/s2orc | v3-fos-license | Dynamic Metrics of Heart Rate Variability
Numerous metrics of heart rate variability (HRV) have been described, analyzed, and compared in the literature. However, they rarely cover the actual metrics used in a class of HRV data acquisition devices - those designed primarily to produce real-time metrics. This paper characterizes a class of metrics that we term dynamic metrics. We also report the results of a pilot study which compares one such dynamic metric, based on photoplethysmographic data using a moving sampling window set to the length of an estimated breath cycle (EBC), with established HRV metrics. The results show high correlation coefficients between the dynamic EBC metrics and the established static SDNN metric (standard deviation of Normal-to-Normal) based on electrocardiography. These results demonstrate the usefulness of data acquisition devices designed for real-time metrics.
Introduction
Metrics of heart rate variability (HRV) are widely used in medical diagnostics, physiological research, and biofeedback training (TaskForce, 1996;Wheat & Larkin, 2010). HRV metrics are typically based on non-invasive measurements of cardiac function using electrocardiography (ECG) or photoplethysmography (PPG). A specific point is identified in each cardiac cycle, and the inter-beat interval (IBI) between those points in adjacent ECG heartbeats or PPG pulsebeats is measured.
Time intervals derived from ECG provide the input to a function that produces the HRV metric. Functions defined over PPG time intervals are often termed metrics of pulse rate variability (PRV).
A broad range of linear and non-linear HRV and PRV metrics have been developed based on statistical, spectral, geometric, neural network, and vector analysis techniques. 1 These metrics offer various tradeoffs in their computational complexity, applicability to real-time analysis, tolerance for physiological artifact and measurement artifact, and 1 An informal survey of published literature conducted by the second author has cataloged 69 distinct metrics, without distinguishing between ECG and PPG variants. significance with respect to particular medical or physiological conditions. Most of HRV and PRV metrics described in published literature operate directly on a set of IBIs taken over a given length of timewe use the term static metrics in this article for these metrics. However, a wide selection of devices are available that provide metrics constructed in fundamentally different ways. These devices are typically designed for realtime reporting of metrics. Hence, we use the term dynamic metrics for the class of metrics provided by real-time data acquisition devices.
This article characterizes dynamic metrics and reports the results of a pilot study which compares one such dynamic metric with established HRV metrics.
ECG and PPG Metrics
In this article, we differentiate ECG-based metrics from PPG-based metrics using e and p postscripts, respectively. For example: SDNNe and SDNNp are variants of the widelyused SDNN metric of HRV. SDNNe is the standard deviation of the intervals between adjacent R peaks of the QRS complex derived from normalized ECG measurements recorded over a given time period. SDNNp is the corresponding metric based on peaks in normalized PPG measurements.
Because of the relative ease of collecting PPG measurements in many situations, PRV metrics are often preferred to ECG-based HRV metrics. A number of studies have examined the relationship between the ECG and PPG variants of specific metrics, in particular SDNN (Johnston & Mendelson, 2005;Selvaraj, Jaryal, Santhosh, Deepak, & Anand, 2008;Teng & Zhang, 2003).
Dynamic Metrics
Dynamic metrics introduce a number of characteristics not typically found in static metrics: Span. This extends the definition of IBI beyond the limitation of using adjacent cardiac cycles. The metric may be based on an IBI where the interval is a span measured between a given number, C, of cardiac cycles apart (span = C cycles) or between the cardiac cycles that are at least a given number, S, of seconds apart (span = S seconds).
An example is a metric defined over a heart rate (HR) derived from an IBI that spans 10 cardiac cycles.
Sampling window.
In the interest of producing real-time metrics, dynamic metrics are often calculated over a sampling window of IBI measurements. The size of the sampling window, typically denoted W, can be defined over time or cardiac cycles.
Static metrics use a sampling window that is typically the size of an overall study period and produce a single metric result. In dynamic metrics where the size of the sampling window is less than the study period, a sequence of metrics can be produced.
Progression. Sampling windows of measurement can be taken by moving the sampling window forward by the size of the window (sequential sampling windows, Figure 1a), by a single time interval or cardiac cycle (moving sampling windows, Figure 1b), or any other amount. In general, we use the term progression for the rule which defines how the sampling window is moved.
Note in Figure 1 that J, the last data point, is not included in the sequential sampling windows but is included in the final moving sampling window.
Also note in Figure 1b that the initial and final W-1 data points of the study period participate in fewer moving windows than the more centrally-located data points. We call this characteristic of moving windows center-bias.
Aggregation. With the introduction of sampling windows, dynamic metrics can produce a sequence of results within a given study period. Those results can then be aggregated into a single metric for the study period. A common aggregation function (also called a second-order function or a functional in other contexts) is to take the mean of the results from each sampling window. However, other functions such as taking the standard deviation of the sampling window results might be useful in some contexts.
Estimated Breath Cycle
This study compared a dynamic metric, called estimated breath cycle (EBC), with some established metrics of HRV. EBC is uses a sampling window whose size is based on an estimation of the length of a breath cycle of the subjects.
This study uses EBC metrics based on PRV (EBCp) with sequential sampling windows of 10 seconds (EBCp 10s ) and 16 seconds (EBCp 16s ) and moving sampling windows of 10 seconds (EBCp 10m ) and 16 seconds (EBCp 16m ). Mean and SD functions are used to aggregate EBC metrics within a study period.
Method
Simultaneous measurements were taken using two independent systems on a single subject that included five periods of varying breathing rates. EBC metrics were derived from a MindDrive™ finger sensor (Discovogue Infotronics, Modena, Italy). PPG levels were sampled at 24 Hz and processed data for HR were recorded at one-second intervals. HR readings on this system are derived from a weighted average of PRV intervals that span the 10 most recent pulsebeats.
PRV metrics other than EBC were derived from a Lightstone™ system by Wild Divine, Inc. (Las Vegas, Nevada). The Lightstone system reports instantaneous pulse rate by interpolating peaks between PPG readings taken at 30 Hz (Matt Cullen, Wild Divine, Inc., personal communication, June 28, 2012).
Analysis
Artifacts were identified by visual inspection in both the MindDrive and Lightstone data streams. Time segments containing artifacts in either data stream were eliminated from the analysis.
Instantaneous pulse rate metrics from the Lightstone system were converted to IBIs, which formed the basis of all PRV metrics other than EBC. Figure 2 plots two of the EBC metrics against two static metrics, demonstrating a strong correlation between SDNNp and EBCp 10s (r = .941) and between SDNNp and EBCp 16s (r = .953). Numerical correlation coefficients are provided in Table 1 for all pairs of metrics.
EBC -SDNN Correlation
The results of this pilot study demonstrated a very strong correlation between SDNNp and all the EBCp metrics.
The correlation between SDNNp and SDNNe (derived from ECG data) has been studied in three references: r = .993 based on the average of the three conditions reported by Gil et al. (2010), Table 2; r = .989 reported by Medeiros (2010), Comparison of estimated breath cycle (EBC) metrics of heart rate variability (HRV) with static HRV metrics. 10s and 16s indicate sequential windows of 10 and 16 seconds, respectively. SDNNp is the standard deviation of the intervals between adjacent peaks over the study period. RMSSDp is the square root of the mean of the sum of the squares of the intervals between adjacent peaks over the study period. All metrics are derived from normalized photoplethysmographic data. Combining the lowest SDNNp -EBCp correlation of r = .925 with lowest reported correlation of r = .989 for SDNNp -SDNNe, we get a value for r a 2 + r b 2 of 1.83. Based on this, we propose that the four EBCp metrics studied correlate very strongly with SDNNe.
EBC Correlation with RMSSD and SDSD
Correlations with two additional static metric were performed: RMSSDp is the square root of the mean of the sum of the squares of the successive differences between adjacent peaks of the PPG data over the study period.
SDSDp is the standard deviation of the successive differences between adjacent peaks of the PPG data over the study period. Both of these metrics are estimates of the short-term / high-frequency components of HRV (Thong, Li, McNames, Aboy, & Goldstein, 2003).
Two factors of the EBC metrics of this study were expected to "smooth" the data and mask high-frequency variations: the span of 10 cardiac cycles inherent in the HR data produced by the MindDrive and the sampling window itself.
As expected, the correlation between all EBC metrics and RMSSD and between all EBC metrics and SDSD is quite low, ranging from -.084 to +.106.
Limitations
This pilot study measured a small sampling of the possible dynamic HRV metrics, using a single subject, and five study periods of unequal length. We would suggest a full study in this area be carried out to further validate correlations found in this pilot study.
Conclusions
This study tested the hypothesis that data acquisition devices based on PPG data and designed primarily for realtime HRV metrics could be useful in research studies. After characterizing the properties of the dynamic metrics gathered by those devices, a pilot study was carried out to compare those dynamic metrics with the metrics of HRV that are widely cited in the literature. | 2013-08-29T15:57:03.000Z | 2013-08-27T00:00:00.000 | {
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12654965 | pes2o/s2orc | v3-fos-license | Retinal Vascular Imaging Markers and Incident Chronic Kidney Disease: A Prospective Cohort Study
Retinal microvascular changes indicating microvascular dysfunction have been shown to be associated with chronic kidney disease (CKD) in cross-sectional studies, but findings were mixed in prospective studies. We aimed to evaluate the relationship between retinal microvascular parameters and incident CKD in an Asian population. We examined 1256 Malay adults aged 40–80 years from the Singapore Malay Eye Study, who attended both the baseline (2004–07) and the follow-up (2011–13) examinations and were free of prevalent CKD. We measured quantitative retinal vascular parameters (arteriolar and venular calibre, tortuosity, fractal dimension and branching angle) using a computer-assisted program (Singapore I Vessel Assessment, SIVA) and retinopathy (qualitative parameter) using the modified Airlie house classification system from baseline retinal photographs. Incident CKD was defined as an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 + 25% decrease in eGFR during follow-up. Over a median follow-up period of 6 years, 78 (6.21%) developed CKD (70.5% had diabetes). In multivariable models, smaller retinal arterioles (hazards ratio [95% confidence interval] = 1.34 [1.00–1.78]), larger retinal venules (2.35 [1.12–5.94] and presence of retinopathy (2.54 [1.48–4.36]) were associated with incident CKD. Our findings suggest that retinal microvascular abnormalities may reflect subclinical renal microvascular abnormalities involved in the development of CKD.
Apart from retinal vascular caliber, newer retinal geometry vascular parameters (e.g. retinal fractal dimension and tortuosity) representing the 'optimality state' of the retinal microcirculation have been shown to be associated with risk factors of CKD including diabetes and hypertension 19,20 . Earlier, we reported cross-sectional association between fractal dimension and CKD in two independent studies 10,21 . Nevertheless, the prospective association between retinal geometry vascular parameters and incident CKD has not been evaluated before.
To address these limitations, we aimed to examine the association of a panel of retinal microvascular imaging markers with incident CKD in a population-based sample of Malay adults aged 40-80 years in Singapore.
Results
Over a median follow-up period of 6 years, 78 participants developed CKD. Of those who developed CKD, 55 (70.5%) had diabetes and 74 (94.9%) had hypertension. Incidence of CKD was significantly higher among those aged 60 years and above and those with diabetes ( Fig. 1). A total of 229 (18.2%) participants had a decline in eGFR of>3 mL/min/1.73 m 2 over the follow-up period. Table 1 compares the baseline characteristics of participants with and without retinopathy. Participants with retinopathy were more likely to be older, had higher prevalence of hypertension and higher levels of HbA1c, blood glucose and systolic BP. Supplementary Table shows the baseline characteristics of those who were included and excluded from the analysis. Table 2 compares the baseline characteristics of participants stratified by those with and without incident CKD. Participants who developed incident CKD were older, less likely to be secondary/ above educated, had higher prevalence of hypertension and diabetes and higher levels of HbA1c, blood glucose, SBP and DBP and lower levels of HDL cholesterol and eGFR compared to those without incident CKD. Table 3 presents the associations between retinal vascular parameters and incident CKD. In tertile analysis, CRAE was not associated with incident CKD in both age, sex adjusted and the multivariable model. In continuous analysis, CRAE showed significant association after adjusting for potential confounders in the multivariable model (hazards ratio [HR] [95% CI] = 1.34 [1.00-1.78]) although the association was not significant in the age, sex-adjusted model. On the other hand, CRVE showed a significant association with incident CKD in both age, sex-adjusted and the multivariable model in tertile analysis but not when analyzed as a continuous variable. Compared to tertile 1 of CRVE, HR (95% CI) of incident CKD was 1.94 (1.11-3.38) in age, sex-adjusted model and 2.35 (1.12-5.94) in the multivariable model. In continuous analysis, although the association of CRVE with CKD was in the positive direction, it did not reach significance. Arteriolar tortuosity showed a positive association in the age, sex adjusted model but lost significance when adjusted for potential confounders in the multivariable model. Presence of retinopathy (HR 2.54, 95% 1.48 to 4.36) at baseline was significantly associated with incident CKD in both age, sex-adjusted and the multivariable models. No significant associations were observed between retinal D f , tortuosity and branching angle with incident CKD in either model. Figure 2 shows the association of retinal vascular parameters with incident CKD stratified by diabetes status. Among those with diabetes (Fig 2A), smaller CRAE (per SD decrease: HR 1.49, 95% CI,1.00 to 2.21), wider CRVE (per SD increase: HR 1.47, 95% CI,1.03 to 2.11) and presence of retinopathy (HR 2.35, 95% 1.30 to 4.23) were associated with incident CKD. After additional adjustment for HbA1c and diabetes duration amongst people with diabetes, the association of retinopathy with incident CKD persisted (HR 3.78, 95% 1.72 to 8.32). However, association of CRAE and CRVE with incident CKD lost statistical significance (data not shown). No significant associations were observed between CRAE, or CRVE or retinopathy with incident CKD amongst participants without diabetes (Fig 2B). In analyses stratified by gender, in women, only presence of retinopathy showed significant association with incident CKD (HR 2.83, 95% 1.40 to 5.75). In men, none of the parameters showed any significant associations with CKD (data not shown).
Supplementary analyses. Incidence of CKD using alternative definition 1 (eGFR 45 + 25% decrease) was 3.1% (n = 39). Repeating the analyses using this definition, associations between retinopathy (HR 3.84, 95% 1.78 to 8.32) and incident CKD remained, but associations between CRAE (HR 1.30, 95% 0.86 to 1.96 per SD decrease) and CRVE (HR 1.22, 95% 0.80 to 1.85 per SD increase) with incident CKD lost statistical significance. Similar to the main analysis, no significant associations were observed between retinal D f , tortuosity and branching angle (data not shown). Incidence of CKD using alternative definition 2 (incident CKD and or rapid decline in eGFR) was 24.4% (n = 307). Repeating the analyses using this definition, associations between CRVE (HR 1.17, 95% 1.01 to 1.35), retinopathy (HR 1.54, 95% 1.07 to 2.22) and incident CKD remained significant. No significant associations were observed between CRAE, retinal D f , tortuosity and branching angle (data not shown).
Discussion
In this population-based study, we observed that smaller retinal arterioles, larger retinal venules and presence of retinopathy were associated with an increased risk of CKD over a median follow up of 6 years, independent of potential confounders. These associations between smaller retinal arterioles, larger retinal venules and presence of retinopathy with incident CKD were stronger in participants with diabetes. To our knowledge, the current study is the first one to examine the prospective association of retinal vascular parameters with CKD in an Asian population.
The association between retinopathy and CKD is well established 14,15 . However, few previous studies have examined the prospective association between retinal vascular parameters and CKD [13][14][15][16] . Smaller retinal arterioles were reported to be associated with incident CKD in whites only in the Multi-Ethnic Study of Atherosclerosis (MESA) 13 . In the the Atherosclerosis Risk in Communities Study (ARIC), both retinal arteriolar and venular narrowing were associated with 6-year change in serum creatinine 15 . On the other hand, in the Beaver Dam Chronic Kidney Disease 16 , and in the Cardiovascular Health Study 14 , retinal vascular calibers were not associated with CKD. It is possible that selective mortality and survival bias may have masked the associations between retinal vascular caliber and renal impairment. Further, previous studies 14, 15 did not assess exposure (retinal imaging) and outcome (CKD) measurements at baseline concurrently. For e.g. in ARIC study, authors compared retinal data collected at the third examination with the change in serum creatinine levels and development of renal dysfunction between the second and the fourth examinations 15 .
In a previous study evaluating the association of retinal microvascular abnormalities with incident ESRD, we observed significant association of retinopathy, but not other retinal parameters with ESRD 18 . The lack of association of retinal vascular parameters could be due to the smaller number of ESRD events (n = 33) or selective mortality of those with smaller retinal arterioles and ESRD 18 . In a recent study by Grunwald et al. 17 , retinal vascular calibre was not significantly associated with incident ESRD in a cohort of patients with CKD. In the current study, in persons with diabetes, smaller retinal arterioles, larger retinal venules and retinopathy were significantly associated with incident CKD. Similar to our findings, the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), reported that retinal venular dilation was associated with gross proteinuria and renal insufficiency in persons with type 1 diabetes 22 .
Retinopathy absent
Retinopathy present Retinal arteriolar narrowing has been hypothesized to represent a dysregulation of the renin-angiotensin and endothelin 23,24 . Importantly, over expression of endothelin, a potent vasoconstrictor secreted by vascular endothelial cells, has been hypothesized to be associated with sclerotic renal changes and progression of kidney disease 25 . Furthermore, experimental data from kidney biopsies of persons with type 1 diabetes have shown that narrower retinal arteriolar caliber is morphologically related to extracellular matrix accumulation 26 , which ultimately leads to a decrease in eGFR 27 . Together, it plausible that these processes may provide a common pathophysiologic link between retinal arteriolar narrowing and decrease in eGFR. The pathological processes underlying the association between retinal venular widening and incident CKD are not clear. Larger venular diameters and increased blood flow has been reported to be associated with diabetic status [28][29][30][31][32][33] . In addition, retinal venular widening and DR have been postulated to be a result of endothelial damage and inflammatory processes 34 . Clinically, retinal venular widening and DR represent thickening of basement membrane, and increased leakage, which is also observed in CKD 35 . Therefore, it is possible that retinal venular widening and DR are reflective of cumulative renal microvascular damage which eventually results in CKD.
The strengths of our study include its population-based sample, quantitative and masked evaluation of retinal vascular parameters, standardized measurement of renal function, and the availability of information on potential confounding factors. Our study has some limitations. First, we could not adjust for albuminuria in the multivariable model since data was not available in most of the participants. Second, since majority of the participants had hypertension (58.4%), we could not stratify the population by hypertension status. Third, antihypertensive medication use was not assessed in detail (e.g. ACE inhibitors). Fourth, since majority of the participants who developed CKD belonged to stage 3 (63 of 78 incident CKD, i.e. 81%), we were unable to evaluate if the addition of retinal vascular parameters aid in risk-stratification of CKD.
In conclusion, a population-based sample of Malay adults, we found that the presence of retinal microvascular changes including smaller retinal arterioles, larger retinal venules and presence of retinopathy were associated with increased risk of CKD. Our findings suggest that retinal microvascular abnormalities may reflect early subclinical damage in the renal microvasculature that is subsequently associated with development of CKD.
Methods
Study Population. We utilized data from the Singapore Malay Eye Study (SiMES), a population-based cohort study. In brief, 3280 adults aged between 40 to 80 years recruited from the community using an age-stratified random sampling method attended the baseline examination in 2004 to 2006 (78.7% response rate). The methodology and objectives of the study population have been reported in detail elsewhere 36,37 . Of the 2636 eligible participants, 1901 returned for the follow-up examination in 2004-06 (response rate: 72.1%). Written, informed consent was obtained from each participant; the study conducted adhered to the Declaration of Helsinki. Ethical approval was obtained by the Singapore Eye Research Institute Institutional Review Board. After excluding participants who had missing data on estimated glomerular filtration rate (eGFR) levels (n = 101), prevalent CKD (n = 307), ungradable baseline retinal photographs (n = 111), missing data on covariates (n = 76) and history of cardiovascular disease (CVD; n = 50), 1256 participants were included for the current analysis.
Measurement of Retinal Vascular Parameters.
Retinal fundus photographs of both eyes were taken after dilating the pupils with 1% tropicamide and 2.5% phenylephrine hydrochloride, using a digital non-mydriatic retinal camera (CR-DGi with a 10D SLR backing; Canon, Tokyo, Japan). We used a semi-automated computer-assisted program (Singapore I Vessel Assessment [SIVA], version 1.0) to measure retinal microvascular parameters quantitatively from digital retinal photographs. Trained graders, masked to participant characteristics, executed the SIVA program to measure the retinal microvasculature. SIVA automatically identifies the optic disc and places a grid with reference to the center of optic disc. Retinal arterioles and venules were also automatically identified. All visible vessels coursing through the specified zone (0.5 disc diameter -2.0 disc diameter) were measured. Graders were responsible for the visual evaluation of SIVA-automated measurements and manual intervention if necessary, following a standardized grading protocol. The intra-and inter-grader reliability was assessed and reported previously 38 . Retinal vascular parameters (arteriolar and venular caliber, fractal dimension, tortuosity and branching angle) were automatically measured and quantified by the SIVA program 38,39 . Based on the revised Knudtson-Parr-Hubbard formula, the retinal arteriolar and venular calibers were summarized as central retinal artery equivalent (CRAE) and central retinal vein equivalent (CRVE) respectively 40 . CRAE represents the average width of retinal arterioles while CRVE represents the average width of retinal venules.
Retinal vascular fractal dimension (D f ) represents a 'global' measure that summarizes the entire branching pattern of the retinal vascular tree. Retinal D f was calculated from a skeletonized line tracing using the box counting method; these represent a "global" measure that summarizes the whole branching pattern of the retinal vascular tree 41 . Larger values indicate a more complex branching pattern. Retinal vascular tortuosity was derived from the integral of the curvature square of along the path of the vessel, normalized by the total path length, taking into account the bowing and points of inflection 20,42 . Estimates were summarized as retinal arteriolar tortuosity and retinal venular tortuosity. Smaller values indicate straighter vessels. Retinal vascular branching angle was defined as the first angle subtended between two daughter vessels at each vascular bifurcation 38,43 . The estimates were summarized as retinal arteriolar branching angle and retinal venular branching angle, representing the average 44 . For each eye, a retinopathy severity score was assigned accordingly and retinopathy was defined as being present if the retinopathy score (a scale modified from the Airlie House classification system) was at level 15 or higher 45 . Ascertainment of Incident CKD. Glomerular filtration rate was estimated from serum creatinine using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation 46 . Serum creatinine was measured using an enzymatic method calibrated to the National Institute of Standards and Technology (NIST) Liquid Chromatography Isotope Dilution Mass Spectrometry (LC-IDMS) method recommended by the National Kidney Disease Education Program and traceable to NIST SRM967 47 . Incident CKD was defined as eGFR < 60 mL/min/1.73 m 2 accompanied by a decrease in eGFR of at least 25% over the follow-up period 48 among subjects free of CKD at baseline. For sensitivity analyses, we used two alternate definitions of incident CKD: 1) eGFR < 45 mL/min/1.73 m 2 accompanied by a decrease in eGFR of at least 25% over the follow-up period among subjects free of CKD at baseline 48 2) composite of incident CKD (our main definition) and/or rapid decline in eGFR. Rapid decline in eGFR was defined as annualized eGFR rate of >3 mL/ min/1.73 m 2 /year where annualized eGFR rate is calculated as the difference in eGFR between the baseline and follow-up visit divided by elapsed time in years 49 . Assessment of covariates. Information on participants' demographic characteristics, lifestyle (current smoking), personal and medical history was obtained by using a standardized questionnaire administered by trained personnel. Education was categorized into primary/below education or secondary/above education. CVD was defined as self-reported history of stroke, myocardial infarction or angina. Age was defined as the age at the time of baseline examination. Height was measured in centimeters using a wall-mounted measuring tape and weight was measured in kilograms using a digital scale. Body mass index (BMI) was calculated as body weight (in kilograms) divided by body height (in meters squared). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using a digital automatic BP monitor (Dinamap model Pro Series DP110X-RW, 100V2, GE Medical Systems Information Technologies Inc, Milwaukee, WI), after the subject was seated for at least 5 minutes. BP was measured twice at intervals of 5 minutes apart in a seated position. If these measurement differed by >10 mm Hg of SBP, or >5 mm Hg of DBP, then a third measurement was made. The mean between the 2 closest readings was then taken as the BP of that individual. Hypertension was defined as systolic BP of ≥140 mm Hg, diastolic BP of ≥90 mm Hg, or self-reported previously diagnosed hypertension. Antihypertensive medication use was defined as self-reported use of antihypertensive medications.
Non-fasting venous blood samples were analyzed at the National University Hospital Reference Laboratory for measuremnt of plasma glucose, serum total cholesterol, glucosylated hemoglobin (HbA1c) high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, and high-sensitivity C-reactive protein (hsCRP). Diabetes mellitus was defined as a casual plasma glucose measurement of ≥200 mg/dL (11.1 mmol/L), self-reported physician-diagnosed diabetes, use of glucose-lowering medication, or HbA1c ≥6.5% 50 .
Statistical Analysis. All statistical analyses were performed using STATA statistical software (Version 10, StataCorp, College Station, Texas). The outcome of interest for our study was incident CKD. The main exposures of interest were CRAE, CRVE, retinal vascular tortuosity, retinal D f , retinal vascular branching angle and presence of retinopathy. Each parameter was analysed separately with outcome CKD. Retinal vascular calibers (CRAE and CRVE) were analysed in tertiles as well as continuous variables (per each standard deviation increase/decrease). As previous studies have shown smaller retinal arteriole (CRAE) 13 and larger retinal venule (CRVE) 22 to be associated with CKD. In tertile analyses, tertile 3 (wider retinal arteriole) was used as the reference for CRAE, and tertile 1 (smaller retinal venule) was used as the reference for CRVE. We compared baseline characteristics between participants 1) with and without retinopathy and 2) with and without incident CKD at follow-up, by employing the chi-squared test or independent t-test as appropriate. Cox proportional-hazards regression was performed to examine the relation between retinal vascular parameters (CRAE, CRVE, retinal tortuosity, retinal D f and retinal branching angle, retinopathy) and CKD, in two models: model 1 adjusting for age and sex; model 2 additionally adjusting for education level, baseline eGFR, glucose levels, SBP, hypertension, smoking status, anti-hypertensive medications, hsCRP, total cholesterol, HDL cholesterol and fellow retinal vessel caliber (e.g. CRAE in models including CRVE and vice versa). P-trend was examined using CRAE and CRVE categories as continuous variables in the corresponding multivariable models. To examine the consistency of the association, we performed subgroup analyses stratified by sex and diabetes status using retinal vascular parameters as continuous variables. To test the robustness of association, we repeated the main analyses using alternative definitions of incident CKD.
Availability of data and material. As the study involves human participants, the data cannot be made freely available in the manuscript, the supplemental files, or a public repository due to ethical restrictions. Nevertheless, the data are available from the Singapore Eye Research Institutional Ethics Committee for researchers who meet the criteria for access to confidential data. | 2018-04-03T00:21:53.491Z | 2017-08-24T00:00:00.000 | {
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229512457 | pes2o/s2orc | v3-fos-license | Anesthetic management of a known case of pemphigus vulgaris with diabetes mellitus posted for surgery
80 yr old diabetic female known case of pemphigus vulgaris, got admitted to our hospital with relapse of fresh lesions. During her hospital stay she fell down in the washroom and developed fracture in intertrochanteric region of left femur for which surgery was planned. She had pancytopenia, low hemoglobin level, low platelet count and deranged blood sugar levels. After optimization she was planned for surgery under spinal anesthesia. Special care was taken during transfer, positioning, placement of intravenous lines and monitors. She was premedicated with intravenous steroids. Part was painted gently with chlorhexidine & spinal anesthesia was administered using hyperbaric bupivacaine with a 26 gauge Quincke’s needle taking care not to puncture the blisters and papules. Pressure points were adequately padded. Intra-operative hemodynamics were stable. Post-operatively, care was taken for the existing lesions along with special care to prevent the development of fresh lesions. © This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Introduction
Pemphigus vulgaris is an autoimmune disease characterized by bullous lesions of skin and mucous membrane. 1 Patients with pemphigus vulgaris can present with numerous issues which can complicate anesthetic management. Placement of intravenous lines and monitors require careful inspection of the site so as to prevent injury to skin lesions. As the patients are being treated with immunosuppressants and high dose of steroids they may have side effects like bone marrow depression and addisonian crisis, posing a challenge to the anesthesiologist. Lesions in the airway can make airway instrumentation difficult and risky. Regional anesthesia is hence commonly chosen in these patients whenever possible. Geriatric patients with this disease may have other co-morbidities which can complicate intraoperative management.
Case Study
80 year old female, known diabetic for 20 years on insulin therapy and Pemphigus Vulgaris for 10 years on oral steroids got admitted to our hospital with relapse of fresh skin lesions. Dermatology consultation was sought after which she received Cyclophosphamide therapy. While being managed in the hospital, she fell down in the washroom and developed fracture in inter-trochanteric region of left femur.
The patient was emaciated; vitals were normal and systemic examinations were within normal limits. There were blisters and papules on the upper limb, chest, abdomen and lower limb. The skin overlying the back had blisters all over except on the skin overlying the L3-L4 inter vertebral space, however there were no active lesions on the skin of the back. There was no deformity of the spine. Airway examination revealed Mallampati Grade I, blisters on the oral mucosa and poor oral hygiene, thyromental distance was 6 centimeters, mouth opening was 3 fingers and she had normal range of neck movement. Blood examination revealed low hemoglobin level, low total white blood cell count (TLC) and low platelet count, peripheral smear revealed pancytopenia, fasting & post-prandial blood glucose levels were high. Patient was administered GCSF (Granulocyte colony stimulating factor) injections 300 mcg subcutaneously once daily for 3 days in view of low TLC count. After the 3 injections her TLC count became normal. In view of low hemoglobin 2 units of packed red blood cells were transfused. Patient also received 4 units of platelet concentrates for low platelet count after which platelet count reached acceptable level for anesthesia and surgery. Endocrinology advice regarding insulin therapy for managing deranged blood sugar levels was followed. After optimization she was accepted for open reduction & internal fixation of left femur under subarachnoid block. Patient was kept nil per oral since midnight, tablet alprazolam was prescribed at dose of 0.25 mg at midnight, high risk consent was taken, morning dose of insulin was skipped. On the morning of surgery fasting blood sugar and electrolytes were within normal limits, urine was negative for ketones.
While shifting to the operation theatre precaution were taken not to injure the blisters and papules. Blood pressure cuff, electrocardiogram leads & pulse oxymeter were attached in blister free area. Vitals were within normal limits. A 20 gauge cannula was inserted on the dorsum of right hand as the skin in that region was free of blisters and papules; while fixing the cannula with a transparent cannula fixing adequate care was taken not to overlap neighboring blisters. Pre-loading was done, the patient was pre-medicated with injection dexamethasone 6 mg intravenous bolus dose.
The procedure of spinal anesthesia was explained to the patient and she was shifted to left lateral position after which the pressure points and blisters were padded. Taking all aseptic measures her back was painted gently with chlorhexidine and draped. L3-L4 inter vertebral space was felt & 26 gauge spinal needle was inserted without giving local anesthesia in order to avoid sloughing of skin. After free flow of CSF 2.4 ml of 0.5 % heavy bupivacaine mixed with 10 micrograms of fentanyl were injected and the patient was made supine and padded properly at the pressure points and skin lesions. There was complete motor blockade of the lower limbs and sensory block assessed by sensory response to cold saline was up to T6 level.
Intra-operative monitoring was done in the form of non-invasive blood pressure, heart rate, oxygen saturation, electrocardiography, end-tidal carbon dioxide concentration, skin surface temperature, blood sugar levels and blood loss. Appropriate care of the skin lesions was taken during traction & counter-traction. Her intraoperative hemodynamic status remained stable and hourly sugar levels were within normal limits. The surgery was uneventful and lasted for 2 hours; blood loss was below the maximum allowable blood loss. Before shifting out her vitals were normal, consciousness was intact and her reflexes were normal.
Post-operatively, endocrine opinion was followed for post-operative blood sugar management. The patient was pain free for 5 hrs after surgery when she started having mild pain for which she was administered paracetamol 1 gram intravenously. She did not have nausea, vomiting, drowsiness, pruritus or any other complaints in the postoperative period. The patient was discharged after 2 days after ensuring that no new lesions had developed.
Discussion
Pemphigus vulgaris is an autoimmune disorder characterized by vesiculobullous lesions & blisters involving the skin and mucous membranes. The clinically characteristic lesions are the presence of flaccid weeping lesions which leave large denuded areas. Blisters rupture very easily and crusting ensures. Oral cavity, pharynx and larynx may also be involved. During pre-anesthetic checkup detailed history of the disease and the treatment of it should be documented including the dose and duration of steroid therapy and immunosuppressant. Careful examination of the site, staging and extent of lesion is required. Perioral region should also be inspected as perioral scarring from previous lesions can limit mouth opening. Inspection of the oral cavity can reveal ulceration, edema and risk of bleeding from existing lesions. Mouth opening and neck movement may be restricted from painful ulcerations. Investigations should include complete blood count (to check for bone marrow depression as an effect of immunosuppressant), kidney function test (to check for side effects of steroids) and serum electrolytes as there can be water and electrolytes loss from raw areas. 2 Tracheal intubation is difficult in these patients because of possible ulcerations and edema from pre-existing lesions in the glottis.
In the operation theatre care should be taken during positioning and transport. The intravenous lines should be placed at skin free of any lesions. Gauze pieces soaked in saline and hydrocortisone should be placed on the arm before tying the BP cuff and pressure points should be padded to avoid development of new lesions (Kobnoer's phenomenon). Electrocardiogram leads should be placed at skin free of lesions. These patients should be adequately premedicated with corticosteroids to prevent addisonian crisis arising from adrenal insufficiency in these steroid dependent patients. At the time of administering subarachnoid block strict asepsis should be maintained and part should be gently prepared to avoid sloughing of skin lesions. Skin areas free of lesions should be chosen for performing lumbar puncture and skin infiltration with local anesthetic should be avoided. 2 Smaller gauge intrathecal needle should be used to minimize trauma. Intrathecal morphine has been successfully used in patients with pemphigus vulgaris. 3 For patients with Diabetes Mellitus use of bupivacaine mixed with fentanyl is associated with less hemodynamic consequences.
Thiopentone should be avoided as it causes porphyrias in bullous skin lesions. 4 Propofol is beneficial as it prevents pruritis, nausea and vomiting by depressing the central nervous system. Neuraxial opioids should be avoided as it causes pruritus resulting in itching leading to the development of Kobnoer's phenomenon and is associated with nausea and vomiting. 5 Oropharyngeal mucosal lesions usually occur in 50%-80% of these patients. Bag and mask ventilation should be gentle, facial lesions should be covered with hydrocortisone cream and soft cotton sponges before placing the face mask. Face mask should be held without pressure which can result in delayed preoxygenation. Airway instrumentation is potentially hazardous in such patients in view of risk of ulceration, edema, and bleeding from these pre-existing bulla. Cicatricial laryngeal lesions causing severe airway obstruction has been reported. 6 Whenever general anesthesia requiring a secure airway (intubation/laryngeal airway) is unavoidable, all protective measures shall be ensued. In patients with documented oral lesions putting in a tracheal tube is a safer technique over LMA for the risk of bleeding from intraoral lesions and aspiration. Suctioning should be done gently.
Post-operatively care should be taken to ensure adequate hydration & oxygenation of the patient and to avoid development of new lesions.
Conclusion
Pemphigus Vulgaris is an autoimmune disease of skin, the treatment options can have impact on major organ systems of the body. Our patient had pancytopenia as a result of the immunosuppressant therapy and deranged blood sugar levels which were optimized before surgery. As these patients are steroid dependent they can develop addisonian crisis in the perioperative period, hence we premedicated our patients with steroids. Administration of general anesthesia could be dangerous in these patients as hazardous complications could arise during bag & mask ventilation and tracheal intubation due to trauma and bleeding of the oral lesions. Spinal anesthesia with a smaller gauge spinal needle was selected as our anesthetic technique as it was a lower limb surgery and the site of administration of spinal anesthesia was free of skin lesions. In order to avoid Kobnoer's phenomenon utmost care needs to be taken in the perioperative period, hence pressure points & skin lesions were adequately padded and care was taken during patient transport, positioning, application of monitors, while inserting intravenous lines and administration of regional anesthesia to avoid trauma to the skin lesions. Post-operatively fresh lesions can develop; hence patient care was extended into the postoperative period while ensuring adequate hydration, oxygenation, post-operative pain management and post-operative blood sugar management of the patient.
Source of Funding
None.
Conflict of Interest
The authors declare that there is no conflict of interest. | 2020-11-26T09:04:37.959Z | 2020-11-15T00:00:00.000 | {
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119200469 | pes2o/s2orc | v3-fos-license | Resolvable heavy neutrino-antineutrino oscillations at colliders
Heavy neutrino-antineutrino oscillations can appear in mechanisms of low scale neutrino mass generation, where pairs of heavy neutrinos have almost degenerate masses. We discuss the case where the heavy neutrinos are sufficiently longlived to decay displaced from the primary vertex, such that the oscillations of the heavy neutrinos into antineutrinos can potentially be observed at the (high-luminosity) LHC and at currently planned future collider experiments. The observation of these oscillations would allow to measure the mass splitting of the respective heavy neutrino pair, providing a deep insight into the nature of the neutrino mass generation mechanism. Introduction: Sterile neutrinos, which are singlets under the gauge group of the Standard Model (SM), are an attractive extension of the SM to explain the observed masses of the light neutrinos. We consider scenarios where the masses of the heavy neutrinos are around the electroweak (EW) scale. Various models with such a low scale seesaw mechanism exist in the literature (see e.g. [1–7]). The models may be classified by how the approximate “lepton number”-like symmetry, which ensures the smallness of the light neutrinos’ masses, is broken: If the breaking happens in the sterile neutrino mass matrix, the models are called low scale “inverse seesaw” models [1,2], and when the breaking happens in the coupling matrix between the sterile neutrinos and the active SM neutrinos, i.e. in the Yukawa sector, then the models are called low scale “linear seesaw” models [3]. In both classes of models, due to the approximate “lepton number”-like symmetry, pairs of heavy neutrinos have almost degenerate masses, i.e. they form heavy pseudo-Dirac particles. For collider studies it is often useful to focus on one of the pairs of heavy neutrinos and to consider the limit of intact “lepton number”-like symmetry, as e.g. in the “SPSS” (Symmetry Protected Seesaw Scenario) benchmark model (cf. [8]). A recent study of the prospects for testing such low scale seesaw models at the LHC and at future colliders can be found e.g. in [9]. The small mass splittings between the heavy neutrinos typical for low scale seesaw models lead to oscillations between the heavy neutrinos and their antiparticles. The time-integrated effect of these oscillations, i.e. when the oscillations cannot be resolved experimentally, has been highlighted in [10–13]. Heavy sterile neutrino oscillations in meson decays have been discussed in [14, 15], and further observable effects at colliders e.g. in [16]. In this letter, we discuss the case where the heavy neutrinos are sufficiently long-lived to decay displaced from the primary vertex. We show that the oscillations of the heavy long-lived neutrinos can potentially be resolved at the high luminosity (HL) phase of the LHC and at currently planned future collider experiments. The observation of these oscillations would allow to measure the mass splitting of the respective heavy neutrino pair, providing a deep insight into the nature of the neutrino mass generation mechanism. Such a measurement would also be very important towards testing leptogenesis in minimal seesaw models, as discussed recently in [17–19]. Low scale seesaw mechanism: After electroweak symmetry breaking the Lagrangian density containing the masses of the light and heavy neutrinos can be written as Lmass = −1/2ΨMνΨ + H.c., where the active neutrinos νL and pairs of sterile neutrinos (N R) , (N R) c have been combined to Ψ = ( νL, (N 1 R) , (N R) c )T . If the approximate “lepton number”-like symmetry mentioned above was exact, 1 ar X iv :1 70 9. 03 79 7v 2 [ he pph ] 1 2 M ar 2 01 9 the neutrino mass matrix is schematically given by Mν = 0 mD 0 (mD) 0 M 0 M 0 . (1) In the following, we will consider one pair of sterile neutrinos for simplicity. In the exact symmetry limit it has a Dirac-type mass M , and mD = YνvEW/ √ 2 with the neutrino Yukawa coupling matrix Yν coupling the three active neutrinos to the sterile neutrino which carries the same charge under the “lepton number”-like symmetry. In order to generate the light neutrino masses, the structure in eq. (1) has to be perturbed. There are two characteristic ways to implement such perturbations, corresponding to two classes of models: • Linear Seesaw: The perturbation is introduced in the 1-3 (and 3-1) block of the symmetric matrix Mν in eq. (1): M lin ν = 0 mD mD (mD) 0 M (mD) T M 0 , (2) where mD has small entries compared to mD. With two sterile neutrinos (one pair), the symmetry breaking term mD can generate masses for two of the three light neutrinos as well as a splitting ∆M lin between the masses of the two heavy neutrinos. With an appropriate choice of mD and mD, the present neutrino oscillation data can be accommodated. We will refer to this scenario as the “minimal linear seesaw model”. It can be shown that in the minimal linear seesaw scenario the mass splitting ∆M lin is predicted in terms of the measured mass squared differences of the light neutrinos, which yields, in the case of normal mass ordering (NO) or inverse mass ordering (IO): ∆M lin NO = 2ρNO √ ∆m21 1−ρNO = ∆m32 = 0.0416 eV (3) ∆M lin IO = 2ρIO √ ∆m23 1+ρIO = ∆m21 =0.000753 eV (4) with ρNO = √ r+1− √ r √ r+ √ r+1 and r = |∆m21| |∆m32| , and with ρIO = √ r+1−1 √ r+1+1 and r = |∆m21| |∆m13| , where ∆mij corresponds to the mass squared differences m2νi −m 2 νj and ∆mij to the mass splitting 1In the general case with more sterile neutrinos, M stands for a n×n Dirac-type mass matrix of n pairs of sterile neutrinos. mνi −mνj , with mνi labelling the light neutrino masses. In the last step, the mass of the lightest of the light neutrino has been set to zero, as implied by the minimal linear seesaw model. • Inverse Seesaw: The perturbation is introduced in the 3-3 block of the symmetric matrix Mν in eq. (1): M inv ν = 0 mD 0 (mD) 0 M 0 M μ , (5) where μ M violates lepton number and introduces a mass for one of the light neutrinos (which may for instance be the lightest or the heaviest of the light neutrinos) as well as a mass splitting between the two heavy neutrinos. We emphasise that in contrast to the minimal linear seesaw model discussed above, additional sterile neutrinos (e.g. additional pairs) have to be introduced in order to be consistent with neutrino oscillation results. To obtain an estimate for the sterile neutrino mass splitting in the inverse seesaw case, we will assume that the considered pair of sterile neutrinos dominantly generates one of the masses of the light neutrinos, labeled mνi . Since for the case of two sterile neutrinos considered here one can choose mD, M and μ real and positive without loss of generality, the mass mνi is (to leading order) related to the squared sum |θ| := (mDmD)/M of the active-sterile mixing angles by mνi = Tr ( μ mDm T D M2 ) = μ mDmD M2 = μ|θ| , (6) The mass splitting ∆M inv then satisfies ∆M inv = μ−mνi ≈ mνi |θ|2 . (7) In the following we will use this relation with mνi in the range mνi = 0.1 eV . . . 10 −4 eV to give example values for ∆M inv as a function of |θ|. We like to emphasise that the resulting ranges for ∆M inv are no strict predictions but at best guidelines for the inverse seesaw scenario. A smaller mass splitting could be a consequence of an even smaller mνi , and a larger mass splitting could be the result of cancellations between the contributions of two pairs of sterile neutrinos to the light neutrino mass matrix. 2 2Furthermore, we note that in addition a perturbation of Mν of eq. (1) in the 2-2 block may be introduced which does not lead to a contribution to the light neutrinos’ masses (at tree-level) but can enhance or reduce the splitting ∆M inv.
Introduction: Sterile neutrinos, which are singlets under the gauge group of the Standard Model (SM), are an attractive extension of the SM to explain the observed masses of the light neutrinos. We consider scenarios where the masses of the heavy neutrinos are around the electroweak (EW) scale. Various models with such a low scale seesaw mechanism exist in the literature (see e.g. [1][2][3][4][5][6]).
The models may be classified by how the approximate "lepton number"-like symmetry, which ensures the smallness of the light neutrinos' masses, is broken: If the breaking happens in the sterile neutrino mass matrix, the models are called low scale "inverse seesaw" models [1,2], and when the breaking happens in the coupling matrix between the sterile neutrinos and the active SM neutrinos, i.e. in the Yukawa sector, then the models are called low scale "linear seesaw" models [3]. In both classes of models, due to the approximate "lepton number"-like symmetry, pairs of heavy neutrinos have almost degenerate masses, i.e. they form heavy pseudo-Dirac particles.
For collider studies it is often useful to focus on one of the pairs of heavy neutrinos and to consider the limit of intact "lepton number"-like symmetry, as e.g. in the "SPSS" (Symmetry Protected Seesaw Scenario) benchmark model (cf. [7]). A recent study of the prospects for testing such low scale seesaw models at the LHC and at future colliders can be found e.g. in [8].
The small mass splittings between the heavy neutrinos typical for low scale seesaw models lead to oscillations between the heavy neutrinos and their antiparticles. The time-integrated effect of these oscillations, i.e. when the oscillations cannot be resolved experimentally, has been highlighted recently in [9]. In this letter, we discuss the case where the heavy neutrinos are sufficiently long-lived to decay displaced from the primary vertex. We show that the oscillations of the heavy long-lived neutrinos can potentially be observed at the high luminosity (HL) phase of the LHC and at currently planned future collider experiments. The observation of these oscillations would have farreaching consequences, e.g. it would prove the existence of lepton number violation, that neutrinos are Majorana particles, and it would allow a deep insight into the nature of the neutrino mass generation mechanism.
Low scale seesaw mechanism: If the approximate "lepton number"-like symmetry mentioned above was exact, the full singlet mass matrix (after EW symmetry breaking) including the light and the heavy neutrinos is schematically given by where for simplicity we consider one pair of sterile neutrinos, which in the exact symmetry limit have a Dirac-type mass M , 1 and where m D = Y ν v EW / √ 2 with Y ν being the neutrino Yukawa coupling matrix that couples the three active neutrinos to the sterile neutrino which carries the same "lepton number".
In order to generate the light neutrino masses, the structure in eq. (1) has to be perturbed. There are two characteristic ways to implement such perturbations, corresponding to two classes of models: • Linear Seesaw: The perturbation is introduced in the 1-3 (and 3-1) block of the symmetric matrix M ν in eq. (1): where m D has small entries compared to m D . The symmetry breaking term m D generates masses for all three light neutrinos as well as a splitting ∆M lin between the masses of the two heavy neutrinos.
It can be shown that this mass splitting is predicted in terms of the measured mass squared differences of the light neutrinos, which yields, in the case of normal mass ordering (NO) or inverse mass ordering (IO): • Inverse Seesaw: The perturbation is introduced in the 3-3 block of the symmetric matrix M ν in eq. (1): where µ M violates lepton number and introduces a mass for one of the light neutrinos (which may for instance be the lightest or the heaviest of the light neutrinos) as well as a mass splitting between the two heavy neutrinos.
Since for the minimal case considered here, without loss of generality, one can choose m D , M and µ real and positive, the mass m νi of this light neutrino is (to leading order) related to the squared sum |θ| 2 := (m † D m D )/M 2 of the active-sterile mixing angles by The mass splitting ∆M inv then satisfies In the following we will use this relation with m νi in the range m νi = 0.1 eV . . . 10 −4 eV to give example values for ∆M inv as a function of |θ| 2 . 2 Heavy neutrino-antineutrino oscillations: When heavy neutrinos are produced from W decays together with charged leptons or antileptons, we refer to them as heavy antineutrinos N or neutrinos N , respectively. When they decay via the charged current, they again produce either a lepton or an antilepton, N → − W + or N → + W − .
In the symmetry limit without "lepton number" violation in the mass matrix of eq. (1), i.e. without light neutrino masses, this would imply that processes with the heavy neutrinos at colliders are lepton number conserving (LNC). For instance at proton-proton (pp) colliders, there would be only LNC processes pp → + α − β jj but no lepton number violating (LNV) processes pp → ± α ± β jj. We will focus on these processes as an example in the following, since they can yield an unambiguous sign of LNV at pp colliders.
In the presence of LNV perturbations in the mass matrix of eq. (1) however, also LNV processes pp → ± α ± β jj are possible. One can view these events as stemming from N (or N ) being produced together with a charged antilepton (or lepton) which then oscillates into a N (or N ), decaying into a lepton (or antilepton), finally producing a lepton-number violating final state.
When the heavy neutrinos have sufficiently small decay widths, they can have macroscopic lifetimes such that their decay occurs displaced from the primary vertex, which allows for powerful searches and opens up the possibility to observe the oscillation patterns in the decay spectra. We show in figure 1 for which parameters M and |θ| 2 macroscopic lifetimes are possible.
Due to heavy neutrino-antineutrino oscillations, following [9,10], the ratio between LNV and LNC events between times t 1 and t 2 after heavy neutrino production will be referred to as R (t 1 , t 2 ) and is given as: where and where Γ is the heavy neutrino decay width. |g − (t)| 2 corresponds to the timedependent probability that a heavy neutrino has oscillated into a heavy antineutrino and vice versa, and |g + (t)| 2 denotes the probability that no oscillation has occurred. 3 From the above formula, we can see that the oscillation period of the heavy neutrinos is given by t osc = 4π ∆M , which yields the following oscillation lengths for the linear and inverse seesaw scenarios in the laboratory system: λ lin,NO osc = 5.96 · 10 −5 γ 2 − 1 m , λ lin,IO osc = 3.29 · 10 −3 γ 2 − 1 m , λ inv osc ≈ 2.48 · 10 −6 |θ| 2 10 −4 Especially when the Lorentz factor is large, the oscillation length in the laboratory system can be large enough to be resolved in an experiment. The case of linear seesaw with IO looks particularly promising in this context. For observability it is also important that the decay of the heavy neutrinos is sufficiently displaced from the primary vertex (cf. figure 1). 3 We note that here we neglect CP violating effects, which can be introduced by perturbations of the mass matrix of eq. (1) and could leave imprints in the distribution of the ± α ± β jj and + α − β jj final states.
Potentially observable oscillations:
As an example, we now consider the LHCb experiment and heavy neutrinos with M = 10 GeV, |θ| 2 = 10 −6 , Lorentz factor of γ = 50 (which is typical for these parameters as discussed in [11]). These parameter values are consistent with the present bounds and within reach of, e.g., the HL phase of the LHC [11]. During the HL phase about 70 events could be detected with displacements between 2 cm and 50 cm from the primary vertex at LHCb. For the case of light neutrino masses from a low scale linear seesaw mechanism with IO, the results for the fractions of LNV and LNC events, as function of the distance x from the primary vertex, are shown in figure 2. Our results illustrate that there are promising conditions to measure the signature of heavy neutrino oscillations at LHCb. To confirm this, a proper analysis at the reconstructed level is necessary and left for a future study. In this context we note that in addition to the oscillation pattern in figure 2, which assumed a fixed Lorentz factor, the distribution of the Lorentz factor has to be taken into account. Results for the distribution can be found in [11]. In such an analysis, it will be important to measure γ for each event and to consider the oscillation pattern as function of time in the proper frame of the heavy neutrinos, where the effect of the γ distribution is corrected for.
We note that for the case of NO of the light neutrino masses the oscillation length is shorter, i.e. 3 mm. This is in principle still resolvable with the LHCb tracking resolution, however we expect the experimental uncertainties to make this more difficult than for the IO case.
For the inverse seesaw case and for the used example parameter point, the oscillations would not be visible since the oscillation length is estimated to be much below the tracking resolution of O(10 −5 ) m.
Regarding pp colliders, of course also the FCC-hh [12] would be very promising for testing these signatures with possible much higher γ factors and higher luminosity. Other colliders where signatures of heavy neutrino-antineutrino oscillations could be studied are the electron-proton colliders, e.g. the LHeC [13] or the FCC-eh [14], since they also provide unambiguous signal processes for LNV (see e.g. [8]).
Relevance of LNV for collider searches: Due to the approximate "lepton number"-like symmetry it is generally expected that the LNV effects are strongly suppressed. We now quantify under which conditions this statement holds when heavy neutrinoantineutrino oscillations are taken into account, even when the oscillations cannot be resolved experimentally, using the estimates for the mass splittings from Eqs. (3), (4) and Eq. (7). For this purpose, we consider the ratio R := R (0, ∞) (cf. [9]) which can be expressed as We calculate the heavy neutrino decay rate Γ in our benchmark model using WHIZARD [15,16]. We show, as examples, the range 0.1 ≤ R ≤ 0.9 for the low scale linear and inverse seesaw scenarios in figure 3. From the figure it can be seen for which parameters M and |θ| 2 sizeable LNV due to heavy neutrinoantineutrino oscillations can occur.
Conclusions:
We have discussed the possibility to observe heavy neutrino-antineutrino oscillations at collider experiments, considering low scale seesaw models with a protective "lepton number"-like symmetry. We showed that the linear and inverse low scale seesaw scenarios naturally feature almost degenerate heavy neutrino masses which can give rise to oscillation lengths in the centimeter range. Especially for the linear seesaw case, the oscillation time (in the proper frame) is fixed by the measured light neutrinos' squared mass differences and is thus a prediction that is different for the two orderings. We focused on signatures of the heavy neutrinoantineutrino oscillations via the processes pp → jj, which result in an oscillating pattern between LNV and LNC dilepton events, i.e. an oscillatory distribution of same-sign and opposite-sign dilepton events. We have shown that for the linear seesaw case, the predicted oscillation length can lead to observable oscillation signatures at the LHCb. We note that also various other processes can be used to probe heavy neutrino-antineutrino oscillations. Examples are the trilepton final state at the LHC [17,18] and basically all final states that feature an unambiguous signal for LNV (for a summary see [8]).
When the heavy neutrino-antineutrino oscillations are fast, such that they are not resolvable experimentally, they can nevertheless give rise to LNV events at colliders. For heavy neutrino masses below the W boson mass, where the HL-LHC and all planned future collider experiments are expected to have best sensitivity (cf. [8]), parameter regions where LNV signals are relevant can be reached. For masses above the W boson mass, only for the inverse seesaw estimate we expect that LNV is potentially observable at the currently planned future colliders, while for the linear seesaw it is safe to assume LNC.
In conclusion, the observation of resolved heavy neutrino-antineutrino oscillations would allow a deep insight into the nature of the neutrino mass generation mechanism: The determination of the oscillation length allows to infer the small heavy neutrinos' mass splitting, which can allow to distinguish the linear and inverse low scale seesaw scenarios. Furthermore, it provides a unique way to identify normal or inverse mass ordering of the light neutrinos due to its predicted oscillation time in the linear low scale seesaw scenario. | 2019-04-23T13:23:45.385Z | 2019-03-12T00:00:00.000 | {
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115591505 | pes2o/s2orc | v3-fos-license | A New Method to Retrieve the Three-Dimensional Refractive Index and Specimen Size Using the Transport Intensity Equation, Taking Diffraction into Account
: Refractive index retrieval is possible using the transport intensity equation (TIE), which presents advantages over interferometric techniques. The TIE method is valid only for paraxial ray assumptions. However, diffraction can nullify these TIE model assumptions. Therefore, the refractive index is problematic for reconstruction in three-dimensions (3D) using a set of defocused images, as diffraction effects become prominent. We propose a method to recover the 3D refractive index by combining TIE and deconvolution. A brightfield (BF) microscope was then constructed to apply the proposed technique. A microsphere was used as a sample with well-known properties. The deconvolution of the BF-images of the sample using the microscope’s 3D point spread function led to significantly reduced diffraction effects. TIE was then applied for each set of three images. Applying TIE without taking into account diffraction failed to reconstruct the 3D refractive index. Taking diffraction into account, the refractive index of the sample was clearly recovered, and the sectioning effect of the microsphere was highlighted, leading to a determination of its size. This work is of great significance in improving the 3D reconstruction of the refractive index using the TIE method.
Introduction
Spectroscopic analysis of microscopic specimens sometimes requires knowledge of their refractive indices. The refractive index is an important biophysical parameter, which is used in many studies. In mineralogy and crystallography [1], the refractive index is taken into account in the characterization of precious stones. In medicine, it can be used as a marker of disease. Zhuo et al. [2] showed that it is possible to diagnose tissue cancers via the refractive index distribution, which contains information about the molecular scale organization of tissue. The refractive index maps of red blood cells are used as an indicator of the morphological alterations of host cells infected by Plasmodium falciparum [3].
Standard devices for the refractive index measurement lead to mean values of this important parameter. Progress is being made in the development of new devices and methods that enable the determination of the refractive index in three-dimensions (3D). These methods should help to raise the accuracy of specimen analysis. The most well-established methods for recovering the quantitative phase and the optical parameters are the interferometric method, such as digital holography. Using this method, Florian et al. [4] performed a 3D reconstruction of refractive index of pollen grain, with a precision of 0.01. The interferometric method needs coherent illumination. Therefore, it induces speckle noise in the measurements, which prevent high quality results [5]. Phase or refractive index retrieval is possible by using the Transport Intensity Equation (TIE) which is a non-interferometric method [6]. TIE has been increasingly investigated over recent years due to its unique advantages over interferometric techniques. It uses partially coherent illumination; it is computationally simple with the need of (at least) three brightfield (BF) defocused images, and it does not require a complicated optical system. However, the TIE method is valid only for paraxial ray approximations [7], and diffraction can nullify these TIE model assumptions. This phenomenon drastically affects the quality of the 3D reconstruction of the refractive index [8] as the diffraction effects become prominent.
In this work, we introduce a method to clearly recover the 3D refractive index of a sample with well-known properties by using TIE and taking the diffraction into account.
Experimental Set-Up
In Figure 1, we can see the set-up of the experimental device. This consisted of a BF horizontal microscope with a 16 bit monochrome Complementary Metal-Oxide-Semiconductor (CMOS) camera with a pixel size of 5.5 µm × 5.5 µm, and an effective image chip size of 11.264 mm × 5.984 mm, presenting 2048 × 1088 pixels. The microscope enabled automated sequential acquisition of the BF images. The images were recorded by defocusing the sample slightly in the z-direction (optical axis) through a stepper motor MTS25-Z8. The motor was monitored in three directions (X, Y, Z) by three servocontrols. Therefore, we were able to scan the region of interest in the sample. The servocontrols were connected to the PC, which enables the control of the defocus distance ∆z and the image acquisition protocol by using a MatLab operation code. determination of the refractive index in three-dimensions (3D). These methods should help to raise the accuracy of specimen analysis. The most well-established methods for recovering the quantitative phase and the optical parameters are the interferometric method, such as digital holography. Using this method, Florian et al. [4] performed a 3D reconstruction of refractive index of pollen grain, with a precision of 0.01. The interferometric method needs coherent illumination. Therefore, it induces speckle noise in the measurements, which prevent high quality results [5]. Phase or refractive index retrieval is possible by using the Transport Intensity Equation (TIE) which is a non-interferometric method [6]. TIE has been increasingly investigated over recent years due to its unique advantages over interferometric techniques. It uses partially coherent illumination; it is computationally simple with the need of (at least) three brightfield (BF) defocused images, and it does not require a complicated optical system. However, the TIE method is valid only for paraxial ray approximations [7], and diffraction can nullify these TIE model assumptions. This phenomenon drastically affects the quality of the 3D reconstruction of the refractive index [8] as the diffraction effects become prominent.
In this work, we introduce a method to clearly recover the 3D refractive index of a sample with well-known properties by using TIE and taking the diffraction into account.
Experimental Set-Up
In Figure 1, we can see the set-up of the experimental device. This consisted of a BF horizontal microscope with a 16 bit monochrome Complementary Metal-Oxide-Semiconductor (CMOS) camera with a pixel size of 5.5 µm × 5.5 µm, and an effective image chip size of 11.264 mm × 5.984 mm, presenting 2048 × 1088 pixels. The microscope enabled automated sequential acquisition of the BF images. The images were recorded by defocusing the sample slightly in the z-direction (optical axis) through a stepper motor MTS25-Z8. The motor was monitored in three directions (X, Y, Z) by three servocontrols. Therefore, we were able to scan the region of interest in the sample. The servocontrols were connected to the PC, which enables the control of the defocus distance and the image acquisition protocol by using a MatLab operation code.
Sample Preparation
In this work, the sample was made up of 1 µm polystyrene microspheres beads (refractive index = 1.586 at wavelength λ = 640 nm [9]) at the temperature = 17 ℃. The solution of microspheres was diluted with distilled water ( = 1.33 at the temperature = 17 ℃). A small drop of the diluted solution was placed and spread on a slide made of the highest purity corrosion-resistant glass with dimensions 75 mm × 25 mm × 1 mm. After drying the sample for 30
Sample Preparation
In this work, the sample was made up of 1 µm polystyrene microspheres beads (refractive index n = 1.586 at wavelength λ = 640 nm [9]) at the temperature T = 17 • C. The solution of microspheres was diluted with distilled water (n = 1.33 at the temperature T = 17 • C). A small drop of the diluted solution was placed and spread on a slide made of the highest purity corrosion-resistant glass with dimensions 75 mm × 25 mm × 1 mm. After drying the sample for 30 minutes, it was used for microscopic examination under partially coherent illumination at a wavelength λ = 640 nm.
Measurement Procedure
We performed the measurements in transmission mode. Three sets of images were recorded in the same conditions: image T d , the contribution of the background and the dark current in the detector; it was achieved with nothing in the microscope and with the source illumination turned off; -image T b , the bright reference; it was achieved with an empty slide in the microscope; -image T s , the raw image of the sample, it was achieved with the sample in the microscope.
Each set of images was recorded for each plane of the sample by moving it along the optical axis. The corrected image T of the sample is defined by Equation (1) (flat-field correction), as is commonly described by Tadrous [10], Brydegaard et al. [11], and Agnero et al. [12]: This operation cancels out the different emissive yields of the light emitting diode (LED) source, electronic gains, and exposures for each illumination, and also the variation in the illumination intensity over the field of view [11].
In practice, one major disadvantage of TIE method is the sequential capture of images. It requires the precise motion of the sample, which can be shifted from a plane to another due to a little vibration in the measurement environment. This induces artefacts in some of the recorded images [13]. Therefore, care was taken to avoid any movement that was able to produce vibration as the image acquisition was running.
Analysis Methods
Optical systems generally are not free of aberrations or imperfections that affect the quality of the recorded images. Consequently, the image of a point object is an extended distribution of intensity called point spread function (PSF). During data analysis, we firstly sought to remove from the corrected images the distortions of observation due to the optical acquisition system and the contributions from out-of-focus planes for the sample. In this approach, we introduced the model of an appropriate PSF required for a deconvolution, which should remove any artefact. At the second time, we used the TIE method to extract the refractive index and the size of the specimen.
Deconvolution requires a realistic model of the phenomena that induce distortions in the image formation process. The term "deconvolution microscopy" generally assumes fluorescence microscopy [14,15], and PSF deconvolution imaging theory has traditionally been linked to fluorescence. A main reason behind this association is that for the case of self-luminous objects, one needs only to consider signal intensity, leading to a unique PSF and making deconvolution a linear process. In general, this is not the case in BF microscopy, where two point spread functions are needed to describe image formation [16]. In fluorescence microscopy, the 3D image i(x, y, z) of an object corresponds to the convolution of the object o(x, y, z) with the PSF(x, y, z) [17]: where B models noise, and the symbol ⊗ denotes the convolution operation. BF microscopy is presented as an alternative to fluorescence in deconvolution image processing, because of the possibility for observing unstained objects. However, its corresponding PSF consists of two separate components, one for phase (PSF p ) and one for absorption (PSF a ). Therefore, the BF-3D image i(x, y, z) is generally described as the sum of the convolutions of the real (P) and imaginary (A) parts of the object scattering potential with the corresponding PSFs [18]: This excludes the application of linear deconvolution processing to remove distortion. However, in the presence of the pure phase object as it is the case in this work, BF imaging essentially reduces to Equation (2), making it possible to perform linear deconvolution [16]. Therefore, knowing the PSF leads to the determination of the object through deconvolution. The PSF can be: computed from the optical properties of the microscope system, -estimated from the measurements of the microspheres.
The experimental PSF takes into account all of the aberrations introduced by the whole image acquisition system [19]. It is determined by recording images of a specimen (assumed to be a point object) in different planes of defocus above and below the focal plane. A point object does not exist in practical applications; the object necessarily has a particular size. Therefore, for the measured data to correspond to the microscope's PSF, it is very important that the object size is smaller than the diffraction limit. If this is not the case, the measured data will instead correspond to the convolution of the PSF with the object shape, according to Equation (2) [20]. Unfortunately, using a very small object introduces the problem of signal detection, due to a very low signal-to-noise ratio. This requires detectors of high sensitivity. The signal to noise ratio is so low that often a bead is used whose size is not smaller than diffraction limit, but of the order of the microscope's resolution [21]. This leads to an overestimated PSF. The experimental PSF is noisy and needs to be devoid of noise, which is a limitation in a deconvolution algorithm. The method of denoising sometimes distorts the real PSF of the microscope. Lai et al. [22] used a denoising method based on singular value decomposition to get a denoised PSF, but it produced small visible artificial ripples to the denoised PSF [19].
The computed PSF, in particular the model of Gibson and Lanni [23], also takes into account the conditions in which the experiment is carried out, and it has an advantage of being estimated for all of the object space. It is free of noise, flexible, and suitable to any specific condition of the experimentation, unlike experimental PSF, which is not modifiable because it is measured to represent a specific condition in which the images of biological sample must be recorded. The BF-PSF model was reported previously by Hernandez and Gutierrez [16]. However, they do not assume a real point object in their model. The model of Gibson and Lanni [23] is more an accurate model for PSF generation that is suitable for the brightfield setting [10], and this was discussed and publicly made available as an ImageJ plug-in by Besson et al. [24]. According to the model, the image of a point object located at X p = x p , y p , z p is defined by Equation (4) [23]: where X = (x, y, z) is a point on the image plane, N A is the numeric aperture of the objective, A is an amplitude constant, ρ is the radius of the microscope's limiting aperture in the microscope's back focal plane, J 0 denotes the Bessel function of the first kind of order zero, and w ρ, X, X p is the phase which enables aberrations to be taken into account in the model, The phase w ρ, X, X p is defined by Equation (5), as there are two layers (immersion medium of refractive index n c and the specimen layer of refractive index n m ) [25]: The parameters n c * and WD are the design refractive index of the immersion medium and working distance of the objective, respectively, and k is the wave number. The model of Gibson and Lanni [23] allowed for the restoration of our BF images.
Defining the wave field traversing the specimen by E z (x, y) = I z (x, y)e −jφ z (x,y) , where I z (x, y) is the intensity, φ z (x, y) the phase, z denotes the position along the optical axis, and (x, y) are the two-dimensional coordinates in the plane orthogonal to the optical axis. Therefore, the denotation I z (x, y) indicates the intensity I(x, y, z). The TIE is obtained by substituting E z (x, y) into the paraxial wave equation and taking the imaginary part of the resulting expression; the TIE is defined by [6]: where λ is the wavelength of the illumination source and ∇ ⊥ denotes the gradient operator in the lateral dimensions (x, y). Due to its non-interferometric nature, the illumination can be quasi-monochromatic and partially-coherent [26]. The numerical solution for the TIE in the case of a pure phase object (transparent object), is defined by Equation (7) [7], resulting from the standard equation of Poisson [27,28]: where TF −1 and TF denote the inverse Fourier Transform and Fourier Transform respectively, and (u, ν) denotes the spatial frequency variables corresponding to the coordinates (x, y). Equation (7) is valid only for paraxial ray assumptions. The phase of the wave field is related to the variation of the image intensity along the optical axis through Equation (7). In practice, the intensity derivative ∂I z (x, y)/∂z in the observation plane z cannot be measured directly; we adopted the following approximation (Equation (8)) using two defocused images, one above the observation plane (I(x, y, z + ∆z)) and one below (I(x, y, z − ∆z)): where ∆z is the defocus distance. Phase distortions induced in the wave field as the wave propagates from point r z 0 (x, y) to r z (x, y) can be related to the refractive index of the specimen through solutions to the eikonal equation [8,29] for the phase: ∆n z (x, y) is the refractive index difference between the specimen and its surrounding medium (specimen layer). Taking the specimen to be located directly above the origin r z 0 (x, y), hence φ z 0 (x, y) = 0, and for points inside the specimen, it is found that [8]: Therefore, if φ z (x, y) is known in multiples planes (with small value of defocus distance ∆z) inside the specimen, its 3D refractive index distribution can be recovered via Equation (11) [30] through sequential method (using finite differences) applied to pairs of consecutive planes (one pair after one sequentially) with the same value of defocus distance: where n m is the refractive index of the specimen layer. To retrieve the 2D phase φ z (x, y) corresponding to the observation plane z, we used Equation (7) and three recorded images of the sample were needed, one in the observation plane, and the both of the others below and above the observation plane with a defocus distance ∆z. This procedure was repeated to obtain φ z (x, y) in multiples planes by changing the observation plane with the same value of ∆z until the whole specimen was scanned along the optical axis. The defocus distance is an important factor in the quality of the reconstruction. When ∆z was large, the hypothesis of linearity that enables the above approximation (Equation (8)) of the intensity derivative ∂I z (x, y)/∂z was not valid, and this nonlinearity affected the accuracy of the reconstruction. For small values of ∆z where the linearity was preserved, there was a reduction of signal to noise ratio due to the term I(x, y, z + ∆z) − I(x, y, z − ∆z), which becomes smaller. This required a compromise between the defocusing, accuracy, and signal to noise ratio. Therefore, when we are in the presence of multiple plane images and we would like to retrieve the 2D phase φ(x, y) corresponding to the focal plane, we cannot simply use pairs of plane images to recover the phase from average of them [13]. In this work, we used the method proposed by Zhong et al. [31], which performs fitting (through Gaussian process regression) on each pixel's intensity in the frequency domain along the optical axis, taking into account all the images in different planes. This method allowed an estimation of the derivative ∂I z (x, y)/∂z, which led us to retrieve the phase in the focal plane. Consequently, the specimen's size t(x, y) was obtained by using Equation (12) [32]: where n f is the refractive index of the specimen in the focal plane.
Results and Discussion
During the computation of the PSF for our experimental device, two parameters were not directly accessible: the depth z p of the point object in the specimen layer, and the refractive index n c of the immersion medium (air) during the experimentation [25]. Our sample of microspheres of diameter 1 µm was allowed to dry during experimentation. This enabled us to assume that the point object was on the interface of air and specimen layer, so z p = 0. The measured pressure and temperature in the measurement environment (P = 992 hPa, T = 17 • C) enabled the determination of the refractive index, n c , of the immersion medium (air) during experimentation; n c = 1.00024 by using Equation (13) [33,34]: where P, T, and λ are in bar, in degrees Celsius and in µm respectively. Figure 2 presents the PSF of our device. The images of the sample in different planes were affected by the contribution of out-of-focus planes (Figure 3a-c). Restoration of these images was achieved by using the Richardson-Lucy algorithm [35,36] implemented in MatLab, taking into account the 3D-PSF. The number of iterations used was 25. This led to a reduction not only in the effects of diffraction but also in the noise and pixel intensity, due to the contribution of the adjacent planes (Figure 3d-f).
Applying the TIE method sequentially on the corrected images led to an extracted refractive index of the specimen (Figure 4a-g). We can observe in Figure 4a-g that the outlines of the microsphere became smaller as we moved away from the focal plane (z = 0 µm). This was due to effect of sectioning. In a plane of the microsphere, the spatial distribution of its refractive index was not homogeneous. The homogeneity of refractive index distribution was drastically damaged as we moved away from the focal plane. This was due to diffraction effects [37] that became prominent. In this case, the paraxial ray assumptions, which were required to apply TIE, were nullified [8]. This induced artefacts in the refractive index reconstruction (Figure 4a-c). TIE method drastically failed to recover the 3D refractive index distribution of the sample for an optical system with a high numeric aperture, as was the case for our device (N A ob = 0.75) [37]. Appl. Sci. 2018, 8, x FOR PEER REVIEW 7 of 11 A reconstruction of the refractive index after the deconvolution of the corrected images by the use of the 3D-PSF is presented in Figure 4h-n. The sectioning effect was more greatly appreciated through the spatial distribution of refractive index. The circular outlines of the microsphere were well highlighted. The artefacts that were observed in the planes = −0.4 µm, = −0.3 µm, = −0.2 µm were removed. In each plane of the microsphere, the homogeneity of refractive index distribution was greatly improved and tended to cover all of the circular outline of the microsphere, mostly in the focal plane. Table 1 presented a comparison between the mean values of the microsphere's refractive index in the presence of the diffraction, and in the absence of diffraction. A mean value of the refractive index for the microsphere was an average over all the points inside the microsphere that corresponded to a plane. Before the deconvolution (presence of diffraction), the mean value of the refractive index seemed to be a function of the z-position. We mainly observed a decrease of refractive index values while we moved away from the focal plane. This was due to the diffraction dependence along the optical axis, which becomes more significant as we moved away from the focal plane. From = −0.1 µm to = −0.2 µm, an increase occurred. This could explain an effect of the vibration which occurred during the measurement. This induced the sample to be shifted from a plane to another during the measurement. This was a drawback for the TIE method, which required the precise motion of the sample. After the deconvolution (taking into account 3D-PSF), the mean value of the refractive index was quasi-constant. The value in the focal plane where diffraction effects are greatly reduced was also in agreement with the refractive index of 1.586, which is the reference value [9] at 640 nm. In Figure 5, we can see the size's map ( , ) of the specimen. It was obtained by using Equation (12), taking = 1.587 as the mean value of microsphere's refractive index in the focal plane and = 1.33 as the refractive index of the distilled water used in the sample preparation. The microsphere's diameter as deduced from the map was = 0.943 µm. This value represents the intensity of the pixel in the middle of the microsphere ( Figure 5). The graph for the mean values of the refractive index as a function of z-position also led to the recovery of the diameter of the microsphere. The diameter was defined by the full width at half maximum (FWHM) indicated in the graph by the double arrow ( Figure 6). The diameter, which was deduced from the graph, is 1.1 µm. These two diameter values were close to 1 µm (the reference diameter of the microsphere used for the microscopic examination in this work), and they were in relative variation, between 6% and 10%. A reconstruction of the refractive index after the deconvolution of the corrected images by the use of the 3D-PSF is presented in Figure 4h-n. The sectioning effect was more greatly appreciated through the spatial distribution of refractive index. The circular outlines of the microsphere were well highlighted. The artefacts that were observed in the planes z = −0.4 µm, z = −0.3 µm, z = −0.2 µm were removed. In each plane of the microsphere, the homogeneity of refractive index distribution was greatly improved and tended to cover all of the circular outline of the microsphere, mostly in the focal plane. Table 1 presented a comparison between the mean values of the microsphere's refractive index in the presence of the diffraction, and in the absence of diffraction. A mean value of the refractive index for the microsphere was an average over all the points inside the microsphere that corresponded to a plane. Before the deconvolution (presence of diffraction), the mean value of the refractive index seemed to be a function of the z-position. We mainly observed a decrease of refractive index values while we moved away from the focal plane. This was due to the diffraction dependence along the optical axis, which becomes more significant as we moved away from the focal plane. From z = −0.1 µm to z = −0.2 µm, an increase occurred. This could explain an effect of the vibration which occurred during the measurement. This induced the sample to be shifted from a plane to another during the measurement. This was a drawback for the TIE method, which required the precise motion of the sample. After the deconvolution (taking into account 3D-PSF), the mean value of the refractive index was quasi-constant. The value in the focal plane where diffraction effects are greatly reduced was also in agreement with the refractive index of 1.586, which is the reference value [9] at 640 nm. In Figure 5, we can see the size's map t(x, y) of the specimen. It was obtained by using Equation (12), taking n f = 1.587 as the mean value of microsphere's refractive index in the focal plane and n m = 1.33 as the refractive index of the distilled water used in the sample preparation. The microsphere's diameter as deduced from the map was d = 0.943 µm. This value represents the intensity of the pixel in the middle of the microsphere ( Figure 5). The graph for the mean values of the refractive index as a function of z-position also led to the recovery of the diameter of the microsphere. The diameter was defined by the full width at half maximum (FWHM) indicated in the graph by the double arrow ( Figure 6). The diameter, which was deduced from the graph, is 1.1 µm. These two diameter values were close to 1 µm (the reference diameter of the microsphere used for the microscopic examination in this work), and they were in relative variation, between 6% and 10%.
Conclusions
In this work, we introduced a method to reconstruct the 3D refractive index distribution by combining TIE and deconvolution. A BF microscope using a camera was constructed, and the proposed technique was applied for a technical sample with well-known properties. The microscope's 3D-PSF, which takes into account optical aberrations, was computed. Its influence on the 3D refractive index recovery, using the TIE method, was demonstrated. Experimental results show that taking the diffraction into account while performing TIE on BF-images can significantly improve the accuracy of the 3D refractive index reconstruction. It was demonstrated that the method is able to infer the size of the sample accurately. Therefore, this approach was used in our laboratory to extract the optical parameters. The proposed technique can be applied, not only to homogeneous specimens, but also to specimens presenting heterogeneous compositions in their structures, such as biological cells. However, they must be pure phase objects, where it is possible to perform linear deconvolution according to Equation (2); otherwise the proposed method cannot be used.
Conclusions
In this work, we introduced a method to reconstruct the 3D refractive index distribution by combining TIE and deconvolution. A BF microscope using a camera was constructed, and the proposed technique was applied for a technical sample with well-known properties. The microscope's 3D-PSF, which takes into account optical aberrations, was computed. Its influence on the 3D refractive index recovery, using the TIE method, was demonstrated. Experimental results show that taking the diffraction into account while performing TIE on BF-images can significantly improve the accuracy of the 3D refractive index reconstruction. It was demonstrated that the method is able to infer the size of the sample accurately. Therefore, this approach was used in our laboratory to extract the optical parameters. The proposed technique can be applied, not only to homogeneous specimens, but also to specimens presenting heterogeneous compositions in their structures, such as biological cells. However, they must be pure phase objects, where it is possible to perform linear deconvolution according to Equation (2); otherwise the proposed method cannot be used.
Conclusions
In this work, we introduced a method to reconstruct the 3D refractive index distribution by combining TIE and deconvolution. A BF microscope using a camera was constructed, and the proposed technique was applied for a technical sample with well-known properties. The microscope's 3D-PSF, which takes into account optical aberrations, was computed. Its influence on the 3D refractive index recovery, using the TIE method, was demonstrated. Experimental results show that taking the diffraction into account while performing TIE on BF-images can significantly improve the accuracy of the 3D refractive index reconstruction. It was demonstrated that the method is able to infer the size of the sample accurately. Therefore, this approach was used in our laboratory to extract the optical parameters. The proposed technique can be applied, not only to homogeneous specimens, but also to specimens presenting heterogeneous compositions in their structures, such as biological cells. However, they must be pure phase objects, where it is possible to perform linear deconvolution according to Equation (2); otherwise the proposed method cannot be used. | 2019-04-16T13:28:49.043Z | 2018-09-13T00:00:00.000 | {
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62814244 | pes2o/s2orc | v3-fos-license | Implementation of Corporate Social Responsibility ( CSR ) of Cement Factory : Partnership Program , Environmental Guidance , and National Company-Care
Corporate Social Responsibility (CSR) of a company is aimed to improve social welfare around the company, then to get good relationship among people in the society as well as between society and the company itself for the sustainability. A research about implementation of Corporate Social Responsibility (CSR) program of PT. Semen Padang, a cement factory, was aimed to identify programs and collaborative model conducted by PT. Semen Padang in implementing the CSR program. This research was conducted in Batu Gadang Lubuk Kilangan (which is categorized as ring I for CSR receiver) located next to limestone hill, a source of raw material for cement Padang. Data were collected by interviewing CSR Bureau of PT Semen Padang, local government (“Kelurahan”) staffs, local organization (“KAN”) staffs, people in Batu Gadang receiving and non-receiving CSR using survey method. Data were analyzed using Descriptive Analyses method. Based on the data collected, it could be concluded that PT Semen Padang totally had allocated fund for the CSR more than the amount (>2%) it had to set aside. The CSR program was implemented through 3 forms, those were Partnership Program (PP), Environmental Guidance (EG) and National Company-Care (NCC), as well as other program which was not included in PP and EG (non-PPEG). Among the programs implemented, partnership program gave better result. If PT Semen Padang fully engaged local organization such as “KAN” as a co-worker, implementation of the CSR program can be more effective and reliable. Keywords— CSR; Corporate Social Responsibility; local organization; Partnership Program; Environmental Guidance; National Company-Care
I. INTRODUCTION
Even though corporate social responsibility (CSR) has been discussed for a long time, but it is relatively new in Indonesia.The importance of CSR is supported by Indonesian Government through its regulations [1], which suggest all of the National Corporate Bodies ("BUMN") to set the profit aside to improve the society welfare around the firms known as "Partnership Program and Environmental Guidance (PPEG).Then, the regulation is explained in "BUMN" Ministry's letter [2], and then become rules of the"BUMN" ministry itself, which explained the implementation of the ministry's decision above.Finally, Indonesian government put it into amendment [3].It is about the responsibility of a company, especially the one which explores natural resources, to implement CSR.
In fact, the CSR concept is meant and therefore implemented different by every company, because it is relatively new for the companies in Indonesia.The CSR is sometimes conducted as a charity program, such as a company contributes some gifts to the people around the company.Actually, the concept of CSR is not the same as charity or philanthropy which are used to have a short term effect rather than long term infestation for the society.The CSR is specifically to empowerment the society, either in terms of economy, social, and culture, therefore, the company can develop sustainability.
One form of the CSR implementation is community development (CD).This program is hopefully able to give opportunity for the target society to actively participate either in planning, conducting, monitoring, or in evaluating the program.In order to be seriously implemented, the CD program should empower and incorporate the social capital available in the surrounding.As stated in a journal [4] that use of social capital in implementing CSR gave good impact either in economy, social, or culture in sustainable way.
Social capital plays an important role in functionalizing and empowering life of modern society [5].It is needed in all aspects of human development, such in economy, social, politic, as well as in democratic stabilization.Any problems and deviation happened in several countries are mainly due to stagnation of the social capital.In a journal [6] stated that weakness of the social capital in a society negatively impacted on social welfare.Then, on a research [7] reported that utilization of social capital of local society was able to solve some problems, to improve family income in the society.Furthermore, it was explained the report [7] that social capital will be meaningful if it is combined with modal financial, such as derived from CSR.
PT Semen Padang as a big company processing cement in West Sumatra has payed some of the profit for the development of the surrounding society through its CSR program.The CSR was implemented in several programs.By those, PT Semen Padang in 2011 got award as "The Most Committed Company in Participating All Categories for Indonesian CSR Awards".In this article it is uncovered the method of PT Semen Padang in implementing the CSR, especially in Batu Gadang, the first ring area of PT Semen Padang.
II. METHODOLOGY
This research was conducted in Batu Gadang, Lubuk Kilangan District, Padang city-West Sumatra, INDONESIA.The area is located in the first ring as priority region for CSR implementation by PT Semen Padang.Research method employed for this study was in form of combination between qualitative and quantitative approach.Combination model used was Dominant-less dominant design [8] with triangulation technique.
Data collected were in form of primary and secondary data.Primary data came from the key informant determined before, either from people in Batu Gadang or from persons in charge for CSR PT Semen Padang.Secondary data, in addition, was got from government and any bodies around and linked to the Batu Gadang, such as data from the local government, local non-government organization (especially "KAN"), and the public schools.
Data collected were analysed using qualitative approach and conducted simultaneously with data collecting (on going analyses) called as triangulation technique.This technique could help data analyses, because this technique was done to gain better meaning about problems being analyzed.Data collected from quantitative approach were analyzed using descriptive statistics like means, percentage, and graphs.
A. Implementation of CSR Program
Implementation of CSR in PT Semen Padang followed the mission which is going to be "The best and environmental safe of Cement Industry", besides CSR itself was also governed in Ministry law.In operation, CSR PT Semen Padang has had its own CSR organization (Fig. 1).
Procedure used to conduct any of the CSR forms was coordinated by the CSR Bureau responsible to Director.The Bureau was divided into ( 1 Partnership Program (PK) could help the productive small to medium work ("UKM") in all economic sectors (such as industry, trade, agriculture, plantation, fishery, etc).Requirements needed for the "UKM" to be helped were explained in regulations stated by CSR of PT.Semen Padang [9].
Program CSR for EG and NCC was a kind of program giving a non-bound help to the people to improve the society welfare.This program included aids for disasters, either formal or informal education, improvement health facilities, development of facilities for religious activity, and for sustainable environment.
Furthermore, besides both previous programs, there was another CSR program called Non-PPEB.This program was a kind of aid aiming to support social activities in the society, such as aid for religious activities, aid for National Event celebration, etc.Then, the fund was also used for any activities planned and executed by PT Semen Padang for the society around, such as sport clubs, traditional art clubs, bazaar activities, as well as collecting and distributing people charity ("zakat"), and many other things.
Besides all above, PT Semen Padang also gave a chance for the people in the society to work at PT Semen Padang through job outsourcing, business cooperation, and local training.Then, CSR PT Semen Padang also distributed scholarships for outstanding students in pursuing their study universities.There was an increase of CSR budget allocation from 2009 to 2012.In 2010, the CSR budget allocated increased by 20% compared to that in 2009.The increase reached >40% for Partnership Program (PP), almost 300% for legacy in PP, while for environmental guidance (EG) as well as for non-PPEG programs the increase was<2%.During the 5 years (2008-2012), the highest CSR budget paid by PT Semen Padang was found in 2012, which was >300% compared to that in 2009.Of three programs introduced by PT.Semen Padang, the partnership program (PP) was allocated for the highest budget which was around 50% (37.5-57.0%),then followed by non-PPEG, and finally EG.
B. Allocation of CSR Budget of Company
Partnership program was a form of program giving loan for people (in person) for their capital in starting or continuing the works.This fund must be returned to the Corporation (CSR of PT Semen Padang), in order to be used by other people.
As partnership program, environmental guidance program was also conducted by PT Semen Padang.This program was aimed to improve the human resource capacity, infrastructure, economy, culture, as well as religious activities of the society.
2) Budget Allocation of CSR
Based on key informant from CSR Program of PT.Semen Padang, there was no specific mechanism aimed to implement CSR Program in Batu Gadang.Globally, CSR of PT Semen Padang has set rules for the CSR program, but not detail.So, every body, or group, or organization must write proposal to gain fund, then the proposal have to be signed by local government ("RT, RW, Lurah"), before getting approval from local non-government organization ("KAN").
However, as reported by "LURAH", Batu Gadang that there is no specific rules to be followed in writing the proposal.Furthermore, PT Semen Padang did not give exact time how long they have to wait for the realization of the proposal.Based on the Batu Gadang society, generally, PT Semen Padang has not yet socialized the programs of the CSR as well as how to get them.People did not know what the difference between programs in PT Semen Padang and other loan sources is.
Based on "Lurah" in Batu Gadang, whether every proposal is approved or not, and how much fund was received by people was not reported to "Lurah".So far, there is no exact data about people or organization receiving and amount of fund got from CSR program of PT Semen Padang in Batu Gadang.
Therefore, synchronization between CSR program of PT Semen Padang and other programs implemented by government is hard to conduct.
In proposing some help especially for PP, people must show the guarantee besides writing proposal.It is good in one part, because people have to work hard.On the other hand, it means that people having nothing will not get the loan from CSR program of PT Semen Padang.It was hard for them to change their life.
Among the three programs (PP, EG, Non-PPEG) implemented in Batu Gadang, partnership program was successful enough.In 2011, from total fund (Rp 260 million) allocated by CSR PT Semen Padang, 84% (Rp.218.4 Million) was dominated by industrial sector.It was noted that almost all of the industrial sectors in Batu Gadang were partners of PT Semen Padang.The number of this program as well as the total of the fund received by Batu Gadang society was almost the same every year since year 2011.It indicated that there was no development of the program, and there was no new programs introduced to improve society welfare in Batu Gadang.
Environmental Guidance (EG) program was divided into 2 sub programs, those were incidental and monthly program.Based on key informant, this program, as Partnership program, did also not change especially for the routine program, either the amount or the form, by time since the last 3 years since year 2011.
For human resource improvement, PT Semen Padang through its CSR program gave scholarship for good students as well as for poor family students from elementary school to university.Moreover, informal training was also conducted for unschooled children and for general society.This scholarship had been distributed by PT Semen Padang far beyond the CSR Program.Process of aid distribution mechanism by CSR PT Semen Padang to Batu Gadang society in education and in culture field was the same.
Besides giving scholarship to students, PT Semen Padang had also developed schools from kindergarten to senior high school for people living around PT Semen Padang including Batu Gadang.The schools were managed by PT.Igasar, a contractor of PT Semen Padang.These schools helped people very much especially for students could not be accepted in public school.Then, the school quality was good.
Based on data in Tabel 4, it can be calculated that amount of scholarship distributed by PT Semen Padang in Batu Gadang in 2013 was 9.61% (of the total allocated with the number of students receiving was 6.23% of the total students in the area.It means that amount of scholarship allocated to students in Batu Gadang was higher than the average.This indicated that PT Semen Padang through its CSR program had given more attention especially in education to people in Batu Gadang.
However, if it is compared between scholarship received and the total people in Batu Gadang, the amount of scholarship received was still far from what the society needed.
Furthermore, to improve the quality of non-student human resources, such as unschooled youg people and housewives, PT Semen Padang through its CSR program had given training for them.Among the materials trained were sewing, and cooking for housewives, electronics, machines, and workshops for unschooled young men.This training was aimed to initiate the entrepreneur feeling of people to be self-sufficient, and then to eliminate poverty in Batu Gadang, as well as around PT Semen Padang in general.
Based on data collected, in general, CSR PT Semen Padang had well implemented the fund for education.This was indicated by the total amount of fund had been distributed for education, either formal or informal, for outsourcing, as well as for work field initiation since year 2011.The treasurer of PT Semen Padang [10] proclaimed that it committed to improve human resources through education, because human resource was a national capital that must be optimally managed and developed.For that, PT Semen Padang reached it through EA and NCC programs in the CSR.
However, even though people in Batu Gadang had received aids for education, they were still unsatisfied.It was described in their activities as published in local daily news.From that, it was concluded that there was no cooperation between Batu Gadang society and PT Semen Padang.Batu Gadang society did not have sense of belonging yet toward the company, on the other hand, PT Semen Padang was not yet seriuous in developing them.
The misunderstanding between Batu Gadang society or people in Lubuk Kilangan in general and PT Semen Padang could be reduced if there was transparency about the CSR program between them.Therefore, people first must be educated in order to be able to accept some rules given by PT Semen Padang as long as it is fair for all.On the other hand, PT Semen Padang had to be fair and wise in deciding something for the people.This was hoped to create harmonized or good relationship between people and the firm as well as the sustainability of the firm.
Furthermore, PT Semen Padang was hoped to be able to develop a new system in implementing the CSR program within Batu Gadang society.The people in Batu Gadang and the PT Semen must sit together to discuss what kinds of problems the society have and what kinds of programs that PT Semen Padang has.What is the priority and what is the secondary could be agreed if there is a transparency and socialization.Decision started with comprehensive study and discussion and then ended with agreement will give good result.Therefore, the CSR program of PT Semen Padang could be effective and efficient.
C. Role of "KAN", a local non-government organization, in
Implementing CSR Program Based on "KAN" its organization must be included in implementing CSR program of the PT Semen Padang.This will help the CSR to be more effective in the implementation.If "KAN" has become a part of PT Semen Padang in implementing the CSR program, it is hopefully the misunderstanding among the people or organizations in the society can be avoided.This could be true that "KAN" knows and understands the people more than the PT Semen Padang does.
In order to increase people education in Batu Gadang, "KAN" hoped that PT Semen Padang through its CSR program can conduct (1) any training, vocational, and TOT for any local organizations such as "KAN, Karang Taruna, Bundo Kanduang", etc, therefore they can also teach other people within or out of the society.Then, for school age children, besides giving scholarship, it is suggested PT Semen Padang through the CSR program adopt the children as they own to be funded and schooled, especially for common children from poor families.
D. Response and Expectation of Batu Gadang Society from CSR Program
Generally, people in Batu Gadang did not know well yet what CSR is.They just said that they used to receive some aids (fund) from PT Semen Padang such as scholarship for children, free medical for manula and widows, religious activities, public facilities, as well as street development, etc.They thought that what PT Semen Padang through its CSR gave so far was a kind of "charity".Through people to people, some knew that CSR PT Semen Padang can give a loan as a capital for people to conduct their business.
Actually, based on the fact, PT Semen Padang through its CSR program has to give extra attention to Batu Gadang society due to some reasons.First, raw material of cement for the cement factory was located in Batu Gadang.It means that PT Semen Padang exploits the area of Batu Gadang for its profit.Second, area of company/firm/factory is also in Batu Gadang.Therefore, negative impact of the factory which is pollution especially in form of sound, ash, tremor, either during the exploitation of the raw material or during the processing of the cement is felt by the society.That is why most of people in Batu Gadang thought that what PT Semen Padang had done in their area was not yet balance, it was still far from what it has to be.Fundamentally, Batu Gadang has a good potency for development of agriculture, either for food crops, animal husbandry, or for fishery.However, the aids given were not yet based on the potency it has.Therefore, to be more effective and efficient, CSR allocation in Batu Gadang must be based on the potency.
IV. CONCLUSIONS
Based on the research conducted, it can be concluded that CSR program of PT Semen Padang followed the rules stated in the government regulation and the technical implementation itself.Three (3) programs of the CSR implemented were "Partnership Program" (PP),"Environmental Guidance (EG) and National Company Care (NCC) Program, and other than both Programs (Non PP-EG).Those had been done in Batu Gadang and other areas since few years ago.Among the program, Partnership Program seemed to be better than other programs in improving society welfare, even though it still needs improvement.
Based on the fact found, it is suggested that in implementing the CSR programs, PT Semen Padang should cooperate with the local organization, such as "KAN, Karang Taruna, Randai" and any other organizations in the Batu Gadang society.The company should sit together with the society to do comprehensive study on the natural and the human resources.
Then, through open dialogue and cooperation it must look for the priority, what the urgent and basic need of the society as a whole.
By these, implementation of the CSR program can be more effective and meaningful.
) Division of environmental guidance (EG) program and National Company Care (NCC) program, (2) Division of partnership Program (PP), and (3) Division of other than EG and PP (Non-PPEG).
1 )
General AllocationAs a big industry in West Sumatra, PT Semen Padang has contributed to the society either local or national through the PT Semen Padang only needed to allocate Rp 12.94 billion (2% of the profit) for the CSR program.However, it had paid for Rp.17.139 billion for CSR in that year.Based on the amount of the budget it had paid, it can be concluded that PT.Semen Padang had not only fulfilled its duty (compliance to laws and regulations) but it also had done the CSR beyond the compliance. | 2018-12-27T10:13:16.298Z | 2015-01-01T00:00:00.000 | {
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228925154 | pes2o/s2orc | v3-fos-license | A study on effectiveness of topical steroid therapy in boys with phimosis in the age group of 5 to 10 years
Background: Phimosis defined as the inability to retract the prepuce over the glans of penis is a common condition affecting boys. Objective was to study the effectiveness of topical steroid therapy (0.05% clobetasol propionate cream) in boys with phimosis in the age group of 5 to 10 years. Methods: This retrospective observational study was conducted at the Department of Pediatrics and Department of Surgery at Arunai Medical College and Hospital, Thiruvannamalai, Tamilnadu, India among 74 boys in the age group of 5 to 10 years with phimosis. The effect of twice daily application of 0.05% clobetasol propionate cream for six weeks on phimosis was studied. Results: out of the 74 boys, 25 (33.78%) were in the age group of 5 to 6 years, 20 (27.02%) in the age group of 7 to 8 years and 29 (39.19%) in the age group of 9 to 10 years. As per Kikiros et al system of grading of retraction of foreskin, majority of the boys 30 (40.54%) were grade 4, followed by grade 2 (24 boys, 32.43%), grade 3 (14 boys, 18.93%) and grade 5 (6 boys, 8.1%). Out of the 74 boys with phimosis, 53 boys (71.62%) had associated complications. After 6 weeks of topical steroid therapy, 39 (52.71%) boys showed complete response, 24 (32.43%) boys showed partial response and 11 (14.86%) boys showed no response to the treatment regimen. There was no significant correlation between age of boys and grade of phimosis with treatment response. Significant correlation was noted between history of urinary tract infection and treatment response. None of the other complications showed significant correlation with treatment response. None of the boys had any side effects to topical steroid therapy. Conclusions: Topical steroid application can be tried as an effective treatment modality in boys with phimosis in the age group of 5 to 10 years.
INTRODUCTION
Phimosis is defined as the inability to retract the prepuce over the glans of penis. It is a common childhood condition affecting boys. Surgical treatment of phimosis by circumcision is being considered as the main stay of treatment in boys with phimosis. Kikiros et al in their study in 1993 proposed that local application of topical steroids can may obviate the need for circumcision. 1 In our study, we aim to assess the effectiveness of topical steroid therapy in boys with phimosis in the age group of 5 to 10 years after a duration of 6 weeks of treatment.
Objectives
Objective was to study the effectiveness of topical steroid therapy (0.05% clobetasol propionate cream) in boys with phimosis in the age group of 5 to 10 years.
This retrospective observational study was conducted at the Department of Pediatrics and Department of Surgery at
Arunai Medical College and Hospital, Thiruvannamalai, Tamilnadu, India among 74 boys in the age group of 5 to 10 years with phimosis. Data were collected retrospectively from hospital records. Boys with phimosis attending the outpatient clinic or admitted as inpatients in the department of Pediatrics and department of surgery from September, 2019 to September 2020 fulfilling the inclusion and exclusion criteria were included in the study.
Retraction of foreskin in boys were graded as per the grading proposed by Kikiros et al (Table 1). 1 Slight retraction, but some distance between tip and glans, i.e. neither meatus nor glans can be exposed 5 Absolutely no retraction
Inclusion criteria
The study were boys with Phimosis in the age group of 5 to 10 years with Kikiros et al grade 2, 3, 4 and 5 whose parents were willing to participate in the study.
Exclusion criteria
The study were boys with Kikiros et al grade 0 and 1, boys with buried penis, Balanitis xerotica obliterans and boys whose parents were not willing to participate in the study.
After obtaining informed consent from parents, demographic details and clinical data regarding grading of phimosis, clinical presentation, symptoms and details of treatment were collected retrospectively from hospital data. Boys were prescribed topical steroid (0.05% clobetasol propionate cream) and advised to apply it twice daily to slightly retracted foreskin and to massage gently while retracting the foreskin. The boys were evaluated after six consecutive weeks of topical steroid application. Treatment success was defined as full retraction (Kikiros et al grade 0 and 1). Those with Kikros et al grade 0 and 1 were defined as complete response, those with improvement in grade of phimosis were defined as partial response and boys with no change in grade or worsening of grade of phimosis were defined as no response. Details regarding potential side effects of topical steroid therapy (striae, pigmentation changes and telangiectasia) were evaluated. Data collected were analyzed by suitable statistical methods using SSPS 25 software. Statistical significance was assessed at 5% level of significance (p<0.05).
RESULTS
A total of 118 boys fulfilled the inclusion and exclusion criteria over the study period, of which 20 children were excluded due to non-adherence to the treatment regimen, 18 were excluded as the boys could not be followed up and 6 of the boys were excluded as they underwent circumcision surgery before the completion of six weeks of topical steroid therapy. Total 74 boys were included in the study and statistical analysis done.
Out of the 74 boys, 25 (33.78%) were in the age group of 5 to 6 years, 20 (27.02%) in the age group of 7 to 8 years and 29 (39.19%) in the age group of 9 to 10 years. As per modified Kuppusamy's socio economic status scale, 9.45% belonged to class I (upper), 22.97% were class II (upper middle), 28.38% were class III (middle), 20.27% belonged to class IV (upper lower) and 18.93% belonged to class V (lower). The demographic distribution as per the boy's age and socioeconomic status is shown in Table 2.
Out of the 74 boys with phimosis, 53 boys (71.62%) had associated complications. The number of boys with associated complications are shown in Table 3. Urinary tract infection was the most common complication observed in our study. Figure 2). None of the boys who underwent topical steroid therapy experienced any side effects of topical steroid therapy.
The distribution of treatment response based on age of the boys is shown in (Table 4). No statistically significant correlation was found between age of the boys and treatment response (p>0.05).
The distribution of the treatment response in the boys based on the grade of phimosis is shown in ( The treatment response to topical steroid therapy in boys with the complications of phimosis is shown in (Table 6). There was statistically significant correlation between treatment response and history of urinary tract infection (p value<0.05). No statistically significant correlation was found between treatment response and other complications. The incidence of pathological phimosis is 0.4 per 1000 boys per year or 0.6% of boys are affected by their 15th birthday. 4 Several studies are being done to find effective medical treatment for phimosis as an alternative to circumcision. Topical steroid therapy and preputial dilatation and stretching are alternate treatment options being tried in phimosis. Prolonged antibiotic therapy, intralesional steroid injection, carbon dioxide laser therapy, and radial preputioplasty alone or with intralesional injection of steroid are all few experimental therapies for phimosis lacking in proper randomized trials. 5 Kikiros et al from the Royal Children's Hospital, Australia studied the effectiveness of topical steroid application for 4 weeks in 63 children with phimosis and observed that 51 children showed improvement to normal or near normal state obviating the need for circumcision. 1 Two important mechanisms are proposed for the effect of topical steroids in phimosis. The first mechanism proposed is an anti-inflammatory and immuno suppressive effect regulated by glucocorticoid activity, stimulating the transcription of anti-inflammatory genes and decreasing the transcription of inflammatory genes. Humoral factors involved in the inflammatory response and leukocyte migration are inhibited. Glucocorticoids also interfere with the function of endothelial cells, granulocytes, and fibroblasts. 1,6 The second mechanism of topical steroids is related to a skin thinning effect caused by the inhibition of collagen synthesis. Glucocorticoids inhibit the synthesis of hyaluronic acid, the main glycosaminoglycan produced by fibroblasts. Thus, the dermal extracellular matrix is reduced and collagen and elastin fibers become tightly packed and rearranged. 6 In our study, 0.05% clobetasol propionate cream was applied twice daily for six consecutive weeks in boys with phimosis in the age group of 5 to 10 years and treatment response assessed after 6 weeks. Of the 74 boys studied, 39 (52.71%) boys showed complete response, 24 (32.43%) boys showed partial response and 11 (14.86%) boys showed no response to the treatment regimen. These results were similar to those observed by Kikiros et al. 1 In a study by Ashfield et al among 228 boys with phimosis, six weeks of topical steroid therapy showed an overall efficacy of 87% at 3 months follow up. 7 Our study is limited by the lack of long term follow up to assess the risk of recurrence of phimosis and future need for circumcision in boys showing complete and partial treatment response. Data regarding risk of recurrence will help us understand if topical steroid therapy for phimosis can be an effective alternative for circumcision in boys with phimosis. Only 0.05% clobetasol propionate cream has been used as the topical steroid in our study.
Comparison of the efficacy of various topical steroid formulations will help to make better treatment recommendation in boys with phimosis.
CONCLUSION
Our study shows that topical steroid therapy is an effective treatment for boys with phimosis and can be tried as a conservative mode of management before surgical interventions. Further studies with more sample size, different topical steroid formulations and long term follow up will help us learn more about the effectiveness of this treatment modality and help to make better recommendations regarding topical steroid therapy for phimosis in boys. | 2020-11-05T09:10:46.861Z | 2020-11-24T00:00:00.000 | {
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12497606 | pes2o/s2orc | v3-fos-license | Detection of anti-Toxoplasma gondii antibodies in carthorses in the metropolitan region of Curitiba , Paraná , Brazil Detecção de anticorpos anti-Toxoplasma gondii em cavalos carroceiros da região metropolitana de Curitiba , Paraná , Brasil
Toxoplasma gondii, the agent for toxoplasmosis, has worldwide distribution. Horses normally play a secondary role in its life cycle, but movement around urban areas, feeding on grass and the increasing use of carthorses for gathering recyclable material in some urban areas of Brazil may increase their exposure to T. gondii infection. The aim of the present study was to investigate the frequency of anti-T. gondii antibodies in carthorses in the metropolitan region of Curitiba, PR. IgG antibodies against T. gondii were detected using the indirect fluorescence antibody test (IFAT) (titers ≥ 64). Seventeen (17.0%) of the 100 horses sampled were seropositive. There were no statistical differences in relation to sex (p = 0.28) or age (p = 0.15). Our findings suggest that carthorses are exposed to T. gondii infections and that no associations with age or sex exist.
Toxoplasmosis is a worldwide infectious disease caused by Toxoplasma gondii, which is an intracellular obligate protozoon capable of infecting any warm-blooded animal, including human beings (DUBEY et al., 2004).Domestic and wild cats are the definitive hosts and can eliminate oocysts in their feces (ARAMINI et al., 1999;LINDSAY et al., 2005).
Toxoplasmosis has been identified in horses since the early 1970s (WEILLAND; DALCHOW, 1970).These animals normally play a secondary role in T. gondii infection (GARCIA et al., 1999).However, the carthorse population has been continuously increasing in some urban and peripheral urban areas in Brazil, mostly due to transportation of recyclable material (LARA et al., 2006).During their daily journeys within the city limits, horses are fed on urban grassy areas in public parks, where cat feces are found more intensely.The objective of this study was to evaluate the frequency of anti-T.gondii antibodies in carthorses in the metropolitan area of Curitiba.A total of 100 carthorses were examined.All of them were crossbred horses, 53 were males and 47 were females.Their ages ranged from 6 months to 22 years .These horses were mostly used for pulling carts containing recyclable material.All of these animals had been spontaneously taken to the Zoonosis Control Center by their owners because of a veterinary program sponsored through a partnership between the city's Animal Services Agency and the Federal University of Paraná.The present study was approved by the Animal Ethics Committee of the Federal University of Paraná(protocol number 027/10).
Blood samples were collected by means of venous puncture using a vacuum tube system.The serum was separated and stored at -20 °C until processing at the Biological Institute, São Paulo, Brazil.The samples were screened for anti-T.gondii antibodies using an indirect fluorescence antibody test (IFAT) for IgG with a previously-established cutoff titer ≥64 (SULZER; HALL, 1967).Associations among occurrence of anti-T.gondii antibodies and sex and age were analyzed by means of the chi-square and Fisher statistical tests, with p < 0.05.
Antibodies against T. gondii were found in 17 (17.0%) of the 100 horses examined, all with a serum titer of 64.Among the positive horses, 58.82% (10) were females and 41.18% (7) were males.There were no associations with sex (p = 0.28) or with age (p = 0.15).The data are presented in Table 1.
Different techniques, cutoff values, geographical locations and management conditions may explain some of the seroprevalence differences among different studies.
In summary, our findings suggest that carthorses in this study region are exposed to T. gondii infections, and that age and sex are not associated with the presence of antibodies.
Table 1 .
Age and sex of carthorses examined and the numbers and percentages of animals positive for the presence of anti-T.gondii antibodies, in the metropolitan area of Curitiba, PR. | 2017-04-26T11:46:16.779Z | 2013-01-01T00:00:00.000 | {
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135651067 | pes2o/s2orc | v3-fos-license | Influence of Glass Fiber wt % and Silanization on Mechanical Flexural Strength of Reinforced Acrylics
The aim is to evaluate the flexural strength of acrylic resin bars depending on the addiction of glass fibers with or without previous 3-methacryloxypropyl-trimethoxysilane (silane) application. Short fibers (3 mm) were treated and added to an acrylic resin powder, being further mixed with acrylic liquid to create bars (25 × 2 × 2 mm) of 11 experimental groups (N = 10), according to the interaction of experimental factors: weight % of glass fibers: (0.5; 1; 3; 4; 6 and 7) and silane application (with silane (S) or without silane (N)). Flexural strength and scanning microscopy evaluation were performed (SEM). Data (MPa) were submitted to ANOVA and Tukey (α = 5%). A significant difference between groups was observed (p = 0.001): S7%(128.85 ± 35.76)a, S6% (119.31 ± 11.97)ab, S4% (116.98 ± 25.23)ab, N4% (107.85 ± 24.88)abc, S1% (96.29 ± 20.65)bc, S0.5% (89.29 ± 7.33)cd, S3% (89.0 ± 11.27)cd, N3% (86.79 ± 17.63)cd, N1% (85.43 ± 16.44)cd, Control (73.29 ± 25.0)de, N0.5% (59.58 ± 19.46)e. For N groups, it was not possible to include more than 4%wt fibers. SEM showed better fiber-resin interaction for S groups, and fractures around fibers on N groups. Previous silane application enables the addiction of greater quantity of glass fibers and better interaction with the acrylic resin resulting in higher flexural strength. Without silane, fibers seem to act as initial crack points due to poor interaction.
Introduction
Acrylics' fracture due to high transitory force caused by an accident or a small force during repeated mechanical load [1] is a direct result of development and propagation of cracks in the areas of stress concentration [2].The chances of fractures can be reduced increasing the strength of polymethyl methacrylate (PMMA), typically used in dentistry for provisional restorations or removable prosthesis.
After many attempts to improve the mechanical properties of PMMA resins, interest has turned to fiber rein-forcement.The incorporation of fibers into polymer matrix has provided substantial improvements on flexural strength and fatigue resistance of composite resin materials [3][4][5].The fiber reinforcing mechanism has been explained by the principle that a relatively soft ductile polymer matrix is fully capable of transferring an applied load to fibers via shear forces at the interface [3].
Studies have demonstrated a direct relationship between the fiber content and the flexural and impact strength of the reinforced resin [14,15], according to the law of mixtures [16].However, through the use of 3% (by weight) of many types of fibers without any surface treatment, Dogan et al. [17] reported no structural reinforcement of acrylic resin bars.
The silane coupling agent treatment (3-methacryloxypropyl-trimethoxysilane) creates a siloxane network with the hydroxyl (OH) of the Si in the glass fiber surface and co-polymerize with the acrylic resin [18].Silanized glass fibers present a higher surface energy and tend to be better impregnated, resulting in better adhesion to polymer matrix [19][20][21].It is expected that more fiber content would be included into the acrylic resin after fiber silane treatment, resulting in better flexural strength.
The aim of this study was to evaluate the flexural strength of acrylic resin bars by varying the weight content of fibers and surface treatment with silane coupling agent.
Materials and Methods
The materials used in this study are listed in Table 1.Eleven test groups (n = 10 per group) were created, representing the combination of factors in study: weight proportion of fibers (0.5%, 1%, 3%, 4%, 6%, 7% wt), surface treatment (with silanization (S) and without silanization (N)) and a control group.For N groups it was not possible to include more than 4%wt fibers.
Preparation of Specimens
Standardized rectangular specimens were created with dimensions of 25 mm (± 2.0) × 2 mm (±0.1) × 2 mm (±0.1), according to ISO4049/2000 [22].Fibers (3 mmlong) were weighted on an analytical balance (HR-200, A&D Company Limited, Japan) and mixed with the acrylic resin powder, either after silane treatment for 1 min at room temperature (S groups) or no silane treatment (N groups).The acrylic resin was then manipulated following manufacturer´s powder/liquid ratio.The specimen mold was covered with a clean glass slab to remove excess resin and kept at room temperature for 20 minutes under 9.8 N load until polymerization of the resin was completed.After preparation specimens were finished with 600, 1000 and 1200 grit SiC paper (Norton, São Paulo, SP, Brazil) under water stream.All specimens were stored in distilled water at 37˚C for 24 hours before testing.
Flexural Strength Test
Specimens were positioned on a 3-point bending flexural strength testing apparatus (K5005 MP; Kratos, Cotia, SP, Brazil) with two supports 20 mm apart, and tested at a crosshead speed of 1 mm/min.The load at fracture was recorded in Newtons and Flexure Strength (FS) was calculated in MPa with the following equation: FS = PL/wb 2 , where "P" is the maximum load at fracture, "L" is the distance between the supports (20 mm), "w" is the sample thickness and "b" the height.The samples' thickness and height were measured with a digital caliper (Mitutoyo, Japan).
Scanning Electron Microscope (SEM) Examination
Random samples were selected from each group and analyzed with a SEM.The samples, fixed on metal stubs, were placed in an ultrasonic bath of deionized water for 10 minutes, and then sputtered with gold (1 cycle of 120 s), under vacuum, in a sputtering device (MED 010; Balzers Union, Balzers, Liechtenstein).The surfaces were analyzed by SEM (LEO 435 VP; LEO Electron Microscopy Ltd., Cambridge, UK), focusing on the fracture features, integrity, and homogeneity along the interfaces between reinforcement material and acrylic resin.Samples were examined under magnification varying from ×250 to ×10,000.The unit operated at 20 kV, WD = 15 -18 mm, and with a spotsize range of 25 pA to 100 pA.
Statistical analysis
Statistical analysis was firstly performed with a factorial analysis (2 × 4) including the fiber surface treatment (S and N) versus fiber wt% (0.5; 1.5; 3 and 4) with a general linear model procedure in SSPS17.0 (SPSS Inc., Chicago, USA).After that all groups were submitted to Kolmogorov-Smirnov test of normal distribution, oneway ANOVA and Tukey Honestly Significant Difference (HSD).All tests were performed at 5% level of significance.
Results
The factorial analysis showed no interaction between factors in study (p = 0.267).Further ANOVA and Tukey tests showed significant difference between groups (p = 0.001; Table 2).The highest reinforcement effect was presented by S7% group, but similar to S6%, S4% and N4%.N4% group was similar to S1%, which in turn was similar to S0.5%, S3%, N3% and N1% groups.N 0.5% presented the lowest strength value, similar to the control group (C).SEM analysis showed N groups with areas of poor interaction between glass fiber and acrylic resin with the presence of empty spaces (Figures 1(B) and 1(D)), suggesting potential sources for crack propagation.In S groups this situation was not found, showing better micromechanical/adhesive interlocking due to resin remnants at the fiber surface (Figure 1(A)) and a multi plane fracture at the fiber seen in Figure 1(C).Images of S groups also showed the fracture line in more than one plane, suggesting better resistance for crack propagation (Figure 2(A)).The opposite occurred in N groups where the rupture line occurred in only one plane, showing lower resistance for crack propagation (Figure 2(B)).
Discussion
Fibers are known to reinforce dental polymers [6,23] and the present study compared the strengthening effect of including different weight proportion of fibers with or without surface treatment with a silane coupling agent.It was initially hypothesized that after silanization, the glass fiber reinforcement would be better wetted by the PMMA resin, facilitating the inclusion of a highest quantity of fiber and improving the final strength.The results of this work showed that the use of silanized glass fiber reinforcement significantly allowed more fibers to be included, increasing flexural properties, confirming this hypothesis.Fiber-to-resin interaction may be the reasons for these results.Glass fiber is an inorganic substance based on alumina-lime-borosilicate, considered to be the predominant reinforcement for a polymer matrix due to their high mechanical properties, low susceptibility to moisture absorption, resistance to chemicals, thermal stability and high melting point [5,20].Besides these characteristics, glass fibers are hydrophobic in nature and have low surface energy so that their natural compatibility with PMMA tends to be poor [21].Untreated fibers could act as foreign bodies in the acrylic resin mixture and, instead of a strengthening effect, they would actually weaken the resin by breaking up the homogenous matrix [24].This fact could be observed in this study, since N3%, N1% and N0.5% groups presented flexural strength similar to C group.N4% group was the only one that had a significant high strength when compared to samples with no reinforcement (C group).This might be explained by the greater amount of fiber included in this group, as described by Behr et al. [16].
The silane coupling agent treatment increases fiber's surface energy [19] resulting on better impregnation by the polymer matrix (Figures 1(A) and 1(C)).This occurrence resulted in significant higher flexural strength obtained in specimens reinforced with silanized fibers compared to non-silanized ones.S7%, S6% and S4% groups had flexural strength values significantly higher than control.The silanization of fibers enabled the inclusion of a greater quantity of fibers when compared to N groups.At N groups, any increase in fiber content beyond 4 wt% affected the flowability of the resin, so that fibers could not be mixed and prevented resin to react itself, producing a dry friable dough [21].
The reinforcing effect of the fibers is based on stress transfer from the polymer matrix to fibers but also the behavior of individual fibers acting as a crack stopper [25].Figure 2(A) shows fracture line in more than one plane, demonstrating that an effective adhesion between fiber and resin can difficult crack development.The same could not be observed at N groups (Figure 2(B)) where a fracture line in only on plane was observed.
In general, acrylic resin reinforcement with glass fibers produced improved fracture strength.Provisional or even definitive prosthesis can successfully employ fiber reinforcement in order to assure better longevity and ease of repair [2].The use of short random fiber presents itself as a less expensive and easy handling option for clinicians.Heat treatment of silanized glass to maximize the bonded strength is routinely performed in the glass industry, so heating the silane coupling agent after application into fibers could strengthening even more the PMMA resin, as seen in other studies for other materials [26,27].Future research may focus on improving adhesion of fibers with heat treatment of silane in order to improve mechanical properties.
Conclusions
According to the results and limitations of the present study, it is possible to conclude: 1.The use of silane allows for greater inclusion of fibers, better interaction with the PMMA resin and higher flexural strength.
2. Without silane treatment, fibers seem to act as crack starter points due to poor interaction.
3. Silanized fibers can act as crack stopper.
Figure 2 .
Figure 2. Rupture plane of fracture specimens.(A) (×250 magnification) S6% group showing a multi plane fracture, suggesting better resistance for crack propagation; (B) (×250 magnification) N4% group with rupture occurring in only one plane, a signal of easier crack development.
Table 2 . Flexural Strength-means and standard deviations (MPa) for different %wt of glass fiber and surface treat- ments. Groups Mean (SD)
EDifferent capital letters mean significant differences within the same reinforcement (p < 0.05). | 2019-01-03T09:00:25.571Z | 2014-01-24T00:00:00.000 | {
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85614057 | pes2o/s2orc | v3-fos-license | Diversity Analysis among Chickpea Genetic Stock as Revealed Through STMS Marker Analysis
Genetic diversity analysis of chickpea germplasm can provide useful information for the selection of parental material and thus help in planning breeding strategies.In the present study, a total of 57 STMS loci were analyzed to discern the variability among 87 chickpea lines consisting of released varieties and elite germplasm. A total of 87 alleles were found for the 19 STMS loci with an average of 4.57 alleles per locus. PIC value ranged from 0.94 to 0.10 and the heterozygosity ranged from 0.11 to 0.94, indicating good variability among the material as well as polymorphism generated. All the genotypes could cluster into six distinct groups with one genotype remaining unclustered. Greater gains can be obtained by crossing lines MPJG-2000-108 with SBD 377 for Desi and PG 0515 with ILC 212 for Kabuli improvement. Base broadening through Kabuli × Desi introgression with greater gains can be obtained by using ICC 4516 and ILC 212 as parents.
Introduction
Chickpea (Cicer arietinum L.; Family: Fabaceae) is a self-pollinated, diploid (2n=16), cool season pulse crop with a genome size of ~738-Mb and an estimated 28,269 genes (Varshney et al., 2013). It is widely grown in more than 50 countries representing all the continents (Upadhyay et al., 2011). Worldwide chickpea ranks third among legumes (Food and Agricultural Organization, 2010) i.e. almost 15% of the total pulse production of world. In the duration of 2010, the worldwide chickpea area was about 12.0 million ha, with 10.9 million metric tons of production with the yield of 911 kg/ha (FAOSTAT, 2012). India is the world's major producer, the annual production is around 7.58 Mt, grown in the area of approximately 8.32 mha, which is the world's 68% production of total chickpea and the average yield is approximately 912 kg/ha (FAOSTAT, 2012). More than 95% of the area of production and consumption of chickpea is shared by the developing countries. Chickpea is grown mainly in South East Asian countries. Kabuli (white seeds) and Desi (brown seeds) are the two main types of cultivated chickpea, presenting two diverse gene pools (Nawroz and Hero, 2011).
Chickpea has a very narrow genetic base which is limiting the genetic improvement of chickpea through breeding efforts. The level of natural variation among cultivated chickpea and wild accessions at molecular level is greatly aids in increasing the efficiency of breeding programme (Bharadwaj et al., 2011). This is because the phenotypic variability is largely an account of 'G × E' interaction where as the variability at molecular level is devoid of the interference by environment. Diversity analysis is essential to understand per se the variability present in germplasm collection that can be practically put to use in plant breeding programmes for recombination breeding. Simple Sequence Repeats (SSRs) are the preferred markers in most areas of molecular genetics as they are highly polymorphic even between closely related lines require very low amount of DNA and are very transferable across populations. SSRs are generally co-dominant markers and are most useful for studies on population genetics and mapping (Jarne and Lagoda, 1996;Goldstein and Pollock, 1997). SSR genotypic data from a number of loci have potential to provide distinctive allelic profiles for establishing genotypes identity (Bharadwaj et al., 2010(Bharadwaj et al., , 2011Chaudhary et al., 2012). Keeping the above points in mind an investigation was planned to discern the variability of diverse chickpea lines consisting of elite germplasm and cultivated lines, so that most diverse parents for crossing programme can be identified and diversity of the material can be analyzed.
Materials and methods
A total of 87 elite chickpea genetic stock obtained from Chickpea Project, Division of Genetics, Indian Agricultural Research Institute, New Delhi 110012 were used in this study. The genotypes were designated as GS-1 to GS-87 (Table 1).
DNA Isolation and Genotyping
Isolation of DNA was carried out by as per Kumar et al., 2013. A total of 50 sequence tagged microsatellite site (STMS) loci were screened in the accessions of which only 19 were polymorphic ( Table 2). The STMS markers were synthesized as per the sequences of Bharadwaj et al., 2010) from Bioneer, Daejeon, South Korea. BioRad MyCycler thermal cycler, Richmond, USA was used to carry out amplifications in 10 mL volume reaction mixture. This mixture contained 1 mL of 20 ng plant genomic DNA, 1.6 mL of 10×Tris buffer (15mM MgCl2 and gelatine), 1 mL of 10 mM dNTP mix, 1.0 mL each of forward and reverse primer and 0.3 mL of 3 UmL -1 Taq polymerase. PCR was performed with following conditions 50s at 90 o C followedby 18 cycles of denaturation at 94 o C for 20s, annealing for 50s at 50 o C (Touch down of 0.5 o C for every repeat cycle) and1 min elongation at 72 o C for 50s. Further 20 cycles of denaturationat 94 o C for 20s, annealing for 50s at 55 o C and 50s elongation at 72 o C were given and final extension at 72 o C for7 min were performed. The resolution of PCR products was done on three per cent metaphor gels (Lonza) (Fig. 1).
The polymorphic bands were scored in a spread sheet format with '0' representing absence of band and '1' representing the presence of band 'Null allele' for any specific marker in any genotype was again considered as absence of band (designated as '0'). The data was analyzed in NTSYS-PC software (version 2.21b). Bootstraps were done using Free Tree and Tree view software. For Clustering, UPGMA was used based on the similarity matrix generated on combined data. Polymorphic information content for each STMS primer pair was calculated.
Results
In the present study, a total of 57 STMS loci were analyzed, covering various bin locations on different linkage groups of which 19 were polymorphic (Table 2). A lower level of polymorphism is expected in chickpea which is having a narrow level of diversity compared to other crops and here it was 33.3%. All the 19 STMS loci, in the genetic material under study were found to be highly polymorphic. Excellent polymorphism was revealed by most of these STMS markers. Data from all the 19 STMS loci were utilized for statistical analysis. A total of 87 alleles were found with an average of 4.57 alleles per locus. The highest numbers of alleles were observed in TA194 (five alleles), TA 14, TA80, TA113, TA117 (four alleles each), TA14, TA71, TA110, CaSTMS2, CaSTMS15 and NCPGR4 (three alleles each).
Polymorphism information content (PIC) of each marker system was calculated for each marker and locus using the polymorphism information content (Lynch and Walsh, 1998) which gives an estimate of the discriminating power of a locus by taking into account not only the number of alleles that are expressed but also their relative frequencies. PIC ranged from 0.11 to 0.94. Highest PIC was observed for NCPGR7 and lowest for TA71.
Some STMS markers were found to have high discriminative power for differentiation of chickpea genetic stocks as the present study demonstrates that 19 out of 57 STMS alleles were found to be unique or rare; unique or rare allele is one with a frequency less than or equal to 0.10. The present findings also indicated instances where the STMS profiles for some of the genotypes displayed maximum variation pattern. Chickpea is highly self-pollinated and should, therefore, reveal lower polymorphism for majority of the genotypes, thus the occurrence of dialleles is also very less with a few primers only and is in concurrence with the reports (Singh et al., 2008;Ahmad et al., 2010;Singh et al., 2014) of a narrow genetic base of chickpea. It has been The STMS data was utilized for estimating pair wise genetic similarities among various entries using Jaccard's coefficient (1908) method. The genetic similarity matrix was further analyzed using UPGMA clustering algorithm by software programme NTSYS pc version 2.21b. The dendogram derived from this analysis is depicted in Fig. 2. The dendogram clearly showed 5 large clusters, 1 small clusters and 1 genotype remained ungrouped (GS39).The Bharadwaj et al., 2010Bharadwaj et al., , 2011a -from Huttel et al.;Varshney et al., 2013. cluster I, II, III, IV, V and VI comprised of 36, 5, 13, 11, 19 and 2 genotypes respectively. Maximum Jaccard's correlation was seen for the genotypes IPC-2000-20 (GS4) and IPC-2000-37 (GS5) while the genotypes BGD-132 (GS83) and BGD-9812(GS84) were having highest similarity index, while the genotypes SBD377 (GS71) and ILC-212(GS72) have shown the highest dissimilarity with all the other genotypes and distinctly formed a separate cluster (cluster VI) (Fig. 2).
Discussions
Among the various DNA based markers, microsatellite or STMS markers are highly accepted and have been used in the diverse crop plants owing to their abundance in the genome (Powell et al., 1996). The application of STMS markers in genetic analysis of chickpea, started with an initial study of (Huttel et al., 1999) and after that, the power and potential of SSR markers for a broad range of applications in genetic and breeding of chickpea has been well demonstrated by a number of researchers (Huttel et al., 1999;Winter et al., 2000;Flandez-galvez et al., 2003;Choumane et al., 2000). Microsatellite genotypic data from a number of loci have potential to give unique allelic profiles or DNA fingerprints for establishing genotypes identity (Bharadwaj et al., 2010). A narrow genetic base in chickpea warrants immediate base broadening efforts. Though morphological diversity is generally used by the breeders as a criteria in making crosses, it is clearly known that the manifestations of 'G × E' interactions make closely related individuals to appear diverse and thus there are greater chances of these being used in crossing programmes. Knowledge of molecular diversity in the material helps discern this diversity and in identification of parents for crossing programme (Bharadwaj et al., 2010).
For acceleration and optimizing the long process of creating new chickpea cultivars molecular markers are included as analyzing tools. Molecular markers are considered as good candidates for classifying genotypes in different groups and thus assessing genetic distances as well as genetic expected gains. RAPD were earlier used. However owing to greater reliability and repeatability, SSR markers are now being increasingly used for discerning genetic diversity.
Nineteen STMS primer pairs could amplify 1-4 loci per primer pair generating 4.57allels per locus on an average. Contrary to the fact that chickpea is a self pollinated crop and should generate lower polymorphism. World chickpea germplasm has a narrow genetic base (Nguyen et al., 2004) and lacks the desirable traits needed for ready utilization in varietal improvement programs. A narrow genetic base and sexual incompatibility with other Cicer wild types, which carry the sources for various desirable traits, contribute to the limited progress in the improvement of chickpea yield (Chaudhary et al., 2012). The presence of multiple alleles may have occurred to the fact that there is a very high residual heterozygotic balance conserved due to Desi × Kabuli introgression that played a major role in chickpea evolution. This may be one of the causes for obtaining multiple bands using SSR markers (Singh et al., 2008). A high degree of molecular polymorphism was exhibited by all the markers studied indicates the markers that have been used for diversity analysis were sound. The PIC ranged from 0.10 to 0.94 and heterozygosity ranged from 0.11 to 0.94. The Jacards similarity matrix dendogram constructed using the UPGMA method showed that all the clusters were dissimilar and grouped into seven major clusters. A critical examination of these clusters with indicates that the grouping was primarily based on seed size. Cluster VI has SBD 377 and ILC 212. SBD 377, a simple leaf mutant developed at IARI had an ICARDA line in its pedigree PRR1, a derivative from FLIP 90-166, an ICARDA line and thus would have got clustered in proximity with GS72 i.e. ILC 212. The market collection GS39, a bold seeded kabuli type remained un-grouped as it was very bold in its size and does not represent a released cultivar but market collection obtained under the ISOPOM trial. Cluster two comprised mostly either bold seeded or erect types. Thus plant architecture also played an important role. However, contrary to earlier workers reports, it is clearly noticed that the place where the cultivars were developed did not play a major role in grouping. This may be due to the fact that, the elite breeding lines included in this study obtained from different centers were developed from germplasm either obtained from ICRISAT or ICARDA. In the previous study of Bharadwaj et al. (2011) specific lines of ICARDA, ICRISAT and IARI were used where all the lines from ICARDA and wild species were grouped as a distinct cluster. The breeder's generally use diverse sources selected based on morphological traits and their observation in making crosses. Similar results were obtained by Choudhary et al. (2012).
Results from the present study support the observations of several workers about the potential utility of STMS in characterizing asparagus lines (Huttel et al., 1999;Winter et al., 2000;Flandez-galvez et al., 2003;Choumane et al., 2000). There was reasonably high rate of polymorphism for at least ten markers namely TA194, TA80, TA113, TA117, TA14, TA110, NC6, NC7, CaSTMS15 and NCPGR4 out of 19 STMS markers loci in the present study. This pointed towards the scope for further utilization of these markers for characterization of different cultivars of chickpea. The STMS polymorphism were assayed using a DNA pooling No. of genotypes Names of genotypes strategy, although it is not supposed to do as all the genotypes under study are pure lines (Flandez-Galvez et al., 2003) demonstrated the power and potential of SSR markers for a wide range of applications in genetic and breeding of chickpea. Molecular markers being easily reproducible they have become favourite tools with breeders and biotechnologists to discern the traits as well as to study diversity among cultivars (Satyavathi et al., 2005). However, no correlation could be derived from PIC and allele numbers in this study. Further, Greater gains can be obtained by crossing lines MPJG-2000-108 with SBD 377 for desi improvement and PG 0515 with ILC 212 for kabuli improvement. These genotypes have been identified as most diverse in the present study. Pre-breeding and Base broadening through kabuli × desi introgression for greater gains is an important activity of the breeders. This increases the diversity obtained in the succeeding generations to carry out further selections as there is enormous amount of variation that is seen in these generations for seed size, seed type and other traits. In the present study greater base broadening can be achieved by using ICC 4516 and ILC 212 as parents in the breeding programme.
This study helped to determine the genetic relationship between elite genetic stock of chickpea based on STMS marker data, and these results greatly contribute to germplasm bank management, conservation programs, and breeding purposes. The occurrence of unique alleles or rare STMS alleles provides an immense opportunity for generation of comprehensive fingerprint database. The present investigation also gives an idea of the interrelationship among the genotypes and highlights the need for helpful supplementation of pedigree data and other morphological data with the database generated by STMS marker to efficiently discover the genetic inter-relationship among the genotypes, fingerprint the varieties for their protection and most importantly select parents for a sound breeding programme. | 2019-03-30T13:12:42.565Z | 2015-01-01T00:00:00.000 | {
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235661930 | pes2o/s2orc | v3-fos-license | Parents' expectations of the outpatient care for daytime urinary incontinence in children
INTRODUCTION
Daytime urinary incontinence (UI) can have an enormous impact on a child's life, lowering both self-esteem and quality of life. Although most children start therapy after their first visit to our outpatient clinic, no studies have reported on parents' or patients' expectations of care for daytime UI in this setting.
OBJECTIVE
We aimed to explore the expectations of the parents of children referred to an outpatient clinic for daytime UI.
STUDY DESIGN
This was a qualitative study that involved performing semi-structured interviews with the parents of children who had been referred for daytime UI (with or without nocturnal enuresis). Interviews took place between July 2018 and October 2018 and continued until saturation was reached. The results were transcribed verbatim and analyzed according to Giorgi's strategy of phenomenological data analysis.
RESULTS
Nine parents of children, aged 5-12 years old, were interviewed, revealing "(Experienced) Health," Self-management," and "Social Impact" as the main themes that influenced parental expectations. All parents wanted to know if there was a medical explanation for UI, some were satisfied when diagnostics revealed no underlying condition, and others wanted treatment. Parents expressed no preferences about diagnostics or the content and duration of treatment, but they hoped that any previously attempted ineffective steps would not be repeated. Some parents defined treatment success as their child becoming completely dry, but most stated that learning coping strategies was more important.
DISCUSSION
This is the first study to explore the expectations of parents when attending outpatient care for children with daytime UI. We employed a strong theoretical framework with a clear interview guide. The main limitations are that we only interviewed parents and that this was a qualitative study, precluding the drawing of firm conclusions. Nevertheless, our results point to the need for quantitative evaluation.
CONCLUSION
Expectations seem to be influenced by (experienced) health, efforts at self-management, and the social impact of UI, making it critical that these themes are addressed. It was interesting to note that parents do not always attend outpatient departments with the goal of completely resolving daytime UI. Instead, some only want to know if there is an underlying medical condition or want to reduce the social impact by learning coping mechanisms. Excluding underlying medical conditions may therefore stimulate acceptance of watchful waiting without the need to start treatment.
Introduction
Daytime urinary incontinence (UI) can have an enormous impact on a child's life, lowering both self-esteem and quality of life.Although most children start therapy after their first visit to our outpatient clinic, no studies have reported on parents' or patients' expectations of care for daytime UI in this setting.
Objective
We aimed to explore the expectations of the parents of children referred to an outpatient clinic for daytime UI.
Study design
This was a qualitative study that involved performing semi-structured interviews with the parents of children who had been referred for daytime UI (with or without nocturnal enuresis).Interviews took place between July 2018 and October 2018 and continued until saturation was reached.The results were transcribed verbatim and analyzed according to Giorgi's strategy of phenomenological data analysis.
Results
Nine parents of children, aged 5e12 years old, were interviewed, revealing "(Experienced) Health," Selfmanagement," and "Social Impact" as the main themes that influenced parental expectations.All parents wanted to know if there was a medical explanation for UI, some were satisfied when diagnostics revealed no underlying condition, and others wanted treatment.Parents expressed no preferences about diagnostics or the content and duration of treatment, but they hoped that any previously attempted ineffective steps would not be repeated.Some parents defined treatment success as their child becoming completely dry, but most stated that learning coping strategies was more important.
Discussion
This is the first study to explore the expectations of parents when attending outpatient care for children with daytime UI.We employed a strong theoretical framework with a clear interview guide.The main limitations are that we only interviewed parents and that this was a qualitative study, precluding the drawing of firm conclusions.Nevertheless, our results point to the need for quantitative evaluation.
Conclusion
Expectations seem to be influenced by (experienced) health, efforts at self-management, and the social impact of UI, making it critical that these themes are addressed.It was interesting to note that parents do not always attend outpatient departments with the goal of completely resolving daytime UI.Instead, some only want to know if there is an underlying medical condition or want to reduce the social impact by learning coping mechanisms.Excluding underlying medical conditions may therefore stimulate acceptance of watchful waiting without the need to start treatment.
Introduction
Daytime urinary incontinence (UI) is a common condition that can lower both self-esteem and quality of life [1].As the child ages, daytime UI poses a greater burden and more often leads to them being bullied, resulting in social withdrawal and even aggression [2].Daytime UI can be diagnosed from 5 years old and requires multidisciplinary treatment [3] by general practitioners (GPs), urologists, and pediatricians in the Netherlands.However, whereas the Dutch associations of pediatricians and urologists have composed a joint guideline for managing UI [4], none exists for Dutch GPs.In the joint guideline, urotherapy is initially advocated after excluding anatomical and neurological deficits.This involves explaining urinary tract function and giving instructions about micturition, but may include treatment of constipation and infection as well as the use of cognitive therapy, behavioral therapy, and physical therapy.When an overactive bladder is diagnosed, antimuscarinic medication can be prescribed [5].
In the Netherlands the GP is the first doctor a patient turns to.It is not possible to consult a pediatrician nor urologist without a referral, except in case of a medical emergency.When GPs decide to refer a child with daytime UI, they can choose to refer to the pediatrician or urologist.In our hospital, we treat some 130 children with daytime UI at the outpatient clinics annually.Although most start therapy after their first visit with the goal of completely resolving symptoms, there has been no structured analysis as to whether parents share this goal in such settings.This need rectifying given that the role of shared decision making has grown in importance in recent decades.Gaining information on children's expectations is relevant in this context, and meeting these has been shown to correlate with overall satisfaction [6].Positive expectations in adults have similarly been associated with better health outcomes, underlining the need to discuss these expectations [7].However, we are aware of no studies on what care parents or patients expect for daytime UI.Studies of nocturnal enuresis indicate that enuresis can be stressful for parents, indicating the need for not only information on the causes and available treatment options but also access to aids and support [8,9].
In this study, we aimed to explore the expectations of parents whose children were referred to our hospital with daytime UI.
Study design
We conducted a qualitative study with semi-structured interviews among the parents of children with daytime UI who were referred to the outpatient departments of pediatrics or urology of our hospital.The Medical Ethics Committee of our hospital approved the study in March 2018 (METC number 180333) and all participants provided written informed consent.
Sample selection
We selected children aged 4e18 years referred for daytime UI, regardless of the presence of nocturnal enuresis or other lower urinary tract symptoms.The selection was made based on information in the referral letter from the referrer.Exclusion criteria were insufficient mastery of the Dutch language, nocturnal enuresis without daytime UI, or urinary tract infections as the explanation for UI.We approached parents before their first visit to explain the study and invited them to participate.Written information about the study was then sent to their home addresses, and after two weeks, parents were called again to check their willingness to participate.
Data collection
We developed an interview guide based on research questions, clinical experiences, and literature review (Appendix Theoretical model showing three main themes influencing expectations of parents concerning the assessment of daytime urinary incontinence.The (experienced) health, social impact and self-management of parents influence each other and together form the expectations of on the assessment of daytime urinary incontinence.
A) [10e13].The guide was used to initiate the interview and stimulate discussion.All interviews took place between July 2018 and October 2018 and were conducted in-person before the first visit at the outpatient clinic.The interviewer (JML) was independent, had no connections with the parents or children, and audio-recorded the interviews.The children were present during the interview and could participate in the discussion when they wanted.
Interviews were transcribed verbatim by an external company.Recruitment and interviewing continued until saturation was achieved (meaning that further interviews revealed no new findings or opinions).We also collected demographic variables from children's medical records (gender, age, type of incontinence, period in life without urine loss, medical history).All data collection and analysis took place concurrently, and during the analysis, the research team met to discuss the ideas emerging from the data.
Data analysis
Data were coded and analyzed in duplicate based on Giorgi's strategy of phenomenological analysis [14], using ATLAS.ti8.3.1 (ATLAS.tiScientific Software Development GmbH).After open coding of the first two interviews, matching codes were merged and differences were discussed (Table 1).The third interview was then coded and the research group met to discuss the emerging codes and themes until consensus was reached.Subsequent interviews were coded accordingly.The research group was multidisciplinary including a GP, (pediatric) urologists, pediatricians, nurse practitioners, epidemiologists and an independent researcher.An extensive description of the data analysis is given in appendix B.
Results
Saturation was reached after nine interviews with parents of patients recruited from the Department of Pediatrics.Each interview lasted 15e30 min, only mothers participated, and all included children were unrelated.There were no new referrals to the urology outpatient department during the study period.Table 2 shows an overview of the participant characteristics.Three main themes emerged from the interviews: ( 1) "(Experienced) Health," (2) "Self-management," and (3) "Social Impact."The themes influenced each other, and together, influenced parental expectations, as illustrated in Fig. 1.
(Experienced) health
The type and severity of UI, insight into the cause of UI, and the experiences of parents and children to date were included in the (experienced) health theme.Parents expressed that the UI and its lack of predictability was a burden.
Quote 1: "The question is not if it's going to go wrong, but when."(P5) A total of 14 children was selected.Two parents could not be reached by telephone and three refused participation.Four of these children were aged 4e7 years old, one child was 12 years old.
Parents' expectations of DUI care in children 473.e3 Not knowing why the loss occurs intermittently also raised questions and doubts, but most parents acknowledged that multiple factors contribute to the urine loss.From age 5e6 years, some parents experienced UI as an increasing problem, but others reported having reached a sustainable situation.
Quote 4: "He has been in school for two years now.In the first period we still thought something like, well not everyone is super potty-trained at this point, so it doesn't matter.But in the past year and a half we did start to feel like, well ."(P1) Quote 5: "You know, it is handleable at the moment.I am not constantly occupied with making sure it doesn't go wrong" (P3)
Self-management
Self-management involved steps already taken by parents to minimize or stop UI, the information sources used, prior experience with treatments and caregivers, and the reason for the current referral.
Parents wanted support when UI persisted.Most had started on the internet, searching for tips, advice, and the experiences of other parents.Relatives were also asked for help.However, some parents avoided searching because they feared it could make them unnecessarily worried.
Quote 6: "My brother and my two nephews also wet their beds for a long time.So, I had, of course, already discussed that with my mom and aunts like, how did you two deal with all of that."(P1) Parents also reported that they felt that they had failed when, despite their efforts, the UI did not improve.They mentioned that it affected daily life and the relationship with their child.
Social impact
The third main theme concerned the social impact of UI on daily life, which related to the reactions of others, interactions with peers, and experiences at school.Parents expressed that UI was an obstacle to the social development of their child.For example, peers noticed wet spots and the smell of urine, often commenting, and parents recognized that this made contact with classmates harder.Children also omitted certain activities to hide their UI, such as sleepovers or playdates.Quote 8: Question: "Do you refrain from doing things because of this?" Child: "Sometimes, sleepovers, for example."Mother: "Oh, yes.School camp .that was quite a thing."(P2).
Many schools have a policy of not changing the clothes of children, forcing the child to handle UI without assistance.Some schools even demand that the child is continent before they are welcome.Parents therefore experienced little support from schools.In addition, support from family members varied between participants.
Quote 9: "The teachers at school don't do anything about that anymore because it isn't included in their job responsibilities.He thus has to be able to do it completely by himself, he doesn't even get small inclinations like, go to the toilet, anymore.Another example is that, when he is wet, he has to be able to redress himself.So, it's a lot different now compared to what I was used to, going to school back in my day."(P4) Fig. 1 Theoretical model showing three main themes influencing expectations of parents concerning the assessment of daytime urinary incontinence.The (experienced) health, social impact and self-management of parents influence each other and together form the expectations of on the assessment of daytime urinary incontinence.
The social impact was also reported to increase as the child aged.In particular, parents expressed concerns that the child would be bullied and excluded from social groups because of the UI.
Expectations on the outpatient care
The three main themes influenced parental expectations of outpatient care.These concerned not only the overall care trajectory but also the specific components, such as diagnostics, treatment, support, information, and treatment outcomes.
All parents wanted to know if a medical condition caused the UI.Parents expected that diagnostic tests would be performed to exclude pathology, but they did not have a clear idea on what diagnostics would be used.Some parents only wanted to know if there was a physical abnormality, and if reassured, they stated that they would be satisfied with no further treatment or support.
Quote 10: "See, if anything is found, you'll be busy for a year before you know it.But if they find nothing, yeah well that's it and we'll undertake action at home.I don't require any further assistance in that case."(P3) Most parents found it hard to say what they expected of the treatment because they did not know the cause of the UI.However, they did not want to repeat steps that had been tried and found to be of no benefit.Quote 11: "I don't really know, because I'm not sure whether it's behavior or something physical, so to speak.I really expect that if it is behavioral, he'll have to start practicing.A sort of physical therapy, specifically for this."(P6) Quote 12: "That the child is being checked, not that every child is being treated the same, that is the starting point.That someone checks what all is going on, so that you don't have to return with the thought 'wow this could all have gone so much faster.'"(P4) Parents wanted accessible information about the cause of the UI, the diagnostic tests, and the treatment to be provided (both orally and in writing).Some mentioned they would prefer informational videos to re-watch at home, and some mentioned that this information was necessary for others involved in their care, such as grandparents or school staff.A number wanted child-centered support, expecting that efforts would be made by practitioners to involve the child in the consultation, providing tailored advice and tips.
It was hoped that hospitals would consider the schedules of children when making appointments to avoid their child needing to attend hospital during school time.This was because the child often did not want to miss fun activities, feel different to their peers, or explain their absence.
Although all parents were confident that outpatient treatment would be successful, the definition of success varied.Some parents defined success as becoming totally dry, but most stated that their main goals were to learn coping strategies and to reduce the social impact.
Quote 13: Mother: "a success, or actually: just being helped to a point where she can be dry during the day."Child: "If everything, really everything, is over."(P6) Quote 14: "Success is him not wearing soaking wet pants during the day anymore.A few drops are fine.But not everything from his socks to his pants.So that other children at least don't see him standing around with wet pants anymore.So that he has that self-confidence of 'hey, I go to the toilet in time; I can hold it in.'For me, that's a real goal: it not being a problem that others see; it being handleable."(P4) Quote 15: Well, I never really have many expectations.I just go in with an open mindset and hope that a solution is found.But I also know that may not be possible and that it maybe just needs time (.)I at least hope to find out if there is something medical going on.And for the rest, possibly, get some tips on how to deal with it (.)I have tried everything at this point, and we really do not know anymore.(P9).
Parents responded differently about whether they wanted to meet other parents to discuss their problems.Some only wanted to speak to other parents if there was something medically wrong.Others said they had enough friends and family to ask for help and tips, and that they did not feel the need to talk to a stranger about UI.
Quote 16: "Possibly, for him, it is depending on the cause.What the origin of this?Is it something physical?Is it just something psychological?Is it something .a lack of interest on his part?Or .? (.)If there is a physical cause, I think you would talk with other parents about it: how do you manage this? (.)If this is something time will resolve, then I don't know."(P4)
Discussion
We are aware of no prior research evaluating the expectations of parents before attending outpatient care for children with daytime UI.We showed that expectations were affected by the (experienced) health, selfmanagement, and social impact of UI.Most parents worry about the health and social development of their child when potty training is unsuccessful, typically only seeking help when self-help strategies do not resolve the problem.The main goal of parents is not always to achieve complete symptom resolution, however, with some only wanting to know if there is a medical cause or how to reduce the social impact by learning coping mechanisms.They do not want to repeat steps or treatments if they have previously been unsuccessful.
It was noteworthy that some parents expressed that they would be satisfied if diagnostic testing showed that there was no physical abnormality causing the UI.These parents stated that they had already acquired the necessary coping strategies to handle the condition and did not want immediate treatment if it was unnecessary.We believe that this is a remarkable finding because, in daily practice, it has been our experience that most health care professionals assume that therapy is necessary and start it Parents' expectations of DUI care in children 473.e5 from the first visit.Given that the prevalence of UI is known to decrease with age [15], overtreatment may be a problem in some children, particularly the young and/or those with potentially self-limiting conditions.If the expectations of these parents and children are addressed at the first visit, confirming the absence of a physical abnormality could encourage watchful waiting.Indeed, this may allow discharge from outpatient follow-up, meaning fewer hospital visits, fewer unnecessary treatments, and a reduction in health care cost.We believe that it is likely that this happens in other hospitals and in other countries, though confirmatory studies will be needed.We were unable to find comparable studies concerning the expectations of the parents of children with daytime UI, but we did find two similar studies about nocturnal enuresis [8,9].First, Cederblad et al. reported that Swedish parents wanted more information and to protect their child from gossip or teasing [9].Similarly, parents in our study expressed that they had sought further help because they were afraid that, as their child aged, they would become a victim of bullying from classmates.Another parallel is that parents mentioned that they had already exhausted all selfhelp options and that they sometimes felt guilty [9].However, among parents of children with nocturnal enuresis in America, Dunlop reported that fewer than half wanted to know more about the causes and that fewer than one-third wanted to know about available treatment options [8].By contrast, our results showed that most parents wanted information on the cause of UI.Another difference is that a surprisingly few parents (12%) in Dunlop's study expressed interest in knowing how to discuss enuresis with their child.This contrasts starkly with our finding that parents wanted to know more about coping mechanisms for their child and themselves.Nevertheless, these differences could be explained by the fact that the studies were performed in different populations.Dunlop included a national probability sample, while we selected parents attending an outpatient clinic after seeking help for UI in their child.Only half of all parents in Dunlop's study reported that they would take the initiative to contact their healthcare provider if their child experienced nocturnal enuresis.It is possible that the parents in our study had suffered more from the experience of dealing with their child's UI, which could explain the need for information and coping strategies.
Finally, parents in our study mentioned that they felt little support from the teachers of their children, consistent with statements by children in a previous study [16].This may explain why parents in our study stated that information should not only target themselves but also those in their wider environments.
Strengths and limitations of this study
The main strengths of this qualitative study are that we employed a strong theoretical framework with a clear interview guide based on current literature and clinical experience.Interviews were carried out by only one interviewer who was familiar with the subject and not involved in the treatment.This gave parents the freedom to speak without affecting the care trajectory.We also continued to interview until we reached saturation, and the results were discussed extensively among a multidisciplinary research team during analysis, ensuring the input of different professional perspectives.
A limitation of this study is, that despite the age range for inclusion of 4e18 years old, we only enrolled children between 5 and 11 years old.Therefore, our studied population may not fully cover the population of our clinic.In another study we performed [unpublished data], we noted that the vast majority of our population consists of young children (median age 6).It might be possible that teenagers have other expectations.Following the current study, we developed a questionnaire, which will be send to the older children as well to also explore their expectations.However, it is possible that not all aspects that older children experience are included in the questionnaire and that teenagers have other expectations.
Another limitation could be that we only interviewed parents and, because of their young age, did not interview children.However, we did ask parents about their child's expectations, and they often mentioned that their child did not know what to expect from the hospital.We therefore feel that the impact of this limitation is small, not least because children typically do not initiate referrals and only attend on the instruction of a parent or GP.In older children, some mothers did involve their child in the interview, but most of the time, the child had no clear opinion or simply agreed with the mother.A study on treatment expectations revealed that half of adolescents aged 13e18 years with chronic musculoskeletal pain agreed with their parents [12].Given that we had younger children in our group (all were aged 12 years), we expect a much higher level of agreement in our study.
All children in our study were referred to a pediatrician, with no referrals to the urologist during the inclusion period.This is in line with the findings of an observational study in our hospital, which revealed that 85% of new referrals for daytime UI were to pediatricians [unpublished data].We believe that there would be no major differences in parental expectations before their first visit to the pediatrician or urologist, because they have not been seen by either specialist.From one of our other studies, we know that parents (and children) are not actively involved in choosing to which specialist they are referred [unpublished data].We assume that the knowledge of the parents about the differences between the pediatrician and urologist is limited, and therefore this will not influence the experience.Our study focusses on the expectations regarding solving the UI and this problem is the same between the two groups.Another limitation was that all participants in the current study were mothers.This is consistent with our experiences at the outpatient clinic, where the vast majority of children is accompanied by their mother, but we do not know if mothers have different expectations from fathers.It could be possible that there are differences.However, although Dunlop found that mothers were more likely to contact a healthcare professional about bedwetting, parents were reported to be equally concerned about the effect of nocturnal enuresis [8].Similarly, Cederblad et al. reported that the patterns of the answers were comparable between fathers and mothers [9].
Quote 2 :
"I'm very busy with everything, except for what counts" (P5) Quote 3: "It just needs its time.The one child is faster than the other."(P4)
Quote 7 :
"Interviewer: Did you also look up information yourself?Mother: No, that only makes you worry."(P9)
Table 1
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54032876 | pes2o/s2orc | v3-fos-license | Relationship Between Prior Knowledge and Creative Thinking Ability in Chemistry
This study aims to determine the relationship between prior knowledge and creative thinking ability of 11 Grade science students. This research is ex post facto. The population for this research was 11 Grade science students at a public school in Takalar, Indonesia, and consisted of 39 classes with a total enrollment of 1,537 students. In selecting the sample, the study employed stratified purposive random sampling technique in order to select 134 of the 11 Grade science students (from SMAN 1 Takalar, SMAN 3 Takalar, SMAN 1 Polongbangkeng Selatan, and SMAN 3 Polongbangkeng Utara). The data was collected by using a prior knowledge test that consisted of 16 items (α = 0.883) and a verbal creativity test consisting of 18 items (α = 0.808). Data were analyzed using correlation and regression analysis. The coefficient correlation between the two variables is 0.619 with p = 0.000 (p <0.05). This value indicates the existence of a relationship between prior knowledge and creative thinking ability in chemistry students and that there is a positive relationship between the two variables.
Introduction
Creativity is indispensable for the development of a nation, as through creativity each nation can compete with other nations.The Indonesian Act No. 20 (2003) on the National Education System detailed the goal to develop students to become people of faith who fear God Almighty, and who are noble, healthy, knowledgeable, skilled, creative, independent, and who are democratic and accountable citizens.Based on the Act, one aspect that needs to be developed in the educational process is the creativity of learners.
Based on the National Education goals, the development of creativity in learners is one part of teaching and learning in schools.Thus, chemistry, as one of the subjects taught in schools, can be used as a means to develop the creativity of learners.Creativity in learning chemistry, among other things, is required in terms of completing challenging problems, problems related to the application of chemistry in everyday life, and in the trials or experiments and scientific methods related to chemistry.This is consistent with the statement of Mulyasa (2009), that chemistry is one of the Natural Sciences that closely matches the trials or experiments, as well as other scientific methods, that can provide experiences for learners to perform hypothesis testing.This can be achieved by designing experiments through the installation of instruments, retrieval, processing, interpretation of data, and presenting the results of experiments both orally and in writing.This statement shows that in studying chemistry, it requires a degree of creativity or creative thinking ability of the students.Therefore, creative thinking abilities can be developed through the study of chemistry, and it is one of the responsibilities of chemistry teachers to develop the creative thinking skills of learners.
In general, chemistry teachers, including those in Takalar, South Sulawesi Province, Indonesia, have made various efforts to develop the creative thinking skills of learners; for example, by using a variety of models and instructional media, such as problem-based learning and animated media in their teaching of chemistry.However, the expected outcome has not yet been maximized.This statement is supported by data from one of the high schools in Takalar which shows that the average value of creative thinking abilities of students in hydrocarbon material is 44.41.
Based on the aforementioned state, it should be noted that efforts made by teachers would be meaningless if the learners themselves, as the subject of study, do not involve themselves or do not actively participate in the learning process.Therefore, studies are needed as to the related variables that contribute to the creative thinking ability of students.
Research by Groncher, Johri, Kothaneth, and Lohani (2009) showed that prior ability contributed to the ability of learners to make new engineering design solutions.Prior ability can be defined as the ability possessed by learners who are used to facilitating the acquisition, organization, and rephrasing of new knowledge (Sanjaya, 2012).However, prior ability of each learner is potentially different.These differences affect how they are presented, interpreted, and managed.The differences in processing and integrating new information can influence remembering, thinking, applying, and creating new knowledge (Yaumi, 2013).Therefore, prior ability affects the capability of learners in thinking, and it can be said that prior ability determines the creative thinking ability of students (Semiawan, Putrawan and Setiawan, 2004;Sugiyono, 2009Sugiyono, , 2011;;Suharnan, 2005;Uno, 2012;Uyanto, 2009;Walgito, 2004;Widyastono, 2009).EDUPIJ • Volume 6 • Issue 3 • 2017 Anwar and Rasool (2012) argue that everyone has differences in their creativity, background, motivation, ability, and also differences in their response.Because of these reasons, Anwar and Rasool (2012) conducted a study comparing the creative thinking ability of students of high and low achievers.The results showed no difference in the ability of creative thinking among learners of high and low achievers, but the students were all female and came from a town which has the ability to think creatively better.The results of the study by Groncher et al. (2009) and the opinion of Anwar and Rasool (2012) can be considered in examining the relationship between creative thinking ability and prior knowledge.
Methodology
Reviewed based on the data, this study employed the quantitative research method.
Based on this research, data retrieval is ex post facto research as this research is used to explain the existence of relationship of each variable.
The population in this research were a total of 1,537 science students attending the 11 th Grade of a State High School in Takalar Regency, South Sulawesi Province, Indonesia, during the 2013-2014 academic year.Sampling employed the purposive stratified random sampling technique.This sampling technique was chosen because the population is not homogeneous and stratified for several high schools in Takalar, and it divisible into highperforming and low-performing schools at different locations both within and outside the city.In order to research data more representative of the population, two schools within the city and two schools outside of the city were selected.
The Department of Education, Culture, Youth and Sports for Takalar advised that the schools located within the city are high-performing schools and schools outside of the city are low-performing schools.Thus, the sample selected for this study was SMAN 1 Takalar, SMAN 3 Takalar, SMAN 1 Polongbangkeng Selatan, and SMAN 1 Polongbangkeng Utara.The 11 th Grade Science 1 students from each school were selected as the sample.The reason for this sample selection is due to existing SMA Takalar determination of students based on their ability level.Thus, the number of samples in this study were 134 students.
The variable in this study is the students' prior ability and creative thinking ability in chemistry.Prior ability is the prerequisite knowledge possessed by students in order to learn the concepts of acid-base measured using a cognitive test that includes the abilities of remembering (C1), understanding (C2), and applying (C3).Creative thinking ability of students is the ability of learners in completing a verbal creativity test which is based on the cognitive dimension, and includes: fluency, and flexible, original and elaborative thinking.
The instrument applied in this study consists of prior ability tests, and tests of verbal creativity.The test for prior ability was arranged in the form of multiple-choice questions which includes materials related to the acid-base concept such as the periodic system and atomic structure, reaction rate, and chemical equilibrium.The verbal creativity test was arranged as an essay test that consists of six sub-tests that assess based on the beginning of the word, arrangement of words, forming sentences of three words, use of the same properties, to various uses, and what the consequences are.Sub-tests are taken as the basis to determine the ability learners' creative thinking, and includes the ability for fluency, flexibility, original and elaborate thinking.A validity test was performed by testing the content validity and the empirical validity.EDUPIJ • Volume 6 • Issue 3 • 2017 Content Validity was tested by seeking the opinion of two experts with regard to the appropriateness of the indicators and the instruments' developed grains; whereas, for testing the empirical validity, one test was carried out with the instrument at one of the schools in the population that was not included in the study sample, and then factor analysis was conducted using product moment correlation.Reliability testing used the Cronbach Alpha formula.
After having tested the validity and reliability, it was noted that the prior ability tests used in the study consisted of 16 items and a Cronbach Alpha reliability coefficient of 0.883.Meanwhile, the verbal creativity test used 18 items and a Cronbach reliability coefficient of 0.808.
The data obtained in this study were analyzed using the technique of Pearson product moment correlation and regression analysis.The technique of the Pearson product moment correlation was used to test the hypotheses, and regression analysis is used to determine the contribution of prior ability on the ability of creative thinking in chemistry.
Description of student prior ability
The result of descriptive analysis for prior ability indicates that the prior ability of learners in Takalar have a minimum score of 6, a maximum score of 100, an average score of 53.5, median 50, mode 38, and a standard deviation of 26.7.The results of a descriptive analysis of the prior ability of students is presented in Table 1.The distribution of data frequency by category of prior ability low, middle, and high can be seen in Table 2.In Table 2, it appears that the highest frequency of prior ability is 71 learners in the middle category.Therefore, it can be concluded that in general, the prior ability of learners in Takalar is situated in the middle category.Description of the average value for each aspect of prior knowledge can be seen in Table 3.It can be seen that the understanding (C2) aspect has the highest score.More clearly, the average value for each aspect for each school can be seen in Figure 1.
Description of creative thinking ability of students
The results of the descriptive statistical analysis of students' creative thinking ability through verbal creativity tests indicate a minimum score of 16, a maximum score of 93, an average score of 48.6, median 42, mode 20, and a standard deviation of 23.9.The results of the analysis of students' creative thinking ability based on verbal creativity tests are presented in Table 4.
The frequency distribution of creative thinking ability through verbal creativity tests with categories of low, middle, and high can be seen in Table 5.It can be seen that the highest frequency of creative thinking skills through the verbal creativity test is 82, which is in the middle category.So it can be concluded that in general, the creative thinking ability of learners in Takalar is in the middle category.Description of the average value for each aspect of the verbal creative thinking abilities can be seen in Table 6.It can be seen that the fluency aspect of creative thinking has the highest average.More clearly, the average value for each aspect of the creative thinking abilities at each school can be seen in Figure 2.
Correlation between prior knowledge and creative thinking ability
From the data processing, the coefficient correlation between prior knowledge and creative thinking ability using the Pearson Product Moment technique is 0.619, with a significance value of 0.000 < α.It means that the presented hypotheses are accepted, or there is a relationship between prior knowledge and the creative thinking ability of students.
Regression analysis showed that the value of the variable constant prior ability and the creative thinking ability is 18.885 and the value of the regression coefficient is 0.556.Thus, the pattern of the relationship between prior ability and creative thinking ability can be expressed in the equation of the regression line: Ŷ = 18.885 + 0.556 X 1
Conclusion and Discussion
The result of analysis descriptive showed that the prior knowledge of Takalar learners was in the middle category.Results of this study illustrates that learners in Takalar have sufficient ability in terms of remembering, understanding, and applying their knowledge in order to gain further knowledge.
Based on the analysis of the average value of each aspect of prior knowledge, it can be seen that the understanding aspect has the highest average and the remembering aspect has the lowest average.This value indicates that learners in Takalar have the ability to EDUPIJ • Volume 6 • Issue 3 • 2017 understand more than the ability to remember.Of the four schools that participated in the study, the average value of achievement for each aspect of prior knowledge at highperforming schools located in the city is higher than that for low-performing schools located outside of the city.The result of descriptive analysis also showed that creative thinking ability of learners in Takalar is in the middle category, and that the fluency aspect of thinking has the highest average whilst the elaboration of thinking aspect has the lowest average.This means that learners in Takalar have fluency of thinking.Each school presented a different average value for each aspect of creative thinking ability, but the high-performing schools located in the city have higher average values than the low-performing schools outside of the city.This is because the high-performing schools located in the city had study groups and performed tutoring, while the low-performing schools located outside of the city offered no form of tutoring.Tutoring affects the ability of learners as they learn through the additional practice and further develop their prior knowledge.The result of this study corresponds to the results of the study by Anwar and Rasool (2012), who stated that students from city institutions had the ability to think more creatively than students from outside the city (Al-Hajjaj, 2010;Cameroon and Bryan, 1992;Mariati, 2006;Munandar, 2009;Prawiradilaga, 2009;Purwanto, 2011;Raehana, 2013).
The results of correlation analysis showed a relationship between the prior ability of students and their creative thinking ability.The coefficient correlation between these variables is 0.619.This value indicates that the direction of the relationship between prior knowledge and creative thinking ability is positive.It means that the learners who have higher prior knowledge have higher creative thinking ability.Conversely, learners who have lower prior knowledge have lower creative thinking ability.
The results of regression analysis indicate that the pattern of the relationship between the variables of prior knowledge and creative thinking ability is Y1 = 18.885 + 0.556 X1.The pattern of the regression equation provides the information that each unit of change in the score of prior ability leads to changes in creative thinking ability score by 0.556.In addition, the coefficient of determination (R square) between the two variables is 0.384.This value explains 38.4% variance of the creative thinking ability variable as explained by prior ability and 62.6% influenced by other variables.
The results of the analysis of the average value for each aspect of the initial capabilities and aspects of creative thinking abilities found that, at the beginning, prior knowledge variables and aspects of understanding had average values that were the highest among the other aspects.The creative thinking ability variable and the fluency aspect of thinking had the highest average value.In terms of these aspects, it can be said that there is a relationship between prior knowledge and verbal creative thinking abilities, as the understanding aspect in prior ability makes students more fluent in expressing words or phrases in verbal creativity testing.In summary, there is a relationship between prior knowledge and creative thinking ability in the learning of chemistry.
Notes
Corresponding author: ANSARI SALEH AHMAR
Figure 2 .
Figure 2. Average value for each aspect of verbal creative thinking abilities at each school
Table 1 .
Descriptive Statistic of Students Prior Ability
Table 3 .
Average Value of Each Aspect of Prior Ability Figure 1.Average value of each aspect of prior ability for each school
Table 4 .
Descriptive Statistics for Students' Creative Thinking Ability
Table 5 .
Frequency Distribution of Students' Creative Thinking Ability
Table 6 .
Average Value Aspects of Creative Thinking | 2018-12-01T00:50:06.614Z | 2017-09-01T00:00:00.000 | {
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226251732 | pes2o/s2orc | v3-fos-license | Coronavirus (SARS-CoV-2) Pandemic: Future Challenges for Dental Practitioners
In the context of the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) pandemic, the medical system has been subjected to many changes. Face-to-face treatments have been suspended for a period of time. After the lockdown, dentists have to be aware of the modalities to protect themselves and their patients in order not to get infected. Dental practitioners are potentially exposed to a high degree of contamination with SARS-CoV-2 while performing dental procedures that produce aerosols. It should also be noted that the airways, namely the oral cavity and nostrils, are the access pathways for SARS-CoV-2. In order to protect themselves and their patients, they have to use full personal protective equipment. Relevant data regarding this pandemic are under evaluation and are still under test. In this article, we made a synthesis about the way in which SARS-CoV-2 spreads, how to diagnose a novel corona virus infection, what the possible treatments are, and which protective personal equipment we can use to stop its spreading.
Introduction
In December 2019, an outbreak of pneumonia appeared in Wuhan City. Wuhan is an important international trading centre in central China. This pathology was concluded to be generated by a novel Coronavirus (nCoV-2019). Since then, the virus infection has spread throughout the world, it has been declared a pandemic by WHO on 12 March 2020 [1][2][3]. It seems that the first COVID-19 (coronavirus disease 2019) cases were connected to a large fish and living animal market in this large metropolis. It was thought that the path of direct transmission came from a food market. Since then, person-to-person transmission has been found be one of the main spreading mechanisms of COVID-19 [1][2][3].
After the identification of the initial cases, the pandemic hit almost all the nations in the world. Now, there are more than 1,113,307 deaths worldwide due to the coronavirus pandemic. The updated data of Johns Hopkins University identified 1,113,307 deaths. On the other hand, 39,964,414 contagions are global. COVID-19 has spread to 189 countries and territories and there are approximately 39,964,414 confirmed cases (as of 19 October 2020) [4].
The WHO (World Health Organization) presented the guidance for case management of COVID-19 in health facility and community Interim on 19 March 2020 [3]. The response interventions proposed by the WHO are presented in Figure 1. Because this pandemic emerged in our lives and has produced a lot of changes, dental professionals have to introduce new strategies to perform dental treatments in order to reduce the risk of cross infection. A study performed by a team of Jordanian dentists showed that dental practitioners have very little information regarding the measures they have to take in order to protect themselves and their patients [5]. In his study, Ing showed that 4% of deaths were dentists because of the lack of protection equipment [6].
In this article, we made a synthesis about the way in which SARS-CoV-2 spreads, how to diagnose a novel corona virus infection, what the possible treatments are, and which protective personal equipment we can use to stop its spreading. Because this pandemic emerged in our lives and has produced a lot of changes, dental professionals have to introduce new strategies to perform dental treatments in order to reduce the risk of cross infection. A study performed by a team of Jordanian dentists showed that dental practitioners have very little information regarding the measures they have to take in order to protect themselves and their patients [5]. In his study, Ing showed that 4% of deaths were dentists because of the lack of protection equipment [6].
Epidemiology
In this article, we made a synthesis about the way in which SARS-CoV-2 spreads, how to diagnose a novel corona virus infection, what the possible treatments are, and which protective personal equipment we can use to stop its spreading.
The first name given to this virus was 2019-nCoV, after a short period of time the name of the virus was changed due to the similarity with the SARS virus into SARS-CoV-2 [7]. The virus comes from the family of Coronaviridae and is made of single stranded RNA viruses [7]. This virus can be secluded from animal species and can determine cross infection, passing the barriers of certain species and infecting animals and humans. The virus has a cover that is composed of glycoproteins that look similar to a solar crown, as shown in Figure 2 [7]. In the literature, there are four genera of Coronaviruses. Two of the genera, γ-CoV and δ-CoV, determine changes in birds, while the other two genera, α-CoV and β-CoV, contaminate mostly mammals and also humans, by determining changes in different systems of the organism like the respiratory, gastrointestinal, and central nervous systems [7][8][9][10][11]. The new virus that determined infections in Wuhan belongs to the β-CoV family of viruses that includes the SARS-CoV (Severe Acute respiratory syndrome coronavirus) and MERS-CoV (Middle East respiratory syndrome), two viruses that are known for the infections they caused several years ago [8][9][10][11][12][13][14].
The nucleotide sequence similarity between SARS-CoV-2 and SARS-CoV is of about 80% and approximately 50% between SARS-CoV-2 and MERS-CoV. This could explain the reason why this novel virus is less deadly than the other two. Hence, its routes of transmission can spread the SARS-CoV-2 faster than the other two viruses [15][16][17][18][19][20]. It has been suggested that the natural host of SARS-CoV-2 may be the Rhinolophus affine bat, due to the similarities in the RNA (ribonucleic acid) sequence of the coronavirus found in the bat (96.2%) and SARS-CoV-2, and the intermediate host is the pangolin, with a genome sequence similarity of 99% between SARS-CoV-2 and the coronavirus found in these species [15][16][17][18][19][20].
As far as the intermediate guest is concerned, there are recent studies that contradict the hypothesis of appearance from the pangolin [21]. Some studies say that the first SARS-CoV-2 found in Wuhan's HU-1 patient, in early December 2019, was perfectly adapted to humans, that is, despite In the literature, there are four genera of Coronaviruses. Two of the genera, γ-CoV and δ-CoV, determine changes in birds, while the other two genera, α-CoV and β-CoV, contaminate mostly mammals and also humans, by determining changes in different systems of the organism like the respiratory, gastrointestinal, and central nervous systems [7][8][9][10][11]. The new virus that determined infections in Wuhan belongs to the β-CoV family of viruses that includes the SARS-CoV (Severe Acute respiratory syndrome coronavirus) and MERS-CoV (Middle East respiratory syndrome), two viruses that are known for the infections they caused several years ago [8][9][10][11][12][13][14].
The nucleotide sequence similarity between SARS-CoV-2 and SARS-CoV is of about 80% and approximately 50% between SARS-CoV-2 and MERS-CoV. This could explain the reason why this novel virus is less deadly than the other two. Hence, its routes of transmission can spread the SARS-CoV-2 faster than the other two viruses [15][16][17][18][19][20]. It has been suggested that the natural host of SARS-CoV-2 may be the Rhinolophus affine bat, due to the similarities in the RNA (ribonucleic acid) sequence of the coronavirus found in the bat (96.2%) and SARS-CoV-2, and the intermediate host is the pangolin, with a genome sequence similarity of 99% between SARS-CoV-2 and the coronavirus found in these species [15][16][17][18][19][20].
As far as the intermediate guest is concerned, there are recent studies that contradict the hypothesis of appearance from the pangolin [21]. Some studies say that the first SARS-CoV-2 found in Wuhan's HU-1 patient, in early December 2019, was perfectly adapted to humans, that is, despite being replicated several times in the following months, it did not undergo genomic transcription changes, remaining practically almost unchanged [17][18][19][20][21][22][23][24]. The bat is now considered to be the first source of the virus and the initial hypothesis of the infection from the pangolin that has been considered to be an intermediate host has been discarded [17]. Tang et al. suggested that the genomic sequence between the pangolin virus and the human one is not 99%, but lower (84%) than that the bat one (96%) [22].
Scientists are now taking into consideration the fact that the virus evolved and is infecting humans that were asymptomatic for three months, and in the same manner it increases the infectious capacity and reduces lethality [21]. This type of virus has the same structure as the common coronavirus, possessing the "spike protein" in the exterior structure of the envelope of the virion, and besides this, in its structure we can find proteins like nucleo, poly, and membrane proteins that include RNA polymerase, papain-like protease, helicase, 3-chymotrypsin-like protease, accessory proteins, and glycoprotein. The S protein from coronavirus can bind with the receptors of the host to encourage viral entry into target cells. Although there are four amino acid variations of S protein between SARS-CoV-2 and SARSCoV, the first can also bind with the human angiotensin converting enzyme 2 (ACE2), the same host receptor for the SARSCoV. SARS-CoV-2 cannot bind with cells without the presence of ACE2. The recombinant ACE2-Ig antibody, SARSCoV-specific human monoclonal antibody, and the serum from a convalescent SARS-CoV-infected patient can neutralize SARS-CoV-2, and thus confirms ACE2 as the host receptor for SARS-CoV-2. Due to the high affinity between ACE2 and SARS-CoV-2 S protein, it has been suggested that the population with a higher expression of ACE2 might be more susceptible to Coronavirus Disease 2019 (COVID-19) [17,18,[25][26][27].
Zhou et al. indicated that the angiotensin-converting enzyme II (ACE2) is likely the cell receptor of SARS-CoV-2, which is also the receptor for SARS-CoV. Moreover, it has been proven that SARS-CoV-2 does not use other coronavirus receptors such as aminopeptidase N and dipeptidyl peptidase 4 [44]. Xu et al. showed that the S-protein of the SARS-CoV-2 supports a strong interaction with human ACE2. Those findings suggest that the ACE2 plays an important role in cellular entry of the SARS-CoV-2, thus, ACE2-expressing cells may act as target cells for susceptible to SARS-CoV-2 infection. Moreover, this research has shown that the ACE2 could be expressed in the oral cavity, which was highly enriched in the oral epithelial cells, especially at a higher level in the tongue than in the buccal and gingival tissues. These findings indicate that the oral mucosa can express a potential high-risk route of SARS-CoV-2 infection transmission [45]. This fact underlines the importance of dentists and dental healthcare workers to wear all the protective measures that are indicated to prevent infection [18].
The connection between the host and the virus is encouraged by the S glycoprotein that integrates the receptors of the host cells to produce the viral infection of the cells. This glycoprotein is part of the class 1 viral fusion proteins and it has more than 1300 amino acids [46].
The SARS CoV 2 can also bind a specific enzyme in the human body that is represented by the angiotensin-converting enzyme 2 (ACE 2), in order to bind the virus to the cells which need this enzyme [47]. The way the entire process takes place is presented in Figure 3. . The connection of SARS-CoV2 to ACE2(figure inspired by https://www.thescientist.com/news-opinion/receptors-for-sars-cov-2-present-in-wide-variety-of-human-cells-67496 [48], and drawn by Giovanna Dipalma).
TMPRSS2(transmembrane protease serine 2) is an enzyme used by the SARS-CoV2 for S protein priming. CTSL2 (Cathepsin L2) is a gene, proteins encoded in this gene are members of the peptidase C1 family. For the SARS-CoV2 virus entering the human cells, Spike (S) protein needs to be cleaved by the cellular enzyme furin [49,50].
Furin is an enzyme, encoded by the FURIN gene, in the cells, belonging to hydrolases, splits proteins (inactive precursors) and transforms them into an active biological state (mature proteins) [44,45].
The S protein allows the virus to transfer the genome into the cell which leads to viral replication. In order to become active, the S protein must be cleaved by proteases. The S protein has two functional domains S1 and S2. S1 is implicated in the initial stage of viral entry, using its receptor binding domain to link to the receptors of the target cell and S2 acts in the second stage, fusing the cell and the viral membrane containing amino acid sequences necessary to continue the infiltration process [50][51][52][53][54].
In the corona virus family, the S protein has the largest variable amino acid sequence. In this virus the furin site is located between S1 and S2 subunits not unlike the pattern found in highpathogenic influenza viruses, but not in other members of the Beta coronavirus genus [50][51][52]. The connection of SARS-CoV2 to ACE2(figure inspired by https://www.the-scientist.com/ news-opinion/receptors-for-sars-cov-2-present-in-wide-variety-of-human-cells-67496 [48], and drawn by Giovanna Dipalma). TMPRSS2(transmembrane protease serine 2) is an enzyme used by the SARS-CoV2 for S protein priming. CTSL2 (Cathepsin L2) is a gene, proteins encoded in this gene are members of the peptidase C1 family. For the SARS-CoV2 virus entering the human cells, Spike (S) protein needs to be cleaved by the cellular enzyme furin [49,50].
Furin is an enzyme, encoded by the FURIN gene, in the cells, belonging to hydrolases, splits proteins (inactive precursors) and transforms them into an active biological state (mature proteins) [44,45].
The S protein allows the virus to transfer the genome into the cell which leads to viral replication. In order to become active, the S protein must be cleaved by proteases. The S protein has two functional domains S1 and S2. S1 is implicated in the initial stage of viral entry, using its receptor binding domain to link to the receptors of the target cell and S2 acts in the second stage, fusing the cell and the viral membrane containing amino acid sequences necessary to continue the infiltration process [50][51][52][53][54].
In the corona virus family, the S protein has the largest variable amino acid sequence. In this virus the furin site is located between S1 and S2 subunits not unlike the pattern found in high-pathogenic influenza viruses, but not in other members of the Beta coronavirus genus [50][51][52].
Several patches of the RBD (receptor binding domain) are similar in SARS-CoV and SARS-CoV-2, an overall amino acid sequence identity of 76.47%. Because of the five important interface amino acid residues, four of these are different in SARS-CoV-2, and its S protein has a significantly higher binding affinity to human ACE2 than SARS-CoV S protein. Regardless of this, the two viruses shared an almost identical 3-D structure of the RBD which demonstrates similar Van der Waals electrostatic properties [50][51][52][53][54].
As treatment options, the effective antibodies against SARS-CoV's spike S protein did not bind to the SARS-CoV-2 S protein. So, a vaccine sounds more tempting, but in order to create a live attenuated vaccine we must limit the replicative capabilities of the virus. In order to achieve this, we could remove the furin activation sequence which is essential for cell-virus fusion, thus allowing it to replicate. By doing so, the immune system can create antibodies in order to neutralise the virus and protect the body from further infections [50][51][52][53][54][55].
Another option would be to isolate antibodies from patients who have recovered from COVID-19 by using the novel S protein structure and mapping its structure in order to mass produce them [50].
In 2012 a new corona virus MERS-CoV (Middle East Respiratory Syndrome Coronavirus) was discovered resulting in a disease that manifests itself as severe respiratory disease with renal failure. The fatality rate was up to 38%. The place of emergencies was the Middle East, specifically countries where dromedary camels were identified as species harbouring the virus. Another outbreak was seen in 2015 in South Korea where the final tally was 36 people killed out of 186 confirmed cases. The SARS-CoV outbreak in southeast China had a worldwide tally of 774 killed from 8096 infected with a fatality rate of 9.6% [56].
Viral Load-Inflammation
The world virus comes from Latin and means "poison". Viruses are small microorganisms whose size varies from 0.2-0.3 µm to 1 µm. They need a host cell for living and reproducing (bacterial, vegetal, or animal). They have a very simple structure with an external cover of glycol proteins and lipids, called an envelope or pericapsid, in which there is a protective coat called capsid surrounding the virus genome. In the literature, there are DNA and RNA viruses, double stranded (DNA virus or dsRNA virus) or single (ssDNA virus or ssRNA) stranded. For the latter there is the "polarity" (consisting in process coding the virus) which can be positive or negative, namely ssDNA, ssRNA-, ssDNA+, or ssRNA+ (coronavirus) [57,58]. The cell replication (cytoplasmic or nuclear) is guided by the genome. The genome of the virus enters the host cell, and in few hours the formation of thousands of viral particles is performed, and they spread in the external environment. The replication of the RNA virus occurs easily with errors, as there is no RNA polymerase during the transcription. The high number of viruses as well as the error high frequency during the transcription are the main factors explaining the fast capacity to evolve proper of the SARS-CoV-2. Resistance to therapy is justified by the RNA mutation, even if it is very small, and allows the virus to avoid the attack of the immune system, continuing to change in terms of response in order to adapt to the constant changes of the genome [57,58].
Corona viruses are classified according to their own nature, structure, genome, and replication. The main feature of viruses is the infection of a special type of cell on which surface there are receptors which are similar to the binding. When binding with the host cell membrane is performed through those receptors, the virus penetrates the cell with its own genome, DNA or RNA. In this way. replication and multiplication of the virus starts. After the virus replication, the host cell usually dies. freeing new microorganisms in the surrounding environment where they can keep on infecting a new host cell having completed their lifecycle [59,60].
The Furin is considerably present in the lung tissue, in the intestine and the liver, this would make those organs as potential target of the 2019-nCoV infection. In dental field, Furin expression has been also revealed by the epithelium of the human tongue and in significant quantities in the squamous cell carcinoma. Researchers have shown a high availability of ACE2 receptors as well as the presence of Furin. Therefore, the tongue has a high risk of coronavirus infection and the SCC increases the risk in case of coronavirus exposure. The cleavage site on the spike similar to Furin plays an important role in spreading the 2019-nCoV virus [61][62][63][64][65][66][67].
At the moment there are some researches in which this site is eliminated, by observing effects or blocking the action of Furin, as issued on Nature [16]. This explains the strategic possibilities which we can sum up in this way:
The activation of TMPRSS2 (Trans Membrane Protease, Serine 2) is fundamental as SARS-CoV-2 infects the lung cells, SARS-CoV-2 can use the TMPRSS2 to trigger the S protein. Some studies highlight that the TMPRSS2 is an important element of the host cell as it is essential for spreading a great number of viruses causing potentially significant infections, as the influenza A and coronavirus. Important data show that the TMPRSS2 is not necessary for the development and homeostasis and so it is potentially and sensible pharmacological target able to inactivate the infection. It is important to underline that the serine protease inhibitor, camostat mesylate, blocks the TMPRSS2 activity. This treatment, or something similar with likely increased antiviral activity (Yamamoto et al., 2016), could be used for treating patients with SARS-CoV-2 infection. Further studies suggest that the activation mediated by Furin on the S1 / S2 site within the infected cells could activate the subsequent access depending on the TMPRSS2 within the target cells [69][70][71][72][73].
An analysis on the real proteolytic elaboration of the protease on the S protein, and on its cleavage in S1 and S2 through detection with the antigenic system, underlined the existence of a band corresponding to the subunit S2 and protein S of the host cells infected by the virus of the vesicular stomatitis (VSV) containing SARS-2-S [61][62][63][64][65][66][67].
In 2011, a study on macaques infected by coronavirus with severe lung infections has been taken into consideration as the saliva droplets were source of infection [81].
It has been confirmed that the epithelial cells of the salivary glands covering the salivary ducts had high ACE2 expression (Angiotensin-Converting Enzyme 2), and therefore the first target cells have been revealed together with the first production source of virus [46,49,50,[82][83][84][85].
The ACE2 expression in human organs has been analyzed by considering data collected by the portal Genotype-Tissue Expression. It is noted that the ACE2 expression in minor salivary glands was higher than the ones found in lungs. As a result, salivary glands are targets for SARS-CoV [82].
Another confirmation derives from the fact that the SARS-CoV-2 may be recorded in the saliva before lungs lesions appear. This explains the presence of asymptomatic infections. Therefore, it is possible to state that the salivary gland is not only the first access site for the SARS-CoV-2, but also one of the main reproduction sources, as it makes saliva highly infective and infecting [81][82][83]. Indeed, the high presence of corona virus SARS-CoV-2 in saliva of COVID19 patients reaches 91.7%, and from their saliva samples it is also possible to easily cultivate the virus in vivo [83][84][85][86][87][88][89].
There is a study analyzing the virus SARS-CoV-2 resistance to the internal surfaces and to the sun light. This study proved that the UV-C light (absent to the natural light) inactivates coronaviruses and that the UVB levels found in sun light may really inactivate the SARS-CoV-2 on surfaces, especially the dry virus on stainless steel specimens. This research provided the first evidence that sun light may quickly inactivate the SARS-CoV-2 on surfaces. Data suggest that the natural sun light may be also effective as a disinfectant for contaminated non-porous materials [90]. Researchers have also revealed that the simulated sun light is quickly able to inactivate the corona virus SARS-CoV-2 on specimens performed on stainless steel. The results of this study highlighted that 90% of the infecting virus was inactivated in a period of time consisting of 6.8 min in the saliva solution. The sun light necessary for those tests is similar to the summer solstice, in a not cloudy day. Researchers stated that the inactivation has been tested when the sun light levels were also lower [90,91].
Clinical Features
The article published by Doremalen has underlined chilblain, urticaria and tremor representing the new symptoms of the COVID-19 patient [92]. Moreover, some isolated cases were recorded, but only three cases in Madrid (two suspected and one confirmed) of herpetic-like vesicular lesions in the oral cavity with pain, desquamative gingivitis, and ulcers [93,94].
The involvement of the oral cavity in SARS-CoV-2 calls dentists to the front line once again in order to be able to perform an early clinical diagnosis.
The first SARS-CoV-2 found in the HU-1 patient of Wuhan, in the first days of December 2019, was perfectly adapted to the human being, as in the following months, despite the fact it reproduced itself trillions of times, the substantial and important mutation of genomic transcription did not occur, and the situation proceeded unchanged [19,20]. While for the SARS-CoVs there has been an increasing infectivity through meaningful mutations during the first months of the epidemic in the transmission from man to man, this is also the reason why scientists have had great difficulty in making a vaccine. Indeed, the SARS-CoV-2 has been a more stable virus than the SARS-CoVs since the beginning, which can also infect the first respiratory tracts, by encouraging the infection.
The affinity SARS-CoV-2 for its receptor in the host, ACE2, is 10-20 times higher than the SARS-CoV. Those two features make the SARS-CoV-2 much more infective than the SARS-CoVs. The SARS-CoV-2 virus has generally a reduced lethality but at the same time a higher risk of infection, it is more resistant to the survivors, while the SARS-CoVs virus is less contagious and very lethal, but it has been easily extinguished. Viruses with a lipid coating are generally more fragile. Thanks to its lipid coating, SARS-CoV-2 virus encourages the disactivating with the use of cleaning agents containing and all cleaning agents using hot water. However, in SARS-CoV-2, ORF8 and ORF3b proteins modulating antiviral and pro-inflammatory responses are very different from other similar SARS coronavirus, and this confers differences of pathogenicity and transmissibility. It has been shown that SARS-CoV-2 is extremely pathogenic, a powerful suppressor of the antiviral immunity, and it is also an activator of the pro-inflammatory response [95,96].
The cytokine storm syndrome leads to the interleukin release (IL) -6, IL-1, IL-12, and IL-18 together with the tumour necrosis factor alpha (TNF-α) and other inflammatory mediators. The increasing lung inflammatory response causes an increasing alveolar-capillary gas exchange, making hard the patients' oxygenation in severe patients. The collapse of lung walls and severe bilateral respiratory insufficiency occur, as well as organs lesions with severe functional deficits. Severe lymphopenia and eosinopenia cause a decay in antiviral immunity. The recommendation is early screening for inflammatory markers, Ferritin, c-reactive protein (CRP) and D-dimer. Helper cells of type 1 mediate the delayed inflammatory response causing IL-6 activation and other pro-inflammatory cytokines. If it is not treated, the inflammatory reaction may lead to severe lung lesions [97][98][99][100][101][102][103][104][105][106][107][108].
The key role of IL-10 is to modulate the hyper-inflammatory response as well as the regeneration of damaged tissues in COVID-19 affected patients. The use of IL-10 tends to finalize the block process of IL-6 by the Tocilizumab and avoid the formation of damaged interstitial lung tissues in fibrotic tissue [99].
IL-10 is one of the last potential biologic therapeutic agents. It is able to regulate the functions of lymphoid and myeloid cells, IL-10 has a great inflammatory activity both in vitro and in vivo.
IL-10 has been identified as a potential therapy for inflammatory diseases involving the helper 1 of type T (Th1) and the responses to macrophages. It is to be noted the occurrence of the secondary bacterial pneumonia during or immediately after the COVID-19 infection, determined by a complex interaction between the viruses, bacteria, and host [109].
The host remains more susceptible to bacterial infections even after several weeks from the elimination of the virus, this greater susceptibility is due to an increased viral virulence, indeed the infection increases the adherence and even for the following colonization with bacterial respiratory pathogenic agents. Bacteria adhere to the basal membrane after the interruption of the epithelial layer by air because of the cytopathic effect of the virus. This is caused by the greater adherence and it is due to the over-regulation of the receptors involved in the attack of bacteria. The SARS-CoV-2 alters the host's immune response to the successive bacterial solicitations by getting more sensitive to the bacterial components, as the staphylococcal enterotoxin B and LPS. Cytokines, such as IFN-γ, TNF-α, and IL-6, are synergistically upregulated by the staphylococcal enterotoxin B or LPS during influenza infections. These data show that the virus SARS-CoV-2 significantly modifies the full immune response to the bacterial infections in a singular and atypical way. The process through the virus modulates the full immune response to the lung bacterial infections must necessarily be focused upon [110].
In vivo, most part of the tissue's destruction is caused by an inflammatory response which is extreme and not-regulated, mainly neutrophil which, if not controlled, generates irreversible damage to tissues by decreasing the protective and regenerative dynamics [85,111,112].
Experiments on knockout deficient IL-10 mice spontaneously develop inflammatory syndromes as Irritable bowel syndrome (IBS). IL-10 in lungs was shown to be expressed by alveolar macrophages and stimulated by bacterial lipopolysaccharide (LPS), TNF-a. Regular bronchial epithelial cells produce anti-inflammatory cytokines interleukin-10, regulated by cystic fibrosis [97,118,119].
The average age of 2019-nCov infected patients is 55.5 years, while for mortality (case fatality rates-CFR), the age is 75 years, and it gets higher in the 80s age group. The number of deaths is higher present in the elder population with comorbidity and it enforces the key role that the Immune system played in the control of persistency of the SARS-CoV-2 virus. The decline of immunity occurs in the aging process, so the SARS-CoV-2 virus may access the respiratory tract in elder patients more easily [120,121].
Men are more affected than women (67%) as there are more smokers among men and because the woman immune system has a better antibody response linked to the X chromosome [122,123].
Some studies on the ongoing of infections of babies, according to some recent epidemiological data coming from Norway, Iceland, South Korea, and China, even if analyzed on different sized samples, all confirm the same infection rate, namely that babies represent 1-5% of the infected population, and most of them are asymptomatic or show a slight or moderate symptomatology that is higher in the male population. A big percentage of babies (90%) with severe evolution of the disease interests the age group of 0-2 years. The presumed less occurrence of infection on babies is linked to the structural and functional immaturity of the cellular receptor ACE2 by offering a less affinity to the virus spike. In contrast on what happens to infected adults, most of infected babies seems to have slighter clinical course. Frequent asymptomatic infections classify the infected babies as asymptomatic infected patients [125][126][127].
The early diagnosis together with prevention methods (social distancing, use of personal protective equipment, such as face masks and wash handing with alcohol solutions) are important to contain and contrast the 2019-nCoV spreading. The 2019-nCoV contagion mainly occurs through saliva droplets and nasal secretions, and tears in less measure in faeces, urine, sperm, and blood [26,67,[85][86][87]112].
Routes of Transmission for the SARS CoV 2
As dentists work in contact with patient, we have to know very well the possible paths of transmission of this virus. We already know that virus has several paths. First of all, there is the direct way from one person to another, by coughing, sneezing in the proximity of another subject, and by inhaling infected droplets (Flugge's droplets). Another way of transmission is by contact with oral and nasal mucosa or conjunctival mucosa after touching an infected surface, as presented in Figure 4. inhaling infected droplets (Flugge's droplets). Another way of transmission is by contact with oral and nasal mucosa or conjunctival mucosa after touching an infected surface, as presented in Figure 4. The way of contamination is by respiratory droplets that are considered to be particles that are made of water and have the ability to fall to the ground. They have a diameter bigger than 5μm. This type of droplets can be produced by breathing, talking, sneezing, coughing, or vomiting, or they can appear artificially being produced by aerosols generated during medical procedures, flushing toilets, touching of surfaces, or other domestic activities [128,129].
It is transmitted both by air, through the breath, and by direct contact with infected surfaces, including entrance via respiratory airways, mouth, nose, eyes. It has been shown that the virus enters through the eye conjunctiva, touching infected surfaces, and then bringing the virus to the face and mouth, as well as through the air conditioning that collects the virus from the air droplets produced by nebulization during dental operations). Hence, droplets containing the virus are collected by air conditioning and then are put back in the same environment, exiting the air conditioner at a minimum distance of eight meters, which is very dangerous compared to the two meters recommended for spacing [129][130][131].
Another route of transmission documented is through contact. All the personal items of the infected patient and the immediate environment can be considered as a potential medium for the transmission of the virus even by the indirect contact with those [9,132,133].
To check this hypothesis, a surgical mask was checked in order to determine the level of contamination and the results showed the presence of the virus on the outer layer even after a period of seven days [134].
Air Transmission
The virus does not travel in the outdoor air alone, but is conveyed through the droplets (Flügge's droplets) issued by a COVID-19 patient who, through sneezing or coughing, reaches over two meters away. Flügge's heavier droplets immediately fall to the ground due to the force of gravity and pollute the most contiguous surfaces to the infected patient, while the light ones, called aerosols, travel as in The way of contamination is by respiratory droplets that are considered to be particles that are made of water and have the ability to fall to the ground. They have a diameter bigger than 5 µm. This type of droplets can be produced by breathing, talking, sneezing, coughing, or vomiting, or they can appear artificially being produced by aerosols generated during medical procedures, flushing toilets, touching of surfaces, or other domestic activities [128,129].
It is transmitted both by air, through the breath, and by direct contact with infected surfaces, including entrance via respiratory airways, mouth, nose, eyes. It has been shown that the virus enters through the eye conjunctiva, touching infected surfaces, and then bringing the virus to the face and mouth, as well as through the air conditioning that collects the virus from the air droplets produced by nebulization during dental operations). Hence, droplets containing the virus are collected by air conditioning and then are put back in the same environment, exiting the air conditioner at a minimum distance of eight meters, which is very dangerous compared to the two meters recommended for spacing [129][130][131].
Another route of transmission documented is through contact. All the personal items of the infected patient and the immediate environment can be considered as a potential medium for the transmission of the virus even by the indirect contact with those [9,132,133].
To check this hypothesis, a surgical mask was checked in order to determine the level of contamination and the results showed the presence of the virus on the outer layer even after a period of seven days [134].
Air Transmission
The virus does not travel in the outdoor air alone, but is conveyed through the droplets (Flügge's droplets) issued by a COVID-19 patient who, through sneezing or coughing, reaches over two meters away. Flügge's heavier droplets immediately fall to the ground due to the force of gravity and pollute the most contiguous surfaces to the infected patient, while the light ones, called aerosols, travel as in a gas cloud for which they are certainly transported further away (even several meters) and for more time [135,136].
SARS-CoV-2 Diffusion in Hospital
The risk of infection from SARS-CoV-2 can be found in any enclosed environment in which there are symptomatic patients, the hospital is a high-risk environment, as well as dental practices where water-cooled rotary and ultrasonic instruments are used. In the hospitals, a lot of manoeuvres and procedures that generate small droplets are performed: such as endotracheal intubation, open aspiration, administration of nebulized treatment, manual ventilation before intubation bronchoscopy, open aspiration, administration of nebulized treatment, disconnection of the patient from the ventilator, ventilation non-invasive positive pressure, tracheostomy and cardio-pulmonary resuscitation.
In environments where many patients are undergoing mechanical ventilation, constant air change becomes fundamental as the rooms become saturated with infected air. For this reason, patients hospitalized in intensive care or who are under ventilation must be kept in rooms with negative pressure. In a closed environment, there is certainly the air spread in the presence of the virus and the risk of contagion certainly enhances for people present in that closed environment. For this reason, constant air exchange is essential. An article published by Ong et al. concerns the search for sites of infection in closed environments [9,136,137].
As studies have shown, regarding the survival rate of SARS-CoV-2 on different surfaces, the management of medical waste can be considered as a crucial factor in order to control the spread of the infection. How we manage the medical waste should consider a manner that has to include various departments and individuals that have to collaborate in order to have specific protocols for every unit regarding SARS-CoV-2 [138].
Diagnostics
The specific symptoms identified are fever, dry cough and also other types of symptoms were identified like fatigue, anosmia, difficulty in breathing, loss of taste, muscle pain, confusion, headache, sore throat, diarrhoea, sweating, and chill, as shown in Figure 5. a gas cloud for which they are certainly transported further away (even several meters) and for more time [135,136].
SARS-CoV-2 Diffusion in Hospital
The risk of infection from SARS-CoV-2 can be found in any enclosed environment in which there are symptomatic patients, the hospital is a high-risk environment, as well as dental practices where water-cooled rotary and ultrasonic instruments are used. In the hospitals, a lot of manoeuvres and procedures that generate small droplets are performed: such as endotracheal intubation, open aspiration, administration of nebulized treatment, manual ventilation before intubation bronchoscopy, open aspiration, administration of nebulized treatment, disconnection of the patient from the ventilator, ventilation non-invasive positive pressure, tracheostomy and cardio-pulmonary resuscitation.
In environments where many patients are undergoing mechanical ventilation, constant air change becomes fundamental as the rooms become saturated with infected air. For this reason, patients hospitalized in intensive care or who are under ventilation must be kept in rooms with negative pressure. In a closed environment, there is certainly the air spread in the presence of the virus and the risk of contagion certainly enhances for people present in that closed environment. For this reason, constant air exchange is essential. An article published by Ong et al. concerns the search for sites of infection in closed environments [9,136,137].
As studies have shown, regarding the survival rate of SARS-CoV-2 on different surfaces, the management of medical waste can be considered as a crucial factor in order to control the spread of the infection. How we manage the medical waste should consider a manner that has to include various departments and individuals that have to collaborate in order to have specific protocols for every unit regarding SARS-CoV-2 [138].
Diagnostics
The specific symptoms identified are fever, dry cough and also other types of symptoms were identified like fatigue, anosmia, difficulty in breathing, loss of taste, muscle pain, confusion, headache, sore throat, diarrhoea, sweating, and chill, as shown in Figure 5. The diagnosis of COVID-19 infection is based on methods of investigation such as the epidemiological data, clinical symptoms, computer tomography (CT) imaging, laboratory tests, and a history of travel to epidemic regions. The detection of the viral RNA can be confirmed via the re-verse transcriptase polymerase chain reaction (RT-PCR) test of upper or lower respiratory specimens or serum specimens. However, the false negative results of this test are not an indicator signaling the patient is virus free. Therefore, the most significant diagnosis tool is chest CT, as it shows a pure ground-glass opacity with consolidation in the posterior and peripheral lungs, which are signs of COVID-19 pneumonia. The chest CT is considered to be a more precise diagnosis tool that RT-PCR, regarding the sensitivity and the early detection of the presence of COVID-19 [139].
The aspects concerning the treatment are controversial and the randomized controlled trials did not validate any specific anti-SARS-CoV-2 treatment or vaccine. The management of the infected patients has been palliative until now, addressing the symptoms, and stabilizing patients 'critical conditions towards full recovery. With the universal approaches toward COVID-19, including the identification of the source that determined the infection, measures taken in order to prevent the cross-infection, and the evolving diagnostic measures, more hospitals are receiving infected patients [140].
For dental professionals, the way they perform classic treatments has changed, and they have to adapt to new strategies in order to be able to perform dental treatments. For this reason, the dentist, before welcoming the patient into his office, must perform a telephone triage. During this triage, the patient is asked several questions as in this way the dentist is able to know if the patient has any specific COVID-19 symptoms, and then it is vital to know if the patient has travelled abroad, if he has been in contact with people that travelled abroad or if he has been submitted to a diagnostic buffer test for SARS-CoV-2. This telephone screening directs the practitioner for further, and if possible, greater precautions before the patient arrives at the dental office to perform the intended treatment [17].
In order to protect the patients needing dental care, we can use instruments that do not generate aerosols while performing treatments. For this reason, the use of lasers can be taken into consideration to replace the conventional use of hand pieces generating aerosols [141][142][143][144].
Until now the best technique for the clinical diagnosis of SARS-CoV-2 is based on the virus detection through a nasopharynx swab and sputum specimens and salivary tests, a technique established by the real time PCR and confirmed by the next-generation sequencing. This last method is useful to find future mutations of the SARS-CoV-2, but difficult to perform. The accurate diagnosis affected by SARS-CoV-2 is important to stop the global COVID-19 pandemic. However, the current diagnosis tests based on the RT-PCR are not so reliable (70%) as they are often not able to find several infected cases and must be performed in specialized laboratories. The delay in diagnosis has encouraged disease spreading. The diagnosis may be confirmed by a combination of clinical, epidemiological elements, as well as chest CAT, even if a negative RT-PCR may not exclude a SARS-CoV-2 infection [145][146][147].
The "Aldo MORO" University of Bari and Pham Chau Trinh University, Danang City (VIETNAM) of Vietnam have filled a license application for a diagnostic procedure for the multiple detection of pandemic Corona Virus. The test is based on the reverse transcriptase polymerase chain reaction RT-PCR, but for the RNA detachment of more viruses of the corona family, such as SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV by analysing respiratory tract samples (nasopharynx and oropharynx swabs) and lung sputum sampled by patients suspected of having COVID-19. As a result, there is a reliability of 99.9% in a very short time (less than 3 h) and it may be performed in any laboratory employing the RT-PCR technique. Usually, during the acute phase, the SARS-CoV-2 virus RNA is detectable in swabs. Positive or negative results constitute a response about the SARS-CoV-2 infection by referring to the clinical condition or asymptomatic patient. With a positive result, this technique does not exclude a combined bacterial infection or in association with other viruses belonging to the Coronavirus family. In this case, the disease cause is due to the result of the pathogen identified [148].
Mathur and Mathur suggest the use of antibodies as a screening test. Antibodies known as immunoglobins are searched for in blood as they mark the immune response to SARS-CoV-2 infection.
In their opinion, these tests are indicated as screenings in areas with high prevalence, whereas in low prevalence areas, a high specificity test must be used [149].
Regarding the serological diagnostic performance, Zainol affirmed the evaluation of serology tests is not quite straightforward, because compared to PCR tests, these ones are capable of detecting the viral nucleic acid compared to the serological tests that are capable only to detect antibodies or host response to the infection [150].
The clinical assessment of the disease is classified in mild forms that present just mild symptoms, with no clinical signs of pneumonia, and the second stage is represented by the moderate form that presents respiratory symptoms accompanied by fever and with the presence of pneumonia, and the last type is represented by severe or very severe form [151].
Personal Protective Equipment
The dentists, but also the dental hygienists and dental assistants are potentially exposed to high degree of contamination with SARS-CoV-2 virus because they perform treatments that generate aerosols by using different devices such as turbines, micromotors and ultrasounds. It is very important to remember that the airways-oral cavity, nostrils, and eye conjunctiva are the access routes of SARS-CoV-2.
Moreover, the salivary gland duct epithelium as well as the respiratory tract cells highly represent ACE2 receptor cells. Hence, while working in close proximity with the patient's oral cavity, GDP is the most likely to contract the virus. In order to avoid this, extra caution and PPEs should be implemented [152,153].
All the professionals during this period have to take protective measures in order not to infect themselves and also their patients. For that the suggested equipment has to cover all the body and also the nose, eyes, and mouth. The protection for the mouth is considered to be the mask. In Figure 6, we present different types of masks in order to protect healthcare professionals [152,[154][155][156][157]. Mathur and Mathur suggest the use of antibodies as a screening test. Antibodies known as immunoglobins are searched for in blood as they mark the immune response to SARS-CoV-2 infection. In their opinion, these tests are indicated as screenings in areas with high prevalence, whereas in low prevalence areas, a high specificity test must be used [149].
Regarding the serological diagnostic performance, Zainol affirmed the evaluation of serology tests is not quite straightforward, because compared to PCR tests, these ones are capable of detecting the viral nucleic acid compared to the serological tests that are capable only to detect antibodies or host response to the infection [150].
The clinical assessment of the disease is classified in mild forms that present just mild symptoms, with no clinical signs of pneumonia, and the second stage is represented by the moderate form that presents respiratory symptoms accompanied by fever and with the presence of pneumonia, and the last type is represented by severe or very severe form [151].
Personal Protective Equipment
The dentists, but also the dental hygienists and dental assistants are potentially exposed to high degree of contamination with SARS-CoV-2 virus because they perform treatments that generate aerosols by using different devices such as turbines, micromotors and ultrasounds. It is very important to remember that the airways-oral cavity, nostrils, and eye conjunctiva are the access routes of SARS-CoV-2.
Moreover, the salivary gland duct epithelium as well as the respiratory tract cells highly represent ACE2 receptor cells. Hence, while working in close proximity with the patient's oral cavity, GDP is the most likely to contract the virus. In order to avoid this, extra caution and PPEs should be implemented [152,153].
All the professionals during this period have to take protective measures in order not to infect themselves and also their patients. For that the suggested equipment has to cover all the body and also the nose, eyes, and mouth. The protection for the mouth is considered to be the mask. In Figure 6, we present different types of masks in order to protect healthcare professionals [152,[154][155][156][157]. The protective equipment that is recommended by the World Health Organization (WHO) [158] includes the following: The protective equipment that is recommended by the World Health Organization (WHO) [158] includes the following:
1.
Respiratory protection: N95 is highly efficient, while filtering masks with FFP2 (filtering face piece 2) or FFP3 (filtering face piece 3) are standard. Protective masks vary according to the production region; appropriate markings specification should be used for the assessment of the facemask. An orientation on the filtration potency of different masks is recommended by Domminiak et al. as shown in Figure 6: Figure 7 [152,158,159].
3.
Body protection such as waterproof, long-sleeved medical gowns and disposable head caps.
Microorganisms 2020, 8, 1704 14 of 33 1. Respiratory protection: N95 is highly efficient, while filtering masks with FFP2 (filtering face piece 2) or FFP3 (filtering face piece 3) are standard. Protective masks vary according to the production region; appropriate markings specification should be used for the assessment of the facemask. An orientation on the filtration potency of different masks is recommended by Domminiak et al. as shown in Figure 6: • FFP1 and P1: at least 80% filtration of all particles that are 0.3 μm in diameter or larger • FFP2 and P2: at least 94% filtration of all particles that are 0.3 μm in diameter or larger • N95: at least 95% filtration of all particles that are 0.3 μm in diameter or larger • N99 and FFP3: at least 99% filtration of all particles that are 0.023 μm in diameter or larger. • P3: at least 99.95% filtration of all particles that are 0.3 μm in diameter or larger • N100: at least 99.97% filtration of all particles that are 0.02 μm in diameter or larger 2. Eye protection, for example glasses and face shield as in Figure 7 [152,158,159]. 3. Body protection such as waterproof, long-sleeved medical gowns and disposable head caps. 4. Hand protection such as sterile surgical gloves that should cover the cuffs of the gown [152,[158][159][160][161][162].
The FFP2 mask is one hundred times more effective, as a protective shield than surgical mask [153]. The protective equipment is represented in Figure 8 and the ways to protect ourselves from infection are presented in Figure 9.
A study performed by Scarano showed that the N95 masks produce a rise in the skin temperature and also discomfort while using them [163]. The FFP2 mask is one hundred times more effective, as a protective shield than surgical mask [153]. The protective equipment is represented in Figure 8 and the ways to protect ourselves from infection are presented in Figure 9. There are specific dental emergencies, according to the ADA, which "are potentially life threatening and require immediate treatment to stop ongoing tissue bleeding or to alleviate severe pain or infection." Those conditions are uncontrolled bleeding, cellulitis, or a diffuse soft tissue
2.1.2.
Routine dental cleaning and other preventive therapies.
Aesthetic dental procedures [154]. The air that is in the treatment room has a very high potential transmission pathway of the virus, if there is no adequate protocol for ventilation. The need to sanitize the environment between one patient and another one should be highlighted as ECDC recommends. For this reason, it is necessary to have rapid and effective sanitizing devices against bacteria and viruses. There are a great number of sanitizing devices, but many of them have limitations caused by the substances that are used for A study performed by Scarano showed that the N95 masks produce a rise in the skin temperature and also discomfort while using them [163].
There are specific dental emergencies, according to the ADA, which "are potentially life threatening and require immediate treatment to stop ongoing tissue bleeding or to alleviate severe pain or infection." Those conditions are uncontrolled bleeding, cellulitis, or a diffuse soft tissue infection with intraoral or extraoral swelling, which can potentially compromise the patient's airway, as well as facial trauma [154].
The ADA [154] has added, as part of the emergency guidance, that urgent dental care should "focus on the management of conditions that requires immediate attention to relieve severe pain and/or risk of infection, in order to alleviate the burden on hospital emergency departments." The conditions requiring dental treatments and should be managed as minimally invasive as possible, are as follows:
The air that is in the treatment room has a very high potential transmission pathway of the virus, if there is no adequate protocol for ventilation. The need to sanitize the environment between one patient and another one should be highlighted as ECDC recommends. For this reason, it is necessary to have rapid and effective sanitizing devices against bacteria and viruses. There are a great number of sanitizing devices, but many of them have limitations caused by the substances that are used for room sanitization. In fact, machines that use Ozone, chemicals, UV rays, or vaporized hydrogen peroxide involve some limitations, such as oxidation of the electronic boards and large sanitization times. These sanitizers described above necessarily involve a work break between one patient and another one of about an hour. For this reason, dental activity would have seriously slowed due to those long breaks, also causing economic damage. From a research of the devices, we discovered that only the machines that sanitize in the presence of operators and patients, during work with a higher standard HEPA filter inserted in sanitization devices, allow for operation in extreme safety without pauses between one patient and the next [152,[154][155][156][157]. Such air sanitizing systems include those used with NASA space technology and certified with the ActivePure ®® name (registered trademark) Beyond (https://www.activepure.com/italy-home/, https://beyond.vithagroup.eu/). This device was created by NASA for spacecraft, transferred for terrestrial use by Aerus, tested in university laboratories, and is validated by all US certification bodies and by the Ministry of Health in Italy as a medical device. This device is shown in Figure 10. room sanitization. In fact, machines that use Ozone, chemicals, UV rays, or vaporized hydrogen peroxide involve some limitations, such as oxidation of the electronic boards and large sanitization times. These sanitizers described above necessarily involve a work break between one patient and another one of about an hour. For this reason, dental activity would have seriously slowed due to those long breaks, also causing economic damage. From a research of the devices, we discovered that only the machines that sanitize in the presence of operators and patients, during work with a higher standard HEPA filter inserted in sanitization devices, allow for operation in extreme safety without pauses between one patient and the next [152,[154][155][156][157]. Such air sanitizing systems include those used with NASA space technology and certified with the ActivePure ®® name (registered trademark) Beyond (https://www.activepure.com/italy-home/, https://beyond.vithagroup.eu/). This device was created by NASA for spacecraft, transferred for terrestrial use by Aerus, tested in university laboratories, and is validated by all US certification bodies and by the Ministry of Health in Italy as a medical device. This device is shown in Figure 10. In order to reduce the presence of aerosols, dental practitioners may use the double surgical suction in order to remove all the water from the oral cavity. On the market, there are also additional external suction devices to the dental chair, this is a third powerful suction transportable from one dental chair to another and additional to the two surgical aspirators already existent as represented in Figure 11. In order to reduce the presence of aerosols, dental practitioners may use the double surgical suction in order to remove all the water from the oral cavity. On the market, there are also additional external suction devices to the dental chair, this is a third powerful suction transportable from one dental chair to another and additional to the two surgical aspirators already existent as represented in Figure 11. It has been published that the use of a solution that contains oxidative agents, such as 1% hydrogen peroxide or 0.2% povidone-iodine, as an antiseptic mouth rinse that should be used at the beginning of every dental treatment, is considered to help decrease the bacterial loading and also the viral one (SARS-CoV-2) in the saliva [17,152,164].
The Resistance of the Virus on Surfaces
The SARS-CoV-2 can survive up to three days on inanimate surfaces at room temperature, with a higher affinity for humid environments. In this context, all the staff at the dental practice need to ensure that all the inanimate surfaces should be disinfected with chemical detergents, which are recently approved for SARS-CoV-2, and a dry environment should be maintained to prevent the spread of SARS-CoV-2 as represented in Figure 12 [152,[162][163][164][165][166][167]. It has been published that the use of a solution that contains oxidative agents, such as 1% hydrogen peroxide or 0.2% povidone-iodine, as an antiseptic mouth rinse that should be used at the beginning of every dental treatment, is considered to help decrease the bacterial loading and also the viral one (SARS-CoV-2) in the saliva [17,152,164].
The Resistance of the Virus on Surfaces
The SARS-CoV-2 can survive up to three days on inanimate surfaces at room temperature, with a higher affinity for humid environments. In this context, all the staff at the dental practice need to ensure that all the inanimate surfaces should be disinfected with chemical detergents, which are recently approved for SARS-CoV-2, and a dry environment should be maintained to prevent the spread of SARS-CoV-2 as represented in Figure 12 [152,[162][163][164][165][166][167]. There is preliminary evidence that environmentally mediated transmission may be possible, in particular, that COVID-19 patients could acquire the virus through contact with abiotic surfaces [92].
Disinfectants
The disinfection of the surfaces is very important and different chemicals can be used in order to perform the surface and equipment measures of asepsis. The chemicals that are recognized to perform this action are: Sodium hypochlorite, the phenolic compounds, disinfectants based on water or a combination of water and ortho-phenylphenol, tertiary amylphenol, or another type of chlorophenol, the O-benzyl, disinfectants at base of ethyl Alcohol, or isopropyl alcohol combined with ortho-phenylphenol or tertiary amylphenol, as well as disinfectants ethanol iodine complex based [69,168].
Sterilization Procedures and Methods of Medical Devices
For the sterilization of dental instruments, the known protocols have to be followed but with more caution and performed with autoclaves. Among the most used methods for the sterilization in health field, there are: • With saturated steam. • With ethylene oxide. • With Hydrogen peroxide.
Sterilisation with Saturated Steam
This is a technique using the fluent steam or saturated (autoclave). It eliminates microorganisms through the denaturation of their proteins and other biomolecules. Sterilisation through autoclave is the most widespread being inexpensive and non-toxic, as well as thanks to its penetrating capacity. Sterilisation temperature (T) is of 1134 °C to the P of 2.1 bar. The time of minimal exposure of devices There is preliminary evidence that environmentally mediated transmission may be possible, in particular, that COVID-19 patients could acquire the virus through contact with abiotic surfaces [92].
Disinfectants
The disinfection of the surfaces is very important and different chemicals can be used in order to perform the surface and equipment measures of asepsis. The chemicals that are recognized to perform this action are: Sodium hypochlorite, the phenolic compounds, disinfectants based on water or a combination of water and ortho-phenylphenol, tertiary amylphenol, or another type of chlorophenol, the O-benzyl, disinfectants at base of ethyl Alcohol, or isopropyl alcohol combined with ortho-phenylphenol or tertiary amylphenol, as well as disinfectants ethanol iodine complex based [69,168].
Sterilization Procedures and Methods of Medical Devices
For the sterilization of dental instruments, the known protocols have to be followed but with more caution and performed with autoclaves. Among the most used methods for the sterilization in health field, there are: With Hydrogen peroxide.
Sterilisation with Saturated Steam
This is a technique using the fluent steam or saturated (autoclave). It eliminates microorganisms through the denaturation of their proteins and other biomolecules. Sterilisation through autoclave is the most widespread being inexpensive and non-toxic, as well as thanks to its penetrating capacity.
Sterilisation temperature (T) is of 1134 • C to the P of 2.1 bar. The time of minimal exposure of devices is from 5 to 7 min or 121 • C at P 1.1 bar. Time for this cycle (also defined autoclaving cycle) is from 15 to 20 min [69,168,169].
Sterilisation with Chemical Means
The only chemical means still in use to sterilise is Ethylene oxide or ethoxide (EtO). It is used above all in hospitals because of its dangerousness. Indeed, it is an explosive gas and inflammable. The ethoxide has the feature of impregnate for long time the treated objects; in order to avoid body damage, before using these objects it is important to store them in ventilated areas or cabinets up to their complete sterilisation. Its action process is due to the alkylation, (that is to say the replacement of a hydrogen atom with an alkyl group) of sulfhydryl, amine, carboxylic, phenol, and hydroxyl groups and spore and vegetative cells. This process leads to the microorganism death. The contraindications of this method are: Toxicity.
•
Long time for sterilisation and ventilation. • It may be installed in an appropriate premise.
•
Staff must have a license for toxic gas manipulation.
•
It is reserved to sterilizable materials meeting the requirements in compatibility (modification of physical kind/levels of residual gas). It is not possible to perform the re-sterilisation of materials previously processed with gamma ray. Those limitations have led hospitals to an external running of the ETO sterilisation.
Another chemical mean used is the peracetic acid. The formaldehyde has been adopted in the past as chemical sterilising, but its use has been strongly limited by the law, having shown indications of carcinogenicity [69,168,169].
Treatment Ways Performed until Now for the SARS-CoV 2 Infection
The management of severe pneumonia determined by the SARS-CoV-2 is very challenging to all the medical teams and intensivists, due to lack of data, as it is a novel virus. Equally, it is very challenging for the infected patient, due to powerful medication protocol, which can cause burden on many organs, in terms of metabolism and excretion along with a series of side effects. At the present, until a vaccine can be procured, prevention always is the best approach for the COVID-19 pandemic [170,171].
Scientists, researchers, healthcare workers, and shareholders are investing all their efforts to produce a vaccine against SARS-CoV-2 to end this pandemic crisis. The current investigations are based on the immunological responses of T and B cells of infected subjects with SARS-COV, and compare the similarities between this virus and SARS-CoV-2. On this note, due to the genetic differences, only 23% and 16% of the known SARS-CoV T cell and B cell epitope of B cells mapping are identical to SARS-CoV-2 [172].
Several studies were conducted on different epitopes, in which major histocompatibility complex (MHC) class I and II epitopes are one of them. However, further experimental studies (T cell and B cell assays) are required to determine the potential of the identified epitopes to induce a positive immune response against SARS-CoV-2. This would help to further refine the reported epitope set, which is based on observed immunogenicity [173,174].
It is important to note that there is a lack of knowledge on the epidemiological and the biological properties, in view of vaccine production of the SARS-CoV-2 at the present time. There is also a lack of information on specific immune responses, against SARS-CoV-2 at this early stage, which represents a challenge towards vaccine development [172].
Searching for effective therapies for SARS-CoV-2 infection is a complex process. The disease can manifest, as upper respiratory tract disease and lower respiratory tract disease, which can be further subclassified as mild, severe, or critical, where patients should receive a support care to alleviate the symptoms and maintain the function of the vital organs in severe cases.
Great collaborative efforts are performed to discover and evaluate effectiveness of antivirals (remdesivir), immunotherapies (hydroxychloroquine, sarilumab), monoclonal antibodies and vaccines, which have rapidly emerged. However, these therapies have not been probably tested and the long-term effects and safety are still unknown [175]. Moreover, the demand for unproven therapies can cause shortages of medications that are approved and indicated for other diseases. Thereby, patients who depend on these medications for chronic conditions have been abandoned without effective therapies.
The association of virus with cytokine storm syndrome (CSS) is a fundamental challenge in its management, due to the severe consequences. The CSS is characterized by the release of interleukin (IL)-6, IL-1, IL-12 and IL-18, along with tumour necrosis factor alpha (TNF-α) and other inflammatory mediators. The increased pulmonary inflammatory response may result increasing in the alveolar-capillary gas exchange, resulted in difficulty in patients' oxygenation with severe illness. Therefore, the recommendation is an early screening for inflammatory markers, ferritin, c-reactive protein (CRP), and D-dimer, and subsequently monitoring the trend of these markers. The goal is to initiate an early and aggressive immunosuppressive therapy. Initially, clinicians should rule out any other causes of an increased inflammation and deterioration of the clinical status such as: secondary infection, pulmonary embolism, or chronic obstructive pulmonary disorder (COPD) exacerbation. Patient with initial low ferritin level should not automatically be ruled out as a low risk for developing CSS, because low iron levels with or without anaemia can also cause reduced levels of ferritin. On the other hand, patient with high level of ferritin can be related to one of the following reasons: secondary infection, hepatitis B, hepatitis C, hemochromatosis, or other liver diseases [175].
This inflammatory response is a delayed response mediated by Type 1 T helper cells, causing an activation of IL-6 and other pro-inflammatory cytokines. On this note, the CSS progression can lead to acute lung injury if untreated.
Moreover, insignificant elevation of serum markers is not an indication for an immediate beginning of treatment with hydroxychloroquine and azithromycin, as this might lead to deleterious side effects. In this context, prior to any therapy, all patients should undergo laboratory evaluation for active tuberculosis disease or invasive fungal disease. Possible agents include IL-6 and IL-1 inhibitors or JAK inhibitors can be proposed, however, randomized clinical controlled trials to ensure the best treatment are not yet accessible. The treatment for severe pneumonia induced by SARS-CoV-2 is very challenging for both clinicians and patients, due to lack of data and the potent medications associated with the metabolism and excretion processes [175].
Some clinical trials have started in the UK, Germany, Switzerland, and USA, at the present time, toward vaccine development. There will not be such thing as a best treatment for COVID-19 until a vaccine is produced.
Currently, many drugs have been tried and used in the treatment of COVID-19 worldwide, but whether or not they are effective is subject to scientific evidence. However, these drugs are useful in a wide range of medical domains, especially in treating other viral infections effectively. These drugs are Chloroquine phosphate and Hydroxychloroquine sulphate, having a common mechanism of action, but caution is recommended. Although Hydroxychloroquine appears to have a higher efficiency. The viral entry can be blocked by inhibiting the glycosylation host receptors, proteolytic processing, and endosomal acidification, as well as by additional immunomodulatory effects inhibiting the cytokine production, autophagy, and lysosomal activity in host cells [176,177]. The association between hydroxychloroquine and azithromycin is showing tantalizing superior viral clearance infection, compared to the hydroxychloroquine monotherapy [108,178]. The general consensus is that an initial loading dose of 400 mg twice daily for one day, followed by 200 mg, twice daily for 5 days is an efficient dosage therapy. In terms of the adverse effects, both agents can cause prolongation of QT interval, which can lead to serious arrhythmia. Moreover, hypoglycaemia, neuropsychiatric effects and retinopathy are among the most frequent side effects. Notably, Azithromycin, as a monotherapy, can produce prolongation QT interval [171]. Hernandez et al. put in question the possibility of administrating hydroxychloroquine or chloroquine as a preventive treatment against COVID-19, but they concluded that the benefit harm ratio is too hard to asses [179].
Lopinavir/Ritonavir are drugs used for the treatment of HIV infection. They have shown to be a potential drug in treating novel coronavirus patients, via 3-chymotrypsin-like protease inhibition. After the endocytosis and uncoating of the virus, the single strand of RNA is translated into polypeptides, which undergo proteolysis to form non-structural proteins, and also these drugs act on the proteolysis link of this chain [180,181]. This medication has showed to have a significant efficiency, due to the multiple side effects (gastrointestinal intolerance, nausea, vomiting, diarrhoea, pancreatitis, hepatotoxicity, and cardiac conduction abnormalities). Nevertheless, for the other antiretrovirals, including protease inhibitors and integrase strand transfer inhibitors, further research consideration is required [171].
Ribavirin is a guanine analogue which inhibits the viral RNA-dependent RNA polymerase. This prevents the virus replication, which reduces viral load. Previous data regarding respiratory syncytial virus showed that inhaled administration of Ribavirin offers no benefit over enteral or intravenous administration. The adverse effects of this drug are hematologic (haemolytic anaemia) and liver toxicity. Ribavirin is also a known teratogen, and therefore it is contraindicated in pregnancy [182,183].
Remdesivir is a monophosphate prodrug, which undergoes metabolic process to an active C-adenosine nucleoside triphosphate analogue. At the present, this drug shows a promising potential for the treatment of COVID-19 due to its broad spectrum, potent in vitro activity against several CoVs [184].
Following its administration, reversible elevations of aspartate amino transferase/alanine amino transferase (ASAT/ ALAT) ratio were observed. The present posology is taken into consideration, as an initial loading dose of 200 mg and then 100 mg daily infusion. An initial dose is not indicated for patients with an estimated glomerular filtration rate less than 30 mL/min [171].
Favipiravir is a prodrug of a purine nucleotide, favipiravir ribofuranosyl-5 -triphosphate. The active form inhibits RNA polymerase; thus, it stops the virus replication. Evidence showed that an experience with influenza viruses and other corona viruses have for some time indicated different dosing variations. At this stage, the consensus is that doses at the higher end of the dosage range should be considered [185]. A loading dose is recommended (2400-3000 mg every 12 h × 2 doses) followed by a maintenance dose (1200 mg to 1800 mg every 12 h). The half-life of Favipiravir is approximately 5 h with mild adverse effects, however experience with higher doses is still limited [186].
As there is no proven specific therapy yet, an adjuvant therapy is still important and significant. In this category drugs to be considered are corticosteroids, anticytokine or immunomodulatory agents and immunoglobulin therapy [187].
Corticosteroids are used to reduce inflammation, especially in the lungs. However, it carries an increased risk of secondary infection and delayed healing from the virus infection. It is observed that corticosteroids do not necessarily improve the survival rate by extrapolating data from MERS, SARS as well as by other viral pneumonias, and also there is an increased risk to develop hyperglycaemia, psychosis and avascular necrosis, as well as, delayed viral clearance from the respiratory tract and blood. Interestingly, they are useful in bacterial infections and COPD exacerbation or refractory shock [171,188,189].
Anticytokine or immunomodulatory agents are based on the theory that they cause significant organ damage by an amplified immune response, therefore they are called "cytokine storm". One of the key players seems to be IL-6. Therefore, treatment options have targeted this interleukin when Tocilizumab is used, which is a monoclonal antibody receptor antagonist. This drug seems to be highly effective, but the small sample size and the lack of a comparative group limits our interpretation. Moreover, Sarilumab is another IL-6 receptor antagonist, which is being studied [190].
Immunoglobulin therapy is based on the use of convalescent plasma or hyperimmune immunoglobulins. The concept of this therapy is to obtain antibodies from patient who completely recovered from the virus. Wang et al. study used the same concept in relation to other viral infections (H1N1) and showed to be successful. The maximum efficiency of this therapy occurs in the first 7-10 days of the infection when the viral load is at its peak and the innate immune response has not struck [191]. The use of extracorporeal membrane oxygenation (ECMO) for patients with respiratory failure, due to acute respiratory distress syndrome (ARDS) caused by SARS-COV-2 infection, is suggested. It is important to note that the use of this advanced therapy needs specific equipment, trained clinicians, along with a good protocol for maximum efficiency [192].
Conclusions
Our research has shown the high risk of infection which can occur in a dental practice, where there is vaporization and a great quantity of aerosol prompted through any dental operation, as well as the use of ultrasound and all rotating tools requiring water for cooling (turbines, micromotors, piezo-surgery). Our document provides some guidelines that doctors and dentists should scrupulously observe in order to prevent the spread of SARS-CoV-2.
Dental professionals should have knowledge regarding the safety procedures, the systems that can be used in order to purify the air, what measures they can take in order not to get infected, and how they have to perform dental treatments from now on. Because this information is changing, it will have to be updated with all the measures and protocols they have to follow. | 2020-11-05T09:05:24.700Z | 2020-10-31T00:00:00.000 | {
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1192440 | pes2o/s2orc | v3-fos-license | Quantum Geometric Description of Cosmological Models
This is a written version of the review talk given at the meeting on"Interface of Gravitational and Quantum Realms"at IUCAA, Pune during December 2001. The talk reviewed the recent work of Martin Bojowald on Loop Quantum Cosmology.
I. INTRODUCTION
Canonical quantization of gravity formulated in terms of connection variable is now at a stage where one can address questions of physical interest. At the interpretational level, one still has the outstanding issues of understanding the classical limit and development of a systematic semiclassical expansion. [1] There are at least two classes of situations in classical GR that call for a quantum elucidation: (i) situations where classical GR predicts 'singularities' eg early universe and collapse, and (ii) situations where GR predicts horizons possessing entropy. In a sense, these can be thought of as 'requiring' a quantum theory of gravity and hence any such theory should lead to better understanding of these situations.
There is another context in which one can look for signatures of quantum nature of space-time. This refers to matter wave propagation in a quantum geometrical background. Looking for such effects experimentally actually seems possible thanks to the GRB sources at cosmological distances. This context is very different from the first two in that QG effects are being probed in very ordinary, highly classical, non-extreme situations. [2] The horizon context has already been analyzed and quantum geometry framework does provide a microscopic understanding of entropy. Hawking effect is however is not yet clear. [3] Of the two singular context, Martin Bojowald has recently analyzed the cosmological context in detail. As this is a first application of the quantum geometry framework with very interesting results, I decided to review this work in some details.
II. STANDARD QUANTUM COSMOLOGY
A version of quantum gravity in the cosmological context was already attempted in the sixties. This was again a canonical quantization approach but using the geometrodynamical variables -metric g ij and a symmetric second rank tensor k ij defined on a three manifold. This is a constrained system with diffeomorphism constraint implementing spatial diffeomorphism and the Hamiltonian constraint implementing the space-time diffeomorphisms. Since one does not know how to carry out the Dirac (or otherwise ) quantization of this constrained system, one looks for highly symmetric class of spacetimes, does the symmetry reduction classically and quantizes the left over finitely many degrees of freedom. In the context of so called Bianchi models, the gravitational phase space is at most six dimensional. This is a 'minisuperspace' quantization and has become synonymous with 'standard quantum cosmology'. [4] Here are its main conclusions.
(i) The Hamiltonian constraint (quadratic in momenta) can be interpreted as an evolution equation which is second order in a suitably chosen 'time' variable. This is intimately connected with the 'problem of time'. In a 4-diffeomorphism invariant theory one has to interpret dynamics in terms of evolution relative to a 'clock' degree of freedom.
For FRW models, the scale factor (a gravitational degree of freedom) is a natural choice of a 'clock'. The evolution equation -Wheeler-De Witt equation -being second order in time has two independent solutions i.e. a non-unique wave function of the universe.
(ii) When the scale factor vanishes, the inverse scale factor and hence the curvatures blow up, implying persistence of classical singularity.
(iii) All geometrical quantities such as areas, volumes etc have continuous spectra.
Note that what is quantized is a finite dimensional phase space as nothing else could be done. One was forced to do the symmetry reduction classically and then proceed to quantization. The Ashtekar reformulation in terms of connection variables offers a different alternative. One can quantize the infinite dimensional kinematical phase space and explore the possibility of doing symmetry reduction after quantization. Further more generically the spectra of areas , volumes etc turn out to be discrete indicating that this quantization is qualitatively different.
III. QUANTUM GEOMETRY FRAMEWORK
Let me briefly recall some of the basic steps. One now chooses to look for a Hilbert space on which these functions are represented by multiplicative operators. This is achieved by using the commutative C * algebra of these functions and choosing one of the representations of this algebra. This gives H kin = L 2 (A/G, µ AL ). In other words, the Hilbert space is the space of square integrable complex functions on the 'quantum configuration space'Ā /G square integrable with respect to the Ashtekar-Lewandowski measure constructed from the Haar measure on SU (2). A convenient description of the Hilbert space is obtained in terms of the so called spin-network functions. [5] In Σ consider closed graphs γ. Associate with each edge e i a representation π i of SU(2) and associate with each vertex v α an intertwiner (contractor/invariant tensor), C α . For each A ∈Ā one has, by definition, g i = A(e i ), the generalized holonomy. Define, These are the spin network functions -functions on A/G -labeled by graphs, representations and contractors. The set of all such functions forms an orthonormal set which is dense in H kin . In practice one defines various operators by their actions on these and extends them to H Σ kin . This dense space is also called the space of cylindrical function.
(c) In order to accommodate the possibility that the zero eigenvalue of constraints could be in a continuum (i.e. a generalized eigenvalue) one introduces a 'rigging' -Ω ⊂ H kin ⊂ Ω * . Ω is the dense space above while Ω * is the space of continuous linear functional on Ω. The important point is that the physical states generically belong to Ω * . One says that physical states are distributional. [6] (d) For matter sector analogous constructions based on suitable C * algebras are made. These are available for all the usual scalar, spinor, gauge fields. The kinematical Hilbert spaces constructed here differ from the usual Fock spaces. The crucial point of these constructions is to have no dependence on background space-time geometry. [7] IV. QUANTUM SYMMETRY REDUCTION One would now like to specialize this frame work to the cosmological context of highly symmetric space-times. Since the configuration space variable are now connections, the notion of symmetry requires that the connection transformed under a symmetry diffeomorphism of Σ to be gauge equivalent to the original connection. The first task is to characterize such symmetric connections precisely. This has already been done and can be summarized as (simplifying a bit for brevity): [8,9] If S is a symmetry group (compact Lie group) and F a (Lie) subgroup of S, then (a) Σ ∼ B × S/F , B ∼ Σ/S is the space of orbits of S-action on Σ ; (b) Symmetric connections on Σ are completely characterized by a (reduced) connection on B together with a set of (Higgs) scalars on B, possibly satisfying further constraints.
For example, for the Bianchi class A models, S is one of the Bianchi groups with structure constants satisfying C I JI = 0 while F = {e}. Σ is the group manifold of S while B is a single point of Σ. In terms of the Maurer-Cartan 1-forms ω I and the corresponding left invariant vector fields X J one has, Φ i I are the scalars, P I i are the conjugate momenta, γ is the Barbero-Immirzi parameter and the indices i (su(2)), I (Lie algebra of Bianchi group) both take three values. If in addition to spatial homogeneity one also has isotropy then the scalars satisfy further conditions whose solution is Φ i I = cδ i I while the conjugate momenta satisfy P I i = pδ I i . The constraint expressions can be likewise simplified and expressed in terms of the scalars and their conjugates. This is the classical symmetry reduction. If one proceeds with quantization in a traditional manner then this is very similar to the usual minisuperspace quantization apart from the phase space variables being different. The results are also similar. [10] However an alternative quantization is possible. Instead of requiring the scalars to be well defined operators one can take their exponentials, the so called point holonomies, as well defined operators. Then, similar to the general framework of quantum geometry (polymer representation), one constructs a new H B kin . One can immediately ask why one should do this? Does this Hilbert have anything to do with the H Σ kin of the full theory? The answer turns out to be yes! Bojowald and Kastrup show that the (cylindrical) states in H B kin can be identified with those distributions in Ω * Σ whose support consists of precisely the classical (smooth) symmetric connections. [9] Recall that the physical states of the full theory are supposed to reside in Ω * Σ . A natural identification of symmetric (distributional) states of the full theory is via the properties of their support. The result shows that such quantum states can be alternatively be dealt with by working with the cylindrical states of H B kin . This provides a justification for the alternative (holonomy based) quantization of the symmetry reduced theory. It also makes available the tools of the general framework in a simplified context. This is what is referred to as 'quantum symmetry reduction'. Note that this is quite general and not restricted to a cosmological context.
V. BIANCHI CLASS A MODELS
The strategy now is step by step adaptation of the general framework. While majority of steps are identical, there are also crucial new inputs needed particularly when additional symmetry such as isotropy is at work. I am including only the minimal details necessary to communicate the final results.
For classical configurations, point holonomies are just the group elements obtained by exponentiating the Lie algebra valued scalars, u(Φ I ) ≡ exp{Φ i I τ i } ∈ SU(2). Distributional scalars do just that, they associate with each vertex, an SU(2) group element. For general anisotropic case the kinematical Hilbert space turns out to be H kin = L 2 (SU(2) 3 , dµ 3 Haar ). [11] There are three copies since I takes three values. For gauge invariant functions, a convenient basis is provided by the usual spin-network functions constructed from graphs γ in the group manifold of S with a single vertex of order 6 and three (closed) edges corresponding to the three left invariant vector fields. Using these one defines the operators corresponding to the conjugate momenta and then proceeds to build the constraint operators. Following the Thiemann approach, the Hamiltonian constraint is expressed in terms of the volume operator together with various commutators of holonomies with the volume operator. So the main problem is to define a volume operator and obtain its spectrum. The full spectrum is not available in the general case. However, for the homogeneous and isotropic models (Bianchi I and IX), spectrum of volume operator has been determined.
When isotropic case is considered, the scalars have to satisfy further conditions. Naively one would expect that since we now have a single scalar, spin-network func- which we now denote as: [13] |n ≡ e i n It is sufficient to note that the matter part of the Hamiltonian operator is diagonal with respect to the (gravitational) states |n . This specifies the physical states of the loop quantum cosmology. [13,16] One therefore takes, the label n ∈ Z as a time label. The physical state condition can now be viewed as specifying a discrete time evolution. [15,13,16] Restricting to the isotropic, flat models, the evolution equation is of order 16 (and consists of the n ± 8, n ± 4 and n terms only). The coefficients are such that A k n = 0 if and only if n + k = 0. Furthermore,Ĥ ϕ (n = 0) = 0 as well. This has two consequences.
The s 0 (ϕ) never appears in the equation. Therefore it is neither determined by nor determines any other s n (ϕ). The state |0 is thus orthogonal to all other physical states (evolving solutions). This is precisely the state which corresponds to the zero eigenvalue of the scale factor operator. Thus one sees that unlike the classical case where evolution through vanishing scale factor is not possible, the quantum evolution equation does not suffer such a breakdown. In this sense the classical singularity is absent in the evolving states. The second consequence is that one gets a conditions on the initial data -the choice of 16 coefficients s −16 , s −15 , ...s −1 (say). This is because the equation can not determine s 0 since its coefficient vanishes. Thus one can conclude that the classical singularity is avoided by the evolving solutions and that there are 15 instead of 16 independent solutions. [13,16] This seems worse than the standard quantum cosmology with only two independent solutions! Quite independently, one has also to understand the discrete time evolution in terms of more familiar continuous time evolution, at least when one hopes to be in classical regime. Clearly, the volume and the scale factor eigenvalues become large compared to the Planck scale values when n ≫ 1. One expects a solution to be classically interpretable when for large n, s n+m (ϕ) can vary significantly from s n (ϕ) when m is also large compared to 1 but remains almost the same when m is comparable to 1. One can now ask: how many solutions exhibit this behavior? In a precise formulation of such a behavior, the Barbero-Immirzi parameter γ come very handy. Note that for large n = 2j + 1, V j → (γℓ 2 p |n|/6) 3/2 ∼ (a 2 ) 3/2 . We can use this to define the scale factor as a function of n as: a 2 (n) ≡ (nγ)ℓ 2 p /6. Now change in a 2 (n) as n changes by 1 will be infinitesimal if γℓ 2 p → 0. Thus we can mimic continuous evolution with respect to the scale factor by considering the formal limit γ → 0, n → ∞ keeping γn a constant. We are keeping ℓ p fixed in this and so are in the quantum domain still. If a solution {s n (ϕ)} has a limiting value in the above limit then it is said to be 'pre-classical'. [16] The question now becomes as to how many evolving solutions are pre-classical? For large n, the equations itself becomes an equation with constant coefficients and is easy to analyze.
The result is that, of the sixteen solutions, only two are pre-classical. There is still the constraint on the initial conditions which reduces these two to a single solution. Thus while there are many evolving solutions (all avoiding the singularity), only one of these can mimic a classical evolution. [16] From the black hole entropy computations, γ is fixed to be a constant of order one.
The γ → 0 limit noted above is to be thought of as a formal device to single out solutions mimicking classical evolution. The presence of the Barbero-Immirzi parameter however offers a new limit to be explored apart from the classical limit with ℓ p → 0. Bojowald has further shown that in the γ → 0 limit, referred to as 'continuum limit' in the sense that the discrete structure of quantum geometry can be ignored, one recovers the standard quantum cosmology. [17] This shows also that it is the specific, discrete structure of quantum geometry that is responsible for avoiding the singularity and selecting a unique (pre-classical) solution and not just any quantization procedure.
VII. CONCLUDING REMARKS
This is a first explicit application of the highly abstract framework of quantum geometry to a context of physical interest, particularly addressing the issue of classically indicated singularity. Not only does it meet the expectation that in a quantum theory of gravity, classically indicated singularities should be absent, it provides a quantitative means to estimate how rapidly quantum geometry picture goes over to the classical picture. In the quantization procedure, many conceptual and technical assumptions have gone in (eg choice of polymer representation). In a sense the classical space-time picture has been 'mutilated' quite a bit. Therefore it could have happened that the none of final set of solutions could be interpreted in classical terms thereby recovering the classical theory. Not only this does not happen but one gets a unique solution displaying the classical picture. It also brings out an intriguing role played by the Barbero-Immirzi parameter. | 2014-10-01T00:00:00.000Z | 2002-05-23T00:00:00.000 | {
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270407699 | pes2o/s2orc | v3-fos-license | MR Molecular Imaging of Extradomain-B Fibronectin for Assessing Progression and Therapy Resistance of Prostate Cancer
Accurate assessment and characterization of the progression and therapy response of prostate cancer are essential for precision healthcare of patients diagnosed with the disease. MRI is a clinical imaging modality routinely used for diagnostic imaging and treatment planning of prostate cancer. Extradomain B fibronectin (EDB-FN) is an oncofetal subtype of fibronectin highly expressed in the extracellular matrix of aggressive cancers, including prostate cancer. It is a promising molecular target for the detection and risk-stratification of prostate cancer with high-resolution MR molecular imaging (MRMI). In this study, we investigated the effectiveness of MRMI with an EDB-FN specific contrast agent MT218 for assessing the progression and therapy resistance of prostate cancer. Low grade LNCaP prostate cancer cells became an invasive phenotype LNCaP-CXCR2 with elevated EDB-FN expression after acquisition of the C-X-C motif chemokine receptor 2 (CXCR2). MT218-MRMI showed brighter signal enhancement in LNCaP-CXCR2 tumor xenografts with a ∼2-fold contrast-to-noise (CNR) increase than in LNCaP tumors in mice. Enzalutamide-resistant C4-2-DR prostate cancer cells were more invasive, with higher EDB-FN expression than parental C4-2 cells. Brighter signal enhancement with a ∼2-fold CNR increase was observed in the C4-2-DR xenografts compared to that of C4-2 tumors in mice with MT218-MRMI. Interestingly, when invasive PC3 prostate cancer cells developed resistance to paclitaxel, the drug-resistant PC3-DR cells became less invasive with reduced EDB-FN expression than the parental PC3 cells. MT218-MRMI detected reduced brightness in the PC3-DR xenografts with more than 2-fold reduction of CNR compared to PC3 tumors in mice. The signal enhancement in all tumors was supported by the immunohistochemical staining of EDB-FN with the G4 monoclonal antibody. The results indicate that MRMI of EDB-FN with MT218 has promise for detection, risk stratification, and monitoring the progression and therapy response of invasive prostate cancer.
■ INTRODUCTION
Prostate cancer is the most common noncutaneous cancer in men and is the second leading cause of cancer deaths in the United States.An estimated quarter million men are diagnosed with prostate cancer in the United States every year. 1 Prostate cancer is a highly heterogeneous disease with significant variations in the molecular and pathological trajectory.Although one in eight men will be diagnosed with prostate cancer in their lifetime, most patients are diagnosed with lowrisk cancer and with an indolent disease course without treatment.However, about one in five patients are diagnosed with high-risk cancer and have a substantial risk of prostatecancer-related death. 2 The five-year survival rate of patients with localized disease is nearly 100%, while it drops dramatically to only 28% for those with distant metastases. 3herefore, accurate early detection, risk-stratification, and active surveillance of primary prostate cancer are critical to identify patients with high-risk disease and to monitor the status and progression of low-risk tumors for precision healthcare of prostate patients.
Currently, prostate specific antigen (PSA) screening, diagnostic imaging, and biopsy-based histology are routinely used for diagnosis of prostate cancer. 2,4−6 PSA testing is inconclusive, 7,8 and imaging and biopsy are followed for those with abnormal levels of PSA. 9 Transrectal ultrasound and multiparametric MRI (mpMRI) are the most commonly used imaging modalities for prostate cancer.However, these strategies have had limited success in localizing and differentiating high-risk prostate tumors from low-risk indolent tumors. 10Prostate biopsy is still the gold standard for prostate cancer diagnosis.Unfortunately, the accuracy of prostate biopsy is often limited by inadequacies of prostate needle sampling due to the multifocal nature of the disease, the coexistence of various grades of tumors within the prostate, and the difficulty in assigning an accurate Gleason grade based on the minute sample size from the needle-biopsy.There is about 20% or higher risk of upgrading the score after radical prostatectomy compared with the original Gleason grade from the biopsy specimen.Consequently, most patients receive treatment even for low-risk disease to avoid potential undertreatment, which also leads to serious treatment-related long-term side-effects.In addition, prostate biopsies can lead to increased risks of hematuria, pain, infections, and septicemia.There is an unmet clinical need for a noninvasive imaging modality for accurate detection of high-risk prostate cancer and active surveillance for low-risk tumors for precision care of prostate cancer patients.
Molecular imaging of the markers associated with prostate cancer could overcome the limitations of the existing diagnostic methods and provide accurate detection, riskstratification, and precision treatment of the disease.Recently, positron-emitting probes specific to prostate-specific membrane antigen (PSMA) 68 Ga-PSMA-11 and 18 F-DCFPyl have been approved for PET imaging of metastatic and recurrent prostate cancer. 11−13 However, their potential for staging or risk-stratification of primary prostate cancer has not been conclusively demonstrated in clinical studies. 14,15In addition, PSMA expression can be absent in localized and metastatic tumors of some patients, which results in a false negative diagnosis with PSMA PET.
Extradomain B fibronectin (EDB-FN) is a cancer-associated isoform of fibronectin (FN) and is highly expressed in aggressive tumors, including high-risk prostate cancer.−18 Elevated expression of EDB-FN is associated with EMT induction, cancer cell invasion, and metastasis. 16−25 EDB-FN is a promising oncotarget for developing molecular imaging technologies for the detection, risk-stratification, and active surveillance of high-risk prostate cancer.
MRI is a noninvasive clinical imaging modality and provides three-dimensional images of soft tissues with high spatial resolution.It is considered to be the most promising tool for prostate cancer imaging and well-suited for detecting localized tumors in the prostate. 26,27mpMRI is currently the preferred imaging method for precision biopsy and surgery planning of prostate cancer. 28,29The capability of MRI for precision diagnostic imaging is restrained by the lack of tumor specificity of the clinical contrast agents. 30We have recently developed small peptide targeted gadolinium-based contrast agents specific to EDB-FN for MR molecular imaging (MRMI) of cancer. 31,32A lead targeted macrocyclic agent ZD2-N3-Gd(HP-DO3A), or MT218, has been identified for clinical translation, Figure 1. 32,33It has a higher T 1 relaxivity (6.54 mM −1 s −1 ) than corresponding nontargeted clinical agent gadoteridol (4.1 mM −1 s −1 ). 32It produces stronger signal enhancement in rodent aggressive tumor models at a substantially reduced dose (0.04 mmol/kg) than the clinical agent (0.1 mmol/kg). 32,34Our previous studies have shown that MRMI of EDB-FN is effective to differentiate high-risk tumors from low grade tumors in animal tumor models. 35,36n this study, we investigated the potential of MRMI of EDB-FN with MT218 for monitoring progression and development of therapy-resistance of prostate cancer.Timely assessment of disease progression and development of therapy resistance are essential for precision care of prostate cancer patients.Demonstration of the capability of MRMI with MT218 could expand its application for active surveillance and imaging-aided precision care in addition to detection and riskstratification.We assessed the expression of EDB-FN in several pairs of prostate cancer cells, including LNCaP and LNCaP-CXCR2, C4-2 and drug resistant C4-2-DR, and PC3 and PC3-DR cells, in correlation with the invasiveness of the cells.We then evaluated the tumor signal enhancement of MRMI with MT218 in different prostate cancer tumor models, including LNCaP and LNCaP-CXCR2, and drug-resistant tumors.The potential of MRMI of EDB-FN for precision imaging, including monitoring disease progression and therapy-resistance, was determined in correlation with EDB-FN expression and the invasiveness of the cancer cells.
MRMI of EDB-FN for Monitoring Progression of Prostate Cancer
C-X-C motif chemokine receptor 2 (CXCR2) neuroendocrine tumor cells are enriched in high-grade and advanced prostate cancer.CXCR2 expression is associated with therapy resistance and progression of the disease. 37It may also play a role in the EMT and ECM remodeling.Since EDB-FN is an oncogenic ECM protein associated with EMT, we evaluated the expression of EDB-FN in the LNCaP and LNCaP-CXCR2 prostate cancer cells to determine the potential correlation of EDB-FN with CXCR2.The CXCR2-positive LNCaP-CXCR2 was obtained by modifying the slow-growing LNCaP cells with stable integration and overexpression of the pro-inflammatory and pro-tumorigenic IL8 receptor CXCR2. 37LNCaP-CXCR2 cells also exhibited the mesenchymal feature, i.e., elongated cellular morphology, in 2D culture and were more invasive, i.e., larger spheroids, than the LNCaP cells, as shown in 3D culture (Figure 2A).Transwell assays showed that more LNCaP-CXCR2 cells (stained with a purple dye) migrated through a basement membrane and invaded through a Matrigel layer, indicating an increased invasiveness of the cells as compared to the LNCaP cells (Figure 2B).These increased aggressive features of LNCaP-CXCR2 cells were consistent with the previously reported results. 37Correspondingly, LNCaP-CXCR2 cells exhibited significantly higher EDB-FN expression, as shown by RT-PCR as compared to the slow-growing parental LNCaP cells (Figure 2C).The staining of the 3D spheroids with the EDB-binding peptide fluorescence probe ZD2-Cy5.5 also showed stronger binding to LNCaP-CXCR2 spheroids (Figure 2D).The EDB-FN expression in LNCaP-CXCR2 cancer cells is correlated with their acquired invasiveness.
To assess the potential of MRMI of EDB-FN with MT218 to monitor the progression of the slow growing LNCaP prostate tumor to an invasive form after its acquisition of CXCR2, we performed MRI studies in mice bearing the LNCaP and LNCaP-CXCR2 prostate cancer xenografts after intravenous injection of MT218. Figure 3 shows the representative MRI images of LNCaP and LNCaP-CXCR2 prostate cancer xenografts established in athymic nu/nu mice.The images were acquired before and 25 min after intravenous injection of MT218 at 0.04 mmol/kg.Strong signal enhancement was observed in the invasive LNCaP-CXCR2 tumors, while only slight signal enhancement was observed in the lowgrade LNCaP tumors (Figure 3A).Quantitative analyses of the signal intensities showed over 3-fold increased CNR in the LNCaP-CXCR2 tumors with MT218, while the LNCaP tumors had only about a 50% CNR increase (Figure 3B).Post-mortem immunohistochemical (IHC) analysis of the tumor tissues with an anti-EDB-FN G4 monoclonal antibody showed strong staining of EDB-FN in the LNCaP-CXCR2 tumors as compared to the low EDB-FN staining in the LNCaP tumor sections, indicating differential expression of EDB-FN in the LNCaP and LNCaP-CXCR2 tumors (Figure 3C).The IHC data corroborated the MRMI results, suggesting that MRMI of EDB-FN with MT218 is able to monitor the progression of slow growing prostate cancer to invasive tumors after acquisition of CXCR2.
MRMI of EDB-FN for Monitoring Enzalutamide-Resistance of Prostate Cancer
The expression of EDB-FN and its potential as a molecular marker for imaging therapy resistance was evaluated in C4-2 and enzalutamide-resistant C4-2-DR prostate cancer cells and tumor models.The C4-2-DR cells were an enzalutamideresistant prostate cancer cell line generated by acquired resistance to 20 μM enzalutamide, an androgen-receptor antagonist. 37Evaluation of the morphological and functional features of the cell lines demonstrated significantly increased propensity of C4-2 cells to form tumor spheroids in 3D culture compared to LNCaP cells (Figures 4A and 2A).Compared to C4-2 cells, C4-2-DR cells showed a similar 3D growth but significantly higher migration and invasion (Figure 4B).The C4-2-DR cells exhibited a significantly increased expression of EDB-FN over the C4-2 cells (Figure 4C).Additionally, in 3D culture, the resistant C4-2-DR cells were more invasive and showed proliferative tumor spheroids with significantly increased EDB-FN secretion compared to the C4-2 spheroids, evidenced by intense ZD2-Cy5.5 staining (Figure 4D).
MRMI showed bright tumor enhancement in C4-2 tumors in the mice at 25 min postinjection of MT218 (0.04 mmol/kg) as compared the precontrast tumors, while brighter signal enhancement was seen in the more invasive enzalutamideresistant EDB-FN-rich C4-2-DR tumors (Figure 5A).Quantitative analyses of the signal intensities showed that MT218 at the same dose resulted in an over 5-fold CNR increase in the C4-2-DR tumors as compared to about a 2.5 CNR increase in the C4-2 tumors.C4-2-DR tumors had about a 2-fold higher CNR increase over their nonresistant C4-2 counterparts, indicating elevated EDB-FN expression in the drug-resistant tumors (Figure 5B).Post-mortem IHC analysis of EDB-FN also showed significantly elevated levels of EDB-FN expression in the drug-resistant tumors over the C4-2 tumors.The results correlated well with the differential signal enhancement in the C4-2 and C4-2-DR tumors, indicating that PC3 prostate cancer cells are a PSMA-negative and highly aggressive cell line that is commonly used as an aggressive tumor model in prostate cancer research.Interestingly, PC3-DR cells, which were generated from PC3 cells by acquired resistance to 200 nM paclitaxel, showed a different trend with decreased ability for invasion as compared to the parental PC3 cells. 38This observation exemplifies the heterogeneous natures of prostate cancer.We investigated the ability of MRMI of EDB-FN for characterizing decreased invasiveness of the PC3-DR tumors in vivo.PC3-DR cells showed no change in 2D and 3D growth and increased migration as compared with PC3 cells (Figure 6A).Interestingly, PC3-DR cells demonstrated a decreased ability to invade Matrigel-coated membranes (Figure 6B).EDB-FN expression was significantly reduced in PC3-DR cells as compared to parental cells (Figure 6C) as well as reduced staining of EDB-FN in the spheroids with ZD2-Cy5.5 (Figure 6D).
Figure 7A shows T 1 -weighted axial spin−echo images of PC3 and PC3-DR tumors before and after the injection of MT218 at 0.04 mmol/kg.PC3 tumors showed robust signal enhancement and about 4-fold CNR increase with MT218, consistent with previous observations. 32,33In contrast, the PC3-DR tumors only showed moderately enhanced signal enhancement and a 1.5-fold CNR increase over the precontrast images (p = 0.07).The invasive PC3 tumors showed significantly higher CNR with contrast enhancement by MT218 than that of the less invasive PC3-DR tumors (Figure 7B).The imaging results were further validated by the strong immunohistochemical EDB-FN staining in the invasive PC3 tumors over the less-invasive PC3-DR ones (Figure 7C), suggesting that EDB-FN upregulation is associated only with the invasiveness of prostate cancer, irrespective of drug resistance.■ DISCUSSION Molecular imaging of the biological signatures of the initiation, proliferation, and invasion of prostate cancer has long been sought for accurate noninvasive characterization of prostate cancer to guide personalized care and treatment.MRI is advantageous over other imaging modalities for diagnostic imaging and treatment planning of localized prostate cancer due to its high spatial resolution for visualizing soft tissues.The challenge for MRMI of cancer is the low sensitivity of contrast enhanced MRI and the low concentration of the protein markers expressed on cancer cell surface.Although targeted macromolecular and nanosized paramagnetic agents have been developed for MRMI of cancer, clinical translation of these agents is hindered by several drawbacks, including slow excretion and potential toxic side effects associated with slow excretion.MT218 is a conjugate of a small linear peptide of seven amino acids (Thr-Val-Arg-Thr-Ser-Ala-Asp) to a clinical macrocyclic contrast agent Gd(HP-DO3A), one of safest GBCAs used in clinical practice. 39MT218 specifically binds to oncoprotein EDB-FN in the ECM of aggressive tumors including prostate cancer.The abundant presence of EDB-FN in the ECM of aggressive tumors allows ready access of freely diffusible, hydrophilic small molecule like MT218.Efficient binding of MT218 and rapid clearance of unbound agent generate robust contrast enhancement in EDB-FN rich tumors in MRMI.Thus, MRMI with MT218 overcomes the limitations of targeting biomarkers expressed only on cancer cell surface.In addition, the small size and moderate binding affinity of MT218 allow rapid excretion after diagnostic imaging to minimize tissue retention of the bound MT218 and avoid any toxic side effects from long-term gadolinium retention.
Heterogeneity of prostate cancer presents a major challenge for the precision healthcare of patients.Accurate riskstratification and active surveillance play a major role in identifying high-risk prostate cancer and monitoring low-grade tumors in the clinical management of the disease.Previously, we demonstrated that MT218-MRMI could effectively differentiate invasive PC3 prostate tumors from slow-growing LNCaP tumors in mice and rats. 32,33This study further explored the potential of MT218-MRMI for active surveillance or monitoring the progression and therapy response of prostate cancer in six tumor models that exhibited a variety of invasivenesses, representing the heterogeneous nature of the disease.Among these models, LNCaP-CXCR2 and LNCaP represented a progression of low-grade prostate cancer into an invasive phenotype associated with an increased expression of CXCR2.C4-2, C4-2-DR, PC3, and PC3-DR represented heterogeneous progression of the disease in response to different treatments.Consistent with our previous observation in other cancer models, 16,36,40,41 the expression levels of EDB-FN correlated well with the invasiveness of the prostate cancer cell lines.EDB-FN expression was significantly higher in the invasive LNCaP-CXCR2, C4-2-DR, and PC3 cells than in the correspondingly less invasive LNCaP, C4-2, and PC3-DR cells.MT218-MRMI provided accurate characterization of the invasiveness of the tumor models based on the contrast enhancement and correlated with the EDB-FN expression levels in the tumors.The results further demonstrated the potential of MT218-MRMI for noninvasive risk-stratification of high-risk prostate cancer and also showed its promise for noninvasive active surveillance of low grade prostate cancer and monitoring tumor response to therapies.Currently, prostate biopsy is still the gold standard for diagnosis, risk-stratification, and active surveillance of prostate cancer in clinical practice.Imaging modalities, including transrectal ultrasound and mpMRI, are routinely used to improve the accuracy of prostate biopsy and diagnosis.However, MRI guided biopsy still suffers from inaccuracy, including underdiagnosis of some tumors. 42Clinical application of a cancer-specific MRI contrast agent may improve diagnostic accuracy.MT218 is able to produce robust contrast enhancement in aggressive tumors and differentiate aggressive prostate cancer from low grade tumors in preclinical models at a substantially reduced dose, 0.04 mmol/kg vs 0.1 mmol/kg of a clinical GBCA. 32The low effective dose of MT218 is clearly a safety advantage over the clinical agents in addition to its specificity for aggressive tumors.The effectiveness of MRMI with MT218 for the precision imaging of high-risk prostate cancer needs to be demonstrated in extensive clinical trials before clinical implementation in diagnostic imaging of prostate cancer.Currently, MT218 is in clinical trials for the MRMI of solid tumors.The phase 1 clinical trial in healthy men showed an excellent safety profile and pharmacokinetics indistinguishable from those of clinical contrast agents. 43Later phase clinical trials are underway to assess its feasibility for detecting EDB-FN positive prostate cancer in patients.
■ CONCLUSION
We have further demonstrated that EDB-FN is an ECM marker of invasive prostate cancer.We have previously demonstrated that MT218-MRMI is promising for the detection and risk stratification of invasive prostate cancer.This study has shown that it also has potential to noninvasively assess progression of a low-grade prostate tumor to an invasive cancer and to monitor tumor response to therapies, whether the tumor becomes a more invasive therapy-resistant phenotype or a less invasive resistant phenotype based on the changes of EDB-FN levels in the tumors.MRMI of EDB-FN with MT218 has promise for detection, risk stratification, and assessment of progression and therapy response of invasive prostate cancer.
Cell Culture
Prostate cancer cell lines LNCaP, C4-2, and PC3 were purchased from ATCC (Manassas, VA).The paclitaxel-resistant derivative of PC3 (PC3-DR) was a kind gift from Dr. Aaron Mohs (University of Nebraska, Omaha, NE).LNCaP-CXCR2 and enzalutamide-resistant C4-2-DR cells were developed in the lab of Dr. Jiaoti Huang (Duke University, Durham, NC) by stable integration of CXCR2 plasmid and with resistance to 25 μM enzalutamide (SelleckChem, Houston TX), respectively. 37The prostate cancer cell lines were cultured in RPMI1640 medium (Sigma, St. Louis, MO).Both of the media were supplemented with 10% fetal bovine serum and 100 units/mL of penicillin/streptomycin.All of the cells were cultured at 37 °C and 5% CO 2 .
Invasion and Migration Assays
Transwell assays, with and without Matrigel, were performed to assess the invasive and migratory properties of the different PCa cells.To evaluate migration, 1 × 10 5 prostate cancer cells (starved overnight) were plated in transwell inserts (VWR, Radnor, PA).The next day, nonmigrated cells were removed by swabbing the insets, and the migrated cells at the bottom of the insets were fixed (10% formalin for 10 min) and stained with 0.1% crystal violet (20 min).The cells were imaged using a Moticam T2 camera after removal of excess stain and overnight drying.To evaluate the ability of the cells to invade through a basement membrane in addition to the transwell membranes, 100 μL of 0.5 mg/mL Corning Matrigel membrane matrix was added atop the transwell membrane and incubated at 37 °C to solidify and form a gel coating (Corning, NY).The cells (2 × 10 5 ) were starved overnight and seeded onto the gel coating, and the assay was performed as described above.
3D Culture and ZD2-Cy5.5 Staining
To test the ability of the prostate cancer cells to grow in a 3D Matrigel culture, 200 μL of Corning Matrigel membrane matrix was added to each well of an eight-well microslide (Ibidi, Fitchburg, WI) and incubated at 37 °C to solidify and form a gel coating.The cells (1 × 10 5 ) were seeded onto the gel coating in each well.3D growth was monitored and imaged for up to 3 days by using the Moticam T2 camera.To evaluate the expression of EDB-FN, the 3D cultures were stained with 100 nM ZD2-Cy5.5 and 5 μg/mL Hoechst 33342 for 30 min.Excess stains were removed with PBS washes, and fluorescence microscopy was performed on an Olympus confocal microscope (Japan).
Xenograft Mouse Models
Nude athymic mice (6-week-old nu/nu males) were purchased from The Jackson Laboratory (Bar Harbor, MA) and housed in the animal facility at CWRU.All the animal experiments were performed according to the protocol approved by the IACUC of CWRU.About (5−6) × 10 6 LNCaP, LNCaP-CXCR2, C4-2, C4-2-DR, PC3, and PC3-DR cells suspended in a Matrigel-PBS mixture (1:1) were subcutaneously injected in the left flanks of nude mice (100 μL per mouse, six mice per group times six models = 36 mice).MRMI was performed on the six xenograft models with a 0.04 mmol/kg dose of MT218 after the tumors reached volumes of about 50−75 mm 3 .After imaging, the animals were euthanized, and the tumors were harvested for post-mortem histology and immunohistochemistry (IHC).
Immunohistochemistry
Staining and IHC services were provided by the Tissue Resources Core Facility of the Case Comprehensive Cancer Center (grant P30 CA43703).The tumor tissues were fixed in 10% neutral buffered formalin.Paraffin-embedded specimens were sectioned and H&Estained to visualize morphology.IHC was performed using an anti-EDB-FN antibody G4 clone (1:100 dilution, Absolute Antibody, UK).All the slides were assessed by a certified pathologist.
Statistical Analyses
All of the experiments were independently replicated at least three times (n = 3), unless otherwise stated.Data are represented as mean ± s.e.m.Statistical analysis was performed using Graphpad Prism.Data between two groups were compared using an unpaired Student's t test.p < 0.05 was considered to be statistically significant.
Figure 2 .
Figure 2. EDB-FN overexpression is associated with invasive prostate cancer cells that evolve from low-risk ones.Compared to the low-risk LNCaP cells, LNCaP-CXCR2 cells showed an elongated mesenchymal feature in 2D culture and high propensity to form 3D spheroids (A), increased migratory and invasive abilities indicated by strong purple color staining of the cells passing through a membrane filter (migration) and Matrigel layer (invasion; B), elevated expression of EDB-FN as measured by qRT-PCR (C), and strong ZD2-Cy5.5 binding (red fluorescence) of EDB-FN in 3D culture (D; *p < 0.05 using unpaired t test).
Figure 3 .
Figure 3. MRMI of EDB-FN with MT218 facilitates noninvasive assessment of the progression of low-risk prostate cancer.T 1 -weighted 2D axial spin echo images were obtained before and 25 min postinjection of MT218 at 0.04 mmol/kg in athymic nu/nu mice bearing LNCaP and LNCaP-CXCR2 xenografts.Compared to the slow-growing LNCaP tumors, the invasive LNCaP-CXCR2 xenografts show brighter signal enhancement (A), significantly higher CNR (B, average of n = 6 mice, **p < 0.05 using unpaired t test), and strong EDB-FN staining in post-mortem IHC (C).
Figure 4 .
Figure 4. EDB-FN overexpression is associated with invasive prostate cancer cells that acquire resistance to androgen-deprivation therapy.Compared to their parent C4-2 cells, the enzalutamide-resistant C4-2-DR cells show comparable propensity to form 3D spheroids in 3D culture (A), increased migratory and invasive abilities (B), and significantly elevated expression of EDB-FN as measured by qRT-PCR (C) as well as ZD2-Cy5.5 binding in 3D culture (D; *p < 0.05 using unpaired t test).
Figure 6 .
Figure 6.EDB-FN overexpression is not associated with paclitaxel-resistant prostate cancer cells that do not acquire invasive abilities.Compared to their high-risk parent PC-3 cells, PC3-DR cells show comparable propensity to form 3D spheroids in 3D culture (A) and increased migratory but decreased invasive abilities (B).The decreased invasion of taxane-resistant PC3-DR cells is accompanied by significant downregulation of EDB-FN, as measured qRT-PCR (C) and ZD2-Cy5.5 binding to EDB-FN in 3D culture (D; *p < 0.05 using unpaired t test). | 2024-06-13T15:07:21.699Z | 2024-06-11T00:00:00.000 | {
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237365329 | pes2o/s2orc | v3-fos-license | Dual challenges of heat wave and protective facemask-induced thermal stress in Hong Kong
During the COVID-19 pandemic, wearing protective facemasks (PFMs) can effectively reduce infection risk, but the use of PFMs can amplify heat-related health risks. We studied the amplified PFM-induced human thermal stress via both field measurements and model simulations over a typical subtropical mountainous city, Hong Kong. First, a hot and humid PFM microenvironment has been observed with high temperature (34–35 °C) and high humidity (80–95%), resulting in an aggravated facial thermal stress with a maximal PFM-covered facial heat flux of 500 W/m2 under high-intensity activities. Second, to predict the overall PFM-inclusive human thermal stress, we developed a new facial thermal load model, SPFM and a new human-environment adaptive thermal stress (HEATS) model by coupling SPFM with an enhanced thermal comfort model to resolve modified human-environment interactions with the intervention of PFM under realistic climatic and topographical conditions. The model was then applied to predict spatiotemporal variations of PFM-inclusive physiological subjective temperature (PST) and corresponding heat stress levels during a typical heat wave event. It was found wearing PFM can significantly aggravate human thermal stress over Hong Kong with a spatially averaged PST increment of 5.0 °C and an additional spatial area of 158.4% exposed to the severest heat risks. Besides, PFM-inclusive PST was found to increase nonlinearly with terrain slopes at a rate of 1.3–3.9 °C/10°(slope), owing to elevated metabolic heat production. Furthermore, urban residents were found to have higher PFM-aggravated heat risks than rural residents, especially at night due to synergistic urban heat and moisture island effects.
Introduction
Recent decades have seen hotter summers with more frequent extreme heat events worldwide due to global warming and excessive anthropogenic emissions, with a consequence of more heat-related mortality and morbidity. Future climate projection studies have shown that the frequency, intensity, and duration of global heat waves are expected to increase considerably, resulting in a greater impact on population health [1,2]. The ongoing COVID-19 pandemic has put the world population into a more dangerous context with dual challenges of infection and heat stress [3], especially for people in densely populated Northeast India and West Africa where are with limited cooling infrastructure and poor public health facilities. Wearing protective facemasks (PFMs) has been proven to be one of the most effective interventions to reduce the infection risk. However, the use of PFMs can amplify heat-related health risks due to impeded heat dissipation through convective and respiratory exchanges. This could be a serious problem for the public, especially in vulnerable populations including people with COVID-19 sequelae, people with respiratory and cardiovascular diseases, outdoor workers, health workers, athletes, pregnant women, the poor, children, and the elderly [4].
Although the PFM-covered facial area is very small compared to the human body surface area, its impact on human thermoregulation and thermal sensation is critical [5]. Firstly, due to very high metabolic activity levels in the human head, the heat flux of bare facial skin area (104 W/m 2 ) is more than twice that of the remaining body surface area (50 W/m 2 ) [6]. The use of PFMs can significantly impede facial heat dissipation via respiration, convection, radiation, and evaporation, resulting in an elevated facial skin temperature. Secondly, the facial area covered by PFMs is very thermosensitive containing a high density of thermoreceptors, thus the elevated facial skin temperature and aggravated hot humid PFM microenvironment will significantly influence human thermal sensations [7]. Previous PFM-related thermal studies mainly focused on investigating PFM-induced facial thermal sensations via field experiments and questionnaires with volunteers standing/sitting indoor under a steady state [8][9][10][11], the quantification of PFM-induced facial thermal load and PFM-inclusive human thermoregulation under different outdoor activities and climatic conditions is still lack of exploration.
In this study, we aim to fill in the gap and conduct a comprehensive investigation on how PFMs will affect human thermoregulation in people's daily life under different activity levels and different climatic conditions. More specifically, we will (1) develop a new PFM microenvironment model by considering the facial skin energy balance, (2) develop a new PFM-inclusive human thermal comfort model by dynamically coupling the new PFM microenvironment model with an advanced human thermoregulation model. To achieve these research objectives, an appropriate human thermal comfort model needs to be first selected. Classical thermal comfort models can be divided into three main categories, including one-node model, two-node model, and multinode model. One-node models (e.g., Fanger's PMV model [12], Jendritzky's advanced PMV model [13], Błażejczyk's MENEX model [14]), are used to quantify the average adaptive steady-state thermal comfort condition of a whole human body by resolving human-environment interactions. On the other hand, two-node models (e.g., Gagge's model [15], Kaynakli and Kilic [16], Foda and Sirén [17], etc.) and multi-node models (e.g., Fiala model [18], Tanabe model [19] and UCB model [20], etc.) are used to quantify more detailed transient thermal comfort conditions of different human body segments by considering a two-layer structure (skin and core) [21] and a multi-segment structure (skin, fat, muscle, bone, etc.), respectively. Here, the one-node MENEX model [14] with high predictive accuracy and small computational cost is selected as the basic framework for further development of our new PFM-inclusive human thermal comfort model, which will be applied in both local and city scales.
The newly proposed methodology will be tested over a stereotyped high-rise compact hot and humid metropolitan area, Hong Kong, where over 95% of people wear PFMs outdoor, for case study. Hong Kong is known for its hot-humid summers with afternoon temperature often exceeding 31 • and nighttime temperature remaining around 26 • C with high humidity, based on past 30 years' meteorological records (1991-2020) [22]. High temperature and high humidity restrain the human body's cooling efficiency via thermal radiation and sweat evaporation, respectively, thus leading to severe human thermal stresses [23][24][25]. On the other hand, it is noteworthy that Hong Kong is featured with rugged terrains with slope ranging from 0 • to 77 • [26], which implies that residents may face greater heat risks from elevated metabolic heat production and expenditure when climbing over inclined terrains compared to walking over flat terrains [27].
Thus, it is of urgent need to quantify the aggravated human thermal stress due to PFM wearing over Hong Kong, so that timely actions can be taken by the government and public. Particularly, it is important to know how PFM aggravates human thermal stress under different activity levels and which neighbourhoods are most vulnerable to PFM-inclusive heat stress. To address these questions, we conducted a comprehensive investigation on PFM-induced facial thermal stress and PFM-inclusive human thermal stress via field experiments and numerical model simulations, respectively. In the field experimental phase, we observed and compared different thermo-physiological responses of subjects with and without PFMs at different activities (standing, walking, and climbing). In the numerical model simulation phase, we developed a new PFMinclusive human-environment adaptive thermal stress (HEATS) model with the consideration of PFM-modified human thermoregulation schemes under realistic climatic and topographical conditions. The newly proposed PFM-inclusive HEATS model is featured with the following advanced characteristics, including the consideration of the intervention of facemasks in heat dissipation and the implication of city topography on human metabolism and energy expenditure. With the HEATS model, we predicted a PFM-inclusive heat stress map during a typical heat wave event over the Hong Kong metropolitan area, which can provide neighbourhood-scale heat risk warnings so that the public can be better prepared in their daily life under the dual challenges of pandemic and heat risks.
A new human-environment adaptive thermal stress model
In this section, we propose a new human-environment adaptive thermal stress (HEATS) model with the capability of considering PFMmodified human thermoregulation schemes under realistic climatic and topographical conditions (Fig. 1). The governing equation of the new HEATS model is human energy balance [28]: where S is the net heat storage, M is the metabolic heat production, R nh is the net radiation of the human body (which considers the absorption and reflection of shortwave and longwave radiation between human body and the environment), W is the mechanical work, C and E is the heat dissipation via convection and evaporation, respectively, with the subscripts of "body" and "res" indicating from body surface and respiration, respectively. All terms are expressed in W/m 2 . It is noteworthy that classic PFM-exclusive human energy balance can be referred to the MENEX model [14,28], for instance, the calculation procedure of the heat dissipation via human body surfaces, i.e., (C + E) body is cited and shown in Appendix 1 [29]. In the following two sub-sections, we focus on introducing two newly proposed modules, i.e., S PFM -HEATS and Top-HEATS, by considering the extra heat burden in the human energy balance equation (Eqn. (1)) due to the wearing of PFM and topography-induced human activity levels, respectively.
S PFM -HEATS module
First, the usage of PFM can suppress facial heat dissipation via convection, evaporation, radiation, conduction, and respiration. To quantify the PFM-induced facial thermal load, we developed a S PFM model (see Fig. 1b) based on skin energy balance. As shown in Fig. 1b, the PFM surface was regarded as the upper boundary of the PFM microenvironment, while the face surface was regarded as the lower boundary. S PFM is the PFM-induced facial thermal load due to suppressed facial heat dissipation, where C, E, R, G denote convection, evaporation, radiation, and conduction, respectively, with the subscripts "fsk" and "res" representing for facial skin and respiration, respectively. (C + E + R) fsk can be calculated according to general skin energy balance equations [14]. G pfm can be calculated according to PFM surface energy balance equation where R n is the net radiation received by the PFM, H is the PFM and LE is the PFM surface latent heat flux. In current default settings, LE = 0; in special settings if the PFM is wet or the PFM is made from phase change materials, LE ∕ = 0. Q is the convective heat exchange between PFM cavity and ambient air due to leakage. (C + E) res is the suppressed heat dissipation from respiration [28]: where T in and p in denote air temperature and pressure inside the PFM, respectively. Then, the modified governing equation of coupled S PFM -HEATS model is: Detailed information about each variable in the S PFM model is shown in Table 1.
Top-HEATS module
Second, due to the spatial variability of terrain gradients (Fig. 1c), people tend to move at different walking speed (v), metabolic rate (M), and mechanical work (W). To better capture the topography-induced thermal stress, we developed a Top-HEATS module equipped with more realistic equations for v, M, and W considering the impact of terrain gradients (Fig. 1c). The walking speed (v, m/s) can be determined by Irmischer-Clarke's modified version of the Tobler hiking function [33,34]: where slope is the terrain gradient (deg o ).
On the other hand, metabolic rate (M) is usually crudely assumed via the conventional ISO method based on typical activity classes, including resting (Level 0: 55-70 W/m 2 ), low metabolic rate (Level 1: 70-130 W/ m 2 ), moderate metabolic rate (Level 2: 130-200 W/m 2 ), high metabolic rate (Level 3: 200-260 W/m 2 ), and very high metabolic rate (Level 4: >260 W/m 2 ) according to BS EN ISO 8996:2004 [35], which cannot represent the spatiotemporal variation of M due to different terrain gradients and walking speeds. To overcome these limitations in the conventional ISO method, we adopt Looney's modified version of Pandolf's predictive equation (Eqn. (6)), which was derived from steady-state exercise protocols (the derivation of Pandolf's predictive equation is detailed in Appendix 2) [33,36]: where m is the average human body weight, kg (m = 60 kg in this study), l is the carriage loads, kg, η is terrain coefficient (1.0 for hard surfaces or pavements).
In addition, to better represent the spatiotemporal variation of W, we determine mechanical work (W, W/m 2 ) based on realistic walking speeds and terrain gradients [28], as where g is the acceleration rate due to gravity (m/s 2 ), A is a standard value of body surface area (A = 1.8 m 2 in this study [28]). Then, the modified governing equation of coupled Top-HEATS model is: Overall, the proposed HEATS model in this section is a new thermal comfort model built upon a basic MENEX model framework of human energy balance [29] and enhanced by adding two new modules, S PFM -HEATS and Top-HEATS, featuring with the capability of predicting human thermal stress due to the wearing of PFMs and the impact of city topography. To drive the HEATS model, meteorological parameters, terrain data, physiological parameters, clothing properties, and PFM thermal characteristics need to be collected as inputs (Table 2). With the HEATS model, we can calculate Physiological Subjective Temperature (PST), i.e., the level of thermal stimuli of a pedestrian after 15-20 min of adaptation to the ambient environment based on the net heat storage S of the body and inner mean radiant temperature under clothing [14,37,38]. According to the PST values, we can further assess thermal stress levels according to PST, i.e., level 0 -comfortable (14.
Table 1
Variables in the PFM-induced facial thermal load model (S PFM ).
Symbol
Unit Source
Field experiments and model evaluation
Field experiments were conducted to investigate the impact of different activity levels on the PFM microenvironment and human thermal stress under realistic hot and humid climatic conditions in Hong Kong between July and September 2020. A typical PFM form, i.e., the disposable surgical mask (Table 4), was selected for the following experiments. A total of five male participants were recruited attending college or graduate school in this study. The age, height, weight were 24 years (±2), 176 cm (±3), 70 kg (±5), respectively. The participants were briefed on the purpose, design, and experimental procedures of the study, and an informed consent was documented. To standardize the thermal burden from clothing other than the respirators, all participants were required to test with similar clothes with 0.4 clo clothing insulation according to ANSI/ASHRAE Standard 55-2017 [39], including a white T-shirt (100% cotton), shorts (65% polyester and 35% rayon), anklet (100% cotton), and sports shoes. The study was approved by the Human Research Ethics Committee, The University of Hong Kong (EA200158).
Each participant was tested in six scenarios (unshaded), including (a) standing still for 50 min, (b) walking on a flat terrain (0 • ) for 50 min, and (c) climbing on 30 • sloped stairs for 50 min, either wearing a PFM or without wearing a PFM. During each experiment, (a) meteorological variables including T a , RH a , v', and R n , (b) skin-based thermo-physiological variables including T fsk , w, TEWL, and H fsk , and (c) PFM microenvironmental variables including T in , RH in , O 2 , and CO 2 were measured simultaneously ( Table 1). The duration of each experiment is 50 min with the first 20 min as the acclimatization period and the latter 30 min as the steady period (Fig. 2). During the experiments, there were two sets of equipment, including (1) small portable devices such as iButtons, heat flux sensors, CO 2 /O 2 sensors, for continuously measuring air temperature & humidity (T in , RH in ), heat flux (H fsk ), CO 2 and O 2 inside the facemask, respectively, and (2) large non-portable devices including the weather station for continuously measuring meteorological information, as well as the infrared camera and DermaLab Combo for discretely recording the thermal infrared images and skin conditions (w, TEWL) in the facial area, respectively. The measurement equipment used in these experiments is depicted in Table 3. In particular, Fig. 3 shows the detailed measurement set-up in the facial area. Fig. 3a shows six measurement sites of skin-based thermo-physiological variables on the facial area, including sites 1-4 inside the PFM and sites 5-6 outside the PFM. In addition, Fig. 3b shows the set-up of two sensors including an iButton sensor and a gas analyser for measuring (T in , RH in ) and (O 2 , CO 2 ), respectively, in the PFM microenvironment. The iButton sensor was fixed on the inner surface of PFM pointing inward with a 0.5-cm distance from the mouth. The gas analyser was connected with a tube positioned inside the PFM so that the air in the PFM microenvironment can be withdrawn and analysed (Fig. 3a). Experimental results can be used for model validation and will be elaborated in section 3.1.
Model simulation scenarios
After model validation, this newly developed HEATS model framework will be applied to simulate the spatiotemporal variations of human thermal stress with and without PFMs over Hong Kong during a typical heat wave event (22-27 June 2016). To drive the model, spatial terrain slope map (30 m × 30 m) (Fig. 4a) and meteorological data at 15 typical meteorological stations (6 rural stations and 9 urban stations) (Fig. 4b) were collected from CEDD [41] and HKO [42], respectively. Significant spatial heterogeneity of terrain slopes and meteorological conditions (e. g., air temperature and absolute humidity) in urban and rural areas can be seen in Fig. 4c and 4d, respectively. First, to investigate the differences of PFM-aggravated human thermal stresses between urban and rural areas due to heterogeneous terrain slopes and meteorological conditions, we conducted a sensitivity test of PST by (1) changing terrain slopes from 0 • to 60 • , (2) changing meteorological input (urban/rural), and (3) changing PFM wearing status (with/without). Second, to investigate the spatial variation of PFM-aggravated human thermal stress over Hong Kong, we selected the most uncomfortable period (1:00 p.m., 26 June 2016) and derived the spatial PST maps with and without PFM via the HEATS model. Detailed results can be seen in section 3.2 and 3.3.
PFM experimental results and model validation
In this section, we analysed the experimental results of facial thermal conditions due to PFMs under three different activity levels (standing, walking, and climbing) as described in section 2.2. First, the use of PFM led to significant increases in the facial skin temperature (up to 2.6 • C) (Fig. 5) and moisture (105.8%~531.7%) under different activity levels (Fig. 5c). Besides, a hot-humid PFM microenvironment can be seen with T a and RH increments of 3.0 • C and 10%~20% respectively, compared to ambient air ( Fig. 8a and Fig. 8a). Furthermore, the PFM microenvironment was significantly aggravated by increasing activity levels (standing-walking-climbing), with T a increasing from 34.3 • C to 35 • C . 8a), and RH increasing from 82% to 91% (Fig. 8b). High temperature and high humidity will further restrain heat convection and sweat evaporation by increasing the facial skin hydration (up to 400%) (Fig. 8c) and reducing the facial TEWL (up to 30 g/m 2 -h) (Fig. 8d). Besides, detailed measured data about the PFM-induced facial thermal stress can be found in Table 5. The suppressed heat dissipation from facial skin and respiration due to PFMs can modify the human energy balance and contribute to an elevated PFM-induced heat load (S PFM ). On the other hand, measurements of facial skin temperature, T a and RH of the PFM cavity, and local meteorological parameters were used to drive the proposed PFM-inclusive HEATS model and to derive facial heat fluxes (S PFM ) under the three different activity scenarios, which were then compared against measurements (Fig. 6e). From Fig. 6e, we can see that the model can capture the temporal variation of S PFM with reasonable accuracy and S PFM increased dramatically from 50 to 500 W/ m 2 with increasing activity intensities and time durations.
Temporal variation of PFM-inclusive thermal stress in urban and rural areas
To investigate the differences of PFM-aggravated thermal stresses in urban and rural areas, we conducted sensitivity analysis for the HEATS model and compared simulated PST values under 28 scenarios (Fig. 7) following the input recipes described in section 2.3. First, since Hong Kong is featured with heterogeneous landscapes with terrain slopes ranging from 0 • to 77 • (Fig. 4c), different terrain slopes can lead to different PST values due to the variation of exertion and activity levels of residents when walking or climbing. It can be seen from Fig. 8 that with a slope increment of 10 • , PST can be increased by 1.3-3.9 • C in both urban and rural areas, implying more heat burden under increased activity intensities. Residents walking on a flat terrain (0 • ) with PFMs may feel equivalent thermal stress as walking on an 30 • inclined terrain without PFMs. On the other hand, it is noteworthy that urban terrain slope significantly varies from 0 • to 60 • in the Hong Kong metropolitan area (Fig. 4c), resulting in that urban residents tend to suffer from aggravated thermal stress with a maximum PST increment of more than 20 • C due to the compound effects of PFM wearing and high-intensity activity levels such as climbing during their daily life (Fig. 7a).
Second, urban area was observed with higher temperature (1.4-2.3 • C) and higher absolute humidity (2.6-4.3 g/m 3 ) than rural area during the selected heat wave event (Fig. 4d), owing to the urban heat island (UHI) and urban moisture island (UMI) effects [23,43]. The synergistic UHI and UMI effects may further constrain the heat dissipation of human body via thermal radiation/convection and sweat evaporation, respectively, leading to aggravated thermal stress. Particularly, severer PFM-inclusive human thermal stress has been identified in urban areas than in rural areas at night, possibly owing to nocturnal UHI and UMI, which are attributed to longwave radiation from massive-engineered surfaces, inhibited condensation due to high temperature, as well as anthropogenic heat and moisture sources [44][45][46][47]. For example, residents walking on the inclined terrain with a slope of 20 • will feel 'hot' (Level-2 stress) in urban areas while feeling 'warm' (Level 1-stress) in rural areas at night (Fig. 7). If wearing PFMs, the daytime maximum PST of residents can be increased from 57 • C to 64 • C in urban areas and from 55 • C to 62 • C in rural areas, while the night-time maximum PST can be increased from 27 • C to 37 • C in urban areas and from 25 • C to 35 • C in rural areas. This offers a caveat for urban residents that there are significantly higher heat risks when doing outdoor activities with PFMs during the pandemic at both daytime and night-time, night-time heat risks cannot be overlooked.
Spatial variation of PFM-inclusive thermal stress at the hottest period
To investigate PFM aggravated heat risks over the whole Hong Kong metropolitan area, we derived the spatial map of PST without and with PFM via the HEATS model for the hottest period (specifically, 1:00 p.m. 26 Jun 2016) during the selected heat wave event (Fig. 9). The spatial variation of PST values over Hong Kong are 37.3-54.8 • C without PFM (Figs. 9a) and 38.7-58.7 • C with PFM (Fig. 9b). By comparing Fig. 9a and b, it is evident that the use of PFM contributes to an average PST increment of 5.0 • C, which leads to an additional percentage of 'sweltering' (Level-4 stress with highest heat risks) by 158.4% over Hong Kong, especially in rugged hills when people are hiking.
Hong Kong has 24 country parks comprising more than 400 hiking trails, frequented by 10 million visitors every year. With many restaurants, beaches, and gyms closed during the pandemic, a near 100-150% increase in the number of hikers was found in the first half of 2020 compared to 2019 [48]. As a result, hikers are facing a tough dilemma that wearing PFM will suffer from intensive thermal stress, while ditching PFMs will increase the infection risk.
To investigate the impact of PFM on the thermal stress of hikers over different hiking trails, we select three hiking trails with the same traveling distance (10 km) but different difficulty levels according to Shenandoah Hiking Difficulty protocol [49], including Wu Kau Tang trail (level 1 with an average slope of 11 • ), Dragon Backtrail (level 3 with an average slope of 17 • ), and Tsing Shan Monastery trail (level 5 with an average slope of 43 • ). Fig. 10 indicates that human thermal stress can be aggravated due to the wearing of PFM and the increase of difficulty levels of hiking trails. During the hottest period, the average PST values of hikers in the level 1, 3, and 5 trails are 44.4 • C, 46.5 • C, and 48.0 • C, respectively, without PFMs, and can be increased to 46.2 • C, 49.2 • C, and 52.5 • C, respectively with PFMs. In addition, the usage of PFMs can lead to increased heat risks in all hiking trails, for example, the probability of "very hot" risks will be increased from 40.3% to 52.1% in the Level-1 trail, from 65.0% to 85.1% in the Level-2 trail, and from 80.0% to 88.8% in the Level-3 trail.
Breathing comfort of PFMs
Extreme heat events add extra heat risks for residents in PFMwearing culture. Many cases of the PFM-related heat stroke or even death was reported in 2020, which causes widespread concern about the side effects of PFMs. During high-intensity activities like hiking, human bodies need to consume more oxygen with accelerated or doubled breathing rates. Since PFMs are not well ventilated and close to the face, they may quickly get damp due to intensive breathing and sweating. Significant CO 2 surges and O 2 reductions were found inside sweaty PFM during our field experiment (Fig. 11), implying that sweaty PFMs can aggravate the breathing discomfort sensation. Although wearing PFMs is uncomfortable in breathing sensation and thermal sensation, it is unavoidable as a necessary personal protective equipment during the pandemic. To deal with the dilemma, hikers sometimes use mask brackets to improve their breathing and thermal comfort, since mask brackets can help keep a constant-volume microenvironment for heat and air exchanges. With the use of mask bracket, we found a decrease in T a but an increase in RH inside the cavity ( Fig. 6a and b), possibly due to enhanced sweat evaporative cooling. However, the accumulated moisture inside the cavity may lead to another possibility of heatstroke due to a reduced hydration level since the damp PFM microenvironment tends to make people feel fewer signs of thirst [50].
Implications on public health and policymaking
The COVID-19 pandemic has amplified health risks in hot seasons for people worldwide, especially for the following groups who are vulnerable to both infection risk and heat stress risk, including people with underlying conditions (respiratory, cardiovascular and cerebrovascular diseases as well as mental health issues), the elderly, children, pregnant women, outdoor workers, athletes, health workers, the economically disadvantaged who might lack of essential cooling and hygiene facilities, and those who have been first infected and then discharged from hospitals after treatment with COVID-19 [1,5,41]. During the pandemic, the use of PFMs in hot seasons can lead to more hospitalization rates of heatstroke due to an aggravated level of thermal stress and a reduced level of hydration since the damp and humid PFM microenvironment tends to make people feel less signs of thirst, according to a report "Wearing masks in summer can lead to heatstroke; Japan doctors urge self-hydration" from the Japan National Daily News [50]. Therefore, it is of great importance for the government and public to be better prepared for the dual challenges of infection risk and heat risk. For example, the Centers for Disease Control and Prevention (CDC) in the US published a document "Employer information for heat stress prevention during the COVID-19 Pandemic" on 26 Aug 2020 [52], which provides heat illness prevention guidelines for employers to better protect outdoor workers from the dual risks of the pandemic and heat stress.
In this study, we predicted a fine-resolution PFM-inclusive thermal stress and heat warning map covering Hong Kong, which can offer significant guidance for the public and the government. For example, the government may build more cooling and rehydration infrastructure in the neighbourhoods that prone to PFM-inclusive heat stress. Urban Renewal Authority (URA) can boost more air-conditioned bus depots [55] in the main public transport interchanges. More pavilions, pergolas and gazebos with hydration stations can be built along the hiking trails to protect the hikers from heat-related risks such as thermoplegia and dehydration. Besides, an early warning system needs to be established via the cross-departmental coordination between Hong Kong Observatory (HKO), Information Service Department (ISD), Centre for Health Protection of Department of Health (CHPDH), and Interactive Employment Service of Labor Department (IESLD). The system can provide heat illness prevention guidelines for employers to better protect outdoor workers from the dual risks of the pandemic and heat stress. The procedures may include adjusting work/rest schedules and daily work completion targets during hot weather, providing more cooling and rehydration stations, and making emergency first-aid plans [52]. On the other hand, the public can also benefit from this study, such as planning or altering their outdoor activity schedules according to the PFM-inclusive heat risk forecasts. For example, traffic policemen and building/road construction workers shall be aware of the aggravated thermal stress in their working areas and make good preparations beforehand, pedestrians can choose paths crossing more comfortable neighbourhoods (shaded areas, green/blue spaces, relatively flat paths) with less heat risks, and hikers can make less dangerous plans such as choosing hiking trails with smaller terrain slopes, more tree canopy shade, or more pavilions, using mask frame to improve thermal and breathing comfort, and bringing spare clean masks to replace sweated ones.
Concluding remarks
In this study, we investigated the aggravated human thermal stress due to PFMs over Hong Kong via both field experiments and model simulations. In the field experimental phase, we conducted detailed observations on facial thermo-physiological responses and the microenvironment within the PFM cavity. It was found that large amounts of facial heat fluxes from skin and respiration (50-500 W/m 2 ) can be trapped in the hot and humid PFM cavity (T a : 34-35 • C, RH: 80-95%) due to restrained heat dissipation, which leads to aggravated facial thermal burden, especially during high-intensity activities. In the model simulation phase, first we developed a new human thermal stress model named HEATS by considering modified human-environment heat exchanges due to PFM wearing under realistic climatic and topographical conditions. This new model was then applied to simulate spatiotemporal variations of PFM-aggravated human thermal stress during a typical heat wave event (22)(23)(24)(25)(26)(27) June 2016) over Hong Kong. Our model simulation results showed that the use of PFMs can significantly aggravate human thermal stress over Hong Kong with a PST increment of 7.0-8.2 • C and additional 158.4% neighbourhoods exposing to the severest heat risks (Level 4-swealtering). Particularly, urban residents may suffer more intensive PFM-aggravated thermal stress at night than rural residents due to synergistic urban heat and moisture island effects. Besides, PFM-inclusive PST was found to increase nonlinearly with terrain slopes at a rate of 1.3-3.9 • C/10 • , owing to increased metabolic heat production when climbing on inclined terrains, which is usually overlooked but very important for residents in mountainous cities. Residents walking on a flat terrain (0 • ) with PFMs may feel equivalent thermal stress as walking on an 30 • inclined terrain without PFMs. In addition, we found that PFMs not only affect people's thermal comfort, but also breathing comfort. Particularly, a high concentration of CO 2 and a low concentration of O 2 were found inside sweaty PFMs, which may cause severe hypoxia and possible disorder of vulnerable people' lungs. It is noteworthy that the use of mask bracket can offer a promising thermal stress mitigation strategy for people during high-intensity activities, since mask bracket is useful to keep a constant volume of the PFM microenvironment for heat and air exchanges. Our timely study on PFM-aggravated thermal stress may offer significant implications for the government, the public, as well as mask design companies under the dual challenges of extreme hot weathers and pandemics.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | 2021-09-01T13:16:08.433Z | 2021-09-01T00:00:00.000 | {
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133120089 | pes2o/s2orc | v3-fos-license | RHODODENDRONS IN UTTARAKHAND : DIVERSITY AND CONSERVATION
Rhododendron is a very widely distributed genus, occurring throughout most of the Northern Hemisphere except for dry areas, and extending into the Southern Hemisphere in southeastern Asia and northern Asia with 1200 species. In India, Rhododendrons are represented by 80 species with 10 subspecies and 14 varieties most of which are widely distributed in the Himalayas at an altitude ranging from 1500 – 5500 m. During the course of a revisionary study of Rhododendrons in Uttarakhand, an attempt has been made to collect information on the uses of different species from the field through personal communications with the inhabitants of the remote villages and through repeated inquiries from local folk. Besides of its immense horticultural importance, about 6 species have been recorded here which are used by the local people in different ways. Some species are also found poisonous. The paper deals with the enumeration of these species, their vernacular names, distribution and abundance, uses and methods of utilization. Natural calamities anthropogenic activities such as deforestation, unsustainable extraction for firewood pose a serious threat to Rhododendrons.
Introduction
The term 'Rhododendron' comes from the Greek word 'rhodo' meaning 'rose' and 'dendron' meaning 'tree', in combination 'rose tree' (Hora, 1981).The genus Rhododendron is an ecologically and economically important group of plants that occur in temperate forests.
Rhododendron is a relatively primitive group of flowering plants that have flourished in the temperate zones of the northern hemisphere for almost 100 million years ( de Milleville, 2002).Towards the equator, this genus is mainly distributed at higher altitudes, and today some species have significant ecological and economic importance (Mao et al., 2001).
Species height range from 2.5 cm (1 inch) alpine plants to 30 m (98 feet) tall trees and are either evergreen, semi-deciduous or deciduous (Hora, 1981).
The aesthetic values of Rhododendrons are significant and it is recognized regional flower in the American States of Washington (Rhododendron macrophyllum) and West Virginia (Rhododendron maximum) and in Japan"s Shiga Prefecture (Rhododendron metternichii var.hondoense), and Rhododendron arboreum is Nepal"s national flower and is depicted on its coat of arms ( de Milleville, 2002).In India, Rhododendron is the state flower of Himachal Pradesh (Rhododendron campanulatum) and Nagaland (Rhododendron arboreum) and is the state tree of both Sikkim (Rhododendron niveum) and Uttarakhand (Rhododendron arboreum, Kant, 2004;Joshi and Sharma, 2005).
Due to human interference natural calamities, the natural populations of Rhododendrons in Uttarakhand and in the entire Himalaya are gradually diminishing.The major threats to Rhododendrons are deforestation, unsustainable extraction for firewood, natural calamities like (Landslides and flash floods) and incense by local people.So, the present task of diversity and conservation status of Rhododendrons of Uttarakhand has made to know the distribution and status in Uttarakhand.
Results and Discussion
Rhododendron has the greatest number of species of all genera in the family Ericaceae, with more than 1200, known to occur throughout the world stretching from the highlands of Nepal, India and China (east of Yunnan and Sichaan) and Malaysia (Leach, 1961;Chamberlain et al.,1996, Rotherham, 1983) Pradesh alone.Uttarakhand is situated in the lap of Western Himalaya famous for its rich heritage, culture, biodiversity, flora, and fauna.Rhododendrons are mainly found at higher altitude, from dominating species all along the cool temperate, subalpine and alpine zones.
Rhododendrons, in general, prefer to grow in regions of high rainfall, high humidity, and a temperate climate, also having a preference for acidic soils.
History of Rhododendrons in world and in India
The first record of a Rhododendron in cultivation in Britain is of R. hirsutumin 1650.(Magor, 2008).Rhododendron arboreum was first species discovered and identified by specialist near Srinagar in 1796 (de Milleville, 2002), but there is no authentic record of its introduction before 1817.R. campanulatum from Nepal followed in 1825, R. barbatum in 1829 and R. formosum from Assam in 1843 (Magor, 2008).Pioneer
Distribution of Rhododendrons
In consideration of many aspects of Rhododendron, the geographical distribution of the genus the large aggregation of species in the great clefts and gorges of W. China, N.E.
Burma, and S.W. Tibet.The genus extends broadly in three directions.Firstly, the least vigorous extension extends westward through the Himalaya and the Caucasus to the Alps of Europe, giving us such well-known species as R. ponticum, R. caucasicum, R. ferrugineum, and R. hirsutum.Secondly, we have an extension eastwards through east and N.E.Asia across to N. America, and thirdly, and at the present time most pertinently, the extension South -a vigorous generic probe terminating with R. lochiae in Queensland and with R.
subpacificum and other species in the Solomons (Black, 2008).The genus Rhododendron includes 850 species mainly in the northern hemisphere (Mabberley, 1997).Rhododendrons are found in a number of humid and cool regions across the northern hemisphere.These plants generally do not grow in low altitudes, preferring mountainous areas that have a temperate climate, although there are some that do flourish in alpine conditions.The rhododendrons are best suited to elevation between 2000-4000m that extends from eastern Nepal to Yunnan ( de Milleville, 2002).The distribution of Rhododendron as natural habitat is found in India, China, Japan, Burma, Malaysia, Borneo, Sumatra, Java and New Guinea, Britain, Bhutan, Europe.All the six species of rhododendron distributed in Uttarakhand and other states of Himalayas have not been yet evaluated for their status so, a thorough study is needed to know the status of these keystone species in Uttarakhand and other states of Himalaya.(Srivastava, 2012).In Uttarakhand, it is widely popular for processed juice of its flowers which have gained market popularity as Rhodojuice/Sharbat.It a small evergreen tree often with a somewhat crooked trunk.Bark soft, easily cut through with a pocket knife, 0.5-1 inch thick, old bark grey, exfoliating in irregular longitudinal plates, exposing the smooth pinkish new bark beneath.The wood is of inferior quality, both as timber and as fuel.
Distribution and habitat: -This is a common tree in western Himalaya, occurring chiefly at 2500 -2800 meter in association with Quercus leucotricophora and Lyonia ovalifolia, and at the lower elevations with Pinus roxburghii, but ascending to 3400 mt. or even higher.It is somewhat rare in hazara, being commonest in the Siran Pinus longifolia forests at 1400 meters and upwards in moist ravines.It extends to the eastern Himalaya, where, it is less common; it is also found in the Khasi hills and the hills of Burma, southern India, and Ceylon.India in the east with populations in adjacent areas of S Tibet.This species is quite common in the wild, sometimes occurring as solid stands in forest openings but more commonly seen as scattered individuals in coniferous and mixed forests.It is found from 8,000 to 12,000 feet
Flowering and
(2,400 to 3,700m) in elevation and typically grows as a large upright shrub or small tree.
R. Campanulatum
It is commonly called as Chimura, simris in Garhwal region and in Hindi it is pronounced as Cherailu.An evergreen shrub or a small tree having height of 3 to 5 meter and girth up to 90 cm.Bark thin, cinnamon-colored or grey, smooth, peeling off in thin, papery flakes; leaves elliptic or ovate, leathery, 7-15 cm x 3-6 cm.undersurface with rusty-brown tomentum.
Distribution and Habitat:-Rhododendron campanulatumspeciesaeruginosum is a wild species rhododendron found in the Himalayan alpine regions of Northern India (Uttarakhand) and Northeastern India (Sikkim), Bhutan, and Nepal.It grows on the stony alpine slopes and ledges at altitudes of 3200 to 3400 meters.This species generally form a bridge between timberline and alpine zone (meadows) known as ecotone.
Flowering and Fruiting:-Rhododendron flowers grow in large trusses, or clusters, which can be up to 10 inches across.Each flower is shaped like a small bell about 3 to 5 cm long.
Flowering: May-Jun, from Jul-Sep. and Badola, 2008).While R. anthopogon leaves are mixed with Juniper species to provide incense that is widely used in Buddhist monasteries.The fruits are the favorites of birds, which also disperse the seeds.Furthermore, the plant provides the very good fuel which results in the degradation of this species in many areas.Ecosystem services are also an important area where Rhododendrons play a vital role.Rhododendrons grow in areas of high rainfall and high humidity on acidic soils; conditions under which few plants would survive.
Therefore their role in slope stabilization and watershed protection should not be underestimated, particularly in the Himalayas where so many of Asia"s major rivers start; nor should we overlook the role of Rhododendrons in providing the structure of plant communities which support a wealth of biodiversity.Thus, the Rhododendron plays a pronounced role as a keystone species, provides an ecological stability to the vegetation communities and associated niche of the region.Therefore, restoration of Rhododendron and their conservation in nature promotes an existence of other biodiversity components.
Similarly on considering subalpine to alpine transition zone that includes timberline is the most fragile ecosystem in the Himalaya.Rhododendron is the only group of plants that has an existence in the aforesaid ecotone and beyond doubt maintains the biological sustenance in this fragile zone.Hence, after knowing the importance of these species which prove its dominant existence in higher altitude vegetation needs conservation as changes in climate particularly in temperature of earth definitely will affect the life cycle of this important species or may resultin extinction.
botanistJoseph D. Hooker (1817-1911) undertook a trip to Nepal but he could not see any Rhododendron blooming and continued his exploration in Northern India.He made an extensive tour of the Sandakphur region and the Singalila range to the northwest of Darjeeling.His famous work, "The Rhododendrons of the Sikkim-Himalaya," (1849) is considered as the standard test for the study of Himalayan Rhododendron.
Fruiting: -The large showy crimson, sometimes pink, flowers in dense corymbs appear usually from March to May, but in certain years only partial flowering takes place then, and a second flowering takes place in June and July; this happened in the Shimla hills in 1916, following an exceptional dry winter and spring, and the flowers of the second bloom were paler in colour than usual.Similar late flowering is also said to take place if the first bloom is checked by hail or other elated injuries.Occasionally trees may be seen in flower in January-February.The fertilization of the flowers is carried out partly by insects.Mr. G.B.F Muir notes on interesting case observed in Tehri Garhwal of Indian martens (Martes flavigula) visiting one cluster of flowers after another and thrusting their noses into the flowers to lick up the nectar; fertilization is thus carried out by their agency, and possibly birds may also be agents in cross-fertilization.The capsules are 2 to 3 cm long by 1 to 1.5 cm diameter, oblong, curved, greenish brown when ripening, and then turning brown.They contain a large number of minute dark brown compressed oblong seeds about 0.05 in.long, with a fimbriate tuft at either end.The capsules open and shed their seeds chiefly from January and March (western Himalaya).The open capsules, as a rule, remain many months on the tree.Plate 1: Rhododendron arboreum in Phairikhal R. barbatum An evergreen tree, up to 15m.in ht.; found in the Himalayas from Kumaun to Bhutan, extending into the Aka hills and Balipara tract of Assam, at altitudes of 2,100-4,000 m.Bark purple red, smooth, peeling off in large flakes; leaves ellipticlanceolate or oblong, 10-20cm.x 4-7cm.; young leaves woolly underneath.Distribution and habitat: -It is native over much of the Himalayan Range, from Uttarakhand in the west, through Nepal, Sikkim & Bhutan to western Arunachal Pradesh, is locally called as Konthya or Dhoop and its height is up to 40 cm.found in bushes habitat.It has a little yellow flower and indicates as a threatened medicinal herb.The leaves of which are used for essence in religious ceremonies by Buddhists all over its distribution range in Himalaya.The leaf of R. anthopogon is also used in tea.The leaves are reported to possess stimulant properties.The plant yields incense.The associate of R. anthopogon is dwarf shrubs Berberis kumaonsis, Juniperus communis, R. lapidotum and Lonicera obovata.Distribution and habitat: -This is a common shrub in western Himalaya Alpine and meadows eco-region occurring chiefly at 3800 meters in association with other species of Rhododendron lapidotum.It is native to the Himalaya from Bhutan to Central Nepal.In India, it mainly found on moist open slopes, hillsides, and ledges of cliffs of Uttarakhand and Sikkim.Flowering and Fruiting: It is known for its brilliant blossoming that covers hillsides.Inflorescence 4-6 (-9) flowered.Pedicel 0.2-0.4cm, scaly; calyx lobes 3-5(-6) mm, elliptic or oblong, persisting to enclose mature capsule, scaly, margin densely ciliate; corolla narrowly tubular-funnel form, pink, or yellowish white, tube 6-12 mm, outer surface not scaly; inner surface densely pilose; lobes spreading, 1.2-2 cm; stamens 6-8 mm, included in corolla tube, filaments glabrous; ovary ca. 1 mm, scaly; style short, thick, straight, as long as ovary, glabrous.Capsule ovoid, 3-5 mm, scaly, Flowering: May-Jun, fr.Jul-Aug.R. anthopogon in TungnathR.lapidotum:-R.lapidotumis growing up to 3800-4500 m and prefer ridges for growing.It occurs a height up to 35 cm.just less than R. anthopogon.R. lapidotumwas commonly observed with pink flowers along open mass covered rocky stone.However, this species has a wide distribution from Pakistan and across Western and Central Himalaya to Southwest China between 2,500-4,500 m where it prefers rocky and stony grassy slopes.The R. lapidotum is most dominant in Kedarnath Wild Life Sanctuary in Tungnath.
International Journal of Environment ISSN 2091-2854 geographical
th state of the Republic of India lies between 28 o 44' & 31 o 28' N area of the state, about 19% are under permanent snow cover, glaciers and steep slopes where tree growth is not possible due to climatic and physical limitations(FSI, 2009).
(FSI, 2015)r of 45.32% of the total geographical area of the state(FSI, 2015).Out of the total Fig 1: Map of the study area
Table 1 : Distribution of Rhododendron in Uttarakhand, India and in IHR Name of the taxa
Evaluated; NG -Nagaland; SK -Sikkim; UK -Uttarakhand; WB -West Bengal Rhododendron
in Uttarakhand and their uses Rhododendron arboreum
Amongst the Indian species, Rhododendron arboreum Smith (Ericaceae) is the most widely distributed and occur from the western to the eastern Himalayan region of India and other neighboring countries.Rhododendron arboreum is the state tree of Uttarakhand.It is called Burans, "Bras" and "Buras" in the local dialect.Rhododendron arboreum holds the Guinness Record for World Largest Rhododendron and is widely popular for its medicinal benefits & economic value
Table 2 : Uses of Rhododendron Species
(Badola, 1992) keystone species of the biodiversityRhododendron plays an important role beyond admiring as keystone species of Himalaya region.It is one of the most important genera of the Himalayan region which has a major use in landscaping, accent, and woodland planting.It has the potential to attract tourism in the Himalayan region through its scarlet blooming in the flowering season which results in generating employment for local people and consequently boost up the needs of local people.Beside flourishing tourism, Rhododendron species has medicinal uses which increase its importance more.R.arboreum flower petals are used in making health juice(Badola, 1992)and to stop excessive bleeding in female when mixed with water (Pradhan | 2018-12-10T23:02:11.251Z | 2017-02-28T00:00:00.000 | {
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240760188 | pes2o/s2orc | v3-fos-license | Use of endoscopic ultrasound‐guided fine needle aspiration of the pancreas to diagnose a case of primary linitis plastica of the colon with retroperitoneal dissemination
ABSTRACT A 54‐year‐old man had previously undergone curative sigmoidectomy for poorly differentiated adenocarcinoma with a signet‐ring cell component of the sigmoid colon, which was characterized morphologically by stenosis and inelasticity of the colon (linitis plastica). Six weeks after surgery, the patient developed stenosis of the right ureter. Disseminated sigmoid cancer was suspected, and chemotherapy was started. Nine months after initiation of chemotherapy, obstructive jaundice was observed which was due to stenosis of the distal bile duct (BD). Although computed tomography showed no evident metastatic lesion that could cause the stenosis, swelling of the entire pancreas was evident compared to that of 11 months earlier. Endoscopic ultrasound (EUS) also did not detect any focal masses in the head of the pancreas, although there was a diffuse hypoechoic change in the entire pancreas. Histopathology of the stenotic BD and biopsy specimen from the head of the pancreas showed no malignant cells. Two months after the initial endoscopic bile duct drainage, the patient was admitted again for epigastric pain. A second EUS fine needle aspiration (EUS‐FNA) of the head of the pancreas was performed and showed poorly differentiated carcinoma with some signet‐ring cells. This finding provided histological confirmation of a disseminated pancreatic lesion of the previously resected linitis plastica of the sigmoid colon. This is a rare case of disseminated pancreatic lesion from primary linitis plastica of the colon diagnosed by EUS‐FNA.
INTRODUCTION
Metastatic pancreatic lesions derived from tumors such as renal cell carcinoma and nonsmall cell lung cancer are known to have well-defined margins, 4 and it has been reported that histological evaluation of a biopsy specimen is useful for definitive diagnosis. 5 However, there have been no reports of metastatic or disseminated pancreatic lesions from linitis plastica of the colon. In this paper, a case of a disseminated pancreatic lesion from primary linitis plastica of the sigmoid colon diagnosed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is reported, together with the imaging characteristics on computed tomography (CT), magnetic resonance imaging (MRI), and endoscopic ultrasound (EUS).
CASE REPORT
A 54-year-old male visited our hospital complaining of constipation and small-caliber stools for 1 month. Colonoscopic examination showed marked narrowing of the sigmoid colon lumen with edematous mucosa (Figure 1a). Histological examination of a biopsy specimen demonstrated poorly differentiated adenocarcinoma. Abdominal CT showed long segmental narrowing of the sigmoid colon (Figure 1b), no metastatic lesions in the lung and liver, and no ascites. The patient was diagnosed with primary linitis plastica of the sigmoid colon with poorly differentiated adenocarcinoma, and sigmoidectomy was performed (Figure 1c). Histopathological examination of the resected specimens showed poorly differentiated adenocarcinoma with signet-ring cells (Figure 1d, arrow) infiltrating into the colonic mucosa with venous and lymph duct invasion and lymph node metastasis. The final diagnosis was pT3, pN3, cM0, stage 3b, according to the 8th edition of the TNM classification, with curative resection.
CT performed 6 weeks after surgery showed right renal pelvis dilation due to stenosis of the right ureter and para-aortic and mesenteric lymphadenopathy, which suggested retroperitoneal dissemination. This prompted initiation of FOLFOX (5-FU, leucovorin and Figure S1a and b). EUS (GF-UCT260; Olympus, Tokyo, Japan) also showed no distinct masses, but the entire pancreatic parenchyma was hypoechoic ( Figure S2). Compared to the head of the pancreas (Figure 3c, square), diffusion-weighted MRI demonstrated slight abnormal diffusion restriction in the body and tail of the pancreas (Figure 3c), but no areas showed focal attenuation of the apparent diffu-sion coefficient (ADC) value in the pancreas (Figure 3d). Histological examination of a BD biopsy performed during endoscopic bile duct drainage (EBD) and cytopathological examination of EUS-FNA found no malignant cells. There were also no malignant cells in a right inguinal lymph node that was excised. Two months after the EBD, the patient was admitted with epigastric pain. CT showed swelling of the adrenal glands and peripancreatic soft tissue infiltration (Figure 2d). In contrast to the atrophic change of the body and tail of the pancreas, swelling and mass formation were evident in the head of the pancreas (Figure S1e and f ). A second EUS-FNA of the head of the pancreas was performed using an Olympus 22-G FNA needle (NA-U200H; Olympus, Tokyo, Japan). The results revealed that the head of the pancreas seemed to be enlarged and its outline changed to being rounder in shape (Figure 3a and b and Supporting Information Video) compared to that of the first EUS. The biopsy specimens showed poorly differentiated carcinoma and some signet-ring cells (Figure 4a and b). Tumor cells were negative for cytokeratin seven and positive for cytokeratin 20, CDX2, SATB2, F I G U R E 3 (a) Image obtained during the second EUS-FNA from the descending duodenum. Head of the pancreas seemed to be enlarged and its outline changed to being rounder in shape compared to that of the first EUS. (Figure 4c, Figure S3), suggesting that the tumor cells originated from the colorectal epithelium. Immunohistochemical evaluation of the biopsied specimen was consistent with the histology of the previously resected linitis plastica of the sigmoid colon. The patient's liver function gradually deteriorated due to multiple stenoses of intrahepatic bile ducts, which appeared within 3 weeks after his second hospitalization. The patient eventually died of liver failure 15 months after initial surgical resection.
DISCUSSION
Laufman et al first reported primary linitis plastica of the colon in 1951. 6 It was described as a scirrhous carcinoma with diffuse thickening and hardening of the bowel wall. The histological characteristics included a scirrhous reaction by poorly differentiated adenocarcinoma to signet-ring cell carcinoma. 2 It is known to develop at an earlier age and metastasize rapidly to lymph nodes and disseminate to the peritoneum.
The reported incidence of patients with colorectal cancer exhibiting a component of signet-ring cells comprising <50% of the tumor mass is 5%. 7 This histological type is known to have poor survival rates compared to common histological types consisting of well-differentiated and moderately differentiated adenocarcinoma cells. The majority of metastatic pancreatic lesions originated from renal cell carcinomas (63.6%, 14/22), followed by colon adenocarcinomas (9.0%, 2/22) and nonsmall cell lung carcinomas (4.5%, 1/22). 8 Sakai et al reviewed a series of 59 Japanese cases of pancreatic metastasis from colorectal cancer and reported that in 91.5% (54/59) of the cases, the histology was well-differentiated and moderately differentiated adenocarcinoma, and the CT imaging showed a lobular mass with poor enhancement, 9 different from the present case. Prior to this, there have been no previous reports of pancreatic metastases or dissemination of primary linitis plastica of the colon.
The usefulness and high accuracy of EUS-FNA in diagnosing the etiology of several diseases have been widely accepted, 5 and it can be used to investigate the etiology of pancreatic lesions. In the present case, biopsy specimens from the head of the pancreas obtained by the first EUS-FNA showed normal acinar cells, whereas those by the second EUS-FNA showed poorly differentiated carcinoma with signet-ring cells. It is suspected that the reason for the success of the second EUS-FNA in arriving at a diagnosis is an increase in the number of tumor cells in the puncture site. Drastic changes in CT findings and rapid disease progression after the second EUS-FNA may reflect the aggressive cell proliferation in the retroperitoneal space. Therefore, further examination of similar cases may be needed to establish the number of biopsies and the required puncture procedures to accurately diagnose metastatic lesions with scirrhous carcinoma.
The characteristics of pancreatic metastases are reported to be lesions with regular borders, lack of retention cysts, and presence of a nondilated MPD. 4 In the present case, despite the MPD and BD that were markedly stenosed in the head of the pancreas, EUS neither showed any well-demarcated lesion in the area nor any masses with poor enhancement on CT. These features are rather reminiscent of so-called mass-forming pancreatitis (MFP). Similar to MFP, there was no focal attenuation of ADC values throughout the pancreas, including the head of the pancreas. 10 These imaging characteristics could suggest the characteristics of disseminated pancreatic lesions of linitis plastica of the colon.
In summary, this is the first report of a case of disseminated pancreatic lesions of linitis plastica of the colon diagnosed by EUS-FNA, with characteristic features on CT, MRI, and EUS. Repeated EUS-FNA might be necessary to make the histological diagnosis because the poorly cohesive cancer cells infiltrate diffusely and do not form masses in the metastatic organs.
C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.
F U N D I N G I N F O R M AT I O N
None.
S U P P O R T I N G I N F O R M AT I O N
Additional supporting information may be found in the online version of the article at the publisher's website.
Supplemental Video. Second endoscopic ultrasoundguided fine needle aspiration of the head of the pancreas is performed from the descending duodenum. | 2021-09-28T18:08:42.137Z | 2021-07-05T00:00:00.000 | {
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5122919 | pes2o/s2orc | v3-fos-license | On the topology preservation of Gneiting's functions in image registration
The purpose of image registration is to determine a transformation such that the transformed version of the source image is similar to the target one. In this paper we focus on landmark-based image registration using radial basis functions (RBFs) transformations, in particular on the topology preservation of compactly supported radial basis functions (CSRBFs) transformations. In [1] the performances of Gneiting's and Wu's functions are compared with the ones of other well known schemes in image registration, as thin plate spline and Wendland's functions. Several numerical experiments and real-life cases with medical images show differences in accuracy and smoothness of the considered interpolation methods, which can be explained taking into account their topology preservation properties. Here we analyze analytically and experimentally the topology preservation performances of Gneiting's functions, comparing results with the ones obtained in [2], where Wendland's and Wu's functions are considered.
Introduction
In medical image analysis, registration is a crucial step to analyze two images taken at different times or coming from different sensors or situations. The problem of image registration can intuitively formulated in the following way: given two images, called source and target images, respectively, find an appropriate transformation between the two images, such that it maps the source image onto the target one. The differences between the two images can derive from different conditions, and for analyzing them we want to make images more similar each other after transformation, for an overview see e.g. [3,4,5,6,7,8]. In particular, in [8], Zitová and Flusser gave an overview of image registration, and presented different methods to solve this problem.
Some methods for image registration are based on landmarks. The landmark-based image registration process is based on two finite sets of landmarks, i.e. scattered data points located on images, where each landmark of the source image has to be mapped onto the corresponding landmark of the target image (see [4,5,6]). The landmark-based registration problem can thus be formulated in the context of multivariate scattered data interpolation. In landmarkbased image registration, the deformed results are sensitive to the displacements of landmarks. If the displacement of one landmark is far enough from neighborhood landmarks, the deformation will be large and the geometrical structure will change after transformation. In such case, topology violation could thus occur.
One of the most used methods in landmark-based image registration is the radial basis functions (RBFs) method, useful to handle various geometric deformations. RBFs are functions whose values depend on the distance between points and centers [13,9]. This important property allows the use of RBFs in interpolation problems, such as image registration. RBFs can be classified in two groups: (i) globally supported such as thin-plate spline (TPS) and Gaussian, and (ii) compactly supported such as Gneiting's, Wendland's and Wu's functions, see [1,10,11,12,14], respectively.
In general, globally supported RBFs (GSRBFs) can guarantee the bending energy be small but the deformed field will occur in the whole image after transformation; conversely, compactly supported RBFs (CSRBFs) make the influence of the deformation local: around a landmark in 2D images on a circle whereas in 3D images on a sphere.
In [1,15], authors analyzed different computational properties of GSRBFs and CSRBFs for landmark-based image registration.
However, if GSRBFs or CSRBFs are chosen to solve the registration problem, topology should be preserved. For some RBFs the presence of a shape parameter is an important characteristic. In fact, a large shape parameter leads to flat basis functions, whereas a small shape parameter results in peaked RBFs [9]. Therefore, we can use it to control influences on the registration result [18].
In [2] evaluation of topology preservation for different CSRBFs in case of landmark-based image registration was performed. The authors compared topology preservation of different CSRBFs using two criteria: locality parameter and positivity of determinant of the corresponding Jacobian matrices for different transformations. Locality parameter is the optimal support size of different CSRBFs under topology preservation condition. In [19], we instead evaluated the performances of topology preservation for a GSRBF family such as Matérn functions in landmark-based image registration.
The family of Gneiting's functions was proposed by Gneiting in 2002 for the first time [11]. Gneiting's functions are oscillatory compactly supported functions and it is known in scattered data interpolation that they achieve good approximation results [9]. Then, they were used also in image registration, obtaining accurate results [1,16]. For this reason in this paper we analize the topology preservation of Gneiting's transformations under the two criteria given above and compare the numerical results with those obtained in the paper [2] using Wendland's and Wu's functions.
The paper is organized as follows. Section 2 introduces the landmark-based image registration problem. Section 3 gives definitions of two kinds of Gneiting's functions and of the associated transformations. In Section 4 we evaluate topology preservation of Gneiting's functions in the case of one-landmark. Computations and numerical results are presented. Section 5 deals the four-landmarks schematic diagram case and the landmark-based registration in brain images. Finally, in Section 6, we report the conclusions and the future work on the topic.
Landmark-based image registration problem
In this paper, we only consider the 2D case. For landmark-based image registration, we define a couple of landmark sets S N = {x j ∈ R 2 , j = 1, 2, ..., N} and T N = {t j ∈ R 2 , j = 1, 2, ..., N} corresponding to the source and target images, respectively. The registration can be described as follows.
The aim is to find a proper transformation R : R 2 → R 2 between S N and T N , such that For image registration, we can interpolate displacements to fulfill the deformation. As we mentioned in Section 1, the influence of deformed field is limited by CSRBFs. Here the displacements can be displayed by a CSRBF interpolant R k : R 2 → R, k = 1, 2, of the form where Ψ stands for a CSRBF, r = x − x j is the Euclidean distance between x and x j , and the coefficient α jk can be calculated by solving two linear systems. In this way we obtain the transformation R. Following [9], when functions Ψ are strictly positive definite, the matrix is invertible since all the eigenvalues are positive. Therefore, we have a unique solution of the two linear systems. Hence in this paper, all of the CSRBFs we consider are strictly positive definite. In the following we list two examples of Wendland's (ϕ 3,1 ) and Wu's (ψ 1,2 ) functions, i.e., where (·) + is the truncated power function and r c ≤ 1, c being the support function size. We remark that the larger (smaller) c is, the larger (smaller) the field is. We will use these functions to compare numerical results of topology preservation obtained by Gneiting's transformations.
Gneiting's functions and transformations
In this section we introduce the definitions of Gneiting's transformations. In 2002, Gneiting obtained a family of oscillating compactly supported functions [11]. Starting with a function ϕ m that is strictly positive definite and radial on R m , for m ≥ 3, and using turning bands operator [17], we get The latter is strictly positive definite and radial on R m−2 [9]. In order to obtain Gneiting's functions, we start with Wendland's functions, for example Using the turning bands operator, we thus obtain the functions which are strictly positive definite and radial on R 2 provided l ≥ 7/2. From this family we list two specific Gneiting's functions in C 2 (R), i.e., Under the landmark-based image registration context we define Gneiting's transformation as follows.
Definition 3.1. Given a set of source landmark points S N = {x j ∈ R 2 , j = 1, 2, ..., N}, and the corresponding set of target landmark points T N ={t j ∈ R 2 , j = 1, 2, ..., N}, Gneiting's transformation G : R 2 → R 2 is such that each its component assumes the following form According to Definition 3.1, the transformation function G k (x) : R 2 → R is calculated for each k = 1, 2, and the coefficients α jk are obtained by solving two systems of linear equations.
Topology preservation: the one-landmark case
From [18], under injectivity of map, the necessary conditions to preserve topology are that the function H : R 2 → R 2 is continuous and the Jacobian determinant is positive at each point.
In this case, the source landmark p is shifted by ∆ x along the x-axis direction and by ∆ y along the y-axis direction to the target landmark q. The coordinates of transformation are where Φ is any CSRBF.
The positivity of the Jacobian determinant requires i.e.
If we set ∆ = max(∆ x , ∆ y ), the value of θ minimizing the determinant in 2D is π 4 ; thus we get With the condition (8), one can show that all principal minors of the Jacobian are positive. It follows that the transformations defined by equation (3) preserve the topology if (8) holds. The minimum of ∂Φ ∂r depends on the localization parameter and therefore on the support size of the parameter c of Gneiting's functions.
In the next subsections we compute the minimum support size of locality parameter of Gneiting's functions (5) and (6), satisfying (8).
Gneiting τ 2,7/2
Considering Gneiting's function (5) with support size c, we look for the minimum value of c such that (8) the value of r minimizing (9) is Thus, evaluating (9) at (10) and replacing its outcome in (8), we obtain that the support size must be c > 3.60 √ 2∆ ≈ 5.09∆.
Gneiting τ 2,5
Focusing now on Gneiting's function (6), whose support size is always c, we search for the minimum c satisfying (8). Here we have while the value of r minimizing (11) is given by Evaluating (11) at (12) and substituting its result in (8), we obtain c > 4.43 √ 2∆ ≈ 6.26∆. Table 1 gives the locality parameters for the two Gneiting's functions (5) and (6), which are compared with those of Wendland's and Wu's functions (see [18,2]). As we mentioned in Section 1, the advantage of having small locality parameter is that the influence of deformed area at each landmark turns out to be small. This property allows us to have a greater local control. From Table 1, we get that the locality parameter of function ϕ 3,1 and ψ 1,2 are very similar. Moreover, the latter are smaller than those of Gneiting's functions. This means that the deformed field of Wendland's and Wu's transformations in one-landmark case is similar and its corresponding transformed area is smaller than the one of Gneiting's functions.
Topology preservation: the four-landmark case
For more extended deformations, we consider much larger supports which are able to cover whole image. Here, the influence of each landmark extends to the entire domain, therefore global deformations will be generated. In the following we compare topology preservation properties of large extended deformations, and we can set locality parameter large to fulfill this purpose. For this aim, we consider four inner landmarks in a grid, located so as to form a rhombus at the center of the figure, and assuming that only the lower vertex is downward shifted of ∆ [2]. The landmarks of source and target images are respectively, with ∆ > 0.
In this case, we explicitly write the two components of a generic transformation H : R 2 → R 2 obtained by a transformation of four points P 1 , P 2 , P 3 and P 4 , i.e.
We transform P i to Q i , with i = 1, . . . , 4, to obtain the coefficients c 1,i and c 2,i . To do that, we need to solve two systems of four equations in four unknowns, whose solutions are given by ∆, ∆, For simplicity, we denote Then the determinant of the Jacobian is det (J(x, y) For analyzing the positivity of the determinant of the Jacobian matrix, we calculate the minimum value at position (0, y), with y > 1. In the following, we analyze the value of the Jacobian determinant at (0, y), with y > 1, for these four CSRBFs. Because, in this case, we choose a very large parameter c, in order to get the value of the Jacobian determinant, we consider || · ||/c to be infinitesimal and omit terms of higher order.
Gneiting τ 2,5
Gneiting's function τ 2,5 can be approximated as Then, α and β can approximatively be represented as follows In order to evaluate (1 + β) 2 − 4α 2 , from approximations of α e β we deduce so we can obtain Referring now to (13), we can compute the following four partial derivatives for τ 2,5 , i.e., Then, replacing such derivatives in (13), we get We compare the approximations of (14) and (16) with the ones acquired by the work [2]. Figure 3 shows the same values of det(J(0, y)), with y > 1, when one uses as CSRBF transformations based on Wendland's, Wu's and Gneiting's functions. This indicates that functions ϕ 3,1 , ψ 2,1 , τ 2,7/2 and τ 2,5 have the same behavior. The equations obtained in [2] using Wendland's and Wu's functions and those deduced in (14) and (16) guarantee the Jacobian determinant is positive for any y > 1. This means all these transformations can easily preserve topology.
Numerical experiments
In this subsection, a schematic diagram of four landmarks and a real case of brain images are evaluated. Firstly, we consider a grid Taking c = 100 as support size, we obtain Figure 5.2 in case of CSRBFs.
In agreement with theoretical results, Figure 5.2 confirms that all these functions can preserve topology and all of them present very similar deformations, with the exception of the τ 2,7/2 function. In this case, we can see that, even if the support is very large, the image is only deformed slightly on local area. This is the best result obtained.
We can conclude that among these four functions, τ 2,7/2 can lead to good result in case of high landmarks density, i.e. when distance among landmarks is very small and each of them influences the whole image. Furthermore, it not only preserves topology, but also changes shape very slightly in the whole image when support is relatively large.
Actually, [1] reported the properties of τ 2,7/2 and τ 2,5 functions in the case of a large number of landmarks for square shift and scaling, and also for circle contraction and expansion. Numerical results showed that these two functions have good performances in those cases. In Figure 5.3, we show the case for c = 0.15 in which topology preservation is not satisfied. The transformed images are deeply misrepresented mainly close to the shifted point.
Moreover, a real medical case of landmark-based image registration is displayed in Figure 5.4, which focuses on two brain images. The image (a) is the source image with the corresponding landmarks marked by •, whereas (b) is the target image with the landmarks * . In this case, we choose two different support sizes for τ 2,7/2 in order to observe the various topology behaviours. More precisely, when c = 20, which is relatively a large value, the transformed image can preserve topology well. However, the opposite situation occurs when c is smaller, for instance equal to 2.
Conclusions
For image registration, we should guarantee that images preserve their original structure and they are not folded after deformation. Hence the transformations we use should be preserved topologically.
In this paper, we evaluated the topology preservation of two kinds of Gneiting's functions, and compared the results with Wendland's and Wu's functions in one-, four-landmark and medical brain image cases. In the first case, all functions have very similar performances. Conversely, in four-landmark and brain image cases, Gneiting's functions have better performances, especially the τ 2,7/2 function provides the best registration result. | 2016-12-14T16:34:03.000Z | 2016-12-13T00:00:00.000 | {
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246405997 | pes2o/s2orc | v3-fos-license | Comment on nhess-2021-399
The paper addresses a topic that is relevant, due to the great vulnerability and exposure that characterizes Rome from a seismic risk perspective. The high number of people who lives, works and spends holydays in this city, the critical infrastructure that characterizes the Capital of Italy, along with the value of its ancient building stock and cultural heritage, makes a detailed evaluation of the seismic hazard of this area very important to be carried out.
The paper addresses a topic that is relevant, due to the great vulnerability and exposure that characterizes Rome from a seismic risk perspective. The high number of people who lives, works and spends holydays in this city, the critical infrastructure that characterizes the Capital of Italy, along with the value of its ancient building stock and cultural heritage, makes a detailed evaluation of the seismic hazard of this area very important to be carried out.
For this reason, the paper is in principle of interest to the nhess audience and would deserve to be published. However, it is not ready for publication, as described below. In general, more care is needed in the drafting of the text, the citation of bibliographical references and the content of the figures.
1.
The general frame depicted in Section 2 would benefit from a wider description of the seismic events that hit Rome in the past, also including those occurred in "the period of ancient Rome, as well in the Early Middle Ages" (lines 84-86). This information can be easily retrieved in the available seismic catalogues and, in general, in the literature. This would show that comparable damage (e.g., intensity VI-VII) has been caused both by strong earthquakes with a far epicentre, e.g., in the Apennines chain, and by moderate events much closer. For this reason, defining the potential size of these moderate events significantly contributes to a better definition of the seismic hazard of the area. I suggest also adding a new figure with a graph or a table representing the seismic history of Rome.
In the same line, it would be useful to know the magnitude of the instrumental earthquakes. Are there any comparable with the 2020 Ml 3.3 event? In case, they could be outlined in Figure 1. Concerning this figure, there are also some details that need to be fixed: the blue star of the 2020 event is not so evident; there are letters A-B and C-D that are not defined in the caption; in the legend, Bulletin has two t; neither in the caption nor in the text a definition of G.R.A. is reported (only in Figure 8, at the end of the paper). Could you add the stream of the Aniene river? It would help compare this one with the other figures.
2.
The description of the structural setting could be more precise, even without being longer, and for this purpose an improved Figure 2 would be of great help. In general, this figure needs to be rethought for an international audience unfamiliar with the tectonics of Central Italy. Which is the age of the tectonic features reported (thrusts, normal and strike-slip faults)? Which are active today and which not? Why the extensional stress field in the Apennines has a different graphic than that along the Tyrrhenian margin? Is the stress field of the strike-slip faults no longer active (the retrieved focal mechanism has an opposite kinematics)? In the text, you talk about the volcanoes of the "Roman Province", whereas in this figure you represent the volcanic districts of the Tyrrhenian Sea margin. Could you homogenize the names, also highlighting the Colli Albani? Could you add a box corresponding to figure 1 and a graphic scale? Could you add any references in the figure caption? I suggest redrafting Figure 2 and then rewriting coherently the structural setting.
Concerning the morphological setting, Figure 3 is not centred on Rome and does not include the 2020 Ml 3.3. I suggest reframing the figure, expanding it to the North and to the West.
3.
The way the seismicity is addressed in the paper should be better organized. The seismicity of the area is described in Section 2. Section 4, called Seismicity, describes the data collection, but it also shows a part of methodological description, in particular the hypocentres relocation. It does not mention, however, the computation of the moment tensor solution, that is addressed directly in the Results, but is shown in Figure 4, to which Section 4 refers. Now it seems that the focal mechanism in Figure 4 comes from the literature.
Moreover, why only two out of the four networks described in the text at page 8 are shown in Figure 4? I suggest including all the networks in the figure 4, distinguishing them with different colours and/or symbols in the maps.
I also suggest adding a table with the list and the parameters of the Ml 3.3 event and the 4 aftershocks (magnitude, depth, etc.) mentioned at page 14. Why only 2 out of these aftershocks are shown in Figure 4?
This example highlights, as a more general point, the need of a clearer organization of the text. I suggest reviewing the Table of Contents of the paper, separating better the introductory framework, the data used, the methodologies adopted, and then results and discussion. Within each of these general topics, subsections regarding the different disciplines (seismology, geomorphology, tectonics, etc.) need to be included. Otherwise, as it happens now, you have a mix of literature, data and interpretations in many parts of the paper, and this does not help the reader.
4.
The morphotectonic analysis of the drainage network (Section 6.3) is a huge work, supported by several detailed figures. However, the tectonic lineaments that are present in all these figures do not allow a proper view of the data analysis, whose details are masked by the black lines. Therefore, on the one hand, I suggest removing the tectonic lineaments from Figure 6 a)
5.
The most critical point, in my view, is in the concept of "seismic intensity" that, according to the Authors, the analysed faults have now compared with that they had in the geological past. This concept, along with the seismic intensity of the area related to the Pleistocene stress field, is present since the beginning of the paper (page 7, from line 153), up to the Discussion.
I think that this concept should be totally revised. In general, seismogenic faults are not characterised by a "seismic intensity" but, rather, by a "seismic rate", that can be estimated if you are able to recognise one or more seismic events that they generated in the past (for instance from historical and/or palaeoseismological record), associated with a "geological slip rate" from structural, geomorphological and stratigraphic data.
In this study, the faults analysed are buried and blind, there are only hints of their activity at surface. Therefore, there is no information to assess which is their current and past activity and seismic rate.
Moreover, there are no data to discuss the "dimension" of the current and previous stress fields, although it is clear that the Middle Pleistocene tectonic activity, also responsible for the development of the volcanic district, was much more developed than the current tectonic activity.
I think that a scheme or a table is needed reporting (with refs) the orientation, kinematics and age of the different stress fields (including the strike-slip one of figure 2) that affected the study area through time and that are relevant to this study. Based on this, the inception, development, segmentation and possible reactivation of faults can be framed and discussed. This will allow the Authors to strengthen their idea that segmented faults with limited tectonic activity can be assigned a seismogenic potential for events with moderate magnitude.
Details
Page 3, lines 79-81 I suggest describing what the Greater Rome is: the Province of Rome?
Page 6, lines from 139 The name of the volcanic complex should be added. | 2022-01-30T16:25:36.739Z | 2022-01-28T00:00:00.000 | {
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246569399 | pes2o/s2orc | v3-fos-license | Approaching Well-Being 2.0: Nephrologists as Humans, Not Heroes
and increased costs to the health care system. Nearly half of practicing physicians met the criteria for burnout in a national study that assessed burnout using the Maslach Burnout Inventory in 2017. According to a survey conducted by the Medscape news website that examined burnout by medical specialty, nephrology fell in the middle of the pack in 2020, with 43% of responding nephrologists reporting burnout. The nephrology literature has seen an uptick in publications on burnout in recent years that emphasize implications of the burnout crisis and propose systemic solutions. Notably, nephrologists may be uniquely at risk of burnout. Our patients rank among the most medically complex, often requiring disproportionally more time and resources; our administrative burden is considerable; and our work is highly protocol-driven. Nephrology leaders have highlighted the corporatization of health care delivery as a potential driver of burnout. Furthermore, nephrology is experiencing a recruitment crisis that both contributes to and may be an unfortunate repercussion of our intense workload. In this issue of Kidney Medicine, Nair et al describe the development and administration of a 15-item survey that aimed to assess the prevalence and drivers of burnout among a sample of 457 practicing US nephrologists in 2019. The survey was developed by educational leaders at the National Kidney Foundation and contains a validated, 2-question measure of burnout adapted from the Maslach Burnout Inventory that asked participants to qualify how often they feel burned out from their work as a nephrologist and how often over the past year they had felt callous toward people. Respondents were queried about the top 3 factors that have contributed to their burnout from a list of determinants relevant to nephrology. These determinants map to 3 key aspects of the work environment that affect an individual’s level of well-being: the demands placed on us in the workplace, the support we receive from supervisors and colleagues, and the degree to which we have control over the work environment. The prevalence of burnout in this sample was 23%. This is considerably lower than that described in the aforementioned surveys, as well as lower than the rate in a 2018 study in which Pawlowicz and Nowicki reported that
and increased costs to the health care system. 1,2 Nearly half of practicing physicians met the criteria for burnout in a national study that assessed burnout using the Maslach Burnout Inventory in 2017. 3 According to a survey conducted by the Medscape news website that examined burnout by medical specialty, nephrology fell in the middle of the pack in 2020, with 43% of responding nephrologists reporting burnout. 4 The nephrology literature has seen an uptick in publications on burnout in recent years that emphasize implications of the burnout crisis and propose systemic solutions. [5][6][7][8] Notably, nephrologists may be uniquely at risk of burnout. Our patients rank among the most medically complex, often requiring disproportionally more time and resources; our administrative burden is considerable; and our work is highly protocol-driven. 9 Nephrology leaders have highlighted the corporatization of health care delivery as a potential driver of burnout. 10 Furthermore, nephrology is experiencing a recruitment crisis that both contributes to and may be an unfortunate repercussion of our intense workload. 11 In this issue of Kidney Medicine, Nair et al 12 describe the development and administration of a 15-item survey that aimed to assess the prevalence and drivers of burnout among a sample of 457 practicing US nephrologists in 2019. The survey was developed by educational leaders at the National Kidney Foundation and contains a validated, 2-question measure of burnout adapted from the Maslach Burnout Inventory that asked participants to qualify how often they feel burned out from their work as a nephrologist and how often over the past year they had felt callous toward people. 1 Respondents were queried about the top 3 factors that have contributed to their burnout from a list of determinants relevant to nephrology. These determinants map to 3 key aspects of the work environment that affect an individual's level of well-being: the demands placed on us in the workplace, the support we receive from supervisors and colleagues, and the degree to which we have control over the work environment. 13 The prevalence of burnout in this sample was 23%. This is considerably lower than that described in the aforementioned surveys, as well as lower than the rate in a 2018 study in which Pawlowicz and Nowicki 7 reported that 49% of practicing nephrologists in Poland experienced high levels of emotional exhaustion, 52% reported high levels of depersonalization, and 32% reported feeling a high level of reduced personal accomplishment. 3,4 The results of the study by Nair et al 12 are similar to those of a 2018 survey of US nephrology fellows, in which the overall burnout prevalence was 30%. 8 All 3 of these studies used validated measures of burnout-versions of the Maslach Burnout Inventory-although Pawlowicz and Nowicki 7 applied a longer instrument with relatively more emphasis on the personal accomplishment component of burnout. 1 Nair et al 12 point to the inability to identify a true response rate as a limitation of their study. Respondents were primarily men, trained in the United States, and practicing in academic settings. This may not accurately reflect the current workforce of practicing nephrologists, and it is difficult to know what we might have seen had they obtained more responses from women, internationally trained nephrologists, and those practicing in nonacademic settings. 14 Nonetheless, nearly a quarter of US practicing nephrologists have recently experienced burnout, and it is worth noting that these data were collected before the coronavirus disease 2019 era. In the years since, this same workforce has been practicing medicine in the midst of a pandemic, which for many of us has generated greater work intensity, an increased burden associated with caring for even more medically complex and critically ill patients, concerns about our safety in the workplace, and relative isolation. Leaders from the National Academy of Medicine Action Collaborative on Clinician Well-Being and Resilience have acknowledged the threat of a parallel pandemic of deteriorating clinician well-being and warned that we have a brief window of opportunity to get ahead of not only the spread of the virus but also the threat to clinicians' mental and physical health imposed by the coronavirus disease 2019 pandemic. 15 The primary drivers of burnout most commonly identified in the study by Nair et al 12 included the number of hours worked per week, electronic medical record requirements, the lack of time with family and friends, and clinic workload. Free text responses showed that burnout was driven by the following factors: challenges related to medical complexity, moral distress that comes from an inability to provide patients the care that they deserve due to a lack of resources, lack of autonomy, administrative demands, financial burdens, lifestyle intrusiveness, and concerns about innovation in the field. The comment "it has become hard to do the right thing" succinctly embodies the frustration felt by so many nephrologists over these issues. Of particular concern is an additional emerging theme captured in the qualitative responses that some nephrologists feel undervalued in their work. This seems to occur at all levels-by patients and family members, colleagues, local administrators, and the wider US health care system. Respondents who commented on the positive aspects of their work highlighted the importance of control over their time and feeling appreciated by patients, colleagues, and their employing organizations.
One cannot overestimate the importance of feeling that the work that we do makes a difference. A sense of personal accomplishment, which goes beyond simply feeling that we are competent at our job, is important for us to thrive in the workplace. Because it is less directly tied to negative outcomes, a sense of low personal accomplishment is often underemphasized as a dimension of burnout compared with emotional exhaustion and depersonalization. However, feeling chronically undervalued at work can lead to emotional exhaustion. Conversely, becoming more intentional about the aspects of work that we find meaningful can mitigate the effects of exhaustion, which may ultimately prevent us from experiencing depersonalization. In the study by Nair et al, 12 the quantitative assessment of burnout incorporates questions related to 2 of the 3 components of burnout. Specifically, they ask about both the subjective experience of burnout, which maps to the construct of emotional exhaustion, and the feeling of callousness toward other people, which corresponds to depersonalization; however, they do not specifically ask about having a sense of low personal accomplishment. The inclusion of a qualitative content analysis, a significant strength of this article, highlights the importance of underappreciation at work for some respondents and thus more completely captures the full picture of burnout faced by practicing nephrologists in the United States.
The antidote to a sense of low personal accomplishment is finding meaning at work, and a growing body of literature supports the notion that drawing our awareness to the aspects of work that are most meaningful is linked to a reduction in burnout. [16][17][18] As humans, we are naturally conditioned to focus on the negative and draining aspects of our life, including our work. To overcome this negativity bias, we should endeavor to notice the things at work that sustain us and bring us gratification and be deliberate in carving out time to do these things. Finding meaning at work, like all drivers of burnout and engagement, should be the shared responsibility of the individual and the system. 19 Health care systems ought to strive to provide a robust mentorship network to support physicians, with an emphasis on matching work to strengths and interests, and should promote an organizational culture that fosters appreciation and acknowledgment of physicians' performance.
Unfortunately, many health care organizations send the message to their physicians that it is the physicians' responsibility to become more resilient and to fix their own burnout. When organizations focus on physicians' personal resilience to the exclusion of addressing organizational factors, our natural response is to be skeptical, or even indignant. In a recently published article, Shanafelt, 20 the chief wellness officer at Stanford Medicine, outlined the evolution of the physician well-being movement. He divided this into 3 distinct eras: the Era of Distress (before 2005), in which there was a lack of awareness, or even neglect, of physician distress; Well-Being 1.0 (2005-2017), a period characterized by increasing evidence and awareness that physician well-being is important; and Well-Being 2.0, the future. 20 Efforts developed in Well-Being 1.0 were primarily focused on the individual and included things such as the expansion of mental health and peer support and efforts to cultivate resilience. In the process, physicians were given the message that they should take care of themselves, engendering a victim mentality and, for many, resentment. The progression to Well-Being 2.0 has brought a more proactive approach characterized by systems-based interventions to address the root causes of physicians' distress and to work to prevent it, rather than finger pointing, which has historically occurred bidirectionally between individual physicians and the systems in which they work.
Well-Being 2.0 is a call to action for 2 groups: administrators, to address system issues, and physicians, to recognize the things that are under our control and to work to incorporate mindfulness, self-compassion, boundaries, and self-care into our professional lives. In the ideal future, as Shanafelt 20 writes, we are not victims, nor are we heroes. We are humans; we are vulnerable; we thrive most when we find meaning and purpose in our work. | 2022-02-06T16:11:22.706Z | 2022-02-01T00:00:00.000 | {
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237821168 | pes2o/s2orc | v3-fos-license | Effectiveness of innovative instructional module for professional competence in health literacy in medical students
Background Physicians should be equipped with professional competence in health literacy to communicate more effectively with patients with limited health literacy. However, the health literacy curriculum has not yet been refined globally, and is scarce in Taiwan’s medical education. We implemented an innovative instructional module to attain professional competence in health literacy among medical students and investigated its effects. Methods We adopted a quasi-experimental design and recruited 204 fifth-year Taiwanese medical students between December 2019 and May 2020. Participants who worked as clerks at the Department of Family Medicine of three medical schools in northern Taiwan were assigned to the experimental group through convenience sampling. A total of 98 students received a three-hour innovative instruction, including medical simulation videos, role-playing, and board games. Both the experimental and control groups completed the online pre-test and mail-in post-test. A generalized estimating equation was applied to measure the effects of the intervention. Results There was a significant difference between the experimental and control groups in terms of professional competence in health literacy in all three aspects. In terms of knowledge, the experimental group improved 12% more than the control group (𝛽=0.12, 95% CI: 0.05 ~ 0.19, p = 0.001). In terms of attitude, the experimental group improved by an average of 0.27 more points per question than the control group (𝛽=0.27, 95% CI: 0.08 ~ 0.46, p = 0.007). As for skill, the experimental group improved by an average of 0.35 more points per question than the control group (𝛽=0.35, 95% CI: 0.14 ~ 0.55, p = 0.001). Conclusion The proposed innovative instructional module significantly improved fifth-year medical students’ professional competence in health literacy, which is expected to benefit their future medical practices.
Background
Health literacy is pivotal for people to engage their knowledge, motivation and competencies to access, understand, appraise and apply health-related information to healthcare, disease prevention, and health promotion [1]. Many studies have suggested a general lack of health literacy among people across different countries. The WHO Regional Office for Europe conducted a large-scale adult health literacy survey and discovered that 29-62% of adults had insufficient or problematic health literacy [2]. 51.6% of adults in Taiwan have inadequate or problematic health literacy [3]. Studies have established a strong association between low health literacy and poor health outcomes, such as increased hospitalisation and utilisation of emergency care, poor use of preventative health services, inappropriate medication use, inadequate comprehension of medical information, decreased self-care ability, higher risk of contracting chronic diseases and poor prognosis, and unstable physical and psychological conditions [4][5][6][7].
Physicians in medical environments should be equipped with the ability to acquire better understanding, strengthen autonomy, and support the self-management of patients, which will in turn enhance their health literacy [8][9][10][11]. However, the gap between the health literacy evaluation of health care professionals and the actual health literacy skills of patients might cause difficulty in communication [12]. Health literacy practices play a key role in effective communication and lead to high-quality patient-centred healthcare [13,14]. A crucial concern to address is the way future physicians can play a better role in providing health literacy. In addition, some barriers to health literacy practice in healthcare workplaces are resultant of lack of knowledge or training about health literacy and related activities [15]. School-based literacy educational interventions can be considered powerful tools to create positive impacts [16]. Therefore, health literacy training is imperative and should be incorporated into the medical student curricula.
Research projects in Taiwan and across the globe all suggest that the health literacy curriculum in current medical education has not yet been refined. Coleman et al. investigated 61 medical schools in the U.S. and found that 70% of them had introduced health literacy into their medical education curriculum, with content emphasis on "oral transmission of knowledge in health literacy" and "association among literacy, health literacy, and patient health status. " [17] Several reviews have reported that professional competence building in health literacy for undergraduate healthcare students and professionals could improve knowledge and skills and equip them to communicate effectively with patients [18][19][20][21]. Previous studies have shown the effectiveness of interventions on healthcare professionals' professional competence in health literacy [22][23][24][25]. However, the lack of a comparative group was noted in previous intervention studies, which revealed insufficient evidence to support the effectiveness of the training curriculum. In the recent years, some research on training in health literacy focused on medical students and revealed a positive effect, but lacked comprehensive intervention approaches or evaluation methods [26][27][28][29].
There is a dire need to develop innovative teaching methods and empowerment dedicated to physicians' professional competence in health literacy as awareness about health literacy issues and courses to mitigate the same are scarce in Taiwan's medical education system. We systematically and rigorously established indicators of professional competence in health literacy, designed a validly structured questionnaire, and developed a theorybased, innovative instructional module to teach medical students how to observe and assess a patient's health literacy, understand its significance, and provide suitable doctor-patient interaction patterns to patients with different levels of health literacy [30,31].
Our goal was to implement an innovative instructional module in the medical school curriculum to teach medical students and investigate whether the intervention helped them improve their professional competence in health literacy.
Subjects and methods of study
We adopted a quasi-experimental design and recruited fifth-year students from 12 medical schools in Taiwan. The inclusion criteria were that the participants were fifth-year medical students willing to participate in the study. The exclusion criterion was that the participants did not complete the questionnaires. The sample size was determined using G-power analysis and based on a previous recent similar study [32]. Given a level of significance of alpha = 0.05, statistical power level of 0.8, and a medium effect size of 0.25, the minimum required sample size was 98, with 49 in each group being sufficient for a significant analysis. The requirement was 196 students in order to account for a dropout rate of 50% in the follow-up measurements. A flowchart of participant recruitment and allocation is presented in Fig. 1.
We employed convenience sampling and assigned 98 fifth-year medical students serving clerkships at the Department of Family Medicine in three teaching hospitals in northern Taiwan as the experimental group. All three hospitals were medical centres and referral hospitals, including two governmental and one private hospital. Participants attended a three-hour course on professional competence in health literacy, and completed a pre-test questionnaire prior to the course and a post-test questionnaire after it. A total of 106 fifthyear medical students from other medical schools were assigned as the control group through social media. They completed a pre-test via an online questionnaire and post-test via an email questionnaire 2 weeks later, and received no treatment regarding health literacy. All participants were informed about the research goal and execution method and provided signed consent. The teaching interventions and data collection were conducted from December 2019 to May 2020. All 98 participants in the experimental group completed the "Innovative Instructional Module for Professional Competence in Health Literacy" course as well as both pre-test and post-test. Of the 106 participants in the control group, 99 completed the pre-test and post-test. Valid response rates were 100 and 93.4%, respectively.
Research instruments
We developed our questionnaire based on the indicators of physicians' professional competence in health literacy, as proposed by Liu et al. [30] We referred to Coleman et al. [33] for health literacy teaching and practices and examined the Objective Structured Clinical Examination (OSCE) curriculum implemented across Taiwanese medical schools for clinical teaching [34]. Medical simulations of doctor-patient interactions for pre-test and post-test evaluation were subsequently designed. The questionnaire comprised of 47 items, including seven knowledge items, eight attitude items, and 32 skill items. The knowledge items were multiple-choice questions with a singular correct answer; a higher correct response rate (range 0-1) indicates a higher level of understanding of health literacy. Both attitude and skill items were on of 5-point Likert scale. Attitude items offered the answer options of "strongly disagree, " "disagree, " "no comment, " "agree, " and "strongly agree, " and scored between 1 and 5 points respectively (range 1-5 for each question), while skill items were answered in "very unconfident, " "unconfident, " "somewhat confident, " "confident, " and "very confident, " and scored between 1 and 5 points respectively (range 1-5 for each question). Higher scores in attitude questions indicate more agreement with the acceptance of health literacy concepts and respect for patient-centred health care. Higher scores on the skill questions indicated greater skill confidence in communication and interaction with patients.
We also submitted our questionnaire for content validity evaluation using the Content Validity Index (CVI). We invited six field experts to review the questionnaire content and evaluate the items and simulations in terms of their adequacy, significance, and clarity. For each item, the CVI was calculated by dividing the number of experts who gave a rating of 3 or higher by the total number of experts. The average CVI value was 0.8 or higher, with a Cronbach's α value of 0.944, which demonstrated that
The instructional module, teaching curriculum, and instructor
Our proposed innovative instructional module and intervention strategies were developed based on the Spreitzer's Psychological Empowerment Scale [31]. The intervention course module included two parts -two 80-min instructional sessions and two 10-min sessions for the pre-test and post-test surveys -for a total of 180 min. Teaching strategies involved didactic teaching, observational learning, inquiry-based learning, roleplay, and game-based learning. We also created simulation videos using original content and invited practising physicians to demonstrate appropriate and inappropriate doctor-patient interactions to inspire student reflection on health literacy competence. Additionally, we developed a set of card-based board games, wherein students enacted the roles of physicians, patients, and observers.
Students practiced teach-back and made shared decisions by performing in groups. The highlights of the teaching content are listed in Table 1. The teaching instructors were three senior attending physicians in family medicine from three different hospitals who were qualified to teach at universities. They all participated in curriculum development and teaching design, and performed pilot teaching and attended evaluation meetings, which created consistency between the teaching module's curriculum and teaching execution.
Data analysis
After collecting and filing the pre-and post-test questionnaires, we used SPSS version 22 (IBM SPSS Statistics for Windows, Armonk, NY, USA) for statistical analysis, utilising mean values and standard deviation to describe the distribution of variables and basic information. Knowledge items are expressed in terms of the average correct response rate while attitude and skill items are expressed in terms of the average score per item. In our statistical -Didactics -Observation learning -Group discussion -Problem solving -Inquiry-based learning 1. Show videos that help students understand the sense of insecurity or embarrassment that patients may experience when receiving medical attention; encourage students to acknowledge a patient's emotional reactions and express empathy so the patient feels supported and on the same front as their healthcare provider; evaluate a patient's health literacy and accordingly adopt an adequate interaction pattern and offer social support. 2. Utilize videos with storylines centered around a crisis conceptual model on health literacy to help students see different medical scenarios that result from different levels of a physician's professional competence on health literacy. The purpose is for students to acknowledge the importance of a physician's ability to observe and assess a patient's health literacy.
1. Present slide decks to summarize and conclude key points to help students acknowledge the importance of a physician's ability to observe and assess a patient's health literacy.
2.New guideline for doctor-patient communication (80 min)
-Empathy and acceptance -Respect and support -Communication environment -Relationship building -Verbal and non-verbal communication -Easy-to-understand patient education materials and human resources -Teach-back -Medical purpose and needs -Shared decision-making -Confirm medical decision 1. Divide students into groups for quiz competition and role play, in which students will choose cards corresponding to the story plot; the purpose is to emphasize how different communication patterns will lead to different doctor-patient interaction and help students acquire adequate communication skills. 2. Present slide decks to conclude that good doctor-patient communication is the most important step towards a good doctor-patient relationship; practice applying "teach back" and "shared decision-making" to help patients obtain adequate medical services and the best healthcare outcomes.
-Didactics -Observation learning -Group discussion -Game-based learning -Role play 1. Have students act out exemplary doctor-patient interaction scenes; the student who plays the observer role follow the checklist items to conduct evaluation and offer feedback; topics include "teach back, " "shared decisionmaking, " and "communication and interaction.
1. Use slide decks to summarize learning objectives for physician's professional competence in health literacy; students should be able to apply relating principles in future medical scenarios to improve doctor-patient relationship and healthcare quality. analysis, we performed an analysis of covariance with pre-test score as the covariate and post-test score as the dependent variable to test for within-group homogeneity of the regression coefficient. We found significant effect in "group*pre-test" interaction, suggesting that the two groups are not homogeneous. Subsequently, GEE was applied to compare the treatment effects between the different groups.
Results
A total of 197 medical school students participated in this study, with 98 in the experimental group and 99 in the control group. Among them, 136 (69.0%) were male and 61 (31.0%) were female; in terms of educational background, 159 (80.7%) were enrolled in public schools and 38 (19.3%) in private schools. The study did not find a statistically significant difference in the distribution of sex or educational background between the two groups. Tables 2 and 3 illustrate the descriptive statistics and analysis results of the two groups' pre-tests and posttests. In terms of knowledge, the average correct response rate for the experimental group was 63% (SD = 18%) in the pre-test, 79% (SD = 20%) in the post-test; for the control group, it was 57% (SD = 16%) in the pre-test and 61% (SD = 19%) in the post-test. We applied GEE to analyse variables that impact the effect of intervention; after controlling for "group" and "test type" variables, the progress margin for the experimental group was 12% higher than the control group, with significant difference (p = 0.001). This suggests that the treatment significantly elevated the professional competence of the research subjects in health literacy ( Fig. 2A).
In attitude questions, the average score of the experimental group was 4.30 (SD = 0.47) in the pre-test and 4.62 (SD = 0.42) in the post-test; for the control group, it was 4.38 (SD = 0.49) in the pre-test and 4.44 (SD = 0.56) in the post-test. We applied GEE to analyse variables that impact effect of intervention; after controlling for "group" and "test type" variables, and the progress margin for the experimental group was 0.27 points higher than the control group, with significant difference (p = 0.007). This suggests that the treatment significantly elevated the professional competence of research subjects in health literacy (Fig. 2B).
In skill questions, the average score for the experimental group was 3.87 (SD = 0.43) in the pre-test and 4.38 (SD = 0.49) in the post-test; for the control group, it was 4.17 (SD = 0.54) in the pre-test and 4.33 (SD = 0.61) in the post-test. We applied GEE to analyse variables that impact the effect of intervention; after controlling for "group" and "test type" variables, the progress margin for the experimental group was 0.35 points higher than the control group, with significant difference (p = 0.001). This also suggests that the treatment significantly elevated the professional competence of research subjects in health literacy (Fig. 2C).
Discussion
This study revealed that medical students in the experimental group showed a significantly increased in professional competence in health literacy for knowledge, attitude and skill compared to the control group after receiving teaching intervention. To our knowledge, this study is the first research project in Taiwan to target medical students in an intervention study on professional competence in health literacy that applies scale-based evaluation instrument, conducts teaching interventions, and achieves significant results.
There are several possible reasons for this effect. First, the proposed curriculum adopted multidimensional Table 2 Mean proportion of correct answers and scores of professional competence in health literacy for post-test and pretest of two groups indicators, such as conception and evaluation, empathy and acceptance, communication and interaction, as well as medical information and shared decision-making. Other previous studies did not include such comprehensive indicators and most lacked shared decisionmaking aspects [26-29, 35, 36]. Second, we applied various student-centric teaching methods and created interesting materials for innovative teaching, including inquiry-based learning, observational learning, group discussion, game-based learning, and role-play. Strategies such as inquiry-based learning and role-play inspire students to think critically about a physician's professional competence in health literacy. Original simulation videos and intriguing board games piqued their interest and motivation in this field. Third, we employed game-based learning in our doctor-patient interaction curriculum to highlight how different communication patterns lead to varied doctor-patient interactions. Meanwhile, the teach-back approach checks whether patients have fully comprehended a medical diagnosis or report. Fourth, according to Nutbeam's model, there are three levels of health literacy -functional, interactive, and critical health literacy [37]. For healthcare professionals, physicians should consider a patient's reading and writing abilities and whether they can exercise functional health literacy in regular doctor-patient interactions. In the course, the program teaches students about available measurements to assess a patient's health literacy and common signs of insufficient health literacy. Interactive health literacy helps physicians and patients extract information from different communication channels, understand its significance, apply the information, and improve doctorpatient communication patterns. Our educational videos were physician centric. They presented outcomes in two-sided arguments to teach students about the consequences of having (positive) and neglecting (negative) professional competence in health literacy. This reinforces the students' concerns and impressions regarding the issue. We also introduced the concept of shared decision-making for the best healthcare outcomes [38]. As for the highest level, critical health literacy, physicians provide patient-tailored teach-back approaches, provide readable materials and social support resources, and help patients execute self-management and disease management [39]. Our findings add to previous review articles and evidence that reveal the effectiveness of teaching module interventions in professional competency among medical students [18][19][20][21].
Other studies on health literacy training in medical students have shown positive effects, even in communitybased service learning, but all of them were one-group pretest-posttest designs [35,36]. Similar concepts of Nutbeam's model were also applied in a study with a randomized controlled trial design, different from our quasi-experimental design [32]. This also emphasised that three levels of health literacy (functional, interactive, and critical) were embedded in the teaching module, so it included the shared decision-making aspect. The primary outcome variables evaluated were knowledge, attitude and skill competency using a valid questionnaire. However, our questionnaire corresponding to health literacy competency was designed with four clinical scenarios of physician-patient communication, and it woud be better to understand of the core meanings for each evaluation item instead of containing only straightforward questions.
Since the COVID-19 pandemic outbreak in early 2020, healthcare professionals and the public have been receiving disease information that caused swift and fundamental influence regardless of their accuracy. In a time like this, health literacy has become more important than ever [40,41]. Misinformation threats have impacted all aspects of our social-ecological model, and the complexities of healthcare systems are increasing [42]. The need to establish health literacy systems of care and increase organizations' health literacy sensitivity is dire [15,43]. This study emphasises that the importance of teaching and intervention effects of health literacy courses in professional healthcare education is a key step in gradually reducing barriers to organizational health literacy practice [15].
.As for research limitations, first of all, since our control group recruited free-willed participants, it is possible that they were students interested in the subject matter, which could have given them better pre-test scores in attitude and skills in comparison to the intervention group. Second, a selection bias might have existed because of convenience sampling. Third, posttest surveys were completed immediately after the teaching intervention, therefore, long-term effects such as medical students' behaviour change in the future medical residents or patients' health outcomes have yet to be observed. Fourth, since our evaluation survey was self-administered, it is possible for self-reported errors or overestimation because participants perceived their capacity to be elevated over the time or from a learning effect to impact performance analysis. Fifth, our findings may not be generalizable to other regions of medical schools because we studied in northern Taiwan.
Based on our findings, regarding the curriculum reform of liberal arts courses, for medical students, we noted that although our current medical education offers extensive liberal arts courses, it falls short on issues concerning physicians' professional competence in health literacy. We recommend transforming our proposed innovative instructional module into themed micro-teaching activities or dividing them into different sessions to streamline class time. We plan to conduct relevant courses in Taiwanese medical schools. We suggest hosting educational training in health literacy to raise physician awareness of the phenomenon of low health literacy in Taiwanese clinical fields. Government departments and academic institutes can also develop teaching materials that physicians across specialties may take advantage of during healthcare services or health education scenarios to improve and raise awareness of patients' health literacy. They will provide access to appropriate medical information to enhance doctor-patient professionals and improve the quality of care.
Conclusions
The proposed innovative instructional module significantly improved fifth-year medical students' professional competence in health literacy, which is expected to benefit their future medical practice. They should be able to observe a patient's health literacy more acutely to help them obtain, understand, evaluate, and apply medical information; achieve efficient utilisation of medical resources; and improve healthcare quality. | 2021-09-28T01:09:08.416Z | 2021-07-13T00:00:00.000 | {
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233838109 | pes2o/s2orc | v3-fos-license | Physical Properties of Films Based on Gelatin and Dialdehyde Starch with Different Oxidation Degrees
In this study, dialdehyde starch (DAS) was prepared by oxidizing corn starch using potassium periodate as the oxidant. The aldehyde contents in DAS were determined using titration with hydroxylamine and were found proportional to the molar ratio of KIO4:C6H10O5. Fourier transform infrared spectroscopy was used to confirm the breakage of the bond between C2 and C3 in anhydroglucose units and the formation of -CHO group in DAS. X-ray diffractometry showed a decrease in crystallinity of starch after periodate oxidation. Thermogravimetric analysis showed reduced thermal stability of DAS in comparison with the native starch. The prepared DAS was blended with gelatin to form films by casting method. Increasing aldehyde contents in DAS from 0 to 12.2% resulted in decreased moisture contents, solubility in water, and the swelling capacity of gelatin-DAS films. The value of tensile strength and elongation at break of the gelatin-DAS films rose when the aldehyde content was low (0-3.5%), but declined when the aldehyde content was high (3.5-12.2%). Slight periodate oxidation is, therefore, an effective way to enhance the mechanical characteristics of blend films from DAS and other biopolymers.
INTRODUCTION
Nowadays, the use of petrochemical-based plastics causes nagative impacts on ecosystem because it take many years for them to decompose. To solve this problem, the development and applications of biodegradable polymeric materials, especially those from renewable resources, are needed. Among natural resources, starch is the most commonly used polymers because it is inexpensive, non-toxic, renewable, and biodegradable 2 . However, practical applications of starch as a plastic material were limited due to its disadvantages, such as poor mechanical properties and hydrophilic character 3 .
Dialdehyde starch (DAS) is an industrial starch derivative, in which the C 2 -C 3 bonds of the anhydroglucose units are cleaved and the CH-OH groups at these atoms are converted to CH=O aldehyde groups 4 . When blended with other biopolymers to form films, aldehyde groups in DAS can crosslink with other functional groups (-OH and -NH 2 ) and hence enhance the physicomechanical properties of the blend films. Moreno et al., reported that the improvement of hydrophobic and mechanical characteristics of the gelatin-DAS films is related to the formation of crosslinking processes in polymeric matrices while increasing the hydrophobicity of the film 5 .
DAS is commonly prepared by periodate oxidizing starch with NaIO 4 as the most widely used reagent 4 . Despite lower price compared to NaIO 4 , KIO 4 is rarely used as the oxidizing agent to prepare DAS, possibly due to its low solubility. In this study, we used KIO 4 to prepare DAS from corn starch and evaluated the influence of the oxidation degrees of starch on physico-mechanical properties of gelatin-DAS blend films.
Preparation of dialdehyde starch (DAS)
The corn starch of 3.0 g and various quantities of KIO 4 were suspended in distilled water to obtain various molar ratios of KIO 4 :C 6 H 10 O 5 = 0.1, 0.2, and 0.3 ( Table 1). The pH of solution was adjusted to 3 by adding 0.1 M HCl. Then the mixtures were vigorously stirred at 35°C for 4 horus. After that, they were filtered and rinsed ten times (10 × 10 mL) with distilled water. The products were dehydrated in a convection oven at 508°C for 48 h to obtain DAS with different aldehyde contents. stirred at 50°C. During the reaction, the pH gradually decreased due to the release of HCl from NH 2 OH.HCl. Therefore, the 0.1 M NaOH was continuously added from a buret to remain pH = 5. After 2 h, pH of the reaction mixture stopped decreasing, indicating the end of the reaction. The total volume of 0.1 M NaOH used VDAS was recorded. A blank determination was also conducted for 0.2 g of the native corn starch instead of DAS and the volume of 0.1 NaOH used was also recorded (Vblank). The content of aldehyde in DAS was evaluated using Eq (1) 1 .
DAS characterization
FTIR spectra of DAS were measured with using PerkinElmer MIR/NIR Frontier. The resolution was at 2 cm -1 . The scanning region was 400-4000 cm -1 .
X-ray diffraction (XRD) patterns of DAS were measured by diffractometer (Brucker D2) at ambient temperature. The scanning region was 10-90° of 2θ. The equipment was operated with CuKα wavelength of 1.54056 angstroms.
Thermal decomposition of DAS was investigated by thermogravimetric analyser TA-200 (TA Instruments). The mass of the sample was continuously recorded while being heated in temperature range of 30 -350°C. The heating rate was 10°C/minute. These experiments were carried out under a nitrogen flow (100 mL/min) to help protect the samples from oxidative degradation.
Preparation of gelatin-DAS films
Gelatin (Ge) of 1.5 g was first completely dissolved in 5 mL distilled water by stirring at 80°C. Then 3 g of DAS, 2 mL of glycerol and the prepared gelatin solution were added to 25 mL of distilled water. The blended Ge-DAS with glycerol were mixed using magnetic stirrer at 100 °C during 30 minute. After that the mixtures were left to cool down during 10 min and then tranfered into Petri dishes (15 g/dish). The Petri dishes with the mixtures were dehydrated at room temperature for 72 hours. The obtained films were peeled off and placed in a desiccator with a saturated NaBr solution to equilibriate the moisture in the films. A film with the same composition but using native corn starch (NS) instead of DAS was used as the control. Determination of aldehyde content A 0.25 M hydroxylamine solution was prepared from NH 2 OH.HCl. The pH value of solution was adjusted to 5 by using sodium hydroxide 0.1 M. Dried DAS of 0.2 g was then added to 25 mL of the prepared hydroxylamine solution and magnetically
Characterization of Ge-DAS films Mechanical testing
The tensile tester (Testometric, UK) was used to investigated the mechanical characteristics of Ge-NS and Ge-DAS films. The instrument was operated with a crosshead speed of 0.8 mm/s (ASTM Standard method D882-12).
Moisture content, solubility and swelling capacity in water
Film samples of 2 × 2-cm 2 were weighed (m o ) and then dehydrated in an oven at temperature of 55 o C for 24 hours. The weight of the prepared film samples were tested (m 1 ). Then these samples were immersed in distilled water during 2 h at ambient temperature. After that, the films samples were removed and the surface water was cleaned using a filter paper. After weighing, the films (m 2 ) were kept dry at temperature of 55 o C during 24 horus. Then these films samples were weighed (m 3 ).
The moisture content (MC) of the Ge-NS and Ge-DAS films was determined using Eq (2): The solubility (S) of the Ge-NS and Ge-DAS films was determined using Eq (3): Swelling capacity (SW) of the Ge-NS and Ge-DAS films was determined using Eq 4: Table 2 shows that the aldehyde content of DAS was proportional to the molar ratio of KIO 4 :anhydroglucose C 6 H 10 O 5 units in corn starch. The aldehyde content of DAS increased as the molar ratio of KIO 4 /glucose increased. In comparison with the study of Yu et al., with using NaIO 4 as the oxidant, it was shown that the oxidation degree of starch in our study was lower, which might be due to the lower solubility of KIO 4 comparing with NaIO 4 6 .
FTIR
FTIR was applied to investigate the changes in structure when native starch was oxidized to DAS (Figure 1).
Fig. 1. FTIR spectra of native and dialdehyde starch
For NS, the main characteristic groups were the secondary -OH groups at the position of C 2 and C 3 , the primary -OH groups at the position of C 6 , and the D-pyranose ring structure. These structural characteristics in the infrared spectrogram are shown as follow. The broad peak at 3310 cm −1 correlates to the -OH stretching. The peak absorption observed at 2930 cm −1 is atributed to C-H stretching vibrations. The bound water in the starch was performed by the peak near 1640 cm -1 4,6-8 . The peaks in range of 1000-1200 cm -1 are attributed to the C-O bond of stretching and vibration 4 . The oxidation of starch by potassium periodate led to the linkage break at the position of C 2 and C 3 of the anhydroglucose units. The hydroxyl groups at C 2 -C 3 was taken place by aldehyde groups. The peak of the C-O bond stretching in C-O-H group in the anhydrogluscose ring of starch was observed near 1150 cm -1 . With the rising in contents of aldehyde in starch, the double peak of C-OH groups turned to a board band and gradually weakened. For DAS12.2 besides those peaks, there was an apperance of the peak around 1720 cm -1 , represented the C=O stretching and vibration 4 . This peak was not found in the spectra of DAS3.5 and DAS7.4 due to the low content of aldehyde groups in these samples.
XRD analysis
The method of X-ray diffractometry was applied to analyse the crystal structure of natural and dialdehyde starch (Figure 2). chain and formation of small crystallites exhibiting amorphous phase in DAS 4,6,9,10 .
Thermogravimetric analysis (TGA) and differential thermogravimetry (DTG)
TGA and DTG curves for NS and DAS12.2 are shown in Fig. 3. For DAS there were two stages of weight loss. The first stage around 30-120°C was related to the loss of water. The second one represented the decomposition of DAS with the maximum decomposition in the range of 249.6-270.8°C. For the native starch there were three stages of weight loss. The first stage around 30-120°C was related to the loss of water. The double peaks were attributed to the stages of starch decomposition. It was shown that the maximum decomposition was at a range of 269.0-292.3°C. With the existence of -CHO groups, the maximum peak shifted to region of lower temperature. It indicated the lower thermal stability of DAS in comparison with NS. This phenomenon might be related to the decline in evarage molecular weight of DAS 12.2 comparing to NS 11 . The hydroxyl groups at the position of C 2 and C 3 of anhydroglucose units are oxidized with the formation of aldehyde groups, hence destroying the hydrogen bonds between starch molecules (Scheme 1) 4 . Moreover, the oxidation using periodate was conducted at pH of 3, which might partially hydrolys the glycosidic bonds between glucose units in starch. These phenomena led to the cleavage in starch
Hydrophilicity of Ge-DAS films
The blend films containing DAS with higher aldehyde contents exhibited lower hydrophilicity, expressed in lower moisture content, solubility, and swelling capacity ( Table 3). The first reason is the lower hydrophilicity of carbonyl groups in DAS compared to hydroxyl groups in native starch 11 . The second reason is the crosslinks between the aldehyde groups in DAS with amino groups in gelatin (to form imine) and with hydroxyl groups in unoxidized fragments in DAS (to form acetal and cetal) 12 . These crosslinks tightened the film structure, hence lowering the the amount of moisture in the samples, hindering dissolution and suppressing the water diffusion into the film maxtric. and -OH groups in starch 13 . However, for Ge-DAS films, the tensile properties are associated to the crosslinking reaction between -CHO and -NH 2 groups in a polymeric matrix. The change in crosslinking reaction in Ge-DAS matrix might not corespond to the change of the level of starch oxidation. It was related to the heterogeneous property due to containing of both hydroxyl and carbonyl in the polymeric chain. This phenomenon might produce hindrances to the formation new crosslinking reaction in the polymeric matrix5. Another possible reason for the decreases in TS and YM at high degree of oxidation is the depolymerization of starch 14 .
CONCLUSION
DAS was successfully prepared in a slightly acidic solution using KIO 4 as the periodate oxidant. The oxidation introduced aldehyde groups into the starch backbone, destroyed the crystallinity of starch, and decreased the thermal stability of starch. DAS was blended with gelatin to produce biodegradable films with lower hydrophilicity due to the crosslinking aldehyde groups in DAS with functional groups of gelatin. The value of elongation at break and tensile strength of Ge-DAS films increased at low and decreased at high degrees of starch oxidation. Therefore, controlling the degree of periodate oxidation is a way of altering the physicochemical properties of film from DAS and other polymers. | 2021-05-07T00:04:39.861Z | 2021-02-28T00:00:00.000 | {
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88521105 | pes2o/s2orc | v3-fos-license | Sparsity-promoting and edge-preserving maximum a posteriori estimators in non-parametric Bayesian inverse problems
We consider the inverse problem of recovering an unknown functional parameter $u$ in a separable Banach space, from a noisy observation $y$ of its image through a known possibly non-linear ill-posed map ${\mathcal G}$. The data $y$ is finite-dimensional and the noise is Gaussian. We adopt a Bayesian approach to the problem and consider Besov space priors (see Lassas et al. 2009), which are well-known for their edge-preserving and sparsity-promoting properties and have recently attracted wide attention especially in the medical imaging community. Our key result is to show that in this non-parametric setup the maximum a posteriori (MAP) estimates are characterized by the minimizers of a generalized Onsager--Machlup functional of the posterior. This is done independently for the so-called weak and strong MAP estimates, which as we show coincide in our context. In addition, we prove a form of weak consistency for the MAP estimators in the infinitely informative data limit. Our results are remarkable for two reasons: first, the prior distribution is non-Gaussian and does not meet the smoothness conditions required in previous research on non-parametric MAP estimates. Second, the result analytically justifies existing uses of the MAP estimate in finite but high dimensional discretizations of Bayesian inverse problems with the considered Besov priors.
1. Introduction. We consider the inverse problem of recovering an unknown functional parameter u ∈ X from a noisy and indirect observation y ∈ Y. We work in a framework in which X is an infinite-dimensional separable Banach space, while Y = R J . In particular, we consider the additive noise model where ξ is mean zero Gaussian observational noise, with a positive definite covariance matrix Σ ∈ R J×J , ξ ∼ N (0, Σ). Here the possibly nonlinear operator G : X → Y describes the system response, connecting the observation y to the unknown parameter u. More specifically, G captures both the forward model and the observation mechanism and is assumed to be known. Inverse problems are mathematically characterized by being ill-posed: the lack of sufficient information in the observation prohibits the unique or stable reconstruction of the unknown. This can either be due to an inherent loss of information in the forward model, or due to the incomplete and noisy observation. To address ill-posedness, regularization techniques are employed in which the available information in the observation is augmented using a priori available knowledge on the properties of candidate solutions.
We adopt a Bayesian approach to the regularization of inverse problems, which has in recent years attracted enormous attention in the inverse problems and imaging literature: see the early 1 work [20], the books [54,27] and the more recent works [36,51,10] and references therein. In this approach, prior information is encoded in the prior distribution µ 0 on the unknown u, and the Bayesian methodology is used to formally obtain the posterior distribution µ y on u|y, in the form (2) µ y (du|y) ∝ exp − Φ(u; y) µ 0 (du), Our work is driven by two types of a priori information: on the one hand, we aim to recover unknown functions with a blocky structure, as is typically the case in image processing [16] and medical imaging applications [15]. On the other hand, we are interested in prior models which promote sparse solutions, that is high-dimensional solutions that can be represented by a small number of coefficients in an appropriate expansion. To achieve these effects, we utilize so-called Besov space priors [36,11], which are well-known for their edge-preserving and sparsity-promoting properties, see e.g. [34,44,39,26,8]. With these goals in mind, our work relates to classical L 1 -type regularization methods such as penalized least squares with Total Variation penalty [46,50].
In practice, the implementation of the Bayesian approach to an inverse problem typically requires a high-dimensional discretization of the unknown and large computational resources. From a computational perspective, it is hence imperative that the probability models and estimators related to finite-dimensional discretizations of the problem scale well with respect to refining discretization. In particular, there is a fundamental need to understand whether specialized prior information used frequently in applications, for example of the type described above, leads to welldefined non-parametric probability models and whether the related finite-dimensional estimators have well-behaving limits; this motivates the study of Bayesian inverse problems in the infinitedimensional function-space setting. A body of literature on this topic has emerged during the last years, centered around two theories: a) the discretization invariance theory [37,36], which aims to ensure that when investing more resources in increasing the discretization level, the prior remains faithful to the intended information on the unknown function and the posterior converges to a well defined limit which is an improved representation of the reality; b) the well-posedness of the posterior theory found in [51,10], which secures that the posterior is well defined in the infinite-dimensional limit and robust with respect to perturbations in the data as well as approximations of the forward model.
Our work studies Bayesian inversion in function spaces especially from the perspective of point estimators. Namely, we study and give a rigorous meaning to maximum a posteriori (MAP) estimates for Bayesian inverse problems with certain Besov priors in the infinite-dimensional function-space setting. A MAP estimate is understood here as the mode of the posterior probability measure, hence our results require a careful definition of a mode in the infinite-dimensional setting. The main challenge is then to establish a connection between the topological definition of a MAP estimate (mode of the posterior) and an explicit variational problem. Succeeding in doing so, opens up the possibility of studying the behaviour of MAP estimators in certain situations. In particular, we are able to prove a weak form of consistency of the MAP estimator in the infinitely informative data limit.
Non-gaussian prior information and the need for MAP estimators. A major challenge in
Bayesian statistics is the extraction of information from the posterior distribution. In Gaussianconjugate settings, such as linear inverse problems, on the one hand there are explicit formulae for the posterior mean which can be used as an estimator of the unknown, and on the other hand one can (in principle) sample directly from a discretized version of the posterior, [40,38,1,42,41]. Nevertheless, draws from Gaussian priors do not vary sufficiently sharply on their domain to have a blocky structure, neither are they sparse.
For this purpose, the so-called TV prior has been used widely in applied literature, e.g. in medical imaging [49,35]. Drawing intuition from the classical regularization literature, the TV prior has a formal density of the form π(u) ∝ exp(−α u BV ), where α > 0 is a (hyper)parameter. Here, the norm of bounded variation, u BV , can be formally thought of as the L 1 -norm of the derivative of u. However, the numerical implementation of Bayesian inversion with a TV prior miss-behaves as the discretization level of the unknown increases, and in particular the TV prior is not discretization invariant, [37,36,34]. For example, depending on the choice of the parameter α as a function of the discretization level, the posterior mean either diverges or converges to an estimate corresponding to a Brownian bridge prior. For alternative types of approaches to edge-preserving non-parametric Bayesian inversion, see [17,24,22]. We consider the family of Besov-priors which were proposed and shown to be discretization invariant in [36]. A well-posedness theory of the posterior was developed in [11]. These priors are defined by a wavelet expansion with random coefficients, motivated by a formal density of the form where · B s p = · B s pp is the Besov space norm with regularity parameter s and integrability parameters p. For p = 2 the Besov space B s 2 corresponds to the Sobolev space of functions with s square-integrable derivatives, H s , and the corresponding family of priors are Gaussian with Sobolevtype smoothness parametrized by s. We are especially interested in the case p = 1, which is highly interesting for edge-preserving and sparsity-promoting Bayesian inversion, [34]. For the case s = 1 this is due to the close resemblance between the way u B 1 1 and u BV work, both dealing with the L 1 -norm of a (generalized) derivative of u, see [34,Section 2]. We define rigorously the class of Besov priors for p = 1 and s ∈ R, called B s 1 -Besov priors, in section 3 below. To probe the posterior in the non-conjugate context of linear or nonlinear inverse problems with Besov priors, one typically resorts to Markov chain Monte Carlo (MCMC) methods. Unfortunately, in practice standard MCMC algorithms become prohibitively expensive for large scale inverse problems, consider e.g. photo-acoustic tomography [43,55]. This is due to the heavy computational effort required for solving the forward problem which is needed for computing the acceptance probability at each step of the chain.
In such situations maximum a posteriori estimates are computationally attractive as they only require solving a single optimization problem. Furthermore, it was shown in [34] that for the B 1 1 -Besov prior defined via wavelet bases, at finite discretization levels the resulting MAP estimators are sparse and preserve the locations of the edges in the unknown. This remains true as discretization is refined, while our work validates that, unlike in the TV prior case, the discretized MAP estimators converge to the MAP estimators of the limiting infinite dimensional posterior distribution.
1.2.
Review of the main results. Our results draw inspiration from previous papers by the authors [12,23] (see also [17]), where concepts that we will call strong and weak MAP estimates were coined. We quote both definitions in section 2 below.
As mentioned earlier, we can formally think of a MAP estimator as a mode of the posterior. In finite dimensional contexts, especially when working with continuous probability distributions, the definition of a mode is straightforward as a maximizer of the probability density function. If the prior has probability density function of the form for a suitable positive function W : X → [0, ∞), the density of the posterior is In this case, a MAP estimator can be interpreted as a classical estimator of the unknown, arising from Tikhonov regularization with penalty term given by the negative log-density of the prior, [19].
In the infinite dimensional setting things are less straightforward due to the lack of a uniform reference measure. An intuitive approach to define a mode in a function space X is as follows: compute the measure of balls with any center u ∈ X and a fixed radius ǫ > 0 and proceed by letting ǫ tend to zero. A modeû is a center point maximizing these small ball probabilities asymptotically (as ǫ decreases) in a specific sense. What distinguishes a strong mode from a weak mode is exactly how 'asymptotic maximality' is perceived: a) in the strong mode case, we look for the maximum probability among all centres in X; b) in the weak mode case, we look for a centre of a ball with the property that it has maximum probability among all shifts of the ball by elements of a dense subspace E ⊂ X.
Natural choices of E turn out to be spaces of zero probability, thus giving one interpretation for the term 'weak'. Note that weak and strong modes coincide for E = X. Each of the two notions of mode gives rise to a notion of MAP estimator termed strong and weak MAP estimators, respectively. Let B ǫ (z) ⊂ X denote the open ball of radius ǫ, centered at z ∈ X. If we can find a functional I defined on an appropriate dense subspace F ⊂ X, such that when µ = µ y , then for any fixed z 1 ∈ F , a z 2 ∈ F maximising the limit or equivalently minimising I is a potential MAP estimator. For weak MAP estimators, if the space E ⊂ F over which we shift the ball centres, can be chosen sufficiently regular so that again for µ = µ y , then it is straightforward to establish the equivalence of weak MAP estimators and the minimisers of I. In the case of strong MAP estimators one needs to work considerably more, the difficulty stemming from the 'smallness' of the subspace F ⊂ X with respect to the prior hence also the posterior. If a functional I satisfying (5) exists, it is called the (generalised) Onsager-Machlup functional, [18,25,12,23]. As for the limit in (6), it is identified with R µ h , the Radon-Nikodym derivative of the shift of µ by h ∈ E with respect to µ itself, provided this derivative has a continuous representative, see [23, lemma 2] (quoted in lemma 2.3 below). In finite dimensions, both the existence of the Onsager-Machlup functional and the statement in (6) follow from the Lebesgue differentiation theorem under very mild conditions on Φ and the density of the prior. In the Banachspace setting, establishing (5) and (6) for the posterior boils down to showing similar results for the prior given that the posterior is obtained by a suitably regular transformation of the prior.
Strong MAP estimators were defined and studied in [12], in the context of nonlinear Bayesian inverse problems with Gaussian priors. In this case, an expression for the limit in (5) was readily available from the Gaussian literature for F being the Cameron-Martin space of µ 0 . The identification of minimisers of the resulting Onsager-Machlup functional with strong MAP estimators, however, required a considerable amount of work, in particular many new estimates involving small ball probabilities under the prior.
Weak MAP estimators were defined and studied in [23], in the context of linear Bayesian inverse problems with a general class of priors. The authors used the tools from the differentiation and quasi-invariance theory of measures, developed by Fomin and Skorohod [5], to connect the zero points of β µ h , the logarithmic derivative of a measure µ in the direction h, to the minimisers of the Onsager-Machlup functional. An essential assumption that makes this possible is the continuity of β µ h over X for sufficiently regular h. The authors considered as examples Besov priors with integrability parameter p > 1 and a conditionally Gaussian hierarchical prior with a hyper-prior on the mean.
In both [12] and [23], the Onsager-Machlup functional was shown to be a Tikhonov-type functional as in (4), where W is the formal negative log-density of the prior, hence MAP estimators are identified with the corresponding Tikhonov approximations in the studied contexts.
In this work, we are interested in defining and studying both the strong and the weak MAP estimates for generally nonlinear inverse problems with B s 1 -Besov priors, that is for Besov priors with integrability parameter p = 1. Since the prior has formal density as in (3), we expect and indeed show that MAP estimates are identified with minimizers of the Tikhonov-type functional (7) I(u; y) = Φ(u; y) + u B s 1 .
In particular, the weak and strong MAP estimates coincide. For the considered prior, the general theory developed in [23] for weak MAP estimators does not apply, due to the fact that the logarithmic derivative of the B s 1 -Besov priors is inherently discontinuous. We will show that the continuity of the Radon-Nikodym derivative R µ h , for h in a suitable subspace E ⊂ X, is sufficient to get the result for weak MAP estimators. For the B s 1 -Besov priors, we prove the continuity of R µ h for shifts h ∈ E ⊆ B r 1 (T d ) for any r > s, and establish the validity of (5) over F = B s 1 (T d ). It is then straightforward to prove our first main result which is the identification of weak MAP estimators by the minimisers of the functional I in (7). Furthermore, we generalise the program of [12] to the assumed non-Gaussian case, in order to prove our second main result which is the identification of strong MAP estimators to minimisers of I in (7). We also consider the theory of local weak modes separately, relying on the Fomin literature.
Consistency of MAP estimators.
Under the frequentist assumption of data generated from a fixed underlying true u † , it is desirable to verify that in the infinitely informative data limit, the Bayesian posterior distribution µ y contracts optimally to a Dirac distribution centered on u † . In recent years, there have been many studies on the rates of posterior contraction in the context of Bayesian inverse problems. The case of linear inverse problems with Gaussian and conditionally Gaussian priors is now well understood [32,1,33,2,29,53,31], and a theory for linear inverse problems with non-Gaussian priors is also being developed [45,30]. For nonlinear inverse problems the asymptotic performance of the posterior is not yet fully understood, with some partial contributions being [42,56,57].
Note that for general nonlinear problems, especially with finite dimensional data as in the present paper, one cannot expect to recover the underlying truth u † in the infinitely informative data limit. Instead the aim is to recover a u * ∈ X such that G(u * ) = G(u † ). Posterior contraction rates for Bayesian inverse problems with Besov priors are studied in ongoing work of a subset of the authors. A form of weak consistency of the strong MAP estimator in the presence of repeated independent observations, for general nonlinear inverse problems with Gaussian priors, was shown in [12]. In the present paper, we prove a similar result for the strong (hence the weak) MAP estimator obtained using the Besov prior for p = 1.
1.4. Notation. Throughout the paper we assume that X is a separable Banach space equipped with the Borel σ-algebra. All probability measures are assumed to be Borel measures. The Euclidean norm in R J is denoted by | · | to distuinguish it from the norm of any general X denoted by · X . We write f ∝ g for two functions f, g : X → R if there exists a universal constant c ∈ R such that f = cg as functions.
Definition 1.1. A measure µ is called quasi-invariant along h, if the translated measure µ h (·) := µ(· − h) is absolutely continuous with respect to µ. We define which is readily verified to be a linear subspace. Notation 1.2. Let h ∈ Q(µ). We denote the Radon-Nikodym derivative of µ h with respect to µ by R µ h ∈ L 1 (µ).
1.5. Organization of the paper. This paper is organized as follows: in section 2 we discuss the definition of modes for probability measures on separable Banach spaces. We introduce novel localized versions of the modes studied in previous work and discuss briefly how different modes can be characterized for log-or quasi-concave measures. The Besov priors are introduced and discussed in section 3 including the key results relating to the Radon-Nikodym derivative of the Besov prior in section 3.2. Section 4 covers the Bayesian inverse problem setup and our main results related to identification of weak and strong MAP estimates as the minimizers of certain variational problem. Moreover, the weak consistency result is given in section 4.2. In section 5 we discuss the logarithmic derivative of the posterior and its use in characterizing the MAP estimates. Finally, all proofs are postponed to section 6.
2. Modes of measures on Banach spaces. In subsection 2.1 we introduce the two existing notions of maximum a posteriori estimator (modes of the posterior measure) proposed in [12,23] in the context of measures on infinite-dimensional spaces. We also define two new notions of local modes and hence local MAP estimates. In subsection 2.2, we focus on log-concave measures, study the structure of the set of modes, and give conditions for local modes to be global.
2.1. Weak and strong, global and local. The following definition of a mode, introduced in [12], grows out of the idea that highest small ball probabilities are obtained asymptotically at the mode.
A mode of the posterior measure µ y in (2), is called a maximum a posteriori (MAP) estimate.
Below we occasionally use the terms strong mode and strong MAP estimator for the concepts introduced in definition 2.1 in order to distinguish them from the following weaker notion of a mode (similarly, the weak mode or weak MAP ) introduced in [23].
for all h ∈ E. A weak mode of the posterior measure µ y in (2), is called a weak maximum a posteriori (wMAP) estimate.
Notice that the definition of a weak mode is dependent on the choice of the subspace E. Therefore, in some contexts it may be more appropriate to discuss E-weak modes. In the following, however, we will suppress this dependence since the key question to our study is whether such a space exists.
The notions of weak and strong mode are related as follows: any strong mode is a weak mode for the choice E = X [23, lemma 3], which is straightforward to see by simply estimating µ(B ǫ (û−h)) ≤ M ǫ . The key motivation to study the weak definition, is the case when small ball asymptotics are not available explicitly or only available in some subspace of translations h. It is then of interest to choose E so that an expression for the limit on the left hand side of (8) exists pointwise. This typically leads to choices of E which have zero probability with respect to µ. The following lemma, which is an immediate generalization of [23, lemma 2], provides further guidance for this choice. Lemma 2.3. Assume that µ is quasi-invariant along the vector h. Let A ∈ B(X) be convex, bounded and symmetric and define for any u ∈ X.
Remark 2.4. According to lemma 2.3, it is desirable to consider a subspace E, such that R µ h is continuous for h ∈ E. A sufficient condition for the continuity of R µ h was given in [23], namely the so-called logarithmic derivative of µ along h needed to be continuous and exponentially integrable with respect to µ. For the sparsity promoting measure that we will consider in this paper, the logarithmic derivative is inherently discontinuous (section 5). We are however able to show the continuity of R µ h over an appropriate subspace E in subsection 3.2 by using its explicit expression.
Both strong and weak mode can also be associated with a natural localization described by the following definitions: (1) We callû a local mode of the measure µ, if there exists a δ > 0 such that the quantity (2) We callû a local weak mode of µ if there exists δ > 0 such that Local modes represent an analogue to local maxima of the probability density function in finite dimensions. In the setting of [23] the local wMAP coincides with the zero points of the logarithmic derivative of the posterior. Especially, regularization techniques in non-linear inverse problems are often known to give birth to local maxima and, therefore, the local wMAP can give statistical interpretation for these points.
2.2.
Modes for log-concave measures. This work studies the Besov prior which is a prototypical example of a log-concave measure. Below we show some general properties regarding the modes of such class of measures. Most of these ideas naturally extend to a larger class called the quasi-concave measures.
It is straightforward to verify that any log-concave measure is also quasi-concave. An immediate result of quasi-concavity is the well-known Anderson inequality [3,7]. Proposition 2.7. Let µ be a symmetric quasi-concave measure on X. For any symmetric and convex set A ⊂ X we have The next result follows from the Anderson inequality.
Proposition 2.8. Suppose that the measure µ on X is symmetric around u and quasi-concave. Then u is a strong mode of µ.
Let us next consider briefly the structure of the set of strong modes of a quasi-concave measure µ. When working in finite dimensions, X = R d , a probability density function f with respect to the Lebesgue measure is called quasi-concave if for all x, y ∈ R d and all λ ∈ [0, 1] we have Clearly a quasi-concave probability density function has a convex set of global modes. For a reference on convexity and unimodality in finite dimensions see [14]. We show that a similar result holds in infinite dimensions for our definition of strong mode.
Proposition 2.9. Suppose that the measure µ in X is quasi-concave (but not necessarily symmetric). Then the set of strong modes is convex.
It turns out log-concavity is a sufficient condition for the global and local modes to coincide.
Theorem 2.10. Suppose µ is log-concave andû is a local mode. Thenû is also a global mode. Similarly, ifû is a local weak mode then it is also a global weak mode.
3. Besov priors with p = 1. The family of Besov priors has been introduced in [36] and studied in [11,23]. In subsection 3.1 we recall the definition and some useful properties of Besov priors with integrability parameter p = 1 and regularity parameter s > 0, termed B s 1 -Besov priors, on which we focus in this work. We also present some straightforward convexity properties of B s 1 -Besov priors. The main results of this section are listed in subsection 3.2, where we compute the Radon-Nikodym derivative R µ h for B s 1 -Besov priors, determine the space E in which h needs to live in order for the corresponding Radon-Nikodym derivative R µ h to be continuous, and finally show that 3.1. Definition and basic properties. We work with periodic functions on a d-dimensional torus, T d . We first define the periodic Besov spaces B s pq (T d ), where s ∈ R parametrises smoothness and p, q ≥ 1 are integrability parameters. We concentrate on the case p = q and write B s p = B s pp . To define the Besov spaces, we let {ψ ℓ } ∞ ℓ=1 be an orthonormal wavelet basis for L 2 (T d ), where we have utilized a global indexing. We can then characterise B s p (T d ) using the given basis in the following way: the function f : T d → R defined by the series expansion (10) f is finite. Throughout, we assume that the basis is r-regular for r large enough in order to consist a basis for a Besov space with smoothness s, [13].
We now follow the construction in [36] to define periodic Besov priors corresponding to p = 1, using series expansions in the above wavelet basis with random coefficients. Notice also the work [21] on defining Besov priors for functions on the full space R d .
ℓ=1 be independent identically distributed real-valued random variables with the probability density function Let U be the random function Then we say that U is distributed according to a B s 1 -Besov prior.
The next lemma determines the smoothness of functions drawn from the B s 1 -Besov prior and shows the existence of certain exponential moments.
Notation 3.3. We denote by ρ ℓ the probability measure of the random variable ℓ −s/d−1/2 X ℓ on R. We identify the random function U in definition 3.1 with the product measure of coefficients We next consider the convexity of the B s 1 -Besov prior.
An immediate consequence of the last lemma, is that by proposition 2.7, the B s 1 -Besov prior satisfies Anderson's inequality. Notice that lemma 3.4 (and hence proposition 2.7) also holds for B s p -Besov measures with p > 1, as defined in [36].
3.2.
Radon-Nikodym derivative R µ h and small ball probabilities. Recall the definitions of quasiinvariance for a measure µ and of the subspace of directions in which µ is quasi-invariant, Q(µ). In general the structure of Q is not known and there are even examples of measures for which Q fails to be locally convex [5, exercise 5.5.2]. The space Q is known to be a Hilbert space for certain families of measures, for example for α-stable measures with α ≥ 1 and for countable products of a single distribution with finite Fisher information, see [5, theorem 5.2.1] and [48] (note that the B s 1 -Besov prior, λ, is not an α-stable measure). Using similar techniques to [48], namely the Kakutani-Hellinger theory, we now show that Q is a Hilbert space also for the B s 1 -Besov measure λ and calculate the Radon-Nikodym derivative R λ h between λ and the shifted measure λ h for h ∈ Q(λ).
We next provide a more detailed view of spaces of shifts h for which R λ h has a continuous representative, which we denote byR λ h (u). This is a crucial result for our study of weak MAP estimators, see lemma 2.3, Remark 2.4 and the discussion in subsection 1.2.
Note that in the expression for the Radon-Nikodym derivativeR λ h (u), h ∈ E and the less regular u ∈ X are coupled component-wise (in the wavelet basis defining the Besov measure) and hence establishing the continuity ofR λ h (u) with respect to u is not straightforward. See section 6 for the proof of above lemma.
We record the following immediate corollary of the last lemma and lemma 2.3.
for any u ∈ B t 1 .
By remark 2.4 and noting that the Besov space for any r > t, the last corollary shows that it is natural to choose E = B r 1 (T d ) for any r > s in the definition of wMAP estimate. It also shows that the origin is the unique weak mode and, therefore, by proposition 2.8 also the unique strong mode.
We also remark that the series above consists of the difference of two terms, whose respective series are not convergent in general for h ∈ B r 1 (T d ). However, they coincide with the difference of the norms in h ∈ B r 1 (T d ) if h and u are elements of this smaller space. Based on this view we close this section with an important building block for the study of the MAP estimate. It extends the last corollary to h ∈ B s 1 (T d ), for balls centered at the origin.
) denote a convex and zero-centered symmetric and bounded set. Then It follows immediately from the above lemma that for z 1 , giving the Onsager-Machlup functional of λ. The space B s 1 (T d ) here, is the largest space on which the Onsager-Machlup functional is defined. This is the space F of the discussion in subsection 1.2 in the case of Besov priors.
It is also worth noting the difference between (13) and (14). The centres of the balls in (13) are in X = B t 1 (T d ), the continuity of the right-hand side in u is due to sufficient regularity of the shift h. In (14), the centres of the balls are more regular than (13), but the shift is less regular in general, i.e. only in B s 1 (T d ).
Main results.
We are now ready to present our main results. We first show the existence of weak and strong MAP estimates in Bayesian inverse problems with B s 1 -Besov priors, and identify both with the minimisers of the Onsager-Machlup functional. Using this characterization, we then prove a weak consistency result for MAP estimators.
4.1.
Identification of MAP estimates for Bayesian inverse problems. We consider the inverse problem of estimating a function u ∈ X = B t 1 (T d ) from a noisy and indirect observation y ∈ R J , modelled as (15) y = G(u) + ξ.
Here G : B t 1 (T d ) → R J is a locally Lipschitz continuous, possibly non-linear operator and ξ is Gaussian observational noise in R J , ξ ∼ N (0, Σ) for a positive definite covariance matrix Σ ∈ R J×J .
We assume that u is distributed according to the B s 1 -Besov measure λ defined in Section 3, so that λ(B t 1 (T d )) = 1 for t < s − d. Under the assumption of local Lipschitz continuity of G, it follows that almost surely with respect to y the posterior distribution µ y on u|y, has the following Radon-Nikodym derivative with respect to the prior λ: and Z(y) is the normalization constant. Indeed, local Lipschitz continuity of G implies measurability of Φ(·, y) with respect to λ; it also, together with non-negativity of Φ, gives the finiteness and nonsingularity of Z. For details see [10].
Following the intuition described in subsection 1.2, we define the Tikhonov-type functional otherwise.
The existence of minimizers for I is classical, however, we include the proof for completeness.
The underpinning of our main results in theorems 4.3 and 4.5 is that the posterior inherits the property of the prior given in lemma 3.6, i.e. there exists a subspace, where the limit of the translated small ball probability ratios has a continuous representative. Moreover, one can show how this limit is connected with functional I. The next result follows directly by combining lemmas 2.3, 3.6 and 3.9, and the local Lipschitz continuity of Φ.
The main theorems below show that the weak and strong MAP estimates of the posterior µ y in (16), are identified with minimisers of functional I. In particular, the weak and strong MAP estimates coincide for the inverse problems considered here. This is remarkable since it is not known in general under which conditions there exists weak MAP estimates that are not strong. We note that the last result implies the existence of weak MAP estimates by lemma 4.1. We next show the existence of strong MAP estimators, and that any strong MAP estimate is a minimiser of I.
Proposition 4.4. Consider the measure µ y given by (16) and (17) with the B s 1 -Besov prior λ and G : B t 1 (T d ) → R J locally Lipschitz for t < s − d. i) For any δ > 0 there exists z δ ∈ B t 1 (T d ) satisfying z δ = arg max z∈X µ y (A δ +z), where A δ := δA with A a convex, symmetric and bounded set in B t 1 (T d ). ii) There is az ∈ B s 1 (T d ) and a subsequence of {z δ } δ>0 which converges toz strongly in B t 1 (T d ).
iii) The limitz is a strong MAP estimator and a minimizer of I(u; y) in equation (18).
In the following theorem we prove that any minimizer of I is a strong MAP estimate and, hence, show the identification of strong MAP estimates and minimizers of I using part (iii) of proposition 4.4. The proof of theorem 4.3 concerning weak MAP estimates is relatively straightforward and relies on lemma 3.6, i.e., the ability to consider the subspace B r 1 (T d ), where the Radon-Nikodym derivative R µ y h has a continuous representative. The proof of proposition 4.4 related to strong MAP estimates is more involved and requires a series of technical lemmas (lemma 6.1 to 6.4) which are stated in section 6.3. Our approach is based on developing asymptotic estimates for the small ball probability ratios by projecting the related measures to finite dimensions, where explicit calculations can be carried out.
The difference in difficulty of the proofs related to the two notions of MAP estimates highlights the flexibility of weak MAP estimators. It seems that explicit calculations are typically required for the proof in the case of strong MAP estimates. For practical purposes, it is a highly interesting future task to develop general conditions under which the two MAP estimate concepts coincide.
Remark 4.6. Proposition 4.3.8 in [5] shows that if Φ is convex in u, then since by lemma 3.4 the B s 1 -Besov prior λ is logarithmically-concave, the posterior µ y is also logarithmically-concave and hence quasi-concave. In that case proposition 2.9 shows that the set of modes is convex. The convexity of Φ depends on the forward operator G: if for example G is linear then Φ is convex, however in the general nonlinear case Φ may be non-convex.
4.2.
Weak consistency of the strong MAP. We consider the frequentist setup, in which ∼ N (G(u † ), Σ), j = 1, . . . , n, for a fixed underlying value of the unknown functional parameter u † ∈ X. As before, we assume that G : X → R J is locally Lipschitz and Σ ∈ R J×J is a positive definite matrix.
For this set of data and with a B s 1 -Besov prior, λ, the posterior measure satisfies Here also the Lipschitz continuity of G implies the well-definedness µ y 1 ,y 2 ,...,yn [10]. Proposition 4.4 then implies that the strong MAP estimator of the above posterior measure is a minimizer of Theorem 4.7. Suppose that G : B t 1 (T d ) → R J is locally Lipschitz and u † ∈ B s 1 (T d ). Denote the minimizers of I n given in (20) by u n for each n ∈ N . Then there exists u * ∈ B s 1 (T d ) and a subsequence of {u n } such that u n → u * in Bs 1 (T d ) almost surely for anys < s. For any such u * we have G(u * ) = G(u † ).
If u † lives only in X = B t 1 (T d ), and not necessarily in B s 1 (T d ), we can only get the convergence of {G(u n )}: Corollary 4.8. Let G and u n , n ∈ N, satisfy the assumptions of theorem 4.7 and suppose that u † ∈ B t 1 (T d ). Then there exists a subsequence of {G(u n )} n∈N converging to G(u † ) almost surely.
Theorem 4.7 states that the true solution is identified in the range of G, which is the natural objective also in regularization theory [19]. The full identification of u † is dependent on further properties of G, e.g., injectivity of G would immediately yield u * = u † .
Connections to logarithmic derivative.
In this section we discuss the logarithmic derivative of the posterior measure. We mainly revisit known results (see e.g. [5]) and also derive the logarithmic derivative of the posterior µ y given in (16). The intuition behind logarithmic derivative is that it roughly corresponds to the Gâteaux derivative of the posterior potential I. If the logarithmic derivative is smooth, then its zero points can determine the weak MAP estimates as shown in [23]. The Besov B s 1 -prior does not meet this criteria due to the discontinuity of its logarithmic derivative at the origin as we show in theorem 5.8. The logarithmic derivative also determines the Radon-Nikodym derivative as recorded in proposition 5.3 below. Moreover, it can be used as a basis of Newton-type algorithms to estimate the weak MAP in case an explicit form of the potential I is not easily accessible, see e.g. Cauchy priors in [52]. It is well-known that if µ is Fomin differentiable along h then the limit d h µ is a countably additive signed measure on B(X) and has bounded variation [5]. Moreover, d h µ is absolutely continuous with respect to µ.
We denote the domain of differentiability by Suppose µ is a Radon measure on a locally convex space X and is Fomin differentiable along a vector h ∈ X. If it holds that exp(ǫ|β µ h (·)|) ∈ L 1 (µ) for some ǫ > 0, then µ is quasi-invariant along h and the Radon-Nikodym density R µ h of µ h with respect to µ satisfies the equality Remark 5.4. Recall that as discussed in Remark 2.4, it is desirable to choose E in the definition of wMAP to be a subspace E ⊂ X such that R µ h , h ∈ E, has a continuous representativeR µ h .
Therefore, the integral 1 0 β µ h (u− sh)ds in (23) has a measurable representative, which is continuous outside the set {u ∈ X |R µ h (u) = 0}. Moreover, a weak modeû of µ can be equivalently defined by condition The construction of the Besov prior in definition 3.1 is a prototypical example of a product measure. By setting λ ℓ = 1 a ℓ π X x a ℓ dx for a n = ℓ −( s d − 1 2 ) we can define the probability law of the Besov prior on (R ∞ , B(R ∞ )) by λ = ⊗ ∞ ℓ=1 λ ℓ . For product measures, the Fomin differentiability calculus reduces to finite dimensional projections in a straightforward manner. . Then µ is Fomin differentiable along h = 0 if and only if has an absolutely continuous density π µ whose derivative satisfies π ′ µ ∈ L 1 (R). In this case, d 1 µ = π ′ µ dx.
The Fomin differentiability of a product measure µ = ⊗ ∞ n=1 µ n on the space X = ∞ n=1 X n assigned with the product topology is characterized by the following theorem: hn is the logarithmic derivative of µ n in the direction h n ∈ X n . The following claims are equivalent: j=1 ∈ X, (ii) the series ∞ n=1 β µn hn converges in the norm of L 1 (µ) and Let us introduce the following subspace which has a natural Hilbert space structure [5, section 5]. Surprisingly, for a large class of product measures H(µ) coincides with D(µ). The following proposition follows from [6, cor. 2] and [5, ex.
5.2.3].
Proposition 5.7. Suppose µ = π(x)dx is a Borel probability measure on the real line such that If we set µ n (A) = µ(A/a n ), where a n > 0, and µ = ⊗ ∞ n=1 µ n , then it follows that Let us record the following direct consequence of proposition 5.3: if t > 0 then . It is rather easy to see that λ 1 (and consequently λ ℓ for any ℓ ∈ N) is Fomin differentiable since for any A ∈ B(R). In more generality, this follows from lemma 5.5 since the density function is absolutely continuous.
is the logarithmic derivative of ρ ℓ (see notation 3.3).
Notice that the previous theorem directly states that for any h ∈ B s 1 (T d ) the logarithmic derivative is bounded |β λ h (u)| ≤ C h B s 1 λ-almost surely. For the posterior distribution µ y we can solve the logarithmic derivative by using properties of the prior and the functional Φ. The following result follows directly from [5, prop. 3.3.12] (see also [17, thm. 5.7]).
Proofs of results in section 2.
Proof of proposition 2.8. Without loss of generality assume that µ is symmetric around the origin and show that the origin is a strong mode. For any u ∈ X the Anderson inequality (proposition 2.7) implies µ(B ǫ (u)) ≤ µ(B ǫ (0)), and so µ(B ǫ (0)) = sup u∈X µ(B ǫ (u)) and the origin is a strong mode of µ.
Proof of theorem 2.10. Let us consider the identity (25) with values u 1 =û − h and u 2 =û. By applying log-concavity we have and, consequently, for any 0 ≤ κ ≤ 1. Now supposeû ∈ X is local mode in a neighborhood B δ (û) but not a global mode. Then there exists δ > 0 such thatû is a local mode in the neighborhood B δ (û) but not in B δ+1 (û). That is, in the larger neighborhood B δ+1 (û) we have for some η > 0 that there exists such a subsequence Then it follows that lim sup Sinceû is a local mode there existsǫ > 0 such that for any ǫ <ǫ we have and some η > 0. In fact, for this choice ofδ we haveû −δ(û − u j ) ∈ B δ (û) and it follows by (26) for any ǫ j <ǫ that This yields a contradiction and proves the claim for strong MAP estimates.
Suppose now thatû ∈ X is a local wMAP but not a global wMAP. Assume like above that B δ (û) is the maximal neighborhood, whereû is a local wMAP. Then there exists an element h ∈ E and h / ∈ B δ (û) such that Again we see that the inequality (26) yields a contradiction. This completes the proof.
Proofs of results in section 3.
Proof of lemma 3.4. Let λ N = N ℓ=1 ρ ℓ . Then it is straightforward to check that λ N converges weakly to λ as N → ∞. By [7, theorem 2.2], for λ to be logarithmically concave, it suffices to show that the measures λ N in R N are logarithmically concave. Note that the measures λ N have density, denoted π N , with respect to the Lebesgue measure in R N , given by 2 are the coefficients in the expansion defining the B s 1 -Besov measure. Since the density π N is a logarithmically concave function, by [4, theorem 1.8.4] λ N is logarithmically concave and the result follows.
Proof of lemma 3.5. We use the Kakutani-Hellinger theory [9, Chapter 2]. We start by calculating the Hellinger integrals H(ρ h,ℓ , ρ ℓ ), where ρ h,ℓ (·) := ρ ℓ (· − h ℓ ): where H N = N ℓ=1 e − α ℓ 2 |h ℓ | 1 + α ℓ 2 |h ℓ | ∈ (0, 1]. By taking the negative logarithm we get By [9, theorem 2.7] the set Q(λ) coincides with the set of h such that − log (H(λ h , λ)) < ∞. The Taylor theorem implies the upper and lower bounds Using the lower bound, we get that which implies that a sufficient condition for the equivalence of λ h and λ is Using the upper bound, and letting x ℓ = α ℓ 2 |h ℓ |, we get that If x ℓ is unbounded, then the sum on the right hand side is infinite and we have that λ h and λ are singular (note that if x ℓ is unbounded then obviously condition (27) does not hold). If x ℓ is bounded, therefore condition (27) is also necessary for the equivalence of the measures λ h and λ.
Note, that condition (27) is equivalent to
for all x ∈ R and ℓ ∈ N, the claimed expression for dλ h dλ follows from [9, theorem 2.7].
Proof of lemma 3.6. We have Let us next define index sets Clearly A j are disjoint and N = ∪ 4 j=1 A j . We utilise the index sets A j to rewrite where We continue by studying the terms I j separately. Let 0 < δ < 1 be a value, which we later fix. For I 1 we have by the Hölder inequality for δ 1 = δ 1−δ , where to bound the second parenthesis in the second to last line we have used that |v ℓ − u ℓ | ≤ 2|h ℓ | for any ℓ ∈ A 1 . For I 2 we have We note that since |v ℓ | > |h ℓ | ≥ |u ℓ |, the following two inequalities hold: As a direct consequence we have where as before δ 1 = δ 1−δ . The term I 3 is very similar to I 2 (only the role of u ℓ and v ℓ is swapped), and we have .
Proof of lemma 3.9. Let A ǫ := ǫA. We first note that by lemma 3.5 we can write This implies that This, using lemmas 3.6 and 2.3 implies that lim sup and letting j → ∞ in the right-hand side gives The above inequality together with (31) give the result.
6.3. Proofs of results in section 4. In some of the proofs below we consider the sequences that converge in the weak*-topology of B t 1 (T d ). We note that the Banach space B t 1 (T d ) is isomorphic to a weighted l 1 space, and hence its pre-dual is the space of functions {v ∈ B −t ∞ (T d ) : lim j→∞ v, ψ ℓ = 0}.
is a minimizing sequence of functional I. Clearly, we can assume {I(u j , y)} ∞ j=1 , and therefore also u j B s 1 to be bounded. By the Banach-Alaoglu theorem there exists a subsequence that converges to someû ∈ B s 1 (T d ) in the weak*topology. Notice that the norm of B s 1 (T d ) is lower semicontinuous in the weak*-topology and consequentlyû ∈ B s 1 (T d ). We now show the strong convergence of the above subsequence in Bs 1 (T d ) for anys < s. We have Noting that û B s 1 + u j B s 1 ≤ C < ∞, given any ǫ > 0, N can be chosen large enough, independently of j, such that the second term in the last line of the above inequality is bounded by ǫ/2. Having convergence coefficient-wise, there is M ∈ N large enough so that for j > M the first term is bounded by ǫ/2 as well. We therefore conclude that u j →û in Bs 1 (T d ) for anys < s and hence in particular fors = t < s − d. By the continuity assumption on Φ, it now follows that Therefore,û must be a minimizer.
Proof of theorem 4.3. Assume that u min ∈ B s 1 (T d ) is a minimizer of I(·; y). By lemma 3.6 and Lipschitz continuity of G we know that R µ y h ∈ C(B t 1 (T d )) for any h ∈ B r 1 (T d ) with r > s. Since u min ∈ B s 1 (T d ), we can study R µ y h pointwise and obtain log(R µ y h (u min )) = I(u min ) − I(u min − h) ≤ 0 for any h ∈ B r 1 (T d ) due to the minimizing property of u min . Therefore, u min is a weak MAP. Consider the reversed claim and assume thatû is a weak MAP to the posterior µ y in (16). Let us also assume thatû Due to the continuity of Φ and lemma 3.6 we have for any h ∈ B r 1 (T d ), r > s. Let us construct a particular function h N = ∞ ℓ=1 h N ℓ ψ ℓ ∈ B r 1 (T d ) by defining its coefficient vector according to for some small ǫ > 0. It follows by inequality (32) and continuity of Φ that where C > 0 is the local Lipschitz constant on the neighbourhood ofû. However, N was chosen arbitrarily and by our assumption on the smoothness ofû the sum on the left hand side of (33) does not stay bounded when N increases. Therefore, inequality (33) leads to a contradiction and we must haveû ∈ B s 1 (T d ). Assuming now that the weak MAPû ∈ B s 1 (T d ), we can separate the sum in (32) and obtain . By continuity of I and density of B r 1 (T d ) in B s 1 (T d ), we find thatû minimizes I.
Proof of proposition 4.4 relies on the following four lemmas giving some properties of the Besov prior measure we have here. We list these lemmas and their proofs first.
By our assumption on non-degeneracy of µ, we have that µ n is absolutely continuous with respect to the Lebesgue measure in R n , and hence we have µ n (∂(hB)) = 0. Noting that µ(h −1 (hB) \ B) ≤ µ(B ǫ ) − µ(B) ≤ ǫ, the result follows.
In the following λ stands for a centered B s 1 -Besov prior on B t 1 (T d ) with t < s − d. Also, we frequently consider projections of λ to a subspace span{ψ 1 , · · · , ψ n } ⊂ B t 1 (T d ). We write P n : B t 1 (T d ) → R n as (34) P n u = ( ψ ℓ , u ) n ℓ=1 and define λ n (A) := (λ • P −1 n )(P n A) for any A ∈ B(B t 1 ).
To consider the limiting case, note that P −1 n (P n A) is convex and symmetric. Therefore, we can use the first part of the proof above, and write (35) λ(A + z) ≤ λ n (A + z) ≤ e with ǫ → 0 as n → ∞ due to weak convergence of λ n to λ and since A is a continuity set by lemma 6.1.
, t < s − d, and z δ converges toz in the weak*-topology of B t 1 (T d ) as δ → 0. Then for any ǫ > 0 there exists δ small enough such that for A δ = δA where A any convex, symmetric and bounded set in B(B t 1 (T d )).
Proof. Below we write u ℓ = u, ψ ℓ for any u ∈ B t 1 (T d ) and without losing any generality assume that A has diameter 1. Sincez / ∈ B s 1 (T d ), for any M > 0 there is an N large enough such that N ℓ=1 α ℓ |z ℓ | > 4M.
Thus, for any z ∈ A δ 1 + z δ 1 , we can write Let δ ≤ δ 1 be sufficiently small so that For λ n we have that for any M > 0 there exist N > 0 and δ 1 > 0 such that for n ≥ N and δ < δ 1 it follows where in the last line we have used the fact that the density in the integrals of the third line is log concave and A is absolutely convex. Similar inequality as (35) generalizes the result for λ. Lemma 6.4. Suppose that {z δ } δ>0 ⊂ B t 1 (T d ) converges in weak*-topology and not strongly in B t 1 (T d ) to 0 as δ → 0. Then for any ǫ > 0 there exists δ small enough such that for A δ = δA where A is any convex, bounded and symmetric set in B(B t 1 (T d )).
Proof. Letα ℓ = ℓ t d − 1 2 and without loss of generality assume that A has diameter 1. For any j ∈ N we have ψ ℓ , z δ → 0, as δ → 0. There exists a subsequence which we relabel {z δ } for which elements there exists κ > 0 such that Let M > 0 be arbitrary and choose N large enough so that α ℓ > Mα ℓ for any j > N . By the weak* convergence there exists δ small enough such that where z δ ℓ = ψ ℓ , z δ . This then, using (36), means that there exists n > N such that n ℓ=N +1α for any x ∈ A δ + z δ and n ℓ=N +1α Having these bounds we obtain where we used the log-concavity of the integrands on the last line. Since κ is fixed, M is arbitrary and δ decreases, the result follows for λ n . In a same way as the previous two lemmas an inequality similar to (35) yields the result.
Proof of proposition 4.4. i) Without losing any generality we assume that the diameter of A is 1. The function z → µ y (A δ + z) for fixed δ > 0 is bounded Suppose the sequence z j is unbounded. Since Φ ≥ 0 we have for any z ∈ B t 1 (T d ) that by lemma 6.2. Now as z j B t 1 → ∞, we have µ y (A δ + z j ) → 0 which yields a contradiction. Therefore, the sequence z j must be bounded.
Next, by the Banach-Alaoglu theorem there exists a limit w ∈ B t 1 (T d ) in the weak-* topology. In particular, we have P n z j → P n w in R n for any n > 0. Let us write µ y n (A) = µ y • P −1 n (P n A) for any A ∈ B(B t 1 (T d )). By weak convergence of µ y n to µ y for any ǫ > 0 there exists N > 0 such that for n > N we have µ y n (A δ + w) = µ y n (A δ + w) ≤ µ y (A δ + w) + ǫ, since A δ is a set of continuity for µ y by lemma 6.1, i.e. µ y (∂A δ ) = 0. In consequence, we have where the convergence to ǫ in (38) appears since λ n is non-degenerate. Since ǫ > 0 was arbitrary, we obtain lim j→∞ µ y (A δ + z j ) ≤ µ y (A δ + w) and by (37) the supremum must be attained at z δ = w ∈ B t 1 (T d ). ii) Since Φ(u) ≥ 0 and G is locally Lipschitz continuous, for any δ ≤ 1 we have with a constant L independent of δ. Suppose that {z δ } is not bounded in X. Then by lemma 6.2, for any ǫ > 0, there exists δ small enough such that µ(B δ (z δ )) µ(B δ (0)) ≤ λ(B δ (z δ )) e −L λ(B δ (0)) < ǫ.
It remains to show thatz is a minimizer of I. Let z * := arg min u∈B s 1 I(u). Suppose thatz is not a minimizer so that I(z) > I(z * ). We first note that by local Lipschitz continuity of Φ, as before we have that there is an L depending on z B t 1 and z * B t 1 such that µ y (A δ +z) µ y (A δ + z * ) ≤ e Lδ e −Φ(z)+Φ(z * ) λ(A δ +z) λ(A δ + z * ) .
Proof of theorem 4.5. Suppose thatz is a strong MAP estimator. Any strong MAP estimate is a weak MAP estimate and hence, by theorem 4.3,z is a minimizer of I. Now let z * be a minimizer of I. By proposition 4.4 we know that there exists a strong MAP estimatē z ∈ B s 1 (T d ) which also minimises I. Therefore, by proposition 4.2, we have lim ǫ→0 µ y (A ǫ +z) µ y (A ǫ + z * ) = 1.
The result follows.
6.4. Proofs of results in section 5. | 2017-05-23T07:03:00.000Z | 2017-05-09T00:00:00.000 | {
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225635164 | pes2o/s2orc | v3-fos-license | Development of Teaching Materials Based on Learning Cycle 5E and Enriched With Augmented Reality for Rate of Reaction Topic
Chemistry is the study that most of the material is abstract. Many students have difficulty learning chemistry especially the reaction rate. Therefore, we need a learning model that can help students think critically, one of which is the 5E Learning Cycle and is supported by a program that can visualize aspects of the reaction rate, such as Augmented Reality. The purpose of this study was to produce Learning Cycle 5E teaching materials assisted by augmented reality on the reaction rate material. The development of teaching materials is carried out in three stages, namely (1) the analysis phase, (2) the design phase, and (3) the development phase. The results showed that the teaching material developed was included in the very feasible category with a percentage of (a) material validation of 89.7%, (b) media validation of 86%, and (c) legibility test of 90%. Based on the results of the validation and readability test, it can be concluded that teaching materials can be used as a support in the process of learning the reaction rate.
INTRODUCTION
Most of the concepts in chemistry are abstract. Observations by the senses directly and indirectly, and are represented required to understand the chemistry [1] [2]. Understanding the concept of chemistry requires three representations include representations of macroscopic, submicroscopic, and symbolic [3]. Learning in school does not involve the third representation so that students do not understand and tend to just memorize theories that exist without understanding it. One chemical material involving three representations to understand that reaction rate. Results by Handayanti [4] showed that students' understanding at the level of sub-microscopic to the material reaction rate is the lowest when compared with other representations. Most of the students had difficulty in learning the material reaction rate. This is shown by the data in SMA Negeri 1 Karanganyar that the average value of daily tests on the material that the reaction rate is still lower than the 68.25 completeness value of 75 on a list of values in 2010/2011 [5]. Other data obtained from SMA Negeri 1 Manyar, the result that as many as 57.7% chemistry is a difficult subject and material that is difficult is the reaction rate with a percentage of 31.43% [6]. To help overcome these difficulties required various forms of representation that can visualize the material. Their learning resources can help improve student understanding. Teaching materials are things that contain a lot of information such as the concept of learning materials, formulas, pictures, and others. The results by Sholahuddin [7] state that class X chemistry textbooks based on didactic reduction are feasible to be used in the learning process with validated data with excellent categories and students' learning completeness classically reaches 80.2%. Kusuma and Siadi [8] also stated that student learning outcomes can be improved by applying chemo-entrepreneurship oriented teaching materials (CEP). Existing teaching materials must be adapted to the curriculum and curriculum used today, the 2013 curriculum. Learning is required to be able to achieve KI-3 where students can understand, apply, analyze factual, conceptual, procedural knowledge with the help of teachers or independently [9]. One learning model that allows students to actively investigate a phenomenon through a scientific process Learning Cycle model that can be abbreviated as LC. This model facilitates inquiry activities and helps students understand scientific processes to develop and deepen understanding of concepts [10]. The stages of learning in this model allow students to play an active role in mastering certain competencies. The research result by Gazali [11] shows that the LC 5E can enhance students' critical thinking skills and science process skills. Research by Silaban [12] suggest that the use of innovative teaching materials on the material chemical reaction rate provides better learning results, the average grade obtained by the 85.00 and the percent increase in learning outcomes by 76%. 5E LC-based teaching materials has been developed and demonstrated that teaching materials valid and enforceable in the school including by Setyaningsih [13] shows the results of expert lecturers validation of 3.9 and 3.2 teachers validation and validation of devices for 3 declared invalid test results, by Nuraisyah [14] shows the validation results of 92.78% student books and teacher books validation by 93.88%. Teaching materials that have been there already alluded submicroscopic level, but only indicated by a twodimensional image is not animated so that students are still difficulties in understanding it. It is, therefore, necessary to develop teaching materials that can represent the 3D shape of the molecule. 3D shapes can be displayed using Augmented Reality (abbreviated as AR) in the application form. AR development has been done but in chemical material is still small. Research by Hafidha & Sudarmilah [15], namely the development of AR on the material Periodic System Elements yield based applications android good look, operational, and material and test results indicate that over 80% of respondents said AR application on the material Periodic System Elements excellent. By Supriono [16] showed that the application can run well, the test results portability of 96.7%, can be used on all smartphones except the resolution widened as tabs, and there are features of the periodic table that can be used directly in applications. The purpose of this development to produce a product of teaching materials based on the AR-assisted 5E LC material reaction rate valid and feasible to be implemented in learning.
METHOD
The research and development model of this teaching material uses the Research & Development model proposed by Lee & Owens namely Multimedia-based Instructional Design. This model has the advantage of systematic and structured steps. The model consists of five stages: (1) assessment/analysis which includes needs analysis and front-end analysis (2) design (3) production/development), (4) implementation, and (5) evaluation [17]. The step-by-step development procedure can be seen in Figure 1. The material development procedure is carried out only until the third stage, this is due to time constraints. These stages are (1) assessment/analysis (2) design, and (3) production/development. Data collection instruments used in the form of expert validation questionnaires and student validity questionnaires. The questionnaire was used in the form of an open questionnaire (for criticism and suggestions) and a closed questionnaire (using a Likert scale). The validation questionnaire consisted of material validation and media validation validated by one high school chemistry teacher and one UM chemistry lecturer, while the questionnaire readability was up to 20 students in class XI of UM Laboratory. The questionnaire instrument was used to obtain (a) quantitative data from the questionnaire attached in the form of students' validation and readability scores, (b) qualitative data from the open questionnaire in the form of comments and suggestions used as consideration for the revision of teaching materials. The Likert scale used was adapted from Riduwan [18] with a range of 1-5 votes. Scores obtained in the form of a percentage scale indicate that the instructional materials feasible/valid use or need to be revised.
RESULTS AND DISCUSSION
Teaching materials have been developed in the form of teaching materials based on the 5E Learning Cycle which is enriched with Augmented Reality technology. Teaching materials are printed in A6 (10.5 cm x 14.8 cm) pocketbooks and Augmented Reality applications with the .apk format. Teaching materials are divided into four main parts, including the pre-introduction, introduction, content section, and closing section. The pre-introduction section consists of a cover page, a student identity page, preface, table of contents, and instructions for the use of teaching materials. The introductory part consists of learning objectives, concept maps, and introductory writers and perceptions. The body part consists of two stages of the learning cycle with the 5E Learning Cycle. The concluding section consists of competency tests, sundries, notes, bibliography, and author biographies.
There are some analyzes done first. The analysis fronf-end result are student analysis consists of psychological analysis and analysis of habits. This analysis was obtained from interviews with several students. Psychological analysis about the way of thinking of students who are still concrete, is not yet abstract so teaching materials are needed that can trigger the process of abstract thinking of students so that the material learned is truly understood by students. Analysis of habits about the habits of students who more often play gadgets and in their lives cannot be kept away from technology, so that teaching materials are made that can be used simultaneously with gadgets or smartphones so that students are more interested in learning the reaction rate material. Schools have the facilities needed to support learning using teaching materials developed, one of which is WiFi. Students are allowed to bring a smartphone that is useful as a support for learning. The types of smartphones students carry are also included in this analysis. Students use a smartphone that has a large storage space and adequate type of android for the use of certain applications so that it can support the teaching materials that are developed. Geographical location will affect the strength of the internet network that will be used to access teaching materials. The condition of the learning environment is used to determine the form of teaching materials to be developed. The situation of student learning is also conducive so that the use of teaching materials will be maximized. Task analysis has been used as a determination of material that has been developed as teaching material. The material that has been used in the development of teaching materials is material that is abstract, so it requires a deep understanding. KD and KI assessment is also needed on the material that has been developed to be able to determine indicators and learning objectives in accordance with the 2013 curriculum. Material that is considered abstract is the rate of reaction because in understanding it takes three representations, so it needs to be reviewed about KD and KI as well as making indicators and also learning objectives so that teaching materials can improve student understanding. Analysis of important events will be used for the manufacture of developed teaching materials. Students are more interested in matters relating to images and animations compared to the text alone so it takes teaching materials that contain images, animations and videos. The problem is that videos and animations that support learning that fit students' needs are difficult to find, so a review of the rate of reaction is needed. The purpose of developing teaching materials is to help students understand more deeply the material reaction rates, especially in some abstract parts so there is a need for videos, 3D animations that can describe the reaction rate material. The media needed for the reaction rate material are phenomena videos, experiments and microscopic shapes and 3D animations, so that the teaching material will contain these things so that they can be utilized maximally. The data available in the form of material in books, videos and some images, needed other video making and also 3D animation. Questionnaire validation generates two types of data, namely quantitative data and qualitative data. Quantitative data obtained from the calculation of the average value of the questionnaire, while the qualitative data obtained from the comments and suggestions of the validator that is used for product revision. The validation test results consist of four main aspects, namely the feasibility of the language, the presentation of the feasibility, the suitability of the content, and the correctness of the concept.
Material Validation Result
Material eligibility consists of three aspects: the first cycle, the second cycle, and understanding tests. Thus obtained six aspects in the results of the validation test of this material with the values as follows: (1) the feasibility of language 92.5%, (2) the feasibility of representation of 85.7%, (3) the feasibility of the contents of the first cycle of 90.7% , (4) the feasibility of the contents of the second cycle 92.2%, (5) the feasibility test for the content of understanding by 7%, (6) the truth of the concept of 100%.
Media Validation Result
On the results of the media, validation test consists of two aspects which include aspects of graphics and aspects of using augmented reality. To get two values including (1) graphic of 92.3%, (2) the use of augmented reality by 80%. Based on the calculation of the overall average value, the final result obtained is a material validation value of 91 and a media validation of 83.
Legibility Test result of Student
The results of the data from the students' readability test obtained a percentage of 90.11% that met the criteria for teaching materials is very valid, so that teaching materials are very appropriate to be used in learning activities at the material and media scales. Based on the results of validation and test the readability of these students, the conclusion that teaching materials have been developed including in the category of very decent so it can be used in learning activities. In general, students and validators give a positive response to instructional materials, including materials that can make learning a more exciting interest in learning, creative, and makes learning more fun.
CONCLUSION
The results of this research and development in the form of teaching materials chemistry A6 sized to print out the form on the material and the rate of reaction with augmented reality applications. The .apk format developed is used on smartphones that have Android operating system specifications. Teaching materials that contain QR Codes and markers. QR Code in the form of books connected to google, pdfs, as well as videos, and 3D animations connected to markers using the downloaded Arce application. Teaching material to get material validity is 89.7%, media validity is 86.1%, and readability is 90.1%. In the teaching material, the material consists of two cycles of learning with Learning Cycle 5E stages comprising the steps of (1) involvement, (2) exploration, (3) an explanation, (4) the elaboration, and (5) evaluation. | 2020-10-30T08:06:41.099Z | 2020-07-13T00:00:00.000 | {
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251693381 | pes2o/s2orc | v3-fos-license | Acute and subacute toxicity studies of a poly herbal formulation used for diabetes
Objectives: PHF-dia (Poly Herbal Formulation Diabetes) is a polyhedral formulation possessing antihyperglycemic and antihyperlipdimic effects. This study aims to assess acute and sub-acute toxicity of PHF-dia in rats. Methods: This is an experimental study conducted in two different phases. Acute toxicity was conducted for 14 days and sub-acute toxicity was conducted for 28 days. Both studies were conducted in animal house of Jinnah University for Women, Karachi, Pakistan. Acute toxicity was evaluated in vivo with single time oral administration of 400 mg/kg and 2000 mg/kg doses for two weeks. Sub-acute toxicity was investigated with the application of repeated doses of 150 mg/kg/day, 250 mg/kg/day and 500 mg/kg/day for 28 days. Results: Acute toxicity study results showed no toxic symptoms, behavioral changes or death in rats up to 2000 mg/kg. Therefore, LD50 of oral toxic dose must be more than 2000 mg/ml. Similarly, sub-acute toxicity studies confirmed the safety of PHF-dia and showed no clinical symptoms nor biochemical or histological variation in rats treated with 150 mg/kg, 250 mg/kg and 500 mg/kg compared to the control group (p <0.05). Conclusion: This indicates safe nature of PHF-dia for the further clinical trials. However, mechanism of action of PHF-dia is not fully understood.
INTRODUCTION
Diabetes Mellitus (DM) is a metabolic disease increasing immensely all over the world. Carbohydrates, proteins and lipids metabolism disturbs that causes many complications. DM is being presumed as the largest worldwide non-contagious disease by year 2025. It is also hypothesized that developing world will be more prone to DM. 1,2 There is lack of systematized health care system in developing countries. 3 Only a tiny population has access to the modern medicine while other mostly relay on native herbs having hypoglycemic potential. 4 A poly herbal formulation (PHF-dia) consisted of Syzigium cumini (seeds), Holarrhena antidysenterica (seeds), Citrullus collocynthis (fruit) having reported antioxidant, hypoglycemic and hypolipidemic effects in streptozotocin induced diabetic rats. 4 All the plants included in PHF-dia have traditional evidence based ant diabetic effects. 5,6 A common belief prevailed is about the harmlessness of natural herbal medicines due to which safety profile and toxicity studies of traditional herbal medicines are scarce. Therefore, we planned this study to investigate acute and sub-acute toxicity of PHF-dia. Ex-vivo, in vitro and animal models can be used as test systems. However, for toxicity evaluation laboratory animals are more suitable than other studies. 7 Male rats were used because they are more sensitive to assess acute and subacute toxicity. 8 The primary objective of this study was to access acute and sub-acute toxicity for safe application of PHF in future clinical trials.
METHODS
This is an experimental study conducted in two different phases. Acute toxicity was conducted for 14 days and sub-acute toxicity was conducted for 28 days. Both studies were conducted in animal house of Jinnah University for Women, Karachi, Pakistan. Plant parts namely Syzigium cumini (seeds), Holarrhena antidysenterica (seeds), Citrullus collocynthis (fruit) were purchased as a dried herb from local market of Bahawalpur, Pakistan and were authenticated in the Department of Botany, Islamia University of Bahawalpur. The voucher specimens were placed at the herbarium of IUB (Voucher no 2201/L.S).Extracts of selected plants were made according to methodology of Abbasi et al 2018. 9 The poly herbal formulation was prepared by taking specific ratios of extracts of selected plant parts based on method of Pari L & Saravanan R et al. 10 This study was carried out on Wistar male rats. Average weight of Wistar rats was 145-250g. They were reserved in normal atmosphere (25°C, 12h/12h light/dark cycle) with standard diet and water ad libitum. Acclimatization was done for one week prior to study. Study design and procedures were performed according to National Toxicology Program 2016. Prior ethical permission was obtained from the animal ethical committee of Jinnah Women University of Karachi (Ethical approval number: EC/EMS/0252020). Animals were grouped in to three and four groups for acute and sub-acute toxicity studies respectively. For acute toxicity studies, two dosages were tested i.e., 400mg/kg and 2000mg/kg while for sub-acute toxicity studies, 150mg/kg, 250mg/kg and 500mg/ kg dosages were tested. General toxicity guidelines for natural traditional medicines are followed. 7 Acute toxicity study test: For this study male Wistar rats were used and were kept fasted for 24 hours. A single dose of PHF 2000 mg/kg was administered to each rat. They were kept under observation for first 30 minutes following dose. They were then observed during first 24 hour and for 72 h respectively. The parameters studied were body weight, drowsiness, lacrimation, nasal bleeding, paralysis, piloerection, salivation, skin, utilization of food, water and death. 11 Sub-acute toxicity study: The animals were separated in four groups, each having six animals. One group was set as control. In control group normal food and water was given. Other three groups received the doses of 150mg/kg b.w/ day, 250mg/kg b.w/day and 500mg/kg b.w/ day respectively. The weight of animal was measured weekly and their behavioral changes, morphological changes were also observed. The animals were anaesthetized on 28 th day of treatment by introducing 5ml/kg of 1% solution of chloralose in 25% urethane (w/v) intra peritoneal. For the collection of blood sample cardiac puncture was performed. Blood sample was collected in EDTA tubes and heparinized tubes it was then sent for hematological and biochemical analysis. 12,13 Biochemical Analysis: Samples of blood were analyzed by CBC machine. The contents include; RBCs count, PLT count, WBCs count, Hb, monocytes, lymphocytes, basophils, neutrophils and eosinophil. Serum urea, uric acid, creatinine, cholesterol, triglycerides, HDL, VLDL, ALT, AST, acid phosphatase, and alkaline phosphatase were calculated. Histopathological Analysis: The heart, liver, adipose tissues and kidney sections were fixed in 10% neutral buffered formalin overnight at room temperature. Histopathology was performed by the method of Loha et al 2019. 14 Statistical Analysis: Data were presented as mean ± SEM. Statistical analysis was performed by using SPSS version 20. One way ANOVA followed by LSD post hoc test. The P value of less than 0.05 was considered significant.
Results of acute toxicity study indicated safe nature of PHF-dia at doses of400 mg/kg and 2000mg/kg. No death or behavioral change was observed in the treated groups at tested doses. So, the LD 50 of the PHF could be greater than 2000mg/ kg body weight. Fig.1 describes weight evolution of rats during acute toxicity studies of PHF. There was no statistical difference of body weights of control and tested groups (400 mg/kg and 2000 mg/kg).
During sub-acute toxicity study, there were no gross pathological changes in the liver, heart, adipose tissues, pancreas and kidneys of the control and experimental groups. Throughout the subacute study, no death or toxic effects were detected on experimental rats (125 mg/kg, 250 mg/kg and 500 mg/kg). There was no significant difference (P > 0.05) observed in the mean rat body weights between control and treated groups (Fig.2).
Herbal formulation used for diabetes There was no statistical difference between control group and PHF different dosages p≤0.05.
In the sub-acute toxicity study RBC, WBC, PLT, HB, neutrophils, lymphocytes, eosinophils, monocytes, basophils of the experimental groups (125 mg/kg, 250mg/kg and 500 mg/kg) were within the reference range for rats. 15 The hematological values in the control group and PHFgroups were not significantly different (Table-I
).
In the sub-acute toxicity study, the biochemical parameters, such as urea, creatinin, uric acid, ALT, AST, acid phosphatase, alkaline phosphatase, triglycerides, HDL, LDL of experimental groups were within the reference range for rats. 15 The biochemical values in the control group and PHF groups were not significantly different (Table-II).
In the histopathological studies hematoxylin and eosin stain technique was used, the sections of the kidneys, liver, adipose tissues, and heart of treated rats showed normal general structure with no significant difference as compared to control. The lense used to capture these pictures from histopathological slides was of 10 magnifications hence is of 100 scale bars (Fig.3).
DISCUSSION
This study evaluates the toxicity profile of PHFdia in rats in two phases. First phase determines acute toxicity while 2 nd phase assesses sub-acute toxicity. Changes in the body weight is used as an index of toxicity evaluation. 16 PHF-dia might be considered relatively safe on acute exposure as it showed no toxic effect or body changes in rats. Sub-acute toxicity study analyses toxicity caused by repeated oral dosing of a substance for 28 days in rodents. This study gives evidence of any change in organ morphology, hematological changes or biochemical parameters in the organism. This study could be used as the basis for the determination of the toxicity effects. 14 Hematological parameters examination can be used to identify any harmful effect of foreign compounds including in poly herbal formulation on blood. 17 Biochemical parameters such as urea, creatinine, uric acid, ALT, AST, acid phosphatase, alkaline phosphatase, triglycerides, HDL and LDL etc. have substantial roles as a marker. Liver and kidney function assessment is of main significance to assess the toxic properties of poly herbal formulations and drugs. 18 Urea measurement is an indicator of kidney function. It could be raised in several acute and chronic renal disorders. 19 Normal range of serum urea in rats is 15-45mg/dl. 2 Creatinine is used as a marker of glomerular filtration rate. 20 Normal value of creatinine in rats is 0.2-0.8 mg/dl. 21 ALT is a sensitive marker for checking any liver cell damage 22 while AST is present in RBCs, heart, kidney and skeletal muscles. 23 PHF-dia showed no toxic effects on hematological and biochemical parameters. The study indicates the safe nature of PHF-dia as it is confirmed by former studies that 10 ± 0.04 mg/ mL is confirmed dose exhibiting α-glucosidase potential in jamun seed extract,0.1mg/ml of the profuse extracts of citrus colocynthis produced remarkable hypoglycemic activity by sensitizing insulin production from pancreas whereas,300 mg/week is the safe dose studied in literature for ant diabetic purpose. So, it can be used safely for treatment of diabetes and might be proved as an effective alternative option for control of hyperglycemia and hyperlipidemia.
Limitation of the study: Study indicates a safe alternative choice of treatment for Diabetes. However, mechanism of action of PHF-dia is not fully understood.
CONCLUSION
The acute toxicity study of PHF-dia showed safe nature of PHF-dia up to 2000 mg/kg. No morbidity and mortality were observed at tested doses. So, oral LD 50 of PHD-dia will be more than 2000mg/kg. Meanwhile, sub-acute toxicity studies of PHF-dia did not produce adverse effects on body weight of rodents, organ weights & morphology, hematological and biochemical parameters. Moreover, histopathological studies indicate no significant difference between control and treated animal heart, liver, adipose tissues and kidney sections. These studies indicate the safe nature of PHF-dia for the further testing of herbal formulation in human clinical trials. | 2022-08-21T05:12:36.623Z | 2022-01-01T00:00:00.000 | {
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250187040 | pes2o/s2orc | v3-fos-license | Cytostatic Effects of Polyethyleneimine Surfaces on the Mesenchymal Stromal Cell Cycle
Polyelectrolytes assembled layer-by-layer (PEMs) are commonly used as functional coatings to build-up biological interfaces, particularly suitable as compatible layers for the interaction with a biological medium, providing suitable conditions to promote or prevent cell seeding while maintaining the phenotype. The proper assessment of the biocompatibility of PEMs and the elucidation of the related mechanisms are therefore of paramount importance. In this study, we report in detail the effect of two different PEM endings, polystyrene sulfonate (PSS) and polyethylenimine (PEI), respectively, on the cell adhesion, growth, and viability of human bone mesenchymal stromal cells (MSCs). The results have shown that PSS-ended substrates appear to be the most suitable to drive the cell adhesion and phenotype maintenance of MSCs, showing good biocompatibility. On the contrary, while the cells seem to adhere more quickly and strongly on the PEI-ended surfaces, the interaction with PEI significantly affects the growth and viability, reducing the cell spreading capability, by sequestering the adhesion molecules already in the very early steps of cell–substrate contact. These results point to the promotion of a cytostatic effect of PEI, rather than the often-claimed cytotoxicity.
Introduction
Cell adhesion is a multistage process involving the attachment, spreading, and formation of stress fibers and focal adhesions at the very early steps [1], being critically dependent on surface characteristics. As is known, the bulk properties of biomaterials are very important, but their surface properties are of utmost importance, as they boost the design of functional materials and are able to drive tissue and cellular events such as protein adsorption, recolonization, adhesion, proliferation, migration, and inflammatory response [2]. Thus, controlling the surface properties of materials is a real challenge on the way to developing new generations of smart biomaterials and the related tissue engineering strategies [3].
Indeed, surface properties, such as topography, stiffness, surface free energy (SFE), roughness, and chemistry (specific chemical functionalities), have been studied in detail, showing that they all play important roles, promoting the cell response to chemical and physical cues that can be "added" or modified by surface modification [4]. One of the wellestablished methods to modify surface properties is to coat them by means of polyelectrolyte Polymers 2022, 14, 2643 2 of 18 multilayers (PEMs), built by a layer-by-layer (LbL) technique, consisting of the alternate deposition of polycations and polyanions that self-organize on the material's surface [5]. This methodology is simple, flexible, effective, inexpensive, and versatile and it has been applied to a large variety of biomedical devices [2,3]. Understanding the specific effect of chemically different PEMs on the initial cell attachment process, as well as analyzing adhered cells and studying the cell behavior and phenotype, may lead to a significant improvement of biomedical devices [6].
In order to better comprehend the interaction mechanisms between cells and LbL thin films, the cell accommodation on the PEM surfaces have to be studied by using a non-invasive and real-time technique with high sensitivity for the interfaces.
In this context, a technique based on the characterization of interface's properties by using surface acoustic waves as sampling tools, i.e., quartz crystal microbalance with dissipation monitoring (QCM-D), can be employed. This technique, indeed, has been widely used to study the interaction process of "soft" matter thin layers with solid surfaces, including the formation of interfaces with ultrathin polymer films, biomolecules, nanoparticles, vesicles and, in general, biological systems [7][8][9]. Moreover, the technique has been successfully extended to the study of cell-substrate interaction events, owing to the specific sensitivity of the technique to the viscoelastic changes occurring in the wall region of the cells interacting with the sensor surface [10][11][12].
The aim of the present work was to study the early steps of cell-surface interaction using two different PEM coatings, to understand the effect of surface physicochemical properties on cell adhesion, morphology, and proliferation. In particular, we studied the early events of the interaction between mesenchymal stem cells isolated from human bone marrow (MSCs) and PEMs of polystyrene sulfonate (PSS) (bearing negatively charged groups) and polyethyleneimine (PEI) (positively charged polymer), both assembled with the LbL deposition technique. It is worth mentioning that while PSS is known to have a good biocompatibility with the biological medium, the literature has reported that polyethyleneimine (PEI), on the other hand, exhibits some form of "toxicity" towards various cell lines [13].
In this context, the mechanical response of the cells to the two PEMs was studied by using the QCM-D technique, while the differential cell response was investigated by means of thorough biological assays, consisting of cell viability tests, morphological changes due to the rearrangement of the actinic and tubulin cytoskeleton, and tests evaluating the formation of focal adhesion complexes. Finally, mitochondrial activity was also examined through the use of a fluorescent probe, sensitive to the variation of the mitochondrial membrane potential.
Our results showed a remarkable difference between the two PEMs on the cells in cytoskeleton reorganization and related different effects on cell adhesion, growth, and metabolism.
More specifically, while on the PSS-ended surface the cells retain the MSC phenotype and show good compatibility, on the PEI-ended surface the cells adhere faster and strongly, blocking the cell growth and spreading, owing to the random and peripheral formation of many focal adhesion sites in connection with the sequestering of the adhesion molecules in the initial cell-substrate contact area. Overall, these results point to a cytostatic effect of PEI-ended coating, rather than the often-claimed cytotoxicity effect, actually mostly reported for PEI in solution, instead of compact anchored layers.
Polyelectrolyte Multilayers
Polyelectrolyte solutions were prepared as follows: PEI (MW 750,000) and PSS (MW 70,000) were purchased from Sigma, Milan, Italy and solubilized in a 0.15 M NaCl solution at a concentration of 1 mg/mL. Before PEM deposition, the surfaces were irradiated with UV-O 3 for 30 min at atmospheric pressure in a Jelight Instruments apparatus (Jelight Company Inc., Irvine, CA, USA) (λ ex of 185 and 254 nm) to remove any carbon moieties, washed extensively with ultrapure water, and dried with blown nitrogen. Then, the deposition of the PSS (−) and PEI (+) polymeric layers on the wells and on the slides to be used for our study using the LbL technique was carried out. Simple immersion in the PEI or PSS solution for 15 min was enough to achieve a complete layer of each polyelectrolyte: PEMs were built-up on the surfaces by alternating PEI and PSS layer deposition to obtain a PEI-PSS film with an anionic surface, and a PEI-PSS-PEI film with a cationic surface. The obtained multilayers were completely dried and incubated for 2 h with antibiotic-antimycotics andm after two washings with PBS, experiments were started by adding the cell suspension to each sample. Culture plates or glass slides without preliminary treatment with PEMs were used as control samples. For clarity, the cell-facing outer layer is used throughout the text as a shorthand for multilayers, with PEI-ended indicating the PEI/PSS/PEI multilayer and PSS-ended indicating the PEI/PSS multilayer.
Cell Culture
Mesenchymal stromal cells were obtained from human bone marrow aspirates from healthy donors after they had given their informed consent. hBMSCs were seeded in 75 cm 3 T flasks and expanded in minimum essential alpha medium (αMEM) supplemented with L-glutamine, nucleosides and Earle's salts, antibiotic-antimycotic (10,000 U/mL penicillin, 10,000 µg/mL streptomycin, 25 µg/mL Fungizon), 10% fetal bovine serum (FBS), and 100 µm of ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA). The culture flasks were maintained in a 37 • C incubator, with a humidified atmosphere of 5% CO 2 . Medium was changed after 3-4 days to remove non-adherent cells and replaced with fresh medium. When the cell monolayer was approximately 80% confluent, the cells were detached by trypsin/EDTA (0.05%/0.2% w/v) and seeded in T25 flasks with complete αMEM, and 10% FBS. Flow cytometric analysis revealed positivity for CD105, CD90W, and CD73 and not for CD34, CD14, and Gly A, [14]. All products were purchased from GIBCO (Life Technologies, Carlsbad, CA, USA).
Scanning Electron Microscopy
MSCs were cultured on PSS-ended or PEI-ended round glass coverslips. At the prefixed experimental time (24 h), cells were fixed in 2% glutaraldehyde in 0.1 M Na cacodylate buffer (EMS), pH 7.4, and then post fixed in 1% osmium tetroxide (EMS) in the same buffer. The samples were dehydrated in graded ethanol followed by critical point drying (Emscope CPD750), mounted on stubs, and sputter coated with gold (Sputter Coater, Polar SC7640, Quorum Technologies, East Sussex, UK). Cell morphologies were observed with a field emission scanning electron microscope (FESEM Hitachi S4000, Fukuoka, Japan).
Indirect immunofluorescence-IF was used to reveal tubulin, integrin α1, integrin α5, FAK-pY397, and paxillin pY31. The cells were initially treated as set out above, but after permeabilization they were incubated in blocking buffer (BSA 5% in PBS) for 30 min and with the primary antibody (1 h). Samples were then washed in PBS and incubated with red or green fluorescent secondary antibody goat anti-rabbit IgG Alexa Fluor 594/488 (2.5 µg/mL) (Immunological Science, Rome, Italy) for 1 h at room temperature. After two washes with PBS, the samples were allowed to dry and were mounted with Fluoro Gel with DAPI (EMS). Fluorescence was observed with an Olympus BX50 fluorescence microscope equipped with a DC500 camera (Leica, Wetzlar, Germany). pFAK and p-paxillin were observed using a laser scanning confocal microscope (Zeiss LSM 700, Oberkochen, Polymers 2022, 14, 2643 4 of 18 Germany). Green and blue signals were detected with laser light at 488 nm and 405 nm, respectively. All acquisitions with the laser scanning confocal microscope were performed using ZEN-2010 software.
α5 Integrin and microfilament double labeling-To better highlight the relationship between microfilaments and integrins, double labeling was carried out with primary antibodies for the cytoplasmic subunits of the two integrins and FITC-phalloidin. Microfilaments were stained after incubation with Alexa Fluor 594 (Thermo Fisher, Waltham, MA, USA).
Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D)
Measurements of adsorption kinetics were performed by using a quartz crystal microbalance with dissipation monitoring (QCM-D) instrument (Q-Sense AB, Gothenburg, Sweden) with AT-cut gold crystal sensors. The simultaneous measurements of frequency, f, and energy dissipation, D, were performed in the fundamental resonance frequency (n = 1, i.e., f = 5 MHz) and the overtones (n = 3, 5, 7, and 9 corresponding to f = 15, 25, 35, and 45 MHz, respectively). The resolution in F and D was ± 0.1 Hz and 1 × 10 −7 , respectively. Each QCM-D experiment started with the sensor running on PBS (outgassed with 30 min sonication), then the addition of cells and, after 180 min, the exchange of the solution being measured with PBS to check both the desorption and stability of the adsorbed layer. All the experiments were performed in water at 37 • C and the flow rate was 150 µL min −1 . Three replicas for each experiment were performed for data reproducibility.
Cell Viability and Cell Cycle Assessment
MSCs were seeded (1 × 10 4 cells/well) on a 24-well plate previously coated with PEMs, and incubated for 3 h, 24 h, 48 h, 72 h, and 120 h at 37 • C. Mitochondrial activity was measured by the MTT assay, incubating the cells with 0.05 mg/mL/well 3-(4,5dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide salt (MTT; Sigma Aldrich, Italy) in serum free medium. After 2.30 h of incubation at 37 • C, formazan salts, produced by succinate dehydrogenase activity in live cells, were solubilized with 0.1 M isopropanol//HCl and quantified spectrophotometrically by a Cary 50 (Varian, Palo Alto, CA, USA). The values obtained are expressed as corrected optical density (∆ OD: λ570−λ650). All samples and experiments were carried out in triplicate. Evaluation of the cell cycle was performed using the Muse ® Cell Cycle Kit (Luminex Corporation, Austin, TX, USA) by the Guava ® Muse ® Cell Analyzer following the manufacturer's instructions. Briefly, cells were trypsinized, washed, and resuspended in fixing solution with 70% ethanol and then stained with cell cycle reagent containing propidium iodide (PI). On average, 5000 events were acquired per sample. Data were generated by the Muse ® Cell Cycle software module.
ATP Evaluation and Mitochondrial Transmembrane Potential (∆Ψ)
Cellular ATP content was measured using a firefly luciferase-based ATP assay kit (ATPlite 1 step Luminescence, Perkin Elmer, Waltham, MA, USA) according to the manufacturer's instructions. Samples were lysed and quantified after 3 h, 6 h, and 24 h of culture. The light emission was directly proportional to the concentration of ATP and is expressed in counts per second (CPS). The mitochondrial transmembrane potential was determined by fluorescence microscopy after incubation with JC-1 (5,5 , 6,6 -tetrachloro-1,1 , 3,3 -tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes, Eugene, OR, USA). MSCs were seeded on glass slides and PEI-ended slides for 3, 24, and 48 h, incubated with 0.1 µM JC-1 fluorescence probe for 30 min, and washed three times with PBS. Mitochondrial membrane potential was observed with an Olympus BX50 fluorescence microscope (Olympus Italia, Segrate, Italy). The green JC-1 signals appeared at 485/535 nm, and the red signals appeared at 590/610 nm. Some experiments were performed to test the effect of exogenous ATP on cell viability, actin cytoskeleton, and ATP content of MSCs on PEI-ended surfaces. The culture medium was supplemented with 20 µM, 10 µM, and 5 µM of ATP (Sigma Aldrich, Italy) and, after intervals of 3 and 24 h of incubation, samples were prepared for MTT assay, ATP content, and FITC-phalloidin assay. After 72 h of incubation, the medium was replaced with fresh α MEM containing the different concentrations of ATP and incubated for another 3 h. All experiments were carried out in triplicate.
Statistical Analysis
Statistical analysis data are expressed as mean ± standard deviation (S.D.) of three independent experiments (i.e., biological and technical triplicates). We evaluated the statistical significance of these data by applying the one-way ANOVA test, as described in the figure legends.
Morphological Analysis
The initial MSC attachment and spreading were visualized using scanning electron microscopy. The morphology of cells after 24 h of adhesion on PEMs was different with respect to the control as shown in Figure 1. Indeed, the cells adhering to the PSS-ended surface had a regular and elongated morphology (Figure 1b), similar to the control (Figure 1a), with long pseudopodia suggesting that they had begun to spread, as shown by the cyan arrows and line, while MSCs on the PEI-ended substrates ( Figure 1c) maintained a more rounded morphology with a flat cytoplasmic edge from which thin filaments branched off towards the substrate, as shown by the cyan circle.
ture. The light emission was directly proportional to the concentration of ATP and is expressed in counts per second (CPS). The mitochondrial transmembrane potential was determined by fluorescence microscopy after incubation with JC-1 (5,5′, 6,6′-tetrachloro-1,1′, 3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes, Eugene, OR, USA). MSCs were seeded on glass slides and PEI-ended slides for 3, 24, and 48 h, incubated with 0.1 μM JC-1 fluorescence probe for 30 min, and washed three times with PBS. Mitochondrial membrane potential was observed with an Olympus BX50 fluorescence microscope (Olympus Italia, Segrate, Italy). The green JC-1 signals appeared at 485/535 nm, and the red signals appeared at 590/610 nm. Some experiments were performed to test the effect of exogenous ATP on cell viability, actin cytoskeleton, and ATP content of MSCs on PEIended surfaces. The culture medium was supplemented with 20 μM, 10 μM, and 5 μM of ATP (Sigma Aldrich, Italy) and, after intervals of 3 and 24 h of incubation, samples were prepared for MTT assay, ATP content, and FITC-phalloidin assay. After 72 h of incubation, the medium was replaced with fresh α MEM containing the different concentrations of ATP and incubated for another 3 h. All experiments were carried out in triplicate.
Statistical Analysis
Statistical analysis data are expressed as mean ± standard deviation (S.D.) of three independent experiments (i.e., biological and technical triplicates). We evaluated the statistical significance of these data by applying the one-way ANOVA test, as described in the figure legends.
Morphological Analysis
The initial MSC attachment and spreading were visualized using scanning electron microscopy. The morphology of cells after 24 h of adhesion on PEMs was different with respect to the control as shown in Figure 1. Indeed, the cells adhering to the PSS-ended surface had a regular and elongated morphology (Figure 1b), similar to the control ( Figure 1a), with long pseudopodia suggesting that they had begun to spread, as shown by the cyan arrows and line, while MSCs on the PEI-ended substrates ( Figure 1c) maintained a more rounded morphology with a flat cytoplasmic edge from which thin filaments branched off towards the substrate, as shown by the cyan circle. After 8 days, cells adhering to the PSS-ended films formed a confluent monolayer (Figure 1e), while on the PEI-ended substrates, they did not form a monolayer, retaining their initial rounded shape and showing an evident irregular cellular morphology ( Figure 1f). Overall, the data suggest a remarkable dependence of cell response on the chemical nature of the two surfaces.
Microfilaments and Microtubules
We analyzed changes in the cell shape and cytoskeletal organization of microfilaments and microtubules during the early events of cell adhesion (3 h, 6 h, 24 h) on polyelectrolyte Polymers 2022, 14, 2643 6 of 18 multilayer films compared with MSCs seeded on control surfaces. Direct immunofluorescence results showed that a noticeable change in actin distribution, cell morphology, and size occur during the adhesion and spreading processes of MSCs ( Figure 2). Again, on the PSS-ended slides, cells showed an actin distribution and organization in microfilaments very similar to the control, even if the adhesion and spreading processes seemed to proceed more slowly.
nature of the two surfaces.
Microfilaments and Microtubules
We analyzed changes in the cell shape and cytoskeletal organization of microfilaments and microtubules during the early events of cell adhesion (3 h, 6 h, 24 h) on polyelectrolyte multilayer films compared with MSCs seeded on control surfaces. Direct immunofluorescence results showed that a noticeable change in actin distribution, cell morphology, and size occur during the adhesion and spreading processes of MSCs ( Figure 2). Again, on the PSS-ended slides, cells showed an actin distribution and organization in microfilaments very similar to the control, even if the adhesion and spreading processes seemed to proceed more slowly. After 3, 6, and 24 h, they assumed an elongated morphology with regular and numerous stress fiber formation (Figure 2b,e,h). On PEI-ended slides, the MSCs, after 3 and 6 h (Figure 2c,f), instead showed a rounded morphology with a distribution of fluorescent protrusions that were extended radially in the periphery. After 24 h some cells spread on the substrate and displayed long protrusions and numerous filopodia, without stress fibers, but with an evident actin cortex (Figure 2i). These results revealed that PEI had a strong, hindering effect on the correct formation of stress fibers, expected, in turn, to severely affect the cell cytoskeletal functions.
As far as motor-driven and polymerization-dependent forces can also be mediated by other cytoskeletal systems, such as microtubules and their associated protein, and cytoskeletal proteins, in view of their importance for intracellular transport, mitosis, and After 3, 6, and 24 h, they assumed an elongated morphology with regular and numerous stress fiber formation (Figure 2b,e,h). On PEI-ended slides, the MSCs, after 3 and 6 h (Figure 2c,f), instead showed a rounded morphology with a distribution of fluorescent protrusions that were extended radially in the periphery. After 24 h some cells spread on the substrate and displayed long protrusions and numerous filopodia, without stress fibers, but with an evident actin cortex (Figure 2i). These results revealed that PEI had a strong, hindering effect on the correct formation of stress fibers, expected, in turn, to severely affect the cell cytoskeletal functions.
As far as motor-driven and polymerization-dependent forces can also be mediated by other cytoskeletal systems, such as microtubules and their associated protein, and cytoskeletal proteins, in view of their importance for intracellular transport, mitosis, and migration [15][16][17][18], we have also investigated the variation of microtubule organization and distribution during the early stages of cell adhesion on PEMs ( Figure 3). Again, on PSSended substrates the MSCs exhibited a thin and well dispersed microtubular network, with a higher fluorescence intensity around the nucleus ( in the microtubule arrangement in the cells on PEI-ended substrate was observed, the microtubules being concentrated and compact, with a felt-like structure, in the perinuclear area and in the cytoplasm, aggregating into a thicker microtubular network (Figure 3c,f,i). migration [15][16][17][18], we have also investigated the variation of microtubule organization and distribution during the early stages of cell adhesion on PEMs ( Figure 3). Again, on PSS-ended substrates the MSCs exhibited a thin and well dispersed microtubular network, with a higher fluorescence intensity around the nucleus (Figure 3e), and at 24 h ( Figure 3h) they showed an elongated shape of healthy cells. At variance with this, a significant alteration in the microtubule arrangement in the cells on PEI-ended substrate was observed, the microtubules being concentrated and compact, with a felt-like structure, in the perinuclear area and in the cytoplasm, aggregating into a thicker microtubular network (Figure 3c,f,i). As a consequence of this failure to organize the actin cytoskeleton, the morphology of the cells on PEI-ended films did not change, remaining rounded and compact.
These results confirm the remarkable effect of the chemical nature of the substrates, for these two PEMs films, on the behavior of the cytoskeletal elements and in particular on their correct assembly. The hindered formation of the appropriate cytoskeletal structures will be responsible for the compromised correct cell morphology and the related cellular functions [19]. It should be stressed that the reported data provide a first explanation of the cell morphologies revealed by means of the SEM analysis above (Figure 1).
Integrins
Adhesion molecules, such as integrins, act primarily to provide localized signals rather than to support physical attachment, and they are directly connected to the actin cytoskeleton with the activation of a signaling cascade that regulates many cellular functions [20,21]. In view of this, it is important to investigate their localization and distribution during the early steps of cell-PEM interactions. Cell-material interactions are mediated by a layer of proteins that in our in vitro experiments are present in the FBS 10% culture medium [22] and absorbed at the cell-substrate interface. As fibronectin and collagen are the two proteins most expressed by MSCs [23], we analyzed the α5ß1 (fibronectin receptor) and α1ß1 (collagen receptor) integrins distribution, and the results are shown in Figure 4. A normal expression of integrins in all samples, but no translocation in the PEI- As a consequence of this failure to organize the actin cytoskeleton, the morphology of the cells on PEI-ended films did not change, remaining rounded and compact.
These results confirm the remarkable effect of the chemical nature of the substrates, for these two PEMs films, on the behavior of the cytoskeletal elements and in particular on their correct assembly. The hindered formation of the appropriate cytoskeletal structures will be responsible for the compromised correct cell morphology and the related cellular functions [19]. It should be stressed that the reported data provide a first explanation of the cell morphologies revealed by means of the SEM analysis above (Figure 1).
Integrins
Adhesion molecules, such as integrins, act primarily to provide localized signals rather than to support physical attachment, and they are directly connected to the actin cytoskeleton with the activation of a signaling cascade that regulates many cellular functions [20,21]. In view of this, it is important to investigate their localization and distribution during the early steps of cell-PEM interactions. Cell-material interactions are mediated by a layer of proteins that in our in vitro experiments are present in the FBS 10% culture medium [22] and absorbed at the cell-substrate interface. As fibronectin and collagen are the two proteins most expressed by MSCs [23], we analyzed the α5ß1 (fibronectin receptor) and α1ß1 (collagen receptor) integrins distribution, and the results are shown in Figure 4. A normal expression of integrins in all samples, but no translocation in the PEI-ended sample, was found at 24 h ( Figure 4). The cell-surface contact in reference samples induced integrin clustering (dot-like fluorescence) that shifted towards the cell periphery with a rod-like organization, finally yielding newly assembled actin filaments (Figure 4a,d). As for the PSS-ended surface (Figure 4b), at 6 h the α5ß1 integrin showed clustering and translocation towards the cell periphery, running parallel to the actin cytoskeleton, indicating a good adhesion and an optimal spreading performance at 24 h (Figure 4e). At variance with this, on the PEI-ended films, the integrins remained punctiform, with a spot-like fluorescence localized and dispersed in the central area and in the periphery of the cell (Figure 4c), which, after 24 h of adhesion, produced a disordered clustering, but without peripheral translocation, pointing to a strong cell adhesion but a very limited spreading (Figure 4f), again matching closely with the morphology findings (see Figure 2). ended sample, was found at 24 h ( Figure 4). The cell-surface contact in reference samples induced integrin clustering (dot-like fluorescence) that shifted towards the cell periphery with a rod-like organization, finally yielding newly assembled actin filaments ( Figure 4a,d). As for the PSS-ended surface (Figure 4b), at 6 h the α5ß1 integrin showed clustering and translocation towards the cell periphery, running parallel to the actin cytoskeleton, indicating a good adhesion and an optimal spreading performance at 24 h (Figure 4e). At variance with this, on the PEI-ended films, the integrins remained punctiform, with a spotlike fluorescence localized and dispersed in the central area and in the periphery of the cell (Figure 4c), which, after 24 h of adhesion, produced a disordered clustering, but without peripheral translocation, pointing to a strong cell adhesion but a very limited spreading (Figure 4f), again matching closely with the morphology findings (see Figure 2). It should be stressed that the strong adhesion of the cells to the PEI-ended surfaces, inhibiting the spreading, could have "physically" prevented the translocation of the integrins through a mechanism involving the lack of actin polymerization, caused by ATP deficiency (see below), which is, thus, unable to generate the traction force needed for the displacement (i.e., translocation) of the integrin/microfilament complexes.
It is known, in fact, that a correctly polymerized actin cytoskeleton is a necessary requirement for the assembly of integrin-dependent focal processes [24] giving rise to mechano-transduction forces involved in various physiological and pathological processes [25].
Finally, as for the α1ß1 integrin ( Figure S1), this was found to be more uniformly diffused in the central area of the cell body after 6 h of adhering to all the surfaces; no significant differences were found among the analyzed samples.
Overall, the reported results show that, on the one hand, the numerous membrane specializations (filopodia, lamellipodia, and blebs) induced by PEI-ended surfaces in mesenchymal cells could indicate a good degree of cell adhesion, while on the other hand, the absence of microfilaments and the lack of translocation of integrins severely limit the correct spreading process, producing the characteristic rounded cell morphology. It should be stressed that the strong adhesion of the cells to the PEI-ended surfaces, inhibiting the spreading, could have "physically" prevented the translocation of the integrins through a mechanism involving the lack of actin polymerization, caused by ATP deficiency (see below), which is, thus, unable to generate the traction force needed for the displacement (i.e., translocation) of the integrin/microfilament complexes.
It is known, in fact, that a correctly polymerized actin cytoskeleton is a necessary requirement for the assembly of integrin-dependent focal processes [24] giving rise to mechano-transduction forces involved in various physiological and pathological processes [25].
Finally, as for the α1ß1 integrin ( Figure S1), this was found to be more uniformly diffused in the central area of the cell body after 6 h of adhering to all the surfaces; no significant differences were found among the analyzed samples.
Overall, the reported results show that, on the one hand, the numerous membrane specializations (filopodia, lamellipodia, and blebs) induced by PEI-ended surfaces in mesenchymal cells could indicate a good degree of cell adhesion, while on the other hand, the absence of microfilaments and the lack of translocation of integrins severely limit the correct spreading process, producing the characteristic rounded cell morphology.
Focal Adhesion Kinase and Paxillin
Focal complexes (FCs), with a characteristic dot-like shape, evolve in focal adhesions (FAs), with a rod-like morphology, being associated with long actin and myosin fibers located peripherally and near the perinuclear region of the cell [26].
The formation of FAs and stress fibers is accompanied by the phosphorylation of many specific proteins, including FAK, paxillin [27], and the FAK-SRC complex that promotes the cell cycle progression in normal and cancer cells [28][29][30][31][32].
Accordingly, the analysis of the localization of phosphorylated FAK and paxillin and their punctiform or rod-like organization in adhesion cells provided the degree of maturation of focal adhesions for the various substrates here investigated. Indeed, in the control and in the PSS-ended substrates, during the first few hours of MSC adhesion, a well-defined maturation of the FCs in FAs was observed, with the characteristic rod-like organization and peripheral localization of pFAK ( Figure 5), both indicating that a correct assembly of the adhesion machinery occurred.
Focal Adhesion Kinase and Paxillin
Focal complexes (FCs), with a characteristic dot-like shape, evolve in focal adhesions (FAs), with a rod-like morphology, being associated with long actin and myosin fibers located peripherally and near the perinuclear region of the cell [26].
The formation of FAs and stress fibers is accompanied by the phosphorylation of many specific proteins, including FAK, paxillin [27], and the FAK-SRC complex that promotes the cell cycle progression in normal and cancer cells [28][29][30][31][32].
Accordingly, the analysis of the localization of phosphorylated FAK and paxillin and their punctiform or rod-like organization in adhesion cells provided the degree of maturation of focal adhesions for the various substrates here investigated. Indeed, in the control and in the PSS-ended substrates, during the first few hours of MSC adhesion, a welldefined maturation of the FCs in FAs was observed, with the characteristic rod-like organization and peripheral localization of pFAK ( Figure 5), both indicating that a correct assembly of the adhesion machinery occurred. On the other hand, for MSCs on PEI-ended surfaces, the phosphorylated FAK appears punctiform and uniformly distributed in the central area of the cell body, but not in the periphery, where only a lower amount of it seems to have translocated.
Also, more pFAK was observed in cells attached to glass or PSS-ended surfaces with respect to those seeded on a PEI-ended multilayer. On the other hand, for MSCs on PEI-ended surfaces, the phosphorylated FAK appears punctiform and uniformly distributed in the central area of the cell body, but not in the periphery, where only a lower amount of it seems to have translocated.
Also, more pFAK was observed in cells attached to glass or PSS-ended surfaces with respect to those seeded on a PEI-ended multilayer.
As is known, the biological function of phosphorylated PXN (pPXN) is to regulate cell spreading and motility through the organization of the actin cytoskeleton, by generating binding sites for other proteins, promoting the mechanical binding to the actin cytoskeleton, and, finally, producing the event cascade to regulate proteins involved in the management of numerous cellular functions [33]. Therefore, the previous observations on the formation of the actin cytoskeleton have to be confirmed by the careful analysis of the fate of pPXN.
In particular, we found that the cells on the control substrates at 3 h showed a dot-like paxillin structure, also evident near the nucleus, finally evolved at 24 h to a dominating rod-like structure as a result of the cell spreading.
As for MSCs on PEI-ended surfaces, it was found that from 3 to 24 h of cell incubation ( Figure 6) the pPXN gradually extended towards the cell periphery following the final localization of the FAK. ating binding sites for other proteins, promoting the mechanical binding to the actin cytoskeleton, and, finally, producing the event cascade to regulate proteins involved in the management of numerous cellular functions [33]. Therefore, the previous observations on the formation of the actin cytoskeleton have to be confirmed by the careful analysis of the fate of pPXN.
In particular, we found that the cells on the control substrates at 3 h showed a dotlike paxillin structure, also evident near the nucleus, finally evolved at 24 h to a dominating rod-like structure as a result of the cell spreading.
As for MSCs on PEI-ended surfaces, it was found that from 3 to 24 h of cell incubation ( Figure 6) the pPXN gradually extended towards the cell periphery following the final localization of the FAK. More specifically, after 3 h pPXN was predominantly localized near the nucleus and in the peripheral adhesion zone of the mesenchymal cells, as in the control (although with less evident fluorescence). However, at 24 h pPXN was found to concentrate around the nucleus, while the previous adhesion zones were no longer appearing and the cells did not spread.
These alterations in the maturation of the adhesion plaques on PEI-ended substrates are perfectly congruent with the failure in the peripheral translocation of pFAK and pPXN, which indeed stops at the dot-like stage, and reflect the disarrangement of actin and microtubules disorder, corresponding to the patterns above reported for integrins on the same substrates.
Finally, in view of the direct connection between the adhesion molecules and the regulation of cellular functions, highlighted by the ability of FAK to translocate to the cell nucleus, further regulating cell proliferation, we have characterized the distribution of pFAK in the MSCs on the three substrates (control, PSS-ended, and PEI-ended).
We have observed that, for control and PSS-ended films, pFAK is mainly localized in FAs and the cell cytoplasm, only in small part being able to translocate to the nucleus. On More specifically, after 3 h pPXN was predominantly localized near the nucleus and in the peripheral adhesion zone of the mesenchymal cells, as in the control (although with less evident fluorescence). However, at 24 h pPXN was found to concentrate around the nucleus, while the previous adhesion zones were no longer appearing and the cells did not spread.
These alterations in the maturation of the adhesion plaques on PEI-ended substrates are perfectly congruent with the failure in the peripheral translocation of pFAK and pPXN, which indeed stops at the dot-like stage, and reflect the disarrangement of actin and microtubules disorder, corresponding to the patterns above reported for integrins on the same substrates.
Finally, in view of the direct connection between the adhesion molecules and the regulation of cellular functions, highlighted by the ability of FAK to translocate to the cell nucleus, further regulating cell proliferation, we have characterized the distribution of pFAK in the MSCs on the three substrates (control, PSS-ended, and PEI-ended).
We have observed that, for control and PSS-ended films, pFAK is mainly localized in FAs and the cell cytoplasm, only in small part being able to translocate to the nucleus. On the contrary, on PEI-ended surfaces there is also a translocation of FAK into the nucleus ( Figure 5), but without any progression in the cycle (block in phase G2/M, see Table 1) and hindering the further proliferation, as far as the action carried out by FAK at the level of the nucleus consists of activating the mitogen-type signals that prompt the cell proliferation, following well-studied pathways [34,35].
Monitoring Cell Behavior with QCD-D Experiments
The quartz crystal microbalance with dissipation monitoring (QCM-D), which is commonly used to detect in situ and real time biomolecular interactions with surfaces, was used here to study the interaction between cells and interface to understand the underlying mechanisms at an early stage of the cell-surface interaction.
However, it is necessary to make some theoretical considerations prompting the further interpretation of the results. Generally, for the adsorption of a rigid film, the Sauerbrey equation can be applied, since it assumes the proportionality between the frequency shift and the deposited mass. This assumption, however, is not valid for the adsorption of a sub-monolayer of cells. The analysis of the response of a QCM-D sensor, when interacting with a cell, is complex, owing to the fact that the decay length for a shear wave in liquid or in dense fluid phases is considerably shorter than cell thickness (i.e., about 250 nm in water with a density of~1 g/mL) and exponentially less for fluid phases of greater density, as a cell may be considered. This means that the quartz sensor may sample only a fraction of the whole cell's mass, yielding a correspondingly low frequency shift. Therefore, each adhered cell can be considered as an added effective mass, much lower than the real cell mass uptake, thus preventing the straight use of the frequency shift. However, the energy dissipation (recorded as D shift) can be related to the mechanical properties of the cell membrane, in contact with the quartz sensor, and the more or less thick fluid portion in the cytoplasm overlaying the membrane. Generally, it is possible to describe the cellsubstrate interaction as follows: the initial cell-substrate physical contact leads to the first QCM-D response, i.e., a decrease of the frequency shift, cell spreading, and modification of the adhesion properties affects the signal and, finally, changes in the cytoskeleton of the cells, which influence their rigidity, involve the dissipation factor. In particular, changes in dissipation give a qualitative measure of how rigidly the cell adheres to the substrate, i.e., an increase in dissipation suggests a relatively softer cell layer [10]. The relevant information for this complex system is provided by the ∆D/∆F ratio (D-F plot), which, in fact, does not depend on the apparent cell mass, as it represents the energy loss per unit of attached mass. In particular, the D-F plots reflect the viscoelastic properties of the "normalized" mass attached to the sensor, involving, in turn, the effect of the shape and internal structure of the cells (i.e., the spreading factor). Accordingly, QCM-D measurements provide a qualitative diagnostic of the internal structure and spread of the cells, i.e., of the rigidity of the cell region in contact with the quartz sensor as a function of the mechano-transduction forces relative to the organization and distribution of actin and their change during early adhesion steps.
In the present work, we simultaneously measured the frequency and dissipation shifts occurring during the very early stage (3 h) of cell adhesion on PEMs and reference SiO 2 surfaces (Figure 7). Following the cell suspension injection, all substrates showed an immediate decrease of the resonance frequency (∆F), indicating an apparent cell mass adhesion to the surface of the sensors, while the dissipation (∆D) increased for all samples, in agreement with the sampling of more or less viscoelastic layers in contact with the surfaces. immediate decrease of the resonance frequency (ΔF), indicating an apparent cell mass adhesion to the surface of the sensors, while the dissipation (ΔD) increased for all samples, in agreement with the sampling of more or less viscoelastic layers in contact with the surfaces. The SiO2 surfaces (Figure 7a) showed a particular trend, consisting of the complete reversal of the initial apparent mass uptake during the very early incubation time (180 min), qualitatively suggesting that an attachment-detachment process was occurring, from the initial cell adhesion event (in the first 50 min) read as a loss of frequency, followed by the re-increase in frequency, which suggests cell detachment. It must be stressed that the frequency data are in no way quantitatively meaningful, but are merely indicative of the qualitative trend of the cell-sensor interaction.
For PEI-ended surfaces (Figure 7b), the apparent cell mass adhesion (associated with a stable frequency loss) still occurs gradually; however, it remains stable over time also after a washing step with PBS. Moreover, dissipation increases in a stable way, yielding slightly spread overtones, diagnostic of a more viscous response of the cell adhered regions with respect to the SiO2 case. The ΔD data indicate that the cells are attached on PEIended substrates, hindering their spreading on PEI substrates, in agreement with the observed rounded morphology and the formation of an evident rigid actin cortex (see Sections 3.1 and 3.2 above).
At variance with this, for cells on PSS-ended surfaces (Figure 7c), an abrupt frequency decrease was observed, qualitatively diagnostic of a diffuse cell adhesion to the substrate, followed by small frequency changes. The related dissipation was found to be significantly higher than the one for SiO2 and PEI, suggesting that in this case, the contact sites were more viscous than in those cases and that the cells are substantially more mobile on the surface, prompting the efficient cell spreading process responsible for the best cell seeding on PSS-ended substrates [36]. Finally, the ΔD/ΔF plots for the cell adhesion to the three surfaces are reported in Figure 7d. In particular, for each substrate, different regions can be seen: region I, common to all substrates, is characterized by a slightly increasing The SiO 2 surfaces (Figure 7a) showed a particular trend, consisting of the complete reversal of the initial apparent mass uptake during the very early incubation time (180 min), qualitatively suggesting that an attachment-detachment process was occurring, from the initial cell adhesion event (in the first 50 min) read as a loss of frequency, followed by the re-increase in frequency, which suggests cell detachment. It must be stressed that the frequency data are in no way quantitatively meaningful, but are merely indicative of the qualitative trend of the cell-sensor interaction.
For PEI-ended surfaces (Figure 7b), the apparent cell mass adhesion (associated with a stable frequency loss) still occurs gradually; however, it remains stable over time also after a washing step with PBS. Moreover, dissipation increases in a stable way, yielding slightly spread overtones, diagnostic of a more viscous response of the cell adhered regions with respect to the SiO 2 case. The ∆D data indicate that the cells are attached on PEIended substrates, hindering their spreading on PEI substrates, in agreement with the observed rounded morphology and the formation of an evident rigid actin cortex (see Sections 3.1 and 3.2 above).
At variance with this, for cells on PSS-ended surfaces (Figure 7c), an abrupt frequency decrease was observed, qualitatively diagnostic of a diffuse cell adhesion to the substrate, followed by small frequency changes. The related dissipation was found to be significantly higher than the one for SiO 2 and PEI, suggesting that in this case, the contact sites were more viscous than in those cases and that the cells are substantially more mobile on the surface, prompting the efficient cell spreading process responsible for the best cell seeding on PSS-ended substrates [36]. Finally, the ∆D/∆F plots for the cell adhesion to the three surfaces are reported in Figure 7d. In particular, for each substrate, different regions can be seen: region I, common to all substrates, is characterized by a slightly increasing ∆F, while ∆D does not change, suggesting that the cells are merely adhered to the surface by gravity. Furthermore, in region II, for the SiO 2 substrate (magenta triangles in the figure) ∆F decreases and ∆D increases steeply, suggesting the occurrence of firm cell attachment, while, in region III, a greater mass removal, with only a smaller increase in dissipation compared to region II, is observed, indicating the detachment of loosely attached cells.
For PSS-ended (negatively charged surface, cyan circles), a very similar behavior for region II is observed, whereas in region III, ∆F continues to gradually decrease and ∆D continues to increase. This overall behavior has been related to ECM remodeling and the related cytoskeletal changes, leading to an increase in the rigidity of the cellular membrane, associated with the establishment of strong cell focal adhesion points [1]. Thus, the overall ∆D/∆F plot indicates that for the PSS-ended surface there is an effective and positive cell adhesion and spreading. Finally, on the PEI surface (positively charged surfaces, blue squares), the slope of region II becomes steeper because cells adhere strongly to the surface, while region III is missing, indicating that ECM remodeling does not occur in these early cell adhesion stages, that is, within 3 h of incubation.
Cell Viability and Cell Cycle
In order to determine whether PEMs affect cell viability, we analyzed their effects on MSCs for 120 h by MTT assay (graphic in Figure 8). Indeed, cell viability is reduced for both polyelectrolytes, with respect to the control, after 3 h to 78% and 39%, for PSS-ended and PEI-ended, respectively, and to 59% and 22% (again, for PSS and PEI) after 48 h. ΔF, while ΔD does not change, suggesting that the cells are merely adhered to the surface by gravity. Furthermore, in region II, for the SiO2 substrate (magenta triangles in the figure) ΔF decreases and ΔD increases steeply, suggesting the occurrence of firm cell attachment, while, in region III, a greater mass removal, with only a smaller increase in dissipation compared to region II, is observed, indicating the detachment of loosely attached cells.
For PSS-ended (negatively charged surface, cyan circles), a very similar behavior for region II is observed, whereas in region III, ΔF continues to gradually decrease and ΔD continues to increase. This overall behavior has been related to ECM remodeling and the related cytoskeletal changes, leading to an increase in the rigidity of the cellular membrane, associated with the establishment of strong cell focal adhesion points [1]. Thus, the overall ΔD/ΔF plot indicates that for the PSS-ended surface there is an effective and positive cell adhesion and spreading. Finally, on the PEI surface (positively charged surfaces, blue squares), the slope of region II becomes steeper because cells adhere strongly to the surface, while region III is missing, indicating that ECM remodeling does not occur in these early cell adhesion stages, that is, within 3 h of incubation.
Cell Viability and Cell Cycle
In order to determine whether PEMs affect cell viability, we analyzed their effects on MSCs for 120 h by MTT assay (graphic in Figure 8). Indeed, cell viability is reduced for both polyelectrolytes, with respect to the control, after 3 h to 78% and 39%, for PSS-ended and PEI-ended, respectively, and to 59% and 22% (again, for PSS and PEI) after 48 h. The cell cycle, analyzed for 48 h and 6 days by flow cytometry, is shown in the Table 1.
After 48 h of treatment with PSS-ended, we did not observe significant changes in the cell cycle with respect to the control, but a reduction of the G2/M phase was observed with PEI-ended (5.1% decrease). After 6 days of treatment, we observed a reduction of the G0/G1 phase and a slight increase in phases S and G2/M in cells cultured on the PSS-ended slides. In the same conditions, we observed on PEI-ended slides a conspicuous reduction of the G0/G1 phase (16.4% decrease) and an increase of the G2/M phase (31.9% increase). It is possible that there is a stimulating effect of PEI-ended on the progression of the cell cycle from the G0/G1 phase towards the G2 phase and mitosis.
These results confirm that PEI worsens cell viability and the cell cycle, in agreement with the abnormal cell morphology that cells show on PEI-ended samples.
The strong reduction of the G0/G1 phase (Table 1) could highlight a blocking at the mitotic or pre/mitosis (G2) phase of the cell cycle, with the consequent decrease of cell The cell cycle, analyzed for 48 h and 6 days by flow cytometry, is shown in the Table 1.
After 48 h of treatment with PSS-ended, we did not observe significant changes in the cell cycle with respect to the control, but a reduction of the G2/M phase was observed with PEI-ended (5.1% decrease). After 6 days of treatment, we observed a reduction of the G0/G1 phase and a slight increase in phases S and G2/M in cells cultured on the PSS-ended slides. In the same conditions, we observed on PEI-ended slides a conspicuous reduction of the G0/G1 phase (16.4% decrease) and an increase of the G2/M phase (31.9% increase). It is possible that there is a stimulating effect of PEI-ended on the progression of the cell cycle from the G0/G1 phase towards the G2 phase and mitosis.
These results confirm that PEI worsens cell viability and the cell cycle, in agreement with the abnormal cell morphology that cells show on PEI-ended samples.
The strong reduction of the G0/G1 phase (Table 1) could highlight a blocking at the mitotic or pre/mitosis (G2) phase of the cell cycle, with the consequent decrease of cell viability observed in cells cultured on PEI slides as reported in the MTT results, that could be assigned to the strong adhesion of cells on the surface.
Cell cycle progression is regulated by a series of signals from the extracellular matrix. The cytoskeletal organization of a cell can have a direct influence on the progression of the cell cycle, as demonstrated in fibroblasts with a not well-structured cytoskeleton [37]. The early variations in the phosphorylation of FAK and paxillin and their correct assembly are a necessary condition to stimulate the transition from the G1 phase to the S phase of the cell cycle following adhesion, through the activation of the RAS-MAP kinase cascade (proteins activated by mitogens) and consequent activation of the retinoblastoma (RB) protein and induction of cyclin D [38]. Strong integrin-mediated attachment to a substrate serves as a checkpoint for cell cycle progression, and there is evidence that signals arising from focal adhesion (FA) directly communicate with the pathway that regulates cell proliferation. The FAK, which is associated with focal adhesion proteins, interacts directly with the GRB2 adapter protein. Since GRB2 is linked to Ras, this explains the anchorage dependence of the cellular response to growth factor-initiated mitogenesis [39].
ATP Evaluation and Mitochondrial Transmembrane Potential (∆Ψ)
In light of the results obtained from cells on PEI-ended slides and to better understand their behavior, we evaluated the content of ATP and the mitochondrial transmembrane potential (Figure 9). Membrane permeability plays a key role in regulating the traffic across the membrane; its alteration generates a deregulation, leading to the hydrolysis of molecules essential for cell survival and for numerous biological functions, such as ATP.
viability observed in cells cultured on PEI slides as reported in the MTT results, that could be assigned to the strong adhesion of cells on the surface.
Cell cycle progression is regulated by a series of signals from the extracellular matrix. The cytoskeletal organization of a cell can have a direct influence on the progression of the cell cycle, as demonstrated in fibroblasts with a not well-structured cytoskeleton [37]. The early variations in the phosphorylation of FAK and paxillin and their correct assembly are a necessary condition to stimulate the transition from the G1 phase to the S phase of the cell cycle following adhesion, through the activation of the RAS-MAP kinase cascade (proteins activated by mitogens) and consequent activation of the retinoblastoma (RB) protein and induction of cyclin D [38]. Strong integrin-mediated attachment to a substrate serves as a checkpoint for cell cycle progression, and there is evidence that signals arising from focal adhesion (FA) directly communicate with the pathway that regulates cell proliferation. The FAK, which is associated with focal adhesion proteins, interacts directly with the GRB2 adapter protein. Since GRB2 is linked to Ras, this explains the anchorage dependence of the cellular response to growth factor-initiated mitogenesis [39].
ATP Evaluation and Mitochondrial Transmembrane Potential (ΔΨ)
In light of the results obtained from cells on PEI-ended slides and to better understand their behavior, we evaluated the content of ATP and the mitochondrial transmembrane potential ( Figure 9). Membrane permeability plays a key role in regulating the traffic across the membrane; its alteration generates a deregulation, leading to the hydrolysis of molecules essential for cell survival and for numerous biological functions, such as ATP. The chemiluminescence ATP quantification assay showed a clear decrease in intracellular ATP in the PEI-ended samples compared to the PSS-ended ones (Figure 9a). After 3 h of adhesion on the PEI-ended slide, MSCs showed a decrease of intracellular ATP by about 50%, which remained constant at subsequent time points, unlike for PSS, in which a resumption of cellular metabolic activity was evident. The chemiluminescence ATP quantification assay showed a clear decrease in intracellular ATP in the PEI-ended samples compared to the PSS-ended ones (Figure 9a). After 3 h of adhesion on the PEI-ended slide, MSCs showed a decrease of intracellular ATP by about 50%, which remained constant at subsequent time points, unlike for PSS, in which a resumption of cellular metabolic activity was evident.
The decrease in ATP shown in cells grown on the PEI-ended surface could be the cause of the dysfunctions of the numerous ATP-dependent cellular functions and of the entire cell metabolism. These low ATP levels of MSCs adhering to PEI-ended could be due to a destabilization of the plasma membrane that makes the mitochondrial component more accessible [40]. The alteration of the plasma membrane is via an SOS protein, which establishes a link between FAK activation and a well-established mitogenesis pathway. Furthermore, cell attachment via integrins controls the cellular sensitivity to the growth factor. This synergy between signals generated by cell adhesion receptors and the growth factor receptors helps to observe the SEM image shown in Figure 9b. The ∆Ψ analysis, carried out in 3, 24, and 48 h (Figure 9c) with the fluorescent JC-1 dye, showed a decrease in mitochondrial activity in PEI samples, maybe due to an impairment of electron transport through the inner membrane coupled to proton pumping. From the observation under the fluorescence microscope, we noted a limited mitochondrial activity in PEI-ended samples in all experimental times analyzed and a greater concentration and distribution of mitochondria at the cytoplasmic level, with respect to the control cells spreading on the uncoated substrate. Given that the decreased mitochondrial activity does not permit the restoration of the intracellular ATP concentration necessary to reactivate a correct metabolic pathway, we studied the effect that extracellular ATP has on MSCs seeded on the PEI coating and supplemented the culture medium with different concentrations of ATP. After 72 h, we replaced the medium with fresh medium containing ATP (20 µM, 10 µM, and 5 µM) for 3 h and measured vitality by MTT ( Figure 10a) and stained microfilaments with FITC-phalloidin ( Figure 10b).
The decrease in ATP shown in cells grown on the PEI-ended surface could be the cause of the dysfunctions of the numerous ATP-dependent cellular functions and of the entire cell metabolism. These low ATP levels of MSCs adhering to PEI-ended could be due to a destabilization of the plasma membrane that makes the mitochondrial component more accessible [40]. The alteration of the plasma membrane is via an SOS protein, which establishes a link between FAK activation and a well-established mitogenesis pathway. Furthermore, cell attachment via integrins controls the cellular sensitivity to the growth factor. This synergy between signals generated by cell adhesion receptors and the growth factor receptors helps to observe the SEM image shown in Figure 9b. The ΔΨ analysis, carried out in 3, 24, and 48 h (Figure 9c) with the fluorescent JC-1 dye, showed a decrease in mitochondrial activity in PEI samples, maybe due to an impairment of electron transport through the inner membrane coupled to proton pumping. From the observation under the fluorescence microscope, we noted a limited mitochondrial activity in PEIended samples in all experimental times analyzed and a greater concentration and distribution of mitochondria at the cytoplasmic level, with respect to the control cells spreading on the uncoated substrate. Given that the decreased mitochondrial activity does not permit the restoration of the intracellular ATP concentration necessary to reactivate a correct metabolic pathway, we studied the effect that extracellular ATP has on MSCs seeded on the PEI coating and supplemented the culture medium with different concentrations of ATP. After 72 h, we replaced the medium with fresh medium containing ATP (20 μM, 10 μM, and 5 μM) for 3 h and measured vitality by MTT ( Figure 10a) and stained microfilaments with FITC-phalloidin ( Figure 10b). The initial addition of ATP slightly improved the condition of the cells with respect to the PEI control, but after 72 h (plus 3 h ATP) the decrease in viability was the same as for PEI. Actin was totally disarranged in all samples and appeared as large fluorescent The initial addition of ATP slightly improved the condition of the cells with respect to the PEI control, but after 72 h (plus 3 h ATP) the decrease in viability was the same as for PEI. Actin was totally disarranged in all samples and appeared as large fluorescent spots, located particularly in the periphery of the cells. The different concentrations of ATP added to the culture medium caused only a slight increase with respect to the PEI control.
The presence of exogenous ATP led to an initial improvement in cellular conditions, supporting the hypothesis that the depletion of ATP prevents cells from carrying out their normal processes. The correct organization of the cytoskeleton is an ATP-dependent process. Previous studies showed that the integrity and rearrangement of the microfilament organization were closely related to the levels of ATP, which plays a key role in the polymerization of actin molecules, regulating the passage from their monomeric form (G actin) to their polymeric form (F-actin) [41,42]. The loss of ATP resulted in a rapid increase in the rigidity of the cytoskeleton; moreover, the addition of ATP immediately reversed this effect, suggesting that the stiffness of the cytoskeleton depends on ATP-dependent molecular transformations, such as the sliding of actomyosin filaments or chemical modifications of cytoskeletal proteins (phosphorylation) [43].
Conclusions
The understanding of the basic cell seeding phenomena is of paramount importance for effective tissue engineering strategies of clinical relevance. The present paper reports the study of the early adhesion events of undifferentiated MSCs on two different polyelectrolyte multilayers: one PSS-ended (anionic surface) and the other PEI-ended (cationic surface) layer.
It was found that cells seeded on the PSS-ended surface presented morphology behavior of the principal cytoskeleton components and of the adhesion molecules (integrins, pFAK, and pPXN) very similar to those of the mesenchymal cells seeded on a reference glass substrate. The pattern of adhesion and the evolution of focal adhesion plaques retrace the classic model known for these cells, implying the cytoskeletal protein translocation from the central area to the periphery of the cell. Furthermore, we observed that, after eight days, the cells growing on the PSS-ended surface were able to form a monolayer, even if a slight decrease in viability and cell cycle progression was observed. Therefore, these results suggested that the PSS-ended surface was substantially biologically compatible with undifferentiated MSCs.
On the other hand, the PEI-ended surface showed a decrease in MSC viability and inhibition of proliferation that were activated by an uncorrected assembly between adhesion molecules and the cytoskeleton. In particular, the absence of microfilaments or stress fibers appears to prevent the translocation of integrins, pFAK, and pPXN to form focal adhesion complexes. Cells that were not spread on the PEI-ended surface maintained, therefore, the initial round-shaped morphology. Furthermore, the alteration that the PEI-ended surface induces on the plasma membrane and the loss of ATP could be the reason for the failure of actin polymerization. The QCM-D results indicate that, already at the early stage of cell-surface interaction, the cell membrane and near-membrane cytoplasm have a different rigidity on the two different PEM substrates, with cell adhesion being remarkably stronger for PEI-ended than for PSS-ended ones. This supports the hypothesis that the cell spreading and the related cytoskeletal reorganization is strongly prevented on the PEI-ended surface, due to the strong adhesion force. On the contrary, the lower rigidity observed for cells on PSS appears connected to correct cell-material interactions, allowing proper cell spreading, which in turn may trigger the cascade of transduction events that promote later regulated growth, cell differentiation, and migration.
In summary, the change in the spreading capability of the cells onto different substrates, already at early steps of interaction, following the sequestering of the adhesion molecules in the initial cell-substrate contact area, appears to control the relative cytostatic effects.
Author Contributions: A.A., investigation and visualization; G.V., investigation, visualization, and data curation; G.M.L.M., investigation, data curation, and review and editing; M.C., investigation and writing-review and editing; C.F., investigation; M.T.C., writing-original draft and writing-review and editing; G.M. and F.S., conceptualization, supervision, writing-original draft, and writingreview and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Projects PIACERI "Starting Grant" (University of Catania-2020-2022) and University funds 2020-2022, University of Catania, Open Access line. | 2022-07-02T15:03:00.027Z | 2022-06-29T00:00:00.000 | {
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119284579 | pes2o/s2orc | v3-fos-license | Entangled Dilaton Dyons
Einstein-Maxwell theory coupled to a dilaton is known to give rise to extremal solutions with hyperscaling violation. We study the behaviour of these solutions in the presence of a small magnetic field. We find that in a region of parameter space the magnetic field is relevant in the infra-red and completely changes the behaviour of the solution which now flows to an $AdS_2\times R^2$ attractor. As a result there is an extensive ground state entropy and the entanglement entropy of a sufficiently big region on the boundary grows like the volume. In particular, this happens for values of parameters at which the purely electric theory has an entanglement entropy growing with the area, $A$, like $A \log(A)$ which is believed to be a characteristic feature of a Fermi surface. Some other thermodynamic properties are also analysed and a more detailed characterisation of the entanglement entropy is also carried out in the presence of a magnetic field. Other regions of parameter space not described by the $AdS_2\times R^2$ end point are also discussed.
Abstract: Einstein-Maxwell theory coupled to a dilaton is known to give rise to extremal solutions with hyperscaling violation. We study the behaviour of these solutions in the presence of a small magnetic field. We find that in a region of parameter space the magnetic field is relevant in the infra-red and completely changes the behaviour of the solution which now flows to an AdS 2 × R 2 attractor. As a result there is an extensive ground state entropy and the entanglement entropy of a sufficiently big region on the boundary grows like the volume. In particular, this happens for values of parameters at which the purely electric theory has an entanglement entropy growing with the area, A, like A log(A) which is believed to be a characteristic feature of a Fermi surface. Some other thermodynamic properties are also analysed and a more detailed characterisation of the entanglement entropy is also carried out in the presence of a magnetic field. Other regions of parameter space not described by the AdS 2 × R 2 end point are also discussed.
Introduction
The AdS/CFT correspondence suggests that interesting connections could arise between gravitation and condensed matter physics. An important class of systems in condensed matter physics which one could try and study using this correspondence consists of fermions at finite density with strong correlations. Landau Fermi liquid theory is one paradigm that often describes such systems, but it can fail. The resulting Non-Fermi liquid behaviour is poorly understood and believed to be of considerable interest, e.g., in the study of High T c superconductors in 2 + 1 dimensions.
On the gravity side, the Einstein Maxwell Dilaton (EMD) system consisting of gravity and a Maxwell gauge field coupled to a neutral scalar (the Dilaton) is of considerable interest from the point of view of studying this problem. Fermions in the boundary theory carry a conserved charge -fermion number-so it is natural to include a gauge field in the bulk. The presence of a neutral scalar allows for promising new phases to arise where the entropy vanishes at non-zero chemical potential and zero temperature, as was discussed in [1], [2], [3]. These phases correspond to compressible states of matter with unbroken fermion number symmetry 1 . It was found that the thermodynamics and transport properties of these systems, while showing the existence of gapless excitations, do not fit those of a Landau Fermi liquid. For example, the specific heat is typically not linear in the temperature (T ), at small temperatures, and the electric resistivity also does not have the required T 2 dependence 2 .
An exciting recent development has shown that for an appropriate range parameters such an EMD system could give rise to an entanglement entropy which reproduces the behaviour expected of a system with a Fermi surface. If we take a sufficiently big region in space in a system with a Fermi surface it is believed that the entanglement entropy goes like S entangled ∼ A log(A) (1.1) where A is the area of the boundary of this region 3 . The log enhancement is believed to be the tell-tale signature of a Fermi surface. Exactly such a behaviour was shown to arise for appropriate choices of parameters in the EMD system in [10], see also [11]. In addition, it was argued that the specific heat, at small temperatures, could be understood on the basis of gapless excitations which dispersed with a non-trivial dynamical exponent. Taken together, these developments suggest that for an appropriate range of parameters the EMD system could perhaps describe phases where a Fermi surface does form but where the resulting description is not of Landau Fermi liquid type. While this is a promising possibility it is far from being definitely established. In fact, as has been known for some time now, at large N (classical gravity) the system does not exhibit some of the standard characteristics expected of a system with a 1 The significance of the compressible nature of the state was emphasised to us by S. Sachdev, see [4]. 2 These results refer to the case when the boundary theory is 2 + 1 dimensional with a 3 + 1 dimensional bulk dual. 3 Strictly speaking this behaviour has only been proven for free or weakly coupled fermions [5], [6] but it is expected to be more generally true due to the locus of gapless excitations which arises in the presence of a Fermi surface. Additional evidence has also been obtained in [7], [8], [9]. Fermi surface. For example there are no oscillations in the magnetisation and other properties as the magnetic field is varied (the de Haas-van Alphen effect) , nor are there any 2k F Friedel oscillation 4 . More recently the non-zero momentum currentcurrent two point function has been calculated and found to have suppressed weight at small frequency [14].
In this paper we will continue to study this class of systems from the gravity side by turning on an additional magnetic field and determining the resulting response. In our work the magnetic field will be kept small compared to charge density in the boundary theory. We will be more specific about what this means in terms of the energy scales of the boundary theory shortly. For now let us note that without a magnetic field the purely electric theory has a scaling-type solution (more correctly a hyperscaling violating solution). The magnetic field is kept small so that its effects are a small perturbation compared to the electric field in the ultraviolet (UV) of this scaling solution.
Key Results
We find that in the dilaton system even a small magnetic field can have an important effect at long distances since the magnetic field can become relevant in the Infrared (IR). The resulting thermodynamic and entanglement entropy can then change significantly. In particular this happens for the whole range of parameters where the entanglement entropy is of the form eq.(1.1).
More specifically, the EMD system we analyse is characterised by two parameters (α, δ) which are defined in 5 eq.(2.2), eq.(2.3). When |α| > |δ| we show that the magnetic field is relevant in the IR and the geometry in the deep infra-red (small values of the radial coordinate r we use ) flows to an AdS 2 × R 2 attractor. As a result the system acquires a non-zero extensive entropy even at zero temperature. The entanglement entropy also changes and grows like the volume of the region of interest 6 (for large enough volume). In particular, this happens for the values α = −3δ where the purely electric theory gives rise to an entanglement of the form eq.(1.1).
We also analyse the thermodynamics and some transport properties of the resulting state. The system continues to be compressible in the presence of a magnetic field and its specific heat is linear at small temperatures. Both these facts indicate the presence of gapless excitations. In general the system has a magnetisation 4 At one loop de Hass-van Alphen type oscillations are seen, [12]. For some recent discussion of Friedel oscillations in (1 + 1) dim. see [13]. 5 The relation of (α, δ) to the parameters (θ, z) now more conventionally used in the literature is given in eq. (2.29). In particular α = −3δ corresponds to θ = d − 1 = 1. 6 A potential confusion with our terminology arises because we are in two dimensions. Thus the volume of the region of interest is actually its area and the area of the boundary of this region is the perimeter. which is linear in the magnetic field and which is expected to be diamagnetic. The AdS 2 × R 2 attractor leads to the magnetisation having a temperature dependence, at small T , which can become important even for small magnetic fields, eq.(4.15).
The summary is that for parameters where the electric theory has an entanglement of the form eq.(1.1), suggesting that it is a non-Landau Fermi liquid, the magnetic field is a relevant perturbation in the IR. As a result even a small magnetic field has a significant effect on the state of the system at long distances. The state continues to be compressible, with a linear specific heat, but the thermodynamic entropy at zero temperature is now extensive and the entanglement entropy scales like the volume of the region of interest, this behaviour also being linked to the extensive ground state entropy 7 . At intermediate length scales for which the relevant region of the geometry is still reasonably well approximated by the hyperscaling violating type metric and the effects of the magnetic field are small, the behavior of the system continues to be essentially what it was in the absence of the magnetic field. In particular the thermodynamics is essentially unaffected by the magnetic field and the entanglement entropy also stays unchanged. Similar results for the existence of an AdS 2 × R 2 attractor and associated changes in thermodynamic and entanglement entropy etc are true in the whole region where |α| > |δ|.
The behaviour mentioned above is roughly analogous to what happens in a weakly coupled system with a Fermi surface 8 . While in this case the introduction of a small magnetic field leads to the formation of Landau levels, at intermediate energies still low compared to the Fermi energy but big compared to the spacing of the Landau levels, and correspondingly at intermediate length scales smaller than the magnetic length, the behaviour continues to be essentially that of a system with a Fermi surface. In particular the thermodynamics is essentially unchanged by the small magnetic field and the entanglement entropy is also expected to have the A log(A) behaviour at these length scales. Going to much lower energies of order the spacing between the Landau level and correspondingly to distance scales of order or longer the magnetic length though the behaviour of the system can change. For example in the free fermion theory, depending on the fermion density, a Landau level can be fully or partially filled, and partial filling would result in an extensive ground state entropy.
In other regions of parameter space where |α| < |δ| the magnetic perturbation is either not relevant in the IR and thus essentially leaves the low-energy and large distance behaviour of the system unchanged. Or it is relevant but we have not been able to completely establish the resulting geometry to which the system flows in the 7 More generally from the fact that the magnetic field is a relevant perturbation in the IR we learn that the compressible state described by the purely electric solution "anti-screens" the effects of the magnetic field making them grow at larger distances. 8 We thank the referee for his/her comments which have lead to this paragraph being incorporated in the revised version of the paper. deep IR.
The paper is planned as follows. We start with a brief description of the dilatonic system and the hyperscaling violating metrics in §2. The effects of a magnetic field are discussed in §3. The resulting thermodynamics is discussed in §4 and the entanglement entropy in §5. We end with a discussion of results and some concluding comments in §6. Appendix A contains important details about the numerical analysis.
Two papers in particular have overlap with the work reported here. While their motivations were different the analysis carried out in these papers is similar to ours. The EMD system with the inclusion of possible higher order corrections was analysed in [36] and it was found that sometimes these corrections could change the behaviour of the geometry resulting in an AdS 2 × R 2 region in the deep IR. This analysis was generalised to the case with hyperscaling violation in the more recent paper [37] which appeared while our work was being completed.
The Dilaton Gravity System
We work in 3 + 1 dimensions in the gravity theory with an action Much of our emphasis will be on understanding the near horizon region of the black brane solutions which arise in this system. This region is more universal, often having the properties of an attractor, and also determines the IR behaviour of the system. In this region, in the solutions of interest, the dilaton will become large, φ → ±∞. The potential and gauge coupling function take the approximate forms along this direction of field space. The two parameters, α, δ, govern the behaviour of the system. For example the thermodynamic and transport properties and also the entanglement properties crucially depend on these parameters. The action in eq. (2.1) has a symmetry under which the sign of φ, α, δ are reversed. Without loss of generality we will therefore choose δ > 0 in the discussion which follows.
Our analysis will build on the earlier investigations in [3] and [26], and our conventions will be those in [26]. We will work in coordinates where the metric is, The horizon of the extremal black brane will be taken to lie at r = 0. The gauge field equation of motion gives, (2.5) The remaining equations of motion can be conveniently expressed in terms of an effective potential [38] 6) and are given by, (2.10)
Solutions With Only Electric Charge
Next let us briefly review the solutions with Q m set to zero which carry only electric charge. The solution in the near-horizon region take the form, where the coefficients C a , γ, β, k and the electric charge Q e are given by . (2.13) It might seem strange at first that the electric charge Q e is fixed, this happens because in the near-horizon metric we work with the the time (and spatial coordinates) which have been rescaled compared to their values in the UV.
The following three conditions must be satisfied for this solution to be valid : Q 2 e > 0, C 2 a > 0, γ > 0. These give the constraints, 2 − δ(α + δ) > 0 (2.14) The last of these conditions follow from the requirement that so that the specific heat is positive. Figure (1) shows the the region in the (δ, α) plane, with δ > 0, allowed by the above constraints. To summarise our discussion, the metric in the purely electric solution takes the form And the dilaton is given in eq.(2.11,2.12) While this solution is not scale invariant it does admit a conformal killing vector. This follows from noting that under the transformation the metric eq.(2.19) remains invariant upto a overall scaling, The dilaton also changes under this rescaling by an additive constant, The two exponents γ, β which appear in the metric are related to the dynamic exponent with which gapless excitations disperse and hyperscaling violations, as was explained in [11]. Under the coordinate change, This is the form of the metric discussed in [11] (upto the overall 1 factor which was set to unity by a choice of scale). The exponent z is the dynamic exponent, as we can see from the scaling weights of the t and x, y directions in eq.(2.21), eq.(2.22). The exponent θ is the hyperscaling violation exponent, we will also explain this further in §4.
Let us end this section with some more comments. In eq.(2.5) the two-form F is dimensionless, so that Q e , Q m have dimensions of [Mass] 2 . The chemical potential µ is related to Q e by Q e ∼ µ 2 (2. 30) and has dimensions of mass. The near-horizon geometry of the type being discussed here can be obtained by starting from an asymptotically AdS space in the UV for a suitable choice of the potential V (φ). This was shown, e.g., in [26], for additional discussion see Appendix A. It is simplest to consider situations where the asymptotic AdS space has only one scale, µ, which characterises both the chemical potential of the boundary theory and any breaking of conformal invariance due to a non-normalisable mode for the dilaton being turned on. In our subsequent discussion we will have such a situation in mind and the scale µ will often enter the discussion of the thermodynamics and entanglement.
Also note that the parameter N 2 which will enter for example in the entropy eq.(4.5) is given in terms of the potential eq.(2.3) by Again to keep the discussion simple we will take the cosmological constant for the asymptotic AdS to be of order V 0 so that N 2 is also number of the degrees of freedom in the UV 9 . The solutions we have considered can have curvature singularities as r → 0, when such singularities are absent tidal forces can still diverge near the horizon, e.g., see 10 , [40]. These divergences can be cut-off by heating the system up to a small temperature as discussed in [2], [26]. Also, as we will see shortly, adding a small magnetic field can alter the behaviour of the geometry in the deep IR again removing the singular region.
The Effect of the Magnetic field
Now that we have understood the solutions obtained with only electric charge we are ready to study the effects of adding a small magnetic field.
The presence of the magnetic field gives rise to an additional term in the effective potential eq.(2.6). The magnetic field is a small perturbation if this term is small compared to the electric charge term, giving rise to the condition (3.1) From eq.(2.11) and eq.(2.12) we see that The examples studied in [26] are of this type. There the full potential was taken to be V (φ) = −2|V 0 | cosh(2δφ) (see eq.(F.1 of [26]). As a result in the asymptotic region r → ∞ the potential goes to its maximum value, 10 Sometimes the geometries can be regular with no singularities or diverging tidal forces, [39]. so eq.(3.1) in fact gives rise to a condition on the radial coordinate By a small magnetic field we mean more precisely choosing a suitable value of Q m and starting at a value of the radial coordinate r where eq.(3.3) is met. We will be then interested in asking if this magnetic perturbation continues to be small in the IR, i.e., even smaller values of r, or if its effects grow 11 .
The requirement that the magnetic field is small can be stated more physically as follows. Consider a purely electric solution which asymptotes to AdS space in the far UV and let µ be the only scale characterising the boundary theory which is dual to this electric theory as discussed in the previous section. Then the magnetic field is small if it satisfies the condition so that its effects can be neglected in the UV and continue to be small all the way to the electric scaling region. Our discussion breaks up into different cases depending on the values of the parameters α, δ. We will choose δ ≥ 0 in the discussion below without any loss of generality. Let us also note that although we do not always mention them for an electric solution to exist the additional conditions eq.(2.14)-eq.(2.17) must also be met.
We now turn to the various cases.
3.1 Case I. −δ < α < 0 In this case the magnetic perturbation is irrelevant in the infrared. From eq.(2.12) we see that αk > 0 so that as r → 0. Thus choosing a value of Q m , r, where eq.(3.1) is met and going to smaller values of r, eq.(3.1) will continue to hold and therefore the effects of the magnetic field will continue to be small 12 . In this range of parameters then the low temperature behaviour of the system and its low frequency response will be unchanged from the purely electric case. Also the entanglement entropy in the boundary theory of a region of sufficient large volume will be unchanged and be given as we shall see in §5 by eq.(5.16). 11 As is clear from eq.(3.3) and we will study this shortly in more detail, the magnetic field is relevant in the IR when αk < 0. It is easy to see from eq.(2.12), eq.(2.13) that when this condition is met the coupling g 2 = e −2αφ in the purely electric solution is weakly coupled in the IR since g 2 → 0 as r → 0. 12 On the other hand the magnetic field gets increasingly more important at large r, i.e., in the UV. However from numerical solutions one sees that for a suitable V (φ), when Q / m µ 2 ≪ 1 its effects continue to be small all the way upto the asymptotic AdS region.
Case II. |α| > δ
In this case the magnetic perturbation is relevant in the infrared and in the deep infrared the solution approaches an attractor of the extremal RN type. The dilaton is drawn to a fixed and finite value φ 0 and does not run-away and the near-horizon geometry is AdS 2 × R 2 with the metric components eq.(2.4), being where b 0 , R 2 2 are constants with R 2 being the radius of AdS 2 .. Note that in this attractor region of the spacetime the effects of the electric and magnetic fields are comparable.
To establish this result we first show that eq.(2.7), eq.(2.8), eq.(2.9) and eq.(2.10) allow for such an attractor solution. Next, starting with this attractor solution we identify appropriate perturbations and establish numerically that the solution flows to the electric scaling solution in the UV.
It is easy to check that the equations of motion allow for a solution of the type described above. Eq.(2.9) and eq.(2.10) are met with φ being constant and b being constant as long as the conditions V ef f = ∂ φ V ef f = 0 are met. This gives rise to the conditions which determine φ 0 , b 0 . Eliminating b 0 between the two equations gives The LHS must be positive, this gives a constraint | δ α | < 1 which is indeed true for Case II. Substituting eq.(3.10) in eq.(3.8) next determines b 0 in terms of φ 0 to be (3.11) Of the remaining equations eq.(2.8) is trivially satisfied while eq.(2.7) determines R 2 to be We see that for α > 0, R 2 → 0 as Q m → 0, making the AdS 2 highly curved, while for α < 0, R 2 → ∞ as Q m → 0.
Appendix A contains some discussion of the two perturbations in this AdS 2 × R 2 solution which grow in the UV. Starting with an appropriate choice of these two perturbations we find that the solution flows to the electric scaling solution in the UV. This can be seen in Fig. (4) and (5). For an appropriate choice of potential going out even further in the UV one finds that the solution becomes asymptotically AdS 4 , as shown in Fig. (7) and (8).
The AdS 2 × R 2 near-horizon geometry changes the IR behaviour of the system completely. As discussed in the introduction there is now an extensive thermodynamic entropy and the entanglement entropy also scales like the volume, for large enough volume. For additional discussion of the thermodynamics see §4 .
Case III. 0 < α < δ
In this case also we will see that the magnetic perturbation is relevant in the IR. Our analysis for what the end point is in the IR will not be complete, however.
We do identify a candidate "run-away" attractor as the IR end point of the system. In this attractor solution the magnetic field dominates and the effects of the electric field are negligible in comparison. As a result a solution taking the hyperscaling violating form eq.(2.11), for an appropriate choice of exponents, exists. We will refer to this solution as the magnetic scaling solution below. Unfortunately, we have not been able to satisfactorily establish that starting with the electric solution of interest one does indeed end in this magnetic scaling solution in the IR. This requires additional numerical work.
To see that the magnetic perturbation is relevant in the IR note that αk < 0 in this region so that eventually, for small enough values of r, condition eq.(3.3) will no longer hold and the effects of the magnetic field will become significant.
To identify the candidate run-away attractor let us begin by noting that the effective potential eq.(2.6) and thus the equations of motion are invariant under the transformation, Q m ↔ Q e accompanied by α → −α with the other parameters staying the same. Under this transformation the region discussed in Case I maps to the region 0 < α < δ. The discussion for Case I above then shows that, with Q m present, in this region of parameter space there is a consistent solution where the effects of the electric charge in the deep IR can be neglected. The solution takes the form, eq.(2.11) and eq.(2.12), eq.(2.13), with Q e → Q m and α → −α. This is the magnetic scaling solution referred to above. Actually, this solution exists only if (−α, δ) meet the conditions eq.(2.14)-eq.(2.17). In Fig.(1) the region for Case III where all the conditions eq.(2.14)-eq.(2.17) are met is shown in green. It is easy to see that for any point in this allowed green region the corresponding point (−α, δ) automatically lies in the allowed blue region.
Assuming that we have identified the correct IR end point we see that the thermodynamic entropy at extremality continues to vanish once the magnetic perturbation is added. It is also easy to see that the entanglement entropy is of the form eq.(5.16).
A more complete analysis of the system in this region of (α, δ) parameter space is left for the future.
Additional Comments
We end this section with some comments. It is sometimes useful to think of the solutions we have been discussing as being embedded in a more complete one which asymptotes to AdS space in the UV. The dual field theory then lives on the boundary of AdS space and standard rules of AdS/CFT can be used to understand its behavior. We take this theory to have one scale, µ, as discussed in §2. In addition the magnetic field is also turned on with Q m /µ 2 ≪ 1. The full metric for this solution will be of the form eq.(2.4) and in the UV will become AdS space 13 : Starting with this geometry for r → ∞ it will approach the electric scaling solution when r µ.
The magnetic field becomes a significant effect when its contribution to the effective potential eq.(2.6) is roughly comparable to the electric field. This gives a condition for the dilaton (3.14) Using eq.(2.12) this happens at a radial location r ∼ r * where Here we have introduced the parameter µ which was set equal to unity in eq.(2.11), eq.(2.12), eq.(2.13). For Case II and III where the magnetic perturbation is relevant in the IR, αk < 0, and the magnetic field continues to be important for all r < r * . In Case II for r ≪ r * the solution becomes AdS 2 × R 2 . In Case III we have not identified the IR endpoint with certainty when r ≪ r * . For Case I the magnetic perturbation is irrelevant in the IR. Second, Figure (2) shows a plot of various regions in the (δ, α) plane, with δ > 0. Region C corresponds to Case I. Regions A, D and E correspond to Case II. And region B corresponds to Case III. The line α = −3δ which is of special interest is the thick black line separating regions E and D. These regions are also described in terms of the parameters β, γ, eq.(2.12), eq.(2.13) in Table (1). The corresponding values of the parameters (θ, z) can be obtained from eq.(2.29). 13 We have set R AdS = 1. Table (
More on Thermodynamics
In this section we will discuss the thermodynamic behaviour in the presence of the magnetic field in some more detail. The introduction of a small magnetic field in the dilaton system can have a significant effect on the IR behaviour as we have already discussed. Here we will study some additional aspects of the resulting thermodynamics.
In our system the role of the Fermi energy is played by the chemical potential µ. Let us start with the purely electric theory first at a small temperature T T /µ ≪ 1 (4.1) As discussed in [3], [26] the entropy density s = S/V goes like Under the scaling symmetry equations, (2.20), (2.21), (2.22), s has dimensions of L θ−2 , where θ is defined in eq.(2.29) and L transforms in the same way as the (x, y) coordinates do in eq.(2.22). Thus θ is the exponent related to hyperscaling violation. Now we can consider introducing a small magnetic field Q m . Since the stress energy of the electromagnetic field is quadratic in Q 2 m this should result in a correction to the entropy which is of order Q 2 m . The scaling symmetry eq.(2.21), eq.(2.22) then fixes the resulting temperature dependence of this correction so that s is given by where k is defined in eq.(2.11), eq.(2.12) and s 1 is a µ independent constant. We see that the magnetic field can be regarded as a small perturbation only for temperatures meeting the condition We have numerically verified that the coefficient s 1 indeed does not vanish for generic values of (α, δ). The condition eq.(4.4) is in agreement with the discussion of section §2 where we found that the magnetic field is irrelevant or relevant in the IR depending on the sign of αk. Since 2γ − 1 > 0, eq.(2.18), we see from eq.(4.3) that when αk > 0 the effects of the magnetic field on the entropy vanish as T → 0. On the other hand when αk < 0 these effects grow as T → 0.
More on Case II.
One region of the parameter space where αk < 0 corresponds to Case II. As discussed in §2 in this case the resulting geometry for T = 0 in the deep IR is of the extreme RN type and the entropy at extremality does not vanish. From eq.(3.11) this entropy is given by where s 0 is a dimensionless constant, V is the volume and we have used eq.(2.30). The remaining region of parameter space where αk < 0 corresponds to Case III. For this case as discussed in §2 our analysis is not complete. If the IR in the gravity theory is an attractor of the magnetic scaling type described in 3.3 then the entropy vanishes at extremality. It is also worth commenting on the behaviour of some of the other thermodynamic variables for Case II. We start with the case where both Q m , T vanish, then first introduce a small Q m /µ 2 ≪ 1 and finally a small temperature. The temperature we consider meets the condition T /µ ≪ 1. In fact it is taken small enough to meet the more stringent condition so that eq.(4.4) does not hold and the near horizon geometry is that of a nearextremal RN black brane at a small non-zero temperature. The discussion of thermodynamics is conceptually simplest if we think of the gravity solution being asymptotic in the deep UV to AdS space with a possible nonnormalisable mode for the dilaton turned on, as was discussed in 2.1. In the absence of a magnetic field the dual field theory is a relativistic theory with the coupling constant dual to the dilaton being turned on and thus scale invariance being broken. The energy density ρ and pressure P for such a system at zero temperature are given by where the ρ 0 term arises due to the cosmological constant induced by to the breaking of scale invariance when the non-normalisable mode of the dilaton is turned on.
On introducing a small magnetic field the geometry changes for Case II significantly in the deep IR. However one expects that the resulting change in ρ, p, which are determined by the normalisable mode of gravity at the boundary, is small. Since the stress-energy in the bulk changes at quadratic order in Q m , as was discussed above, this correction should be of order Q 2 m . Thus the pressure, working still at zero temperature, would become where a 1 is a dimensionless constant. The resulting magnetisation can be obtained using the thermodynamic relation SdT + Ndµ − V dP + MdQ m = 0. (4.10) Keeping T = 0 and µ fixed gives We expect this magnetisation to be diamagnetic. Introducing a small temperature next will result in a temperature dependence in the pressure and the magnetisation. The change in the pressure keeping µ, Q m fixed and increasing T slightly is given from eq.(4.10) by where we have used eq.(4.5). Adding this to eq.(4.9) gives the total pressure to be The resulting magnetisation also acquires a linear dependence on temperature (4.14) Notice that in Case II |α| > δ and therefore the exponent δ−α 2α in the second term on the RHS is negative. Since Q m /µ 2 ≪ 1 this means that the coefficient of the term linear in T in the magnetisation is enhanced. As a result at a small temperature of order this term will become comparable to the zero temperature contribution. A case of particular interest is when α = −3δ. This corresponds to θ = 1, eq.(2.29), and gives rise to the logarithmic enhancement of entropy eq.(1.1). The pressure and magnetisation etc can be obtained for this case by substituting this relation between α, δ in the the equations above.
Let us end this section some comments. It is important to note that after turning on the magnetic field the state is still compressible. The compressibility is defined by κ = − 1 V ∂V ∂P | T QmN and can be related to the change in charge or number density n as µ is changed, From eq.(4.10) and eq.(4.13) we see that where the first term on the RHS arises from the first term in P in eq.(4.13) and the ellipses denote corrections which are small. Thus the charge density is only slightly corrected by the addition of Q m and therefore the state remains compressible. Our discussion above for the magnetisation etc has been for Case II. The analysis in case I where the magnetic field is irrelevant in the IR is straightforward. For Case III we do not have a complete analysis of what happens in the gravity theory in the deep IR. A candidate attractor was identified in §3, if this attractor is indeed the IR end point then starting from it the resulting thermodynamics can be worked out at small Q m , T along the lines above.
Entanglement Entropy
The entanglement entropy for the hyperscaling violating metrics we have been considering has already been worked out in in [10], see also [11]. Knowing these results, the behaviour of the entanglement entropy for our system of interest, in the presence of a small magnetic field, can be easily deduced.
To keep the discussion self contained we first review the calculation of the entanglement entropy for hyperscaling violating metrics and then turn to the system of interest.
Entanglement Entropy in Hyperscaling Violating Metrics
We will be considering a metric of the form (this is the same form as eq.(2.11) except that we have dropped the constant C 2 a by appropriately scaling the metric). In the discussion below it will be useful to think of this metric as arising in the IR starting with an AdS metric in the UV. This could happen for an appropriately chosen potential as was discussed in §2, [26]. The field theory of interest then lives on the boundary of AdS space. For simplicity we will restrict ourselves to a circular region R in the field theory of radius L. The boundary of this region in the field theory ∂R is a circle of radius L. To compute the entanglement entropy of R we work on a fixed constant time slice and find the surface in the bulk which has minimum area subject to the condition that it terminates in ∂R at the boundary of AdS. The entanglement entropy is then given by [34], [35].
where A min is the area of this surface. We will work in a coordinate system of the form eq.(2.4) which as r → 0 becomes eq.(5.1) and as r → ∞ becomes AdS space Replacing (x, y) by (ξ, θ) the metric eq.(2.4) can be rewritten as We expect the minimum area bulk surface to maintain the circular symmetry of the boundary circle. Such a circularly symmetric surface has area where ξ(r) is the radius of the circle which varies with r. To obtain A min we need to minimise A bulk subject to the condition that as r → ∞, ξ → L. The resulting equation for ξ(r) is Let us note that the circle on the boundary of R has area 14 It is easy to see that as r → ∞ the b 2 ( dξ dr ) 2 term in the square root in eq.(5.5) cannot dominate over the 1 a 2 term. The contribution to A bulk from the r → ∞ region can then be estimated easily to give where r max is the IR cutoff in the bulk which should be identified with a UV cutoff in the boundary. This is the expected universal contribution to entanglement which arises from very short distance modes entangled across the boundary of R. Now one would expect that as L is increased the bulk surface penetrates deeper into the IR eventually entering the scaling region eq.(5.1). For large enough L one expects that the radial variable ξ stays approximately constant in the UV and undertakes most of its excursion from L to 0 in this scaling region. We will make these assumption here and proceed. These assumptions can be verified numerically for the interesting range of parameters and we will comment on this further below.
With these assumptions the contribution from the scaling region for the minimal area surface δ 2 A bulk can be estimated by a simple scaling argument. We can neglect the change in ξ before the surface enters the scaling region and take its value at the boundary of the scaling region 15 which we denote as ξ 0 to be L. Under the scaling symmetry eq.(2.20)-eq.(2.22), which takes we see from eq.(5.5) that Now by choosing λ in eq.(5.9) to be λ = L 1 γ+β−1 (5.11) 14 As mentioned earlier we will persist in calling this the area although it is of course the perimeter. 15 By the boundary of the scaling region we mean the region where the metric begins to significantly depart from eq.(5.1). This happens as we go to larger values of r; for even larger values the metric becomes AdS space.
we can set the rescaled value for ξ 0 to be unity. In terms of the rescaled variable the minimisation problem has no scale left and δ 2 A bulk must be order unity. This tells us that when Note also that with ξ 0 set equal to unity the surface would reach a minimum value at a radial value of r min which is of order unity. Thus before the rescaling Now we are ready to consider different regions in the (γ, β) parameter space. From eq.(2.12) we see that β > 0 and from eq.(2.18) that γ > 1/2.
• γ > 1 + β: In this case we see from eq.(5.12) and (5.8) that δ 2 A bulk > δ 1 A bulk for sufficiently big L and fixed UV cutoff r 0 . Thus the dominant contribution to the area for sufficiently big L comes the scaling region. The entanglement is then given by with an additional term proportional to L in units of the UV cutoff r 0 . In eq.(5.14) we have introduced the scale µ to make up the dimensions. We remind the reader that this scale stands for the chemical potential which is the only length scale in the boundary theory. We see from eq.(5.14) that the entanglement grows with L with a power faster than unity. Also notice that from eq.(5.13) r min decreases with increasing L in accord with our expectation that the surface penetrates further into the scaling region as L is increased.
• A special case of importance is when 1 + β = γ. Here the term (Lµ) γ−β−1 γ+β−1 is replaced by a log, [10], resulting in eq.(5.14) being replaced by S EE ∼ N 2 (Lµ) log(Lµ) (5.15) • 1 + β > γ > 1 − β : In this case we see from eq.(5.12) and eq.(5.8) that the contribution δ 2 A bulk grows with L with a power less than unity and therefore the contribution made by the scaling region to the total area is less significant than δ 1 A which is linear in 16 L. In this region of parameter space the entanglement entropy is therefore dominated by the short distance contributions and given by where a = 1 rmax is an UV cutoff in the system.
• γ < 1 − β: In this case r min does not decreases with increasing L, actually the surface stops entering the scaling region and our considerations based on the scaling symmetry are not relevant. The entanglement entropy is again given by eq.(5.16).
One can calculate the minimal area surface numerically for cases when γ > 1 + β and also 1 + β > γ > 1 −β. This gives agreement with the above discussion including the scaling behaviour for r min eq.(5.13) in these regions of parameter space.
Let us make some more comments now. In the case where the near horizon geometry is of extreme RN type, i.e., AdS 2 × R 2 we have β = 0, γ = 1. This case needs to be dealt with separately. Here the entanglement entropy scales like the volume and equals the Beckenstein-Hawking entropy of the corresponding region in the boundary theory.
The discussion in §2 was in terms of the parameters (α, δ) while here we have used (β, γ). The relation between these parameters is obtained from eq.(2.13) and summarised in Table 1. We see that γ > 1 + β corresponds to Region D. The line γ = 1 + β corresponds to α = −3δ at the interface between D and E. The condition 1 + β > γ > 1 − β corresponds to A and E and finally γ < 1 − β to B and C.
Entanglement with a Small Magnetic Field
We are now ready to consider the effects of a small magnetic field. The behaviour of the gravity solutions was discussed in §2 where it was shown that the analysis breaks up into various cases. In all cases we will take the solution to approach AdS space in the UV. The resulting behaviour of the solution was discussed for the various cases in subsection 3.4. For r ≫ µ the solution is AdS space while for r * ≪ r ≪ µ it is of electric scaling type (r * is defined in eq.(3.15)). What happens for r ≪ r * depends on the various cases.
• Case II. |α| > δ: In this case the geometry for r ≪ r * is AdS 2 × R 2 . Let us start with a boundary circle of very small radius and slowly increase its size. When the radius Lµ ≪ 1 the entanglement entropy is given by eq.(5.16). When Lµ ∼ 1 the surface begins to penetrate the electric scaling region and as L increases we see from eq.(5.13) that r min decreases. When r min reaches r * the surface begins to enter the region where the magnetic field has an appreciable effect on the geometry. Using eq.(5.13) this corresponds to . (5.18) the entanglement entropy is given by the calculation in the electric solution. Thus for −3δ < α < −δ it grows faster than L with an additional fractional power while for α = −3δ it is logarithmically enhanced. For other values of (α, δ) which lie in this region the entanglement entropy is proportional to L and is dominated by the UV contribution.
Finally, when L ≫ L * the surface enters into the near-horizon AdS 2 × R 2 geometry. Now the entanglement entropy grows like L 2 and is given by This is an expression analogous to the Beckenstein-Hawking entropy, eq.(4.5), but with L 2 now being the volume of the region of interest. For the case of special interest, α = −3δ, this becomes .
(5.21)
• Case III. 0 < α < δ: In this case the magnetic field is important at small r. For L * ≫ L ≫ 1 µ the entanglement is given by the electric theory, it goes like eq.(5.16) and arises dominantly due to short distance correlations. The geometry in the deep IR could be the magnetic scaling solution discussed in subsection 3.3. If this is correct for L ≫ L * the entanglement will continue to go like eq.(5.16).
• Case I. −δ < α < 0: In this case the magnetic field is not important in the IR and the the entanglement entropy is given by eq.(5.16) both when L < 1 µ and L > 1 µ .
Concluding Comments
In this paper we have studied a system of gravity coupled to an Abelian gauge field and a dilaton. This system is of interest from the point of view of studying fermionic matter at non-zero charge density. Some of our key results were summarised in §1. We end in this section with some concluding comments.
• For the case |α| > δ (Case II in our terminology) we saw that the magnetic field is a relevant perturbation in the IR and the inclusion of a small magnetic field changes the behaviour significantly making the zero temperature thermodynamic entropy extensive and the entanglement grow like the volume. In particular this happens along the line α = −3δ (θ = 1, eq.(2.29)), where the electric theory has an entanglement entropy of the form eq.(1.1) suggesting the presence of a Fermi surface.
• It is well known that an extensive ground state entropy can arise in the presence of a magnetic field due to partially filled Landau levels. When this happens in a free fermionic theory the entropy scales like Q m while, in contrast, for the dilaton system the dependence on the the magnetic field is typically more exotic, eq.(5.20). E.g., with α = −3δ the entropy goes like Q 1/3 m , eq(5.21). Such a non-trivial exponent suggests that the ground state is more interesting and strange.
• A notable feature about how the entanglement entropy behaves in all the cases we have studied is that it never decreases in the IR, i.e., as one goes to regions of larger and larger size (L)in the boundary. For instance, consider the case where −3δ ≤ α < −δ. In this case for very small L is it given by eq. . We see that as L increases the entanglement increases monotonically 17 . In other cases while the detailed behaviour is different this feature is still true. These observations are in agreement with [42] where a renormalised version of the entanglement entropy was defined and it was suggested that in 2 + 1 dimensions this entropy would monotonically increase. It is easy to see that the behaviour of the entanglement entropy we have found implies that the renormalised entanglement entropy of [42] is monotonic and increasing.
• In [26] the behavior of a probe fermion in the bulk in the electric hyperscaling violating geometry was discussed. This corresponds to calculating the two point function of a gauge invariant fermionic correlator in the boundary. It is notable that the region in parameter space where Fermi liquid behaviour was found to occur for this correlator is exactly the region |α| > δ for which we have found that the geometry flows to an AdS 2 × R 2 endpoint in the IR 18 . It would be worth understanding this seeming coincidence more deeply. It is also worth mentioning that in [26] marginal Fermi liquid behaviour was found when |α| = δ. This region lies at the boundary of the region |α| > δ where an AdS 2 × R 2 endpoint arise 19 .
17 Our scaling argument does not directly fix the sign of the entanglement entropy in eq.(5.14) and eq.(5.15). However it is clear that the sign must be positive since the corresponding contribution to the surface area is bigger than the contribution from the UV for fixed r max as L → ∞, and the total surface area must be positive. 18 The remaining region |α| < δ does not exhibit Fermi liquid behaviour, this includes Case I of §2 where the magnetic field is irrelevant and also Case III of §2 where it is relevant but where we have not identified a definite IR end point to which the solution flows. 19 In fact the nature of the attractor changes at this boundary. E.g. in the purely electric case • Our focus in this paper was on taking an electrically charged system and including a small magnetic field. However, it is worth pointing out that the magnetic solutions with no electric field present (Q e = 0) are also of considerable interest in their own right. These solutions can be obtained by taking and α → −α in the solutions eq.(2.12), eq.(2.13). For a choice of parameters, which now meet the condition α = 3δ, the resulting entanglement entropy has the form eq.(1.1) which suggests the presence of a Fermi surface even though the charge density is now vanishing. It would be worth understanding the resulting state better in the field theory. The transformation eq.(6.1) is an electromagnetic duality transformation, and should act by exchanging charged particles with vortices in the field theory [43], [44]. These vortices perhaps form a Fermi surface resulting in the logarithmic enhancement of the entropy.
• We have not included an axion field in our analysis. Such a field is natural to include once the dilaton is present and it can have important consequences once the magnetic field is also turned on as was discussed in [23]. For example it was shown in [23] for the case δ = 0 that in the presence of the axion the entropy at extremality continues to vanishes in the presence of a magnetic field. Once the axion is included we need to allow for the potential to also depend on it, this leads to considerable choice in the kinds of models one can construct.
To remove some of this arbitrariness it would be worth including the axion within the context of models which arise in string theory or at least gauged supergravity.
• More generally, the time seems now ripe to systematically embed models of this type in string theory and supergravity. Some papers in this direction have already appeared [45], [46], [47], [48], [49], [50]. It would be worth understanding these constructions better and also gaining a better understanding of their dual field theory descriptions.
In this appendix we consider Case II solutions which were discussed in section 3.2 and establish that the deep IR solution is indeed AdS 2 × R 2 . We establish this numerically by integrating outwards from the AdS 2 ×R 2 near horizon solution discussed in subsection 3.2 and showing that the system approaches the electric scaling solution. For a suitable potential we find that the electric scaling solution in turn finally asymptotes to AdS 4 in the UV. Our numerical work is done using the Mathematica package.
We divide the discussion into three parts. In the first part we identify two perturbations in the AdS 2 ×R 2 region that grow towards the UV. In the second part, by choosing an appropriate combination of these two perturbation, we numerically integrate outwards taking the scalar potential to be V (φ) = −|V 0 |e 2δφ , eq.(2.3). At moderately large radial distances we get the electric scaling solution. In the last part, taking the potential to be of the form V (φ) = −2|V 0 | cosh(2δφ) 20 , we continue the numerical integration towards larger r and show that the geometry asymptotes to AdS 4 .
A.1 The perturbations
To identify the perturbations in the AdS 2 × R 2 solution discussed in 3.2 we consider perturbations of this solution of the following form for the metric components and the dilaton, Note that this is a perturbation series in r ν which is valid in the near horizon region where r ≪ 1. Equations (2.7-2.10) can be solved to leading order in r ν to find perturbations which are relevant towards the UV, i.e with ν > 0. We find two such perturbations parametrized by their strengths φ c1 which are given below : Note that b (1) c1 vanishes in the first of the above perturbations, as a result the first correction to b(r) starts at second order. Actually, we found it important to go to second order in the first of the above perturbations for carrying out the numerical integration satisfactorily. We will not provide the detailed expressions for these second order corrections here since they are cumbersome.
The first scaling symmetries can be used to set 21 a (2) c1 = −1. The second scaling symmetry can then be used to set Q e = 1. In addition we can choose units so that |V 0 | = 1. With these choices the system of equations has two parameters, φ c1 needs to be tuned very precisely to ensure that the solution flows to the electric scaling solution. Otherwise, for example with the modified potential considered in subsection A.4 below, the solution can flow from the AdS 2 × R 2 region in the IR directly to AdS 4 , as r → ∞, without passing close to the electric scaling solution at intermediate values of r.
A.3 Numerics : AdS 2 × R 2 to the Electric Scaling Solution We will illustrate the fact that the solution evolves from the AdS 2 × R 2 geometry to the electric scaling solution once the values of φ (1) c1 is suitably chosen with one example here. Similar behaviour is found for other values of (α, δ), which lie in Case II 22 The example we present here has α = 1, δ = 0.6 (satisfying |α| > δ). We will present the data here for the case when Q m = 10 −4 the behaviour for other values of Q m ≪ µ 2 is similar. It turns out that in this case we have to fine tune the value of 21 We cannot change the sign of a (2) c1 by using the symmetries. The above sign is necessary for the solution to flow to the electric scaling solution in the UV. 22 The example we choose here has α, δ > 0. Similar results are also obtained when α < 0, δ > 0.
this parameter to be near φ (1) c1 = −0.3173 so as to obtain an electric scaling solution at intermediate r.
Evidence for the electric scaling solution can be obtained by examining the relative contributions that various terms make in the effective potential eq.(2.6). In the electric scaling region the contribution that the Q 2 m dependent term makes must be smaller than the Q 2 e dependent term and the scalar potential which in turn must scale in the same way. Fig.(3) shows the different contributions to V ef f made by the terms, e −2αφ Q 2 e , e 2αφ Q 2 m and b 4 (r) e 2δφ 2 in a Log-Log plot. Clearly the Q 2 e term is growing as the same power of r as the scalar potential e 2δφ term and Q 2 m is subdominant. 5) show the plots of metric components a(r), b(r) and the scalar φ(r) obtained numerically. Each of them is fitted to a form given in eq (2.12). We see that the fitted parameters agree well with the analytic values for β, γ and k obtained from eq.(2.13) with (α, δ) = (1, 0.6). This confirms that the system flows to the electric scaling solution.
Here we show that on suitably modifying the potential so that the IR behaviour is essentially left unchanged the solution which evolves from the AdS 2 × R 2 geometry in the deep IR to the electric scaling solution can be further extended to become asymptotic AdS 4 in the far UV.
We will illustrate this for the choice made in the previous subsection: (α, δ) = (1, 0.6), (Q e = 1, Q m = 10 −4 ). For this choice of (α, δ) it is easy to see from eq.(2.12), eq.(2.13) that φ → ∞ in the IR of the electric scaling solution, and from eq.(3.10) that it continues to be big in the AdS 2 ×R 2 geometry once the effects of the magnetic field are incorporated. We will modify the potential to be V (φ) = −2|V 0 | cosh(2δφ) (A.12) instead of eq.(2.3). For φ → ∞ we see that this makes a small change, thus our analysis in the previous subsection showing that the solution evolves from the AdS 2 × R 2 geometry to the electric scaling solution will be essentially unchanged. However, going to larger values of r the modification in the potential will become important. This modified potential has a maximum for the dilaton at φ = 0 and a corresponding AdS 4 solution with 23 R 2 AdS = − 3 V 0 . We find by numerically integrating from the IR that the solution evolves to this AdS 4 geometry in the UV.
To see this first consider a plot of the three different contributions to V ef f proportional to e −2αφ Q 2 e , e 2αφ Q 2 m and b 4 (r) cosh(2δφ) shown in Fig(6). We see that there are three distinct regions. In the far IR AdS 2 × R 2 region, the three contributions are comparable. At intermediate r where we expect an electric scaling solution on the basis of the discussion of the previous subsection the magnetic field makes a subdominant contribution and the other two contributions indeed scale in the same way. Finally at very large r, in the far UV, the cosmological constant is dominant as expected for an AdS 4 solution.
We also show the metric components a(r), b(r) in a Log-Log plot in Fig (7) and (8). Once again, we can see three distinct slopes for a, b, corresponding to three different regions in the solution. In the AdS 4 region, as r → ∞, numerically fitting the behaviour gives a(r), b(r) ∼ r 0.99 which is in good agreement 24 with the expected linear behaviour. Finally, Fig(8) shows the scalar function φ(r) settling to zero with the expected fall-off as r → ∞. These results confirm that the system evolves to AdS 4 in the far UV. | 2018-12-10T21:58:49.268Z | 2012-08-09T00:00:00.000 | {
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1509881 | pes2o/s2orc | v3-fos-license | Quantifying the Impact of Accessibility on Preventive Healthcare in Sub-Saharan Africa Using Mobile Phone Data
Supplemental Digital Content is available in the text.
I n Sub-Saharan africa, a large number of deaths in children under 5 years of age result from a small number of common causes, such as diarrhea, malaria, and poor prenatal care. 1 these deaths are largely preventable through health services that are often available at local health facilities, including childhood immunizations and antenatal care for pregnant women. [2][3][4] Variable uptake of available health interventions can undermine healthcare programs, however, and plays an important role in child and maternal mortality, particularly in Sub-Saharan africa. [5][6][7][8][9] Poor physical access to health facilities is thought to be a critical determinant in these outcomes. Households further from health facilities have higher incidence of child mortality and malaria, for example, and are less likely to seek treatment for fevers, although these relationships are heterogeneous and appear to be stronger in very rural areas. [5][6][7][8][9][10][11][12][13][14][15][16][17] the relation between the travel time to healthcare facilities and actual travel behavior of a community is unclear, however, and 2 contradicting hypotheses have been suggested. First, a broad spatial analysis of the determinants of poverty in Kenya found a positive correlation between access to health facilities, frequency of travel, and wealth, suggesting that more wealthy populations are both more able to travel frequently and have easy access to health facilities. 18 However, it has also been suggested that the inverse is true; poor physical access imposes a higher travel burden on people to reach the resources they need, whether economic or health related. Persons in areas with poor access to health services, which are associated with poor health outcomes, may need to travel frequently and relatively far. 19 Human mobility-the spatial range and frequency of individual travel-reflects a range of basic needs, social incentives, and economic constraints and inherently encompasses multiple modalities. therefore, it remains unclear how measures of mobility, which reflect more than just geographic location and physical infrastructure for travel, correlate with access to healthcare or resulting health outcomes. 16 Disentangling the relationships among healthcare access, travel behavior, and health outcomes is challenging. Household surveys and travel diaries are often used to measure variation in travel times to public resources such as health facilities, [20][21][22] but these are limited in scope and hard to generalize across regions. geospatial techniques have been used to derive maps of approximate distances and travel times to nearby health facilities, 20 but these do not provide insights into how physical access affects human behavior. Due to the conflicting hypotheses described above and the lack of evidence available, identifying the factors underlying poor healthcare uptake and outcomes hinders the ability of policy makers to design programs that help those most in need.
the availability of de-identified mobile phone call detail records provides a new source of data on human mobility on unprecedented scales. 23,24 Mobile phone operators log cell tower locations whenever a person uses a mobile phone. these call detail records, once anonymized, provide detailed, longitudinal information about the travel patterns of millions of subscribers and offer unique opportunities for directly measuring the impact of geographic isolation on human travel behavior. Here, we combine an analysis of the geographic variation in mobility patterns of 14,816,521 anonymous mobile phone subscribers in Kenya with modeled estimates of travel times to health facilities, generated through standard cost-distancebased spatial analysis methods on various scales. We compare these with direct, geocoded measures of childhood immunization and antenatal care uptake in Western Kenya, measured by household surveys during a home-based HiV counseling and testing program. 25 this provides a direct measure of the impact of physical access to healthcare on mobility and health outcomes and a cost-effective new approach to mapping human behaviors underlying health outcomes.
Mobile Phone Data
Mobile phone data were provided by the incumbent mobile phone provider during the time of data collection. anonymized call detail records included the cell tower location (from one of 11,920 routing tower locations), sender, receiver, and timing of calls or SMS communications from 14,816,512 subscribers from June 2008 through June 2009 (with February 2009 missing from the data set). Subscribers were represented as unique anonymized hashed iDs. in total, over 12 billion mobile phone communications were recorded. From these data, we modeled the longitudinal travel behavior of each subscriber. We used a previously developed measure of individual mobility, the radius of gyration, to measure the mobility of each subscriber. this measure encompasses both the range and frequency traveled by a person into a single measure, with higher values characterizing more mobile subscribers and lower values resulting from subscribers who do not travel as frequency or as far. We calculated a primary location (center of mass) for each subscriber using the tower location of each mobile phone communication, based on the most common location over the course of the year. For each of the subscriber's records in the call detail records, we then consider the frequency and distance to other towers. We calculated average radius of gyration from subscribers assigned a primary location at each mobile phone tower (eappendix, http://links.lww.com/eDe/a870). 23
Travel-time Data
geospatial techniques were used to estimate the travel time to the nearest health facility. the population distribution was calculated using land cover and census data, as was done by linard and colleagues. 20 in brief, travel times between pairs of locations was calculated using a cost-distance algorithm that computes the "cost" of traveling on a regular raster grid based on information about the transportation network (obtained from the Kenyan national Bureau of Statistics), land cover data (http://www.africover.org), and topography data (http://srtm.csi.cgiar.org/). 20 the locations of all national health facilities were obtained from noor et al. 27
Survey Data
Survey data were collected from 5 districts in western Kenya that are part of the catchment area for the academic Model Providing access to Healthcare. 25 Between 2009 and 2011, a door-to-door HiV counseling and testing campaign was conducted in western Kenya. Most individuals (95%) agreed to be counseled and tested across 5 districts. the program collected geocoded information about pregnancy, attendance at antenatal clinics, immunization of children, and basic socioeconomic information. individual surveys from these study sites (n = 273,213) were aggregated to the household level (n = 78,882). Households were then aggregated to sublocations (n = 89) where the mean mobility variable and travel time to a health facility were calculated.
We considered 2 primary outcome variables related to the uptake of preventive healthcare interventions-completed childhood immunizations and antenatal care for pregnant women (see etable S1; http://links.lww.com/eDe/a870). For a child to be fully immunized, they should have received Bacillus calmette-guérin vaccine at birth and measles at 9 months. a household was considered to be missing immunizations if there was at least 1 child over 1 year who had not had any of these immunizations. Households were considered to be missing antenatal care if there was a pregnant woman who reported not receiving antenatal care. For each sublocation, we calculated the percentage of eligible households (ie, households with children or pregnant women) who were lacking either immunizations or antenatal care.
We aggregated household-level point estimates of both mobility and travel times to obtain sublocation averages. to aggregate mobility values to sublocations for comparison, each household was assigned the mobility value of the nearest mobile phone tower and a corresponding travel-time estimate.
RESULTS
On large spatial scales, people living in counties with poor physical access to healthcare were also the most mobile, in terms of both distance and frequency. Figure 1a and B shows the Kenyan road network (see eFigure 1; http://links.lww.com/ eDe/a870 for continuous maps), colored by the observed mobility of local populations, and the relationship between mobility and travel times to health facilities aggregated at the county level (eFigures 1 and 2; http://links.lww.com/eDe/ a870 for similar analyses for population centers and schools). radius-of-gyration values decreased nonlinearly as county population density increased and travel times to reach resources were reduced (eFigure 2; http://links.lww.com/eDe/a870: fit for population density; adjusted R 2 = 0.5, sum of squared errors = 3.3, root-mean-square error = 0.29; correlation with average travel time to the nearest health facility, adjusted R 2 = 0.5). eFigure 3 (http://links.lww.com/eDe/a870) shows the impact of mobile phone tower density on radius gyration values. this finding is consistent with the hypothesis that remote communities experience an increased burden of travel.
Substantial heterogeneity in the relationship between radius of gyration and travel time to health facilities was observed on the smaller spatial scale of the individual cell tower, however. For example, Figure 2a and B compares observed mobility with estimated travel times to the nearest health facility for a region of Western Kenya (eFigure 4; http://links.lww.com/eDe/a870 for similar analyses comparing health facilities to schools). Here, we observed that populations with high and low average mobility are adjacent to each other, despite similar estimated travel times to the nearest health facility. thus, while communities can be defined as equally remote mobility, patterns are locally heterogeneous. to examine whether these heterogeneities in mobility were associated with health outcomes, we analyzed income and health data from 5 districts in western Kenya (78,882 households). We identified households missing preventive care-either incomplete immunization among children age 1-5 years or a lack of antenatal care among pregnant women. Most children completed their immunization schedule in the majority of eligible households (2% of eligible households had children missing immunizations, n = 686). almost 40% of households with pregnant women were missing antenatal care (39%, n = 1,374). the households missing care were geographically heterogeneous, with higher percentages found in 2 of the study districts (Figure 3a). We aggregated household data to sublocations (n = 89) to compare with the mobile phone data. (On average, 1% sublocations were missing immunization, and 31% were missing antenatal care.) Mobility estimates derived from mobile phone data were better able to predict the percentage of households in a sublocation missing immunizations or antenatal care than were estimates of travel times to health facilities ( Figure 3B and etable S2; http://links.lww.com/eDe/a870). the correlation coefficients for immunizations and health-facility The relationship between mobility, travel times, and households missing preventive healthcare. A, The percentage of households (HHs) within each area's sublocation who reported missing antenatal care (ANC). The entire study area and a sample study site are shown. B, The predicted percentage of eligible HHs missing ANC per sublocation using travel times to the nearest health facility (HF) (red) or the mobility values from the mobile phone data (blue) (reduction in deviance from the mobility model, 3%; from the travel-time model, 0.41%). The travel-time data would predict that nearly every sublocation is missing the same percentage, whereas the mobile phone data provides more accurate estimates over a wider range.
travel time was 0.06 (P = 0.6), whereas average radius of gyration was correlated with a coefficient of −0.23 (P = 0.03). For antenatal care, there was no relationship between either measure of travel and uptake, although the relationship with mobility was somewhat stronger ( Figure 2B) (health facility travel time, −0.06 [P = 0.56] and mobility, −0.14 [P = 0.2]). in sublocations of similar physical access to health facilities, increased mobility was associated with a higher percentage of households accessing preventive healthcare. (For sublocations within 30 minutes of a health facility, correlation coefficient = −0.21 [P = 0.24]; for sublocations between 40 minutes and 2 hours of a health facility, correlation coefficient = −0.38 [P = 0.067].) the strength of this relationship was not related to the average household wage hours and was stronger in areas further from health facilities, suggesting that the impact of access and mobility is greater in more remote communities. thus, mobility can help explain heterogeneities in accessing care for populations with comparable physical travel times to health facilities, highlighting the ability of new data sources to describe variable uptake of preventive healthcare.
DISCUSSION
We propose that mobility measured by mobile phone data may reflect more about economic conditions and behavior than travel-time estimates, making it a more comprehensive measure that takes into account various aspects of health care access in addition to geography. Our results are consistent with the hypothesis that poor physical access places a high travel burden on persons living in remote regions, but that the most vulnerable among them are the least mobile. We propose that mobile phone call records provide valuable insights into human travel behaviors associated with poverty and access to healthcare and could be used to identify vulnerable populations where households are at risk for missing basic preventive care. Measuring and understanding the relationship between geographic isolation and travel is key to the targeting of development assistance that aims to alleviate health disparities and identify the role of travel and physical accessibility to health resources. 16 although mobile phone data have inherent biases, analytic tools for adjusting estimates are improving, and in this case, they may not have a major impact on estimates. 26,28 We have previously shown that mobile phone ownership is generally still biased toward wealthier, urban-dwelling males. However, we observed mobile phone ownership in all income brackets, and these biases are rapidly diminishing. 26 Mobile phone data are increasingly being analyzed for epidemiologic studies through individual agreements between the operator and researchers, with anonymization and aggregation often taking place before sharing. the value of these approaches is likely to be most profound in low-income settings where data are scarce, but methods of analysis are still in their infancy, as are effective protocols for sharing the data more widely. [29][30][31] Despite the obstacles to the routine use of mobile phone data in public health research, however, we believe they represent a valuable and inexpensive source of information that can be used to identify areas facing substantial travel burden and to identify local variability in access that can exceed the spatial resolution of conventional access measures. | 2016-05-04T20:20:58.661Z | 2015-02-03T00:00:00.000 | {
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6069616 | pes2o/s2orc | v3-fos-license | Frequency Domain Kernel Estimation for 2nd-order Volterra Models Using Random Multi-tone Excitation
We consider the problem of frequency domain kernel estimation using random multi-tone (harmonic) excitation for 2nd-order Volterra models. The basic approach is based on least squares minimization of model output error, and results for the Volterra kernel estimations with random multi-tone inputs and random Gaussian input are compared. We show that kernel estimation with multi-tones are very accurate and efficient compared to the latter. As an illustration, the proposed method is applied to a discrete input–output system obtained from the numerical simulation of a representative hydrodynamic system for modeling semiconductor device transport. We also consider the effect of noise in the kernel estimation.
INTRODUCTION
The Volterra (functional) series approach has been widely used to model the forced response of weakly nonlinear systems [1,2]. In practice, many nonlinear systems can be modeled approximately using only the first few terms of the Volterra series. Identification of such a finite Volterra model requires measurement or estimation of the associated kernel functions from a finite set of representative input -output data (realizations) of the actual system. It is important that the identification be carried out with fewer realizations (to reduce the cost) while yielding maximum information about the kernel functions with a good accuracy. The objective of the present study is to develop an efficient and practical approach, in this sense, for identification of kernel functions of a 2nd-order Volterra model in the frequency domain. The approach is applicable to the analysis of physical systems or numerical simulations, and the focus of the present work is its applications to the analysis of nonlinear mathematical models of physical systems using numerical simulations. In particular, we show that, precise and controlled random multi-tones can be used effectively to obtain realizations.
There is an extensive literature of studies proposing various identification methods using different kinds of inputs for certain systems. In particular, methods for identification of Volterra kernel functions in the frequency domain † (i.e. Volterra transfer functions, VTFs) can be divided into two main groups based on the use of tonal excitation or random excitation. In the latter approach, methods to estimate the linear, quadratic and cubic transfer functions for orthogonal and non-orthogonal models with both non-white Gaussian and non-Gaussian random inputs can be found in the works of Hong et al. [3 -6]. In these studies, kernel estimation with non-Gaussian excitation is based on the least-square minimization (LSM) of the model error. Some drawbacks of using random inputs for estimating VTFs are due to aliasing. First, recall that, it is impossible to generate random input signals with perfect cut-off in its power spectrum. This certainly affects the accuracy of the estimation using random input. Moreover, the number of realizations required for an accurate estimation may be quite large depending on system characteristics. This significantly increases the expense of obtaining realizations. This subject is of general interest in many application areas of nonlinear analysis, including fluid and structural dynamics or circuit and device modeling. For example, in the nonlinear identification of structural systems, the disadvantages of using strictly random or burst-random Inputs have been reported [7]. In practice, the steady-state sine, fast sine, or burst sine are found to be more optimal. The preliminary motivation for our work is to extend LSM techniques for estimation of VTFs with random multi-tone inputs, which eliminates aliasing effects. With this in mind, we, in fact, aim to obtain an estimation method using random multi-tones, which has certain advantages over the conventional techniques to measure the VTFs.
The actual frequency domain formulation of a Volterra model gives the amplitude and phase relationships between sinusoidal tones contained in the inputs and outputs of a system in terms of possible tonal interactions along with the associated VTFs [1,8 -10]. Efficient methods for measuring VTFs by using multi-tone sinusoidal inputs have been reported by several researchers. In these studies, Boyd et al. [11] presented a "quick method" for calculation of the transfer functions of a second-order Volterra system in the frequency domain. Chua and Liao [12] extended the method to third order models, which includes a cubic term. Each multitone input is constructed from the tones at carefully chosen frequencies so that the outputs of different order Volterra operators contain the tones at distinct interaction frequencies, at which the VTF values can be measured explicitly. Thus, the number of frequency coordinates of measured VTFs is maximized for a finite number of multi-tone excitations. On the other hand, with this approach, explicit computation of transfer function values is possible for only some scattered isolated frequency points, not for all discrete spectral points in a given frequency range. (For example, any quadratic Volterra kernel value associated with the DC contribution of the tones cannot be computed explicitly with one multi-tone input.) Furthermore, for multi-tone inputs containing tones at all discrete frequency values within a frequency range, it is not possible to measure the VTFs explicitly since the tonal interactions overlap. The discrete VTF values can be obtained only by solving linear algebraic systems of equations (constructed from the input -output realizations). In principle, the number of realizations can be taken to be equal to the number of unknowns in the algebraic systems. However, in practice, it is desirable to solve over-determined systems (with more realizations), as done in estimations with random inputs. In this way, the effects of imperfections in the Volterra model can be minimized. This is in essence what is done in kernel estimations with random inputs. Our estimation procedure follows this approach and uses multi-tone inputs whose tones are chosen at each consecutive discrete frequency with uniformly distributed random phases and Gaussian distributed random amplitudes. The utilization of random amplitude and phase represents an essential difference from previous multi-tone methods, which, for the most part, used deterministic tones. Sampling the steady-state harmonic outputs to these multi-tone excitations within a record length, which exactly equals one period, prevents aliasing and it also enables a sharp cut-off in the input power spectrum and estimation at each discrete frequency value of the domain. Applying LSM to estimate the VTFs using random realizations makes it possible to check the goodness of the model by using conventional tools such as the power spectrum and coherency. Because of the parallelism in the present approach and conventional techniques using random inputs in kernel identification, we use the term "estimation" rather than "measurement".
The outline of the paper is as follows: We first briefly describe the LSM approach and the harmonic excitation strategy for obtaining the necessary time series of the system inputs and outputs. Then, we compare the frequency domain estimations of the linear and quadratic kernels of the 2nd-order Volterra model using the LSM principle with Gaussian random and random multi-tone inputs. As an illustrative test case, we consider a 2ndorder Volterra model for voltage-current dynamics of a semiconductor device, simulated by a numerical hydrodynamic code. In particular we explore the effects of noise in the system input and output signals, which would be of concern mainly in physical experimental applications as opposed to numerical experimental applications. (However, the effects of approximation error in numerical input can also be interpreted as "noise" and the influence of this on the output and kernel estimation is relevant.) The output of the approximate 2nd-order Volterra model obtained using the present method is compared with the actual output of the numerical model. Finally, we summarize and discuss our findings in the conclusion.
2ND-ORDER VOLTERRA MODEL IN THE FREQUENCY DOMAIN
The 2nd-order Volterra model v 2 consists of linear and quadratic operators acting on an input function x (t ). That is, where l(t ) and q(t,t 0 ) are the associated linear and quadratic kernel functions in the time domain. In the frequency domain, Eq. (1) transforms to Qðg; f 2 gÞXðgÞXð f 2 gÞ dg ð2Þ where X( f ), V 2 ðXÞð f Þ; L( f ) and Q( f, f 0 ) denote the Fourier transform of x(t ), v 2 ðxÞðtÞ; l(t ) and qðt; t 0 Þ; respectively. Here, the quadratic kernel may be assumed to be symmetric without loss of generality.
If the 2nd-order Volterra model in the frequency domain is written more compactly in terms of the respective linear and quadratic operators in Eq. (2) as then the power spectrum of V 2 (X ) can be expressed as where k·l denotes the expectation operator implying the average value of the spectral quantity applied to a large number of signal realizations. R denotes the real part of the associated expression and symbol * implies the complex conjugate. The first two terms in the right-hand side of Eq. (4) represent the individual contributions to output power from the linear and quadratic operators. The last term is the interference term, and vanishes when L(X ) and Q(X ) are orthogonal. The orthogonality condition is satisfied if the bispectrum kX* ð f ÞX* ð f 0 ÞXð f þ f 0 Þl of the input x(t ) is zero. This corresponds to the fact that the sampling frequency to collect the time series for input and output should be chosen such that, the Nyquist frequency f s /2 is smaller than 2f M to avoid aliasing in the output, i.e. f s $ 4f M : When f M ¼ f s =4; for a given N, the largest spectral range is attained with M ¼ N=4 for the identification domain of the quadratic kernel. Now, assume that a single input -single output (SISO) relationship between an input x and an output y of a nonlinear system can be approximately characterized by a 2nd-order Volterra model in the frequency domain. That is, , corresponding to a set of sampled system input -outputs (realizations). Then, the Volterra kernels given in Eq. (5) can be calculated approximately from the LSM of the power spectrum for the model error. That is we seek the discrete values of the Volterra kernels in Eq. (5) such that the least squares functional is minimized for each k. Here, k·l denotes the expectation operator, which is to be approximated by an ensemble average over N r realizations. Minimizing Eq. (7) requires for each k and m in the identification region. This leads to an algebraic system of the following block-matrix form for each k: where the scalars S XX [k ] and S XY [k ] are the values of discrete auto and cross power spectra defined by The entries in the vectors q (k ) , b (k ) and y (k ) and in the matrix C (k ) are, respectively, Remarks In practice, the symmetry of the quadratic kernel, i.e. Q½m; m 0 ¼ Q½m 0 ; m; is taken into account. This implies we need to consider the kernel values only for m $ m 0 ¼ k 2 m (on the solid part of the line in Fig. 1), so the dimension of the algebraic system is reduced approximately by half.
For Gaussian inputs, the orthogonality condition implies b ðkÞ ¼ 0 so Eq. (9) is decoupled and L[k ] and q (k ) can be obtained independently. Furthermore, it can be shown that the 4th-order spectral moment c i 0 j 0 vanishes for i 0 -j 0 ; making C (k ) diagonal. This permits the values of q (k ) to be calculated explicitly [3]. However, for a finite number of realizations, the conditions c i 0 j 0 ¼ 0 for i 0 -j 0 and b ðkÞ ¼ 0 only approximately hold, so calculations that do not make these assumptions are expected to produce more accurate results for the transfer function estimates [13].
Coherency
Coherency is defined as the ratio of the model output power to actual output power.
It can be shown that the coherency function, ‡ calculated using the estimated kernels, is always bounded by zero and unity. Based on the model output as given in Eq. (3), the coherency function may be also written as a sum of partial coherency functions, i.e. g 2 ¼ g 2 l þ g 2 q þ g lq . When orthogonality holds the interference term g lq vanishes, and the individual linear and quadratic coherency terms are less than unity. Partial coherencies are useful in understanding the "strength" of the linear and quadratic mechanisms in terms of their contribution to the total power as a function of frequency [3]. In this study, we are concerned with the total coherency g 2 and the coherency of the first order model g 2 l for comparing the 1st and 2nd-order Volterra model outputs.
Identification With Multi-tone Excitation
In harmonic excitation, multi-tone inputs may be prescribed in the time domain in the form where M is the total number of (tones) harmonics. The selection of amplitudes {A m } determines the class of input signals for the identification. In particular, here, we consider the amplitudes {A m }, and the phases {f m }, taken to be random numbers with uniform distributions in the ranges ½2A; A and ½0; 2p; respectively, where A is the reference input amplitude. Here, we stress the fact that, in conventional methods using multi-tone inputs, constructing the tones with deterministic amplitudes and phases can result in some useful simplifications in measuring the Volterra kernels explicitly. In the present method, however, choosing the amplitudes and phases randomly as described above actually makes the matrix C (k ) in Eq. (9) much more diagonally dominant, ‡ Note that, with this definition, the coherency function reduces to the well known form g 2 ¼ jS XY j 2 =S XX S YY for linear systems.
and therefore, yields more robust computations for the kernel estimations, as confirmed in our numerical experiments.
In kernel identification using random multi-tone inputs, each distinct system realization corresponds to a pair of a multi-tone inputs with a different set of {A m } and {f m } and the steady-state harmonic response of the system to this input. For accurate identification (or estimation) of Volterra kernels, it is essential that the output data from the simulations be collected after the transient dynamics of the system die out.
The complete kernel identification procedure is outlined below: 9) and solve for the L and Q values at the discrete points in the associated range. 7. Having obtained the discrete kernels L and Q in the entire identification domain, calculate the model output from Eq. (5) for the same inputs. 8. Compute the coherency from Eq. (15) to check the "goodness" of the model.
COMPARISONS OF ESTIMATIONS WITH RANDOM INPUTS AND RANDOM MULTI-TONE INPUTS
In this section, we are interested in the performance of the LSM procedure with random and random multi-tone inputs. For simplicity and to isolate the effects due to higher order nonlinearities, we consider the following pure quadratic discrete SISO system which is stable when jaj , 1: This difference equation has a delay effect on the input -output relationship between x and y. If x[n ] and y[n ] are assumed to be sampled with sampling frequency f s ¼ 1; in the frequency domain the input -output system given by Eq. (17) can be easily shown to be represented by a quadratic Volterra operator, whose kernel function is known analytically as Thus, the accuracy of the estimations made with Gaussian random and multi-tone input functions can be compared directly. In our "numerical" experiments, Eq. (17) was sampled with N ¼ 64 points for each realization.
In the random excitation case, the entire set of realizations was obtained from the solution to the difference equation in Eq. (17) with the numerically generated Gaussian random input x[n ]. The input power spectrum is band-limited to f =f s ¼ 0:25: Figure 2 shows the power spectra of the input and output signals obtained for the discrete system Eq. (17) with a ¼ 0:85: Here, the number of realizations was taken to be 100 where each system realization corresponds to the non-overlapping data records (of N ¼ 64 points) for the entire time series of x[n ] and y[n ].
In the random multi-tone (harmonic) excitation case, 16 multi-tones with randomized amplitudes and phases were used in the input signal ðM ¼ 16Þ with the same number of sampled points ðN ¼ 64Þ: Figure 3 gives the power spectra of the input and output signals obtained with the number of realizations N r ¼ 100 for the same discrete system ða ¼ 0:85Þ: Note that the case of M ¼ 16 The magnitude of the quadratic kernels estimated from Gaussian and random multi-tone inputs with N r ¼ 100 realization are compared in Fig. 4. With multi-tone input, the exact quadratic kernel is recovered within the machine accuracy, being independent of number of realizations used for the estimation. The error in the quadratic kernel estimated using random input can be reduced by increasing the number of realizations; however, the convergence to the exact kernel is rather slow. Figure 5 shows the errors in the quadratic kernels with estimated random input of 100 and 3200 realizations. In this figure, it is clear that, even though, the error in the estimation generally reduces with more realizations, the decrease in the maximum local error is small for a ¼ 0:85 with even large N r . One should notice that, the maximum error in the estimated kernel occurs on the frequency coordinate ð0:25; 20:25Þ: This implies that the lack of a sharp cut-off in the power spectrum of the input near f ¼ 0:25 causes this error in the estimation. This situation is more apparent when the discrete system is influenced more and more by the time delay as the parameter a approaches unity. In Fig. 6, we compare the normalized L 2 norms of the errors in the quadratic kernels estimated using Gaussian random input for different number of realizations with a ¼ 0:75; 0.85 and 0.95. As can be seen in this figure, it becomes very difficult to obtain accurate estimations as the parameter a approaches unity using a practical number of realizations with Gaussian random inputs. (See also the test studies presented in Ref. [13]. On the other hand, using random multi-tone inputs recovers the exact kernels within machine accuracy (1.0E 2 6) for any jaj , 1: As expected, additive input noise in the multi-tone realizations result in inaccuracies in the kernel estimation. By using more (multi-tone) realizations, one can anticipate decreasing the error in the kernel estimation. This aspect will be discussed in more detail in "Noise effects in kernel estimation".
APPLICATION TO SEMICONDUCTOR DEVICE MODELING
In this section, we demonstrate an application of the proposed random multi-tone input method for the identification of a 2nd-order Volterra model in the frequency domain to approximately represent the transient FIGURE 4 Quadratic kernels esimated using Gaussian random (left) and multi-tone (right) inputs with 100 realizations. voltage-current dynamics of a simple n þ 2 n 2 2 n þ diode about an operating (steady state voltage-current) point. The identification is carried out using computer simulations of the system. There are several models and numerical techniques in use and under investigation for the steady-state device problem [14 -16]. Our interest is in the nonlinear system identification for the transient behavior. We believe that this topic has not previously been investigated. Here we consider a representative model for transient simulation of a very simple but standard test device [17]. We emphasize that the focus is the nonlinear identification problem so the numerical scheme and test problem is of less importance.
The computer simulation of the device dynamics is based on the numerical solution of the one-dimensional hydrodynamic transport equations [18] using the Mac-Cormack scheme [19]. In these hydrodynamic transport equations, the heat transfer effect is not included while the full convection model for drift kinetic energy is adopted [20] (the approach can be applied to other device models [14]). In the case study, we consider a 0.3 micron device with lattice temperature 290 K. The permitivity of the device is taken to be e ¼ 1:0536 £ 10 210 C 2 /J m. The electron concentration at the gates of the device is 5 £ 10 5 /m 3 while the doping is 2 £ 10 3 =m 3 : Here, a moderate doping profile has been chosen in order to decrease the time-step in the simulations and to ensure weak nonlinearity in the system (more demanding device scales and doping could be similarly studied but we have not elected to do this because out objective is specifically to demonstrate the identification problem). The steadystate solution to the device equations obtained for the nominal voltage V o ¼ 1:6 V gives the corresponding nominal current value I o . Then, a time-dependent voltage perturbation vðtÞ ¼ VðtÞ 2 V o is applied as an input function. The current perturbation from the nominal current, i.e. iðtÞ ¼ IðtÞ 2 I o ; is computed from the numerical solution of the hydrodynamic transport equations, and taken as a response to this voltage perturbation. Thus, we define our "system" as the SISO relationship between temporal perturbations from the nominal voltage v(t ) and current i(t ), as observed in the numerical simulations, and obtain a 2nd-order Volterra model approximating this SISO system. Since the characteristic time scale of the system is in the order of 10 212 s, we present and discuss the results of this analysis in terms of scaled frequency ( f 0 ) and time (t 0 ) which are related to the physical frequency and time by f ¼ f 0 £ 10 12 Hz and t ¼ t 0 £ 10 212 s; respectively. The kernels of the 2nd-order Volterra model were estimated using 200 input -output realizations obtained from the numerical solutions of the hydrodynamic transport equations. The input signals included 16 tones, and the frequency of the maximum tone was 2.5 £ 10 12 Hz. The reference input amplitude for the randomly selected multi-tones is A ¼ 0:05 V: The power spectra of the multi-tone inputs and outputs (averaged over the 200 realizations) used for the estimation are given in Fig. 7 in the scaled frequency range f 0 [ ½0; 5: The resulting estimated linear and quadratic kernels are shown in Figs. 8 and 9, respectively. The high coherency of the model output as seen in Fig. 10 indicates the Volterra model estimated at this amplitude level represents the dynamics of the numerically simulated device quite closely. This is mainly because the estimation has been carried out at small amplitude levels and the input does not strongly excite the higher order nonlinearities.
The estimated 1st-and 2nd-order Volterra models (based on A ¼ 0:05 V) were tested with new sets of higher amplitude random multi-tone inputs. The 1st-order model includes only the linear operator associated with the estimated linear transfer function while the 2nd-order model also includes both the linear and quadratic operator associated with the estimated quadratic transfer function. The frequency range of the test inputs is half that used for the kernel estimation; that is the number of tones in the test inputs is M ¼ 8: Comparisons of power spectra and coherency were made for the outputs in response to the inputs whose reference amplitudes are A ¼ 0:1 and 0.3 V. Note that these amplitudes are two and six times as large as the reference amplitude ðA ¼ 0:05 VÞ used to determine FIGURE 7 Power spectra of the input and output semiconductor device. ð f ¼ f 0 £ 10 12 HzÞ: the 2nd-order Volterra model. Figure 11 shows the power spectra of the outputs of the actual system (which is based on the numerical solutions of hydrodynamic equations) and the estimated Volterra model for these two amplitude levels of the input. In this figure, we see that the cut-off frequency of the input signals is f 0 c ¼ 1:25 while the system outputs have power in the entire frequency range. The 1st-order model gives outputs in the frequency range f 0 [ ½0; 1:25 whereas the 2nd-order model gives outputs in the frequency range f 0 [ ½0; 2:5 since it contains the contribution of the quadratic operator, too. As one can see in this figure, the system output power in the range f 0 [ ½2:5; 1Þ is due to the cubic and higher order nonlinearities in the system, which are not included in the 2nd-order model. As the amplitude level of the inputs are increased from A ¼ 0:1 to 0.3 V, the system output power in the range f 0 [ ½2:5; 5 increases significantly. Of course, the higher order nonlinearities also affect the system output in the range f 0 [ ½0; 2:5: Coherency functions of the 1st-and 2nd-order Volterra models are given in Fig. 12 for the two amplitude levels and indicate this effect. For A ¼ 0:1 V; the coherency function is close to unity for the two models in the associated frequency ranges. The 1st-order model is quite accurate in the range f 0 [ ð0; 1:25; whereas in the 2ndorder model, which contains the quadratic operator, the total system power is recovered in the entire range f 0 [ [0,2.5]. For, the higher amplitude case A ¼ 0:3 V; the coherency is degraded in ½0; 1:25; and with more significant departures in ½1:25; 2:5: This shows that, at this high amplitude, the total system power cannot be recovered by the linear and quadratic operators in these truncated models. One should note that the estimated 2ndorder Volterra model gives the best coherency (as shown in Fig. 10) for inputs at the same level of amplitude as the inputs that are used in the estimations; that is (A ¼ 0.05 V). The Volterra models are expected to become less accurate at the higher amplitude levels of inputs, and results in Fig. 12 demonstrates this.
The comparisons of the system output and model output in time, were made for an arbitrary input -output realization after the inverse DFT of the model output in the frequency domain were carried out. Figure 13 shows the actual and model outputs to a typical multi-tone input in the time domain. The reference amplitude of the input is A ¼ 0:3 V: The differences between the model outputs and actual output are plotted in Fig. 14 for the 1st-and 2nd-order Volterra models. The superiority of FIGURE 11 Power spectra of the input and outputs of the actual system and Volterrs models at the amplitude levels A ¼ 0:1 (left) and A ¼ 0:3 (right). the 2nd-order Volterra model to the 1st-order (linear) model in approximating the actual output locally is evident in this figure. As Fig. 11 indicates, the power of the actual output signal is low by a factor . 10 23 at high frequencies ð f 0 . 2:5Þ: Therefore, the output from the 2nd-order Volterra model approximates the actual output to a considerable accuracy in the time domain, even for this high amplitude input case. Although the 2nd-order Volterra model approximates the actual output waveform quite well, two singular exceptions occur at t 0 < 4:5 and 10.5 (see Fig. 14). Note that at these times the input amplitude is at its maxima, thereby possibly "exciting" higher-order nonlinearities. In such a case, the 2nd-order model cannot capture these higher-order effects.
Finally, in Fig. 15, the L 2 norms of the errors in 1st-and 2nd-order Volterra model outputs in the time domain are plotted for increasing reference amplitude of the excitation. When the reference input amplitude is increased, the effect of higher-order nonlinearities in the system, which are neglected in the truncated 2nd-order Volterra model, becomes stronger. Since the 2nd-order model takes into account the quadratic nonlinearity as well as the linear part in the system response, it gives a more accurate approximation to the system output compared to the 1st-order model as can be seen clearly in Fig. 15. However, for the higher amplitude levels, limitations of the 2nd-order Volterra model are also evident.
NOISE EFFECTS IN KERNEL ESTIMATION
Although noise is not a primary issue in system identification using numerically generated data, it may be present to a significant extent in experimental studies and affect the kernel estimation adversely. Here we consider a SISO system for which a 2nd-order Volterra model representation is sought by estimating the Volterra kernels using random multi-tone inputs. In this process, even though pure multi-tone inputs are to be applied to the system directly, the actual inputs on the system are expected to contain some noise due to imperfections in the exciter. Furthermore, the measured output signals also contain some noise. Figure 16 is a schematic for the kernel estimation process applied to a physical system with independent noises n 1 (t ) and n 2 (t ) added to the input and output signals, respectively. We now examine the (independent) effects of additive noise in the input and output signals on the kernel estimation of a 2nd-order Volterra system. Our 2nd-order test system to be identified is where the coefficient a controls the relative linearity. In the frequency domain, the discrete system given by Eq. Table I gives the calculated errors in the estimated kernels of the 2nd-order Volterra series representation of Eq. (19) with the above a values for different N r and SNR when n 1 or n 2 is present in the system (without being applied simultaneously). Here, the errors are normalized as where E L and E Q are the (complex) differences between the estimated and the exact kernel functions, L and Q, and the domain of integration is the frequency domain in which the kernels are estimated. The four sections of the table correspond to the following cases, respectively: ðaÞ a ¼ 0:1; n 1 -0; n 2 ¼ 0; ðbÞ a ¼ 0:1; n 1 ¼ 0; n 2 -0; ðcÞ a ¼ 0:001; n 1 -0; n 2 ¼ 0; ðdÞ a ¼ 0:001; n 1 ¼ 0; n 2 -0: In Table I, we see that, for these four cases, as the SNR and the number of realizations increase, the errors in both kernels decrease regularly although the effect of increasing N r is quite weak. It is interesting to observe that the number of realizations needed for accurate estimation of kernels is quite high when the noise is present. This is obviously similar to the case of kernel estimation using random noise excitation described previously.
Here we can summarize our qualitative observations as follows: . The number of realizations needed for accurate estimation of kernels is quite high when the noise level is high. This is similar to the case of kernel estimation using random noise excitation described previously. . When the linear operator of the system is dominant, the linear kernel is estimated more accurately compared with the quadratic kernel. In this case, the accuracy of the estimated kernels are less for input noise relative to output noise at the same signal-to-noise-ratio (SNR) and N r . . When the system has a dominant quadratic nonlinearity, the estimation of the quadratic kernel is more accurate compared to the linear kernel. . Compared to output noise, input noise has a slightly more adverse effect on the accuracy of the estimated linear kernel. . For the same number of realizations, as compared to output noise, input noise decreases the accuracy in the quadratic kernel less when SNR is low, and more when SNR is high. . The accuracy of the quadratic kernel is not influenced by the relative strength of the linear operator when noise exists in the output.
Note that the above observations are valid when the identified system is actually a 2nd-order Volterra system.
FREQUENCY DOMAIN KERNEL ESTIMATION
If a system has higher-order nonlinearities, noise effects in the estimation of the kernels of a 2nd-order Volterra model will generally be different from those observed here, depending on the strength of higher order nonlinearities in the system input -output.
The methodology presented in this paper is well suited for numerical experiments. The fact that controlling inputs and observing output with high precision is essential in the present method raises questions about its applicability to physical experimental studies where noise can be an important issue. Naturally, it can be expected that the present method would give accurate and efficient estimations when the noise level is low. On the other hand, our study suggests that, when large amplitude noise is present in the input -output, considerably more realizations are needed in order to reduce the noise effect on the kernel estimation by ensemble averaging. In this case, the advantage of multi-tone inputs over random inputs diminishes drastically since both are expected to require a large number of realizations.
CONCLUSION AND DISCUSSION
In this work, we have presented an extension of the LSM approach using random multi-tone inputs for identification of the frequency domain kernels of a 2ndorder Volterra model. The present method employs a collection of independent multi-tones with random amplitudes and phases at the consecutive frequencies within a frequency range. Comparisons of the kernel estimations using random and random (amplitude and phase) multi-tone inputs for a simple test case (in the absence of noise) show that the latter gives considerably more accurate results. This is due to the fact the present method uses the steady-state responses to the multi-tone inputs and sampling the signals within their period.
The method has been successfully illustrated for a discrete SISO system for the voltage-current dynamics of a semi-conductor device (about an operating point) based on numerical solution of the hydrodynamic transport TABLE I Errors in the kernels of the 2nd-order Volterra model while noise is in input ðn 1 -0; n 2 ¼ 0Þ or noise in output ðn 1 ¼ 0; n 2 -0Þ for a ¼ 1 and a ¼ 0:001 cases (a) Noise in input signal (n 1 -0; Err. in Err. in L 0.97E þ 0 0.68E þ 0 0.81E-1 0.68E-2 Err. in Q 0.10E þ 0 0.10E-1 0.10E-2 0.11E-3 system of partial differential equations. It has given estimations for VTFs for regularly distributed frequency points on a discrete domain with a desired frequency resolution. Based on this experience, we found the present method very efficient, and therefore, promising for estimation of VTFs using realizations obtained from computer simulations of more challenging systems. The present method can also be extended to estimate cubic transfer functions for more accurate Volterra models. Note that the approach can be easily utilized with multiple realizations on parallel PC cluster systems or applied across a GRID composed of heterogeneous clusters. | 2018-04-03T03:28:33.427Z | 2002-12-01T00:00:00.000 | {
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2832630 | pes2o/s2orc | v3-fos-license | Near-Field Enhanced Plasmonic-Magnetic Bifunctional Nanotubes for Single Cell Bioanalysis
surface-enhanced Raman scattering (SERS). More than 2 × SERS enhancement is observed from the hollow section compared to the solid section of the same nanotube. The substantial SERS enhancement on the hollow section is attributed to the dual-sided coating of Ag NPs as well as the near-fi eld optical coupling of Ag NPs across the nanotube walls. Experimentation and mod-eling are carried out to understand the dependence of SERS enhancement on the NP sizes, junctions, and the near fi eld effects. By tuning the aspect ratio of the embedded nanomagnets, the magnetic anisotropy of nanotubes can be readily controlled to be parallel or vertical to the long directions for nano-manipulation. Leveraging the bifunctionality, a nanotube is magnetically maneuvered to a single living mammalian cell amidst many and its membrane composition is analyzed via SERS spectroscopy.
Introduction
Bifunctional plasmonic-magnetic nanoparticles (PM-NPs) are unique hybrid nanomaterials consisting of both optical and magnetic components in a rationally designed nanoscale architecture and have recently attracted intense research interest. [ 1 ] Possessing both enhanced optical and magnetic properties, PM-NPs can be extremely useful for biomedical applications that require either optical sensing/imaging/heating, magnetic stimulation/manipulation, or both functionalities. [ 1a , b , d , 2 ] For instance, PM-NPs can attach to biological entities such as cells and molecules to separate those entities under external magnetic fi elds and simultaneously detect their chemical nature via optical sensing. [ 3 ] The PM-NPs can also be deployed to study the mechanical properties of deeply embedded biological tissues by magnetic fi eld-induced mechanical stimulation [ 4 ] and monitoring the responses by in-situ optical imaging. [ 5 ] However, current available bifunctional PM nanostructures have been largely limited to quasi-zero-dimensional (0D) nanostructures, such as nanospheres and nanoshells. [ 1a-d , 2a ] To our knowledge, there are few reports on quasi-one-dimensional (1D) PM nanostructures, although 1D PM nanostructures provide exclusive advantages for biomedical applications that are unavailable for 0D nanostructures.
In this report, we present a unique type of 1D PM nanotubes and demonstrate their application in targeted singlecell sensing. The PM nanotubes consist of silica nanotubes with embedded solid Ni nanomagnets and uniformly dual-surface-distributed plasmonic Ag NPs. The PM nanotubes provide a high hotspot density (approximately 1200/ μ m 2 on the outer surface) at the junctions of Ag NPs for SERS biodetection. The solid embedded sections of nanotubes provide single-molecule sensitivity with an enhancement factor up to 7.2 × 10 9 . More than 2 × SERS enhancement was observed from the hollow sections than that from the solid section of the same nanotube. This substantial SERS enhancement is induced by the double sided coating of Ag NPs on the nanotubes as well as the near-fi eld optical coupling between Ag NPs on the inner and outer surfaces of the nanotubes. The dependence of SERS enhancement on the particle sizes, junctions, and the near fi eld effects was carried out by both experimentation and modeling. The magnetic anisotropy of the nanotubes, due to the embedded nanomagnets, can be readily tuned to be parallel or vertical to the long direction of the nanotubes for controlled manipulation. Leveraging the nanotubes' unique bifunctionality, we magnetically maneuvered a nanotube to a living Chinese hamster ovary cell and detected the membrane composition of the specifi c cell with SERS spectroscopy. These bifunctional nanotubes are desirable for multiple-task applications in single-cell bioanalysis, biochemical detection, imagingcontrast enhancement, magnetic manipulation and separation, and biosubstance delivery.
There are three outstanding features provided by the 1D PM nanotubes: First, the unique longitudinal geometry of nanotubes is compatible with both biological cells and biomolecules in terms of length and diameter. For instance, the lengths of magnetic anisotropies (thickness of 30 nm) can be readily embedded in the nanotubes (to be discussed) (Figure 1 d). On the entire outer surface of nanotubes, arrays of Ag NPs were uniformly distributed (Figure 1 e). The Ag NPs also grew on the interior surfaces of the nanotubes as shown in the SEM images obtained by cross-sectional focused ion beam (FIB) milling (Figure 1 f). The Ag NPs were semi-spherical and densely arranged, yet overlapping NPs were rarely found.
The Ag NP sizes and junctions can be optimized for highly sensitive SERS detection. With fi xed volume of nanowire suspension (5.7 × 10 8 /mL, 400 μ L) and PVP (10 mL of 2.5 × 10 − 5 M in ethanol), we systematically varied the total volume of AgNO 3 (0.06 M) and NH 3 · H 2 O (0.12 M) (v:v 1: 1) from 20 μ L to 1200 μ L, i.e. 20 μ L (0.2 × sample), 600 μ L (6 × sample), 800 μ L (8 × sample), and 1200 μ L (12 × sample). The morphologies of the as-synthesized nanotubes showed distinctive particle and junction sizes (Supporting Information Figure S2). The average diameters of the Ag NPs increased from 10.2 ± 2.4 nm (0.2 × sample) to 24.8 ± 6.7 nm (8 × sample) with the volume of the AgNO 3 /NH 3 · H 2 O solution. The density of Ag NPs reached a maximum in the 6 × sample (2050/ μ m 2 ). The detailed characterization is given in Supporting Information nanotubes can be adjusted to tens of micrometers for efficient attachment, manipulation, and separation of cells. [ 6 ] The nanoscale diameters restrict the number of molecules that one nanotube can interact with, which is important for moleculelevel biosensing and drug delivery. [ 7 ] Second, substantially enhanced plasmonic properties are sensitively obtained in the nanotubes. The plasmonic NPs coated on the entire surfaces of the nanotubes provide large and uniform SERS EFs, similar to those frequently reported in patterned substrates, [ 8 ] which are provided by traditional 0D plasmonic NPs or their aggregates. Third, by controlling the aspect ratio of the embedded Ni nanosegment, the magnetic moment and anisotropy can be facilely tuned to the desired value, which is important for effi cient magnetic separation and manipulation. [ 9 ] 2. Results and Discussion
Design and Fabrication
In order to synthesize such PM nanotubes, a rationally designed four-step approach has been used: 1) multi-segment Ag/Ni/ Ag (3/3/3 μ m) nanowires were electrodeposited as growth templates for silica nanotubes ( Scheme 1 a and Figure 1 a); 2) a layer of silica, with controlled thickness of 70 nm, was uniformly plated on the outer surface of the Ag/Ni/Ag nanowires (Scheme 1 b) via hydrolysis of tetraethyl orthosilicate; [ 10 ] 3) the Ag segments were selectively etched, resulting in the hollow silica nanotubes with magnetic Ni embedment (Scheme 1 c and Figure 1 b); 4) plasmonic Ag NPs were uniformly coated through PVP assisted catalysis [ 11 ] on the inner and outer surfaces of the nanotubes with optimized particles and junction sizes (Scheme 1 d and Figure 1 c,e). For detailed fabrication process, see experimental section.
Scanning electron microscopy (SEM) images show that hollow nanotubes with embedded cylindrical solid segments were successfully synthesized (Figure 1 c). Energy dispersive X-ray spectroscopy (EDS) confi rmed that the cylindrical solid was Ni (Supporting Information Figure S1). By using the same method, multiple Ni nanodisks with controlled increased with the concentration of R6G (Figure 2 a,b from the 8 × sample). As low as 100 fM R6G can be decisively determined with a signal-to-noise ratio of 7.2 (Figure 2 c). The SERS EF values were estimated by following a well-established approach. [ 14 ] The highest EF of 7.2 × 10 9 was obtained from the 8 × sample on the Ni embedded sections (Supporting Information Figure S6). According to bianalyte studies, an EF value of above 10 9 is suffi cient for single-molecule sensing of various biochemical species. [ 3,15 ] The sensitivity of our nanotubes is already on the single molecule levels.
Interestingly, we also noticed that Ag NPs not only grew on the outer surface of the nanotube, but also on the interior surface of the nanotube as shown in the SEM images of crosssections obtained by focused ion beam (FIB) milling (Figure 1 f). Therefore, there were double-layer Ag NPs on the hollow parts of the PM nanotubes and single-layer Ag NPs on the solid part of the nanotubes (Scheme 1 inset). Due to characterization diffi culties, we cannot directly measure the size and distribution of the inner layer Ag NPs. However, the effect of the inner surface coating of Ag NPs, can be known from SERS measurement. When a laser beam scanned along a single nanotube with 70 nm thick silica coating, the hollow segments of nanotubes with double-layer Ag NPs exhibited more than two-time SERS intensity than those from single-layer Ag NPs on the outer surface of the Ni embedded section of nanotubes (Figure 2 g). Similar results were also observed when we replaced the Ni segment with Pt (Figure 2 h). The experimental details are provided in Supporting Information Figure S5.
Plasmonic Simulation
It is interesting to understand how the EF depends on Ag particle and junction sizes, as well as how the Ag NPs on the Figure S3, S4 and S7. The variation of the particle sizes may be attributed to the dynamic competition between nucleation and crystalline growth of Ag NPs, which has been commonly observed in NP growth. [ 12 ]
SERS Characterization
The SERS performance of the nanotubes were characterized and understood by experimentation and numerical simulation. It is known that a laser-beam focused on two closely neighboring Ag NPs can generate high-intensity electric ( E ) fi eld in the narrow junction (a few nm) due to localized surface plasmonic resonance as a result of coherent electron oscillation in the Ag NPs. The junction with enhanced E -fi eld is often referred as "hotspot". If a molecule is in the vicinity of a hotspot, its Raman scattering signals can be signifi cantly amplifi ed with | E | 4 dependence. This phenomenon is the so-called surface enhanced Raman scattering (SERS). [ 13 ] SERS EF is largely determined by E -fi eld intensity and thus the sizes of junctions. If we only consider extremely narrow junctions ( < 2 nm) on the nanotubes, which contribute most to SERS enhancement, the density of hotspots on the outer surface of nanotubes went up from ≈ 0/ μ m 2 for the 0.2 × sample to the maximum of 1200/ μ m 2 for the 8 × sample (Supporting Information Figure S6). The corresponding average hotspot sizes can also be determined. The 8 × samples provided the smallest average size of hotspots (1.16 nm) and the 6 × sample gave the largest (1.4 nm) as shown in Figure 2 d and Supporting Information Figure S6.
To characterize the SERS performance of nanotubes, we employed standard Raman probes including Rhodamine 6G (R6G) (1 μ M to 1 fM) and 1,2-bi-(4-pyridyl) ethylene (BPE) (1 mM). All of the nanotubes can detect R6G and BPE molecules except the 0.2 × sample (Figure 2 a,e). SERS intensity It is known that the SERS enhancement can be attributed to two factors: E -fi eld enhancement due to the plasmonic resonance of NPs and chemical enhancement due to charge transfer between the molecules and metal particles. [ 3 ] The E -fi eld enhancement can be approximated as where ω L is the resonant angular velocity of the local fi eld E in an external fi eld of E 0 . Due to the quadruple dependence on the local E fi eld, the E -fi eld enhancement is usually considered to be a major contributor to SERS. We used a 3D fi nite difference time domain (FDTD) method to simulate this effect. We simulated the normalized electric fi eld ( | E | / | E 0 | ) close to the Ag NPs. The excitation wavelength was set at 532 nm with inplane polarization perpendicular to the nanotube surface. To fi nd the effect of NP junctions on SERS EF, we began with the simulation for a single particle, a dimer, and then a 4 × 4 array ( Figure 3 a-c, the shape of Ag NPs was assumed to be cylindrical with a uniform thickness of 20 nm. The junction was set at 3 nm). A single silver NP provided electric fi eld enhancement of | E | / | E 0 | = 2.3, hence the SERS EF = 28 due to the E 4 dependence; metal dimers and a 4 × 4 array provided | E | / | E 0 | = 8.6 (EF SERS = 5470) and | E | / | E 0 | = 9.1 (EF SERS = 6857), respectively. This simulation confi rms that a group of metallic particles on the same surface can provide stronger E fi eld enhancement than that of single particles or dimers.
A group of NPs with controlled distributions should further enhance the E fi eld. Therefore we simulated the E -fi eld enhancement depending on junctions of the particle arrays. As plotted in Figure 3 d, narrower junctions between NPs exhibit a strong electric fi eld for an array with Ag NPs of 25 nm in diameter. At a fi xed junction of 1 nm, the highest electric fi eld enhancement is obtained in particle arrays with diameters between 20-50 nm as shown in Figure 3 e. The simulation results excellently agree with previous work, which refers to such results as proof of the "extrinsic size effect". [ 16 ] For NP with larger sizes ( > 40 nm), the E -fi eld enhancement reduces because the plasmonic resonance shifts to longer wavelength. When such NPs are excited by short-wavelength lasers, they induce higher-order electron cloud distortion of conduction electrons and thus degrade the plasmonic resonance. For NPs that are less than 25 nm, scattering of electrons from the particle's surfaces produces a damping term that is inversely proportional to the particle diameter. This means that more optical energy is converted to heat instead of being scattered to produce local fi eld enhancement. As a result, by comparing the electric fi elds among NPs with different diameters and junctions, we have optimized the design of the Ag NPs. The result of simulation readily explained our experimental observation: among all the samples, the 8 × samples with the largest diameters (24.8 ± 6.7 nm) and smallest gaps ( ≈ 1.16 nm) offered the highest SERS EF. To understand how the dual-side-Ag-coated hollow nanotubes can further enhance SERS than the single-side-Ag-coated nanotubes with solid embedment do, we carried out numerical simulations by Comsol 3.5a RF module. In our modeling, a 3D silica nanotube is constructed (illustrated in Scheme 1 d and insets): the inner cylinder radius is 150 nm, and the shell thickness is 70 nm. The densely coated Ag NPs are simplifi ed by a 2D conformal array attached to the outer and inner surfaces of the silica nanotubes. The Ag NP diameter is 25 nm and the gap between them is 2 nm. The silica nanotube is placed on top of a glass substrate, and is excited by a surface normal Gaussian beam with beam diameter of 1 μ m at 532 nm wavelength. The polarization direction is perpendicular to the axis of the cylinder. Table 1 lists our simulation results corresponding to our experimentally measured devices and Figure 4 shows the cross sectional views of the electric fi eld distribution of the four devices as listed in Table 1 .
In Figure 4 a, we surprisingly fi nd that the hot-spot with maximum electric fi eld enhancement is actually at the bottom of the nanotube, not at the top. This is because the bottom hot-spot is surrounded by high-index silica; while the other hot-spots have only one side contacted with silica (other side is exposed to low-index air). Additionally, the inter-particle coupling through the NP chain at the outer surface enhances the electric fi eld at the bottom. [ 17 ] Comparing Figure 4 a,b, we are able to clearly conclude the contribution of inner layer Ag NPs: the presence of inner layer not only adds more hot spots for SERS sensing, but also signifi cantly increase the intensity of Moreover, we noted that the thickness of silica also affects the enhancement of SERS. We observed near-fi eld enhancement effect on nanotubes with silica coating ranging from 70 to 150 nm. However, when the thickness of silica was increased to 300 nm, the near-fi eld enhancement effect was not observed, which may be attributed to the reduced plasmonic coupling between Ag NPs across the silica shell.
Magnetic Characterization
Not only plasmonically sensitive, the unique nanotubes also offered tunable magnetic properties for controlled manipulation. The magnetic anisotropy of Ni segment is dominated by its shape anisotropy [ 9 ] as opposed to its weak crystalline anisotropy. When the aspect ratio of Ni segment is high, e.g. 10/1 (Ni length 3 um, diameter 300 nm), the anisotropy direction and the easy axis is along the nanotube long axis as measured by vibrating sample magnetometry (VSM) ( Figure 5 a). A hysteresis loop along magnetic easy axis demonstrates higher magnetic remanence and squareness than those measured perpendicular to the nanotubes. When the aspect ratio of the Ni segments is below 1, e.g., a stack of thin Ni disks with diameters of 300 nm and thickness of 30 nm, magnetic anisotropy is generally transverse to the nanotubes with essentially zero remanence due to the anti-parallel coupling of the magnetizations in neighboring nanodisks (Figure 5 b). This fascinating way of controlling the magnetic anisotropy has been vividly demonstrated by manipulating nanotubes in suspension with a magnetic fi eld. As shown in Figure 5 , nanotubes with magnetic anisotropy along the long axis align with the magnetic fi eld during transport, but those with transverse magnetic anisotropy align perpendicular to the magnetic fi eld. The transport speed was 2-7 μ m/s, which can be controlled by the magnetic fi eld gradient.
Single-Cell Bioanalysis
Although single complex biological samples can be investigated with standard Raman microscopy, a detailed investigation of specifi c components on the cell surface is not possible with this approach. [ 19 ] In our work, we demonstrated the utility of the bifunctional nanotubes in revealing the the hot spots in the outer layer NPs, which is due to the nearfi eld effect [ 18 ] of inter-layer coupling between NP chains in the inner and outer surfaces of the nanotubes. When the air core is fi lled with Pt, which has no surface plasmonic resonance at visible wavelength, we observed interesting phenomena: fi rst, the EF of the hot-spots on top of the nanotube is enhanced roughly 2 × , which is due to the refl ected light from the Pt core; second, the hot-spots at the bottom of the nanotube, however, is signifi cantly reduced because almost no light can penetrate the Pt core to excite the surface plasmons. And moreover, the interparticle coupling through the NP chain can be weakened by evanescent fi eld absorption of Pt. Similar electric fi eld enhancement is observed in Ni-fi lled nanotube as well.
If we assume there are enough molecules so that every hotspot can contribute to SERS measurement, we can calculate the total SERS signals by ∑ | E i | 4 , where E i is the electric fi eld in each hot spot. The total SERS signals of these four devices are 0.8 × 10 5 , 2.5 × 10 6 , 1.4 × 10 5 and 1.3 × 10 5 a.u., respectively. It is seen that device 2 will be able to provide more than 10 × higher SERS signals than device 3 and 4. However, in reality, the SERS signals from hot-spots at the bottom to the nanotubes are more diffi cult to collect due to NP scattering. Experimental we obtained more than 2 × differences (Figure 2 g,h). The comparison of single-side and dual-side Ag NP coated segments in the nanotubes is summarized in Supporting Information Figure S8. membrane composition of a single Chinese hamster ovary (CHO) cell amidst many. We chose CHO cells because they are widely used in biological research, especially in studies of genetics, toxicity screening, gene expression, and expression of recombinan proteins. Here, leveraging the unique bifunctionality of the nanotubes, we can precisely transport a nanotube to a specifi c living CHO cell (see Experimental Section) amidst many and detected its membrane chemistry with SERS spectroscopy. A PM nanotube was transported and aligned in the direction of the magnetic fi eld and precisely landed on the membrane of a CHO cell (overlapped images in Figure 6 a,b and Supporting Information Video S9). From SERS spectra (Figure 6 c), which was taken from the nanotube with an integration time of 5 s, shows strong characteristic peaks of lipids. [ 7 , 19 , 20 ] The peak position 1511 cm − 1 can be assigned to amide II, which is from protein (blue bar in Figure 6 c). This results revealed that the cell membrane in contact with the nanotube consists mostly lipids and some protein molecules, which is consistent with real cell membrane composition. [ 19 ] Without nanotubes, no Raman signals can be detected from the cell, this clearly demonstrating the highly desirable bifunctionality of the nanotubes for precision and ultrasensitive single-cell bio-analysis. This technique is generally applicable to any adhesive live cells. It can be readily applied to hamster cells as well as to mouse or human cells.
Conclusions
In summary, we have successfully designed and synthesized a unique type of near-fi eld enhanced bifunctional PM-active nanostructure that consists of a hosting silica nanotube, a magnetic segment embedded within the nanotube, and Ag NPs uniformly coated on the dual surfaces of the nanotube. By controlling the fabrication conditions, both the diameter and junction of Ag NPs can be precisely controlled for ultrasensitive molecular sensing. The 3D FDTD simulation of E -fi eld enhancement agrees with the experimental results. Higher SERS intensity is found on hollow than the solid parts of the PM nanotubes, and it is confi rmed to be from the near fi eld coupling between the inner and outer layer of Ag NPs. The embedded nanomagnets with tunable magnetic anisotropy allow fl exible manipulation of the nanotubes with external magnetic fi elds. Such bifunctional nanostructures can be transported to a living Chinese hamster ovary cell amidst many other cells to reveal the membrane composition. The PM nanotubes are suitable for single-cell bioanalysis as well as various biological applications, such as biochemical sensing, magnetic manipulation, separation, MRI contrast, and biosubstance delivery.
Experimental Section
Fabrication of Plasmonic-Magnetic Nanotubes : A series of strategies was implemented to synthesize the unique bifunctional nanotubes using the above approach. In brief, the multisegment Ag/Ni/Ag nanowires were fabricated by electrodeposition in nanoporous anodized aluminum oxide (AAO) templates as reported elsewhere. [ 11,21 ] In brief, a Cu layer of about 500 nm in thickness was thermal evaporated onto the back of the template to seal the pores and serve as the working electrode in a three-electrode electrodeposition system. The electrodeposition of metal materials gradually fi ll the bottom of the nanopores working electrode to form nanowires. Finally, the AAO template was dissolved in 2 M NaOH solution to release the free-standing nanowires. The amount of electric charge passing through the circuit controls the length of nanowires to 7 nm. [ 22 ] The pore size of nanoporous template controls the diameters of the nanowires from 20 to 400 nm, with different compositions along the lengths (e.g., Ag/Ni/Ag nanowires) (Figure 1 a), and here we prepared 300 nm diameter 3/3/3 μ m Ag/Ni/Ag nanowires ( − 1.1 V for Ag and − 1 V for Ni).
Next, the Ag/Ni/Ag nanowires were used as templates for fabricating Ni-embedded silica nanotubes. We fi rst coated an amorphous silica layer on the surface of the Ag/Ni/Ag nanowires. Silica was used due to its porous structure with a high surface area, biocompatible properties, and drug carrier capability. [ 10a ] Here, it also serves as a support substrate for plasmonic Ag NPs. The reaction was accomplished by hydrolysis of tetraethyl orthosilicate for 2-5 h with a controlled thickness of a few to hundreds of nanometers. [ 10 ] Next, the Ag segments were selectively etched with a mixture (4:1:1) of methanol (99%), hydrogen peroxide (30%), and ammonia hydroxide (28-30% as NH 3 ), which result in Ni-embedded nanotubes as shown in Figure 1 b.
Finally, arrays of plasmonic Ag NPs were uniformly synthesized on the surface of silica nanotubes by reduction of Ag ions with PVP (10 mL, 2.5 × 10 − 5 M in ethanol) from a mixed solution of silver nitrate (0.06 M, 400 μ L) and ammonia hydroxide (0.12 M, 400 μ L) at 70 ° C for 7 h.
Cell Culture and Reagents : CHO cells (ATCC) were cultured in RPMI medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). Cells were maintained in a humidifi ed 37 ° C, 5% CO 2 incubator. Before the experiment being conducted, CHO cells were washed by phosphate buffered saline (PBS) followed by addition of nanotubes (dispersed in PBS).
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author. Figure 6 . a,b) Transport a nanotube to a single CHO cell amidst many. c) SERS spectrum from the CHO cell membrane is dominated by lipid contribution, and a few peaks can be assigned to protein (blue bar). | 2016-02-20T08:33:50.931Z | 2013-09-20T00:00:00.000 | {
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231607397 | pes2o/s2orc | v3-fos-license | Identifying Candidate Genes Involved in the Regulation of Early Growth Using Full-Length Transcriptome and RNA-Seq Analyses of Frontal and Parietal Bones and Vertebral Bones in Bighead Carp (Hypophthalmichthys nobilis)
Growth, one of the most important traits monitored in domestic animals, is essentially associated with bone development. To date, no large-scale transcriptome studies investigating bone development in bighead carp have been reported. In this study, we applied Isoform-sequencing technology to uncover the entire transcriptomic landscape of the bighead carp (Hypophthalmichthys nobilis) in early growth stage, and obtained 63,873 non-redundant transcripts, 20,907 long non-coding RNAs, and 1,579 transcription factors. A total of 381 alternative splicing events were seen in the frontal and parietal bones with another 784 events simultaneously observed in the vertebral bones. Coupling this to RNA sequencing (RNA-seq) data, we identified 27 differentially expressed unigenes (DEGs) in the frontal and parietal bones and 45 DEGs in the vertebral bones in the fast-growing group of fish, when compared to the slow-growing group of fish. Finally, 15 key pathways and 20 key DEGs were identified and found to be involved in regulation of early growth such as energy metabolism, immune function, and cytoskeleton function and important cellular pathways such as the arginine and proline metabolic pathway (p4ha1), FoxO signaling pathway (sgk1), cell adhesion molecules (b2m, ptprc, and mhcII), and peroxisome proliferator-activated receptor signaling pathway (scd). We established a novel full-length transcriptome resource and combined it with RNA-seq to elucidate the mechanism of genetic regulation of differential growth in bighead carp. The key DEGs identified in this study could fuel further studies investigating associations between growth and bone development and serve as a source of potential candidate genes for marker-assisted breeding programs.
Identifying Candidate Genes Involved in the Regulation of Early Growth Using Full-Length Transcriptome and RNA-Seq Analyses of Frontal and Parietal Bones and Vertebral Bones in Bighead Carp (Hypophthalmichthys nobilis)
INTRODUCTION
Growth is one of the most economically important traits monitored in domestic animals and therefore is the main objective of most genetic selection programs. Growth rates of aquatic animals, as well as of most organisms, are regulated by multiple genetic and environmental factors (Cleveland et al., 2020). Over the past few decades, the genetic basis of growth in aquatic species has been widely studied. Genetic linkage analyses for genes regulating growth in aquatic animals have been performed using DNA-based molecular markers such as amplified fragment length polymorphism and simple sequence repeat (SSR) (Liu and Cordes, 2004;Yue, 2014). With the development of next-generation sequencing technology, quantitative trait loci (QTL) mapping and genomewide association study have allowed for the identification of genetic markers associated with the traits of interest. Additionally, transcriptome analyses can be used to identify biomarkers and candidate genes regulating growth. A total of 92 differentially expressed genes (DEGs) were identified between fast-growth and slow-growth families of blunt snout bream (Megalobrama amblycephala) in transcriptomes of the liver and gill (Li et al., 2015). Cleveland et al. (2020) compared liver and muscle transcriptomes of a rainbow trout (Oncorhynchus mykiss) line, selectively bred for fast growth, to that of a contemporary randomly mated control line and identified 145 and 36 DEGs in the liver and white muscle transcriptomes, respectively. Our laboratory has also identified 173 DEGs in the hypothalamus-pituitary and 204 DEGs in the liver of bighead carp with differential growth rates (Fu B. et al., 2019). However, these studies have mainly focused on gene expression patterns in the muscle, liver, and brain tissues. Growth, however, is essentially associated with skeleton and bone development and leads to consequent increases in body weight (BW) and body length (BL). The frontal and parietal bones are important components of the skull in vertebrates, serving to protect the brain and permit skull expansion during development (Quarto et al., 2009;Topczewska et al., 2016). The presence of vertebral bone discriminates vertebrate organisms from invertebrates. It supports the BW of the animals and generates force in the muscles for locomotion and physiological activities (Ibaraki et al., 2015). To our knowledge, no large-scale transcriptomic study investigating the differential expression of genes, regulating differential growth rates, in the bones of bighead carp has been previously reported.
Bighead carp (Hypophthalmichthys nobilis), one of the most important fish in Asia, improves the water quality for human consumption and thus has been introduced in many non-Asian countries (Szabo et al., 2019). Principally produced in China, the global production of bighead carp reached 3.1 million tons in 2017 (FAO). The size of the head in aquaculture fish species is important not only for understanding evolution and biological adaptation but also for predicting the economic value, as it directly affects filet yield (Geng et al., 2016). For most aquaculture fish species, smaller head and uniform body conformations provide a greater proportion of carcass yield; thus, selection for smaller head and streamline body conformation is of great value in aquaculture (Rutten et al., 2005). However, the bigger head of bighead carp harvest fish is more popular among Chinese consumers and is used as a source of nutrition in soups (fish head and tofu soup) or delicious dishes (steamed fish head with diced hot red peppers and so on). Therefore, to meet the demands of markets and farmers, selection for fast-growing and bigheaded varieties of bighead carp is prevalent among Chinese breeding programs.
Transcriptomic analyses, widely used in genetic research, uncover the type and expression levels of transcripts and identify physiological and biochemical processes that regulate transcription (Jia et al., 2018). However, owing to technical limitation, obtaining the full-length transcripts and quantifying the isoforms are not possible with RNA sequencing (RNA-seq) (Steijger et al., 2013). Thus, single-molecule real-time (SMRT) sequencing (Pacific Biosciences of California, Inc., Menlo Park, CA, United States) has been developed (Korlach et al., 2010), and it provides a third-generation sequencing platform that is widely used to sequence genomes by generating kilobase-sized sequencing reads (Chaisson et al., 2015;Gong et al., 2018). Moreover, recent studies have reduced the high error rate (up to 15%) of SMRT sequencing by self-correction, via circularconsensus sequencing reads (Li et al., 2014) and validation with high-throughput and high-accuracy short-read data (Au et al., 2013). SMRT sequencing comprehensively analyses splice isoforms of each gene and improves the annotation of existing model organisms by producing full-length transcripts (Tilgner et al., 2014;Wang et al., 2016;Mehjabin et al., 2019). In this study, we applied SMRT sequencing to uncover post-transcriptional regulatory events in bighead carp and coupled it with bone RNA-seq to investigate the genetic regulation of early growth at the transcript level. This is the first study to report the application of SMRT sequencing in bone tissues and provides a comprehensive view of transcriptome complexity in bighead carp at the early growth stage. Furthermore, we identified a set of differentially expressed unigenes (DEGs) from bone development and body growth-related signaling pathways, which potentially services as valuable resources for future molecular breeding studies.
Morphological Measurement
In total, six bighead carp individuals in juvenile stage, including three fast-growing [big group, (BG)] and three slow-growing [small group (SG)] fish, were selected for transcriptome analysis in this study. The BL, head length (HL), head height (HH), head width (HW), and BW were measured ( Table 1). The ratios of HL and BL (HL/BL) from the BG and SG were 0.3239 and 0.3533, respectively. The average BLs for BG and SG were 21.30 and 16.13 cm, respectively. BL from BG was significantly larger than that from SG (p < 0.05). Other phenotypic parameters from BG were significantly larger than those from SG, including HL, HH, HW, and BW (p < 0.05). 16 terms in the molecular function (MF) category, binding (19,616 transcripts), catalytic activity (11,868 transcripts), and transporter activity (1,940 transcripts) had the highest number of transcripts. Three putative growth-related GO terms, including growth (involving 565 transcripts), immune system process (involving 1,931 transcripts), and metabolic process (involving 13,193 transcripts), were successfully annotated. A total of 69.48% annotated transcripts (30,394 of 43,747 transcripts) were annotated in the KOG database, which can be assigned to 25 categories (Supplementary Figure 1). The largest number of functional categories was signal transduction mechanisms reaching 6,286 transcripts. The following four functional categories were general function prediction only (5,947 transcripts), post-translational modification, protein turnover, chaperones (2,882 transcripts), transcription (2,325 transcripts), and function unknown (2,240 transcripts), respectively.
In the KEGG classification, a total of 279 pathways annotated from 25,955 non-redundant transcripts were extracted from the bone transcriptome of bighead carp. The results showed that endocytosis (880 transcripts), mitogen-activated protein kinase (MAPK) signaling pathway (753 transcripts), focal adhesion (723 transcripts), regulation of actin cytoskeleton (709 transcripts), and herpes simplex infection (489 transcripts) were the top five pathways with the most abundant unigenes. Notably, we paid attention to 25 KEGG pathways (Figure 2), which may be closely associated with differential growth and involved in the physiological functions of immune, metabolism, and cytoskeleton of bighead carp, such as insulin signaling pathway, cytokine-cytokine receptor interaction, peroxisome proliferatoractivated receptor (PPAR) signaling pathway, transforming growth factor β (TGF-β) signaling pathway, and regulation of actin cytoskeleton. Among these 25 KEGG pathways, endocytosis (880 unigenes), MAPK signaling pathway (753 unigenes), and regulation of actin cytoskeleton (709 unigenes) were the top three pathways with the most abundant unigenes, which provided some reference value for studying the early growth regulation and bone development in bighead carp.
Prediction of Gene Families and Coding Sequences
We utilized the Coding Genome Reconstruction Tool (COGENT) to further partition these non-redundant transcripts
De novo Detection of Alternative Splicing Events and Long Non-coding RNA Prediction
A total of 381 and 784 pairs of FL transcripts from F01 and F02 that might represent alternative splicing (AS) events were detected (Supplementary Table 3). Additionally, because no reference genome was available for SMRT sequencing of transcriptome in bighead carp, we could not determine the types of AS events.
Identification of Transcription Factors and SSRs
In our Iso-Seq, a total of 1,579 non-redundant transcription factors (TF) transcripts were identified, and their detailed information is shown in Supplementary Table 4. Based on the Animal TFDB 2.0 database classification, these TFs belong to more than 56 families, and a large number of TFs were dominant in zf-C2H2, miscellaneous, and TF_bZIP (Supplementary Figure 3). It was the first time to extensively identify TFs using transcriptome dataset in bighead carp, which provided a useful foundation for TFs studies in the future.
Simple sequence repeats, also known as microsatellite DNAs, have a tandem repeat motif of 1-6 bp in length. The characters of high polymorphism (mainly due to the differences in the number of tandem motifs), stability, and reliability for SSR enable it to be an ideal molecular marker that is widely used in genetic map construction, QTL mapping, and genetic diversity assessment. We searched for SSRs in the 50,694 bighead carp transcripts longer than 500 bp, and a total of 53,508 SSRs were identified, with 13,270 transcripts containing more than one SSR. Most of the SSRs identified were mononucleotide repeats (31,758, 59.35%), followed by the dinucleotide repeats (15,540, 29.04%), trinucleotide repeats (4,989, 9.32%), tetranucleotide repeats (1,009, 1.89%), hexanucleotide repeats (199, 0.37%), and pentanucleotide repeats (13, 0.02%). Among the 53,508 SSRs, 9,085 SSRs presented in compound formation. All SSRs and their primers are listed in Supplementary Table 5.
Identification of DEGs From Bone Tissues
To capture transcript-level expression differences related to different growth rates of bighead carp, the Illumina RNA-seq data of bone tissues, including the frontal and parietal bone and vertebra tissues, were aligned to the refined full-length transcripts of the Iso-Seq database for quantification. The DEGs of the frontal and parietal bone and vertebra tissues between the big and SGs were explored by using DEseq2 with the criteria of | log2 ratio| ≥ 1 and FDR (false discovery rate) < 0.01. Supplementary Table 6 was used as an input file to run DESeq2. In total, the numbers of DEGs from the frontal and parietal bone and vertebra tissues between BG and SG were as follows: 27 from the frontal and parietal bone tissues (15 up-and 12 down-regulated in BG) and 45 from vertebra tissues (24 upand 21 down-regulated in BG). When we compared DEGs from bone tissues, 12 DEGs were shared in the frontal and parietal bones and vertebral bones between BG and SG, which were mainly associated with metabolic process, biological regulation, and immune system process, such as Golgi apparatus protein 1 (glg1), serine/threonine-protein kinase Sgk1(sgk1), and β2microglobulin (b2m).
To elucidate the biological events of the DEGs from the frontal and parietal bone and vertebra tissues, which would be mainly involved in different growth rates of bighead carp at early growth stage, GO term enrichment analyses were conducted. First, GO terms were obtained on the basis of numbers of the DEGs assigned to each GO term (Figure 4). The result was as follows: Under the CC category in both the frontal and parietal bone and vertebra tissues, cell (GO: 0005623) and cell part (GO: 0044464) had the most abundant GO function items. Within the biological process (BP) category in both the frontal and parietal bone and vertebra tissues, higher percentages of genes were commonly clustered into cellular process (GO: 0009987), biological regulation (GO: 0065007), and metabolic process (GO: 0008152). In the MF category in both the frontal and parietal bone and vertebra tissues, most genes were assigned to binding (GO: 0005488), catalytic activity (GO: 0003824), and MF regulator (GO: 0003674). Second, GO terms of the DEGs were also identified according to top three p values of CC, BP, and MF categories, respectively. Under the CC category, the top three GO terms in both the frontal and parietal bone and vertebra tissues were extracellular exosome were the top three GO terms of MF category in both the frontal and parietal bone and vertebra tissues. These common GO terms of DEGs from these two tissues might suggest the frontal and parietal bone, and vertebra tissues played roles in differential growth of bighead carp cooperatively.
Kyoto Encyclopedia of Genes and Genomes pathwaybased analyses help to identify the biological pathways that are related to DEGs. Pathway enrichment analyses identified the enriched pathways from frontal and parietal bone and vertebra tissues, respectively ( Figure 5). Among these pathways, metabolic pathways were the most frequently represented pathways in bone tissues of bighead carp with different growth rates, followed by organismal systems, environmental information processing, and genetic information processing pathways. These observations disclosed the vital implications of energy metabolism, cytoskeleton, and immune regulation in bone tissues between BG and SG of bighead carp, such as citrate cycle (TCA cycle) [ko00020, q = 1 (frontal and parietal bone)/q = 1(vertebra)], regulation of actin cytoskeleton [ko04810, q = 1 (vertebra)], and cell adhesion molecules (CAMs) [ko04514, q = 0.00761 (frontal and parietal bone)/q = 0.12807(vertebra)] pathways. These enriched pathways were not all significant because the number of DEGs was too few, which were considered as potential pathways associated with differential growth of bighead carp at early growth stage.
Key DEGs Related to Differential Growth
In total, 15 key pathways associated with the early growth regulation of bighead carp were identified from frontal and parietal bone and vertebra tissues (Table 3), which played important roles in the physiological functions of energy metabolism, cytoskeleton, and immune regulation. Twenty key DEGs (Figure 6) were selected by three strategies simultaneously, which were considered to be related to the differential growth of bighead carp: (i) 10 important DEGs that showed high participation frequencies in the 15 key pathways; (ii) another seven important DEGs that appeared more than two times in biological regulation, immune system process, and metabolic process of GO term enrichment analyses; (iii) three important DEGs that were commonly identified in frontal and parietal bone and vertebra tissues. Then, these 20 key DEGs were used to construct the correlation network by GeneMANIA (Figure 6).
Validation of DEGs by Quantitative Real-Time Polymerase Chain Reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 12 selected genes and the internal control gene βactin (Supplementary Table 7), to validate the DEGs identified in bone tissues of bighead carp by RNA-seq. Fold changes from qRT-PCR were compared with the RNA-seq expression profiles (Figure 7). The expression patterns revealed by qRT-PCR analysis were similar to those revealed by RNA-seq for the same gene. Thus, RNA-seq could provide reliable data for mRNA differential expression analyses.
DISCUSSION
Transcriptome reconstruction and annotation have significantly improved with the advent of new sequencing technologies. These techniques play an important role in gene discovery, genome annotation, and detection of genomic signatures, particularly in species without a reference genome (Li et al., 2017). In recent years, traditional short-read RNA-seq has been commonly used to investigate RNA expression patterns in several tissues such as that of the brain, liver, and muscle Cleveland et al., 2020). However, short-read RNA-seq has certain limitations in regard to precise reconstruction and reliable sequencing of isoforms due to the complexity of AS mechanisms in eukaryotes (Tilgner et al., 2013;Yi et al., 2018). In contrast, SMRT sequencing is a superior strategy, directly generating a comprehensive transcriptome with accurate sequencing of AS isoforms and novel genes, and its advantages have been extensively documented in previous studies. In this study, the full-length transcriptome of bighead carp was performed using the Iso-Seq technique.
Previously reported transcriptomic studies of the bighead carp were performed using Illumina sequencing platforms (Fu B. et al., 2019;Li et al., 2019). In comparison, our study produced a comprehensive transcriptome with several features. First, accurate full-length transcripts (63,872 transcripts) were generated, serving as a valuable resource for various gene structures and sequences, which can be directly used in genefunction studies without using additional gene clones. Second, a total of 20,907 lncRNAs were identified, which could be useful for investigations of potential lncRNA functions in the bighead carp. Third, the full-length transcripts could serve as a reference for genome assembly and gene annotation of bighead carp. Finally, reliable ORFs identified in this study are essential for the identification of orthologous genes, aiding in the better understanding of culture and breeding techniques used for bighead carp.
A total of 279 pathways were annotated from 25,955 nonredundant transcripts extracted from the bone transcriptome of the bighead carp, using the KEGG classification system. Notably, we focused on 25 KEGG pathways (Figure 2), which are potentially associated with regulation of early growth and are involved in physiological functions of the immune system, metabolism, and cytoskeleton of bighead carp. These KEGG pathways included the insulin signaling pathway, cytokine-cytokine receptor interaction, PPAR signaling pathway, TGF-β signaling pathway, Wnt signaling pathway, and regulation of actin cytoskeleton and were also the focus of previous reports on growth and bone development (Nie et al., 2019;Cleveland et al., 2020), suggesting that they play important roles in growth modulation and bone development of bighead carp at early growth stage.
To identify growth-related genes in the bighead carp, the DEGs in the big and the SGs were identified and functionally analyzed. In total, 15 key enriched pathways (Table 3) and 20 key DEGs (Figure 6), of the frontal and parietal bones and the vertebra, were screened and found to be mainly involved in the physiological functions of metabolism, cytoskeleton, and immunization. Arginine and proline metabolism, biosynthesis of unsaturated fatty acids, PPAR signaling pathway, citrate cycle, oxidative phosphorylation pathway, and carbon and fatty acidmetabolizing pathways are responsible for metabolizing amino acids, lipids, and carbohydrates, producing energy for vital functions of an organism (Jiao et al., 2020). Most of these metabolic pathways are also found in reports of growth-related transcriptomes in fish, such as the PPAR signaling pathway (Lu et al., 2019), carbon-metabolizing pathways (Lu et al., 2020), and oxidative phosphorylation pathway . The eight other aforementioned pathways also play an important role in growth, by regulating the actin cytoskeleton, cell adhesion molecules, and FoxO signaling pathway (Pang et al., 2018;Lu et al., 2019).
involved in protein synthesis and degradation (Chakravarthi et al., 2014;Lu et al., 2019). Moreover, several immune-related DEGs were identified in the bone tissue transcriptome of bighead carp, such as β2-microglobulin (b2m), MHC class II β precursor, receptor-type tyrosine-protein phosphatase C (ptprc), and dopamine β-hydroxylase (dbh) (Kountikov et al., 2004;Das et al., 2011;Cheng et al., 2017). It has been previously shown that these immune system-related genes are essential for maintaining normal growth and physiological functions (Cleveland et al., 2020). In addition to the genes of energy metabolism and immune function, cytoskeletal genes have been identified as candidate growth-related genes in fish (Salem et al., 2012). In this study, we found a differential expression of cytoskeletal genes such as myc target protein 1 homolog (myct1), Golgi apparatus protein 1 (glg1), zinc finger protein 574 (znf574), and actin filament-associated protein 1-like 2 (afap1l2) in the two groups with different growth rates (Mourelatos et al., 1995;Berg et al., 2010;Tie et al., 2016;Wu et al., 2016), suggesting that they play important roles in cell growth, proliferation, apoptosis, and transformation. The protein tyrosine phosphatase 4A (PTP4A) family, consisting of PTP4A1/PRL1, PTP4A2/PRL2, and PTP4A3/PRL3, has been implicated in cell proliferation and tumorigenesis (Kobayashi et al., 2014). Ptp4a is a critical promoter of TGF-β signaling pathway in primary dermal fibroblasts (Sacchetti et al., 2017) and might also regulate the growth of the bighead carp. Serine/threonine-protein kinase (Sgk1) is a serum glucocorticoid kinase that is involved in the regulation of fat storage, body size, and development in Caenorhabditis elegans (Jones et al., 2009). Sgk1 has also been identified as a growth-related gene in the Arctic charr (Salvelinus alpinus), at a specific developmental stage, and is correlated to the size of the organism (Beck et al., 2019). Although reports regarding DEGs pleckstrin homology domaincontaining family J member 1 (plekhj1) and uncharacterized protein (LOC101883013) are rare, we consider them as novel candidate growth-related genes that need further functional investigation and verification. Among the aforementioned DEGs (Figure 6), several DEGs have been identified as candidate genes regulating growth in previous transcriptomic and genomic studies, including scd, ube4b, atp1b, myct1, b2m, and mhcII (Xu et al., 2013;Wu et al., 2016;Lu et al., 2019;Zhou et al., 2019). These reports provide additional support to our findings of these genes being significantly involved in the modulation of differential growth in different domestic animals. However, compared with other transcriptomes of bone tissues in fish, certain wellknown pathways and genes involved in bone formation and differentiation have not been identified in DEG analyses of frontal and parietal bones and vertebral bones in bighead carp, such as calcium, MAPK, TGF-β, and osteoclast pathways (Lu et al., 2019;Nie et al., 2019). For our study, this may be because the bighead carp used were 6 months old, and bone formation and differentiation might have already been completed. Bone formation and differentiation in fish usually occur during early growth and development, approximately 30 days post-fertilization or even earlier, such as in Epinephelus lanceolatus (Lv et al., 2019), M. amblycephala (Nie et al., 2019), and Cynoglossus semilaevis (Ma et al., 2019). Although well-known pathways and genes involved in bone formation and differentiation were not identified in this study, we found several growth-related DEGs that have been previously reported to regulate bone development, such as p4ha1 (Zou et al., 2017) and sgk1 (Beck et al., 2019), suggesting that differential growth of bighead carp is associated with bone development, to an extent.
Ethics Statement
All experimental protocols involved in fish in this study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institute of Hydrobiology, the Chinese Academy of Sciences, China. All efforts were made to minimize suffering of the fish.
Fish Sample Collection
Fish samples of bighead carp at early growth stage used in this study originated from one full-sib family, which were cultured at one pond of the Zhangdu Lake Fish Farm (Wuhan, China). In November 2018, BL, HL, HH, HW, and BW of 6-month samples were measured after being anesthetized with MS-222 (tricaine methanesulfonate; Sigma). The ratios of HL and BL (HL/BL) were calculated. Three samples with larger BW were clustered in fast-growing group (BG). Another three samples with smaller BW were clustered in slow-growing group (SG). The frontal and parietal bones were separated from the skull, removed skins, washed with pure water, cut into pieces, mixed together, and then immediately placed in liquid nitrogen and stored at −80 • C refrigerator before total RNA extraction. The vertebrae were separated from the body, removed muscles, also washed with pure water, cut into pieces, mixed, and then immediately placed in liquid nitrogen and stored at −80 • C refrigerator before total RNA extraction. The layout plan of the study design is shown in Figure 8.
RNA Extraction and Quality Evaluation
Total RNA was extracted from each bone tissue (the frontal and parietal bones or vertebral bones) using RNAiso reagent (Takara, Tokyo, Japan). To prevent genomic DNA contamination, RNA samples were treated to digest DNA using RNase-free DNase I during extraction of total RNA. RNA degradation and contamination were verified by ethidium bromide staining of 28s and 18s ribosomal RNA on a 1% agarose gel. RNA purity and concentration were checked using a Nanodrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was assessed using an Agilent RNA 6000 Nano reagents part I Kit in an Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, United States). The RNA quality criteria for the RNA samples were RIN ≥ 8.0 (RNA Integrity Number), and 1.8 < OD260/280 < 2.2. The qualified RNAs were used for further PacBio and Illumina library construction, respectively.
PacBio Library Construction and Sequencing
To construct the library for PacBio sequencing, the qualified RNAs of bone tissues from BG and SG, including the frontal and parietal bone and vertebra tissues, were mixed in equal amounts, respectively. The mixed RNA sample from BG or SG was reverse-transcribed for mRNA using the SMARTer TM PCR cDNA Synthesis Kit. PCR amplification was performed using the KAPA HiFi HotStart PCR Kit. Then, the PCR product for the SMRTbell library was constructed using the SMRTbell template pre kit. The concentration of the SMRTbell library was measured using a Qubit 3.0 fluorometer with a Qubit TM 1× dsDNA HS Assay kit (Invitrogen, Carlsbad, CA, United States). The quantified criteria of library quality were concentration >10 ng/µL with dispersive but continuous distribution in the range of 1-6 k bp. A total of 2.5 ng of the library was sequenced for each SMRT cell using the binding kit 2.1 from the PacBio Sequel platform, producing 20 h of movies. In the sequencing process, two SMRT cells were prepared on the PacBio RSII platform, including one SMRT cell from mixed bone tissues in BG and the other SMRT cell from bone tissues in SG.
Illumina RNA-Seq and de novo Assembly
The Illumina library for each tissue sample was constructed using the TruSeq RNA Sample Prep Kit (Illumina) following the manufacturer's instructions. Briefly, the polyA mRNA was fragmented using divalent cations at elevated temperature. The RNA fragments were reverse transcribed into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis, end repair, and ligation of the adapters. The ligated fragments were purified and enriched through PCR to generate the final cDNA library. Finally, 12 transcriptomic libraries (six libraries from each group) were sequenced on Illumina HiSeq × Ten platform to obtain 150 bp pair-end reads. Raw RNA-seq reads in fastq format were first filtered through in-house perl scripts to filter out the low-quality reads. Reads with a Q30 percentage greater than 85% were retained as high-quality reads; the rest of the reads as lowquality reads were filtered out. Then clean reads were obtained by removing reads containing sequencing adapters, ploy-N, and low quality. The clean paired-end reads from each library were merged together and then de novo assembled by using Trinity 2.8.4 software with the default parameters. The clean short reads were then mapped to the PacBio reference sequence using Tophat2 tools.
PacBio Iso-Seq Data Processing and Error Correction
According to PacBio's protocol, the raw polymerase reads were first processed using SMRTlink 5.0 software. Briefly, after removing the SMRTbell TM adapter and the low-quality data, postfilter polymerase reads were obtained. The CCS was generated from the subreads BAM files, also known as the reads of insert (ROIs). All the ROIs whose the number of full passes was >1 were further classified into full-length (FL) and non-full-length (nFL) transcript sequences based on whether the 5 primer, 3 primer, and poly A tail could be simultaneously observed. We employed a three-step strategy for error correction to improve the accuracy of the full-length transcripts produced by the PacBio Iso-Seq platform. First, the circle sequencing with >1 pass provided an opportunity for ROI self-correction. Second, full-length, non-chimeric (FLNC) reads were subjected to non-redundant and clustering treatments by the ICE Quiver algorithm and to arrow polishing with the nFL sequence, producing high-quality and polished fulllength consensus sequences. Finally, these polished consensus sequences were further subjected to correction and redundancy removal with Illumina short reads using the Proovread tool and the CD-HIT program with a-c 0.99 parameter cutoff, respectively. The above three corrections resulted in nonredundant, non-chimeric, full-length transcripts (isoform level) with high accuracy for subsequent analyses.
Functional Annotation
For comprehensive functional annotation, the transcripts were searched against seven databases using BLAST software (version 2.2.26): NCBI NR, COG, Pfam, KOG, Swiss-Prot (A manually annotated and reviewed protein sequence database), GO, and eggNOG using an e value of 1e −5 . The KEGG orthology results were obtained by comparing with KEGG database using KOBAS2.0 (Xie et al., 2011). After predicting the amino acid sequence of transcripts, the software HMMER (Eddy, 1998) was used to compare them with Pfam database to get the annotation information of transcripts.
Gene Family and Coding Sequence Prediction
Coding Genome Reconstruction Tool v1.3 1 used K-mer similarity profiles to partition full-length coding sequences into gene families, after which it reconstructed subreads containing the full coding region. To predict the ORFs in transcripts, we used the TransDecoder v2.0.1 program 2 to define putative coding sequences (CDSs). The predicted CDSs were searched and confirmed by BLASTX (E value < 1 × 10 −5 ) against the protein databases of NR, Swiss-Prot, and KEGG. Those transcripts containing complete ORFs were designated as full-length transcripts.
Prediction of LncRNA and AS
Transcripts with a length of more than 200 bp and having more than two exons were selected as lncRNA candidates. The lncRNAs were predicted by four computational approaches, including CNCI (v2), CPC (v1), CPAT (v1.2), and Pfam (v1.5) with default parameters. These approaches can effectively distinguish protein-coding and non-coding transcripts. Transcripts were removed that did not pass any of these analyses; the intersection of the four results was then selected as lncRNAs.
Based on the BLAST method (Altschul et al., 1997), all the transcripts were used for pairwise alignment. BLAST alignments were considered products of candidate AS events (Liu et al., 2017), which met three criteria simultaneously: (i) the length of two transcripts was both greater than 1,000 bp, and there were two high-scoring segment pairs in the alignment; (ii) the AS gap between two aligned transcripts was greater than 100 bp and at least 100 bp from the 3 end and 5 end; and (iii) a 5-bp overlap could be allowed.
Detection of TF and Microsatellite Markers
Transcription factors-related transcript sequences were predicted using the BLAST method with the AnimalTFDB database (Zhang et al., 2015).
Microsatellite markers (also known as SSRs) were identified using MISA 3 with parameters as default. SSR detection was only conducted on non-redundant transcripts that were larger than 500 bp in size. The minimum repeat time for core repeat motifs was set as follows: 10 for mononucleotide, six for dinucleotides, and five for trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides. Based on the structural organization of the repeat motifs, SSRs were classified into perfect and complicated (compound or interrupted) SSRs.
Screening Differentially Expressed Unigenes and GO and KEGG Enrichment Analyses
The expression levels of all the unigenes in 12 samples were assayed based on the Illumina short reads dataset, and reference sequences were the full-length transcripts yielded from PacBio Iso-Seq. The transcripts were quantified using RSEM software. Relative gene expression levels of each unigene were determined by FPKM (fragments per kilobase of transcript per million mapped reads). DEGs of bone tissues (the frontal and parietal bones and vertebral bones) in BG and SG were screened using DESeq2 package. Supplementary Table 6 was used as an input file to run DESeq2. The FDR (<0.01) adjusted by Benjamini-Hochberg method was adopted for screening DEGs. DEGs were defined as by parameters of FDR < 0.01 and the absolute value of the log2 ratio ≥ 1. DEGs were also employed for the enrichment analyses of GO and KEGG pathways in order to determine the potential functions and metabolic pathways.
Validation of Differentially Expressed Unigenes by qRT-PCR
In order to examine the reliability of the RNA-seq results, 12 DEGs randomly selected and the internal control gene β-actin were used for validation by qRT-PCR. Total RNA from 12 samples (each of the frontal and parietal bones and vertebrae from BG and SG) was extracted individually using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. The cDNA was synthesized from 1 µ g of total RNA for each sample using PrimeScript TM RT reagent Kit (TaKaRa, Dalian, China). The selected DEGs and specific primer sequences used for qRT-PCR are listed in Supplementary Table 7, qRT-PCR was performed on a StepOne TM Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). The qRT-PCR reaction solution consisted of 6.5 µL Power SYBR Green PCR Master Mix (Applied Biosystems), 0.2 µM of each forward and reverse primer, 1.2 µL diluted cDNA, and 4.5 µL sterile distilled water. The PCR reaction condition was performed at 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s, 58 • C for 30 s, and 72 • C for 45 s. RNA samples in bone tissues of bighead carp from big and SGs were run in three times' biological replicates and three technical replicates for qRT-PCR. The expression level of each DEG was normalized to that of the reference gene β-actin by using the 2 − CT value method (Livak and Schmittgen, 2001) to validate the results of RNA-seq.
CONCLUSION
In summary, we obtained full-length transcriptome sequences of the frontal and parietal bones and vertebral bones by PacBio Iso-Seq in bighead carp at early growth stage known to exhibit different growth rates. Coupling the RNA-seq data with the Iso-Seq results of the big and the SGs, 27 and 45 DEGs were identified from skull bones and vertebral bones, respectively. A total of 15 pathways and 20 DEGs potentially regulate differential growth in bighead carp and were found to be mainly involved in physiological functions of energy metabolism, immune function, and cytoskeleton function, such as arginine and proline metabolism (p4ha1), fatty acid metabolism (scd), oxidative phosphorylation (sdhdb), FoxO signaling (sgk1), and cell adhesion molecules (b2m, mhcII, ptprc, and glg1). Our study represents the first step in establishing a full-length transcriptome resource of the head and vertebral bones in bighead carp at early growth stage and sheds light on the genetic association between growth and bone development.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/, BioProject ID PRJNA661719.
ETHICS STATEMENT
The animal study was reviewed and approved by the Guide for the Care and Use of Laboratory Animals of the Institute of Hydrobiology, the Chinese Academy of Sciences, China.
AUTHOR CONTRIBUTIONS
JT conceived and designed the experiments and modified the manuscript. WL, YZ, JW, and XY performed the experiments. WL analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. | 2021-01-15T14:12:14.418Z | 2021-01-15T00:00:00.000 | {
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53615644 | pes2o/s2orc | v3-fos-license | Microwave Admittance of Gold-Palladium Nanowires with Proximity-Induced Superconductivity
We report quantitative electrical admittance measurements of diffusive superconductor--normal-metal--superconductor (SNS) junctions at gigahertz frequencies and millikelvin temperatures. The gold-palladium-based SNS junctions are arranged into a chain of superconducting quantum interference devices. The chain is coupled strongly to a multimode microwave resonator with a mode spacing of approximately 0.6 GHz. By measuring the resonance frequencies and quality factors of the resonator modes, we extract the dissipative and reactive parts of the admittance of the chain. We compare the phase and temperature dependence of the admittance near 1 GHz to theory based on the time-dependent Usadel equations. This comparison allows us to identify important discrepancies between theory and experiment that are not resolved by including inelastic scattering or elastic spin-flip scattering in the theory.
DOI: 10.1002/aelm.201600227
measurements. [8,9] Shapiro steps, supercurrent enhancement, and other non-equilibrium effects under intense microwave irradiation have also been extensively studied. [1,[10][11][12][13][14][15][16] In contrast, the near-equilibrium response of superconductor-normalmetal-superconductor (SNS) junctions to weak microwave radiation has become an active area of investigation only recently. [17][18][19][20][21] While the adiabatic contribution to the kinetic inductance can be calculated from the dc current-phase relation, at high frequencies both reactive and dissipative contributions arise also from other mechanisms, such as driven transitions between quasiparticle states and oscillation of the Andreev level populations. [18] Surprisingly, however, little experimental data has been published on the topic thus far. [22,23] The parameter regimes and materials studied in the published experiments are very sparse, hence limiting the extent to which theoretical predictions [17][18][19][20][21] can be tested. In practice, data on the effective inductance and losses also expedites the process of designing high-frequency SNS-junction-based circuits, such as the SNS nanobolometer. [24,25] Previous experimental studies [22,23] have probed flux-and temperature-dependent changes in the linear microwave response of a superconducting ring with a gold normal-metal inclusion. The superconducting ring consisted of ion-beamdeposited tungsten in the first experiments, [22] and sputterdeposited Nb with a thin Pd layer at the SN interface in the later experiments. [23] A single SNS ring was biased with a dc magnetic flux and coupled weakly to a multi-mode microwave resonator. By measuring flux-dependent shifts in the quality factors and resonance frequencies, the authors determined how the complex-valued electrical susceptibility χ changes. [22,23] The change in χ, as a function of flux and temperature, was reported to be in excellent agreement with theoretical predictions based on Usadel equations and numerical simulations. However, the implicit offsets in both the real and imaginary parts of the reported susceptibility prevent a comparison to the theoretically predicted absolute values of Re [χ] and Im [χ]. They also prevent the accurate prediction of the effective inductance (Re[χ] −1 ) and the loss tangent (Im[χ]/Re[χ]), which are the key quantities for practical high-frequency applications of SNS junctions.
In this article, we present measurements of the SNS junction admittance Z -1 (ω) = χ/iω for gold-palladium based junctions at angular frequencies ω of order 2π × 1 GHz. We use a chain of
Introduction
The transport of direct current (dc) between two superconductors (S) separated by a diffusive normal-metal (N) link is in general well understood both theoretically and experimentally. [1][2][3][4][5][6] At low temperatures and currents, Andreev reflection [7] leads to the formation of a gap in the density of quasiparticle states in N and allows a dissipationless supercurrent to flow. This gap has been directly observed in tunnel spectroscopy SNS superconducting quantum interference devices (SQUIDs) with a strong capacitive coupling to a multimode microwave resonator with a typical mode spacing of 0.6 GHz. Each chain consists of 20 SNS SQUIDs in series. The strong coupling and the large number of SQUIDs lead to significant changes in the frequencies and quality factors of the resonator modes, which allow determining Z -1 without an offset. The absence of an offset enables us to show that the Usadel-equation-based theory we consider cannot simultaneously explain the observed real and imaginary parts of Z -1 .
Samples
We study the linear electrical response of SNS junctions at millikelvin temperatures in two samples: Sample 1 and 2. The gold-palladium nanowires used as the normal-metal are deposited simultaneously in the same fabrication steps as for our recent nanobolometer circuits. [25] We determine the Au:Pd atomic ratio of the alloy to be approximately 3:2 using energydispersive X-ray spectroscopy (see Experimental Section). The nominal junction length l is 300 nm and the nominal cross-sectional area is 30 nm × 120 nm. The normal-state resistance of a single junction R N = 15 Ω is estimated based on four-wire dc measurements of reference samples. From the reference measurements, we also estimate the upper bound for the contact resistance R B to be approximately 1 Ω. We only give an upper bound for the contact resistance because of the uncertainties introduced by the crossed-wire geometry [26] of the reference samples. Reference samples are similar to those in Figure 1b in Reference [25].
The key parameter determining the strength of the proximity-induced superconductivity is the Thouless energy E T = ħDl -2 , where D ≈ 22 cm 2 s -1 is the diffusion constant. We obtain the diffusion constant by measuring the resistivity of the nanowires and scaling the result according to literature values for the proportionality D ∝ ρ -1 . [27] The result we obtain is approximately 30% higher than that in Reference [27]. In our samples, the Thouless energy is E T ≈ k B × 190 mK ≈ h × 3.9 GHz, where h = 2πħ is the Planck constant and k B is the Boltzmann constant. The superconducting sections are 100-nm-thick aluminum, which implies that the energy gap Δ in the superconductors is much larger than the Thouless energy (Δ/E T ≈ 13). Both S and N parts are fabricated using electron beam litho graphy and evaporation. Further fabrication details are reported in the Experimental Section.
The SNS junctions are arranged into a chain of SQUIDs, as shown in Figure 1. Each SQUID loop has a relatively small area of 20 µm 2 in order to minimize sensitivity to external magnetic field noise. In addition, we measured Sample 2 in a doublelayer magnetic shield. The geometric inductance of each loop is small (L G < 10 pH) compared to the effective inductance of the SQUID, as verified by the results below. A dc magnetic flux bias is applied by an external coil that provides a uniform flux bias Φ for each SQUID in the 100-µm-long chain. Assuming identical junctions, the field biases each SNS junction at a phase difference π(Φ -mΦ 0 )/Φ 0 at dc, where Φ 0 is the magnetic flux quantum h/2e and m is the integer that minimizes |Φ -mΦ 0 |. We note that the flux bias we label as Φ = 0 may be offset from the actual zero flux condition by an integer multiple of Φ 0 .
The SQUID chains in the two samples are nominally identical, except for the addition of heat sinks to Sample 2 (see Figure 1c). The heat sinks are designed to reduce the hot-electron effect, [28] i.e., the increase of the quasiparticle temperature above the phonon bath temperature T b that we measure. For each SNS junction, the heatsink consists of two large (0.5 µm 3 ) reservoirs of gold-palladium that are thermally strongly coupled to the junction.
In addition to the SQUID chain, the chip contains a transmission line resonator of length 10 cm (see Figure 1a and Figure 1. a) On-chip circuit layout: feedline (FL) between ports 1 and 2, transmission line part of the resonator, and termination. The resonances are externally damped by the capacitive coupling (C C ) to the feedline, and internally damped by the capacitively coupled SQUID chain. See Table 1 for further details on parameter values. b) Schematic detail of the device under test at the termination: 20 SNS SQUIDs in series. c) Micrograph of a single SQUID from a sample fabricated identically to Sample 2. The wide Au-Pd extensions (light-colored regions) are heat sinks. Table 1. Resonator and coupling capacitor parameters for Samples 1 and 2: the internal (external) load capacitance C i (C C ), the transmission line resonator length l r , the fundamental frequency f 0 , and the resonator chacteristic impedance Z 0 shown in Figure 1. The last column emphasizes that Sample 2 includes additional large gold-palladium heat sink reservoirs for enhancing electron-phonon coupling. termination, i.e., samples without the SQUID chain. From the control measurements, we extract the fundamental frequency of the transmission line resonator f 0 ≈ 635 MHz, and confirm that the internal quality factor Q i,n > 10 4 for the resonances we consider (n ≈ 3). The latter implies that we can neglect the losses in the transmission line part of the resonator, and in the Al 2 O 3 used as the dielectric material in the lumped element capacitors (C C and C i ). This is valid because introducing the SQUID chain lowers Q i,n to the order of 10 2 , as observed below. We also deduce the characteristic impedance Z 0 ≈ 39 Ω of the transmission line from the measured f 0 , the length of the resonator, and the design value for the inductance per unit length.
Measurement Scheme and Sample Characterization
We determine the admittance of the SQUID chain by embedding it as the termination of the transmission line microwave resonator, as illustrated in Figure 1a. We first determine the resonance frequency f n and the internal quality factor Q i,n of each mode n by measuring the frequency-dependent transmission coefficient S 21 (f) through the feedline. By comparing f n and Q i,n to values measured in control samples with no SQUID chains, we can determine the admittance of the SQUID chain at multiple frequencies. Specifically, we use a circuit model ( Figure 1a) that allows extracting the admittance of the SQUID chain chain 1 Z − from the response of the combined resonator/SQUID-chain system. The admittance of each individual SNS junction is then given by 10 chain 1 Z − , assuming that the junctions are identical and that geometric inductance is negligible. Figure 2 shows the normalized transmission through the feedline at frequencies near 1.4 GHz, probing the third (n = 3) mode in Sample 2. The normalization (defined precisely in the Experimental Section) removes all spurious features in the transmission data that do not depend on flux. This procedure reveals the oscillatory flux dependence of the resonance frequency f n with a period we identify as Φ 0 . As the flux bias is increased away from integer multiples of Φ 0, we measure a decrease in both the resonance frequency and the loaded quality factor Q 0,n . This behavior is more clearly visible in Figure 2b,c with individual slices of transmission data for Φ/Φ 0 = 0 and Φ/Φ 0 = 0.3. These changes in the resonance indicate that both the inductance and the losses are maximized in the SQUID chain near half-integer values of Φ/Φ 0 .
To extract f n and Q i,n quantitatively, we fit the measured normalized transmission for the nth mode to the model [29,30] where δf is a fit parameter that characterizes asymmetry, and the external quality factor Q C,n is governed by the coupling (C C ) to the feedline. From the obtained fit parameters Q 0,n and Q C,n , we compute the contribution of losses due to the SQUID chain as i, Figure 3 shows the extracted values of f n and Q i,n for frequencies up to 12 GHz (n ≈ 20).
In the low-frequency and low-temperature regime, the SQUID chain behaves like an inductor, i.e., most of the admittance is reactive ( Z ω − varies slowly as a function of the angular frequency ω = 2πf. Consequently, we parametrize the admittance chain 1 Z − as a parallel combination of a resistor and an inductor, such that Figure 3 demonstrates that this is a good parametrization by showing qualitative agreement between the experimental data and predictions for the mode shifts and quality factors using a simplified model where L and R are constant. Let us now discuss the extraction of the admittance chain 1 Z − from the measured f n and Q i,n values. For an ideal transmission line resonator with open-circuit conditions at both ends (C C = C i = 0), the nth mode is located at frequency nf 0 . In contrast, for the samples with the SQUID chains, the frequencydependent reactive, i.e., imaginary parts of the termination admittances iωC C and [(iωC i ) - using the parameters given in Table 1. We derive this equation from the circuit model shown in Figure 1, assuming that Q i,n is dominated by losses in the SQUID chain. Table 2 shows the L and R extracted for two example resonances near 1 GHz. The reported values provide an important reference for designing high-frequency devices based on gold-palladium SNS junctions. That is, they imply that an effective inductance of a few hundred picohenries per junction and a loss tangent of a few percent are observed around 1 GHz at milli kelvin temperatures.
The accuracy of the admittance measurement is limited by uncertainty in the fit parameters f n and Q i , and the systematic device parameters measured in control samples (e.g. f 0 , C i ). The error bars of the R and L values throughout this article are propagated from the uncertainties in the fitted f n , Q i values at the 68% confidence level. There remains a 10% relative error in the reported R and L values from the systematic device parameter uncertainties at 1 GHz. This arises predominantly from chipto-chip variations in the unterminated resonance frequency, Δf 0 ≈ 3 MHz, based on measurements of control samples. As frequency increases, Δf 0 has a larger effect on the uncertainty of L in Equation (2). For example, in Sample 2, the uncertainty Δf 0 ≈ 3 MHz leads to a relative error in L of 70% at 10 GHz. For this reason, we concentrate on resonances near 1 GHz in the discussion below.
Theory
In the next section, we compare the experimental results to theoretical predictions [18] based on the time-dependent Usadel equation. [2] In the low-frequency and low-temperature regime ħω, k B T E T considered below, the imaginary part of the admittance of the junction is expected to be mostly determined by the adiabatic Josephson inductance associated with the supercurrent, i.e., the Φ derivative of the dc supercurrent. The real part, on the other hand, mainly arises from driven quasiparticle transitions in the junction. The availability of such transitions is sensitive to the density of quasiparticle states. In particular, the presence of a proximity-induced energy gap E g ≈ E T in the density of states should lead to an exponential increase in the resistance as k B T decreases below E g .
However, the low-temperature values of L and R -1 we measure (Table 2) are dramatically larger than those predicted using the parameters considered in Reference [18]. This is evident from a cursory comparison of Figure 1 in Reference [18] to our (ωL/10) -1 ≈ 6 N 1 R − and (R/10) -1 ≈ 0.3 N 1 R − . The inductance per junction L/10 ≈ 300 pH is also an order of magnitude higher than the expected adiabatic Josephson inductance L J = [2∂ Φ I s (Φ)] -1 ≈ 50 pH, where we approximate the dc supercurrent I s (Φ) as I c sin(πΦ/Φ 0 ) and the critical current I c as the ideal value 6.7 E T /eR N for Δ/E T ≈ 13. [4] Moreover-in the results below-we observe a weak temperature dependence of R(Φ = 0) measured near 1 GHz, which is in stark contrast to the theoretically predicted exponential dependence.
The observed values of L and R imply that the proximityinduced superconductivity is significantly weaker than expected. We consider two distinct scattering mechanisms as potential explanations for this. First, we include dephasing due to inelastic scattering by choosing a phenomenological relaxation rate Γ. [18] Second, we include a spin-flip scattering rate Γ sf which could arise from dilute magnetic impurities in the weak link. [31] Specifically, we include the spin-flip scattering as an additional self-energy Table 2. SQUID chain admittance parameters R -1 + (iωL) -1 and corresponding loss tangent ωL/R measured at T b = 10 mK and Φ = 0 for the second (third) resonance in Sample 1 (2). The effective inductance (resistance) per single SNS junction is L/10 (R/10). Both frequencies are in the regime ω ≈ E T /h -. in Reference [18]. Considering spin-orbit impurity scattering in a similar approximation [32] would not affect the superconducting proximity effect within the model considered here. Although quantitative details differ, both of the scattering mechanisms generally lead to increased dissipation and increased inductance. Increased dissipation occurs mainly due to the suppression of E g , while increased inductance occurs mainly due to the increase in L J . Theoretical work on the microscopic origin of the scattering rates in disordered metals is reviewed in References [33] and [34]. Experiments have also been carried out with high-purity metal wires. [35,36] However, we are not aware of measurements on the gold-palladium alloy used here, which prevents direct comparison to existing literature. Instead, our goal is to estimate the scattering rates required for a qualitative match to the experimental results. We find that in order to reproduce the experimentally observed L or R, the phenomenological rates Γ and Γ sf must be large, i.e., comparable to E T /ħ and k B T/ħ.
Inelastic scattering and spin-flip scattering are not the only possible explanations for observing proximity-induced superconductivity that is weaker than what is predicted by the ideal Usadel-equation-based theory. Although we do not attempt to exhaustively cover all candidates, we note that the SN contact resistance in our samples is much smaller (R B < 1 Ω) than the normal-state resistance (R N = 15 Ω). While the smallness of the ratio R B /R N does not conclusively exclude explanations based on imperfect interfaces, it limits them significantly. [5,37]
Temperature and Flux Dependence near 1 GHz
Below, we compare the predicted and observed dependences of Z chain on the bath temperature and magnetic flux. We choose to analyze two low-n resonances near 1 GHz, mainly because the L values we extract for them suffer the least from the uncertainty in f 0 . Figure 4 shows the measured flux dependence of R and L for the third (n = 3) resonance in Sample 2. The bath temperature is T b = 195 mK, which should be high enough for neglecting the hot-electron effect, i.e., for assuming that T ≈ T b . As expected, we observe that R and L are periodic in flux, and that the inductance L and the loss tangent ωL/R are minimized (maximized) at integer (half-integer) values of Φ/Φ 0 . Figure 4 also includes theoretical predictions for two different rates of inelastic scattering. The weaker of the two rates (Γ = 2.3 k B T b /ħ) reproduces R(Φ = 0) well and gives a reasonable prediction for its flux-dependent oscillations. Furthermore, if we could only measure changes in L, we might conclude that the predicted flux modulation of L is in fair agreement with the experimental data for this moderate value of Γ. However, the absolute value of the prediction for L(Φ) is several times smaller than the observed value at nearly all flux values. This highlights the importance of measuring L and R without offsets if theories are to be rigorously tested. Note that we can improve the agreement between the predicted and measured L(Φ), especially around integer values of Φ/Φ 0 , by using a very strong inelastic scattering rate of Γ = 8k B T b /ħ in the theoretical calculation. However, this value of Γ leads to a clear disagreement in the amplitude of the oscillations in R(Φ) as shown in Figure 4b. Figure 4 also shows the theoretical predictions that include strong spin-flip scattering. By choosing Γ sf appropriately, the predictions become nearly identical to the case of strong inelastic scattering. Therefore, the conclusions of the previous paragraph also apply to predictions where scattering is spin-flip dominated. Furthermore, the similarity of the predictions shows that, in this parameter regime, the source of additional dephasing is unimportant.
To gain further insight, we study the temperature dependence of R(Φ = 0) and L(Φ = 0) for one resonance from each sample near 1 GHz (see Figure 5). In addition to the measured data points, Figure 5 shows theoretical predictions with scattering parameters that-at 195 mK-are identical to those in Figure 4. However, we note that considerable freedom remains in choosing the temperature dependence of the scattering rates. Rigorously justifying a particular temperature scaling would require knowledge of the specific microscopic mechanism responsible for the scattering. However, as the theoretical predictions already disagree with the measured results at the phenomenological level at a fixed temperature (Figure 4), identifying any specific microscopic mechanism seems implausible.
As instructive examples, we choose Γ ∝ T and a constant Γ sf in Figure 5. Unsurprisingly, none of the predictions simultaneously matches the observed temperature dependence of L and R. Nevertheless, the experimental data in Figure 5 may serve an important role in testing alternative theories in the future.
Conclusions
The main discrepancy between theory and experiment can be summarized as follows. The proximity effect at T E T /k B and ω ≈ E T /ħ is weaker than what is predicted by theory based on the Usadel equation. [18] This disagreement manifests itself experimentally as measured R values that fall below theoretical predictions and measured L values that exceed theoretical predictions. As potential candidates for such loss of coherence, we considered inelastic scattering and spin-flip scattering in the weak link. However, we did not find choices of Γ or Γ sf that would provide simultaneous agreement in L and R, neither in terms of flux dependence at a fixed bath temperature, nor in terms of temperature-dependence at zero flux bias. Furthermore, the scattering rates required for a match in either L or R are larger than expected for, e.g., electron-electron scattering in disordered systems. [34] We note that the discrepancies shown here are not in direct contradiction with the previous experiments [22,23] and that both the SNS junctions and the measurement scheme presented here are very different from these preceding studies. Firstly, the weak link material is different than in the previous experiments. We cannot rule out the possibility of effects specific to gold-palladium [38] that reduce coherence in the weak link. Secondly, we measure both the reactive and dissipative components of the electrical admittance without arbitrary offsets. In contrast, only changes in the admittance have been reported previously. Thus, our experimental technique provides a more stringent test of the accuracy of the theory and reveals quantitative disagreements more easily.
In conclusion, we reported measurements of microwave frequency admittance for gold-palladium SNS junctions, together with a comparison to quasiclassical theory for diffusive SNS weak links. These discrepancies between measurement results and theoretical predictions suggest that dephasing caused by inelastic scattering, or elastic spin-flip scattering, is probably not the correct mechanism for explaining why the proximityinduced superconductivity is weaker than expected in our gold-palladium SNS junctions. Further theoretical work is required for reaching simultaneous agreement for the magnitude, temperature dependence, and flux dependence of both the dissipative and reactive parts of the admittance. Mechanisms that may need to be taken into account include imperfect interfaces, [5,37] electron-electron and fluctuation effects in low-dimensional superconducting structures, [39,40] and paramagnon interaction. [41] Magnetic effects could be particularly important in SNS junctions that include palladium, which is paramagnetic in bulk and can even become ferromagnetic in nanoscale particles. [42,43] In general, the relationship between microscopic materials properties and coherence at microwave frequencies in normal-metal Josephson junctions should be clarified, both experimentally and theoretically. A productive experimental approach may be to first investigate systems such as Nb/Cu weak links that, based on previous experiments, [4,44] are expected to behave in an ideal fashion at dc.
Experimental Section
Resonator fabrication: The substrates were 4" (0.5-mm-thick) highresistivity (>10 4 Ω cm) Si wafers with 300 nm of thermal oxide. First, a niobium thin film (thickness 200 nm) was sputter-deposited on the entire wafer. Next, the coplanar waveguide (CPW) structures were defined with AZ5214E positive photoresist that was reflowed in air at 150 °C for 1 min to ensure a positive etch profile of the resulting Nb features. Then CPWs were etched with an rf-generated plasma under a constant flow of SF 6 (40 sccm)/O 2 (20 sccm) gases at constant power. [45] The remaining resist was removed with solvents and an additional O 2 -plasma cleaning step. The 4" wafer was then coated with a protective layer of resist and pre-diced with partial cuts along device pixel outlines on the back of the wafer.
Capacitor dielectric fabrication: The Al 2 O 3 dielectric for the on-chip Nb-Al 2 O 3 -Al capacitors C C , C i , and C gnd was formed by atomic layer deposition with 455 cycles in a H 2 O/TMA process at 200 °C, resulting in a thickness of 42 nm. The thickness was verified in ellipsometry using an index of refraction = 1.64 Al O 2 3 n . Measurements of reference Nb/Al 2 O 3 / Al capacitors yielded a capacitance per unit area of 1.4 fF µm −2 .
Nanostructure fabrication: The gold-palladium nanowires and aluminum superconducting leads were fabricated by electron beam lithography in two separate evaporation and liftoff steps. In the first step, gold and palladium pellets were evaporated from the same crucible with an electron beam heater. Afterward, unwanted AuPd was lifted off with organic solvents. Prior to the evaporation of the Al leads, samples were cleaned in situ with an Ar sputter gun. Finally, after liftoff of the Al film, individual resonator pixels were snapped along the pre-diced lines and packaged for measurement.
The chemical composition of the gold-palladium material was determined with energy-dispersive X-ray spectroscopy for incident electron beam energies 5 keV, 10 keV, and 20 keV (Figure 6). The average Au:Pd atomic ratio (weight ratio) was approximately 3:2 (3:1).
Cryogenic Measurements: Measurements were carried out in a commercial cryostat with a base temperature of 10 mK. The transmission coefficient was probed with a vector network analyzer. The device input line had >100 dB fixed attenuation. For all measurements, the output signal was amplified by a broadband low-noise cryogenic amplifier and by additional room temperature amplifiers. For some measurements (e.g. Sample 2, n = 3) two cryogenic isolators were placed on the base cooling stage between the low-noise cryogenic (1), each with a different set of fit parameters. These fit parameter determine the S 21, fit (Φ ref ) used in the normalization process described above.
In practice, the scan used as the reference alternates between Φ ref = Φ 0 /2 and Φ ref = 0, changing from one to the other whenever Φ crossed Φ 0 (1/4 + k/2), where k ∈ ℤ. This kept the resonance in the reference far from the resonance frequency at the Φ value being analyzed. These changes in Φ ref caused the apparent discontinuities in the background color in Figure 2.
As shown in Figure 2b,c the normalization procedure revealed clear Lorentzian-like lineshapes for S 21 , as one would expect for a resonator of this type. Raw ′ Φ = ( 0) 21 S and ′ Φ = ( 0.3) 21 S data are shown in Figure S3 of the Supporting Information for reference. Disentangling the SQUID chain response from the rest of the circuit is also aided by the fact that the SQUID chain is the only circuit element that responds to flux periodically, with a period that is identical for all of the identified resonances. In general, the external circuit and background noise do not have such a response as we confirmed in measurements of control samples with no SQUIDs.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author. | 2017-03-09T05:28:19.000Z | 2016-07-29T00:00:00.000 | {
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125444713 | pes2o/s2orc | v3-fos-license | Numerical study and experimental validation of a Roots blower with backflow design
ABSTRACT A three-dimensional computational fluid dynamics (CFD) model of a Roots blower with a backflow design was established to analyse the effects of the backflow on the Roots blower's performance. A prototype of Roots blower with a backflow design was manufactured to validate the CFD model through the pressure distribution and the mass flow rate. The results showed that the proposed CFD model agreed well with the experimental data. The effects of the sizes and directions of the backflow passage on the Roots blower's performance were then investigated using the validated CFD model. It was found that under a properly sized backflow passage, the pressure pulsation and the shaft power can be decreased by 80% and13%, respectively; However, the mass flow rate was reduced by 12% under the same size of backflow passage. Although the direction of the backflow passage affected the shaft power, it had no effect on the mass flow rate or pressure pulsation. The shaft power consumption of a Roots blower with a vertical backflow was 4% lower than a horizontal backflow.
Introduction
The Roots blower is a type of gas transport machine comprising a pair of meshed but non-contacting rotors in the cylinder. The rotors rotate in opposite directions and are driven by two identical timing gears. The fluid is transferred from the inlet side to the outlet side when the rotors rotate as shown in Figure 1. Roots blowers are widely used in the pharmaceutical industry, chemical industry, and sewage treatment because of their high reliability, low cost and easy maintenance. Roots blowers can be applied to numerous devices such as middle-pressure blowers, low-and middle-vacuum pumps, and hydraulic pumps. In recent years, the Roots blowers were used in mechanical vapour recompression systems to increase the pressure and temperature of the vapour and to recycle hydrogen as well as supply the compressed air in hydrogen fuel cells.
The development of Roots blowers primarily involves designing the profile of the rotor, which affects the meshing and performance of the blower. Yang (2000, 2005) and Yang and Tong (2002) presented a method for designing a new profile and established a function for analysing the main factors affecting the efficiency of high-sealing Roots blowers. Wang, Fong, and Fang (2002) discussed the constraints for the tooth profile of five-arc CONTACT Xiao-Han Jia jiaxiaohan@mail.xjtu.edu.cn Roots vacuum pumps. Mimmi and Pennacchi (1999) proposed an explicit three-dimensional (3D) analytical model of the screw rotor considering all the surfaces which form the rotor and their parametric expressions. Litvin and Feng (1996) proposed an approach for the design and generation of the planar cycloidal gearings for screw Roots blowers and developed an improved design to avoid the surface singularity. Kang, Vu, and Hsu (2012) developed a Roots blower with an epicycloid curve, which demonstrated a better performance over circular ones. They also found that multi-lobe pumps did not exhibit a higher performance than two-lobe pumps but provided a more stable output. Hwang and Hsieh (2006) and Hsieh and Hwang (2007) designed smooth trochoid ratio profiles by high-order polynomials which improved the sealing performance and volumetric efficiency of the Roots blower. Burmistrov, Belyaev, Ossipov, Fomina, and Khannanov (2001) proposed an angularcoefficient method for calculating the channel conductance in Roots blowers. Yao, Ye, Dai, and Cai (2005) proposed a new type profile which could create a higher air flow and a lower peak of the blower pressure compared to the conventional straight tooth. Valdes, Barthod, and Perron (1999) presented a calculation method which was able to accurately calculate the inside leakage for oil-less Roots vacuum pumps by semi-empirical Knudsen-Dong law. Many researchers analysed the complex flow phenomena inside positive-displacement machines such as screw compressors and Roots blowers by CFD technology, as it showed a high accuracy in engineering practice. A new type of numerical grid generation for twin screw rotors was proposed based on rack generation in order to develop the 3D CFD model of the screw compressor (Arjeneh, Kovacevic, Rane, Manolis, & Stosic, 2015;Broatch et al., 2015;Kovacevic & Smith, 2002;Kovacevic, Stosic, & Smith, 2007;Stosic, Smith, Kovacevic, & Mujic, 2011) These studies improved the performance of the twin-screw compressor and expander by analysing the pressure, temperature, leakage, and velocity through CFD simulations. In addition, the complex flow field inside the cylinder of the Roots blowers was also investigated by CFD simulations in many studies. Li, Jia, Meng, Shen, and Sang (2013) analysed the effects of different pressure angles on the velocity and pressure of oil as well as the turbulent kinetic energy by 2D numerical modeling. Joshi, Blekhman, Felske, Lordi, and Mollendorf (2006) analysed the clearance between the rotors, showing that the clearance remains constant for 96% of the angles of the rotation in a working period. They also investigated the leakage of the Roots blower through a quasi-steady state analysis. Hsieh and Deng (2015), and Hsieh and Zhou (2015) compared the differences in the mass flow rate and pressure between Roots blowers with screw type rotors and cylindrical rotors using the CFD solver PumpLinx. They also analysed how the rotor phase affects the flow rates and pulsation of a serial multi-stages and a parallel multi-stage Roots blower. Huang and Liu (2009) identified and studied the characteristics of flow in a positive displacement blower through the CFD simulation of an involute-type blower with three-lobe positive discharges containing an unsteady compressible flow.
A large amount of aerodynamic noise and air pulsation is often generated by Roots blowers because of their unique compression process which limits the application field of Roots blowers. The working principle of the Roots blower is shown in Figure 2. When the rotation angle is between 0°and 120°, the volume of the working chamber increases, and the air flows from the intake cavity to the working chamber. After 120°, the working chamber is disconnected from the intake cavity, and the moving chamber transfers air by the rotor rotation. When the rotor rotating angle reaches 180°, the working chamber meets with the exhaust cavity. The pressure in the working chamber is increased by the air with a higher pressure in the exhaust cavity, and this often causes the airflow impact and air pulsation. With rotor rotation over 180°, the volume of the working chamber starts to be reduced, which reaches 0 at the rotation angle of 300°. Ohtani and Iwamoto (1981) experimentally demonstrated the air backflow could effectively reduce the aerodynamic noise by reducing outflow pulsation. It is necessary to analyse how the pressure pulsation was affected by the backflow. Although the backflow design for the performance of Roots blowers can hardly be completed in the traditional way, the development of CFD and dynamic mesh technology provided a new way to examine how the backflow design affects the performance of a Roots blower during the working process.
In the present study, 3D CFD models of a Roots blower with and without a backflow design were established to analyse the inside flow field in their working process. A new type Roots blower identical to the simulation one was designed and manufactured. The flow rate and the pressure distributions in the cylinder and intake/exhaust cavities were measured to validate the result from simulations. Based on all the results from experiments and simulations, the performances of the Roots blowers that have and do not have a backflow design were investigated and compared. Finally, the effect of various backflow passage sizes and directions on the flow field were studied through the validated CFD models. Figure 3 shows the structure of a Roots blower with backflow design. There are eight holes symmetrically distributed on the upper and lower working chambers, through which the higher-pressure gas can afflux into the cylinder earlier. Four of these holes are horizontally located at the rotation angle of 120°while the other four are vertically distributed at the same rotation angle.
Structure of backflow design
Each rotor in this Roots blower has three lobes, which creates six sections with a profile curve consisting of arc line, straight line, involute, straight line, and two arc lines successively (Peng, 2000). The cylinder has a diameter and length of 52.5 and 70 mm, respectively. The double rotors' centre distance is 70 mm; the sizes of the clearances in the cylinder are summarised in Table 1. Between the two rotors 0.14 Between the rotor and the cylinder in the circumference direction 0.07 Between the rotors and the cylinder in the axial direction on the axis side 0.03 Between the rotors and the cylinder in the axial direction on the cover side 0.11 The fluid field in the Roots blower, taking account of the effect of backflow, was investigated by CFD simulations, the geometry of which is shown in Figure 4. The geometry has the same size as the Roots blower, except for the clearances. In the CFD model, the clearances between the rotors and the cylinder in the axial direction are ignored because the dynamic mesh is hard to apply in them. The clearances between the rotor and the cylinder in the circumferential direction in the CFD model are increased to 0.15 mm in order to keep the total leakage area unchanged. A narrow pipe was set on the outlet piping throttle of the flow to make the pressure in the outlet piping reach the exhaust pressure, as shown in Figure 5; to obtain a higher precision of the pressure change in the exhaust cavity (Sun, Zhao, Jia and Peng, 2017).
Governing equations
The continuity equation, the energy conservation equ ation and the momentum equation comprise the governing equations. In this study, the simulation was carried out through Fluent (a commercial CFD software), where In these equations, p is the static pressure, F is the external body force, ρ g is the gravitational body force, and τ is the stress tensor.
The turbulence equation is critical for the simulation of Roots blowers as the backflow causes high rotating shear flows in the blower's working process. Separated flows are generated when the air flows across the top of the rotating rotor. Li, Zhang, Zhu, and Hu (2007) compared the simulation results of the Realizable k-mode, the RNG k-model and the standard k-model for a centrifugal pump. It was found that the best simulation result was obtained by the Realizable k-model. In consideration of the high speed of the lobe of the Roots blower and centrifugal pump, the Realizable k-model was then chosen to work as the turbulence equation. The transport equations of the Realizable k-turbulent model are as follows: where where C 2 , C 1ε , σ ε , σ k are constants with values of them being 1.9, 1.44, 1.2 and 1.0, respectively; Y M is the effect of fluctuating dilatation on dissipation rate; G kb is the turbulence kinetic energy produced by buoyancy; and G kv is turbulence kinetic energy produced by average velocity gradients.
Boundary conditions
Both the inlet and outlet pressure were 0 kPa (gauge pressure). The inlet pressure was set to 0 kPa which means that suction happened under atmospheric pressure. The exhaust pressure was set at 50 kPa because the narrow pipe in front of the outlet buffer area throttled the flow and increased the pressure in the outlet piping. The size of the narrow pipe was determined using the trial calculation method. The interface method was used for connections between the cylinder and the backflow piping. Each interface could be set as a wall boundary condition, whereby the number of working backflow pipes was controlled. Air was set as the working fluid, which is assumed as an ideal gas. Figure 5 shows the grids of the numerical simulation model. The mesh type and the number of elements for each part are summarised in Table 2.
Grid generation and dynamic mesh
Since the Roots blower works as an unsteady process, the shape of its inside flow field changes during its working period. Therefore, a dynamic mesh was required for the CFD simulation. The grids of the rotor wall were driven by a UDF (user-defining function), which rotated as rigid bodies. The spring-based smoothing and 2.5D local-remeshing dynamic mesh methods were used for simulations due to the constant deformation of the cylinder.
The time-step was determined as t = ((CFL • x)/(λ max )), which was set to 1 × 10 −5 s. The case was calculated by a computer with 32 cores, 2.6 GHz, and 128 Gb of operating memory. It took 109 h to reach convergence and another 56 h to complete the simulation.
The grid and time step independence study
The grid independence study was carried out to compare the results of the simulations obtained under different grids. As shown in Figure 6 (a), the difference in the results between the element number in the range from 7.8×10 5 to 1.75×10 6 was less than 1.5%. Therefore, it was concluded that the independent results of the grid could be confirmed in a grid of 7.8×10 5 cells.
A time-step independency study was performed to compare results of the simulations obtained under different time-steps. Figure 6 (b) shows the results of the simulation with the element number of 7.8×10 5 for meshes with different time-steps. The difference between the results of the simulation with different time-steps was less than 0.5%. It was therefore concluded that the independent results of the time-step could be confirmed in the time-step of 1×10 −5 s. When the time-step was longer than 1×10 −5 s, a negative grid was generated and the simulation was unable to process.
Experiment on a Roots blower
To verify the accuracy of the simulation, experimental testing equipment was established, as shown in Figure 7. This equipment consisted of inlet/outlet pipes, inlet/outlet valves, pressure gauges, backflow piping, a flow meter, and pressure sensors. The valves in the inlet/outlet piping were used to adjust the intake/exhaust pressure, with pressure gauges installed to measure pressures. A flowmeter was installed to measure the average flow rate. There were five pressure sensors on the cylinder, which are all located on the upper side as the shape of the two rotors was identical and the installation locations were symmetrical with a 60°phase difference. Three of these pressure sensors were used to obtain the variation of the pressure in the working chamber while the rest were installed to measure the pressure in the intake/exhaust cavity. When the rotor rotated, the working chamber passed through these five sensors in turn. The pressure variation of the working chamber during a working period was obtained by collecting the pressure data from all five sensors. The backflow holes were connected to the outlet piping by the backflow piping, where a valve was installed in each to conduct the operation of each backflow pipe separately.
The pressure variations in the cylinder and the flow rate were obtained through the aforementioned experimental equipment. Operating conditions in experiments were as follows. The rotation speed of the motor was 2,400 r/min. The intake/exhaust pressure was adjusted by valves on the inlet/outlet piping, which were the same as the simulation. The sampling rate of the pressure sensors and the measurement range of sensors were 10 kHz and 100-250 kPa (gauge pressure), respectively.
Validation of simulation results with experimental data
To verify the accuracy of the simulation results, two cases were chosen for comparison with the experimental one regarding the mass flow rate and pressure variations. The first case was a Roots blower without a backflow design. In this case, the wall boundary condition was used for all the connections between the cylinder and the backflow piping. In the experiment, all eight valves in the backflow pipes were closed. The second case was a Roots blower with a backflow design. The boundary conditions of the connections between the cylinder and the vertical backflow piping were changed to an interface in this case, and the valves in the vertical backflow pipes were opened, while the horizontal ones had the wall boundary feature and were closed. The CFD simulation of the Roots blower is an unsteady case with an initial value. Calculations for a great many time-steps are required before the simulation reaches a steady state. One indicator of the simulation reaching steady state was whether the difference in the average mass flow during a working circle between the inlet and the outlet was reduced to less than 5%. Table 3 shows that the difference was 1.6% without backflow and 3.3% with backflow, suggesting that both the cases were periodically steady until the third working circle. The experimental results for the mass flow rate are also shown in Table 3. The results indicate that the pumping rate in the simulation was 5.3% lower than in the experiment for the Roots blower without the backflow design and 2.3% lower for the one with the backflow design. The difference was small; thus, the simulation results for the mass flow rate are accurate.
The pressure variations for the Roots blowers with and without the backflow design are shown in Figure 8. Both simulation cases agree well with the experimental results. Figure 8 also shows that the pressure variations for the Roots blower with a backflow design were almost the same as those for the Roots blower without the backflow design in the range of 0°to 120°. This is because the working chamber is still disconnected from the backflow piping, so the backflow showed little impact. However, at angles larger than 120°, the difference in the pressure variation became enhanced. The working chamber of the blower without the backflow design was closed from 120°to 180°, so the high-pressure gas in the exhaust cavity could only leak into the working chamber through the clearances, which slowly increased the pressure in the working chamber. At angles larger than 180°, as Figure 9 shows, the working chamber would open to the exhaust cavity; the air in the exhaust cavity reflowed to the working chamber driven by the pressure difference. The direction of the exhaust process and the rotor rotation was inverse to the reflow, which leads to air pulsation and impact. The pressure pulsation amplitude was about 30 kPa. Although the working chamber was separated from the intake cavity from 120°and disconnected from the exhaust cavity until 180°in the blower containing a backflow design, the pressure in the working chamber was increased by the high-pressure air influx through the backflow piping. In this manner, the pressure difference between exhaust cavity and the working chamber was reduced before the working chamber opened to the exhaust chamber at 180°. The flow impact was reduced because of the small pressure difference. Thus, the pressure pulsation in the exhaust cavity was only 20 kPa, which is less than that of the Roots blower having no backflow design. That the pulsation in the exhaust pressure could be reduced effectively by the backflow design was proved by both simulation and experiment. Figure 10 shows the frequency characteristics of the pressure pulsation in the exhaust cavity. It showed the fundamental frequency of the pressure pulsation was 300 Hz, which was six times more than the frequency of the rotor rotation, because there were six exhausting periods during the 360°rotor rotation. The backflow design can reduce the amplitude of both the fundamental frequency and multiple frequencies.
Effect of backflow on pressure pulsation in the exhaust cavity
To determine the effect of the backflow passage area on the operation of the Roots blower, different areas were simulated. With specific backflow pipes closed or open, the effects of the backflow direction on the operation of the Roots blower were examined. Figure 11 shows the pressure difference between the exhaust cavity and the working chamber at 180°for different backflow passages. As shown in Figure 11, the cases of only horizontal backflow holes, only vertical backflow holes, and both horizontal and vertical backflow holes yielded a horizontal backflow, a vertical backflow, and both horizontal and vertical backflows, respectively. The pressure difference decreased with the increase of the backflow area until the pressure in the exhaust cavity and working chamber were equal. Then, the pressure in the chamber exceeded the exhaust pressure, but it increased slowly. This is because the backflow came from the outlet piping, and pressure remained in the outlet piping. Figure 11 also shows that the variation of the pressure difference was independent of the backflow direction. Figure 12 shows the variation of the pressurepulsation amplitude in the exhaust cavity with respect to the pressure difference. A larger pressure difference caused a larger amplitude of the exhaust pressure pulsation. The pressure pulsation was independent of the backflow direction. A proper size of the backflow passage could lower the pressure pulsation in amplitude by 820%.
Effect of backflow on mass flow rate
How the pressure difference changes with the variation in the mass flow rate is shown in Figure 13. As the pressure difference decreased, the mass flow rate also decreased. This was because the pressure was increased due to a small difference in pressure in the working chamber, which led to a greater air leakage from the working chamber to the intake cavity and reduced the mass flow rate. However, this reduction was slow. The mass flow rate decreased by approximately 12% -from 0.93 to 0.84when the pressure difference decreased from 40 to 0 kPa. Figure 13 shows that the variation of the mass flow rate was independent of the backflow passage direction.
Effect of backflow on shaft power
To determine how the shaft power changed in the working circle, the torque caused by flow force of the upper rotor in the Roots blower was monitored during the CFD calculations. Figure 14 compares the Roots blowers with and without a backflow. The vertical backflow and the horizontal backflow were the same size. The backflow could significantly reduce the peak shaft torque. The torque of the rotor showed a maximum value at 60°( 180°) in the Roots blower without backflow, because the working chamber connects to the exhaust cavity after 180°, which reduces the pressure difference on the lobe and reduces the torque of the rotor and shaft. The pressure difference between the two sides of the lobes in a Roots blower featuring a backflow design was relatively low compared to that which had no backflow feature. This is why the backflow could significantly reduce the peak shaft torque. Considering the rotation rate and the downer rotor, the average shaft powers are shown in Table 4. The results show the vertical backflow yielded the least shaft power, which was 4.1% lower than the shaft power for the horizontal backflow and 13% lower than that for no backflow. This is because the vertical backflow direction agreed with the rotation direction of the rotors.
Conclusion
Based on the numerical simulation and experimental studies above, the following conclusions can be drawn.
(1) The pressure pulsation in the exhaust cavity was reduced by the backflow. As the area of the backflow increased, the pressure pulsation decreased until the pressure in the working chamber reached the same value of the exhaust pressure at a rotor rotation angle of 180°. At this time, the pressure pulsation in the exhaust cavity was reduced by 80%. Then, as the area increased, the pressure pulsation increased slightly. The pressure pulsation was independent of the backflow direction.
(2) The backflow reduced the Roots blower's mass flow rate because it increased the leakage. However, the amplitude reduction of the mass flow rate was not large (approximately 3-12%, depending on the size of the backflow holes). It was helpful for reducing the pressure pulsation even though the mass flow rate was reduced. The mass flow rate was independent of the backflow direction.
(3) The backflow could reduce the power consumption of the shaft in the Roots blower. The power consumption of the shaft in the Roots blower containing a vertical backflow was 4% lower than the blower which had a horizontal backflow because the backflow direction of the first pump agreed with the rotation direction of the rotors.
This study mainly considered the effect of the backflow passages on the pressure pulsation, mass flow rate and power consumption of the shaft. It was treated as a diabatic process without any thermal transmission. Sometimes, the air from the outlet piping was cooled first and then transferred to the cylinder through the backflow passage. In future, it is recommended to analyse how the cool backflow affects the performance of the Roots blower.
Disclosure statement
No potential conflict of interest was reported by the authors .
Funding
This study was supported by the National Natural Science Foundation of China (Research Project 51306136). | 2019-04-22T13:08:50.009Z | 2018-01-01T00:00:00.000 | {
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269303507 | pes2o/s2orc | v3-fos-license | Progress in endophytic fungi secondary metabolites: biosynthetic gene cluster reactivation and advances in metabolomics
Background Fungal endophytes exhibit symbiotic relationships with their host plants but have recently emerged as sources for synthesizing important varieties of secondary metabolites (SMs). Many of these metabolites have shown significant importance as antibacterial, antifungal, antitumor, and anticancer drugs, leading to their explora‑ tion in medicine and pharmaceuticals. Main body of the abstract The endophytes’ biosynthetic gene clusters (BGCs) are responsible for encoding enzymes that produce these SMs. The fungal endophytes’ ability has been challenged due to their inability to trigger cryptic BGCs and their loss of ability to produce secondary metabolites over an extended period in an artificial culture medium. This review investigates the array of SMs produced by endophytic fungi. It identifies methods for awakening and exploiting silent BGCs to produce novel natural metabolites and explores recent advancements in metabolomics platforms used to profile SMs. Silent BGCs can be activated using various methods, including co‑cultivation, one strain of many compounds, epigenetic modification, heterologous expression, and cluster‑specific transcription factor methods. Short conclusion These methods reviewed effectively enhance the production of silent BGCs, leading to a signifi‑ cant increase in secondary metabolite production. Meanwhile, metabolomics profiling using liquid or gas chromatog‑ raphy coupled with mass spectrometry could provide several chances to discover bioactive compounds’ complexity and chemical diversity. This review has, thus, given insight into the significance of methods used to reactivate BGCs from endophytes and the importance of varying techniques of their metabolomic profiling
several secondary metabolites (SMs) similar to those produced by their host plants (Singh and Kumar 2023).
In addition, SMs produced by these organisms provide defence against predators as virulence factors and as transporting and differentiation agents (Kjaerbølling et al. 2019;Jha et al. 2023).The most explored and effective bioactive compounds include antibiotics, antifungal agents, anticancer agents, and immunosuppressants (Zhgun 2023;Lv et al. 2024).However, these metabolites also include toxic compounds such as aflatoxins, known to be highly carcinogenic, and gliotoxins, known to suppress immune function and prevent angiogenesis (Kjaerbølling et al. 2019;Ye et al. 2021).
Most of these SMs are produced in response to genetic signals coordinated by several genes arranged in continuous biosynthetic gene clusters (BGCs) (Pfannenstiel and Keller 2019;Shen et al. 2022).However, only a small fraction of BGCs produce the current naturally derived antibiotics and pharmaceutical compounds.Harvesting these gene clusters has been a major research challenge because they are mostly silent or cryptic, particularly when cultivated under laboratory conditions (Keller 2018;Qi et al. 2021;Zhang et al. 2024).Since many of these inactive genes have been unexplored, studies have suggested that they could represent the largest reservoir of SMs (Okada and Seyedsayamdost 2017;Figueiredo et al. 2021).
To this effect, researchers have devised various methods of activating cryptic BGCs (Scherlach and Hertweck 2021;Hur et al. 2023).Additionally, proper separation and identification of bioactive compounds are essential in discovering and optimizing various bioactive components.Mass spectrometry coupled with LC or GC and NMR-based analysis paves the way for accurate profiling of these metabolites (Panda et al. 2021).Therefore, this review investigates methods of awakening and exploiting silent BGCs to produce novel natural metabolites and explores recent advancements in metabolomics platforms used to profile SMs.
Endophytic fungi and their associated secondary metabolites
The diversity of endophytic fungi with respect to their host plants is as diverse as the variety of individual plant species present globally.Endophytic fungi in plants are primarily Ascomycetes and their anamorphs, although they can also be Basidiomycetes, Zygomycetes, and Oomycetes (Tan et al. 2018;Gong et al. 2019).
Several antimicrobial compounds produced by endophytic fungi are of importance in their effectiveness against pathogens that have developed resistance to antibiotics.However, Hashem et al (2023) have reported that secondary metabolites from fungal endophytes are strongly affected by many factors, such as the sample collection time, environmental conditions, and site or habitat location of plants (extreme habitats were preferred as saline habitats, very high altitudes, rainforests deserts, swamps, and marshes), source of nutrition, tissues of host plant (root, foliar, seeds), types of plant (angiosperms and gymnosperms).Some of the most essential fungal endophytes and their bioactive properties are given below (Table 1): The population of Endophytic fungi is not static, and it has been reported that medicinal plants tend to have a higher incidence of these pharmaceuticals-producing organisms than their nonproducing counterparts (Gioia et al. 2020).Some of them have even been implicated as having the underlying blueprint for the medicinal activity of some of these plants.They are, hence, essential to the existence of these organisms (Gioia et al. 2020).
Mechanisms of secondary metabolites production by biosynthetic gene clusters
Biosynthetic gene clusters (BGCs) are organized in several ways to synthesize secondary metabolites in fungi.According to Zhgun (2023), these gene clusters can be arranged to contain one or more backbone genes or core enzymes responsible for producing the core structure of the resulting metabolites or as genes that encode enzymes which tailor the core to obtain varying products.Thus, the types of SMs produced mainly depend on the type of core enzymes assembled by the genes.These core enzymes, according to Keller (2018), include synthase or synthetase, e.g.terpene synthase, nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), etc., while the tailoring enzymes include hydroxylases, epimerases, methyltransferases, etc.The author further opined that BGCs might contain transcriptional factors that regulate other genes within the cluster, genes that encode a protein that mitigates toxicity and genes with unknown functions (Keller, 2018).
In endophytic fungi, there are four main types of secondary metabolites produced by BGCs: polyketides, terpenoids, alkaloids, and nonribosomal peptides (Pusztahelyi et al. 2015).The mechanisms of production are as discussed below:
BGCs with core genes for nonribosomal peptide synthetase and polyketide synthase
By this mechanisms, nonribosomal peptides and polyketides are created using large modular megasynthases known as NRPS and PKS.These compounds contain catalytic domains assembled into a polypeptide chain required to polymerize amino acids and acyl groups (acetyl-CoA to malonyl-CoA) for NRPS and PKS, respectively (Zhgun 2023).
Core catalytic domains of NRPS
The assembly of a nonribosomal peptide by an NRPS involves a series of repeating steps that are catalysed by the coordinated actions of three core catalytic domains: adenylation, thiolation (or peptidyl carrier proteins, PCP), and condensation (peptide bond formation).Within a module of NRPS are catalytic domains that incorporate a single amino acid.The adenylation, which is the (A) domain, activates the substrate as an aminoacyl-AMP intermediate and subsequent transfer of the amino acid to the 4'-Ppant of the neighbouring thiolation domain; the aminoacyl-AMP once formed is attached to thiolate (T) (peptide carrier protein) domain to form the aminoacylthioester intermediate and transferring them to the condensation (C) domains for catalytic peptide bond formation or chemical modifications.Together, these three core domains comprise a minimal NRPS module.Lastly, they are delivered to a thioesterase (Te) domain that leads to the release of the final product (Miller and Gulick 2016).This fourth domain, a thioesterase, is often found at the C-terminus of the NRPS and catalyses the release of the peptide from the NRPS.This domain catalyses either the hydrolysis of the nonribosomal peptide from the NRPS or the intramolecular cyclization and release of the peptide from the NRPS (Fig. 1).
Core catalytic domains of polyketide synthase
PKS I and II iterative types are more common in fungi.They are called iterative because they can catalyse similar reactions on different substrate sites (Hang et al. 2016).Elaborating further, Zhgun (2023) asserted that types I and II PKS contain a single module that uses catalytic domains cyclically to produce a metabolite.An amino acid is attached to the module by an acyl transferase domain and then polymerized in the acyl transfer protein domain (Fig. 2).The product is then transferred to the start of the module, and the next amino acid is attached (Zhgun 2023).The diverse organisation of modules in NRPS and PKS results in the production of different metabolites from fungi.Thus, future research can investigate how these processes can be exploited to produce medically important natural products.
BGCs with core genes for terpene cyclase
Terpenoids represent the largest group of naturally occurring metabolites with diverse applications and over 80,000 currently known (Bian et al. 2017).They are produced using terpene cyclase (TPC) as the core enzyme.
Hybrid BGCs with genes for different backbone enzymes
Studies have shown that mixed-BGCs contain genes that encode enzymes for the production of hybrids, for example, NRPS/PKS hybrids, which may contain NRPS module parts and PKS module parts on the gene (Zhgun 2023).Robey et al. (2021) noted that the mechanism of action of BGCs with this property is based on inter-polypeptide linkers on the NRPS and PKS ends of the gene terminal.For the PKS region, the KS domain uses malonyl CoA loaded on ACP and supported by AT to elongate an acetyl-CoA starter unit.Once each elongation is completed, the KS, DH, and ER domains begin the b-keto processing steps (Boettger and Hertweck 2013).For the NRPS region, the major steps of the NRPS catalysis occur first.Once completed, the C (condensation) domain of the NRPS catalyses the binding of the polyketide to the amino acid, producing an amide (Fig. 4).This amide can then be released by undergoing a reductive release through Knoevenagel condensation catalysed by the reductase domain (R) to form a pyrrolinone or a Dieckmann cyclization catalysed by the terminal domain (D) to form a tetramic acid derivative (Fig. 5) (Boettger and Hertweck 2013).
Tailoring enzymes
These include a wide array of enzymes that modify the products obtained from the biosynthesis of secondary metabolites.These modifications occur through enzymatic activities, including methylation, epimerisation, oxidative hydroxylation, and translocation (Zhgun 2023).Thus, after the production of the core structure, BGCs also encode these genes that tailor the products into the known biosynthetic compounds.
Reactivation of fungal silent biosynthetic gene clusters
Endophytic fungi exist in diverse tissues and organs of healthy plants and retain an association with their hosts for at least a portion of their life cycle without causing evident symptoms of infection in the host plant.They asymptomatically colonize living tissues of healthy plants (Li and Lou 2017).These characteristics of endophytic fungi provided a new approach to producing bioactive secondary metabolites compounds via industrial fermentation, as well as new ideas and methods for improving the accumulation of bioactive components in medicinal plants and ensuring the long-term development of traditional medical resources.Endophytic fungi directly create beneficial secondary metabolites by fermentation under controlled settings (Wang et al. 2011), but separation from their hosts invariably results in degradation of this capacity (Kusari et al. 2014).These characteristics usually get eroded and are challenging to regain due to endophytic fungi being cultivated alone for a long time.Many studies have shown that the biosynthetic potential of fungi is far from being exploited, with enormous potential for the development of compounds with more chemical novelty and intriguing bioactivities (Gakuubi et al. 2021).The fact that most biosynthetic gene clusters (BGCs) are not transcriptionally expressed is one barrier to describing these undiscovered SMs and that secondary metabolic gene clusters are silenced under standard laboratory conditions (Kusari et al. 2014), accounting for why only a minority of the potential chemical structures is being produced.Such silent genetic loci are called "cryptic" or "orphan" pathways.
Fortunately, numerous techniques, including the one strain many compounds (OSMAC) (Schwarz et al. 2021), heterologous expression technique (Meng et al. 2022), promoter engineering approach (Yu et al. 2020), and other genetic engineering method (Ochi and Hosaka 2012) strategies, have been effectively devised and used for interrogating these silent BGCs and increasing chemical diversity of fungal SM.A growing body of research suggests that microbial co-culture has a higher impact on microbe growth and metabolism than axenic culture (Pan et al. 2019).
Co-cultivation
Microorganisms do not exist in isolation in nature but rather coexist in all settings.They generally interchange and share chemical signals, including SMs.There is widespread agreement that the interaction of microbes is a driving force in creating SMs (Caesarea et al. 2020).One famous example is the unanticipated discovery of penicillin in a culture of Staphylococcus aureus contaminated by Penicillium notatum (Fleming 1929).Since then, hundreds of co-cultivation investigations have been described for mining natural products, particularly novel compounds not previously found in mono-cultivation.
Co-culture, as one of the most widely utilized OSMAC approaches, is a simple and highly efficient strategy for activating silent BGCs for the synthesis of novel SMs.Microbial co-culture approach can boost antibiotic efficacy in crude extracts, raise the yield of known SMs, develop analogues of recognized metabolites, and initiate hitherto undetected bioactive component pathways by replicating naturally existing conditions (Li et al. 2021).
The co-culture strategy is a successful method for awakening quiet BGCs in fungal strains to produce cryptic SMs, and it typically consists of three approaches: fungal-fungal, fungal-bacterial, and fungal-host co-cultures.
Fungal-fungal co-culture
Fungal-fungal co-cultivation activates silent gene clusters and stimulation of natural product production.Citrifelins with a distinct tetracyclic framework were isolated from a co-culture of Penicillium citrinum and Beauveria felina (Meng et al. 2015).The co-culture of two marine fungus activated a rare type of 2-alkenyl-tetrahydropyran and deactivated the antifungal metabolite pyridoxatin, giving methyl-pyridoxatin, revealing a complicated offensive and defensive fungal-fungal interaction (Shang et al. 2017).According to studies by Wang et al. (2022), who carried out fungal-fungal cocultivation of the endophytic fungus Epicoccum dendrobii with the model fungus Aspergillus nidulans or other filamentous fungi, identification and genetic characterization of SMs resulted in the discovery that a partial loss-of-function mutation of VeA is required for mediating coculturewide SM change in A. nidulans.Meanwhile, HPLC analysis and subsequent separation enabled the identification of 14 aspernidine derivatives, including eight new ones, encoded by the active pkf gene cluster.Comprehensive data from the transcriptome and subsequent genetic alterations also demonstrated that the transcription factor SclB regulated SM synthesis via VeA1.This genomic evidence suggests that a VeA1-containing velvet complex mediates SclB activation of quiet gene clusters in the fungal-fungal system.
Moreover, the work by Schroeckh et al. (2009) showed that physical interactions between Streptomyces hygroscopicus and Aspergillus nidulans were significant in activating polyketide biosynthesis, resulting in the creation of orsellinic acid and its anti-osteoporosis derivatives F-9775 A and B.
Fungal-host co-culture
Endophytic microbes are common and have been found in all plant species investigated.The interaction between the host plant Dendrobium officinale and Paraphaeosphaeria verruculosa was found to have a significant influence on the generation of anti-phytopathogenic metabolites (Hu et al. 2020).
In the work of Shipley et al. ( 2020), a new antifungal 2,4-cyclopentadiene-1-one from the co-culture of endophyte-host (Nigrospora oryzae, Irpex lacteus, and the host plant Dendrobium officinale) in PDB and six new anti-feedant polyketides from the co-culture of Paraphaeosphaeria verruculosa and Dendrobium officinale in PDB. Figure 6 is a typical example of the activation of BGC from fungal co-culture.
One strain many compounds (OSMAC) method
One strain many compounds method (OSMAC) is an important method used to improve the production of secondary metabolites and increase the chances of obtaining novel compounds of medical value (Hashem et al. 2023).The principle behind OSMAC lies in the knowledge that microbial strains are diverse and capable of producing several compounds when cultivated under differing conditions, which can lead to the discovery of novel bioactive metabolites (Romano et al. 2018).According to Hewage et al. (2014), endophytic fungi are most suited for this method because several strains of these fungi have been shown to change their metabolite profiles after long storage periods in culture media.Several cultivation parameters must be changed to utilize the OSMAC approach successfully (Zahroh et al. 2022).Hashem et al. (2023) grouped these parameters into physical properties (temperature, aeration, pH) and media composition (carbon and nitrogen source, salinity, trace elements, metal ions).
To demonstrate the efficiency of OSMAC, Bode et al. (2002), who introduced the concept, were the first to show that changing the temperature, aeration, and shape of the culture flask to cultivate Aspergillus ochraceus improved the yield of SMs.This organism would normally produce one metabolite known as "aspinonene", which later produced fifteen new metabolites.More recently, Supratman et al. ( 2019) isolated the endophytic fungus Clonostachys rosea B5-2 and cultivated it on a solid rice media supplemented with apple juice.This resulted in a change in the metabolic processes of the fungus and induced the production of four new compounds, together with the (-)-vertinolide, which it normally produces.Subsequently, research has improved, and fermentation techniques have become advanced, allowing for the combination of different parameters under varying conditions to induce the production of novel compounds (Supratman et al. 2019).
Epigenetic modification
Genome mining has been a significant advancement in detecting cryptic metabolic pathways producing secondary metabolites (Ramesha et al. 2021).Epigenetics is a study area that deals with reversible changes in gene expression without changing the DNA sequence (Singh 2023).Research has led to the use of epigenetics to change the expression of genes that encode the secondary metabolites in microorganisms to produce novel compounds or increase the production of secondary metabolites (Bind, 2021).The process of using genetic mechanisms to change the gene expression of microbes for specific purposes is known as epigenetic modification (Singh 2023).Epigenetic modification includes DNA methylation, RNA interference, and histone posttranslation (Bind et al. 2022).This process involves the use of small molecules (epigenetic modifiers) that inhibit DNA methyltransferase (DNMT), histone deacetylase (HDAC), and histone acetyltransferase (HAT), thereby inducing changes in the chromatin, and activate silent biosynthetic gene clusters to produce a variety of secondary metabolites (Xue et al. 2023).The mechanism of epigenetic modification is shown in Fig. 7. metabolites, while valproic acid produced 10.Although these chemicals have been successful, research needs to focus on understanding biosynthetic genes and pathways to select and maintain the most effective pathways that can produce the best metabolites.
Chromatin remodelling
As with epigenetic modification, chromatin remodelling involves changes or mutations in the heterochromatin structure of microbial genes.These mutations have been exploited to influence secondary metabolism (Pillay et al. 2022).Studies have shown that changes in histone deacetylase activity can lead to the transcription of genes that encode for metabolites of medical and pharmaceutical importance (Ding et al. 2020).Shwab et al. (2007) conducted a study using A. nidulans to determine the impact of histone deletion on secondary metabolism.The authors found that the deletion or inactivation of HDACs activated silent BGCs that utilized novel biosynthetic pathways to produce bioactive secondary metabolites.In A. nidulans, the deletion of hdaA, an HDAC-encoding gene, bypassed the need for laeA (loss of aflR expression), thereby leading to the expression of Penicillin and sterigmatocystin.
This research further expanded the idea that the hdaA gene was a suppressor of BGCs in several filamentous fungi (Pillay et al. 2022).This occurrence has been studied in Calcariporium arbuscular (Mao et al. 2015) and Penicillium (Ding et al. 2020).However, it is essential to note that deletion of the HDAC gene also negatively impacts the growth, differentiation, and survival of fungal cells (Mao et al. 2015).Therefore, strategies should be carefully adopted to ensure that chromatin is remodelled in such a way that the development and proliferation of microorganisms are not affected in the process.
Heterologous expression of BGCs
Heterologous expression of BGCs has been identified as an important process for identifying gene clusters on organisms that are difficult to culture in the laboratory or not easily manipulated (Kjaerbølling et al. 2021).Liu et al. (2021a, b) note that heterologous expression is used to activate silent gene clusters with the potential to produce novel SMs or increase the production of known SMs.According to the authors, the process of heterologous expression involves three steps: cloning the BGCs, engineering them, and transforming them into the desired heterologous hosts.Further, Kang and Kim (2021) noted that selecting a suitable heterologous host remains a critical factor for ensuring the success of a heterologous expression of natural BGCs.The selected host depends on the products targeted and the aim of its application.
To select a suitable host, Xu et al. (2022) asserted that the physiological characteristics of the original host strains, the characteristics of the BGCs, and the required substrate must be considered.Assessing the selected heterologous host based on these criteria helps to determine the closeness to its original strain, which is important as the closer they are, the more likely they are to share similar codon patterns and the more efficient the transcription factors will work (Xu et al. 2022).
In the same vein, Pham et al. (2021) opined that other elements are necessary to ensure the gene expression is successful for the production of secondary metabolites from microorganisms.These include the engineering of new hosts as well as the construction of new clusters DNA constructs, promoter engineering, vector and cloning methods, and ribosomal binding site (RBS) tuning (Knaerbølling et al., 2019;Pham et al. 2021).Traditionally, heterologous gene expression involves constructing large DNA libraries and screening them using PCR to identify clones with important BGCs (Kang and Kim 2021).However, recent advancements in genomic engineering, such as CRISPR-Cas9 and structural biology, have developed new and highly efficient strategies that allow BGCs to be cloned from DNAs without constructing the libraries (Liu et al. 2021a, b).
Cluster-specific transcription factors
Transcription factors (TF) are a group of DNA-binding proteins specific to a genetic sequence and required to modulate gene expression (Wang et al. 2021).Therefore, cluster or pathway-specific genes are transcription factors (CSTF or PSTF) found on specific BGC and are useful for regulating the SMs produced by that BGC.Thus, studies have investigated the possibility of using cluster-specific transcription factors to activate the specific BGCs they're located on (Kjaerbølling et al., 2021).
Over-expression of a CSTF has been successfully used to activate BGCs in filamentous fungi.For this, a study was conducted by Bergmann et al. (2007), who integrated a CSTF of A. nidulans known as apdR on a regulated alcohol dehydrogenase promoter (alcAp) that allowed for the transcription of the target BGC (apd) under controlled conditions.Two novel products were obtained, Aspyridones A and B, and the cryptic PKS-NRPS hybrid pathway involved in their production was elucidated (Wang et al. 2021;Bergmann et al. 2007).However, wang et al. ( 2021) asserted that proper understanding of the regulatory mechanisms of these TFs is important to discovering new SMs, as overexpression does not always activate BGCs.
Other molecular-based strategies that have been demonstrated include RNA polymerase and the manipulation of transcriptional activators and repressors (Begani et al. 2018;Mozsik et al. 2022).Table 2 provides an overview of other strategies used to activate cryptic BGCs and induce the production of metabolites.
Metabolic profiling: mass spectrometry (LC, GC) and NMR methods in the identification of endophytic metabolites
Metabolic profiling provides some chances for discovering the complexity and chemical diversity of bioactive compounds (Gupta et al. 2021).Mass spectrometry (MS)-based metabolic profiling allows for the most costeffective and sensitive elucidation and characterization of known and undiscovered fungal bioactive compounds (Amberg et al. 2017).MS can be used alone by the direct infusion of samples or can be coupled to chromatographic techniques such as liquid chromatography (LC) and gas chromatography (GC) for a high-throughput analysis.LC-MS, GC-MS, and NMR constitute the primary and predominant methods for metabolic profiling.The techniques, analytical software, instrumentation, statistical methods, or computational techniques employed in these analyses are constantly evolving.Therefore, the studies highlighting advances in this field are essential in the discovery of novel bioactive compounds.A study in 2021 by Spina and colleagues demonstrated the identification of compounds in very low concentrations from endophytic extracts in Leucojum aestivum using LC-MS and GC-MS.To fully leverage MS detection, additional orthogonal chromatographic separation is required to distinguish between isomeric and isobaric structures, which cannot be distinguished by MS or MS/MS alone (Harrieder et al. 2022).This section focuses on the methodology and new advancements in the different chromatographic techniques employed in mass spectrometry and nuclear magnetic resonance, highlighting their advantages and shortfalls.
LC-MS
Liquid chromatography-mass spectrometry is one of the most popular and major techniques used in the analysis and profiling of metabolites.The coupling of LC-MS has led to the identification of a wide variety of molecules with varying polarity in complex biological samples.The current preference employed is the reverse phase (RP) separation method representing the most dominant technique used in LC-ESI-MS, however, only covering mid-to nonpolar metabolites.Some examples include nonpolar amino acids like glycine, alanine, tryptophan, tyrosine, and valine.In contrast, hydrophilic interaction chromatography (HILIC) works better for polar metabolites analysis.While HILIC presents a noteworthy alternative for the separation of these metabolites, its adoption remains less prevalent compared to RP chromatography (Harrieder et al. 2022;Gupta et al. 2021).
A mass spectrometer is typically made up of an ion source, a mass analyser, and a detector.The ion source converts sample molecules into ions; the mass analyser resolves these ions in a time-of-flight tube or an electromagnetic field before being evaluated by the detector (Zhou et al. 2012;Plumb et al. 2023).LC-MS was not a vastly considered method due to the limitation that the MS ion sources were not in compatibility with the continuous liquid stream.The development of the electrospray ion source (ESI) resulted in a quantum jump, and several other options are available as ion sources including atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and fast atom bombardment (FAB) (Zhou et al. 2012;Gupta et al. 2021).
According to a study by Laaniste et al. 2019, comparing four ion sources for LC-MS analysis, ESI is the ion source of choice for trace analysis, with ESI obtaining the lowest limits of detection (LoDs), widest linear range, and was less affected by matrix effect (ME).It also offers desorption and ionization of a broad spectrum of molecules directly from the liquid phase, and the large number of ions are created as a result of charge exchange in solution caused by the "soft ionization" provided by ESI; thereby, minute residual energy is retained by the analyte, preventing fragmentation from occurring during ionization (Bowen and Northern, 2010; Banerjee and Mazumdar 2012).Tienaho et al. (2019) reported the use of electron spray ionization source mass spectrophotometry using ultrahigh performance liquid chromatography to identify 220 compounds out of 318 metabolites from hot water extract of endophytic fungi.
Notably, in certain ESI sources, the application of heat is employed to optimize the efficiency of the dehydration process (Clarke 2017).Some shortcomings in using ESI include uneven, compound-specific responses, restricted ionization of nonpolar compounds, and vulnerability to signal alteration, influenced by the sample's matrix.The matrix effect phenomenon can be regarded as an increase (ion enhancement) or decrease (ion suppression) in response, caused usually by an altered ionization efficiency of compounds of interest due to co-eluting analytes in the same matrix.Although low in ESI, ME influences the reliability, linearity, and accuracy, potentially leading to unreliable quantification.In ESI, matrix effect can manifest through various mechanisms, like the competition between matrix constituents and target analytes for the accessible charges in the liquid phase (Beccaria and Cabooter 2020).
The sensitivity and resolution of a mass spectrometer are largely dependent on the mass analyser employed in ion separation.There are two categories of mass analysers: the high (Orbitrap) and low resolution (mainly quadrupole analysers).The distinction lies in their capability to discern compounds with minute mass difference.High-resolution analysers are particularly vital in untargeted metabolomics, where they play a crucial role in elucidating the chemical composition of complex and unknown mixtures.On the contrary, targeted metabolomics emphasizes the quantification of a specific product ion, leading to heightened sensitivity and specificity by focusing on one or a few more m/z values.This distinction in mass analysers is a powerful tool in advancing our understanding of bioactive compounds and their role in various applications (La Barbera et al. 2017;Segers et al. 2019).Nagarajan et al. (2021) also reported that using high-resolution mass spectrophotometry with liquid chromatography (LC-HRMS) to identify metabolites from endophytic fungi has the unique advantage of accounting for both target and nontargeted metabolites.
On the other hand, tandem mass spectrometry (MS/ MS) involves the integration of multiple mass analysers within a single mass spectrometer.This configuration enables the consecutive separation of ions, followed by their fragmentation in between the separations.The most commonly used MS/MS analyser for quantification is the triple quadrupole (QqQ).The first quadrupole quantifies the precursor ions generated by the ionization source; isolated precursor ions are then moved to the collision cell or second quadrupole, where they undergo fragmentation, and then the third quadrupole selects a particular or an array of product ions.The Orbitrap analyser may also operate in MS/ MS mode; it operates by using an oscillating field to store ions between external electrodes, which combines a nonselective full-scan MS spectrum with high mass accuracy.According to a recent study by Pan et al. 2020, a high-resolution Orbitrap MS can produce approximately 500,000 resolving power (at m/z 200).Generally, a quadrupole time-of-flight (Q-TOF) is the most commonly used mass spectrometer in untargeted metabolomics and can also be used for quantification.Another recent and emerging technique is employing the ion mobility spectrometer (Niessen and Falck 2015;Rozanova et al. 2021).
LC-MS-based methods make up for some shortfalls in GC-MS analysis in its compatibility with highly volatile and thermally unstable metabolites.LC-MS/MS offers the advantage of streamlining sample processing, bypassing complexity, and time-consuming sample preparation.Furthermore, it facilitates the separation and identification of elusive drug metabolites, thereby enhancing analytical specificity, and it significantly improves signal-to-noise ratios and sensitivity through the application of multi-reaction detection (Liu et al. 2021a, b).
The components in RP-LC are separated based on their hydrophobicity through adsorption to the stationary phase.The stationary phase is nonpolar, typically made of either porous silica linked to alkyl chains (C4, C5, C8, and C18) or divinylbenzene (DVB), an inert nonpolar compound.For the more intact proteins, the shorter alkyl chains (C4 and C8) are preferred as they are considerably less retentive than the longer alkyl chains, and longer chains like C18 are typically used for the low hydrophobic proteins (≤ 10 kDa); this is because larger proteins have higher hydrophobicity and as such, interact strongly with the C18 matrix (Boone and Adamec 2016;Gupta et al. 2021).The hydrophobic stationary phase in the polar mobile phase adsorbs to the hydrophobic stationary phase as the sample is added to it, allowing the more hydrophilic molecules to be eluted first.Upon increasing the proportion of organic solvent, chloroform, water, ethanol, acetonitrile or methanol, acetonitrile being the most commonly used in the mobile phase, the polarity decreases, leading to a reduction in hydrophobic interaction between the stationary phase and solutes.This alteration allows for the effective elution of solutes.When dealing with more hydrophobic solutes, a higher concentration of organic solvent is essential in the mobile phase in order to achieve successful elution.
GC-MS
GC-MS stands out as another effective, consistently dependable, and robust tool extensively utilized in metabolic profiling and research.Its reliability stems from the electron impact (EI) hard ionization technique, which ensures consistent molecular fragmentation, making GC-MS a unique asset for identifying metabolites.GC stands as the foremost analytical technique for the separation of volatile compounds (Zeki et al. 2020).
The separation process is dependent on the disparities in both the boiling points and polarity of the analytes.Subsequently, the separation of analyte ions is executed based on their mass-to-charge ratio (m/z).The stationary phase employed is important to a selective analysis and is based on the polarity of the analytes; the stationary phase can be polar, semi-polar, or nonpolar, and the nonpolar stationary phase is the predominant phase used in GC-MS (Zeki et al. 2020;Prodhan et al. 2019).In all EI devices, ionization occurs at 70 electronvolts (eV) and this is achieved when the output flows into a heated ionization source under high vacuum conditions, where collector voltage extracts electrons from a tungsten filament, the voltage applied to the filament dictates the energy of the electrons.These high-energy electrons excite the neutral analyte molecules, leading to ionization and fragmentation (Gupta et al. 2021).Kanjana et al. (2019) carried out the GC-MS analysis of ethyl acetate extracts of fungal endophytes Chaetomium globosum, Cladosporium tenuissimum, and Penicillium janthinellum; the study confirmed the presence of biologically active phytocompounds in these endophytes and the pharmacological abilities of the host medicinal plants: Passiflora foetida, Memecylon edule, and Justicia adhatoda.
Chemical ionization (CI) technique is also utilized, albeit, not as frequent or preferred compared to the electron impact technique.This is because 7 fragments obtained in CI are limited, and libraries available for the analysis and identification are also limited.Its utilization in untargeted metabolomics is due to the soft ionization technique, applying low energy to the molecules, thereby revealing critical information on the molecular weight of the metabolite (Prodhan et al. 2019).
The mass spectra are generally regarded as consistent across instruments made by various manufacturers and across instruments equipped with different types of mass analysers, such as quadrupoles, time of flight, and so on (McNair et al. 2019) The spectra acquired are then carefully compared with established standards such as National Institute of Standards and Technology (NIST) library, Golm library, Metlin, MassBank, and Fiehn library for precise assessment (Schauer et al. 2005).Matyushin et al. (2020) also highlighted the application of deep learning ranking for the identification of small molecules using low-resolution electron ionization mass spectrometry.
The synergy between GC and MS systems is highly harmonious.A study by Farhat and colleagues (2022) using GC-MS analysis resulted in the identification of several compounds, some of which were not reported before from Fusarium solani.The capability of GC-MS to examine a vast number of compounds while being furnished with a standardized library of metabolite spectra, facilitating rapid and precise qualitative assessment of metabolites has made it a frequently used method in metabolomics.The hard ionization, electron impact utilized leads to the production of unique fragmentation patterns for identifying metabolites.The development of commercial and in-house libraries for metabolite identification gives GC-MS an edge over LC-MS.The primary challenge of the GC-MS systems lies in the necessity to decrease the atmospheric pressure within the GC to a vacuum level ranging from 105 to 106 Torr before introducing samples into the MS.This coupling process, characterized by a significant pressure reduction, is achieved through the use of an interface.A common interface in use today is the fused silica tubing.Other drawbacks in the GC-MS systems include its incompatibility with less volatile compounds (McNair et al. 2019;Liu et al. 2021a, b).
NMR
Nuclear magnetic resonance (NMR) spectroscopy entails the examination of nuclei by observing how they interact with electromagnetic radiation of radiofrequency when positioned within a robust magnetic field.NMR analyses compounds by utilizing hydrogen spectrum ( 1 H NMR), carbon spectrum ( 13 C NMR), or phosphorus spectrum ( 31 P NMR), thereby providing insights into molecular properties (Wang et al. 2023).
NMR simplifies chromatographic separation, chemical derivatization, and sample treatment.It is easily measurable, nondestructive, and impartial, making it essential for identifying new compounds (Talukdar et al. 2021).In addition to its other advantages, NMR offers exceptional reproducibility, requiring minimal sample preparation.It employs a quantitative approach without targeting specific compounds, is highly automatable, and enables high-throughput analysis of large-scale compounds without the need for standards.Compound like organic acids, polyols, alcohols, sugar, and many other highly polar compounds with low detection using LC-MS technique can be detected using NMR.NMR has proven to be highly valuable in the provision of detailed insights into the chemical composition, structural elucidation, and molecular identification of endophytic fungal metabolites.Notably, NMR exhibits remarkable detection efficiency, capable of identifying metabolites in solution at concentrations greater than 1 µM, even for compounds not previously reported and with little or no prior documentation.However, these advantages are sometimes overturned by the fact that analytical techniques, like LC-MS and GC-MS, are comparatively more sensitive than NMR, with a lower limit of detection (10 to 100 times better) (Emwas et al. 2019;Gupta et al. 2021).The advantages and limitations of MS and NMR spectroscopy as used as an analytical tool in endophytic metabolite profiling are shown in Table 3.
Conclusions
In conclusion, while there may have been deliberate attempts by various scientists to elucidate more on endophytic fungi, it remains that it may not be completely exhaustive as they are diverse as the individual species of plants exist in all parts of the world.However, for the benefits they hold, continuous research into the benefits they possess, the organisms responsible for such antimicrobial action, and the nature of such compounds remain work infinitum.Extensively, researchers reported reactivation using approaches of co-culture and OSMAC probably because of the ease and cost compared to other genetic-based methods, which are more expensive and require appropriate expertise.NRPS and PKS could be organized in varied ways in fungi, resulting in diverse metabolites that can be produced.Furthermore, since all the metabolic profiling methods discussed are effective, albeit with varied shortcomings, the method chosen will depend on the desired results.Indeed, endophytic fungi present an interesting field of research, leaving us to continually explore the benefits inherent in this beautiful piece of nature.
Fig. 1
Fig.1Mechanism of action of the core domains in the synthesis of NRPS(Felnagle et al. 2008)
Fig. 5
Fig.5Release mechanisms of polyketide-amino acid hybrids (tetramic acids, black) and (pyrrolinones, grey)(Boettger and Hertweck 2013) Furthermore,Hashem et al. (2022) asserted that epigenetic modification also involves the overexpression of the activator or repressor genes or the deletion of some genes, which leads to genetic changes.The most common and successful HDACs and DNMTs used include suberoylanilide hydroxamic acid (SAHA), sodium butyrate, valproic acid, and 5-azacytidine, respectively (Makhwitine et al. 2023).Ramesha et al. (2021) conducted a study to determine the effect of epigenetic modification on developing secondary metabolites by Nigrospora sphaerica.Sodium butyrate showed the highest induction of cryptic metabolites (22); SAHA produced 19 new
Table 1
Endophytic fungi and their bioactive properties
Table 2
Strategies used to activate silent BGCs
Table 3
Advantages and limitations of MS and NMR spectroscopy as an analytical tool in profiling of endophytic-derived metabolites | 2024-04-24T13:06:37.450Z | 2024-04-23T00:00:00.000 | {
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118732495 | pes2o/s2orc | v3-fos-license | Determination of neutrino masses from future observations of CMB B-mode polarization and the growth of structure
Constraints on neutrino masses are estimated based on future observations of the cosmic microwave background (CMB) including the B-mode polarization produced by CMB lensing using the Planck satellite, and baryon acoustic oscillations distance scale and the galaxy power spectrum from all-sky galaxy redshift survey in the BigBOSS experiment. We estimate the error in the bound on the total neutrino mass to be $\Delta\sum m_{\nu} = 0.012$ eV with a 68% confidence level. If the fiducial value of the total neutrino mass is $\sum m_{\nu}= 0.06$ eV, this result implies that the neutrino mass hierarchy must be normal.
Terrestrial experiments such as tritium beta decay [3] and neutrinoless double-beta decay [4] give upper bounds on the absolute neutrino masses. Cosmological observations could further constrain neutrino properties by providing a more stringent bound on the total neutrino mass m ν and the effective number of neutrino species N ν . Cosmic microwave background (CMB) anisotropies are generated mainly until eras before the last scattering surface of the decoupling epoch (z ∼ 1089). Therefore, if neutrinos are as massive as m ν > ∼ 1.5 eV, they become nonrelativistic before the recombination epoch. If so, a finite-mass neutrino significantly affects the CMB spectrum. For masses below m ν < ∼ 1.5 eV, the neutrinos alter the CMB spectrum chiefly through their effect on the angular diameter distance out to the last-scattering surface. The effect is degenerate with other cosmological parameters such as the matter energy density parameter Ω m0 and the Hubble constant h [5]. In that case, other cosmological probes complementary to CMB are needed to break the parameter degeneracy in order to study the small mass scales of neutrinos.
The B-mode polarization due to CMB lensing provides detailed information. It has better sensitivity to neutrino masses smaller than 0.1 eV. This resolution is indispensable to distinguish between a normal and an inverted hierarchy.
The main effects of massive neutrinos on the growth of matter density perturbations arise from two physical mechanisms [6]. First, a massive neutrino becomes nonrelativistic at the transition temperature, and contributes to the energy density of cold dark matter. That changes the matter-radiation equality time and the expansion rate of the universe. Second, the matter density perturbations are suppressed at small scales by neutrino free-streaming. Neutrinos travel at the speed of light as long as they are relativistic, and the free-streaming scale is nearly equal to the Hubble horizon. Therefore, the free-streaming effect suppresses perturbations below such scales.
Neutrino masses from cosmology have been studied by combining observations of CMB anisotropies with Galaxy Clustering [7][8][9], Weak Lensing [10], and the Lyman-α Forest [11,12], and so on. The Planck CMB temperature power spectrum with WMAP polarization constrains the sum of the neutrino masses to be m ν < 0.933 eV (95% C.L.) [13]. By combining the Planck temperature data with WMAP polarization and the high-resolution CMB data and the distance measurements from the baryon acoustic oscillations (BAO), an robust upper bound of m ν < 0.230 eV has been reported [13]. In Ref. [14], by focusing on ongoing and future observations of both the 21-cm line and the CMB B-mode polarization, sensitivities to the effective number of neutrino species, total neutrino mass, and neutrino mass hierarchy are studied.
In this letter, as the most effective means of using the effect of massive neutrinos in cosmology, Planck data [15,16] from ongoing CMB observations is used, including the B-mode polarization from CMB lensing, and the BigBOSS experiment [17,18] is adopted for future observations of BAO and the galaxy power spectrum with an all-sky galaxy redshift survey. By comparing the observational data with models, the errors in the bounds on the total neutrino mass ∆ m ν are accurately estimated.
Here a flat ΛCDM model with two additional parameters of the total neutrino mass m ν and the effective number of neutrino species N ν is assumed. The fiducial values of the parameters are listed in Table I. For the fiducial value, we adopt the value of the maximum likelihood parameters obtained from the Planck temperature data with WMAP polarization at low multipoles [13] for the six parameters other than m ν and N ν . For the latter two, standard values are adopted. The neutrino mass is related to the neutrino density parameter by Ω ν h 2 = Σm ν /(93.04 eV). The CMB temperature is taken to be T CMB = 2.7255 K [19]. The primordial he-lium fraction Y P is a function of Ω b h 2 and N ν using the Big Bang nucleosynthesis consistency condition [20,21]. Here Ω b h 2 is the baryon density today. Ωch 2 is the cold dark matter density today, 100θMC is 100 × approximation to r * /DA (CosmoMC), τ is the Thomson scattering optical depth due to reionization, ns is the scalar spectrum power-law index, ln (10 10 As) is the log power of the primordial curvature perturbations, Σmν is the sum of the neutrino masses in eV, and Nν is the effective number of neutrino-like relativistic degrees of freedom.
Planck is the third CMB observation satellite, following COBE and WMAP. It is possible to take all of the information in the CMB temperature anisotropies, to measure the polarization of the CMB anisotropies to high accuracy. Planck also provides the thermal history of the universe during the formation of the first stars and galaxies. Polarization measurements may detect the signature of gravitational waves generated during inflation [22].
This letter uses data from Planck for CMB observations including B-mode polarization due to CMB lensing. The satellite was launched in May 2009. In March 2013, initial cosmology results based on the first 15.5 months of operations were released [13,15,16,, with analysis of temperature data but not of detailed polarization results. Detailed polarization data are scheduled to be released in 2014. Here we use mock data of the polarization of the CMB anisotropies generated by the code FuturCMB [48]. Experimental specifications assumed in the computation are summarized in Table II Here f sky is the observed fraction of the sky, ν is the observation frequency, θFWHM is the angular resolution defined as the Full-Width at Half-Maximum, ∆T is the temperature sensitivity per pixel, and ∆P is the polarization sensitivity per pixel.
BigBOSS [17,18] is a Stage IV ground-based experiment to probe BAO and the growth of large-scale structures with a wide-area galaxy and quasar redshift survey. The experiment is designed to map the large scale structure of the universe. The resulting 3D sky atlas will contain signatures from primordial BAO that set "standard ruler" distance scales. Using the BAO signature, BigBOSS will measure the cosmological distance scale to better than 1% accuracy, revealing the expansion history and growth of structure in the universe at the time when dark energy began to dominate. Co-moving volume and number of galaxies for BigBOSS is an order of magnitude larger than those for BOSS, and will measure the Hubble parameter H(z) and angular diameter distance d A to < 1% accuracy. The BigBOSS BAO experiment will provide a distinct improvement in the Dark Energy Task Force figure of merit over all previous Stage III BAO experiments combined. As a precision cosmological probe, Dark Energy Science will be enhanced, and the neutrino mass and inflation will be studied.
In this letter, for observations of BAO distance scales and the galaxy power spectrum, we adopt BigBOSS parameters. We make mock data of the BAO distance scale and the galaxy power spectrum according to the projected error in the BAO distance scale and the galaxy power spectrum from BigBOSS shown in Fig. 1 of Ref. [49].
Using the above mock data, the Markov-Chain Monte-Carlo (MCMC) method [50,51] is used to search cosmological parameter estimations in the multidimensional parameter space of cosmological observables. The error bounds on the cosmological parameters are estimated. Recently, the Fisher matrix has become standard for estimations of errors in cosmological parameters for future observations. However, when the phenomena are not Gaussian distributed (such as in the case of strong parameter degeneracies), the Fisher matrix formalism loses validity, as described in Ref. [48]. Because all parameter likelihoods not always possible to approximate a Gaussian distribution, we use Monte Carlo simulations based on the publicly available CosmoMC code [50,51] with mock observational data.
The cosmological parameter ranges to be explored with MCMC are listed in Table III. "High accuracy default" and "accuracy level" 5 are implemented in CAMB [52,53]. We use HALOFIT [54,55] to include nonlinear effects in the evolution of the matter power spectrum. The chains have 1,000,000 points in CosmoMC.
In Fig. 1, the probability distribution of the total neutrino mass m ν in eV is plotted from observational data of CMB including B-mode (Planck), with the addition of BAO (BigBOSS), and finally with the addition of both BAO and the galaxy power spectrum (BigBOSS). The fiducial value of the total neutrino mass is m ν = 0.06 eV, whereas other parameters are marginalized. Figure Here fν is the fraction of the dark matter that is in the form of massive neutrinos. Other symbols are the same as in Table I. 2 enlarges the red curve from Fig. 1. (CMB + BAO + Galaxy Power Spectrum) The parameter degeneracies are efficiently broken by adding the CMB including B-mode (Planck) data to the BAO and the galaxy power spectrum (BigBOSS) data. In summary, from the combination of the ongoing CMB observations, including the B-mode polarization due to CMB lensing (Planck), and the future observations of the BAO distance scale and galaxy power spectrum (BigBOSS), the error in the bound of the total neutrino mass is estimated to be ∆ m ν = 0.012 eV with a 68% confidence level. This prediction is the most stringent bound ever, portending accurate determination of neutrino masses.
Our results of the errors of the bounds are slightly tighter than the estimations using Fisher matrix in Refs. [17] and [56]. The Monte Carlo simulations tend to predict slightly tighter bound on the parameters than the case of the Fisher matrix formalism, as seen in Table 1. of Ref. [48].
It is known that the total neutrino mass is m ν > ∼ 0.1 eV in case of an inverted hierarchy. Hence, if the fiducial value of the total neutrino mass is m ν = 0.06 eV, as seen in Fig. 2, our result implies that the neutrino mass hierarchy must be normal. This work is supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant Number 25400264 (KH)). * Electronic address: k hirano@tsuru.ac.jp | 2013-09-11T15:41:35.000Z | 2012-12-27T00:00:00.000 | {
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8045829 | pes2o/s2orc | v3-fos-license | Arrangement of subunits in microtubules with 14 profilaments.
The structure of 14-protofilament microtubules reassembled from dogfish shark brain tubulin was analyzed by high resolution electron microscopy and optical diffraction. The simultaneous imaging of the protofilaments from near and far sides of these tubules produces a moiré pattern with a period of approximately 96 nm. Optical diffraction patterns show that the 5-nm spots that arise from the protofilaments for the two sides of the tubule are not coincident but lie off the equator by a distance of 1/192 nm-1. These data provide evidence that in reassembled microtubules containing 14 protofilaments, the protofilaments are tilted 1.5 degrees with respect to the long axis of the tubule, giving a left-handed superhelix with a pitch of 2.7 micron. The hypothesis is that the tilt of the protofilaments occurs to accommodate the 14th protofilament. It is determined that when the 14th protofilament is incorporated, the 3-start helix is maintained, but the pitch angle changes from 10.5 degrees to 11.2 degrees, the angle between protofilaments measured from the center of the microtubule changes by 2 degrees, and the dimer lattice is discontinuous. These observations show that the tubulin molecule is sufficiently flexible to accomodate slight distortions at the lateral bonding sites and that the lateral bonding regions of the alpha and beta monomers are sufficiently similar to allow either alpha-alpha and beta-beta subunit pairing or alpha-beta subunit pairing.
The number of protofilaments in a microtubule assembled from temperature-cycled tubulin (17) can differ from the number of protofilaments in the native microtubule (22). Several studies have shown that the predominant population of reassembled microtubules contain 14 protofilaments (9,17) . The arrangement of subunits in the native microtubule that contains 13 protofilaments is known to be a 3-start left-handed helix with a helical pitch angle of 10.5°(2, 3, 7,8,14) . The change in this arrangement of subunits when the 14th protofilament is incorporated into the tubule is not known . Our observation that negative contrast electron microscope images of reassembled shark brain tubules display a moiré pattern and that these tubules contain 14 protofilaments prompted a study of the helical surface lattice of the tubulin subunits (12).
The results show that when the 14th protofilament is incorporated into reassembled tubules, it causes the tubule to twist into a shallow superhelix. The 3-start helical family is retained but the pitch angle ofthe helix changes by 0.7°to accommodate the additional protofilament. The retention ofthe 3-start helix requires that the dimer lattice be discontinuous. Therefore, the reassembled microtubule with 14 protofilaments contains elements ofboth the A-tubule and the B-tubule dimer lattice .
These fmdings are consistent with the recently reported data of McEwen and Edelstein (l5). These authors used electron microscopy, in conjunction with computer analysis based on THE JOURNAL OF CELL BIOLOGY -VOLUME 87 NOVEMBER 1980 521-526 C The Rockefeller University Press -0021-9525/80/11/0521/07 81 .00 Fourier transforms, to obtain evidence in favor of a 3-start monomer helix and a discontinuous dimer lattice which combines elements of both the A-and B-type lattices.
MATERIALS AND METHODS Dogfish brain tubulin was prepared by the temperature-dependent assemblydisassembly procedure of Shelanski et al . (18) as previously described (13) . The polymerization solution contained 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.60, 1.0 mM EGTA, 0.5 mM MgCl2, and 1.0 mM GTP. Glycerol (25%) was added to the crude extract to enhance polymerization because very little polymerization occurred without it . Because polymerization proceeds without glycerol in subsequent polymerization steps, it was not used for assembly after the first step of the purification process.
SDS-polyacrylamide gel electrophoresis was performed according to the procedures of Bryan (5) and Stephens (20). 5% gels, 12 cm in length, were run in 25 mM Tris-glycine buffer, pH 8.3, containing 0.1% SDS. The gels were stained with Coomassie Brilliant Blue and scanned at 560 nm with a Zeiss PM 10 spectrophotometer (Carl Zeiss, Inc., New York).
The electron microscope grids used in this study were coated with only a carbon film by a procedure described by Linck and Amos (14). 400-mesh grids were coated first with a plastic film made from 0.25% Formvar and then a thin film of carbon. The Formvar film was removed by floating the Formvar-carboncoated grids over dichloroethane, which dissolves the Formvar, leaving only a carbon film over the grid holes. Such carbon-coated grids gave much improved resolution by the negative-staining procedure . To stain for microtubules, a drop of the solution containing tubules was placed on the carbon-coated grid for 20 s, after which the grid was rinsed with four to five drops of buffer and stained with several drops of 1% uranyl acetate. Excess stain was removed by blotting the edge of the grid with filter paper. Stained grids were observed as soon as possible in a 1EOL-1005 electron microscope (EM) . The tannic acid staining procedure described by Tilney et al. (22) was used to establish the number of protofilaments in these tubules.
For optical diffraction studies, images of microtubules were transferred from the original EM film to photographic glass plates. Segments of microtubules were selected and prepared for diffraction by masking out unwanted areas of the plate. The diffraction analysis was made with an optical difractometer (10,11) in the laboratory of R. W. Linck, Harvard Medical School, Boston, Massachusetts. The optical transforms were recorded on photographic film, using a Hasselblad 2 x 2 single-lens reflex camera . Optical filtering was performed by the method of Klug and DeRosier (11).
RESULTS
Tubulin Assembly Without Accessory Proteins Dogfish brain tubulin preparations contain little or no accessory protein after purification by two cycles of the temperature-dependent assembly-disassembly procedure of Shelanski et al. (l8) . Such twice-cycled tubulin preparations (C 2S tubulin) analyzed for purity by SDS-polyacrylamide gel electrophoresis (13) show a-and ß-tubulin bands but no protein bands in the high and intermediate molecular weight ranges corresponding to microtubule-associated proteins (l, 4,6,16,19,23). A typical gel profile is shown in Fig. l. C2S tubulin preparations, which contained no detectable amounts of accessory proteins, were warmed to induce polymerization and the reconstituted microtubules were negatively stained for use in the study that follows.
Structure of Reconstituted Microtubules
Dogfish brain microtubules negatively stained and examined in the electron microscope display a twist of shallow pitch. The twist can be seen by sighting along the axis of the tubule or by tracing along the length of a protofilament at the edge of the tubule (Fig . 2) . One sees that the protofilaments lie at a slight angle to the longitudinal axis of the tubule .
In addition to the twist, a moiré pattern is seen along the axis of the tubule. The pattern consists of a set of two alternating bands or striations that repeat at regular intervals along the tubule . The striation pattern represents regions along the tubule where the protofilaments appear alternately distinct and indistinct. The striation containing obscure protofilaments appears SDS-polyacrylamide gel of dogfish brain tubulin after two cycles of purification (C zS tubulin) . A photograph of the gel is shown below the densitometer tracing. a-and J3-tubulin bands are present but no accessory protein bands. Electron micrographs of negatively stained dogfish brain microtubules . The tubules contain a shallow twist with pitch between 1 and 3 pm . The twist can be seen by sighting along the axis of the tubules or by following the path of protofilaments at the edge of the tubules. The tubules are flattened and measure 34-36 nm in width. Six to seven protofilaments can be counted across the width of the tubule . Bar, 50 nm . a-d, x 125,000 . as though the protofilaments at the back of the tubule project exactly between those at the front. When this happens, the grooves between protofilaments are not discernible and the path of individual protofilaments and the tubulin subunits are difficult to identify . The adjacent striation, containing distinct protofilaments, appears as though the protofilaments on the front of the tubule are exactly superimposed on protofilaments at the back . In the latter stripe, the globular substructure of the protofilaments is very evident and individual tubulin monomers are seen . The two bands of the moiré pattern alternate along the axis of the tubule (Fig. 3) . Each band is -48 nm long, inclined at an angle of -40°, and repeats at -96-nm intervals along the length of the tubule .
The presence of the moiré pattern is supporting evidence that the protofilaments lie at an angle to the long axis of the tubule . The period of the pattern is a measure of the degree of Electron micrograph of a tubule that shows several periods of the 96-nm axial repeat . A periodic banding pattern can be seen in the wall of this tubule (sight along the axis of the tubule) . The pattern appears as helical bands or striations containing protofilaments that appear alternately distinct and indistinct at regular intervals along the tubule . Bar, 50 nm . x 250,000.
tilt of the protofilaments. The moiré pattern occurs because the protofilaments from both sides of the microtubule are imaged simultaneously. The tilt of the protofilaments, such that they no longer lie parallel to the longitudinal axis but follow a helical path along the tubule, is defined in this paper as the superhelix of the microtubule.
The superhelix is thought to arise because these tubules contain one more than the usual number of protofilaments found in native microtubules . Tannic acid staining of reassembled shark brain tubules shows that -r95% of them contain 14 protofilaments (Fig. 4) . The other 5% of the microtubules contain either 13 or 15 protofilaments in roughly equal numbers . The hypothesis is that the twist in microtubules with 14 protofilaments occurs to accommodate the extra protofilament . It is not known whether a twist is present in reassembled microtubules with 15 protofilaments. The assumption is that in vitro assembled tubules with 13 protofilaments, like flagella Atubules, do not contain a twist . Optical diffraction was used to FIGURE 4 Electron micrograph of a thin section of a pellet of microtubules stained with tannic acid . The protofilaments making up the wall of the tubules can be seen . 95% of the tubules in a typical section contain 14 protofilaments . The protofilaments do not appear with equal clarity around the entire circumference of the tubule. This occurs because the protofilaments are inclined at a shallow angle to the long axis of the tubule. Theretore, when the plane of section is perpendicular to the protofilaments on one side of the tubule, it is at an oblique angle to the protofilaments on the opposite side.
determine the changes that take place in the arrangement of subunits when the 14th protofilament is incorporated into the reconstituted microtubule .
Optical Diffraction of Microtubules
A microtubule that is twisted into a superhelix should give rise to a diffraction pattern that differs in a predictable way from the normal microtubule . In a 13-protofilament microtubule, the protofilaments from the near and far sides are parallel to the cylinder axis and the equatorial diffraction spots that arise from the protofilaments are coincident. However, if the protofilaments have a helical orientation with respect to the cylinder axis, as appears to be the case for 14-protofilament tubules, the diffraction spots that arise from the protofilaments should appear off the equator (11) .
The pitch angle of the superhelix that is computed based on measurements from the diffraction patterns should agree with the pitch angle determined from measurements of the period of the moir6 pattern . Specifically, for a moiré repeat of 96 nm, the equatorial spots should be split into two spots at a spacing of 1/192 nm-' above and below the equator . This corresponds to a slight twist or superhelix of the protofilaments, at an angle of tan -' (5/192) = l .5°from the microtubule axis.
The diffraction pattern obtained from the tubule shown in Fig . 3 is shown in Fig. 5 . The diffraction pattern shows two orders of the 5-nm equatorial spots and has four spots on the 4-nm layer line . These aspects of the pattern are typical of a 13-protofilament microtubule, but the equatorial spots arising from the protofilaments for the two sides of the 14-protofilament tubule are not coincident but are separated and lie above and below the equator . They are separated from each other by a distance of 1/96 nm-' and from the equator by 1/192 nm-' as predicted by the moiré pattern .
The off-equatorial spots for the two sides do not image at precisely the same distance from the origin. This difference in position occurs because one side of the tubule partially collapses, squeezing the filaments on that side together . Erickson (7) has shown that the near side or the side away from the carbon film is the one that flattens and shrinks . On this basis the off-equatorial reflections that arise from the near side and the ones that arise from the far side of the tubule can be established . Based on this determination and on the assumption that the basic monomer helix in the reassembled tubule is lefthanded, as is the case in the native microtubule that contains 13 protofilaments, it is possible to establish the handedness of the superhelix. The diffraction pattern shows that the equatorial reflections for a given side lie in the same quadrant of the diffraction pattern as the 4-nm spots for the monomer helix of that side. This is proof that the superhelix and the monomer helix are of the same hand, i .e., left-handed.
In summary, the diffraction patterns show that in reassembled shark brain tubules containing l4 protofilaments, the protofilaments are tilted 1 .5' with respect to the long axis of the tubule, giving a left-handed superhelix with a pitch of 2 .7 fm.
The electron microscope images and the optical diffraction data are consistent with the interpretation that microtubules with 14 protofilaments are slightly twisted into a superhelix of shallow pitch. The hypothesis is that the tilt of the protofilaments occurs to accommodate the 14th protofilament . These data provide a means to establish the changes that take place in the subunit lattice when the 14th protofilament is incorporated into the tubule . There are three parameters that may change when the 14th protofilament is incorporated into the tubule : (a) the number of starts in the monomer helical family; (b) the pitch angle of the monomer helix; and (c) the tilt of the protofilaments . Changes in these three parameters do not require changes in the spacing of the subunits along the protofilaments or the spacing between the protofilament but they do require changes in the lateral bonding between subunits .
If one assumes, as a first approximation, that the pitch angle of the monomer helix in the 14-protofilament tubule is the same (10 .5°) as the pitch angle for the monomer helix in the 13-protofilament tubule, one can calculate the tilt of the protofilaments and the corresponding moiré pattern required to accommodate the 14th protofilament . The calculated tilt of the protofilaments and the period of the moiré patterns are shown in Table I for the 2-, 3-, and 4-start helices . These three helices are chosen because they require the smallest departure from the lattice parameters of the 13-protofilament tubule . It is seen that neither the 2-, 3-, or 4-start helix generates a moiré pattern with a period of 96 rim, the period of the observed moiré pattern. Therefore, one can conclude that the native subunit helix pitch angle of 10.5°must be distorted slightly in the 14protofilament tubule . This is reasonable because the lateral bonds that define this helix are relatively weak and may be easily distorted .
If one fixes the value of the protofilament tilt at 1 .5°(the Table II . It is seen that only the 3-start helix gives both a lefthanded superhelix and a pitch angle for the basic subunit helix that is reasonably close to the 10 .5°pitch of the normal, 13protofilament microtubule . By this process of elimination, it is possible to conclude that the 3-start helical family is maintained in the 14-protofilament tubule but the pitch angle changes from 10 .5°to 11 .2°. This model is diagramed in Fig . 6 . These data support the conclusion that the angle of the lateral bonds between subunits in the reassembled tubules with 14 protofilaments is distorted slightly from the lateral bond angle in native 13-protofilament tubules. In addition, it should be noted that the angle between protofilaments measured from the center of the microtubule must change from 27 .69°(360*/ 13) to 25 .71 0 (3600/14) when the 14th protofilament is incorporated . This 2°distortion represents a somewhat larger angular distortion than the distortion of the helix angle . Apparently, the tubulin molecule is sufficiently flexible to accommodate these distortions of the lateral bonding site .
The dimer lattice in reassembled microtubules is not known . The lattice could be either like the dimer lattice in the Asubfiber (21) or the B-subfiber (15) of flagella outer doublet microtubules. Amos and Klug (2) showed that whereas in the A-tubule the dimers in adjacent protofilaments are in a halfstagger arrangement (along the 5-start helix), the dimers in adjacent protofilaments of the B-tubule are lined up obliquely at a shallow angle (along the 3-start helix). Because the Atubule is a complete cylinder and because the B-tubule dimer lattice will not allow for the formation of a 13-protofilament tubule with a symmetric dimer lattice, it is argued that cytoplasmic singlet microtubules must have the same dimer lattice as flagella A-tubules. The model of Amos and Klug (2) shows that for a 13-protofilament tubule, an odd-start (e.g., 3-start) The table shows the tilt of the protofilaments and the corresponding period of the expected moiré pattern for the 2-, 3-, and 4-start helical families when the pitch angle of the helical family is 10 .5°and the number of protofilaments is 14 . The table shows the pitch angle of the basic subunit helix and the hand of the superhelix when the number of protofilaments is 14, the tilt of the protofilaments is 1 .5*, and the corresponding moiré pattern is 96 nor . * The only model of the microtubule that fits the data . Model of the 14-protofilament microtubule. The protofilaments are tilted 1 .5°to the tubule axis giving rise to a left-handed superhelix with a pitch of 2.71am. Superposition of near and far sides of the tubule gives rise to a 96-nor moiré pattern . A 3-start helix is the basic subunit helix with a pitch angle of 11 .2°. One turn of the 3-start helix is unwound at one end of the tubule to illustrate the pitch angle. In this model, the dimers are arranged in the A-type lattice. monomer helix is required to generate a symmetric dimer lattice if the dimers are arranged in the A-tubule lattice . However, an even-start monomer helix is required to generate a symmetric dimer lattice if the dimers are in the B-tubule lattice . But for a 14-protofilament tubule, an even-start monomer helix is required to generate a symmetric dimer pattern for both the A-and B-tubule dimer lattice patterns. Consequently, the 3-start monomer helix in 14-protofilament tubules is a surprising fording because it requires the dimer lattice to be discontinuous . For example, in a 14-protofilament tubule that forms from a ribbon with dimers in the A-tubule lattice (a-,ß lateral pairing), the pair of protofilaments forming the seam will have the B-tubule dimer lattice (a-a and ,B-,B lateral pairing) and vice versa .
This observation suggests that the lateral bonding regions of the a and ß monomers are sufficiently similar to allow either a-a and ,ß-/3 subunit pairing or a-/3 subunit pairing . Therefore, the reconstituted, 14-protofilament, brain microtubule that retains the 3-start monomer helix must contain elements of both the A-tubule and the B-tubule dimer lattice .
The determination that the monomer helix in reassembled microtubules with 14 protofilaments is a 3-start helix agrees with the data of McEwen and Edelstein (15) . These authors used computer analysis based on Fourier transforms and helical diffraction theory, to determine n, the Bessel order that corresponds to the number of starts in the helical lattice . The tilt of the protofilaments reported here further defines the subunit arrangement and has permitted a determination of the pitch angle of the 3-start helix . The relative amounts of A-and Btype dimer lattices in the reassembled tubule remains a matter of conjecture (15), because reflections in optical transforms corresponding to the dimer repeat are not seen. | 2014-10-01T00:00:00.000Z | 1980-11-01T00:00:00.000 | {
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260261 | pes2o/s2orc | v3-fos-license | Cell cycle perturbations induced by human herpesvirus 6 infection and their effect on virus replication
In this study, we demonstrate that infection of HSB-2 cells with human herpesvirus 6 (HHV-6) resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell-cycle-regulatory proteins indicated that the levels of cyclins A2, B1, and E1 were increased in HHV-6-infected cells, but there was no difference in cyclin D1 levels between mock-infected and HHV-6-infected cells. Our data also showed that inducing G2/M phase arrest in cells infected by HHV-6 provided favorable conditions for viral replication.
Human herpesvirus 6 (HHV-6) is a ubiquitous pathogen of the subfamily Betaherpesvirinae, which includes cytomegalovirus and HHV-7, and it primarily infects CD4 ? T cells [32]. Like other herpesviruses, HHV-6 establishes latency after the initial productive infection and thus is never cleared from its host [28]. Two subtypes of HHV-6 have been identified: variants A and B [30]. Although these two variants are similar in sequence and genome organization, they are associated with different pathogenesis. HHV-6B causes exanthema subitum in young children [34]. HHV-6A is associated with several adult diseases. It is a cofactor in AIDS progression [18] and is involved in various neurological disorders, including encephalitis, multiple sclerosis, seizures, and chronic fatigue syndrome; however, the causal link between human disease and virus infection remains to be fully elucidated [1,6].
Many viruses, including DNA viruses, retroviruses, and RNA viruses, can perturb the cell cycle during their infections [2,4]. Recently, there has been increasing evidence demonstrating different levels of cell cycle arrest in HHV-6-infected cells. It has been shown that HHV-6A infection induces cell cycle arrests at the G2/M phase in infected cord blood mononuclear cells [5]. Furthermore, recent studies have suggested that HHV-6A and HHV-6B infection can also alter E2F1/Rb pathways and cause cell cycle arrest in the G2/M phase in infected SupT1 T cells [20] and that HHV-6B infection of MOLT 3 cells causes cell cycle arrest at the G1 phase concomitant with p53 phosphorylation and accumulation [25]. In addition, G1/S arrest induced by HHV-6 infection has been observed in the other types of cells, such as epithelial cells and neural cells [7,26]. Although HHV-6 has been implicated in halting cell cycle progression, the precise mechanisms behind this phenomenon have not yet been fully understood.
The aim of this work was to investigate the effects of HHV-6 on the cell cycle, with emphasis on the patterns of expression of cyclins as the infection progressed. We demonstrate that HHV-6 induced elevated protein levels of cyclin A2, cyclin B1, and cyclin E1 following virusinduced cell cycle arrest. Furthermore, we show here for the first time that the G2/M arrest provides a favorable environment for viral replication.
The GS strain of HHV-6 variant A was propagated in cord blood mononuclear cells (CBMCs), as described previously [33]. HHV-6A viral DNA loads were quantified by SYBR Green real-time PCR. Primers were specific for the HHV-6 late gene U22. The forward primer was 5'-CGCTCGGAAAGGAAACATTA-3', and the reverse primer was 5'-AAGTGGAACTGCTTGGTGGC-3'. A standard curve was generated by amplification of an HHV-6A U22 DNA sequence incorporated into the pMD TM 19-T vector, designated as pMD19T-U22. A multiplicity of infection (MOI) of 10 virus DNA copies per cell was used for all of the experiments. After infection with HHV-6A, HSB-2 cells showed typical cytopathic effects (CPE), rounding up like balloons, and fusion of infected cells to form giant multinucleated syncytia (Fig. 1A). To further demonstrate HHV-6A infection in HSB-2 cells, expression of a late protein gp60/110, which is essential for viral propagation in infected HSB-2 cells, was analyzed by western blot using an anti-gp60/110 monoclonal antibody (Chemicon). Prominent expression of HHV-6 gp60/110 was detected in HHV-6A-infected HSB-2 cells compared with that in control mock-infected cells, and the levels of this marker increased in a time-dependent manner following HHV-6A infection (Fig. 1B). We also investigated the extent of viral genome replication in HHV-6A-infected HSB-2 cells by real-time PCR at 24, 48, and 72 h postinfection. As shown in Fig. 1C, viral loads in cell lysates from HHV-6A-infected cells increased from 1.17 9 10 7 copies/10 6 cells at 24 h and 2.42 9 10 7 copies/10 6 cells at 48 h to 5.15 9 10 7 copies/10 6 cells at 72 h after infection.
These results showed that HHV-6A could efficiently infect HSB-2 cells.
Cell cycle and nuclear DNA content were determined using propidium iodide (PI) staining and measured by flow cytometry, as described previously [14]. Representative cell cycle profiles in mock-and HHV-6A-infected cells are presented in Fig To address the molecular mechanism of HHV-6Ainduced cell cycle arrest at the G2/M phase, we examined the expression kinetics of the cell cycle regulatory molecules cyclin A2, B1, D1, and E1 in HHV-6A-infected cells compared to mock-infected cells. At various intervals after infection, HSB-2 cell lysates were collected, and cyclin protein expression was determined by western blot analysis using anti-cyclinA2, anti-cyclinB1, anti-cyclinD1, anti-cyclinE1 and anti-b-actin antibodies (Cell Signaling Technology). As expected, a more drastic increase in cyclin A2, B1, and E1 levels was observed in HHV-6Ainfected cells compared with mock-control. At 72 h postinfection, densitometric analysis indicated that cyclin A2, B1 and E1 levels were sixfold, eightfold and sevenfold higher, respectively, in HHV-6A infected cells than that in Manipulation of cell cycle progression is an important strategy exploited by many viruses to create conductive cellular conditions for viral replication. Our data indicated that HHV-6A infection caused an increase in the population of G2/M-phase cells. Therefore, we investigated whether this status of the cell cycle was advantageous for the virus by comparing virus DNA replication and protein expression. Asynchronously growing HSB-2 cells were treated with either DMSO or 0.5 lg/ml nocodazole (Sigma-Aldrich) for 24 h. In asynchronous HSB-2 cells approximately 46 % of cells were in the G0/G1 phase, 48 % in the S phase, and 6 % in the G2/M phase, whereas using nocodazole treatment, approximately 48 % of cells were synchronized in the G2/M phase (Fig. 3A). Then, asynchronously dividing cells and G2/M-phase-synchronized cells were infected with HHV-6A at an MOI of 10 in fresh medium, and virus production was measured by realtime PCR and western blot analysis at 72 h postinfection. As shown in Fig. 3B and C, both the amounts of U22 gene (2-fold) and the expression levels of gp 60/110 protein (2.5-fold) were higher in G2/M-phase-synchronized cells than in asynchronously replicating cells.
Manipulation of the cell cycle in infected cells is a common strategy used by many viruses to regulate their infection. Our previous studies showed that HHV-6A can infect human embryonic fibroblasts and induce G2/M arrest and cell death [13]. We also found that HHV-6A infection inhibits cell cycle progression in HSB-2 cells, resulting from the inhibition of Cdc2-cyclin B1 kinase activity, which is involved in the activation of p53/p21 and Chk1/ Chk2/Cdc25C pathways, as well as elevated Wee1 expression [14]. In this study, we demonstrated that HHV-6A infection promotes cell cycle arrest in the G2/M phase and alters the expression of key cell-cycle-regulatory proteins cyclins in HSB-2 cells. A manifest accumulation of cyclins A2, B1, and E1 was observed in HHV-6A-infected cells. However, the levels of cyclin D1 were not obviously changed in infected cells compared to mock-infected cells. Cyclin B1 is an important regulator during the normal cell cycle progression. It accumulates in the S and G2 phases to form a mitosis-promoting factor (MPF) with Cdc2 and then is ubiquitinated and degraded by the anaphase-promoting complex (APC) after the cells pass through mitosis [22,27]. It is noteworthy that the levels of cyclin B1 remain high in HHV-6A-infected cells, suggesting that an absence of degradation through the ubiquitin pathway might contribute to the cell cycle arrest observed in infected cells. Cyclin E1 has been shown to be rate limiting for S-phase entry, and when overexpressed, to accelerate the G1/S transition [23]. In our study, changes in cyclin D1 expression were not detected after infection, suggesting that the majority of the infected cells do not return to G1 phase after infection, as would be expected with normal cycling and division. In support of our findings, Jault et al. [11] reported HCMV-induced G2/M arrest in cycling cells and found that high levels of cyclin E were induced, with cyclin A appearing only at late time points. De Bolle and coworkers [5] also reported that cyclin B1, cyclin A, and to a slight extent, cyclin E accumulated after HHV-6A infection in cord blood mononuclear cells. Recently, Morita et al. [21] also reported that B19-virus-infected UT7/ Epo-S1 cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2, resulting in cell cycle arrest at the G2 phase; however, the Cdc2-cyclin B1 kinase activity remained high in B19 virus-infected cells.
For successful propagation, viruses may manipulate cell cycle progression to create a more conducive environment for replication [24,29,31]. A recent study suggested that influenza A virus may create favorable conditions in infected cells for viral protein accumulation and virus production by inducing a G0/G1 phase arrest in infected cells [10]. EBV could block the host response and actively promote an S-phase-like environment for viral lytic replication [12]. It was also discovered that viral protein expression and progeny virus production were greater in G2/M-phase-arrested cells. For example, it was reported that avian reovirus (ARV) p17 protein facilitates virus replication through initiation of G2/M arrest and host cellular translation shutoff [3]. HIV infection is also favored in the G2/M phase. Groschel and Bushman [9] observed a three-to fivefold increase in HIV transduction compared to other stages of the cell cycle. In addition, the avian coronavirus infectious bronchitis virus (IBV) induces a G2/M phase arrest in infected cells to promote viral replication [8]. In this study, we found that the efficiency of HHV-6A infection is greater in G2/M-synchronized cells than in asynchronously replicating cells. Therefore, we inferred that HHV-6 induces G2/M phase arrest to provide conditions for progeny virus production. The benefits for virus output may be explained by several A Asynchronously growing HSB-2 cells were treated with DMSO or 0.5 lg/ml nocodazole for 24 h, and the cell cycle profiles were then analyzed by flow cytometric analysis. The 2N (diploid) and 4N (Tetraploid) DNA contents represent the G1 and G2/M phases of the cell cycles, respectively. B and C Synchronously growing HSB-2 cells and asynchronously growing control cells were infected with HHV-6A at an MOI of 10, followed by incubation for 72 h. The amount of viral U22 gene was determined by real-time PCR (B). Viral protein accumulation was analyzed by western blot with anti-HHV-6 gp60/ 110 antibody, and the expression level was quantitatively analyzed and compared to b-actin expression using a densitometer (C). The data shown are the mean ± SD from three independent experiments. **P \ 0.01 compared with the control hypotheses, such as increasing the efficiency of transcription, translation and virus assembly. For example, Lin and Lamb [16] proposed that enveloped RNA viruses could arrest the cell cycle before mitosis to prevent disruption of the Golgi apparatus and endoplasmic reticulum (ER), favoring viruses whose assembly occurs in these structures. It is known that coronaviruses such as IBV also utilize the Golgi apparatus and ER for protein processing and assembly [15,17,19].
The studies presented here, coupled with previous work from our lab and others, demonstrate that HHV-6 infection causes dramatic changes in the expression of host-cell regulatory proteins, leading to the cell cycle arrest. Clarification of the molecular mechanisms by which HHV-6 disrupts the cell cycle machinery will not only be important in the study of the cell cycle changes that occur in HHV-6 replication but will also be crucial for better understanding of the processes by which HHV-6 causes disease and for development of novel vaccines and/or therapeutic agents to inhibit HHV-6 infection. | 2017-08-02T20:46:38.945Z | 2013-09-08T00:00:00.000 | {
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57274854 | pes2o/s2orc | v3-fos-license | ADULT ONSET IDIOPATHIC LOWER BACK PAIN: A STUDY OF CAUSATIVE FACTORS
Orthopedic practice involves a major burden of patients presenting with back pain which may involve upper or cervical region, mid back or thoracic region and lower back or lumbosacral region. The pain may remain restricted to specific region or radiate downwards depending on the specific root involved. The causes of pain vary depending on various parameters like age of patient, lifestyle habits, occupation, gender related, geographical distribution and pathology involved. A study is being conducted to analyse the various factors involved in generation of symptomatic pain in adult population. Both male and female patients are being involved in study. The age group being studied includes ages between 20 years to 45 years. Both developmental and degenerative pathologies are being excluded from the study. The results are analysed in terms of average age at presentation, sex distribution of patients, occupational distribution and primary treatment modalities being used.
INTRODUCTION:
Back pain in adults is one of the most common complain increasing day by day. [1] The incidence appears to have increased with time and treatment costs have a direct impact on patients. [2] It can be short lasting or persists for a long time. With the advent of present lifestyles the problem appears to have progressed with time. [3] A wide variety of causes may be involved in generation of LBA. Pediatric causes are chiefly contributed due to various congenital and developmental problems. [4] In elderly age group the chief cause being degenerative changes involving axial skeleton. Adult population contributes a major burden of problem but pathology is still not fully clear. Due to this the treatment modalities remains unclear. The increased load of cases reported now a days in developing countries like India is due to improved healthcare facilities among the rural areas and also increased awareness among population towards the healthcare facilities. Increasing number of specialist services is also one of the contributory factor in increased reporting of LBA burden.
MATERIALS AND METHODS:
A study is being conducted between January 2014 to December 2014 to analyse the trends involved in adult onset idiopathic low back pain. A total of 400 cases are being included in study consisting of 260 females and 140 males. A detailed history is explored consisting of all possible causative factors involved in problem. History of lifestyle habits and working habits is also explored. [5] Routine general and systemic examination is performed in all cases to rule out any illness. Radiographic analysis is also performed in all cases to rule out any vertebral abnormality. All cases with neurologic involvement are excluded from study. Cases with systemic pathologies and metabolic problems affecting back are also being excluded from study. All cases are recorded and various treatment modalities are used to control the pain and response rates are analysed. MRI study was done in about 164 case due to severe symptoms and long duration of pain to rule out soft tissue pathologies. [6] Treatment modalities for all cases included in study are purely non-operative consisting of patient counseling about disease, posture control, short period of bed rest in acute attacks, drug therapy and physiotherapy in long period. [7] RESULTS: On analysis of various factors, results are interpreted and grouped. The various parameters analysed involves sex distribution, average age at presentation, occupation. [8] treatment modalities taken and response rates. Activity modification+ Physiotherapy + Drug Treatment 364 Table 3: Treatment and Response rate DISCUSSION: On analysis of results, numerous inferences are drawn. It is seen that number of female (240/60%) affected are more as compared to number of male (160/40%) patient affected. Our study shows two peaks in progression of symptoms. In males it involves a) age between 31-35 b) age between 41-45 but in females it shows different trend with affected cases belong to a) age between 26-30 b) age between 36-40. We can see that both females and males shows two peaks of progression but females shows a trend towards early age of onset of symptom progression. Table 2 shows the distribution of occupation among the affected cases. On analysis it is seen that among the affected male cases, the maximum number of cases belongs to hard working group. It can be labour group involved in lifting heavy weights problem but number of person affected are less as compared to other ones. Among the females the maximum affected ones are those engaged in heavy labour work in addition to routine household work. All factors contributes to continuous abnormal postures and abnormal loading of spine.
Sl. No. Age Distribution
The vertebral column may respond by adaptive changes but in low socioeconomic strata group due to added dietery deficiencies the degenerative changes supervene. It results in lower weight lifting capacity and thus adaptive changes in soft tissues like spasmodic contractions of paraspinal muscles. Table 3 shows the various treatment modalities taken with time and their response rates. Like every other pathology the table shows same trends of maximum relief in cases in which the chief cause of symptoms is controlled. As our study deals with idiopathic nature of lower back pain in which all routine blood investigations and radiographs shows normal study. All cases with associated systemic, metabolic or neurological are excluded from study.
The chief cause common to maximum affected cases being routine abnormal posture and heavy loading of vertebral column. The relief rate is found to be better in cases where modifications in activity is performed.
CONCLUSION:
Back pain is one of the orthopedic problem growing day by day and unpleasant to both patient and doctor. Complexity of contributory factors leads to variable outcomes. Causative factors vary with age. Both extremes of age including pediatric and elderly shows a defined trend towards the disease but middle age group shows a different pattern of presentation with no apparent cause seen. The problem appears to be increasing with time more due to increased number of cases reporting due to improved healthcare facilities in developing countries. Our study shows a analysis of about 400 cases with ages between 20 to 45 years. Various parameters are studied including sex distribution, occupation and treatment measures taken.
On discussion it is seen that there is increased trend towards increase symptoms in cases assuming abnormal postures over long time and heavy work loading of vertebral column. Females performing labour work are affected at an early age as compared to males. The symptoms are also more debilitating in females as compared to males. Counseling of patient about the problem is one of the most important step to get rid of disease. It chiefly aims at assuming correct postures and avoiding heavy loads. Adding muscle strengthening exercises and drug therapy further improves the course of disease. The problem appears to follow a mulidisciplinery approach towards combined efforts of a psychologist, orthopedician and physiotherapist. | 2019-01-03T14:13:05.082Z | 2015-10-12T00:00:00.000 | {
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255497125 | pes2o/s2orc | v3-fos-license | Trends of rhinoplasty research in the last decade with bibliometric analysis
Background As rhinoplasty (RP) with different requirements is becoming more and more popular in the latest decade, this study aims to quantitatively and qualitatively explore the trends in RP research, depict research hotspots, and point out the future direction with a bibliometric analysis. Methods All RP literature studies in the last decade (from 2012 to 2021) were retrieved from the Web of Science Core database. Annual output, institutions, authors, journals, and most-cited literature studies were analyzed by bibliometric tools, including CiteSpace, bibliometric online platform, bibliometrix R language kit, BICOMB, and gCLUTO. Results A total of 2,590 RP research studies dated between 2012 and 2021 were included according to our criterion. As for the country, the United States, Turkey, and Korea maintained the top three in RP research. As for the institutions, the University of California, Irvine, Stanford University, and University of Ulsan ranked top three in RP research publications based on article counts. Professor Rhorich RJ, Most SP, and Jang YJ were the most contributed authors according to article counts and citation number. The top journals were The Plastic and Reconstructive Surgery, JAMA Facial Plastic Surgery, and Aesthetic Surgery Journal. The 10 most-cited literature studies were also listed explicitly in this study. Finally, biclustering analysis on the most frequent keywords were conducted which helped us to identify seven hotspot clusters in RP research. Conclusions We comprehensively summarized the publication information of RP literature studies in the past decade, highlighted the current status and trends over time, and provide guidance for in-depth research direction on RP for the future.
Introduction
The nose is considered the most noteworthy feature in the face. Rhinoplasty (RP) is one of the most popular surgical cosmetic procedures performed worldwide, which improves the appearance or/and function, within the domain of the plastic surgeon as well as the otorhinolaryngologist (1). Ever since the first thesis that illustrated nose surgery by Edwin Smith Papyrus in 1930 (2), literature studies concerning RP are increasing with the rapid growth of surgical procedures being performed, including new methods, basic research, outcome measurement, artificial intelligence, and so on. However, it is still an inspiring task to imply macroanalysis based on a large amount of literature data to grasp the development trend of a particular field accurately, especially in such an extremely intricate field as RP.
In recent years, bibliometric analysis has gained great attention for it can evaluate quality trends through literature metrology. Meanwhile, research trends or hotspots within a certain field can be predicted. However, very few bibliometric studies on RP are available now. Only Sinha et al. summarized the 100 most cited articles in RP (3) and Lalezari et al. evaluated global trends in rhinoplasty research spanning 20 years between 1994 and 2013 (4). The former lacks a fully and comprehensively analysis due to the limited scope of included literature. The latter lacks updated information in the latest decade. Both of them mainly focus on publication information instead of analysis and prediction of research hotspots. Therefore, research hotspots verified through co-occurrence keywords biclustering in the last decade were highlighted in this study (5), providing a reference for in-depth research direction and clinical practice related to RP.
Data sources and search strategy
All literature studies are searched and downloaded from the Web of Science (WOS) core collection. Search phrases are as follows: topic = (rhinoplasty) AND publication date = (January 1, 2012-December 31, 2021)) AND language = (English) AND document type = (Article, Review). All data were extracted in one day (December 31, 2021).
Data recording
Two independent reviewers (XZ and BZ) both recorded the original data and conducted the primary search, with a coincidence rate of 95% or more. Any differences were raised and discussed until the reviewers reach a consensus.
Publication analysis
Publishing characteristics including countries, institutions, authors, journals, and most-cited articles are presented in this analysis, which were all carried out by a bibliometric online platform (https://bibliometrix.org/).
Network map presentation
CiteSpace is an optimal tool to depict collaboration network according to different publication characteristics. The principle of the CiteSpace operation is based on co-citation analysis and pathfinder network multidimensional scaling (6,7). In this article, particular aspects, including connections and influence among institutions, authors, and co-cited authors are being vividly represented by CiteSpace network analysis. Centrality is the most representative indicators to value the importance of nodes in the network. Typically, higher centrality means the greater importance of the node among the whole network. Meanwhile, high citation keywords, which are also called burst words, within years are revealed to illustrate the research frontiers and focal point.
Thematic map illustration
The thematic map was illustrated by bibliometrix R language kit; the four quadrants drawn represent the following: The first quadrant (upper right corner): motor themes; it is an important and well-developed theme. The second quadrant (upper left corner): highly developed and isolated themes; it has developed well but is not important for the current field. The third quadrant (lower left corner): degrading themes and marginal themes, which have no good development and may just emerge or disappear. The fourth quadrant (lower right corner): basic and transitional themes; generally, it refers to basic concepts which are important in the field, but have not been well developed.
Hotspots detection and clustering
BICOMB (Bibliographic Item Co-Occurrence Matrix Builder) is a software used to construct a co-occurrence keyword binary matrix that reveals the connections between the extremely frequent keywords. Once the matrix is built, we then imported the matrix into software gCLUTO (5,8) and set appropriate parameters to get a matrix visualization as well as mountain visualization, which represented the semantic relationship between keywords and source literature studies. All mountain image features including colors, plane, altitude, peak, and volume are the reflection of associated clusters. The volume of each peak is directly proportional to the number of keywords appeared in this category, and the altitude of each peak shows the positively correlation of keywords within the same category. Also, the closer the peaks are, the more similar the clusters they represent. The internal standard deviation of keywords is revealed by the color of each peak. Red indicates a low deviation, while blue indicates the high one. The matrix values are represented graphically by colors, The color of each reseau paints the proportional emergence frequency of a major keywords in a literature. The colors gradually deepen from white to red, indicating that the keywords are less important to more important. Finally, the framework of RP research hotspots was generated based on the above visualization, and further studies were carried out according to representative papers in each cluster.
Results
The output of literature studies From 2012 to 2021, a total of 3,128 publications were retrieved, according to our inclusion criteria, 538 publications were excluded, and 2,590 publications were used for bibliometric analysis; among them, 2,374 are articles and 216 are reviews ( Figure 1). Figure 2 shows the successively increasing trend in the numbers of RP-related publications.
The international cooperation map reveals that the United States cooperates most frequently with other countries, especially with Canada, Iran, and Germany. Turkey ranks the second according to publication number; however, it cooperates much less frequently with other countries (Figure 4). A low-density (density = 0.0064) map of the rhinoplasty research network ( Figure 5) represents that the research groups were sparsely distributed in various institutions worldwide, thus communications and cooperations need to be intensified. For 7 out of 10 institutions, the central indexes are below 0.1, indicating that most institutions had a relatively low level of influence worldwide and did not cooperate closely enough in the recent 10 years. While the output of University of Pennsylvania was not very high, it had the greatest influence (0.13) among the RP region.
The top 10 prolific authors on RP research
The 10 authors who published the most literature studies in this study are listed in Table 2 Figure 6, which means that the darker the color is or the larger the circle is, the more articles are produced in that year. Meanwhile, we calculated the citation information for authors, visualizing them in a network by CiteSpace. Rhorich RJ, with 644 literature studies, ranked first in the top 10 co-cited authors, followed by Daniel RK (486), Guyuron B (413), Toriumi DM (406), and Gunter JP (338) ( Table 3). The top 10 specialists conducted a huge quantity of research and laid a solid foundation for the development of RP. Also, the centrality of the top 10 authors was more than 0.15, suggesting that they had formed a very influential core scholar group in the domain of rhinoplasty research. Then we use CiteSpace to map the integrated information of authors ( Figure 7) and co-cited authors ( Figure 8).
The top 10 cited literature studies about RP
The details of the top 10 cited articles are listed in Table 4. Clustering analysis of the RP hotspots Figure 9 illustrates the detailed thematic map in the field of RP. In order to get a more comprehensive view, we screened the keywords with frequency higher than 25 (25 included), which accounted for 28.03% of all words, and used BICOMB Flow chart of literature filtering in this study. WoSCC, Web of Science Core Collection. The annual number of publications worldwide. Frontiers in Surgery combined with gCLUTO to sort out 7 distinctive clusters. Thus, closely connected keywords will be identified and categorized into one cluster and systematical knowledge structure and trends in RP field will be reorganized. Moreover, mountain ( Figure 10) and matrix visualizations ( Figure 11) are depicted to visualize the correlations between keywords and source literature. The above highfrequency keywords are divided into seven categories, and representative literature studies of each category were deeply studied and further summarized. In the end, we have concluded seven hotspots as follows: Cluster 0: Augmentation rhinoplasty; Cluster 1: Cleft rhinoplasty; Cluster 2: Functional rhinoplasty; Cluster 3: Osteotomy; Cluster 4: Crooked/deviated nose; Cluster 5: Nonsurgical rhinoplasty; Cluster 6: Outcome and PROMs.
Discussion
Though the research directions of rhinoplasty are relatively extensive, summary and analysis of relating hotspots are still hard to find. Keywords can represent the research content of literature studies and the order of magnitude keywords can depict the current research status and trend of the region. According to a qualitative and co-word biclustering analysis by bibliometric software, analogical keywords can be recognized and classified into clusters. Paying attention to and digging deeply into these clusters can help identify valuable future directions. The worldwide country collaborative map in the field of RP. The network map of institutions that involved in rhinoplasty research (density = 0.0064). In this article, we get seven clusters through the biclustering analysis. Cluster 0 mainly concentrates on augmentation rhinoplasty. Since many Asians have a short nose that is generally characterized as having low dorsum, short columella, poorly defined nose tip, flare nostril shape, and wide alar base (9), augmentation surgery has become increasingly popular to correct above features for esthetic purposes. In the past, simple augmentation of the dorsum using an implant has been widely operated and reported; with the improvement of esthetic cognition, recent literature studies show that this procedure will result in disharmony of the nasal base, leading to visible nostrils, a longer supratip lobule, and shorter columella (10)(11)(12). Accordingly, it is commonly believed that Asians need to augment and elongate the nose simultaneously to achieve satisfying augmentation rhinoplasty (13).
Cluster 1 mainly concentrates on cleft rhinoplasty. Since primary rhinoplasty should be performed at the time of cleft lip repair, it becomes a common practice among cleft surgeons (14). After that, more related publications from 2012 to 2021 focuses on secondary cleft rhinoplasty. Key elements in successfully secondary rhinoplasty are the replacement of bony structures and reconstruction of the absent/asymmetric (15). Other keywords in this cluster deserve attention is anthropometry. Over the years, surgeons have been realizing that it is essential to know how nasolabial features change over time after the primary repair and subsequent surgical and orthodontic interventions, which provides references about residual deformities that may need further revision, and 3D anthropometric technology has been developed rapidly and widely used nowadays in cleft anthropometry (16). Series of longitudinal studies have been implemented according to different interventions (17)(18)(19)(20). Cluster 2 mainly concentrates on functional rhinoplasty. The functional rhinoplasty patient requires improvements in breathing and olfaction instead of shape (21). Internal nasal valve obstruction, external nasal valve collapse, and septal deviation are the three major causes of nasal airway obstruction. Among them, septal deviation is the most common reason and a prevalent problem in the general population (22). Many septoplasty techniques including endonasal, endoscopic, and open procedures have been described. Much of the literature in the latest 10 years focus on comparing the above approaches. However, each of the septoplasty techniques contains advantages and disadvantages, but none of them has been proved as the most successful means to correct septal deviation (23, 24).
Cluster 3 mainly concentrates on crooked/deviated nose and osteotomy. Both extrinsic forces and intrinsic forces can lead to nasal structure distortion and nasal deviation; nevertheless, majority of them share septal deviation in common. Key operative principles for correcting the crooked nose include rectification of deviated septum and nasal osteotomy. Generally, hybridization of preservation and structural rhinoplasty is required (25,26). Due to the emergence of potential negative implications of excision techniques, surgeons nowadays pay much interest to dorsal preservation. The maintenance of the structural integrity at the nasal keystone, dorsal esthetic lines, and the patency of the internal nasal valve are points of dorsal preservation (27, 28). In addition, osteotomy is closely associated with postoperative eyelid edema and ecchymosis. Though much publications report above complications in different surgical methods, there is no consensus on which method is the most effective in reducing them (29).
Cluster 4 mainly concentrates on nasal reconstruction and reconstruction surgery. The missing tissue of nasal is categorized as lining, support, and cover, corresponding to mucosa, cartilaginous and bony skeleton, and the overlying skin (30). Literature studies about reconstruction published from 2012 to 2021 still concentrate on the above three aspects, especially on the flaps. Apart from the cases of classical flaps, many newly flaps such as the prelaminated temporoparietal osteofascial flap (31) and modified flaps such as the nasomentolabial flap (32) are reported.
Cluster 5 mainly concentrates on nonsurgical rhinoplasty (NSR). Comparing to the golden standard-surgical rhinoplasty for nasal correction-NSR possesses the advantages including lower cost, less downtime, and immediate effect, and thus are more and more favored by modern people (33). Also, NSR is more easily carried out by a wider range of practitioners and has a more mild learning curve than RP (34). The most well-known types of fillers include hyaluronic acid (HA), collagen, paraffin, and liquid silicon (35). Experienced doctors report their unique injection The network map of most productive authors. The network map of most co-cited authors.
Zhu et al. 10.3389/fsurg.2022.1067934 Frontiers in Surgery methods (36-38) without reaching a consensus. Typically, injection rhinoplasty is fairly safe but complications may occur occasionally. So many surgeons will introduce the technique they use during the procedure to avoid the complications. However, serious complications such as dermal necrosis due to vascular obstruction are rare but still reported (39). Cluster 6 mainly concentrates on outcome and patient satisfaction of the rhinoplasty. In recent years, there is an increasing trend to use health-related quality of life (QoL) questionnaires or multiple patient-reported outcome measures (PROMs) to assess patient satisfaction after surgical procedure. An ideal assessment of RP outcome should have objective means of evaluation covering shape, function, and psychology (40). Currently available and most widely used outcome measures in rhinoplasty include Rhinoplasty Outcomes Evaluation (ROE), The Nasal Obstruction Symptom Evaluation (NOSE), FROI-17, Rhinoplasty Health Inventory and Nasal Outcomes Scale Description (RHINO), FACE-Q, and The thematic map of keywords plus.
FIGURE 10
Mountain visualization of biclustering of highly frequent keywords and source literature studies on rhinoplasty research. In addition, the Body Dysmorphic Disorder (BDD) Questionnaire has also become essential to surgeons for assessing patients before surgery. It can reveal the psychological state of the patient and help understand the demands of patient, serving as an effective way to avoid the outcome of dissatisfied patients and depressed doctors (41). Though we have analyzed the publications on RP from 2012 to 2021 as comprehensively as possible, some limitations still exist. First, since the RP databases update incessantly from 1870 to date, we only selected the publications from 2012 to 2021. Therefore, a discrepancy may exist between this bibliometric analysis and real publication conditions. In addition, the amount of RP literature may increase rapidly with the breakthrough of treatment methods or novel concept. Take Turkish delight, for example, a few relative literature studies were published between 2000 and 2011; however, 19 articles about Turkish delight were published in 2011-2021 and thus making it a highly frequent major keyword. Since it lacks the tight and continuous correlation with other conventional keywords, the biclustering method did not classify it into any prominent cluster. Most of the published rhinoplasty series are the final works of very prestigious experts working in a stationary location for decades, which limit the chances of collaborative work of different institutions and are also reflected in the calculated centrality and density. The majority of the augmentation RPs are from East like Korean and China, while most osteotomies that aim at hump nose are from NA and Europe. Surgical techniques and research trends can vary differently among regions.
Conclusions
We conducted a comprehensive summary of the publication information of RP-related literature studies in the latest 10 years from 2012 to 2021, pointing out the research trends over time. In general, the purpose of RP is mainly divided into two aspects: the appearance (including esthetic deficiency and deformity) and the function of nose. The literature studies about esthetic RP mainly focus on augmentation RP, which are usually reported by Asians. As for deformity, it mainly concentrates on cleft RP and nasal defect. 3D anthropometry has been used more popular than ever for cleft rhinoplasty, but systematic and large-scale measurement data have not been reported yet, which deserves multiple medical centers to cooperate in the future. The nasal defect mainly involves nasal reconstruction. Both classic flaps and innovative flaps have been reported incessantly; however, existing surgical methods still could not meet the needs of special types of patients or patients with higher requirements for appearance. More suitable ways need to be explored. For the function of nose, nasal obstruction that resulted from septal deviation accounted for over 90% of all related literature studies. The three mainstream surgical techniques including endonasal, endoscopic, and open procedures have been elaborated. However, the comprehensive advantages and drawbacks about the above three still need FIGURE 11 Matrix visualization conducted by gCLUTO (version1.0, University of Minnesota).
Zhu et al. 10.3389/fsurg.2022.1067934 Frontiers in Surgery further studies. Another hotspot deserves scholars' attention lies in the valuation of patients' outcome. The patient self-reported outcome evaluation scale has become a very popular evaluation method. However, the scale is scored by the patients themselves, which has subjective consciousness defects. How to evaluate the postoperative outcome more reasonably, objectively, and comprehensively is a problem that doctors need to think about carefully.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. All authors contributed to the article and approved the submitted version. | 2023-01-07T17:17:14.358Z | 2023-01-06T00:00:00.000 | {
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6744961 | pes2o/s2orc | v3-fos-license | Fumigant Activity of the Psidium guajava Var. Pomifera (Myrtaceae) Essential Oil in Drosophila melanogaster by Means of Oxidative Stress
The guava fruit, Psidium guajava var. pomifera (Myrtaceae family), is a native plant from South America. Its leaves and fruits are widely used in popular medicine in tropical and subtropical countries. Drosophila melanogaster has been used as one of the main model organisms in genetic studies since the 1900s. The extensive knowledge about this species makes it one of the most suitable organisms to study many aspects of toxic compound effects. Due to the lack of studies on the effects of the bioactive compounds present in the P. guajava var. pomifera essential oil, we performed a phytochemical characterization by CG-MS and evaluated the toxicity induced by the essential oil in the D. melanogaster insect model. In order to understand the biochemical mechanisms of toxicity, changes on the Nrf2 signaling as well as hallmarks of oxidative stress response were followed in the exposed flies. Our results showed that exposure of insects to the P. guajava oil increased mortality and locomotor deficits in parallel with an oxidative stress response signaling. Therefore, it suggested a bioinsecticidal activity for P. guajava volatile compounds by means of oxidative stress. Further studies are ongoing to identify which oil compounds are responsible for such effect.
Introduction
With the continual increase in the human population worldwide, one of the most challenging situations is to provide enough food to the human population. There are two possibilities to reach such endeavor: (1) increase the agricultural area or (2) optimize the production of the already cultivated fields. Insect pests are one of the most important threats for the cultivated crops causing a serious reduction in the global production [1].
Synthetic insecticides are widely used to control insect pests. However, the chemical properties of these products make them dangerous for both humans and the environment [2]. Moreover, the plasticity of insect pests makes them prone to develop resistance to many of these compounds [3]. Searching new insecticides that offer no or low risks and that are decomposed to safe compounds after its action is needed in order to overcome these issues. Plant derived insecticides can be a suitable alternative, since vegetables species have evolved molecular mechanisms that protect them against herbivorous insects and other animal species [4]. Essential oils from plant species have been reported as acting on digestive and neurological enzymes as well as with insects tegument [5,6]. Some authors suggested that such insecticide effect is probably due to the secondary metabolites as terpenoids and phenylpropanoids [7]. An insecticidal activity of some monoterpenes as -pinene, -pinene, 3-carene, limonene, 2 Oxidative Medicine and Cellular Longevity myrcene, -terpinene, and camphene had been demonstrated in literature [8].
Psidium guajava (Myrtaceae family) is a native bush species from South America known as "goiaba. " There are two more common cultivated varieties of P. guajava: P. guajava var. pomifera and P. guajava var. pyrifera. The P. guajava var. pomifera produces a fruit highly appreciated in the tropical and subtropical culinary and also is used in the popular medicine [9]. Extracts from leaves and fruits of this species presented several pharmacological properties as antispasmodic, antimicrobial and anti-inflammatory [10]. Moreover, these extracts also have been used as hypoglycemic [11]. Despite the available reports on benefits of guava to human health, little is known about its potential in biotechnological applications (e.g., fumigant activity) of guava extracts, oils, and derived compounds.
In the last decade, Drosophila melanogaster became a model for testing toxicity in vivo. It is due to the fact that this species has many homologous genes with humans and can be easily kept at the laboratory allowing many assays to be performed [12][13][14][15]. Therefore, D. melanogaster model can be widely used for evaluating fumigant activity screenings.
In summary, considering (i) the undesired adverse effects of synthetic means of pest control to humans and the environment, (ii) the ability of plant metabolites to induce toxicity to insects, and (iii) the lack of studies on the biotechnological potential of guava fruit derived compounds, the main goal of this work was to evaluate the biological activity of the essential oil from Psidium guajava var. pomifera and investigate the mechanism by which this oil promotes toxicity using the model organism D. melanogaster. Toxicity was evaluated as mortality and locomotor deficits. In parallel, oxidative stress signaling markers were determined in order to search for potential mechanisms of toxicity induced by the essential oil in Drosophila.
Collection of Essential
Oil. Leaves of Psidium guajava var. pomifera L. were collected, chopped into pieces of approximately 1 cm 2 , and placed in a 5-liter glass flask. The leaves were extracted with a clevenger apparatus, according to the method described by de Matos [16], giving a yield of 0.05%.
Essential Oil Exposure and Flies Survival
Assay. The exposure of flies to the essential oil was performed by a fumigation protocol as described: adult flies (males and females) were placed in 330 cm 3 glass vials, containing a filter paper soaked with 1% sucrose in distilled water at the bottom. A counter-lid of polyethylene terephthalate (PET) was introduced on the screw cap of the vial, to which a filter paper was fixed at the inner side of the cap for application of different doses of essential oil. By doing this, the flies feed and hydrate on sucrose solution at the bottom of the vials and the essential oil is allowed to volatilize from the top in order to reach flies' respiratory system. The vials received the following treatments: 1% sucrose (control) and 3, 7.5, 15, 23.5, and 30 g/mL of essential oil. The final concentration of the essential oil was estimated by approximation, taking into account the volume (in microliters) of the oil applied to a glass vial with a final volume equivalent to 330 mL. Readings of flies' survivorship were taken at 6, 12, 24, and 48 h. Results are presented as percentage (%) of live flies (mean ± SD) obtained from three independent experiments.
2.6. Locomotor Assay. The locomotor capacity was evaluated by following the negative geotaxis behavior as described by Coulom and Birman [17] with some modifications. Twenty adult flies (1-4-day old; both genders) were subjected to essential oil exposure as detailed above. After treatments were finished, flies were immobilized on ice for 1-2 minutes and placed separately in vertical glass columns (length, 25 cm; diameter, 1.5 cm). After 30 min recovery, flies were gently tapped to the bottom of the column and the number of flies that reached 6 cm of the column (top) and flies that remained below this mark (bottom) were registered. The assays were repeated three times for each fly. Results are presented as number of flies on top (mean ± SD) obtained from three independent experiments.
Oxidative Stress Markers.
Oxidative stress was determined by measuring lipid peroxidation, reactive oxygen species formation (ROS), nonprotein thiols (NPSH), and protein thiols (PSH). Byproducts of lipid peroxidation were quantified by the thiobarbituric acid reactive substances method (TBARS) following Ohkawa et al. [18] with few modifications. Briefly, 20 flies from each treatment were homogenized in 1 mL of phosphate buffer 0.1 M pH 7.0 and centrifuged at 1000 g during 5 min at 4 ∘ C. Immediately after centrifugation, the supernatant was incubated in acetic acid 0.45 M/HCl buffer pH 3.4, containing thiobarbituric acid 0.28%, SDS 1.2%, at 95 ∘ C during 60 min for color development, and then absorbance was measured at 532 nm. Malondialdehyde (0-3 nmol) was used as standard. The 2,7-dichlorofluorescein diacetate (DCFDA) oxidation was used as a general index of ROS formation following Pérez-Severiano et al. [19]. The fluorescence emission of DCF resulting from DCFDA oxidation was monitored at an excitation wave length of 485 nm and an emission wavelength of 530 nm in a multimode plate reader (EnsPire PerkinElmer, USA). Protein and nonprotein thiols were determined according to the method described by Ellman et al. [20] and adapted to our lab conditions. In summary, after treatments were finished, flies were homogenized in 0.5 M perchloric acid and centrifuged at 5000 g for 5 min at 4 ∘ C. The NPSH content was determined in the supernatant while the pellet was used for PSH measurement. Total protein was quantified according to Bradford [21].
Enzymatic Assays.
For antioxidant enzymes activity, groups of 20 flies were homogenized in 1 mL 0.1 M phosphate buffer, pH 7.0, and centrifuged at 20.000 g for 30 min. The resulted supernatant was used for determination of glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) according to methods described earlier [22]. Glutathione S-transferase (GST; EC 2.5.1.18) activity was assayed following the procedure of Habig and Jakoby [23] using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The assay is based on the formation of the conjugated complex of CDNB and GSH at 340 nm. The reaction was conducted in a mix consisting of 100 mM phosphate buffer pH 7.0, 1 mM EDTA, 1 mM GSH, and 2.5 mM CDNB. Catalase (CAT; EC 1.11.1.6) activity was assayed following the clearance of H 2 O 2 at 240 nm in reaction media containing 50 mM phosphate buffer pH 7.0, 0.5 mM EDTA, 10 mM H 2 O 2 , and 0.012% TRITON X100 according to the procedure of Aebi [24]. Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed following the procedure of Kostyuk and Potapovich [25]. The assay consists in the inhibition of superoxide-driven oxidation of quercetin by SOD at 406 nm. The complete reaction system consisted of 25 mM phosphate buffer, pH 10, 0.25 mM EDTA, 0.8 mM TEMED, and 0.05 mM quercetin. All enzyme activities were performed at room temperature (25 ± 1 ∘ C) using a Thermo Scientific Evolution 60s UV-vis spectrophotometer. Total protein was quantified according to Bradford [21].
Western Blot Analysis of Nrf2/NQO-1/HSP70 Signaling
Pathway. Protein expression was determined by Western blotting according to Posser [26] with minor modifications. Thirty flies were homogenized at 4 ∘ C in 300 L of buffer (pH 7.0) containing 50 mM Tris, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 20 mM Na 3 VO 4 , 100 mM sodium fluoride and phosphatase inhibitor cocktail (Sigma, MO). The homogenates were centrifuged at 1000 g for 10 min at 4 ∘ C and the supernatants (S1) collected. After protein determination (following Bradford [21]) using bovine serum albumin as standard, -mercaptoethanol and glycerol were added to samples to a final concentration of 8 and 25%, respectively, and the samples were frozen until further analysis. Proteins were separated using SDS-PAGE with 10% gels and then electrotransferred to nitrocellulose membranes as previously described by Posser [26]. Membranes were washed in Tris-buffered saline with Tween (TBST; 100 mM Tris-HCl, 0.9% NaCl, and 0.1% Tween-20, pH 7.5) and incubated overnight (4 ∘ C) with different primary antibodies (Santa Cruz Biotechnology, TX), all produced in rabbit (anti-Nrf2, anti-NQO-1, anti-HSP70 anti--actin; 1 : 1000 dilution in TBST). Following incubation, membranes were washed in TBST and incubated for 1 h at 25 ∘ C with HRP-linked antirabbit-IgG secondary specific antibodies (Sigma, MO). The immunoblots were visualized in the Image Station 4000MM PRO using ECL reagent (Santa Cruz Biotechnology, TX). Immunoreactive bands were quantified using the Scion Image software and expressed as a fold change of the mean relative to control group (treated only with sucrose).
Statistical Analysis.
Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc test when necessary. Differences were considered statistically significant when < 0.05. LC50 values were determined by the Trimmed Spearman-KArber method (v 1.5).
Toxicity in D. melanogaster.
The exposure of fruit flies to P. guajava essential oil by fumigation caused a significant increase in mortality. Such an effect was dependent on time and oil concentration. The calculated LC 50 at 48 h was 13.8 g/mL (Figure 1). The concentrations of 23.5 and 30 g/mL had the most evident biocide effect, a result that could be compared with a food deprivation treatment (water only; data not shown). The highest concentrations tested killed almost the totality of flies at 48 h, showing a potent insecticide action for the essential oil. In Figure 2 the results from the locomotor activity tests are depicted. In agreement with the mortality results, a significant decrease in locomotor activity of D. melanogaster in the first 6 hs of treatment at 15, 23.5, and 30 g/mL can be observed. Moreover, at 48 h of exposure, the highest concentrations tested caused almost completely loss of motor ability in flies (Figure 2).
Oxidative Stress Markers and Antioxidant Response.
In order to clarify potential mechanisms by which D. melanogaster is affected by the P. guajava essential oil, flies were exposed to 15 g/mL of oil during 3, 6, and 12 h. Then, oxidative stress markers and the activity of antioxidant enzymes were determined (Table 2). This concentration is below the LC 50 48 h for D. melanogaster. It was possible to observe a significant increase in ROS formation at 3 h exposure to the essential oil, a result that was maintained after 6 and 12 h as well. Our results showed an increased level of TBARS after 12 h of exposure indicating that lipid peroxidation took place. The levels of protein thiols (PSH) were not changed, but nonprotein thiols (NPSH) significantly increased after 3 h of exposure, returning to basal levels at 6 and 12 h. We also evaluated the activity of three enzymes involved in the antioxidant metabolic route: GST, SOD, and CAT, as well as the expression of protein targets involved in stress response and antioxidant signaling (Nrf2, NQO-1 and HSP70). A significant increase in the activity of GST and CAT was observed when compared to control at 6 and 12 h (Table 2). However, the activity of SOD was not significantly different from the control at the time periods analyzed. As demonstrated in Figure 3, flies exposed to the essential oil presented a significant increase in the expression of NQO-1 at 3 h of exposure, indicating an early activation of the Nrf2-ARE signaling pathway. The protein levels of Nrf2 and HSP70 were not changed at the analyzed time points.
Discussion
Chemical pesticides used for insect control may be dangerous to humans and wild life. In addition, these compounds may induce insect resistance and other adverse effects, which have motivated the search for alternative forms of control [3]. In the present study we demonstrate the toxicity induced by the Psidium guajava var. pomifera essential oil in Drosophila melanogaster. The exposure of flies by the fumigation method induced substantial decreases in survivorship as well as locomotor activity. As a mechanism for the observed toxicity, the results suggest the establishment of a prooxidant condition after flies were in contact with oil derived volatile compounds. Such an effect is confirmed by increased production of reactive species and accumulation of lipid peroxidation byproducts. In addition, a clear adaptive response to oxidative stress was apparent in the oil exposed flies, since it was possible to observe an activation of antioxidant signaling pathways and increased activity of key cellular antioxidant enzymes. Plant derived compounds are reported to induce toxicity to a wide range of insects and may interfere directly with all developmental stages of fruit fly, Drosophila melanogaster, and cockroaches [27,28]. Compounds such as terpenes, flavonoids, alkaloids, steroids, and saponins are important phytochemicals when considering the insecticide activity of plant extracts [29]. In addition to acute toxicity and mortality, terpenoids and flavonoids have been also studied for their insect repellent activity [29,30]. There are a variety of chemical compounds present in the P. guajava essential oil asterpineol, -humulene, -caryophyllene and -guaiene, 1,8cineole, caryophyllene oxide, -bisabolene, aromadendrene, p-selinene, -pinene, among others [31][32][33][34]. Leal et al. [35] showed the insecticidal activity of the 1,8-cineole compound obtained from the S. aromaticum, H. martiusii, and Lippia sidoides essential oils. Some authors suggested that most of the monoterpenes are nontoxic for mammals and can be considered an alternative to synthetic insecticides [36,37]. Oxidative Medicine and Cellular Longevity We observed, in this study, that the P. guajava oil presented mono and sesquiterpenoid compounds, with the 1,8-cineole being the second most abundant (Table 1). Although we did not perform essays to evaluate the insecticide activity of each compound, the presence and abundance of the 1,8-cineole suggest that it may be one of the compounds responsible for such effect. Studies are ongoing in order to clarify the role of the different compounds presented in the essential oil tested here. According to Ennan et al. [7] some compounds from essential oils as terpenoids and phenylpropanoids can alter the insect neurotransmitters system, including the dopaminergic and cholinergic apparatus [38,39]. We observed a significant change in the negative geotaxis behavior of flies treated with P. guajava oil, which reflects in a locomotor deficit. Although we were not able to directly evaluate changes in the dopaminergic and cholinergic systems in our experimental design, some of the effects observed may be linked to a potential interaction between oil components and flies neurotransmitters pathway. In this context, it has been shown that many terpenes are known as inhibitors of the acetylcholinesterase (AchE) [39]. As reported by the same authors the -terpinene found in Salvia leriifolia showed an AChE inhibitor effect. In general, it suggests that the terpenoid compounds found in P. guajava may be involved in the fumigant effect and in the damage to the locomotor apparatus.
In parallel with the induced mortality and locomotor deficits, flies exposed to P. guajava also showed signs of oxidative stress, including ROS and TBARS formation as well as changes in important antioxidant response systems. The cellular response to oxidative stress is mostly regulated by the Nrf2 nuclear transcription factor [40]. ROS/xenobiotics induced alterations in the cellular redox state constitute an important signal to promote adaptive responses mediated by Nrf2 [41,42]. The upregulation of detoxifying enzymes by natural compounds appears to be related to activation of Nrf2-ARE pathway [41,42]. The Nrf2 nuclear translocation and subsequent binding to the DNA sequence known as the "antioxidant response element, ARE" may be triggered by dissociation from the inhibitory protein Keap1 as well as by phosphorylation of serine residues at the Nrf2 protein by upstream kinases such as PKC and MAPK [42]. Among proteins that are usually involved in response to oxidative stress-driven Nrf2 activation, the NAD(P)H dehydrogenase, quinone 1 oxidoreductase (NQO-1), glutamate cysteine ligase (GCL), GST, and CAT play central role [43]. Our results showed a time dependent activation of key factors on the regulation of an antioxidant response. Since a high mortality rate at almost all doses of essential oil was apparent at the first 24 h of exposure, we measured oxidative stress markers up to 12 h, in order to have a profile of the antioxidant response in animals under P. guajava oil treatments. Apparently, in response to the toxicity induced by oil compounds, flies presented increased ROS levels and a peak of GSH and NQO-1 (Table 2) at the first 3 h of treatment, a phenomenon that is consistent with an early activation of the Nrf2-ARE pathway [44]. While ROS continued to increase from 3 h up to 12 h, lipid peroxidation took place only at 12 h time point ( Table 2).
The antioxidant enzymes GST and CAT were increased during the period of 6 h up to 12 h after essential oil treatments. Despite the increased activity of antioxidant enzymes from 6 to 12 h after administration of P. guajava oil, such effect did not protect flies against the late onset of lipid oxidative damage. These results clearly suggest a two-phase adaptive response to oxidative stress induced by P. guajava oil derived compounds. An early phase triggered by ROS induction, resulting in activation of the master regulator of cellular antioxidant response, the Nrf2 transcription factor, and a late phase, characterized by oxidative damage and increased ROS/xenobiotic detoxifying enzymes (CAT and GST). Later on, mortality and locomotor deficits accomplished the toxicity induced by the essential oil.
Glutathione S-transferase is an important antioxidant enzyme involved in phase II detoxification systems [45]. GSTs belong to a family of multifunctional enzymes that catalyze the conjugation of GSH to various other molecules and play a role in mechanisms of intracellular detoxification of endoand xenobiotic compounds [46,47]. The observed increase of GST activity in Drosophila melanogaster exposed to P. guajava oil can be related to an adaptive response related to enhanced elimination of toxic plant derivatives [48,49]. Singh et al. [50] demonstrated that natural compounds are able to increase the expression of GST that together with endogenous GSH favors the elimination of plant metabolites from organisms. Catalysis plays a crucial role in the clearance of hydrogen peroxide from cells as well as for oxidative stress defense [24]. Our results demonstrated a significant increase in CAT activity in flies treated with guava essential oil ( Table 2). This effect was in parallel with a rise in ROS production. The method used in the present study to detect ROS was based on the oxidation of the fluorescent dye DCFDA, which is considered a general reactive species indicator; however, hydrogen peroxide is one major species detected by this probe [51]. The observed rise in GST and CAT activity by P. guajava in fruit flies may be explained by a potential activation of the Nrf2 signaling pathway. In fact, an early activation of this signaling pathway was noted in flies exposed to the essential oil, by means of increased NQO-1 expression as well as a rise in GSH (Figure 3 and Table 2).
Conclusion
According to our results, the essential oil of P. guajava var. pomifera showed a fumigant action by compromising survivorship and locomotor activity of D. melanogaster. As a potential molecular mechanism of toxicity, oxidative stress appeared to be central, since markers of oxidative damage of biomolecules and a clear adaptive antioxidant response were observed in exposed flies. Therefore, our results point out to the potential application of P. guajava essential oil and/or its compounds as an alternative to the synthetic insecticides in agricultural and pest control practices. Additional experiments are necessary to clarify the exact mechanisms of toxicity induced by P. guajava oil in insects and to identify candidate compounds derived from this oil. | 2016-05-04T20:20:58.661Z | 2014-11-12T00:00:00.000 | {
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231982010 | pes2o/s2orc | v3-fos-license | Health-based evaluation of ambient air measurements of PM2.5 and volatile organic compounds near a Marcellus Shale unconventional natural gas well pad site and a school campus
Background Limited air monitoring studies with long-term measurements during all phases of development and production of natural gas and natural gas liquids have been conducted in close proximity to unconventional natural gas well pads. Objective Conducted in an area of Washington County, Pennsylvania, with extensive Marcellus Shale development, this study investigated whether operations at an unconventional natural gas well pad may contribute to ambient air concentrations of potential health concern at a nearby school campus. Methods Almost 2 years of air monitoring for fine particulate matter (PM2.5) and volatile organic compounds (VOCs) was performed at three locations between 1000 and 2800 feet from the study well pad from December 2016 to October 2018. PM2.5 was measured continuously at one of the three sites using a beta attenuation monitor, while 24-h stainless steel canister samples were collected every 6 days at all sites for analysis of 58 VOCs. Results Mean PM2.5 concentrations measured during the different well activity periods ranged from 5.4 to 9.5 μg/m3, with similar levels and temporal changes as PM2.5 concentrations measured at a regional background location. The majority of VOCs were either detected infrequently or not at all, with measurements for a limited number of VOCs indicating the well pad to be a source of small and transient contributions. Significance All measurement data of PM2.5 and 58 VOCs, which reflect the cumulative contributions of emissions from the study well pad and other local/regional air pollutant sources (e.g., other well pads), were below health-based air comparison values, and thus do not provide evidence of either 24-hour or long-term air quality impacts of potential health concern at the school.
Introduction
There has been a proliferation of air monitoring data collected at major U.S. shale gas plays to understand the potential air quality impacts of the recent expansion of unconventional natural gas development (UNGD) activities, including horizontal drilling and hydraulic fracturing. Air measurement studies have been conducted by academic researchers [1][2][3][4], governmental agencies [5][6][7][8][9], industry scientists and industry-funded consultants [10][11][12], and environmental advocates and non-profit groups [13,14]. Air pollutants that have been commonly measured in these studies include both US Environmental Protection Agency (US EPA) criteria air pollutants (e.g., fine particulate matter [PM 2.5 ], nitrogen dioxide [NO 2 ]), and volatile organic compounds (VOCs) classified by US EPA as air toxics (e.g., benzene, ethylbenzene, formaldehyde, n-hexane, toluene, and xylenes).
The Health Effects Institute (HEI)-Energy Research Committee [15] recently published a review of published air quality studies relevant to potential UNGD-related human exposures, identifying the need for additional studies to address important gaps in knowledge. In particular, the HEI-Energy report [15] highlighted the need for more research to characterize the spatial and temporal variability in airborne exposure levels and the conditions contributing to this variability, including more air monitoring data representing a range of geographic locales, meteorological conditions, UNGD operational conditions, and exposure durations (e.g., from acute durations of hours to weeks to chronic durations of a year and longer). In our review of air quality data available for the Marcellus Shale region [16], we observed that the majority of datasets consist of short-term measurements collected over time periods of days to weeks, thus providing insufficient data to evaluate long-term exposure conditions for the full life cycle of well pad development. In addition, most of the available measurement data are for monitoring locations between 0.2 and 1 miles from the nearest UNGD site, with fewer data for closer monitoring locations.
Pennsylvania's Washington County is one area in the Marcellus Shale region that has experienced rapid unconventional natural gas development in the last 10-15 years. In Washington County alone, nearly 1700 unconventional wells have been drilled in the last decade, the most of any Pennsylvania county [17]. Public concerns have been raised regarding potential health risks posed by the proliferation of well pads and other associated natural gas infrastructure (e.g., compressor stations and processing facilities) in Washington County, with air emissions and exposures being particular concerns [7][8][9].
This air monitoring study was conducted in a part of Washington County with extensive Marcellus Shale development [18] (Figure S.1). The primary objective of the study was to investigate whether development activities and production operations at an unconventional natural gas well pad site may be contributing to ambient air concentrations of potential health concern at a nearby school campus. Almost 2 years of measurements for both PM 2.5 and individual VOC species were made at three monitoring locations, including two locations between the well pad and the school campus and all between 1000 and 2800 feet from the well pad site (Fig. 1), during all phases of development and production of natural gas and natural gas liquids. Wind data (direction and speed) were also continuously collected at two of the monitoring sites. Thus, this dataset is notable for the lengthy duration of air quality measurements in close proximity to a well pad during all phases of development and production, and the collection of local wind data for assessing the contribution of the well pad to measured air concentrations.
We conducted a public health evaluation of this air monitoring dataset by comparing short-term (24-h) and long-term (>1 year) average PM 2.5 and VOC concentrations to acute and chronic health-based air comparison values developed by public health agencies to serve as conservative and health-protective benchmarks. In addition, we compared PM 2.5 and VOC measurements to air Fig. 1 Map of the three air monitoring sites relative to the study well pad site, the school campus, and other local producing well pads. Wind roses are also shown for Sites 1 and 2 where meteorological stations were operated. concentrations measured at a background Washington County site more distant from oil and gas development activities and considered to be representative of regional background air quality. Although the study was designed to identify potential air quality impacts at the nearby school campus associated with operations at the study well pad site, the collected dataset reflects the cumulative contributions of air emissions from both the study well pad site and other local and regional sources.
Ambient air measurements
Three air monitoring sites were selected to address the primary study objective of evaluating air quality impacts at a nearby school campus associated with the development and operation of a UNGD well pad. Air monitoring sites 1 and 2 were located at distances of ≈2800 and 1000 feet, respectively, from the study well pad in the direction of the school campus (Fig. 1). Site 1 was the closest to the school campus (≈1500 feet to the southeast). The third monitoring site (site 3) was located about 1000 feet to the southwest of the well pad -i.e., upwind of the well pad for winds blowing in the direction of the school-to help evaluate whether other local sources, including the large number of other UNGD wells in the area (Figure S.1), may be important contributors to the site 1 and 2 measurements. Although an initial evaluation of the wind direction in the area indicated that winds were predominantly from the southwest, a monitoring site was not established to the northeast of the well pad because the area is wooded and inaccessible.
The monitoring program began in December 2016 during the site construction and set-up period of the study well pad and continued through October 2018 and after a full year of measurements were collected with all wells (six in total) in production. Table 1 shows the study air monitoring period relative to the different well pad activity periods, which included each of the typical well pad development phases, periods of lesser activity between the development phases that we have termed interlude periods, and the period when all wells were in production.
Ambient air measurements were made for PM 2.5 and 58 VOC species (see Table 1 for numbers of PM 2.5 measurement hours and VOC canister samples collected during each well pad activity period). Monitoring site 1 was chosen for the PM 2.5 measurements given that it was between the study well pad site and the school campus and in closer proximity to the school campus than site 2. Hourly average PM 2.5 measurements were collected continuously from February 2017 to October 2018 using a Met One Instruments Model This expanded set of VOC analytes was selected based on prior experience of the well pad operator regarding typical air emission sources at its well pads. VOCs not known to be associated with UNGD activities (e.g., chlorinated solvents like carbon tetrachloride and methylene chloride) were retained as analytes. Acrolein was one of the 58 target VOCs, but we have not reported or evaluated the acrolein measurements based on determinations by both PADEP and US EPA that acrolein measurements obtained using this method are unreliable [8,19,20]. Wind speed and direction were also measured at both sites 1 and 2 using solar-powered portable met stations from February 8, 2017 to October 31, 2018, and December 16, 2016 to October 31, 2018, respectively; additional meteorological parameters (e.g., relative humidity, barometric pressure, and temperature) were also collected at site 1.
Data analysis
Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA), SigmaPlot (Systat Software, Inc., San Jose, CA, USA), R (R Core Team, Vienna, Austria), and ProUCL version 5.1 (US EPA, Washington, DC, USA) were used for statistical and graphical data analysis. For PM 2.5 , we analyzed the hourly data, and also calculated 24-h daily average concentrations for days with 18 or more monitoring hours. For VOCs detected at least once, we substituted onehalf the limit of detection (LOD) for non-detects (LODs were typically 0.06 parts per billion). The 95% upper confidence limits (UCLs) of mean concentrations were calculated for VOCs detected at least twice using US EPA's ProUCL software, with reporting of UCLs for the methods recommended by the software. Correlations between measured concentrations at each site were examined using Spearman rank correlations. We conducted statistical testing to compare concentrations between sites, well activity periods, and wind directions using non-parametric tests that included the Kruskal-Wallis H Test and the Mann-Whitney rank sum test. Statistical significance was defined as a p value less than 0.05. For VOCs, we focused statistical testing on a subset of 14 of the 58 target VOCs that were consistently detected (i.e., detection frequencies >75%) at each of the monitoring sites.
The wind direction data collected at sites 1 and 2 were evaluated in several ways. Wind roses were prepared using WRPLOT View (Lakes Environmental, Waterloo, Ontario). To allow for the evaluation of wind directions on a daily basis corresponding to the VOC sampling periods, average daily wind directions were calculated, categorized according to an 8-point compass, and the percent of days in which the winds arrived from each of these directions was calculated. Given the hourly averaging time of the PM 2.5 measurements, the percent of hourly wind measurements in each of the eight directions was also calculated.
The PM 2.5 and VOC measurements were also compared to air concentrations measured at a monitoring site ≈10 miles away in Florence, PA, which has been used by PADEP as a background Washington County comparison site [8]. PADEP has described this rural monitoring site as being impacted primarily by regional transport [8]. PM 2.5 data for the study monitoring period were obtained for the Florence site from US EPA's Air Quality System (AQS). No VOC data are available from the Florence site for the study monitoring period, but maximum 24-h and mean VOC concentrations for 24-h canister samples collected every sixth day between October 2012 and December 2013 at the Florence monitoring site were obtained from PADEP [8] data summaries.
Health-based evaluation of ambient air measurements
We identified acute and chronic health-based air comparisons values (HBACVs) for this evaluation that are healthprotective benchmarks developed by public health agencies. The US EPA PM 2.5 primary National Ambient Air Quality Standards (NAAQS), which are developed to be protective of the health of the general public as well as sensitive populations such as asthmatics, children, and the elderly, were used as PM 2.5 acute and chronic benchmarks. We compared the maximum 24-h daily average concentration to the level of the NAAQS (35 μg/m 3 ), a conservative comparison given that the standard is intended to be compared to a 3-year average of the 98th percentile of 24-h measurements at a site. The annual PM 2.5 NAAQS requires that the mean annual PM 2.5 concentration at a site, averaged over 3 years, remains below 12.0 μg/m 3 . Given that PM 2.5 measurements were not available for a 3-year period, the mean concentration from the entire PM 2.5 sampling period was calculated and compared to the annual NAAQS.
The maximum 24-h measurement of each VOC detected at the three air monitoring sites was compared to acute HBACVs. We employed a tiered approach to identify acute HBACVs because there was not a single HBACV source inclusive of all measured VOCs. Agency for Toxic Substances and Disease Registry (ATSDR) acute inhalation Minimal Risk Levels (MRLs) were considered to be the preferred source of HBACVs because they are developed to be protective of 24-h exposure durations according to a well-documented and conservative process based on the most sensitive substance-induced end point of relevance to humans [21]. ATSDR acute inhalation MRLs are derived for 1-14 day exposure durations, and therefore comparison to the 24-h air monitoring site measurements is conservative. If an ATSDR acute inhalation MRL was not available for a VOC, acute inhalation reference concentrations (RfCs) from the Department of Energy Oak Ridge National Laboratory (ORNL) Risk Assessment Information System (RAIS) were used. When neither a ATSDR MRL nor a RAIS RfC was available, we derived an acute HBACV by multiplying a US EPA chronic reference concentration (RfC) by 10 [22]. For ethanol, the US National Institute for Occupational Safety and Health (NIOSH) timeweighted average recommended exposure limit (REL) was selected as the acute HBACV. We were not able to identify acute HBACVs for 11 VOCs, however, the majority of these were not detected in any samples.
We evaluated chronic health risks by comparing 95% UCLs of mean VOC concentrations (or for VOCs detected just once, mean concentrations that were calculated using half of the LOD for non-detects) at each site to chronic HBACVs. We consider 95% UCLs to represent conservative estimates of chronic air exposure levels at the monitoring sites given not only the likelihood that they are overestimates of true long-term average concentrations, but also due to the transient nature of the well pad development phases. For non-carcinogenic VOCs, US EPA non-cancer RfCs were used as chronic HBACVs, and for known or suspected human carcinogens, the lower value of either the non-cancer US EPA RfC or the cancer-based estimated continuous lifetime concentration was used. Using US EPA inhalation unit risk (IUR) estimates, we calculated the cancer-based estimated continuous lifetime concentrations for a 1-in-10,000 excess lifetime cancer risk, consistent with the US EPA residual risk program and with long-term comparison levels developed as part of US EPA's School Air Toxics Initiative [22,23].
Wind measurement data
Wind roses constructed from wind data collected at monitoring sites 1 and 2 indicate that the prevailing local winds were from the west and southwest (Fig. 1), and thus did not generally blow emissions from the well pad towards the monitoring sites and the school campus. However, it is expected that winds blowing from the southerly and southeasterly directions would have transported study well pad air emissions to monitoring sites 1 and 2, and a detailed evaluation of wind directions at these sites confirmed that winds blowing from southeasterly and southerly directions were relatively common during each of the well pad activity periods (Table S.
1).
Summary of PM 2.5 measurement data Table 2 provides a summary of the hourly PM 2.5 measurement data collected from February 2017 to October 2018 at monitoring site 1, showing an overall mean PM 2.5 concentration of 7.1 μg/m 3 and mean concentrations for the different well activity periods that ranged from a low of 5.4 μg/m 3 for the vertical air drilling phase to a high of 9.5 μg/ m 3 for the interlude III phase. Kruskal-Wallis H Tests identified statistically significant differences in hourly PM 2.5 concentrations between some of the well activity periods, including statistically higher concentrations for the interlude III and hydraulic fracturing periods and statistically lower concentrations for the vertical air drilling and production periods. When data were stratified by hours with winds from the south and southeast (i.e., from the direction of the study well pad site) versus winds from other directions, we observed statistically significant increased hourly PM 2.5 concentrations for the hours with southerly and southeasterly wind directions for all well activity periods except the interlude II and interlude III periods (Figure S.2); however, as illustrated by Fig. 2 which compares 24-h average PM 2.5 concentrations measured at monitoring site 1 with the corresponding 24-h average PM 2.5 concentrations measured at the PADEP Florence background site, highly similar PM 2.5 levels and temporal changes were observed as for a regional background site. Statistical testing showed no statistical difference between the two datasets (Mann-Whitney rank sum test, p value = 0.82). Table S.2 provides a comprehensive set of summary statistics for the VOC measurement data by monitoring site, showing that the majority of the target VOC species were either detected infrequently or not at all. Only 14 VOCs were consistently detected (i.e., detection frequencies >75%) at each of the three monitoring sites-acetone, benzene, 2-butanone, carbon tetrachloride, chloromethane, dichlorodifluoromethane, ethanol, Freon 113, methanol, methylene chloride, n-hexane, propylene, toluene, and trichlorofluoromethane. While median concentrations for these VOCs were frequently less than 1 ppb and all were less than 10 ppb, maximum detected 24-h concentrations exceeded 100 ppb for a few of the VOCs (acetone, ethanol, and methanol). As shown in Table S.2, there were no consistent patterns with respect to when maximum VOC concentrations were detected across VOCs and monitoring sites. For example, maximum detected concentrations for both acetone and methanol occurred in the interlude II, production, and interlude III periods for monitoring sites 1, 2, and 3, respectively; for toluene, maximum detected concentrations occurred in the horizontal drilling, production, and interlude II periods for monitoring sites 1, 2, and 3, respectively. For a limited number of VOCs, maximum concentrations occurred within the same well activity period for either all three sites or two out of three sites (e.g., benzene: site construction and set-up period for two sites; n-hexane: flowback period for all three sites; ethanol: production period for two sites; propylene: flowback period for two sites).
Summary of VOC measurement data
Correlational analysis revealed consistent moderate to strong correlations (Spearman's rank correlation coefficients r s between 0.36 and 0.90) across the three monitoring sites between several groups of VOCs, suggesting that they may have common sources (Tables S. 3a, b, and c). These groupings included 2-butanone, acetone, ethanol, methanol, and toluene (for 2 of the 3 sites, also methylene chloride); chloromethane, dichlorodifluoromethane, and Freon 113; n-hexane and propylene (for 2 of the 3 sites, also toluene); and carbon tetrachloride and trichlorofluoromethane. Benzene exhibited statistically significant weak to moderate N/A signifies that no data were collected for the period.
Conc concentration, PM 2.5 fine particulate matter less than 2.5 micrometers in diameter correlations (r s between 0.31 and 0.44) with propylene and toluene for all sites and with n-hexane for 2 of the 3 sites. For site 1, we also examined correlations between 24-h daily-average PM 2.5 concentrations and VOC concentrations, finding statistically significant weak correlations (r s between 0.23 and 0.37) with benzene, carbon tetrachloride, methanol, n-hexane, and toluene. Although suggestive of possible common sources, the correlational analysis do not allow for the identification and apportionment of sources, such as any contributions from the study well pad site relative to other local well pads and area air emission sources (e.g., industrial sources and traffic). Statistical testing using the Kruskal-Wallis H Test demonstrated no statistically significant differences in measured concentrations across the three monitoring sites for 9 of the 14 consistently detected VOCs. For the five VOCs where statistically significant differences by site were found (acetone, ethanol, methanol, methylene chloride, and toluene), multiple comparisons conducted using Dunn's Method consistently showed statistically significantly higher concentrations at monitoring sites 2 and 3 versus monitoring site 1, but no statistically significant differences between the site 2 and site 3 concentrations.
Focusing on sites 1 and 2 where there were concurrent wind direction measurements, Tables S.4 and S.5 compare summary statistics for the 14 consistently detected VOCs for sampling days with frequent winds from the southerly or southeasterly direction (i.e., from the study well pad site in the direction of the monitoring sites and the school campus) versus for other wind directions. These tables show relatively small difference in concentrations for the two sets of wind conditions (i.e., typical <1 ppb differences in median concentrations). For a limited number of the 14 VOCs, statistically significant increased concentrations were observed for sampling days with frequent winds from south and southeasterly directions versus other wind directions, including for acetone (site 1), benzene (site 2), 2-butanone (site 2), ethanol (site 1), n-hexane (sites 1 and 2), propylene (site 2), and toluene (sites 1 and 2). However, for site 3 (which is to the southwest of the study well pad), most of the same VOCs (all but ethanol and 2-butanone) were found to have statistically significantly higher concentrations for days with frequent southerly and southeasterly winds versus other wind directions, suggesting that other local/regional sources rather than the study well pad site may be responsible for the higher concentrations at monitoring sites 1 and 2 with southerly and southeasterly winds (the wind data for monitoring site 2 were used in this analysis due to the lack of site-specific wind data for monitoring site 3).
Additional statistical testing was conducted on the VOC data to investigate whether measured VOC concentrations were related to study well pad activity period. Given the small number of samples for some of the shorter duration well activity periods, well development and interlude periods were grouped together to form three broader activity periods-active well development periods (encompassing the site construction and set-up, vertical air drilling, horizontal drilling, hydraulic fracturing, and flowback periods), interlude periods (encompassing the three interlude periods), and the production period. This statistical analysis identified some statistically significant differences in VOC concentrations for these activity periods, although the results were not consistent across VOCs and monitoring sites and are thus difficult to interpret. For example, no statistically significant differences across the three activity periods were observed in the Kruskal-Wallis H Test for benzene (p values of 0.562, 0.379, 0.086), ethanol (p values of 0.061, 0.551, 0.347), or n-hexane (p values of 0.396, 0.170, 0.464). However, for both methanol and propylene, statistically significant differences were observed for the activity period factor for each of the three sites, with pairwise multiple comparisons on ranks (Dunn's Method) showing statistically significant lower methanol concentrations for the production period relative to the interlude periods for each site and to the active well pad development periods for one of the three sites, and statistically significant lower propylene concentrations for the production period relative to the active well pad development periods for all three sites. Table S.6 compares summary statistics for VOCs measured at the three study monitoring sites with the corresponding values for 2012-2013 sampling conducted at the PADEP Florence background site [8]. As shown in this table, the same set of 12 VOCs was consistently detected at the Florence background site as at the study monitoring sites (note that neither ethanol nor methanol was monitored at the Florence site). Summary statistics were very similar between the two datasets for seven of the 12 VOCs, including benzene, 2-butanone, carbon tetrachloride, chloromethane, dichlorodifluoromethane, Freon 113, and trichlorofluoromethane. Six of these 7 VOCs are not well established to be associated with UNGD activities; although benzene is known to be present in UNGD site emissions, both mean and maximum benzene measurements for the study monitoring sites were generally lower than the Florence background site measurements. The 24-h maximum measurements for at least one of the three study monitoring sites were noticeably higher than the maximum measured Florence site concentrations for acetone, methylene chloride, n-hexane, propylene, and toluene. Although the study well pad site may have contributed to some of these maximum 24-h concentrations, an examination of the wind measurement data indicated that some of the maximum measurement days had few, if any, winds from the direction of the study well pad site, suggesting the role of other sources unrelated to the study well pad site.
Comparison with health-based air comparison values (HBACVs)
The maximum 24-h PM 2.5 concentration for the entire PM 2.5 dataset was 24.6 μg/m 3 (based on data from 587 days with at least 18 hours of PM 2.5 data), which is well below the acute PM 2.5 HBACV of 35 μg/m 3 . The overall mean PM 2.5 concentration plus or minus one standard deviation was 7.1 ± 4.7 μg/m 3 , which is below the chronic PM 2.5 HBACV of 12 μg/m 3 , even when including one standard deviation. Therefore, measured PM 2.5 concentrations near the study well pad are below established regulatory levels of both acute and chronic health concern.
For VOCs, Tables S.7 and S.8 provide the full set of comparisons to acute and chronic HBACVs. As shown in these tables, maximum measured 24-h VOC concentrations for each site were consistently below the acute HBACVs, while 95% UCLs of mean VOC concentrations calculated from all measurements and from only the production phase at each site were all below chronic HBACVs. Figures 3 and 4 illustrate the large differences that are typical between the measured VOC concentrations and the acute and chronic HBACVs for the BTEX compounds (benzene, toluene, ethylbenzene, and xylenes). There were four compounds detected at one or more of the air monitoring sites, but for which no appropriate acute or chronic benchmarks were identified: hexachloro-1,3-butadiene, m-dichlorobenzene, p-ethyltoluene, and trichlorofluoromethane. These VOCs are not expected to present either acute or chronic health risks due to the infrequent detections (for all but trichlorofluoromethane) and the low, sub-ppb detected concentrations (all).
Discussion
Given the long duration of air monitoring, our study provided a dataset that reflects a range of well pad development phases and operating conditions, meteorological conditions, and exposure durations in the Marcellus Shale region. For PM 2.5 , the similar levels and diurnal trends between the study monitoring site and Florence background site indicate local/regional air quality as the dominant contributor to measured concentrations. Our analysis of PM 2.5 measurements across the different well activity periods suggest possible small PM 2.5 contributions at the measurement site from emissions at the study well pad site, such as for the hydraulic fracturing period; however, it bears mentioning that seasonal PM 2.5 trends are a likely confounder for data comparisons between well activity periods, and our analysis of PM 2.5 concentrations stratified by wind direction cannot differentiate between contributions from the study well pad and other local PM sources to the south and southeast. Both period-average and maximum 24-h concentrations for the well pad activity periods remained well below the US EPA NAAQS, indicating that if there were any PM 2.5 air quality impacts from development activities at the study well pad site, they did not contribute to NAAQS exceedances at the monitoring site. Given the location of the monitoring site between the study well pad site and the school campus, it is thus unlikely that the study well pad site caused any PM 2.5 NAAQS exceedances at the school campus.
Of the 14 consistently detected VOCs, seven (acetone, benzene, ethanol, methanol, n-hexane, propylene, toluene) have been associated with UNGD activities [9,19,[24][25][26]. Of these seven VOCs, there were known sources of all but ethanol and methanol at the study well pad. Some of our study findings, including statistically significantly higher VOC concentrations (e.g., acetone, ethanol, methanol, methylene chloride, toluene) at the two monitoring sites closest to the study well pad site (sites 2 and 3) relative to the third site (1) and higher maximum 24-h VOC comparisons (e.g., acetone, methylene chloride, n-hexane, propylene, and toluene) at the study monitoring sites relative to data for the PADEP Florence background site, may indicate small and transient VOC contributions from the study well pad site at the monitoring sites. However, overall, there was significant variability in measured concentrations across different VOCs, sites, and sampling periods, and other findings suggest contributions from other local and regional sources. These findings include the measurement of maximum 24-h concentrations for a number of VOCs (e.g., acetone and methanol) during the nonactivity interlude periods at the study well pad site, and the similar statistically significant differences in VOC concentrations at monitoring site 3 with southerly and southeasterly winds as for monitoring sites 1 and 2. Half of the consistently detected VOCs (2-butanone, carbon tetrachloride, chloromethane, dichlorodifluoromethane, Freon 113, methylene chloride, trichlorofluoromethane) were frequently detected by PADEP during its short-term air monitoring studies conducted at UNGD sites in southwestern, northeastern, and northcentral PA, and attributed to either regional or global air quality rather than Marcellus Shale development activities [19,24,25]. Other target VOCs reported to be associated with well development activities (e.g., 1,3-butadiene, ethylbenzene, xylenes, trimethylbenzenes) were infrequently detected despite the use of sensitive detection limits.
Regardless of VOC sources, the measured concentrations, which reflect the cumulative contributions of both air emissions from the study well pad site and from other local and regional air pollutant sources including other area well pad sites, are consistently below levels of acute and chronic health concern. Given that two of the air monitoring sites are located between the study well pad site and the school campus, the VOC and PM 2.5 measurement data do not provide evidence of either 24-h or long-term average concentrations of potential health concern at the nearby school campus. More study is needed to confirm their broader generalizability, but these study findings supporting the lack of elevated chronic exposure levels when PM 2.5 and VOC concentrations were averaged across measurements made during all phases of well pad development may apply to other locales in the Marcellus Shale region with similar types of UNGD sites and operations.
These findings are consistent with operator efforts to control air emissions through continued refinement of best practices, as well as evolving governmental regulations focused on air emissions. Operators have made continuous improvements to improve drilling performance, completion design, and production efficiency [27]. For example, during drilling, VOC emission rates are kept relatively low since hydrocarbon zones have not been stimulated, and emissions are combusted as required for safety. Range Resources has developed an enhanced flowback process using updated equipment and processes that is estimated to reduce air emissions during flowback by more than 80% [27]. Design changes, including a transition from flare stacks and enclosed burner units to vapor recovery compression and closed-loop systems, and upgrades to thief hatches on tank batteries [27], have been implemented to eliminate episodic high emission rates. In addition, operators such as Range Resources have deployed advanced technologies, including supervisory control and data acquisition software, remote telemetry monitoring systems, and infrared optical methane cameras, in order to oversee production and quickly respond to potential problems [27]. As discussed in Seguljic and Martin [28], both federal and Pennsylvania state regulations have evolved in recent years to target air emissions from well pad development and production operations.
Other recent studies in the Marcellus Shale region have similarly reported measured air pollutant concentrations to be generally below levels of human health concern for air sampling conducted in proximity to UNGD sites [4,8,9,16,19,24,25,29,30]. In particular, the Maskrey et al. [30] study was conducted in the same community as this study to investigate air quality impacts of development activities at another local UNGD well pad at the same school campus. Conducted on behalf of the local school board, this study made continuous measurements of total volatile organic compound (TVOC) concentrations and collected canister samples for individual VOC analysis at two monitoring sites (on the high school campus and at a private residence) over an ≈3-month period during four well pad activity periods: a baseline period before hydraulic fracturing commenced, the hydraulic fracturing period, the flaring period, and an inactive period following flaring. None of the VOC concentrations measured at either the high school or the private residence exceeded health-based benchmarks, and therefore the study investigators concluded that there was no measurable health impact from the well pad at either site.
The Allegheny County Health Department (ACHD) collected one of the few other long-term datasets for the Marcellus Shale region that included monitoring during all phases of development at nearby well pads. ACHD installed the Deer Lakes and Imperial Pointe temporary monitors in 2014 ≈0.85 and 0.3 miles, respectively, from the nearest well pads. The 4 years of VOC data available for each of these sites prior to their decommissioning in May 2017 have been categorized by ACHD according to activity time periods (baseline, site construction, drilling, fracking, and production) at the nearest well pads [5,6]. All measured VOC concentrations are consistently low and below healthbased benchmarks; for example, the highest 24-h benzene concentration measured during the ACHD monitoring was 0.8 ppb, while study-average benzene concentrations of 0.17 and 0.26 ppb were measured at the two sites [16].
Some studies have reported findings of elevated episodic air pollutant concentrations near UNGD sites during specific phases of development [31,32]. As part of the West Virginia University (WVU) Air, Noise, and Light Monitoring Study, McCawley [31] reported elevated maximum 72-h benzene concentrations ranging from 8.2 to 85 ppb at four UNGD sites during drilling (horizontal or vertical) or hydraulic fracturing/flowback activities. In comparison, maximum 24-h benzene concentrations for this study ranged from 0.29 to 0.95 ppb and were either lower than or only slightly above measured benzene concentrations for the PADEP Florence background site (Table S.6). Differences in these findings may be due in part to the closer proximity (between 492 and 1312 feet [33]) of the monitoring sites to well pads in the WVU study, as well as differences in well pad design and operations and processes. In addition, as mentioned previously, our study did not have a monitoring site in the prevailing wind direction, and it is thus not possible to rule out the presence of higher benzene (or other VOC) concentrations associated with the study well pad site at other non-monitored locations.
While we did not identify any clear, consistent patterns in short-term PM 2.5 and VOC concentrations across the study well pad development phases, we acknowledge some important study limitations that have bearing on future studies investigating the temporal and spatial variability of air quality nearby to UNGD activity. Measurements in this study were focused on PM 2.5 and VOCs, which are important classes of air pollutants that have been associated with UNGD. However, there are a number of other air pollutants that have also been associated with UNGD via primary emissions or secondary atmospheric formation, including other criteria air pollutants (NO 2 , carbon monoxide [CO], sulfur dioxide [SO 2 ], ozone [O 3 ]), and air toxics (e.g., acetaldehyde, formaldehyde, and hydrogen sulfide) [15,16]. Hydrogen sulfide was not measured in this study based on prior analysis conducted by the site operator that indicated that this is not a sour gas region with significant hydrogen sulfide emissions. While it is thus not expected that hydrogen sulfide emissions at the well pad would have posed potential health risks at the school, this study did not address other air pollutants besides PM 2.5 and VOCs. Similar to other air monitoring studies, this study was limited by the small number of air monitoring sites, and for VOCs, by the 24-h sample averaging time and every 6th day sampling frequency. The PM 2.5 and VOC measurement data provide estimates of air exposure levels at the monitoring sites themselves and may not be representative of other locations or time periods. For example, the limited number of air monitoring sites did not allow for the characterization of the full range of potential air exposure levels associated with the well pad development. However, for the primary study objective of evaluating air quality impacts of the study well pad at the school campus, the study design, and specifically the location of two of the monitoring sites between the study well pad site and the school campus, provided reliable evidence that air quality impacts of potential health concern were unlikely at the school. It is possible that higher VOC concentrations may have occurred on non-sampling days, however, it bears mentioning that the collection of 24-h samples every 6th day is the standard US EPA sampling design for air toxics [34]. Moreover, with greater than 1 year of air sampling during the production phase, there were more than 60 air samples collected for VOC analysis at each of the three air monitoring sites, and thus a sizable dataset to represent both 24-h peak and longterm average VOC concentrations during the production phase of the study well pad.
It is recommended that future air monitoring studies conducted in proximity to UNGD well pads include higher resolution sampling (e.g., 1-h) for VOCs, as the standard 24-h sample duration does not allow for the characterization of episodic peak air pollutant events. These data are needed to assess whether brief, intermittent exposures (i.e., 1-h or less) may pose acute health risks. Only a small number of studies conducted in the Marcellus Shale region have measured VOC concentrations for sampling frequencies of 1-h or less [1,2,31,34,35]. Given the difficulty of disentangling the contributions of a specific local well pad site from other area oil & gas development sites, it is also recommended that studies be designed to facilitate source apportionment modeling.
Our study results indicated some higher PM 2.5 concentrations during the hydraulic fracturing phase when ≈100 temporary diesel-powered combustion sources (e.g., generators, light towers, pumps, pressure washers, heaters, and air compressors) are typically utilized at well pad sites. Although mean and maximum 24-h PM 2.5 concentrations remained below the corresponding NAAQS during this time period, our findings indicate a need for additional PM 2.5 monitoring during well completion activities to investigate possible off-site impacts of the combustion emissions. It bears mentioning that the industry has transitioned to greater direct use of natural gas in place of diesel fuel, or co-firing of natural gas with diesel fuel, for both drilling and well completion equipment [27,[36][37][38]. Flowback is generally recognized as a source of hydrocarbon emissions, and the high concentrations of some hydrocarbons (e.g., hexane and propylene) were detected during the flowback phase of the study well pad. Recognizing the recent transition to reduced emissions completions, our findings suggest that additional VOC monitoring during the flowback phase could be helpful to confirm the efficacy of reduced emissions completions for mitigating off-site VOC impacts.
In conclusion, this air quality and public health evaluation, which was designed to identify air quality impacts of potential health concern at a nearby school campus associated with operations of a Marcellus Shale unconventional gas well pad, showed that measured PM 2.5 and VOC concentrations were consistently below acute and chronic health-based air comparison values. While the nearly 2 years of data collected at the three monitoring sites between 1000 and 2800 feet from the study well pad include some episodic short-term concentration increases that may be associated with the transient well pad development phases, the PM 2.5 and VOC measurements do not provide evidence of elevated long-term average concentrations at the three monitoring sites relative to a Washington County background site more distant from Marcellus Shale development. The study measurement data, which reflect not only any air emissions from the study well pad but also air emissions from other local and regional Marcellus Shale development, do not provide evidence indicating that the study well pad was a source of either acute or chronic PM 2.5 or VOC concentrations of potential health concern at the school campus; however, the study design did not include monitoring sites in the predominant wind direction or closer than 1000 feet from the well pad, and thus did not allow for the characterization of the full range of potential air exposure levels associated with the well pad development.
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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. | 2021-02-22T14:43:54.884Z | 2021-02-22T00:00:00.000 | {
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270821262 | pes2o/s2orc | v3-fos-license | Symptom response and episodic disability of long COVID in people with spinal cord injury: A case-control study
Background Spinal cord injury (SCI) is a consequence of significant disability and health issues globally, and long COVID represents the symptoms of neuro-musculoskeletal, cardiovascular and respiratory complications. Purpose This study aimed to identify the symptom responses and disease burden of long COVID in individuals with spinal cord injury. Methods This case-control study was conducted on patients with SCI residing at a specialised rehabilitation centre in Bangladesh. Forty patients with SCI with and without long COVID symptoms (LCS) were enrolled in this study at a 1:1 ratio according to WHO criteria. Result Twelve LCS were observed in patients with SCI, including fatigue, musculoskeletal pain, memory loss, headache, respiratory problems, anxiety, depression, insomnia, problem in ADL problem in work, palpitation, and weakness. The predictors of developing long COVID include increasing age (p<0.002), increasing BMI (p<0.03), and longer duration of spinal cord injury (p<0.004). A significant difference (p<0.01) in overall years of healthy life lost due to disability (YLD) for non-long COVID cases was 2.04±0.596 compared to long COVID (LC) cases 1.22±2.09 was observed. Conclusion Bangladeshi patients of SCI presented 12 long COVID symptoms and have a significant disease burden compared to non long COVID cases.
Introduction
Long COVID (LC) or long COVID symptoms (LCS) can be defined as a persistent clinical symptoms that is manifest after 12 weeks of SARS-CoV-2 infection, persist for two months and cannot be explained by any other clinical diagnosis [1].However, the symptoms can be episodic with relapsing-remitting symptoms that cause episodic disabilities [2].Globally, 43% of post-COVID cases are prominent, and the rate was 50% in Asia [3].Studies from Bangladesh revealed two important cohorts, reflecting that 16% to 24% of post-COVID-19 patients having long COVID [2,4].Considering the WHO clinical case definition globally, the prevalence of long COVID cases varies from 7% to 43% depending on the definition considered as long COVID [5].In Bangladesh, another prevalence study showed that 22% of patients with LCS require rehabilitation services [6].Moreover, according to couple of studies, the prevalence of LCS ranges from 71.21% to 77.7% [7,8].The most common symptom was respiratory manifestation, followed by fatigue at 51.4%.During the early phase of long COVID, fatigue, respiratory problems, headache, and joint pain were the prevalent symptoms [9].
Spinal cord injury (SCI) is a neurological disorder that occurs as a result of traumatic or pathological events and leads to substantial disability and health problems worldwide, including in Bangladesh [10].With neurological deficits and spinal cord injury, people have challenges performing daily activities because of functional limitations, activity limitations, and physical, environmental, and socioeconomic barriers [11].Approximately 19% of individuals who suffer from spinal cord injuries die within two years after being discharged from the hospital [12].Cardiorespiratory involvement issues are a significant factor in predicting death for individuals with spinal cord injuries, along with pressure ulcers and other genitourinary complications [13].Cardiorespiratory tract complications include cough, dyspnea, aspiration pneumonia, and severe respiratory infections [13].Complications post-SCI are well-established, and these patients are vulnerable if they are infected by COVID-19, which commonly causes cardiovascular and respiratory complications.
In Bangladesh, cardiovascular and respiratory complications are common among patients with long COVID, and the presentations includ dyspnea, chest pain, fatigue, weakness, and palpitations being prominent symptoms [4,5].In addition, in spinal cord injury patients, there is a pre-existing comorbidity of respiratory complications [13].Individuals with long COVID who have respiratory disorders, as well as those with spinal cord injuries, have a considerable burden of pre-existing cardiorespiratory and physical complications, in addition to the challenges posed by long COVID [5,13].People with more severe cervical SCI are more vulnerable to severe respiratory problems, and even in post-COVID-19, they had a risk of developing respiratory, immune, and other clinical complications that can lead to significant co-morbidities, mortality, and severe disease burden [14][15][16].
Since there are limited data on SCI patients with long COVID and disease burdens, a significant research gap is exsist both globally and in Southeast Asia.Previous scholarly literature has established a recommendation regarding the disease burden of people with vulnerable issues [5].Disease burden can be expressed through disability-adjusted life years (DALYs), a time-based measure that combines the years of life lost due to premature disability, premature mortality (YLLs), and years of life lost due to time spent in the state of health that is less than complete or years of healthy life lost due to disability (YLDs).This study bridged a significant research gap by revealing the symptom response and episodic pattern of disability and its significant disease burden for long COVID in people with spinal cord injury.Hence, the objectives of this study were 1) to determine the symptom response of long COVID in people with spinal cord injury and their episodic disability and 2) to predict the factors contributing to a significant disease burden for long COVID in people with spinal cord injury.
Study design
The study was a matched case-control study among patients with spinal cord injury who had long COVID and who resided at a specialised rehabilitation centre in Bangladesh [17].
Sample size
We used Epi info software version 7.2.0 to calculate the sample size.We used matched pair case-control study calculations.A total of 17.3% [18] of the SCI patients who had long COVID cases were exposed.A total of 22.4% of non-SCI patients with long COVID [6] were considered controls.In another study in the US [19], 6% of people with physical disability who recovered from COVID-19 were not exposed, and 14% of people with physical disabilities who had no COVID-19 were not exposed.The estimated OR was 0.2679 (95% CI; 0.1080, 0.6595).The McNemar test (χ2 9.4704, P<0.01) indicated a minimal sample size of 28 pairs.According to the specified timeline and from a population-based inception cohort [6], 28 patients with SCI and long COVID were screened between July and December 2021.The second screening was performed between July and August 2023.According to the WHO Working Group criteria, 20 of the screened patients had long COVID [1].Therefore, our sample size was 20 pairs of cases and controls.
Eligibility criteria
The primary eligibility criteria for the case were people with SCI aged more than 18 years with long COVID symptoms screened by a COVID-19 Yorkshire Rehabilitation Scale (C19-YRS).The eligibility for controls were people with SCI aged more than 18 years with no long COVID symptoms screened by a C19-YRS.
Data collection
Data collection and clinical examination were performed by the trained researchers.Skilled researchers conducted the data collection process to ensure accuracy and consistency.Before data collection, practical training is provided to sound clinical screening and minimise potential bias.After that, during data collection, the researcher closely monitored the data collection process.
Study procedure
To enhance the rigour of the study, we followed the Strengthening of the Reporting of the Observational Studies in Epidemiology (STROBE) guidelines for case-control studies.
Tools
The neurological status of the patients with spinal cord injury was determined using the American Spinal Injury Association (ASIA) ASIA impairment scale (AIS), and their motor scores of key muscles, sensory scores, and total scores were recorded.The long COVID symptoms were diagnosed according to the C19-YRS, a 22-item questionnaire with definite answers.The questionnaire includes several symptoms to screen for long COVID cases, and the question is further sub-categorized based on symptom severity scores (0-100), functional disability scores (0-50), additional symptom scores (0-60), and overall health scores (0-10).The tool is valid and reliable for screening long COVID symptoms [20].The disease burden was calculated according to disability-adjusted life years (DALYs), which was calculated by the sum of the years of life lost due to early death (YLLs) from that cause and the years of healthy life lost due to disability (YLDs) for individuals, not in a healthy as a result of the particular cause.The formula for YLLs is (the number of deaths) x L1 (life expectancy at death age), and the YLDs are calculated by the number of incidence cases for cause (I) x a disability weight (DW) x the average duration in years a person experiences with the case until recovery or death (L2) [21].
Data analysis
The data was analysed using Statistical Package for Social Sciences version 20.0 (IBM SPSS 20.0).The significance level was set as an alpha value less than 0.05.Descriptive statistics were performed according to the nature of data for sociodemographic information, health-related information, spinal cord injury-related information, and long COVID-related information.The mean and standard deviation of the duration of symptoms have been expressed by weeks.The differences in parameters between case and control groups were compared and analysed by using independent t-test, chi-square, and Fisher exact test depending on the independent variable as bivariate, trivariate, and multivariate.The predictors of Long COVID cases that have been diagnosed have been determined through an unadjusted odds ratio with a 95% Confidence Interval (CI).
Sociodemographic characteristics
The overall mean age of the respondents was 41±13.81years, and that of the participants without long COVID symptoms was 33.05±10.9years, with an LCS of 50.6±8.3years.The majority of the patients (72.5%) were males (n = 29).Out of 40 participants, 21 individuals (52.5%) agreed to receive a third dose of COVID-19 vaccine.Among those who received the third dose, 11 out of 20 individuals without long COVID symptoms (55%) and 10 out of 20 participants with long COVID symptoms (50%) chose to be vaccinated again.
Clinical presentation
The majority of respondents participating in the study were traumatic paraplegic 26 (65%), and non-traumatic paraplegic was 2(5%).The patients without LCS were both traumatic paraplegic and tetraplegic, 80% (16) of patients with LCS had non-traumatic paraplegic, and the rest of the patients were both traumatic tetraplegic and non-traumatic tetraplegic.The people with long COVID symptoms were diagnosed with AIS-B 5(25%), AIS-C 11(55%), and AIS-D 4(20%).The total motor score on the ASIA Impairment Scale was higher in people with long COVID symptoms 59.45±7.39than those without long COVID symptoms 43.7±20.2.In the sensory score, people with LCS were also higher at 140±8.41 compared to those without long COVID symptoms at 116±5.7.The mean BMI score of the respondents was 26.48.The details of the socio-demographic parameters of people with or without LCS are presented in Table 1.There was a significant relationship between the AISA impairment scale and motor and sensory scores among the groups of people with and without LCS p<0.001.
Long COVID symptoms in spinal cord injury patients
The long COVID symptoms in people with spinal cord injury had a diverse presentations.According to the COVID-19 Yorkshire Rehabilitation Scale, 12 symptoms have been observed in people with spinal cord injury having long COVID, including fatigue, musculoskeletal pain, memory loss, headache, respiratory problems, anxiety, depression, insomnia, palpitations, and weakness problems in the activity of daily living, and difficulty in work.The symptom of fatigue lasted for a more extended period of time (63.3±24weeks), followed by musculoskeletal pain, which lasted for 57.6±16 weeks.Moreover, people with pre-existing depression, insomnia, problems with ADL, and problems in work long COVID had a more devastating effect.The people with long COVID experienced depression that lasted for more than 61.45±4.3weeks, and limitations in the activity of daily living and participating at work lasted for 72±24 weeks, and 62.9±30 weeks respectively (Table 2).
Disability adjusted life years (DALYs)
The mean age at which people with SCI without long COVID had a healthy lifestyle was 33.05 years compared to people with long COVID as 50.6 years.The mean duration of SCI for nonlong COVID patients was 0.75, and the mean duration of comorbidities was 0.25 years.For long COVID patients, the duration of SCI was two years, and the duration of comorbidity was 0.5 years, with a mean duration of long COVID of 1.
Discussion
This groundbreaking study aimed to determine the symptom response and predictors of long COVID symptoms among vulnerable communities such as people with spinal cord injuries.In this matched case-control study design, 40 patients were selected; 20 participants with a spinal cord injury with long COVID symptoms and 20 without long COVID symptoms were determined and found that 12 of the symptoms were observed in SCI patients with long COVID and these findings were similar to those in the general population [4,5].The predictors of long COVID symptoms in patients with spinal cord injury were age, BMI, motor score, and duration of spinal cord injury.People with long COVID symptoms and spinal cord injury have a significant additional disease burden that hampers their memory, cardiorespiratory function, mental health issues, participation in daily living activities and work, and general lifestyle, which are common throughout the duration of the long COVID symptoms.This study answers the frequently asked questions about the vulnerability of long COVID in spinal cord injury patients to long COVID.
No similar case-control study has been conducted on patients with SCI with long COVID symptoms and their disease burdens.Previous studies on long COVID in the general population revealed that fatigue was one of the most important long COVID symptoms [3][4][5][6]10].The population proportion of fatigue ranges from 7% to 50% in the Asian region [17], and in Bangladesh, the percentage is 13% to 18% [4,5].In addition to fatigue, musculoskeletal pain is another long COVID symptom, with nearly 7% to 33% of the global and Asian people experiencing musculoskeletal pain [3], and in Bangladesh, the rate was nearly 10%.Interestingly, people having spinal cord injuries have multiple long COVID symptoms.Eight symptoms significantly impact multiple long COVID symptoms, such as fatigue, musculoskeletal pain, memory loss, headache, respiratory problems, anxiety, depression, insomnia, problems in ADL problems, work problems, palpitations, and weakness, each having multiple significant disease burdens [2-6, 10, 11].Respiratory problems in patients with spinal cord injuries may worsen the respiratory symptoms of long COVID.This can result in longer recovery periods and increased use of healthcare services [22].Another study noted that individuals with SCI have a higher risk of respiratory complications and increased respiratory symptoms associated with long COVID [23].Additionally, individuals with SCI who already have cardiovascular health issues and compromised autonomic regulation may be at a greater risk of experiencing cardiovascular symptoms associated with long COVID [24].In the general population, these long COVID symptoms had a significant impact on activity limitation, participation restriction, social participation, and environmental and personal factors in ICF domain.It was assumed that people with a spinal cord injury have an additional impact of long COVID in ICF domain [5].
The disease burden of a long COVID in patients with spinal cord injury is significant.Fig 1 presents a significant impact of the DALYs parameters in non-long COVID and long COVID symptoms for people with a spinal cord injury.Similar studies showed that long COVID can cause this additional disease burden according to DALY parameters [25,26].
This study identified four predictors of long COVID spinal cord injury cases, including their age, body mass index, motor score, and the duration of spinal cord injury.The duration of spinal cord injury is among the important parameters that longer duration of spinal cord injury can increase the risk of developing long COVID.Similarly, with increasing age, increasing BMI and motor score, people with spinal cord injury can experience long COVID symptoms.The previous study in Bangladesh on the general population found [4] the predictors of long COVID gender, smoking, and severity of COVID as predictors.Another study [27] found that the predictors were subsequently similar.Still, people with set occupations, for example, a healthcare professional, a law enforcement agency, female, and older age have more chance of developing long COVID symptoms.In our study, age and BMI were matched with the population courses.Still, motor score and duration of spinal cord injury are new predictors of long COVID in spinal cord injury cases.A study found no increased mortality risk from COVID-19 among those with a history of SCI, but diagnosis-associated LCS symptoms may worsen disability after infection [28].It is necessary to establish a long COVID rehabilitation protocol for vulnerable people with significant neurological issues, including spinal cord injury, and to monitor the additional disease burden of long COVID in spinal cord injury cases as a routine protocol.
Strength and limitation
The strength of this study was exploring a recommended research question that can significantly impact long COVID rehabilitation, especially in spinal cord injury cases.The study had some limitations, such as a small sample population from the largest rehabilitation hospital in Bangladesh.The reasons are stringent inclusion and exclusion criteria with fewer patients with SCI with long COVID symptoms.This is because there has no clearcut statement of the prognosis of SCI with long COVID.Each patient has a different prognosis and outcome.Therefore, the inclusion and exclusion criteria confirmed, we extracted the maximum number of homogeneous data from the population.However, the sample size with 20 people in each group was sufficient for a significant effect.We overcame the limitation by collaborating with patients in the organisation and strictly following the WHO Working Group criteria while recruiting cases from skilled and experienced healthcare professionals to ensure valid and justified answers to scientific queries.We recommend further study on the long-term consequences of long COVID and its impact on survivor-ship in spinal cord injury patients.
Conclusion
Twelve long COVID symptoms were observed in individuals with Spinal Cord Injury (SCI) in Bangladesh.The most prominent adverse effects on individuals with SCI and long COVID symptoms included fatigue, neuro-musculoskeletal discomfort, mental health issues, and limits in physical activity.Factors associated with the development of long COVID were advanced age, elevated BMI, and a prolonged period of spinal cord damage.Bangladeshi individuals with a spinal cord injury who had LCS experienced a substantial disease burden in comparison of non-long COVID cases, necessitating additional medical attention.
Fig 1 shows the flow diagram of the study process.The rehabilitation science researchers underwent a structured training program to become familiar with the diagnosis of long COVID symptoms before the study.Senior researchers constantly supervised by the study period.
Table 1 . Presenting sociodemographic parameters with or without LCS.
b Chi-Squire c Fisher Exact test; LCS: Long COVID symptoms https://doi.org/10.1371/journal.pone.0304824.t001 4 years.The non-long COVID cases had 38.8 years till average life expectancy, and the long COVID patients had 21.7 years till average life expectancy.Fig 2 shows the DALYs in long COVID and non-long COVID cases.The YLL for non-long COVID patients was 38.9±10.9years, and for long COVID patients, 21.4±8.2years.A significant difference was observed in overall YLD for non-long COVID patients (2.04±0.596)compared to long COVID patients (1.22±2.09).DALYs in long COVID and nonlong COVID patients for people with SCI are presented in Fig 2. | 2024-06-30T05:09:57.695Z | 2024-06-28T00:00:00.000 | {
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244194753 | pes2o/s2orc | v3-fos-license | Certain Coefficient Estimate Problems for Three-Leaf-Type Starlike Functions
In our present investigation, some coefficient functionals for a subclass relating to starlike functions connected with three-leaf mappings were considered. Sharp coefficient estimates for the first four initial coefficients of the functions of this class are addressed. Furthermore, we obtain the Fekete–Szegö inequality, sharp upper bounds for second and third Hankel determinants, bounds for logarithmic coefficients, and third-order Hankel determinants for two-fold and three-fold symmetric functions.
Introduction and Preliminary Results
Let the family of all the functions f that are analytic in U = {z ∈ C : |z| < 1} be represented by A and have the series form f (z) = z + ∞ ∑ n=2 a n z n , z ∈ U. (1) By convention, S represents a subfamily of class A containing all the functions that are univalent in U and satisfy the normalization property f (0) = 0 = f (0) − 1. In geometric function theory, a key problem of analytic functions is their connection with coefficient estimates for these functions. In 1916, Bieberbach conjectured that |a n | ≤ n, n = 2, 3, . . . This famous coefficient problem, the "Bieberbach conjecture" played an important role in research in this field for decades until, in 1984, Louis de Branges proved this result; see [1]. During 1916During -1984, researchers used different techniques and established a lot of coefficient results for various subclasses of S. The subclasses worth mentioning here are the class S * , of starlike functions; the class K , of convex functions; and R, known as the functions of bounded turning. They are defined as below: respectively. These classes can also be defined with the help of the subordination relation. We say that, for analytic functions, f 1 (z) is to be subordinated to f 2 (z) in the region U and denoted mathematically as f 1 (z) ≺ f 2 (z) if a function u(z), known as the Schwarz function, satisfies the conditions |u(z)| ≤ 1 and u(0) = 1, such that f 1 (z) = f 2 (u(z)). Moreover, if f 2 (z), belongs to S, then due to [2,3], the following equivalent conditions will be true Thus, one can define S * (ψ), K(ψ) and R(ψ) as: In (2), if the right hand side is changed, the several well-known subfamilies are originated. For example, if we put ψ = 1+Az 1+Bz , we obtain the Janowski-type class of starlike functions; see [4] for details. Meanwhile, if we change the parameters A and B by 1 − 2α ans −1, respectively, then we obtain a family of starlike mappings of order α; these were defined and discussed in [5]. Additionally, for the choice of ψ = 1 + 2 we obtained a corresponding class of starlike functions, introduced by Ronning; see [6]. Furthermore, if ψ = √ 1 + z, we obtain the class starlike function related to the lemniscate of the Bernoulli domain, defined by Cho et al. [7,8]. Goel and Kumar,in [9], defined the class S * SG , the family of starlike mappings connected with a type of mapping known as modified sigmoid functions. Moreover, if we use ψ = 1 + sin(z), we obtain a subclass of starlike mappings in relation to the sine function; for details, see [10]. Mendiratta et al. The authors of [11] obtained a subfamily of strongly starlike mappings connected with the exponential function for the choice of ψ = e z . Sharma et al. [12] derived a subfamily of starlike mappings associated with a cardoid domain.
In a similar way, one can find various important subclasses of these functions in [13][14][15][16][17][18][19][20][21] for some specific value of ψ. Of these, some well-known ones are the mappings associated with and related to Bell numbers, curves that are shell-like in association with Fibonacci numbers, and mappings associated with the conic domains. Lately, utilizing the techniques of Ma and Minda [22], Gandhi [23] defined a family of starlike functions associated with a three-leaf function, i.e., and characterized it with some important properties.
For the function f that has the form (1), Pommerenke [24,25] defined the Hankel determinant H q,n ( f ) with the parameter q, and n ∈ N = {1, 2, 3, · · · }, as follows: For some subclasses related to the class A, the bounds of H q,n ( f ), for any fixed integer q and n, are evaluated. Almost all the subclasses related to the class S were investigated for the sharp estimates of H 2,2 ( f ) = a 2 a 4 − a 2 3 by Janteng et al. [8,26]. However, for the family of close-to-convex functions, the sharp estimates are still not known (see [27]). On the other hand, Krishna et al. [28] proved the better estimate of |H 2,2 ( f )| for a subfamily of Bazilevič functions. More detailed work on H 2,2 ( f ) can be seen in [29][30][31][32][33] and also the references cited therein.
The determinant is known as the third-order Hankel determinant, and an estimate of this Hankel determinant |H 3,1 ( f )| is more difficult than the second Hankel determinant; that is why a lot of researchers have focused on this field. In 1966-1967, Pommerenke defined the Hankel determinant, but it was not evaluated till the year 2010. In 2010, Babalola [34] was the first researcher who worked on H 3,1 ( f ) and successfully obtained the upper bounds of |H 3,1 ( f )| related to the classes S * , K and R. Following this result, a few researchers extended this work for the various subcollections of univalent and holomorphic functions; see [35][36][37][38][39][40][41][42][43][44][45][46][47].
Additionally, he asserted that the inequality above is not sharp. For sharpness, he considered the subfamily of S * , C and R functions to define them with m-fold symmetry, acquiring a sharp bound. In 2018, Kowalczyk et al. [49] and Lecko et al. [50] obtained sharp inequalities, which are for the classes K and S * (1/2), where the symbol S * (1/2) represents the subcollection of starlike functions of order 1/2. In [51], an improved bound |H 3,1 ( f )| ≤ 8/9 for f ∈ S * was given, which is not the best possible. Our main purpose in this article is to first study four sharp coefficient estimates, the Fekete-Szegö inequality and sharp second Hankel determinant, the third-order Hankel determinant, the bounds for logarithmic coefficients, and the two-and three-fold symmetric functions.
The Sets of Lemmas
Let P be the subclass of mappings p that are analytic in D with p(z) > 0 and its series form, as follows: Lemma 1. If p(z) ∈ P and it is of the form (6), then |c n | ≤ 2 for n ≥ 1, and and for real λ For the results in (7) and (8), see [52]. Additionall, see [53] for (9) and [22] for (10).
). Let p ∈ P have the representation of the form (6); then, for any real numbers α, β and γ
Upper Bound
Theorem 1. Let f (z) ∈ S * 3L be of the form (1); then: All these bounds are sharp for the functions defined below, respectively.
Theorem 2. Let f (z) ∈ S * 3L be of the form (1). Then, The result is sharp for the function defined in Equation (19).
Proof. Since from (24) and (25), we have by applying (9) to the above equation, we obtain the desired result.
For ζ = 1, we obtain the corollary stated below: The bound is sharp for the function defined in Equation (19).
Theorem 3. Let f (z) ∈ S * 3L be of the form (1). Then, Proof. Since from (24) and (25), we have by applying (10) to the above equation, we obtain the desired result.
Theorem 4. Let f (z) ∈ S * 3L be of the form (1). Then, The estimate is sharp for the function defined in Equation (20).
Proof. Since from (24)-(26), we have now, the implementation of Lemma 2 to above equation leads us to the desired result.
Theorem 5. Let f (z) ∈ S * 3L be of the form (1). Then, The result is sharp for the function defined in Equation (19). Applying a triangular inequality along with |z| ≤ 1 and |x| = b with b ∈ [0, 1], we have Since H(c, b) is an increasing function with respect to b so H(c, b) ≤ H(c, 1), putting b = 1 in the above, we obtain
For function f of class S, we denote the logarithmic coefficients with γ n = γ n ( f ), and they are defined by the following series expansion: The logarithmic coefficients of function f given in (1) are as follows: Theorem 7. Let f (z) ∈ S * 3L be of the form (1); then, The first two bounds are sharp.
Proof. From Equations (33) to (35), we obtain The bounds of |γ 1 |, |γ 2 | follow from Lemma 1, and |γ 3 | follows from Lemmas 1 and 2. By S (m) , we mean the set of m-fold symmetric univalent functions having the following series form:
Bounds of H 3,1 ( f ) for Two-Fold and Three-Fold Symmetric Functions
The subclass S * (m) 3L is a set of m-fold symmetric starlike functions associated with a modified sigmoid function. More precisely, an analytic function f of the form (38) belongs to class S * (m) where the set P (m) is defined by 3L is of the form (38), then Proof. Since f ∈ S * (2) 3L , there exists a function p ∈ P (2) such that Using the series form (38) and (40), when m = 2 in the above relation, we have Now, using (42) and (43), we obtain Now, using (7) and (8) with the above, we obtain the required result.
Theorem 9. If f ∈ S * (3) 3L is of the form (38), then The result is sharp for the function defined in (20).
Proof. Since f ∈ S * (3) 3L , there exists a function p ∈ P (3) such that Using the series form (38) and (40), when m = 3 in above relation, we have Using (7), we obtain the desired result.
Conclusions
In the present article, we find four initial sharp coefficient bounds, the sharp Fekete-Szegö inequality, the sharp second Hankel determinant, the third Hankel determinant, and the bounds for logarithmic coefficients, and at last, we find out the bounds of H 3,1 ( f ) for two-fold and three-fold symmetric functions for the class S * 3L . Obtaining a sharp estimate for the third Hankel determinant is still an open problem for a considered class. Additionally, there is an opportunity for researchers to investigate the generalized Zalcman conjecture, Krushkal inequality and fourth-order Hankel determinant for this class. | 2021-10-18T18:25:42.988Z | 2021-09-24T00:00:00.000 | {
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235652070 | pes2o/s2orc | v3-fos-license | Archaeoastronomical study of Christian churches in Fuerteventura
We present an archaeoastronomical study of the orientations of the colonial Christian churches on the island of Fuerteventura, in the Canary Islands, Spain, mostly built from the period of the Norman conquest in the 15th century to the 19th century. Our goal is to analyze the possible astronomical influence on the orientation of these churches. Preliminary results suggest that the vast majority of the island's religious constructions have their axes oriented within the solar range, between the extreme azimuths of the annual movement of the Sun as it crosses the local horizon. This differs from what was found on the islands of Lanzarote and La Gomera (also in the Canaries) previously studied.
Introduction
From the very beginning, Archaeoastronomy has mainly focused on the study of historical constructions, such as temples, megalithic monuments and structures built by different cultures around the world, and on the analysis of the possible influence of celestial objects and phenomena on their design (Ruggles, 2015). In this context, one of the most relevant aspects to approach in this field is the study of the orientation of the constructions, which might provide valuable information regarding the intentionality of the people who built them. In particular, we are interested in studying the orientation of Christian churches in Fuerteventura, one of the Canary Islands in Spain, from the time of the Norman conquest in the fifteenth century to the nineteenth century. Ancient texts state that Christian churches should be oriented with their apse towards the east. In other words, their main axis, that is, the line from the entrance to the altar, should point in the direction from where the Sun rises in a particular day of the year, in this case, the equinox (McCluskey, 1998). In this work we present a brief summary of our analysis and very preliminary results obtained from data previously collected by our research group to test if this is the general case in Fuerteventura (Muratore & Gangui, 2020). Our goal is to investigate if the data supports a possible relationship between the churches orientations and astronomical phenomena. As a future project, we also aim to investigate whether the pre-Hispanic population already existing on the island at the time of the conquest might have had influence on the construction of the first Christian churches. This research is part of a larger project that includes previous work on other islands of the same region (Gangui et al., 2016;Di Paolo et al., 2020).
Data and methodology
In order to analyze the orientation of the churches, onsite measurements taken in a previous field work were used. These consist of the azimuth and the altitude of the horizon corresponding to the direction of the main axis of each church. In those cases where the elevation was difficult to determine, for instance due to the presence of modern buildings, the on-line tools available at heywhatsthat.com allowed us to reconstruct the horizon using a digital model of each site.
The values of the azimuths, measured using a high precision compass, were then corrected by magnetic declination, allowing us to achieve a precision of 0.5 • in the astronomical azimuths. These directions are in general indicated in circular azimuth diagrams as the one shown in Fig. 5 of Muratore & Gangui (2020), in order to help visualize the orientations obtained.
Given that there are 48 churches distributed in approximately 1700 km 2 , we consider the sample to be representative and significant for this type of study.
In order to have a measure of the orientations which is independent of the geographical location and topography across the island, the azimuth and the altitude, together with the latitude of each site under study, are used to determine the astronomical declination corresponding to the direction of the main axis of the church. The declination histogram for the whole group of constructions is given in Fig. 1. The quantities thus obtained may then be compared with the declination of the Sun for different days around the date of construction of each particular temple. This will allow us to investigate if there is a possible connection between the Sun and the architecture of the structure, which in turn might help us to find a possible explanation for the orientation of the church. A thorough statistical analysis of the declination distribution that we found is currently in progress, as it is necessary to confirm the general pattern that was observed in the azimuth diagram. Fig. 1 shows a clear accumulation of declination values within the solar range (delimited by the short vertical bars in the picture), supporting the trend already found in the azimuthal orientation of the churches in Fuerteventura (Muratore & Gangui, 2020). While some of the constructions do not follow this trend, 35 out of the 48 churches do show orientations that fall within the solar range. Moreover, most of them point towards directions not far from east, corresponding to the rise of the Sun near the equinoxes. This is now confirmed by our declination histogram, where we see that the frequency of orientations increases remarkably between the extreme positions of the Sun at the solstices. This result is consistent with the pattern of orientations found in historical churches built before the conquest in the regions where the colonizers were originally from.
Results
On the other hand, some of the temples in our sample present orientations outside the solar range. This may be due to different reasons. For example, some historical churches were built as private temples by landowners, which means that their position and orientation were subject to the distribution of the existing buildings. In a few cases the construction of the structures was limited by, or planned according to, the topography of the island, including coastlines, gorges and mountains. In the case of some modern churches built within big cities, there was probably no possibility or intention to choose their orientation according to the early Christian texts.
Discussion
As mentioned in the Introduction, according to early prescriptions the apse of Christian churches should face east, that is, point towards the rising Sun during equinoxes. Since the definition and determination of the date corresponding to the equinox may be affected by different factors (Ruggles, 1997), this kind of study must include not only the reconstruction of the sky in the epoch when the construction was planned, but also the possible influence of the equinox used as reference.
On the other hand, one of the main difficulties regarding the knowledge about the churches is to find reliable information about them, and sometimes, no information can be found at all. For example, to delimit the epoch when they were planned or when their construction began the information is not always easy to find, and in some cases, different sources make reference to different years or even different centuries. A detailed analysis of historical documents from different periods since the colonization, such as letters, official and informal reports, and records kept by the people in charge of the churches themselves will be extremely important. The search for other sources of information will also be necessary.
The uncertainty in the date of construction of the churches actually leads to a range of possible declinations for the Sun, making it difficult to establish if there is a correlation, for example, between the orientations and the dates of the equinoxes and solstices.
Once the dates are determined, the change from the Julian to the Gregorian Calendar introduced in 1582 has to be taken into account, not only for establishing the epoch when the construction was planned, but also for determining the dates, for example, of the equinox corresponding to that particular epoch.
The influence of the original island inhabitants on the orientation of the first temples might also be expected, since this is the case in other parts of the Canarian Archipelago (Belmonte, 2015). However, pre-Hispanic orientations would be mainly solstitial, while our first results seem to indicate that most of the temples face towards directions relatively close to the equinox. During the first years after the conquest of Fuerteventura, the original inhabitants and the colonists BAAA, 62, 2020 gave rise to a new society, where both coexisted. This might have resulted in a majority of temples oriented within the solar range (although not always strictly towards the equinox), with some of the churches orientations corresponding to important dates for the community, which includes both groups, each one with its own religious customs.
On the other hand, a thorough orographic study of the island has to be carried out. A complete analysis must include detailed topographic maps, which will allow us to study the geographical profile and the altitude of the horizon around the temples. The direction of strong winds also has to be taken into account, since it could influence the orientation of the churches in some parts of the island.
Let us note that in La Gomera and in Lanzarote, two of the islands of the Canarian Archipelago where the orientations of the churches and their possible origin were thoroughly studied, we find a completely different scenario than the one suggested by the preliminary results obtained in Fuerteventura.
Even though further studies might reveal new information that leads to alternative interpretations, the results obtained so far for the orientations of the historical Christian churches of Fuerteventura strongly suggest that in this island, the prescriptions for canonical orientations were followed.
Future work will include the study and comparison of these results with those obtained for other islands, as well as with the orientations of historical Christian churches built in the European continent during the same period as the ones built in the Archipelago. | 2021-06-28T01:16:09.377Z | 2021-06-25T00:00:00.000 | {
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261979251 | pes2o/s2orc | v3-fos-license | Human enhancement technologies and the arguments for cosmopolitanism*
According to political minimalism , a debate is considered political when it revolves around the question “What shall we do?” This account suggests that certain issues related to human enhancement technologies (HETs), which have traditionally been addressed in the realm of applied ethics, could be better approached from a political standpoint. However, this raises the question of who constitutes the “we” – the communities that face the political challenges posed by HETs. We argue that there is a global human community that directly faces at least some of these challenges, and this fact underscores the relevance of a cosmopolitan perspective. While some authors have already advocated for a cosmopolitan approach in addressing issues such as poverty or climate change, they often do so from a moral outlook, without adequately distinguishing between ethics and politics. In contrast, we assert that HETs present compelling arguments in favour of cosmopolitanism as a political stance. In support of this claim, we consider two cases: the pills that would allow people to eat at will without gaining weight, and the choice between different types of cognitive enhancers.
Political minimalism
A student who is deliberating whether to invest their savings in a hair implant to enhance their appearance or in a programming course that aligns with their goal of securing a well-paid job is engaging in what is commonly referred to as "prudential" reasoning.Individuals involved in prudential deliberation seek to clarify their own interests and determine how best to fulfil them.When engaged in prudential deliberation, individuals do not consider the goals of others, unless such goals impact the calculation of the most effective means to achieve their own ends. 1 In contrast, an individual living in a putative future society who is considering whether to spend their savings on a genetic modification aimed at enhancing the intelligence of their offspring or to contribute those funds to vaccination programmes in poor countries is grappling with a moral dilemma.In this case, they see the interests of others as legitimate ends that they consider alongside their own when making decisions.21.The characterisation of the prudential point of view and its differentiation from the moral perspective are classic themes of ethical theory.In this article we are not going to discuss the different proposals to define the prudential and to distinguish it from the moral, which can be found in the literature.Instead, we will use the definition that we have developed in other works (Bermejo-Luque and Rodríguez-Alcázar, forthcoming; Bermejo-Luque, forthcoming).2. This way of characterising the realm of morality may recall certain conceptions of ethics in terms of impartiality (such as, for example, the ideal observer theory: cf.Firth, 1952;Hare, 1981: 44).However, our metaethical claim here is more modest: saying that the moral point of view must consider the interests of others is not the same as prescribing to what extent those interests must be taken into account, whether one must be completely impartial when Let us remain in this future era.Now, envision a future parliament engaged in a debate on whether to pass legislation authorising genetic enhancement interventions such as the one described above.What kind of inquiry would this parliament be undertaking?It might be argued that it is facing, once again, moral questions: are not the members of this parliament contemplating decisions that could impact the interests of others?Yes, but we must differentiate between two types of questions that these MPs could explore in this context.On one hand, indeed, the MPs could pose moral questions to themselves.Such questions arise whenever an MP ponders what they should do personally in a situation like the one described: should the MP vote in accordance with their convictions or their interests?Should the MP adhere to the party's rules and vote with the majority, or should they break the voting discipline if the majority vote contradicts their conscience?These are questions that MPs can certainly ask themselves as individuals concerned with their own interests or as moral agents who take the interests of others into consideration.However, MPs, as members of the parliament, will also typically raise other types of questions, including: -Is the increase of the IQ of a few individuals whose parents can afford gene editing beneficial for the whole community?-Should the freedom of those parents wishing to increase the IQ of their offspring be limited, to prevent the growth of inequality within the political community?-Should the state subsidise gene editing to increase the average IQ of the population?-Should the state make this kind of gene edition compulsory for every couple intending to procreate?-How would the state deal with the possibility that other countries authorise this gene editing if it is not allowed in ours?
These are political questions, and there exists a metapolitical perspective, namely political minimalism, that clarifies why we should perceive them as political. 3According to political minimalism, politics is a practice that seeks to address the question "What shall we do?"This question is approached as an exercise of prudential rationality, with the subject being a political community.As a result, politics, morality and individual prudential deliberation diverge as they seek to answer different questions.The questions above can be judging the interests of others, or whether it is legitimate to prioritise the interests of certain people (for example, members of our own family or compatriots).These are questions that are to be answered by a normative ethical stance, not by a meta-ethical proposal like the one we take for granted here.In any case, it is not the aim of this article to provide arguments in favour of a certain characterisation of morality.Here we will take for granted the rough characterisation above, which we argue elsewhere (Bermejo-Luque and Rodríguez-Alcázar, forthcoming; Bermejo-Luque, forthcoming).3. See Rodríguez-Alcázar (2017a) and Bermejo-Luque and Rodríguez-Alcázar (forthcoming).
interpreted as inquiries that individuals may pose when trying to determine the suitable course of action for a specific political community, such as a community comprised of state citizens.
The political community in our example, represented by the parliament, focuses on the aims of its members and considers the best means to coordinate the pursuit of those aims, rather than considering the interests of others.Thus, the parliament addresses a question resembling the one asked by the student.The difference lies in the fact that the parliamentary debate involves a group -a political community -that, through its representatives, deliberates on what actions to take.While the student seeks to provide a correct answer to the question "What shall I do?", the MPs strive to find the appropriate answer to the question "What shall we do?"In other words, we have transitioned from individual prudential rationality to collective prudential rationality, which can be deemed "political" when the collective entity is a political community.Both perspectives differ from morality in that they consider the interests of the subject -be it an individual or a community -as ends, without considering the interests of others.
Some philosophers have conflated the political and moral perspectives by arguing that politics involves fulfilling the interests of others.This confusion is characteristic of what Williams (2005) referred to as "political moralism".Political moralism is a metaphilosophical stance that asserts the legitimacy of political decisions and institutions based on their pursuit of certain moral values or adherence to moral constraints.According to Williams, the main fallacy of political moralism lies in attempting to impose moral aims or restrictions on politics, which, in fact, has its own distinct goal: "the securing of order, protection, safety, trust, and the conditions for cooperation" (Williams, 2005: 3).Yet, defenders of political minimalism have pointed out that proposing order as the essential goal of all political communities, past and future, as Williams and other realists do,4 is as arbitrary as proposing any moral end -such as liberty, wellbeing or virtue -as the permanent and supreme goal of all political communities (Rodríguez-Alcázar, 2017a).
In our view, the primary flaw of political moralism is not merely proposing a specific moral objective as the primary and enduring goal of politics (although that is indeed a mistake), but rather overlooking the existence of political communities.These communities are distinct from individual subjects who grapple with moral and prudential issues.It is important to distinguish between the perspective of MPs who, as individuals, contemplate their actions within a political context, and the perspective of an MP who, as a representative of a political community, ponders how best to coordinate the interests of the members of that community.For instance, the latter perspective may involve considering appropriate legislative measures regarding potential human enhancements through gene editing.
The confusion of these two perspectives surely responds to a political ontology according to which only individuals can be political subjects.But, even if it is true that only individuals can have political rights and duties, there are communities for which the question "What shall we do?" makes perfect sense, and we surmise that this is, precisely, the question that provides access to the realm of politics.This question is triggered by the type of coordination problems that communities face, 5 and it can be formulated by a group of people (for example, a local council or an assembly), as well as an individual (for example, an MP, a mayor or an ordinary citizen), provided that they adopt the community's perspective, which consists of the set of goals of all of its members and the resources at its disposal to achieve them.
Human enhancement technologies (HETs): moral and political questions
An issue in which it is common, as in many others, to conflate the ethical and the political perspectives is that of the debate about human enhancement. 6 Following Cabrera (2012: 3), we understand human enhancement in a broad sense, as "any intervention or activity by which we improve or augment in any sense (e.g., performance, appearance) our abilities, bodies, minds and wellbeing."Enhancement activities thus understood have accompanied humanity down the centuries, from the invention of clothing to the introduction of compulsory education.However, the widespread use of contemporary technologies has opened new possibilities for human enhancement and has accelerated the development and application of the required technologies.We will call those technologies that are necessary for the realisation of human enhancements, in the broad sense established above, human enhancement technologies (HETs). 7 In some instances, these technologies already exist, such as cosmetic surgery and distance learning, while in other cases, their emergence is anticipated within the next few years or decades.Examples include moral enhancement through chemical means or gene therapies, and IQ enhancement through gene editing.Present-day societies, particularly the wealthier ones, allocate substantial economic and human resources to the development of HETs, through both public and private initiatives.This investment is expected to further increase in the future.Ethics rightly concerns itself with the moral questions that existing and future HETs may raise for individuals.However, it is equally important, if not more so, to address the diverse political debates that var-5.By "coordination problems" we mean problems resulting from either the need to coordinate actions to achieve some common goal, or the need to resolve conflicts of interests among individuals or subgroups in the community.6.On the drawbacks of adopting an exclusively moral perspective on HETs and the advantages of claiming an autonomous political perspective, see Rodríguez-Alcázar (2017b).7.With the term "technology" we do not refer narrowly to artifacts or processes developed from scientific knowledge, but to networks and processes connecting human and non-human beings in social and legal environments (Latour, 2005).
ious communities will face regarding the authorisation, promotion, financing, design and utilisation of HETs.While the effective implementation of some of these HETs may still be several decades away, we should not only be concerned about the consequences of already fully developed technologies but also about the potential outcomes of those currently being designed or envisioned.Future technologies are influenced by present social decisions (Bijker, Hughes and Pinch, 1989), and their social and environmental impacts will vary depending on how they are shaped (Winner, 1986).Therefore, it is crucial for the affected political communities to anticipate the configuration processes of forthcoming HETs well in advance, and to take measures to prevent possible negative consequences arising from ill-conceived designs. 8According to the distinction between ethics and politics previously outlined, these are typically political tasks that require answering essentially political questions.
What political community?
We have emphasised the significance of political deliberation in guiding and regulating the design, development and utilisation of HETs and other technologies.As previously stated, political reasoning occurs when one contemplates the goals of the members of a community and determines the most effective means to attain them.Now the question arises: which communities are pertinent to the discourse surrounding HETs?In other words, which communities encounter the coordination problems triggered by HETs?Until the end of the 19th century, it was usual in political theory to identify the sphere of politics with that of the state (Alexander, 2014).In this context, the political community par excellence was the one delimited by the borders of a nation-state.However, contemporary political theory often omits reference to the state when defining politics.There are at least two reasons for this exclusion.First, we now acknowledge the existence of political communities beyond the state, such as tribes, guilds, religious orders and others.Some of these communities predate the emergence of states, and many coexist alongside them.Second, certain political ideologies, like anarchism and communism, advocate for the establishment of stateless societies, demonstrating that politics without a state is a conceivable concept, regardless of the feasibility of these political projects.We argue that a comprehensive understanding of our perceptions regarding politics necessitates considering all possible communities that give rise to political questions, including various types of human groups such as university departments, chess clubs and neighbourhoods.These groups encounter coordination problems that cannot be 8.While all technologies are socially shaped, this social shaping is not always done in democratic or participatory ways.Although we are in favour of the involvement of political communities in the shaping of socially crucial technologies (by means such as consensus conferences and others), we shall not develop here our arguments for this stance.For a discussion on the importance of adopting a political perspective on the shaping of technologies, see Rodríguez-Alcázar, Bermejo-Luque and Molina-Pérez (2021).
resolved solely through personal interactions, and consequently enact some rules to tackle them.All political communities have a beginning, undergo changes, and eventually may cease to exist.Associations are established for specific purposes but they may dissolve over time.Similarly, states are formed and undergo alterations in their borders and internal structures; and based on historical induction, we may conclude that no state will endure indefinitely.Even the institution of the state itself may eventually disappear.The cohesion of a political community is based on its members sharing common interests and goals, and their collective pursuit of them.However, these interests and goals can differ among different political communities.Some communities may have a singular purpose, such as organising a biannual conference or supporting a football team.In contrast, other communities encompass a multitude of goals.For example, modern democratic states are expected to promote welfare, safeguard rights and freedoms, uphold justice, maintain order and security, protect external borders, and more.Nonetheless, none of these goals serves as the defining purpose of politics, despite the common mistake of associating the latter with a typical state function. 9We maintain that the constitutive aim of politics is to respond adequately to the question "What shall we do?"Moreover, we assert that effective policymaking entails understanding the true objectives of a political community and determining the most suitable methods to attain them.While the goals may be explicit in certain political communities, such as an association formed to safeguard the interests of espadrille manufacturers in the region of Murcia, they may be ambiguous in other instances.Nonetheless, the challenge of identifying goals does not invalidate the assertion that politics aims to address the question "What shall we do?" through collective prudential rationality.The primary criterion for assessing the appropriateness of a political community's decisions is their efficacy in serving the community's aims.
We propose the following definition: a political community is a group of people whose coordination depends on the possibility of enacting rules.Granted, it is not always easy to establish the limits of a given political community or determine what the interests of its members are.Members of specific communities, such as a philatelic society or the citizens of a nation-state, need to coordinate themselves to achieve their goals.This also applies to the members of the global political community.These various communities can coexist and overlap with one another.Certain issues are clearly relevant to a particular political community due to its unique goals and available resources.For instance, the manufacturers of espadrilles in Murcia may discuss the implications of the withdrawal of European subsidies for esparto grass cultivation, while the members of the Cordoba Society for the Advancement of Ancient Philosophy may focus on selecting suitable venues for their next congress on the thought of Seneca.However, these communities may also share concerns 9. Thus, for Kant the end of politics in general, and particularly of the state, was freedom; for utilitarians it is well-being; for realists, order and security (Rodríguez-Alcázar, 2017a).
that bring them together.For instance, both societies might collaborate to address a hypothetical decision by the Spanish Government to impose an annual tax on all Spanish associations.To which political communities do the political debates surrounding HETs correspond?Undoubtedly, these debates involve numerous communities, varying in type and scale, ranging from professional and consumer associations to nation-states.However, we contend that certain debates, which are currently dominated by states, necessitate adopting the perspective of a broader political community -humankind.If this holds true, we aim to demonstrate that HETs, like other contemporary technologies, provide an argument in favour of cosmopolitanism.Specifically, our contention is that the global community indeed faces coordination problems arising from HETs, and that responses to this challenge that benefit the global community will also benefit other communities, provided they must coexist with one another.
Cosmopolitanism
Cosmopolitanism can be defined as the claim that all human beings "are (or can and should be) citizens in a single community" (Kleingeld and Brown, 2019), and indeed the word "cosmopolitan" derives from a Greek word meaning "citizen of the world".We contend that the typical association of cosmopolitanism with the concept of global citizenship renders it a political stance, rooted in the presumption of a global "us", rather than a moral view.Consequently, the community invoked by this definition would be a political community, and the notion of a global community would serve as a response to the political inquiry regarding the identity of the "we" in the question "What shall we do?" Furthermore, if we incorporate our definition of a political community, it follows that, according to cosmopolitanism, certain facets of policymaking pertain to all human beings and necessitate global coordination and shared rules.
It makes sense to assert, then, that cosmopolitanism is a political standpoint that may be defended on political grounds.Nevertheless, it is customary to distinguish various forms of cosmopolitanism and to argue for them with arguments that are not always of a political kind.For instance, Kleingeld and Brown (2019) distinguish between moral, political, economic and cultural varieties of cosmopolitanism, while Pogge (1992) distinguishes between ethical and legal cosmopolitanism, and several varieties within ethical cosmopolitanism.What Pogge (1993) calls "ethical cosmopolitanism", and Pogge (1992) simply "cosmopolitanism", would include three components: (1) the thesis that persons are the ultimate units of concern (individualism); (2) the claim that the status of ultimate unit of concern attaches to every person equally (universality); and (3) the conviction that persons are ultimate units of concern for everyone, and not only for their compatriots, etc. (generality) (Pogge, 1992: 48-49;Pogge, 1993: 316; in this latter work, the universality criterion is split into two: impartiality and all-inclusiveness).But we think that calling this cluster of thesis "cosmopolitanism", or even "ethical cosmopolitanism", is misleading.While it makes sense to characterise cosmopolitanism as a political stance, this "ethical cosmopolitanism" is nothing but a widespread moral stance whose components can be spelled out in the usual vocabulary of metaethics and normative ethics.For instance, when Peter Singer (2016) defends a "global ethics" (very close to Pogge's ethical cosmopolitanism as far as the general goals of the stance and its justification are concerned), he appeals to the principle of impartiality (see chapter 4), common to many ethical traditions, and to usual arguments against moral relativism.On these grounds, he can claim that individuals, regardless of their wealth, age, gender, abilities or skin colour, have a right to equal consideration of their interests (Singer, 2009).The assertion that we have the same moral obligations towards every starving individual, irrespective of their nationality, can be supported on these grounds, as Singer does, as a specific application of the same overarching principles, without relying on the concept of cosmopolitanism. 10 Therefore, we argue that it is analytically beneficial to view cosmopolitanism as a political doctrine that can be justified on political grounds.This perspective is evident in the works of certain prominent proponents of cosmopolitanism, such as Cloots (1792); although in the case of others, such as Kant (1795), it is less clear whether cosmopolitanism should be understood as a political or moral position.The ambiguity arises from the prevalent acceptance of political moralism, a metaphilosophical standpoint that, as discussed earlier, blurs the distinction between politics and morality.From a moral outlook, we would inquire about our obligations towards individuals living in other countries (as Beitz (1979), Pogge (2008) and Singer (2009) do), or about the obligations of certain societies towards others (as Rawls (1999) does).When Rawls considers the duties between societies, he adopts the perspective of societies demarcated by the boundaries of the nation-state and concludes that the obligations of well-ordered societies towards burdened 10.Similarly, we could easily dispense with the terms "cultural cosmopolitanism" and "economic cosmopolitanism", if both are to be understood as labels for two varieties of a general kind, cosmopolitanism, of which political and moral cosmopolitanism would be other variants.
On the one hand, what Kleingeld and Brown (2019) call "cultural cosmopolitanism" encompasses a cluster of theses ranging from cultural anti-relativism to the political defence of the right of individuals to build their own cultural identity independently of the country where they are born.Some of these theses can be seen as loosely related to cosmopolitanism, understood as a political stance, while others belong to ethics or anthropology, and can be labelled using well-known terms from these fields.On the other hand, "economic cosmopolitanism" is defined as the defence of "a single global economic market with free trade and minimal political involvement", a stance favoured by some politicians and economic theorists that "tends to be criticised rather than advanced by philosophical cosmopolitans", as Kleingeld and Brown acknowledge.This is not surprising, since this "economic cosmopolitism" is notably at odds with the main theses commonly associated with cosmopolitanism, especially if understood as a political stance.Consequently, it sounds strange to call "cosmopolitan" this defence of free trade and economic globalisation, accompanied by a weakening of political (global or local) regulation.
societies are limited to a duty of assistance aimed at facilitating the latter's transition to becoming well-ordered societies: The role of the duty of assistance is to assist burdened societies to become full members of the Society of Peoples and to be able to determine the path of their own future for themselves.It is a principle of transition.(Rawls, 1999: 118) According to Rawls, peoples' obligations do not extend to ensuring that the residents of other societies have their basic needs met.In essence, Rawls rejects the idea of a principle mandating permanent distributive justice among societies.Contrastingly, Beitz, Pogge and Singer's moral positions assert that both affluent individuals and affluent communities have a responsibility to assist citizens of less privileged societies who live in extreme poverty.
However, both perspectives -ethical cosmopolitanism and Rawls's anti-cosmopolitan internationalism -address the question of the type and extent of obligations we have towards others.In doing so, they overlook a genuinely political approach.
Let us now shift towards what Pogge calls "legal cosmopolitanism", which amounts to the thesis that humanity should be governed by a single global state.One paradigmatic defender of this stance was Anacharsis Cloots ( 1792), who proposed a global republic that would eliminate interstate struggles.This proposal aroused in Kant (1795) and in Rawls (1999: 36) the fear of a global despotic state, an entity that would be more fearsome than any smaller despotic state, because in the case of the latter the possibility of escaping to another country is at least conceivable. 11 Legal cosmopolitanism is not, though, the only possible version of cosmopolitanism, understood as a political stance.At least, it is not the only form of cosmopolitanism we are doomed to if we previously adopt political minimalism as our framework for understanding the relationship between ethics and politics.The reason is this: political minimalism accounts for the existence of a plurality of political communities that are not mutually exclusive.In particular, the defence of a global political community, encompassing humanity, is not necessarily incompatible with the existence of national political communities or other political communities of various kinds, and this would weaken some of the fears traditionally invoked by legal cosmopolitanism.Cosmopolitanism, as we understand it, entails that some aspects of policymaking concern all human beings, so it makes sense to admit the existence of a global community, but it does not require us to believe either that all political questions are to be solved by this global community, or that this global community is the only existing political community, and even less that a global community necessarily calls for a global state.
Political minimalism and cosmopolitanism are theses located at different levels.Political minimalism is a metapolitical thesis.It characterises political value in terms of constitutive criteria to assess politics, hence accounting for the possibility of an autonomous political normativity.Contrastingly, cosmopolitanism is a normative political proposal.In other words, it is an ideology.Consequently, it might be possible to accept political minimalism without accepting cosmopolitanism, and vice versa.However, political minimalism may favour a certain understanding of cosmopolitanism that might avoid the problems of others, and in doing so it may lend greater credibility to cosmopolitanism.Indeed, by broadening the notion of political community to groups other than those that coincide with the limits of the state, political minimalism prevents the cosmopolitan/anti-cosmopolitan debate from being reduced to the choice between a global state and the nation-states of the modern world.
Next, we will argue that the foreseeable advancement of certain HETs provides compelling reasons to acknowledge the existence of a cosmopolitan political community, because the use of these HETs may have positive or negative impacts on the shared interests of the community's members.Similar arguments can be made regarding other technologies, such as those related to climate change production and mitigation.However, focusing on HETs is particularly interesting as these are often scrutinised within the realm of bioethics.Perhaps more than in other domains, it is necessary to revive the political perspective in this area and pave the way for explicitly political discussions.Among these discussions, one prominent issue revolves around determining which aspects of HET regulation should be entrusted to states (or even smaller political entities), which should concern supranational entities such as the European Union, and which should be regulated and enforced by global institutions.
HETs and the arguments for cosmopolitanism
Above, we propose that certain decisions related to HETs should be taken by the global political community.What specific types of decisions are we referring to?Kamm (2009: 127-128) highlights key debates surrounding HETs, including the relative prioritisation of human enhancements in resource-constrained societies and the equitable distribution of benefits and risks associated with HETs.In both cases, these issues are inherently political.It is easy to envision the necessity for states to implement measures aimed at achieving optimal allocation of scarce resources for HET research and the societal utilisation of HETs.Similarly, states should monitor the distribution of benefits and risks stemming from HETs and rectify potential imbalances through legislative actions.Moreover, we can contemplate reasonable measures within these domains that could be more easily justified from the perspective of a global political community than from the perspectives of national-level political communities.
Below, we provide two examples to illustrate this point.The first demonstrates how certain HETs, which may be perceived as improvements from an individual standpoint or may even receive positive evaluation within a national political community, could give rise to more problems than benefits on a global scale.Consequently, both the national community and the global political community (i.e., humanity) would have strong grounds to establish legal frameworks to prohibit such HETs.The second illustrates how the perspective of the global community may necessitate different priorities for resource allocation in relation to HETs compared to those dictated by smaller political communities.Consequently, it would be prudent for those smaller political communities to consider the global perspective and incorporate it into their decision-making processes.
The wonder pills enabling you to eat as much as you wish without gaining weight
Research has shown that deletion of the RCAN1 gene in mice results in them being able to maintain their weight despite increasing their fat intake (Rotter et al., 2018).These studies suggest the potential for managing obesity in humans through gene-inhibiting pills.These pills would allow individuals to consume high-calorie foods without concern for the negative health and aesthetic impacts of obesity.If these pills were commercially available, it is foreseeable that many individuals, particularly those who are overweight or at risk of obesity, would have compelling reasons to purchase and utilise them.By doing so, they could avoid obesity while still indulging in their desired foods without restrictions and without the need for extensive physical exercise.Consequently, it is likely that a significant number of individuals, when considering the prudential implications, would readily choose to take these pills without much hesitation.If these individuals were to consider the possibility of using such HETs from a moral standpoint, they would likely have more reservations.Various ethical theories would assign different priorities to criteria such as individual autonomy, overall well-being, and the potential implications of nutrient misuse on food scarcity.Some individuals who would be unwavering from a prudential perspective might deem it immoral to take the pills.Others might perceive themselves morally justified in using them, arguing that while the personal benefits to their health and well-being are evident, the impact of their individual actions on global food prices and availability would be minimal.Although the discussion on determining the morally correct response to these ethical questions is highly engaging, it falls beyond the scope of this article.
In addition to the prudential and moral judgment on certain HETs, the political assessment of that same technology is also important, and this assessment may vary depending on the political community that produces it.A prosperous country, whose main problem is not the malnutrition of its inhabitants but their obesity, could perhaps consider it appropriate not only to allow the manufacture and sale of these pills, but even to use public funds to finance the research leading to their development.On the other hand, if the global political community could legislate on these same issues, it would surely have good reasons to ban the design of these technologies.Indeed, an increase in food intake by people who do not need this extra supply could have one of the following effects, or perhaps both simultaneously: (i) diminished accessibility to food by people in need (because food prices would soar and the supply would diminish as the demand for food from the wealthy increases); (ii) escalation in global food production, leading to negative effects on the environment. 12 We do believe that even an affluent community may have good reasons to limit or even prohibit the development and use of this HET.After all, that affluent community will also suffer the effects of global warming and other negative environmental effects of unnecessary food production.Furthermore, it may be profitable for that affluent community to allocate resources to fighting famine, rather than overfeeding those who are already sufficiently fed, to avert political instability in poorer regions and prevent the forced and unplanned migration of millions of hungry people.Given the relative volatility of national public opinion and the negative effects that the authorisation of this HET in a single country might have for the rest, it can be considered in the interests of a national community to transfer the legislative capacity on this type of technology to the global political community.Hence, the mere possibility of developing HETs with global consequences provides a plausible argument to attest the need for a cosmopolitan perspective that responds to the question "What shall we do?" regarding the regulation of these technologies from the point of view of the global community.
Cognitive enhancement. What cognitive enhancement?
Our second example pertains to cognitive enhancement and the development of HETs specifically designed for this purpose, known as cognitive enhancers.Bostrom and Roache (2011: 138) define cognitive enhancement as "the amplification or extension of core capacities of the mind through improvement or augmentation of internal or external information-processing systems."When it comes to cognitive enhancers, a distinction is commonly made between "conventional" and "unconventional" enhancers (Sandberg and Savulescu, 2011: 94).While the former (such as education, mental techniques and epistemic institutions) are generally accepted without controversy, there is greater 12.According to the Food and Agriculture Organization of the United Nations (2019), around 30% of the food produced worldwide is wasted every year.In turn, this wasted food accounts for 8-10% of global greenhouse gas emissions (United Nations Environment Programme, 2021).The food consumed by users of weight loss pills might not be considered "wasted food", because these users would eat it.But, just like wasted food, it would be food that, while not contributing to alleviating hunger in the world or improving the quality of nutrition for human beings, is nevertheless produced at very high environmental costs (besides greenhouse gas emissions, other impacts would have to be added, such as land use and water waste).Therefore, there could be just as good reasons to avoid unnecessary food consumption due to the use of pills as there are to avoid food waste.
distrust towards the latter (which include drugs, implants and gene editing).Some authors attribute this distrust to our unfamiliarity with these technologies (Bostrom and Roache, 2011: 148), while others caution against potential undesirable side effects (Colzato, Hommel and Beste, 2020).As with other HETs and technologies in general, we argue that it is necessary to address questions of a purely political nature concerning cognitive enhancers, while also acknowledging the importance of moral reflection.Furthermore, we contend that adopting this political perspective on cognitive enhancers reinforces a cosmopolitan outlook.
Advocates of unconventional cognitive enhancers usually recognise their social dimension and the right of the political community to limit their use considering possible risks.(On some of these risks, see, for example, Sharif et al., 2021).Besides, some authors point out that society may prioritise certain enhancers over others when designing its policies (Blank, 2016).However, the justification of political interventions usually invokes moral reasons, and this fact reveals an underlying political moralism.Specifically, some proponents of novel cognitive enhancers commonly argue that individuals should have the freedom to assess the trade-offs between risks and benefits, with limited paternalistic intervention from the state to safeguard against grave dangers (Bostrom and Roache, 2011: 144;Sandberg and Savulescu, 2011: 107).This perspective aligns with a broadly liberal moral tradition that allows for moderate constraints imposed by the state on individual liberties, which may be justified by utilitarian considerations, among others.However, it is not evident that the political regulation of cognitive enhancers should be guided by this ethical framework, as there are numerous alternative frameworks that propose different limits on individual agency.A non-moralistic political perspective such as ours would approach this issue differently.According to our view, it should not be assumed that the primary role of the state is to safeguard individual freedom, or any other moral value emphasised by moral philosophers.Rather, the aim of a political community, be it the state or any other, is to advance the diverse goals of that community, including the well-being and liberty of its members, while prioritising certain aims over others to best serve the overall interests of the community.Therefore, determining the appropriate level of paternalism within a political community cannot be solely based on ethical arguments but should consider both the moral and non-moral goals of the community, including its desire for autonomy.
On the other hand, identifying the political community that faces a particular coordination problem is crucial in evaluating the effectiveness of a proposed response to the question "What shall we do?"In the context of cognitive enhancers, we argue that there are compelling reasons to assert that, in many instances, the relevant political community is the entirety of living human beings.Given the global implications of the use or inadequate application of cognitive enhancers, it is desirable that policies in this domain be formulated with the interests of the global political community in mind.For instance, significant disparities in educational opportunities between different regions of the world have adverse effects not only on the most disadvantaged individuals but also worldwide.Consequently, there are strong justifications for developing global policies aimed at promoting the dissemination of the most beneficial cognitive enhancers.This line of reasoning aligns with the initiatives of international organisations such as UNESCO, which advocate for global education, and it corresponds with the inclusion of universal primary education as the second of the Millennium Development Goals established by the United Nations in 2000 (United Nations, 2015).
A factor to consider by any political community (including, of course, the global political community) is the principle of diminishing marginal utility (Stigler, 1972).By virtue of this, the utility of cognitively enhancing (for example, through transcranial magnetic stimulation or nootropic drugs) those who have already enjoyed cognitive enhancements throughout their lives (for example, by having fulfilled all the educational levels) will normally be lesser than the utility of enhancing those who have had little previous contact with cognitive enhancers.Therefore, from the perspective of the global community, it is sensible to ensure that the most effective cognitive enhancers are provided to the entire population before allocating resources to cognitively enhancing those who are already cognitively enhanced to a high degree.
Another reason to consider that the coordination challenges arising from cognitive enhancers impact the global community is their potential to exacerbate economic inequality among different regions of the planet.For it seems plausible that the average cognitive enhancement of the wealthiest peoples, who would be able to invest more resources in the use of HETs, would further increase the gap between the wealth of those peoples and that of the poorest, among whom the use of HETs would be less widespread.Given that this inequality is frequently linked to detrimental social, economic and ecological consequences that impact all communities, adopting a cosmopolitan perspective becomes preferable as a means of addressing this challenge.
Of course, the question of which cognitive enhancers should be prioritised by the global community, which should merely be allowed, and whether some should even be discouraged or banned is an empirical question, depending on considerations such as their efficacy and cost.Cosmopolitan public policies on cognitive enhancers cannot, therefore, be solely based on philosophical arguments.We dare, however, to venture a final observation that may be closer to the concerns of philosophers: we are struck by the emphasis that some authors place on the increase in IQ as a measure of the efficacy of cognitive enhancers (Sandberg and Savulescu, 2011: 97-98).It is true that these same authors usually recognise that the extension of education is one of the most effective ways to improve the IQ of individuals and the average IQ of entire societies (Sandberg and Savulescu, 2011: 94).To this recognition is sometimes added, however, the observation that when the balance of pros and cons between the various forms of social enhancement (including education) and biological enhancement procedures favour the latter, then these should be preferred (Sandberg and Savulescu, 2011: 105).Bostrom and Roache (2011: 139), for their part, ask us to consider "the cost-benefit ratio of a cheap, safe, cognition-enhancing pill compared to that of years of extra education."Although the discussion here concerns public policies, including those at a global level, it is inevitable to detect a certain individualistic and moralistic bias in the judgments of the mentioned authors.On the other hand, if we accept, with Cabrera (2012: xiv-xv;85), a paradigm of social improvement that incorporates a relational conception of the individual, the result could be the prioritisation of interventions aimed at society, among which education and other forms of social cognitive enhancement would stand out.Indeed, universal education is not only valuable because it increases the IQ of individuals.Societies need to build shared projects, train individuals for group work, foster the debate on its goals as a political community and, in general, pursue the best means to achieve the goals of the community.Formal education, which includes processes of interaction with teachers and classmates, contributes to all these goals in ways that drugs or gene editing cannot.While acknowledging the need for a wide range of cognitive enhancers, both conventional and unconventional, it would be misguided to prioritise their potential for increasing individuals' IQ as the primary criterion when evaluating their authorisation, regulation and financing. | 2023-09-17T15:14:55.419Z | 2023-09-15T00:00:00.000 | {
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59224920 | pes2o/s2orc | v3-fos-license | Relapse prevention with levomilnacipran ER in adults with major depressive disorder: A multicenter, randomized, double‐blind, placebo‐controlled study
Background Levomilnacipran extended release (ER) is a serotonin and norepinephrine reuptake inhibitor approved for major depressive disorder (MDD) in adults. This study was designed to evaluate relapse prevention with levomilnacipran ER in patients with MDD. Methods Patients (≥18 years) with MDD (N = 644) received 20 weeks of open‐label treatment with levomilnacipran ER 40, 80, or 120 mg/d (8 weeks flexible dosing; 12 weeks fixed dosing). Patients with a Montgomery–Åsberg Depression Rating Scale (MADRS) total score ≤12 from the end of week 8 to week 20 were randomized to 26 weeks of double‐blind treatment with levomilnacipran ER (same dosage; n = 165) or placebo (n = 159). The primary efficacy endpoint was time to relapse, defined as insufficient therapeutic response (≥2‐point increase from randomization in Clinical Global Impression of Severity score, risk of suicide, need for hospitalization due to worsening of depression, or need for alternative antidepressant treatment as determined by the investigator) or an MADRS total score ≥18 at 2 consecutive visits. Results In the double‐blind intent‐to‐treat population, levomilnacipran ER‐treated patients had a significantly longer time to relapse compared with placebo (hazard ratio = 0.56; 95% CI, 0.33–0.92; P = 0.0212). Crude relapse rates were 14.5% (levomilnacipran ER) and 24.5% (placebo). Double‐blind treatment‐emergent adverse events (AEs) were reported for 58.8% and 56.0% of levomilnacipran ER and placebo patients, respectively; 3.0% and 1.3% discontinued due to AEs, and 1.2% and 0.6% had serious AEs, respectively. Conclusion Levomilnacipran ER (40–120 mg/d) was effective in preventing relapse in patients with MDD. Safety and tolerability results were consistent with levomilnacipran ER acute studies.
INTRODUCTION
The majority of patients with major depressive disorder (MDD) have more than one major depressive episode, and at least 50% of patients who experience one episode are likely to experience another (APA, 2010;Kessler & Walters, 1998;Kessler, Zhao, Blazer, & Swartz, 1997).
A higher number of previous episodes (Berwian, Walter, Seifritz, & Huys, 2017;Kendler, Thornton, & Gardner, 2000), more residual symptoms (Berwian et al., 2017;Nierenberg et al., 2010), and failure 4 to 9 months of antidepressant treatment after initial response is achieved and at least 3 years of continual treatment for maintenance therapy, especially in patients who experience recurrent MDD (APA, 2010;Bauer et al., 2015). Meta-analyses of relapse-prevention studies demonstrated that longer initial and continuation treatment resulted in lower rates of relapse for antidepressants compared with placebo (Geddes et al., 2003;Glue et al., 2010;Sim et al., 2015). Continual antidepressant treatment has also been shown to reduce the risk of relapse by 70% relative to placebo (Geddes et al., 2003;Kaymaz, van Os, Loonen, & Nolen, 2008). However, no medication is effective in all patients, and relapse prevention is an area of unmet need (Stahl, Grady, Moret, & Briley, 2005).
Study design
This was a multicenter, randomized, double-blind, placebo-controlled, relapse-prevention study with levomilnacipran ER in patients with
Participants
The study included male and female outpatients (≥18 to ≤70 years) who met the following eligibility criteria: Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria for MDD (APA, 2013), confirmed by a Mini International Neuropsychiatric Interview; ongoing major depressive episode (duration ≥8 weeks to ≤18 months); ≥3 lifetime depressive episodes (including current episode), with 2 episodes (including current) occurring within the past 5 years; MADRS total score ≥26; and body mass index between ≥18 and ≤40 kg/m 2 .
Patients who met any of the following criteria were excluded from study participation: DSM-5 Axis I diagnosis of a disorder other than MDD within 6 months of screening (secondary diagnoses of comorbid generalized anxiety disorder, social anxiety disorder, and/or specific phobias were allowed); history of mania, psychotic disorder, panic disorder, obsessive-compulsive disorder, bulimia or anorexia nervosa (past 5 years), borderline or antisocial personality disorder, or neurocognitive disorder; alcohol or other substance abuse disorder (past 6 months); nonresponse to adequate treatment with ≥2 antidepressants (i.e., 8-week duration at recommended dose) during the current episode; and suicide risk defined as suicide attempt (past 12 months), MADRS Item 10 score ≥5, or determined by the investigator based on the psychiatric interview or information collected in the Columbia-Suicide Severity Rating Scale (C-SSRS; Posner et al., 2011). Concomitant treatment with psychoactive medications was prohibited, except for medications for insomnia.
Treatment, randomization, and blinding
During the 8-week, open-label RIP, levomilnacipran ER was titrated to a dose of 40, 80, or 120 mg/d based on tolerability and response to treatment. Patients started at 20 mg/d, and the dosage was increased to 40 mg/d after 2 days. The dosage could be increased to 120 mg/d or decreased in 40-mg increments up to the end of week 6 based on investigator judgment and dose-limiting adverse events (AEs). Patients who completed the RIP with an MADRS total score ≤12, a threshold frequently used to define remission, and no tolerability issues entered the 12-week, open-label SP taking the same levomilnacipran ER dose; no dose adjustments were allowed. Patients who completed the SP and met the following randomization criteria were allowed to enter double-blind treatment: stable clinical response (MADRS total score ≤12 at weeks 8, 10, 12, 14, 16, 18, and 20; 1 or 2 modest excursions [i.e., MADRS total score > 12 but ≤16] at weeks 10, 12, or 14 were permitted); no significant tolerability issues as determined by the investigator; and no MADRS total score ≥17 at any time during the SP. During the 26-week double-blind treatment, eligible patients were randomized (1:1) to placebo or levomilnacipran ER at the dose received during the SP. Randomization codes were generated electronically and implemented using an interactive web response system; identical packaging was used for all study medications. All patients, investigators, and the study sponsor were blinded to treatment assignment. Breaking the blind at the study center level disqualified the patient from further study participation. During the study, however, it was not necessary to unblind any treatment code. F I G U R E 1 Study design. a Patients remained on their final effective and tolerated open-label levomilnacipran ER dose; patients randomized to placebo were down-tapered from the levomilnacipran ER dose they received in open-label treatment. b Includes patients completing double-blind treatment or prematurely discontinuing from the study. c During the run-in phase, dose adjustments were allowed up to week 6. d During the stabilization phase, no dose adjustments were allowed
Efficacy assessments
The primary efficacy parameter was time to first relapse during the double-blind treatment phase. Relapse was defined as meeting one or more of the following criteria: insufficient therapeutic response (e.g.,
≥2-point increase from randomization in Clinical Global Impression
of Severity [CGI-S] (Guy, 1976) score, investigator-determined risk of suicide, need for hospitalization due to worsening of depression, investigator-determined need for alternative antidepressant treatment) or an MADRS total score ≥18 at two consecutive visits occurring within 7 to 14 days.
Additional efficacy parameters included CGI-Improvement (CGI-I; Guy, 1976) score relative to open-label baseline and changes from baseline or randomization in MADRS total score, CGI-S score, and Sheehan Disability Scale (SDS; Sheehan, Harnett-Sheehan, & Raj, 1996) total and subscale (work/school, social, and family) scores.
Safety assessments
AEs, serious AEs (SAEs), and vital signs were recorded at all study visits. Clinical laboratory tests and electrocardiograms were administered during screening, at randomization, and at the end of doubleblind treatment. The C-SSRS was administered at all study visits to monitor suicidal ideation or behavior.
Statistical analyses
Sample size and power calculations were based on historical relapse data, with assumed relapse rates at the end of double-blind treatment of 30% in the placebo group and 15% in the levomilnacipran ER group, and a common discontinuation rate of 20% for both groups. It was estimated that 308 randomized patients (154 per group) would be needed for 85% power to detect a difference in the time to relapse between levomilnacipran ER and placebo using the log-rank test at the 0.05 significance level. To attain 308 randomized patients, it was estimated that approximately 640 patients would be needed for enroll-ment, based on an assumption that response rates during the RIP and the SP would be 65% and 75%, respectively. The primary efficacy parameter, time to relapse, was analyzed using the log-rank test with estimates of the hazard ratio and 95% confidence interval (CI) based on the Cox proportional hazards model with treatment group as an explanatory variable. Patients who did not meet any relapse criteria were censored at study completion or upon early discontinuation for any reason other than relapse. The cumulative distribution function of time to relapse was characterized using Kaplan-Meier curves.
To additionally evaluate factors that may have affected time to relapse, post hoc analyses were conducted. To assess the possibility that the presence of a secondary anxiety disorder (agoraphobia without history of panic disorder, generalized anxiety disorder, or social anxiety disorder) may have confounded time to relapse, Kaplan-Meier estimates were generated excluding patients with these secondary diagnoses. Additionally, the potential for withdrawal symptoms to affect results during the first 28 days after randomization was evaluated by a Kaplan-Meier analysis, in which all relapses during the first 28 days after randomization were considered censored. P values were determined by the log-rank test.
For all additional efficacy parameters, between-group differences were analyzed using an analysis of covariance model with treatment group and pooled study center as factors and the baseline value as the covariate; missing values were imputed using a last observation carried forward (LOCF) approach. All analyses were two-sided hypothesis tests performed at the 5% level of significance.
Safety parameters were summarized descriptively using the safety populations. AEs were classified as treatment-emergent AEs (TEAEs) if AEs were classified as newly emergent AEs (NEAEs) if they were not present before the first dose of double-blind treatment or if they increased in severity during double-blind treatment.
Patient disposition
Patient disposition and reasons for study withdrawal are presented in There was no significant between-group difference for any reason for premature discontinuation. Patient characteristics in the double-blind safety population were generally similar between treatment groups and consistent with characteristics in the open-label safety population (Table 1).
Primary efficacy
Time to relapse was significantly longer in patients continuing on levomilnacipran ER compared with patients on placebo (P = 0.0212; BMI, body mass index; ER, extended release; RIP, run-in phase; SD, standard deviation. F I G U R E 3 Cumulative rate of relapse (double-blind ITT population). The 25th percentile for time to relapse was 168 days in the placebo group and was not able to be estimated for the levomilnacipran ER group using the Kaplan-Meier method. ER, extended release; ITT, intent-to-treat when patients with secondary anxiety disorders (levomilnacipran ER = 18 patients; placebo = 14 patients) were excluded from analysis (P = 0.0171). When evaluating whether early relapse events were related to withdrawal symptoms, it was noted that rates of relapse were low and similar (∼5%) for both levomilnacipran ER-and placebotreatment arms in the first few weeks following randomization (Figure 3). Post hoc Kaplan-Meier analysis found that when all relapses during the first 28 days after randomization (i.e., when withdrawal events were likely to occur) were considered censored, the significant benefit of levomilnacipran ER versus placebo in prolonging time to relapse during double-blind treatment was maintained (P = 0.0140).
Additional efficacy
At the end of open-label treatment with levomilnacipran ER, decreases (improvement) were observed in MADRS total score, CGI-S score, SDS total score, and SDS subscale scores (
Adverse events
No deaths occurred during the study. During the open-label treatment period, TEAEs were reported in 85.9% (553/644) of patients (Table 3).
TEAEs reported in ≥10% of patients were nausea, headache, heart rate increased, and constipation. Most events (> 95%) were mild or moderate in severity. SAEs were reported in 9 patients; no preferred term for an SAE was reported in > 1 patient. Only one SAE (severe mania during the RIP) was considered related to levomilnacipran ER.
C-SSRS assessments
During the open-label treatment phase, C-SSRS-assessed suicidal ideation and behavior were reported in 180 (28.1%) and 12 (1.9%) patients, respectively. Suicidal ideation was reported as a TEAE in two patients. During the double-blind treatment phase, the C-SSRSassessed incidence of suicidal ideation was 17.6% and 12.1% in the placebo and levomilnacipran ER groups, respectively. One event of suicidal ideation was reported as a TEAE during double-blind treatment period in the placebo group.
Other safety parameters
The mean changes in liver enzyme, metabolic, or hematologic parame- ters during the open-label or double-blind treatment phases were not clinically meaningful relative to baseline or placebo, respectively. At the end of the double-blind treatment phase, patients in the levomilnacipran ER group had greater mean increases relative to placebo in systolic blood pressure, diastolic blood pressure, and pulse rate (
DISCUSSION
Levomilnacipran ER, an SNRI, preferentially inhibits norepinephrine reuptake at a rate two-fold more than serotonin reuptake, whereas other SNRIs (i.e., venlafaxine, duloxetine, and desvenlafaxine) preferentially inhibit serotonin over norepinephrine reuptake (Bruno, Morabito, Spina, & Muscatello, 2016). In patients who respond to antidepressant treatment, subsequent depletion of norepinephrine in the brain is correlated with the return of depressive symptoms and increases the risk of relapse (Moret & Briley, 2011). Thus, levomilnacipran ER has the potential to be an effective antidepressant for maintenance treatment and prevention of relapse in patients who have responded to acute treatment.
In this relapse-prevention study in adult patients with MDD, patients who responded to 20 weeks of open-label treatment with levomilnacipran ER were randomized to 26 weeks of doubleblind treatment with levomilnacipran ER or placebo. The primary endpoint, time to relapse, was significantly longer for levomilnacipran ER-treated patients versus placebo-treated patients. The risk of relapse in patients in the placebo group was approximately twice that in the levomilnacipran group. The relapse rate was 14.5% in the levomilnacipran ER group compared with 24.5% in the placebo group, yielding an absolute difference of 10 points. At the end of treatment, increases in MADRS total score, CGI-S score, SDS total, and subscale scores were significantly greater in the placebo group than in the levomilnacipran ER group; mean CGI-I scores decreased significantly less for placebo-versus levomilnacipran ER-treated patients. Collectively, these changes suggest that patients who switched to placebo at randomization had greater worsening of depressive symptoms and functional impairment compared with those who continued treatment with levomilnacipran ER.
Because a randomized withdrawal study design was used in this relapse-prevention trial, there is a potential that the higher rate of relapse in the placebo arm may have been due to withdrawal symptoms as opposed to true relapse events. If this were so, an excess of relapse events would be expected in the placebo arm during the first few weeks of treatment when withdrawal symptoms are likely to occur.
On the contrary, our study found low and similar rates of relapse in levomilnacipran ER-and placebo-treated patients in the first few weeks of treatment, which suggests that withdrawal events were not an issue for placebo-treated patients. Further, a post hoc Kaplan-Meier Change at EOT −51.5 (124.9) −133.5 (120.9) n = number of patients with available analysis value at both baseline and a specific time point during double-blind treatment or double-blind downtaper in the double-blind safety population. EOT, end of double-blind treatment; QTcB, QT interval corrected for heart rate using the Bazett formula; QTcF, QT interval corrected for heart rate using the Fridericia formula.
analysis of time to relapse in which relapse events were considered censored during the first 28 days found that the significant advantage for levomilnacipran ER over placebo in improving time to relapse was maintained.
Relapse prevention with antidepressant treatment was evaluated in meta-analyses of 31 trials (Geddes et al., 2003) and 15 clinical trials using data submitted to the FDA between 1987 and 2012 (Borges et al., 2014). The relapse rates for levomilnacipran ER and placebo in the current study were lower than the average rates reported in the meta-analyses. These meta-analyses yielded relapse rates of 18% for antidepressant treatment and relapse rates of 37% to 41% for placebo, with absolute drug-placebo differences in rates ranging from 10 to 31 points. Many of the trials included in the analyses by Geddes et al. (2003) were conducted in secondary care settings in patients at a high risk of relapse. These analyses found no notable impact of total duration of treatment prior to randomization, which may be related to a wide variance in prerandomization treatment duration in the included studies (i.e., as short as 6 weeks to greater than 1 year). Additionally, the studies included in the Geddes et al. (2003) meta-analysis did not routinely include a fixed-dose stabilization period following response to flexible-dose acute treatment, which is a recent requirement by FDA's Division of Psychiatry Products (Borges et al., 2014).
A prior relapse-prevention study in levomilnacipran ER 40-120 mg was conducted before this positive study; it included a 12-week flexible-dose open-label phase followed by a 24-week double-blind phase in which patients who responded to open-label treatment were randomized to continued levomilnacipran ER or placebo (Shiovitz et al., 2014). Although analysis of relapse rates showed that the time to relapse was slower for levomilnacipran ER than for placebo, the treatment effect was not statistically significant for the primary parameter, time to relapse (HR = 0.68; relapse rates: levomilnacipran ER = 13.9%, placebo = 20.5%). Of note, actual relapse rates were lower than the anticipated rates in the statistical analysis plan (levomilnacipran ER = 20%; placebo = 38%), compromising the projected power to demonstrate a difference between groups. Given the reduced power to detect a significant treatment effect, as well as the low placebo relapse rate, this may more likely be characterized as a failed study than as (Asnis et al., 2013;Bakish et al., 2014;Gommoll et al., 2014;Montgomery et al., 2013;Sambunaris et al., 2014) and long-term (Mago et al., 2013) levomilnacipran ER studies; nausea is one of the most commonly reported TEAEs in both short-term and long-term trials.
The percentage of patients reporting increased heart rate as a TEAE declined between randomization and end of double-blind treatment.
Given the long open-label treatment period, including the 12-week SP, a study of longer duration may have enabled us to observe more relapse events; however, because this patient population had characteristics that made relapse likely (e.g., high baseline MADRS and SDS scores, average of 5 prior depressive episodes, suicide history), enough events were observed to determine that levomilnacipran ER is effective in preventing relapse. Additionally, although the initial phases of the study were open-label, which could be considered a limitation, they may have provided some descriptive measures of drug effectiveness; the double-blind phase did not include an active comparator.
Further, because the dose of levomilnacipran ER was optimized for each patient, no conclusions can be drawn about specific doses. Other limitations of this study included the lack of ability to generalize to a broader patient population because of the strict eligibility criteria, including the exclusion of patients with comorbid psychiatric disorders or a history of a nonresponse to antidepressant treatment. Also, while an MADRS score ≥18 is a common and appropriate criterion for determining relapse, this high cutoff score may have lacked the sensitivity to fully detect relapse in this study given the low (∼5) mean MADRS baseline score at randomization.
CONCLUSIONS
Levomilnacipran ER (40-120 mg/d) was effective in preventing relapse in patients with MDD who responded to acute treatment. Long-term treatment with levomilnacipran ER was generally safe and well tolerated, and side effects were consistent with those found in shorter studies.
ACKNOWLEDGMENTS
The study was sponsored by the Forest Research Institute, an affiliate of Allergan (Madison, NJ). The sponsor was involved in the study design, collection, analysis and interpretation of data, and the decision to present these results. The authors would like to thank the investigators and subjects who participated in the study. Writing and editorial support was provided by Jill Shults, PhD, of Prescott Medical Communications Group (Chicago, IL), funding for which was provided by Allergan. | 2019-01-25T14:03:02.961Z | 2019-01-23T00:00:00.000 | {
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144910737 | pes2o/s2orc | v3-fos-license | A Possible Potentiating Antidepressant Effect of Venlafaxine by Recombinant Rat Leptin in a Rat Model of Chronic Mild Stress
The present study was designed to investigate the possible changes in forced swimming test (FST), prefrontal cortical glutamate and gamma amino-butyric acid (GABA) contents by leptin and/or venlafaxine in chronic mild stress (CMS)-induced anhedonia in male albino rats. They were divided into 5 groups: the first group was not exposed to CMS, the second group received normal saline with exposure to CMS, the third group received leptin 1 mg/kg/day intraperitoneally (ip) for 3 weeks after CMS induced anhedonia was assesed by sucrose consumption test, the fourth group received venlafaxine 8 mg/kg/day ip for 3 weeks after CMS protocol, and the fifth group was received both medications for 3 weeks. Leptin and/or venlafaxine restored the changes in sucrose consumption test, behavioural assessment by forced swimming test (FST) as well as prefrontal cortical GABA and glutamate contents in the control stressed group. Furthermore, combination of both treatments seems to be more efficacious than venlafaxine alone in these parameters. In conclusion,these results showed a potential antidepressant role of leptin and beneficial therapeutic interaction with venlafaxine by affecting the GABA and glutamate level in prefrontal cortex. These actions could make leptin a potentially valuable drug for the treatment of depression.
INTRODUCTION
Depression is the most common mental disorder with prevalence rate of about 20% of the population worldwide.It is often associated with alteration of neurochemical markers [1].Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is one of the key biological abnormalities in 30-50% of depressed subjects [2].Abnormalities in glutamate and gamma-aminobutyric acid (GABA) signal transmission had been postulated to play a role in depression [3].Antidepressants exert their therapeutic effects via increasing corticolimbic monoamines [4][5].The limited efficacy of antidepressant, delayed onset of action, and undesirable side effects lead to ongoing efforts to identify improved therapeutic agents.Leptin, antiobesity hormone, is a hormone secreted from adipocytes and enters the brain by a saturable transport mechanism.By binding to its receptors in the hypothalamus, it act as negative feedback adiposity signals [6].Some studies suggested that leptin may be a novel antidepressant [7], however further investigations are needed to confirm the leptin's antidepressant efficacy and its possible interaction with antidepressant drugs and to identify its possible mechanism.Chronic stress is considered as a predisposing factor in the onset of depression in humans.Rats exposed to chronic unpredictable stress showed decrease in plasma leptin independent of body weight alteration [8].Leptin treatment reduced both *Address correspondence to this author at the Pharmacology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt; Tel: 0020224186992; Fax: 00202-26837673; E-mail: saharkamal2003@hotmail.com dopamine release and tyrosine hydroxylase concentrations in the nucleus accumbens of ob/ob rats [9].These findings are suggestive of that leptin enhances the mesolimbic dopamine activity.However, additional studies are necessary to clarify the exact role of leptin on other neurotransmitters.
Monoaminergic theory of mood disorders has yielded a broad range of pathophysiological insights over a long time [3].Meanwhile, the possible involvement of GABAergic system, in pathophysiology and treatment of mood disorders, is an important target of on-going psychiatric studies [10].GABA is synthesized in a single step from its precursor glutamate by glutamic acid decarboxylase.GABA is metabolized by successive transamination and oxidation to yield succinic semialdehyde and succinic acid, respectively.As a part of the transamination reaction, a recycling system is formed in whichketoglutaric acid is converted to the GABA precursor glutamate by GABA-glutamic acid transaminase [11].The cornerstone of the GABA hypothesis in bipolar disorder is that GABA provides inhibitory action to both norepinephrine [NE] and dopamine systems [12].Although this widely expressed neurotransmitter has been thought to exert a tonic inhibitory effect on norepinephrine[NE] systems, recent data suggested that GABA may in fact facilitate NE activity [13].There was a high evidence for depressed patients to have lower levels of GABA in their blood plasma.These low plasma levels are thought to reflect lower brain levels [14].Accordingly, The present study was designed to assess the possible antidepressant effect of leptin and/or venlafaxine and if it is related to changes in glutamate and GABA concentrations in frontal cortex as an area of cognition.
Animals
Sixty male albino rats weighing 180-200 g, were purchased from the National Research Center in Egypt.The rats were housed in cages under standard experimental conditions: room temperature 24 ± 1°C and 12-h light-dark cycle (lights on at 8:00 AM).Food and tap water were freely available.Rats were allowed to have seven days acclimation before any experimentation.After acclimation, rats were randomly divided into 5 groups [n in each = 12 rats].
Materials
Recombinant rat leptin [Sigma chemicals Co], Venlafaxine HCl (Wyeth-Ayerst) were purchased as powders and were dissolved in saline.
Chronic Mild Stress Procedures (CMS): Three-Week Application of Stressors Procedure
It was conducted according to the method of Willner et al. and Solberg et al. [15][16].Rats in the stressed groups were subjected to different stressors over 3 weeks without treatment in groups (2,3,4,5) to induce anhedonia in rats which simulate human depression, then groups (3,4,5) continued to expose to CMS model with treatment either with venlafaxine, leptin or both.
CMS model involved the exposure of tested albino rats to 16-h water deprivation (water bottles were removed from the cages during this time), 5 min.-tailsuspension (animals were held upside down by their tail with metal tongs), one to two hours restraint (animals were placed in a 50 ml conical tube with breathing holes), 30-45 min.paired housing (the mouse was placed in the cage of another mouse of the stress group, each week the home cage mouse was alternated), soiled cage: 100 ml (16-18°C) water was poured into the cage and 5-min forced swim in cold water (16-18°C).Each week, the stressors were presented in a different order and given at different times of the day.
Sucrose Consumption Test
The development of anhedonia in rats was tested by sucrose consumption test.The test was carried out once every week after stress.Six hours after light out, animals were given a bottle of 2% sucrose for a 1-h period (because the pilot study revealed that rats consumed more water during their active period, thereby, enhancing the chance of seeing a difference in sucrose consumption).After 1-hour, this bottle was removed and total sucrose consumption was calculated.The stressed animals when they become anhedonic consumed less sucrose in comparison to the control group.Preliminary data have shown that control rats prefer a 2% sucrose solution over regular unsweetened water (pilot study).
Experimental Groups of Rats
Group 1 Control: neither exposed to CMS model nor to treatment, only ip injection of saline during the therapeutic period of treated groups.
After exposure for 3 weeks stressors, rats were divided into 5 groups (each group=12 rats) with daily administration of saline or drugs for another 3 weeks as follows: Group 2 exposed to CMS + ip injection of saline during the therapeutic period of treated groups i.e. stressed, saline-treated group Group 3 CMS + leptin 1 mg/kg/day ip for another 3 weeks during exposure to CMS model Group 4 CMS + venlafaxine 8 mg/kg ip for another 3 weeks during exposure to CMS model Group 5 CMS + leptin 1 mg/kg ip + venlafaxine 8 mg/kg ip for another 3 weeks during exposure to CMS model
Forced Swimming Test (FST)
At the end of the study, the FST was done to assess the immobility according to the method of Detke et al. [17].It was done by placing rats into individual glass cylinders (46 cm height , 20 cm diameter) containing 23-25°C water 30 cm deep, so that rats could not support themselves by touching the bottom with their paws.
Two training swimming sessions were conducted initially one session (15-min) of pretest followed 24 h later by a second session (5-min) of test.After each swimming session, the rats were removed from the cylinders, dried with paper towels and returned to their home cages.A single observer, who was blind to the treatment conditions, did all the behavioral scoring.
The immobility is defined as floating in water without struggling, and doing only those necessary movements to keep the head above water.For each rat, the immobility time is calculated in seconds over a period of 5 minutes.
Determination of Glutamate and GABA Concentrations in Homogenates of Prefrontal Cortices of Tested Rats by High Performance Liquid Chromatography
Following the behavioural test, rats were sacrificed by decapitation.The whole brain tissues were removed rapidly on an ice-plate.The tissues were washed with cold saline.The prefrontal cortex from each rat was homogenized and samples were centrifuged in a cooling (4 °C) centrifuge at 15,000 rpm for 10 minutes.The supernatant was aspirated and transferred to an Eppendorf tube.The pellet was kept at -70°C until assayed for total protein content according to the method of Bradford [18].
High performance liquid chromatography (HPLC) with pre-column phenyl-iso-thio-cyanate (PITC) derivatization was used for determination of glutamate and GABA levels in homogenates of the prefrontal cortex of the brains of rats from different groups according to the method of Gunawan et al. [19].Each sample was derivatized by drying 100 l of the aspirated supernatant in a centrivap under vacuum.The residue was dissolved in 20 l of ethanol-watertriethylamine (2:2:1) and evaporated to dryness under vacuum.30 l of ethanol-water-triethylaminephenylisothiocyanate [PITC] (7:1:1:1) was added to the residue and allowed to react for 20 min at room temperature to form the PITC derivatives of the amino acids.Excess reagent was then evaporated under vacuum.The mobile phase of HPLC consisted of solvents A & B [solvent A: 0.1 M sodium acetate buffer (pH= 5.8); solvent B: acetonitrile:water (60:40, v:v)].A mixture of 80% solvent A and 20% solvent B was adjusted for "isocratic" HPLC separations.Flow rate was set at 0.6 ml/min.The injected sample was 20 l.The peaks were detected at a wavelength of 254 nm.Standard curves for glutamate or GABA and norvaline were plotted using norvaline 2 nmol/20 l as an internal standard.The ratio of the peak area of each concentration of each standard to the peak area of the internal standard was determined and entered against the concentration of the standard in a simple regression procedure.
Analysis of the Data
The data obtained are presented as mean ±SEM [Standard error of mean] and evaluated using one-way ANOVA, followed by Tukey's post hoc determination, using GraphPad Prism version 3.00 for Windows 97 (Graph Pad Software, San Diego, CA, U.S.A.).
Ethics
All procedures were in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals, as well as the guidelines of the Animal Welfare Act.
Effect of Leptin and/or Venlafaxine on Sucrose Consumption in CMS-Induced Anhedonia in Rats
Figure 1 demonstrates the reversal of anhedonia after 3 weeks of ip administration of leptin and/or venlafaxine in male albino rats continuously exposed to CMS protocol.Sucrose consumption in mL of the different groups was calculated.In comparison to the control non stressed group, control stressed group was associated with a significant (p<0.01)decrease in sucrose consumption by 84.33 %.This decrease was reversed in the leptin, venlafaxine and both-treated to be -35.34%,-10.64%and +13.25% respectively as compared to the control group level.Venlafaxine and leptin were statistically more effective (p<0.01)than either of the medications alone.
Effect of Leptin and/or Venlafaxine on the Forced Swimming Test (FST)
Reduction of immobility time (in the FST) was observed after treatment of rats suffering from CMS with either leptin and/or venlafaxine (see Table 1).Combination of both medications caused statistically significant reduction in the immobility time more than each one alone (Table 1).
Effect of Leptin and/or Venlafaxine on the Prefrontal Cortical Glutamate Level of CMS-Induced Anhedonia in Rats
Figure 2 represents the changes in glutamate concentration in the prefrontal cortex of the control non stressed, CMS, CMS+either leptin and/ or venlafaxine in male albino rats.CMS increased significantly (p<0.01) the glutamate concentration in the prefrontal cortex.Glutamate concentration of CMS treated rats was decreased significantly (p<0.001) by leptin and/or venlafaxine.Administration of a combination of both medications was more effective than either one alone (p<0.01) in reducing glutamate level of CMS-treated rats.
Effect of Leptin and/or Venlafaxine on the Prefrontal Cortical GABA Level of CMS-Induced Anhedonia in Rats
Figure 3 depicts the significant (p<0.01)decrease in the GABA concentration in the prefrontal cortex in CMS-non treated group.GABA concentration of CMS rats was increased significantly by leptin and/or venlafaxine.Both medications was more effective than either drug alone (p<0.01) in increasing GABA level of CMS-treated rats.
DISCUSSION
In the present study, 3-weeks daily dose of leptin (anti-obesity hormone) and/or venlafaxine (serotonin norepinephrine reuptake inhibitor) induced a statistically significant increase in sucrose consumption in albino rats exposed to 6-weeks of CMS model that simulates human depression.They also reduced the immobility time in the FST as a screening test for antidepressant effect.Additionally, they increase the GABA and reduce the glutamate contents of the prefrontal cortex of these albino rats.The present experiment showed that, co-administration of leptin and venlafaxine induced the above changes in the tested parameters more than that induced by administration of either one alone and the difference was statistically significant.Furthermore, leptin alone was statistically efficient in improving these parameters but to a lesser extent than venlafaxine.These results are supported by findings of Lu et al. [6], which demonstrated that leptin is a novel antidepressant.Moreover, it could potentiate the antidepressant effect of venlafaxine, as demonstrated by results of the present study.
Previously, systemic administration of leptin ameliorated the chronic stress-induced decrease in sucrose preference and decreased the duration of immobility of both FST and tail suspension test (TST) in a dose-dependent manner [20].Sucrose preference test is regarded as an analog of anhedonia, a key symptom of depression in human [7,20].FST and TST are analogue to behavioral despair in human and have high predictive validity for antidepressant activity and have been widely used for screening antidepressant drugs [17].Available information about leptin signaling in human depression is limited and controversial.One study described no differences in the leptin level between depressed patients and healthy controls [21].
Another studies, verified that plasma leptin levels were decreased in depressed patients with larger sample size [22][23][24].As the leptin receptors are highly expressed in the limbic system, it was suspected to be the site of actions for leptin.Another study suggests that the hippocampus might be a target site for circulating leptin to exert its actions [20].
A clinical study showed that there was a low concentration of GABA in plasma and cerebrospinal fluid (CSF) of individuals with major depression [25].In addition to that, low occipital cortical GABA concentration had also been found in depressed patients and when these patients were treated with selective serotonin reuptake inhibitor [SSRI], results revealed a normalization of its value, suggesting a role of GABA in the mechanism of antidepressant action [13].Venlafaxine treatment was associated with increase in GABA level in prefrontal cortex of cocainedependant patients.Its effect was more potent than paroxetine being acting on both serotonin and norepinephrine [25].Hypothalamic norepinephrine (NE) is involved in many of the neuroendocrine effects that are associated with leptin.An experimental study demonstrated that leptin induced changes in NE efflux through GABA.It was concluded that leptin could probably produce its central and neuroendocrine effects by modulating NE and GABA levels in the hypothalamus [26].Zang et al. [27] recommended the use of cell culture systems instead of conventional animal tests for studying the pharmacological profile of new compounds that could be used as remedies.The study pointed to the expensive and obscure results that might be obtained from animal tests.It describes cell culture to be a faster and more reliable screening method for all expected biological effects of different drug candidates and phytochemicals.
CONCLUSION
Collectively, These results showed a potential antidepressant role of leptin and beneficial therapeutic interaction with venlafaxine by an increase in GABA and a reduction in glutamate levels in prefrontal cortex.These actions could make leptin a potentially valuable drug for the treatment of depression.
Figure 1 :
Figure 1: Influence of exposure to chronic mild stress (CMS) on sucrose consumption in male albino rats of the different groups; control saline-treated, chronic stress -with and without treatment.Data are means± SEM from 12 animals per group.*p<0.01=significant decrease vs saline control group 1. ** p<0.01 = significant increase versus control stressed group 2.
Figure 2 :
Figure 2: Influence of CMS with and without administration of either leptin or venlafaxine or both medications on glutamate in Pf Cx of male albino rats of the different groups; control stressed and non-stressed as well as CMS-treated albino rats.Data are expressed as the mean ± SEM from 12 animals per group.*P < 0.01 significant elevation versus control-non-stressed group 1. **P < 0.01 significant reduction versus control stressed group 2.
Figure 3 :
Figure 3: Influence of CMS with and without administration of either leptin or venlafaxine or both medications on GABA in Pf Cx of male albino rats of the different groups; control stressed and non-stressed as well as CMS-treated albino rats.Data are expressed as the mean ± SEM from 12 animals per group.*P < 0.01 significant reduction versus control-non-stressed group 1. **P < 0.01 significant elevation versus control stressed group 2. | 2019-05-05T13:06:38.778Z | 2013-03-31T00:00:00.000 | {
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213524769 | pes2o/s2orc | v3-fos-license | Relation between occupant perception of brightness and daylight distribution with key geometric characteristics in multi-family apartments of Malmö, Sweden
Focusing on subjective evaluations of daylight conditions, the present paper explores the relation between room geometric characteristics and perceived brightness and daylight distribution in residential spaces. The study was conducted in 35 apartments consisting of 105 different rooms in the city of Malmö, and selected with respect to typical Swedish architectural building typologies. Questionnaires were distributed by mail to participants, and apartments were surveyed to deduct key geometric characteristics. Results include a reliability analysis of the utilized questionnaire and a correlation study that showed the prevalence of both window size and number as key factors relating to perceived daylight conditions.
Introduction
Following the adoption of the European Daylight Standard EN-17037 [1] by the Swedish Standards Institute [2], policy makers in Sweden are considering updating the daylight requirements stated in the current building code BBR -BFS 2011:6 [3]. Implications of daylight regulations internationally have previously been investigated [4], and there seems to be consensus in founding guidelines on climatebased daylight evaluations [5]. Such evaluations imply the use of photometric benchmarks to ensure adequate daylighting, which in turn relates to occupant health and well-being [6]. Tregenza and Mardaljevic conclude in a seminal paper [7] that daylighting is not a single research topic, but rather a multi-disciplinary field consisting of several distinct strands, such as mathematical sky models, indoor daylight availability, glare analysis, window technology and user preferences. The latter involves, among other things, assessing when occupants perceive a room as sufficiently bright or evenly daylit, which are key daylight qualities in residential spaces where tasks are not strictly defined, compared to office spaces. The assessment may be made by comparing subjective assessments to photometric measurements or to geometric measurements. The present study focuses on the latter, with the aim to provide information regarding key geometric characteristics of multifamily residential spaces and their relation to perception of brightness and daylight distribution by inhabitants. The goal is to link occupant
Method
The study involved three distinct stages: 1) selecting, surveying and modelling the apartments with their actual geometric characteristics, 2) developing and distributing a questionnaire that pertains to perception of brightness and daylight distribution and 3) performing a statistical analysis on the retrieved data to derive correlations between geometric characteristics and perception of daylight conditions. The following sections describe each step in more detail.
Selected apartments and assessed geometric characteristics
The studied apartments are located in the central and metropolitan region of Malmö (Lat: 55,61°), and were selected to belong to six typical building typologies, identified in relevant Swedish planning research [8,9]. Figure 1 shows the evaluated buildings, the amount of apartments included in the present study and the urban density of surroundings for each building type. Surrounding objects (buildings and trees) were considered in a 200 m radius around each investigated building, and were modelled as LOD2 volumes. Their footprints were retrieved in vector format [10] and processed in ArcGIS ArcMap [11] in conjunction with recent Lidar data available from Swedish Surveying and Cadastral Agency [10] to retrieve 3D geometries. The geometries were further processed in the programming environment of Grasshopper [12] 3 process has previously been described in detail in [13]. The evaluated apartment plans were modelled according to documentation drawings retrieved in raster or pdf format from Malmö City Planning Office [14], and apartment dimensions were validated with on-site measurements in at least three apartments per building. Following the Swedish regulation, rooms only included spaces where people stay more than occasionally: bedrooms (B), living rooms (L) and kitchens (K).
For each room, geometric characteristics included the window area (Awin) and its relation to the external wall area (WWR), to the floor area (WFR) and to the room walls area (WRoomWR). Façade characteristics included the number of fenestrated walls (NrWallsWin) and the area of the external wall (AextWall). To assess the effect of surroundings on each room, the Vertical Sky Component (VSC) was calculated based on the method defined by Littlefair [15], which involves a CIE Overcast Sky. The Radiance raytracer [16] via Honeybee [17] was used to compute the VSC, for a 0.1 m grid of points on the window surface, and an -ad rendering setting of 16384 for a high spatial resolution in the raytracing of the sky dome.
Observer-based environmental assessment
The questionnaire items were based on seven-grade bipolar rating scales according to the method developed by Johansson et al. [18]. The questionnaire focused on two perceived daylight qualities: 1) brightness and 2) distribution. Figure 2 shows the segment of the questionnaire that referred to the living room (the same items were used for the kitchen and for the largest bedroom of the apartments). Four items referred to the perception of brightness and three to daylight distribution. The semantic differentials defined by these bipolar adjectives were used in this study (and have been developed) in the Swedish language, which makes them suitable in the context of Scandinavian culture. They are presented here in English to facilitate reading. The participants were asked to 1) be inside the specific room when filling the corresponding items, 2) answer during daytime, 3) switch off electric lighting and 4) pull all curtains/blinds fully open prior to answering. The questionnaire was distributed by normal mail to 945 addresses, on March 13, 2018, to ensure it would be filled during a period close to the spring equinox. The response rate was approximately 11 % (108 questionnaires). Among the 108 returned questionnaires, 80 were complete and 75 of them were used in this study as they corresponded to apartments that included all three room types (N=225 rooms, for 75 respondents). The majority of questionnaires were filled during March. quality. The degree of reliability for the correlation between the initial raw scores and the derived factors was evaluated using Cronbach's Alpha. Associations between the derived factors and the geometric characteristics were then tested with Spearman's rank correlation.
Exploratory factor analysis and reliability analysis of questionnaire items
The results of the exploratory analysis on the items of the returned questionnaires (N=225) are shown in Table 1. The process was conducted for each room type, where the letters K, L, B correspond to Kitchen, Living room and Bedroom respectively. Factor loadings of items transformed to final factors used for correlating with geometric parameters are shown in bold, and the corresponding Cronbach's alpha value of the reliability analysis is shown next to them. The PCA returned two factors per room type. The Brightness factor consistently included the following four items: dark -light, clear -drab, strong -weak, subdued -brilliant. The analysis showed a good internal reliability (α > 0.85) for all four items, in all three room types. Therefore the average of these four items was used for correlations with the geometric characteristics. The Distribution factor consistently included two items: scatteredconcentrated, unfocused -focused. The reliability of their factor was also high (α > 0.85) for all items and rooms. Item uneven -even distributed was not included in the same factor for all room types and was therefore excluded from the study.
Associations between geometric parameters and observer-based environmental assessment
The correlation between perceived daylight conditions (factors Brightness and Distribution) and the geometric characteristics of rooms is shown in Tables 2 & 3. The rooms characterized for the present paper were 135 in total, and were divided into two samples, based on the existence of a balcony obstruction. Samples A (N = 105) and B (N = 30) include rooms without and with balcony respectively. In sample A, Brightness correlates strongly with the window area (Awin, r = 0.531, p < 0.001) and with the window-to-wall ratio (WWR, r = 0.481, p < 0.001). However in sample B, it is more strongly correlated with the number of different walls that are fenestrated (NrWallsWin, r = 0.431, p < 0.02). This can be attributed to the fact that the spaces with an opening to a balcony are shaded by the balcony of the upper floor, but when they have a second window (e.g. on a different wall or away from the balcony), the shading of the first window is not perceived as negative anymore. An interesting outcome for both samples is that Brightness is associated less with the VSC than with the window size (Awin). This practically implies that even in denser urban areas (low VSC), a sufficiently fenestrated room may be perceived as adequately bright, perhaps due to the compensation offered by a generous view out. This was the case with building type "Large Courtyard", where larger window sizes compensated for its dense surroundings (79 % of rooms were graded neutral or higher along the Brightness scale). The Distribution factor was shown to correlate more strongly with the number of fenestrated walls in Sample A (NrWallsWin, r = 0.329, p < 0.005). In addition, Distribution was found to correlate stronger with measures pertaining to window size than with the WFR, which is a value related to floor area. However, the small size of Sample B (N = 30) may explain why no significant correlation was found between Distribution and all geometric characteristics, for the rooms with balconies. Table 2. Spearman's rank correlation between the Brightness and Distribution factors and the geometric characteristics of rooms without balcony (Sample A, N = 105). Table 3. Spearman's rank correlation between the Brightness and Distribution factors and the geometric characteristics of rooms with balcony (Sample B, N = 30).
Conclusion
This article presents a study about perceived daylight conditions in multi-family apartments located in the city of Malmö. The study compares the subjective evaluations of occupants to the geometric characteristics of these dwellings. The main conclusions are stated below: The observer-based assessment used here displays high internal reliability for the two dimensions studied (brightness and distribution). This consistency means than the assessment is trustworthy and usable for this purpose. A number of outcomes can be used for the development of simplified design rules. The results suggest that window size has the highest correlation with the perception of brightness for all rooms, including rooms in dense urban contexts. This might indicate the importance of view out as a key parameter. Another design-related aspect pertains to the balcony element. The results indicate that lower daylight levels resulting from the presence of a balcony may be compensated for by adding 6 an extra opening either on a different wall or in an unshaded portion of the fenestrated wall. Brightness perception was also found to correlate more strongly with the window size than with the Vertical Sky Component. Since the VSC is related to the surrounding urban density, this unexpected result implies that a simple view out may be sufficient in some cases, in absence of a view to the sky. It also reinforces the observation that a more generous view out somewhat compensates for lower daylight levels. This may have important implications in future developments of building regulations. Regarding perceived daylight distribution, it was found to have no correlation to the window-to-floor ratio. A stronger correlation was observed between distribution and the amount of fenestrated walls. This was an expected result since a higher amount of fenestrated walls should provide a better daylight distribution.
Overall, the results reveal a significant association between simple geometric characteristics and perceived daylight conditions. This could be considered prior to the development of future building regulations, as simple design rules can constitute complimentary material or alternatives to photometric requirements for specific cases. Illuminance information about these apartments could not be collected to a significant degree, due to time constraints and limitations in accessibility. Correlating perceived daylight conditions with photometric data is part of our future investigations. | 2019-11-22T00:55:12.329Z | 2019-11-01T00:00:00.000 | {
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224989578 | pes2o/s2orc | v3-fos-license | Oceananigans.jl: Fast and friendly geophysical fluid dynamics on GPUs
Oceananigans.jl is a fast and friendly software package for the numerical simulation of incompressible, stratified, rotating fluid flows on CPUs and GPUs. Intended for wide use, it is simple enough to be used for educational purposes yet fast and flexible enough for research use. It is being developed as part of the Climate Modeling Alliance project for the simulation of small-scale ocean physics at high-resolution that affect the evolution of Earth’s climate.
Summary
Oceananigans.jl is a fast and friendly software package for the numerical simulation of incompressible, stratified, rotating fluid flows on CPUs and GPUs. Oceananigans.jl is fast and flexible enough for research yet simple enough for students and first-time programmers. Oceananigans.jl is being developed as part of the Climate Modeling Alliance project for the simulation of small-scale ocean physics at high-resolution that affect the evolution of Earth's climate.
Oceananigans.jl is designed for high-resolution simulations in idealized geometries and supports direct numerical simulation, large eddy simulation, arbitrary numbers of active and passive tracers, and linear and nonlinear equations of state for seawater. Under the hood, Oc eananigans.jl employs a finite volume algorithm similar to that used by the Massachusetts Institute of Technology general circulation model (Marshall, Adcroft, Hill, Perelman, & Heisey, 1997). (Right) Simulation of instability of a horizontal density gradient in a rotating channel using 256×512×128 grid points. A similar process called baroclinic instability acting on basin-scale temperature gradients fills the oceans with eddies that stir carbon and heat. Plots made with matplotlib (Hunter, 2007) and cmocean (Thyng, Greene, Hetland, Zimmerle, & DiMarco, 2016).
Oceananigans.jl leverages the Julia programming language (Bezanson, Edelman, Karpinski, & Shah, 2017) to implement high-level, low-cost abstractions, a friendly user interface, and a high-performance model in one language and a common code base for execution on the CPU or GPU with Julia's native GPU compiler (Besard, Foket, & De Sutter, 2019). Because Julia is a high-level language, development is streamlined and users can flexibly specify model configurations, set up arbitrary diagnostics and output, extend the code base, and implement new features. Configuring a model with architecture=CPU() or architecture=GPU() will execute the model on the CPU or GPU. By pinning a simulation script against a specific version of Oceananigans, simulation results are reproducible up to hardware differences.
Performance benchmarks show significant speedups when running on a GPU. Large simulations on an Nvidia Tesla V100 GPU require~1 nanosecond per grid point per iteration. GPU simulations are therefore roughly 3x more cost-effective than CPU simulations on cloud computing platforms such as Google Cloud. A GPU with 32 GB of memory can time-step models with~150 million grid points assuming five fields are being evolved; for example, three velocity components and tracers for temperature and salinity. These performance gains permit the long-time integration of realistic simulations, such as large eddy simulation of oceanic boundary layer turbulence over a seasonal cycle or the generation of training data for turbulence parameterizations in Earth system models.
Oceananigans.jl is continuously tested on CPUs and GPUs with unit tests, integration tests, analytic solutions to the incompressible Navier-Stokes equations, convergence tests, and verification experiments against published scientific results. Future development plans include support for distributed parallelism with CUDA-aware MPI as well as topography.
Ocean models that are similar to Oceananigans.jl include MITgcm (Marshall et al., 1997) and MOM6 (Adcroft et al., 2019), both written in Fortran. However, Oceananigans.jl features a more efficient non-hydrostatic pressure solver than MITgcm (and MOM6 is strictly hydrostatic). PALM (Maronga et al., 2020) is Fortran software for large eddy simulation of atmospheric and oceanic boundary layers with complex boundaries on parallel CPU and GPU architectures. Oceananigans.jl is distinguished by its use of Julia which allows for a scriptbased interface as opposed to a configuration-file-based interface used by MITgcm, MOM6, and PALM. Dedalus (Burns, Vasil, Oishi, Lecoanet, & Brown, 2020) is Python software with an intuitive script-based interface that solves general partial differential equations, including the incompressible Navier-Stokes equations, with spectral methods. | 2020-01-20T22:44:20.999Z | 2020-09-22T00:00:00.000 | {
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256924627 | pes2o/s2orc | v3-fos-license | Serum anion gap at admission as a predictor of mortality in the pediatric intensive care unit
An accurate method to predict the mortality in the intensive care unit (ICU) patients has been required, especially in children. The aim of this study is to evaluate the value of serum anion gap (AG) for predicting mortality in pediatric ICU (PICU). We reviewed a data of 461 pediatric patients were collected on PICU admission. Corrected anion gap (cAG), the AG compensated for abnormal albumin levels, was significantly lower in survivors compared with nonsurvivors (p < 0.001). Multivariable logistic regression analysis identified the following variables as independent predictors of mortality; cAG (OR 1.110, 95% CI 1.06–1.17; p < 0.001), PIM3 [OR 7.583, 95% CI 1.81–31.78; p = 0.006], and PRISM III [OR 1.076, 95% CI 1.02–1.14; p = 0.008]. Comparing AUCs for mortality prediction, there were no statistically significant differences between cAG and other mortality prediction models; cAG 0.728, PIM2 0.779, PIM3 0.822, and PRISM III 0.808. The corporation of cAG to pre-existing mortality prediction models was significantly more accurate at predicting mortality than using any of these models alone. We concluded that cAG at ICU admission may be used to predict mortality in children, regardless of underlying etiology. And the incorporation of cAG to pre-existing mortality prediction models might improve predictability.
To improve the quality of intensive care unit (ICU) care, mortality prediction is important 1 . However, estimation of mortality is difficult in critically ill patients whose condition may deteriorate. There are some invasive methods to assess the status of patients, such as pulmonary artery wedge pressure measurement, but these take time to institute and have side effects, such as infection. Recently developed assessments based on physiologic variables have limitations related to the high proportion of missing data, because the variables required are not collected for all patients admitted to ICU or not required for direct patient management 1 . Therefore it is necessary to identify noninvasive, easy tools for mortality prediction in ICUs, especially for pediatric ICU (PICU) patients.
Anion gap (AG) is traditionally one of the most commonly used biomarkers. It is the simplest means of evaluating the acid-base status of patients, and is calculated from the difference between the measured concentration of serum cations and anions. It helps to identify the presence and causes of metabolic acidosis 2, 3 . In addition, calculation of an initial serum AG in adult patients admitted to ICU has been suggested as a sensitive and specific tool to predict prognosis or mortality [4][5][6] . It has been shown that patients with a high AG have increased admission rates to hospital, increased rates of admission to ICU, and increased severity of illness, independent of concomitant electrolyte abnormalities 4 . An elevated AG is also associated with increased in-hospital mortality compared with patients having a normal AG 5,6 . However, there is lack of data related to pediatric patients. In pursuit of better mortality prediction and proper management of patients in PICU, research on AG as a mortality predictor is meaningful.
In this study, we researched several factors associated with mortality in children admitted to PICU. Specifically, we investigated whether serum AG measured at the admission to PICU could be strong predictor of mortality, and what its predictive value is compared with other mortality prediction models.
Clinical Characteristics of survivors and non-survivors. Patients were divided into survivors and
non-survivors on the basis of in-hospital mortality ( Table 2). Survivors were younger than non-survivors were (median age, 2.4 vs. 5.8 years). Median LOS in PICU was statistically different between survivors and non-survivors, 9 days and 14 days, respectively. Non-survivors tended to require mechanical ventilation within 24 hours of PICU admission. The two groups were distinct in their reasons for PICU admission. The percentage of children with respiratory failure, neurologic problems, and requiring intensive monitoring was higher in survivors than in non-survivors. Meanwhile, a higher proportion of non-survivor had sepsis, post-resuscitation status, and renal failure. Scoring measurements for mortality prediction used in PICU (PIM2, PIM3, and PRISM III) were statistically different between survivors and non-survivors (p < 0.0001).
More than 60% of patients in both groups had metabolic acidosis, but this had no statistical meaning. According to the summary data for measured and calculated variables, non-survivors had higher levels of sodium and lactate and lower levels of albumin, pH, SBE, and HCO3, compared with survivors. Both AG and cAG at admission were significantly higher in non-survivors than in survivors (p < 0.0001). There were significant differences in the median values of delta neutrophil index (DNI), coagulation indices (platelets and partial thrombin time), and C-reactive protein (CRP) between survivors and non-survivors (p < 0.0001).
Corrected anion gap as a predictor of mortality in PICU patients. In the univariable analyses, 15
variables were correlated with in-hospital mortality (p < 0.01). Of these, 9 variables (age, sex, pH, DNI, platelets, cAG and pre-existing mortality prediction models) were introduced into the multivariable logistic regression analysis. cAG was identified as an independent factor associated with in-hospital mortality [odds ratio (OR) 1.110, 95% confidence interval (CI) 1.06-1.17, p < 0.001]. PIM3 and PRISM III were also found to be independently associated with mortality (Table 3).
We also considered relationship between cAG at admission and pre-existing mortality prediction models used in PICU. Each model was significantly correlated with cAG, but the correlation coefficients were relatively low. However, receiver operating characteristic (ROC) curves of cAG, PIM 2, PIM3, and PRISM III showed that there were no differences in mortality prediction. The area under the ROC curve (AUC) was 0.728 for cAG, 0.779 for PIM2, 0.822 for PIM3, and 0.808 for PRISM III score (Table 4, Fig. 1).
The incorporation of cAG to pre-existing mortality prediction models improved their ability to predict mortality ( Table 5). The incorporation of cAG to PIM2 gave an NRI of 17.5% (p = 0.001) and IDI of 7.9% (p < 0.001). The PIM3 with cAG provided an NRI of 16.7% (p = 0.002) and IDI of 7.3% (p < 0.001). And the incorporation of cAG to PRISM III yielded an NRI of 9.4% (p = 0.043) and IDI of 5.2% (p < 0.001). All cAG-incorporated models showed good discrimination but, the models of PIM3 with cAG showed was revealed poor calibration in Hosmer-Lemeshow goodness-of-fit test (p = 0.0004) (Supplementary Table S1).
And comparing AUCs between pre-existing models and cAG-incorporated models, all cAG-incorporated models showed improved performance in mortality prediction. But the model of PIM3 with cAG showed no statistical significance as well in AUC comparison, even though AUC of PIM3 with cAG (0.855) was greater Table S2). In this study, the best cutoff value of cAG to predict mortality was 18.0 mEq/L (sensitivity 46.2%, specificity 87.0%). Figure 2 shows survival curves based on data from all 461 patients stratified by cutoff value of cAG (cAG < 18.0 vs. cAG ≥ 18.0 mEq/L, p < 0.001 by log-rank test).
Discussion
In this study, serum cAG calculated at the time of PICU admission was higher in non-survivors than in survivors. Increased cAG was associated with in-hospital mortality, and was an independent predictor of mortality in PICU patients. And cAG was correlated with pre-existing mortality prediction models for children. cAG could be a mortality predictor in critically ill children, regardless of the presence of metabolic acidosis or their underlying etiology. Acid-base derangements are common in critically ill patients. Although the pathogenesis is not fully understood, it is well-known that ongoing acid-base disequilibrium could reflect the severity of disease and is associated with a poor prognosis of the patient 7 . Many studies have observed a strong association between acidosis and increased organ dysfunction and mortality. However, it remains difficult to ensure accurate measurement of in ICU patients because of their complex and mixed clinical situations of each 8, 9 . In recent years, quantitative approaches to acid-base disturbances have been increasingly applied to clinical practice. These aim to give information about unmeasured anions or strong ion differences for quantitative evaluation of acid-base derangements in the ICU [10][11][12] . Previous studies compared traditional biomarkers, such as pH, base excess, or lactate, as means of assessing acid-base disorders and predicting prognosis in critically ill patients [13][14][15][16] . However, their reliability has not been established yet. Therefore, reassessing the clinical application of AG, the easiest and most readily available way to calculate acid-base disequilibrium, is useful and meaningful.
AG is a traditional tool used to assess acid-base status and aid the differential diagnosis of metabolic acidosis. Because hypoalbuminemia could affect its interpretation, AG should be corrected for serum albumin level. An elevated cAG usually reflects the presence of metabolic acidosis caused by the overproduction or decreased excretion of organic acids. In addition, elevated cAG has been reported as a predictor of mortality in critically ill patients 5,6,10,11 . In patients with Streptococcus pneumoniae bacteremia 5 and acute myocardial infarction 6 , the presence of an increased cAG acidosis was associated with increased mortality.
Mortality is considered the most reliable endpoint of clinical management in ICU setting. Therefore, estimating mortality risk is an important of ICU care. Several models are available to predict the risk of death of children in PICU. By keeping these models updated, the clinical meaning and validity of each prediction model have been proved. However, with ongoing revision and updating, these models have become more complicated. They require at least 10 variables to be collected from patients, have different coding rules, and each model relies on complex calculations, making their application difficult 1,17,18 . Therefore, there is an increasing need to establish an easy clinical method for use in PICU settings. Several biomarkers such as DNI 19 , CRP 20 , procalcitonin 21 , thrombocytopenia 22 and eosinopenia 23 have been suggested for use in PICU.
We conducted this study to test whether cAG at admission can predict mortality or morbidity in PICU patients. Comparing survivors and non-survivors, we determined that an increased cAG at admission to PICU was strongly associated with in-hospital mortality, regardless of the underlying etiology. In terms of the multivariable logistic regression analysis for mortality prediction, cAG at admission was identified as the strongest independent factor associated with in-hospital death. Furthermore, incorporation of cAG at admission to pre-existing mortality prediction models improved their ability of to predict mortality in this study. Considering the complex characteristics of physiology and disease etiologies in children, measurement of cAG at admission to PICU could aid understanding of current status and the clinical outcome of pediatric patients. This study shows that cAG, which is a simple method to calculate, does not require arterial puncture and is readily available, could be a marker of mortality prediction in PICU. The limitation of this study is that data were collected retrospectively. We assessed only the initial cAG at admission to PICU, and obtained the results associated with mortality. And considering incorporation of cAG to pre-existing mortality prediction models, the model of PIM3 with cAG showed good discrimination but poor calibration. There was still a clinical significant improvement in mortality prediction, even if not statistical significant when comparing AUCs between PIM3 and cAG-incorporated PIM3. We think that because predictability of standalone PIM3 is already very powerful and the sample size was relatively small. But owing to the results of NRI, IDI, and an increase in AUC from 0.822 of PIM3 to 0.855 of PIM3 with cAG, cAG is still a useful tool to add on to other mortality prediction models in general. Further studies might be needed as a prospective design in a large multicenter setting to validate the ability of cAG to predict clinical outcome. And serial assessment of cAG could provide much more information about prognosis in PICU.
In conclusion, an elevated cAG at admission was associated with higher mortality in PICU. We suggest that cAG at admission may be used to predict mortality in PICU, regardless of underlying etiology. And the incorporation of cAG to pre-existing mortality prediction models could improve their performance.
Methods
Study population. This was an observational study using data collected retrospectively from the medical records of children admitted to the PICU at Severance Hospital, Seoul, Korea between December 2009 and February 2015. Patients were over 1 month and under 18 years of age and were in the care of the Department of Pediatrics. Patients were admitted to PICU from the emergency department or the general ward. Patients who were discharged or died within 24 hours of PICU admission were excluded. Neonates, patients with cardiac diseases, and patients requiring post-operative care were admitted to and treated in separate specialized units, and were thus excluded from this study.
Data collection. All data were collected and analyzed retrospectively. Blood samples were obtained from indwelling arterial catheters or by venipuncture on admission to PICU. The samples were immediately transported to the central laboratory based in the hospital. Initial arterial blood gases, complete blood cell counts, coagulation indices, serum electrolytes, and levels of albumin were analyzed. The AG was calculated as: 2 Compensation for abnormal albumin levels was achieving the following equation: Normal cAG values were defined as 3-12 mEq/L, using the ion-specific electrodes method (Hitachi 747 Manual; Roche Diagnostics, Sydney, NSW, Australia) 8 . Metabolic acidosis was defined as a standard base excess (SBE) < −2 mEq/L, using following formula 7 : .
. 3 Age, sex, in-hospital mortality, length of stay in PICU, underlying etiology, reasons for admission, and requirement for mechanical ventilation within 24 hours of PICU admission were recorded. Concomitant metabolic problems that could affect acid-base status and cAG, such as liver or renal failure, were also recorded. For all patients, Pediatric Index of Mortality (PIM) 2 18 and PIM 3 1 were recorded at admission, and the Pediatric Risk of Mortality III (PRISM III) 17 was recorded at 24 hours after admission to PICU. PIM and PRISM III scores were calculated on the basis of required variables of each. And then PIM2 and PIM3 were expressed as 'logit_probability' based on the logistic regression equation for the derived scores 17 . But PRISM III was expressed as calculated scores 24 . Statistical analysis. Baseline characteristics of patients were compared using Mann-Whitney U test or Fisher's exact test, as appropriate. Data were reported as numbers (percentages) or medians (inter-quartile range). Groups were compared by the chi-square test or Fisher's exact test for categorical variables, and the Mann-Whitney U test, as appropriate. Among variables with P < 0.01 in the univariable logistic regression analyses, less than 9 variables without missing values were introduced into multivariable logistic regression analysis. Multivariable logistic regression model was used to identify independent predictors of mortality and to examine the relation between cAG and mortality. The correlation between cAG and pre-existing mortality prediction models was assessed using Spearman's method. Survival curves were determined using Kaplan-Meyer method, and the differences in survival according to cutoff values of cAG were analyzed by means of the log-rank test. The optimal cutoff value of cAG to predict mortality was chosen by the Youden index and the AUC. Statistical analyses were performed with SPSS 20.0 (SPSS Inc., Chicago, IL).
The predictive values of cAG and PIM2, PIM3, PRISM III for predicting in-hospital mortality were compared by calculating the AUC. Between pre-existing models and cAG-incorporated models (PIM2 with cAG, PIM3 with cAG, and PRISMIII with cAG), Hosmer-Lemeshow goodness-of-fit test for calibration to assess applicability and ROC curve for discrimination and comparisons of AUCs were used by SAS 9.4 (SAS Inc., Cary, NC).
And the net reclassification improvement (NRI) and integrated discrimination improvement (IDI) statistics are introduced to assess the incremental predictive impact of new models that integrate a candidate marker (cAG) to pre-existing models (PIM2, PIM3 or PRISM III) 25 . Subjects are considered separately who develop and who do not develop events; patients who are dead or survived in this case. For NRI, each subject is assigned to a risk category based on the death probability calculated by the pre-existing mortality prediction models. And then each subject is reassigned to a risk category using new mortality prediction models, constructed by incorporation of cAG to pre-existing mortality prediction models. After calculating the net proportion of subjects with death reassigned to a higher risk category (NRI evemt ) and of subjects without events reassigned to a lower risk category (NRI nonevent ), the NRI is the sum of NRI event and NRI nonevent . And the IDI considers separately the actual change in calculated risk for each individual for those with and those without events. The IDI event is the difference between the mean probability of a new mortality prediction model for those with the event and the mean probability of the existing models for those with the event. The IDI for those without events (IDI nonevent ) is the difference in mean probability for those who do not have the event between the reference and new models. The IDI is expressed as the total of IDI events and IDI nonevents . If the value of NRI and IDI are over 0, it means that the performance of new models to predict mortality is improved than existing models in sensitivity and specificity 26 . NRI and IDI were performed using R software (R version 3.0.1). P < 0.05 was considered statistically significant.
Ethics statement. This study was approved by the institutional review board of Severance Hospital [Seoul,
Korea, IRB No. 4-2012-0369]. All protocols and methods in this study were carried out in accordance with relevant guidelines and regulations. And this study was exempted from the informed consent due to retrospective observational study followed by the institutional review board of Severance Hospital. | 2023-02-17T14:32:19.027Z | 2017-05-03T00:00:00.000 | {
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220792918 | pes2o/s2orc | v3-fos-license | Population Data-Driven Formulation of a COVID-19 Therapeutic
This study is designed to utilize computer modeling of the US population through NHANES to reduce the need for preclinical formulation and toxicology studies of an Ebola anti-viral (BSN389) being repurposed for COVID-19, and to thereby speed the candidate therapeutic to the clinic.
Introduction
Coronavirus disease of 2019, or COVID-19, is one of six identified coronavirus types to cause disease in humans (Repici et al., 2020). The disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was first identified in patients in Wuhan, China (Repici et al., 2020). Major symptoms include fever, cough, shortness of breath, fatigue, new loss of taste and smell, sore throat, congestion or runny nose, nausea or vomiting as well as diarrhea (Repici et al., 2020;Tina, Saey, 2020a). Over half of patients infected report difficulty breathing, with some reported cases of acute respiratory distress syndrome (Repici et al., 2020;Wu, 2020). COVID-19 is known to be transmitted from person to person through virus-containing droplets that may remain in the air or on surfaces for extended amounts of time (Ghose, 2020;Tina, Saey, 2020b). The exact length of time the virus can survive in the air or on a given surface seems to be highly dependent on the surface itself (Volkin, 2020). Regardless, COVID-19 is thought to be particularly transmittable due to ridged Spike (S) proteins it contains (Saplakoglu, 2020). These S proteins have been found to bind well to the angiotensin-converting enzyme 2 (ACE2) found on the human cell surface, thereby serving as an entry point for the virus (Balfour, 2020;Gray, 2020). The virus can spread asymptomatically and has been predicted to infect millions of people, with some experts predicting up to 70% of the US population will have been infected by August 2021 (Woodward and Miller, Medaris, 2020).
With millions expected to be infected, it is critical to ensure proper testing and diagnosis of COVID-19 to maintain some control of infections. Currently, two major types of tests are being utilized to identify COVID-19, molecular tests and serological tests. The following two sections provide descriptions of each type.
Molecular testing techniques
Molecular based testing for COVID-19 aims to detect genetic material from SARS-CoV-2. Although different types of molecular testing exist, they all tend to follow the process of first detecting the RNA material, then making copies of the material until an output measurement is produced if the RNA material was present (Kobokovich et al., 2020).
Laboratory Corporation of America was the first of the large national clinical laboratories in the USA to offer a SARS-CoV-2 nucleic acid test (Haskins, 2020). This test utilizes polymerase chain reaction (PCR) technology to detect the virus via nasopharyngeal (NP) or oropharyngeal (OP) aspirates and washes, NP or OP swabs, and bronchoalveolar lavage (Haskins, 2020). RealTime, a different molecular COVID-19 test manufactured by Abbott, also utilizes PCR technology. However, these PCR tests can be time-consuming to perform, so to address this slowness issue, Abbott released ID Now, the first 5-minute test that utilizes isothermal nucleic acid amplification technology to speed up the process (Billingsley, 2020;Koval et al., 2020). However, the accuracy of the ID Now has been questioned (Spaulding, 2020). In addition to these authorized tests, multiple companies such as EverlyWell and OraSure have also developed take-home test/collection kits, with LabCorps Pixel being the first to gain FDA authorization (Hale, 2020;Koval et al., 2020;Spaulding and Mueller, 2020).
. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not certified by peer review)
The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10. 1101 Numerous research efforts have been initiated to improve serological testing, such as the suggested serological assay for detecting SARS-CoV-2 seroconversion proposed by Amanant and colleagues (Amanat et al., 2020). It should be noted that antibody testing is not meant to diagnose active COVID-19 and can lack specificity, raising questions regarding its reliability (Cairns, 2020;Hale, 2020e;McKinney, 2020;Patel, 2020). These two aspects, along with the difficulty of scaling PCR testing among other factors, prompted the FDA to authorize a new category of testing based on antigens (McKinney, 2020;Patel, 2020). These antigen tests aim to identify the virus by detecting fragments of proteins found on or within the virus, and provide high specificity for COVID-19. However, these tests are not as sensitive as molecular testing methods (McKinney, 2020). Nevertheless, antigen testing could prove to be cheaper and have a higher throughput (Patel, 2020). The first EUA went to Quidel Corporation's Sofia 2 SARS Antigen FIA (Burton, 2020).
Because of the possibility of false negatives, physicians are told not to rely solely on these COVID-19 test results alone, but also to evaluate their patients and consider diagnostic tests such as CT (computed tomography) scans. CT scans of COVID-19 patients often exhibit some percentage of ground-glass opacity (GGO) patterns (Belfiore et al., 2020;Dai et al., 2020). GGO's are not, however, unique to COVID-19, and physicians should still take a holistic approach to diagnose COVID-19, i.e., considering patient history or risks of exposure to COVID-19.
Along with properly testing and diagnosing COVID-19, significant effort has been spent on developing a vaccine to combat the virus. The first expected COVID-19 vaccine in China was available for clinical testing by the end of April (Cahill, 2020). Another player in vaccine development is Inovio Pharmaceuticals, which expected to launch clinical trials in April using their INO-4800 vaccine, with results anticipated in September of 2020 (Cahill, 2020). Regeneron is also working diligently to move its clinical trials of REGN3048 and REGN3051 vaccine and treatment up to early summer (Cahill, 2020). Moderna began testing their mRNA-1273 vaccine clinically beginning in April; this vaccine in particular is designed to target the Spike (S) protein of the virus (Cahill, 2020). Additionally, an intranasal vaccine is being developed via animal studies by the biopharmaceutical company Altimmune (Cahill, 2020).
However, vaccines provide no help to those already infected, so significant effort has also been expended on developing pharmaceuticals to combat COVID-19 in those already infected. A novel treatment may take a significant amount of time to develop so many efforts have been focused on drug repurposing. Examples of this include Gilead's Remdesivir (GS-5734) a general antiviral drug formulated to treat Ebola. This drug treated 761 patients with the virus in a Wuhan hospital (Cairns, 2020) and has performed better than most drugs to date. Another example is Olumiant, a r heumatoid arthritis drug which was identified as a possible treatment of COVID-19 by means of artificial intelligence (Sagonowsky, 2020) . Additionally, Avigan an influenza antiviral drug has seen promising results and has moved into phase III clinical trials (Balfour, 2020b;Lewis, 2020). However, some repurposing drug attempts, like that of the anti-malaria drug hydroxychloroquine, are failing to show efficacy under scientific scrutiny . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101/2020.07.24.20161547 doi: medRxiv preprint (Allassan, 2020;Hopkins, 2020;Sagonowsky, 2020). Efforts to identify a possible treatment among other experimental drugs are also being conducted. EIDD-2801 is a compound originally developed for treating influenza. EIDD-2801 has shown to interfere with the virus' reproduction cycle and has shown promising results in the animal testing (Sheahan et al., 2020). A similar story can be told for the compound EDP-1815, which was developed for anti-inflammatory diseases and is in phase 1b trials for psoriasis (Cotrone, 2020;Verbanas, 2020). EDP-1815 has had success with COVID-19 thus far and is in phase 2 trials at the time of writing this paper (Verbanas, 2020). The RECOVERY trial of dexamethasone showed that the drug could reduce COVID-19 patient death risk by one-third in ventilated patients (from 40% to 28%), and by 1/5 in other patients receiving oxygen only, from 25 percent to 20 percent. There was no benefit in the RECOVERY trial to patients who did not need respiratory support (Mahase, 2020).
As these various compounds begin to enter later phase trials, developers must move away from fit-for-purpose (FFP) formulations and move toward development of a final formulation (Marsac, 2019). Formulation is critical in allowing the compound to scale and become a drug product. One method to cut down on formulation development time and cost is to begin with better-informed initial formulations.This paper uses computer population modeling to aid in the development of a smart formulation from the start, one that obviates the need for extensive preclinical formulation and toxicology studies, thereby significantly reducing development time. This study examines the use of such a data-driven approach for a new candidate COVID-19 treatment, BSN389. BSN389 is an FDA designated orphan drug currently in development as an Ebola treatment (Lodder, 2017). The goal of the present study is to determine the current daily exposure of the population to beta cyclodextrin (BCD), and then keep the amount of BCD in the formulation below that level, so drug use does not add significantly to BCD exposure of human subjects.
Methods
This study is designed to use computer population modeling to reduce the need for extensive preclinical formulation and toxicology studies, and to thereby speed a candidate COVID-19 therapeutic to the clinic. This study updates a previous study with data collected from the latest NHANES (Lodder, 2017). The data compilation and statistical methods used are described in (Cambell, 2020) and summarized below.
NHANES Data Mining
Data from the Centers for Disease Control (CDC) National Health and Nutrition Examination Survey (NHANES) (ClinicalTrials.gov Identifier: NCT00005154) are combined in this computational experiment with data on shipments from food ingredient manufacturers to evaluate exposure of the US population through foods to a possible pharmaceutical formulation ingredient, beta cyclodextrin (Lodder, 2017). Formulation is indispensable in drug delivery. An . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101/2020.07.24.20161547 doi: medRxiv preprint inferior formulation can render a drug product inefficacious. A considerable amount of preclinical research must be conducted before a new drug candidate can be evaluated in humans. The cost of these IND-enabling cGxP studies is usually in the range of $3-$5 million. If a misleading drug product formulation is tested preclinically, new iterations of the formulation must be tested again at added cost. BSN389 was devised for treatment of Ebola virus infections. BSN389 is not particularly soluble in water. Therefore, the objective of formulation is to improve the solubility and bioavailability of the active pharmaceutical ingredient (API).
Data-driven computational science can help to lessen the expense of preclinical work. In the absence of extant human exposure data, an array of tests involving acute and chronic toxicology, cardiovascular, central nervous system, and respiratory safety pharmacology must be completed in at least two species before FDA will allow experiments in humans. Nonetheless, for many compounds (such as those originating as natural products) there is a record of human use. In such situations, computer modeling of a population to determine human use may be sufficient to allow phase 0-1 studies with a candidate formulation in humans.
The CDC's National Health and Nutrition Examination Survey (NHANES) is a group of studies created to assess the health and nutritional levels of adults and children in the United States. NHANES is distinctive in that it incorporates both interviews and dietary information with physical examinations with laboratory results. The NHANES database can be analyzed to determine the distribution of exposures to a food ingredient, and preliminary human formulation experiments conducted at exposures less than those to which the US population is normally exposed through food. These data can be integrated with data extracted from international ingredient shipments to validate the usage model. This report details the data-driven formulation investigation process employing a novel COVID-19 treatment candidate that, unlike vaccines, can be utilized after a patient has been infected with the disease. BSN389's mechanism of action allows it to be potentially employed against all strains of the virus, an attribute that vaccines might not share.
Consumption data from individual dietary records, detailing food items ingested by each survey participant, were collated by computer in Matlab and used to generate estimates for the intake of BCD by the U.S. population as previously described (Lodder, 2017). A more complete description of the methods appears in β-Cyclodextrin Uses in Food and Pharmaceuticals Cyclodextrins (CDs) can be procured in three common forms: α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin, which are called the first generation (or parent) cyclodextrins. These cyclodextrins comprise six (α), seven (β) and eight (γ) -(1,4)-linked glycosyl units formed into a ring. The ring-shaped molecule is hydrophilic on the outer surface (permitting the cyclodextrin to dissolve in water) and has a nonpolar cavity on the inside, which provides a hydrophobic . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101/2020.07.24.20161547 doi: medRxiv preprint environment. Cyclodextrins are able to organize inclusion complexes with a diverse array of hydrophobic guest molecules using this hydrophobic cavity. One or two guest molecules can be entrapped by one, two or three molecules of cyclodextrin.
Because β cyclodextrin is used in both food and pharmaceuticals, BCD exposure estimates are more complicated than estimates made just for food. Manufacturers have to be careful when adding what is apparently a minor amount of BCD to a product because that trivial mass may be enough to push a consumer already ingesting BCDs from other sources higher than the Acceptable Daily Intake (ADI) limit.
Every year cyclodextrins are discussed in numerous scientific research articles and meeting abstracts. Many of these reports are about drugs and drug-related products. Table 1 provides a summary of some of the commercially marketed pharmaceuticals formulated with cyclodextrins. In addition, an abundance of novel inventions continue to incorporate cyclodextrins. CDs are well understood from a regulatory point of view, and a monograph for BCD appeared decades ago in both the US Pharmacopoeia/National Formulary and the European Pharmacopoeia (Del Valle, 2004). is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020.
Voltaren Ocular Europe
Data collected from (Jambhekar and Breen, 2016;Kishore Shete et al., 2017;Loftsson et al., 2014;Loftsson and Duchêne, 2007;Nerurkar and Naringrekar, 2013) Evaluation of β-Cyclodextrin Use An evaluation of the utilization of BCD by the U.S. population through the approved uses of BCD was conducted. Appraisals of the intake of BCD were calculated using the approved food uses and maximum use level in conjunction with food consumption data included in the National Center for Health Statistics' (NCHS) 2015-2016 National Health and Nutrition Examination Surveys (NHANES) (USDA, 2012;Bodner-Montville et al, 2006). Estimates of the mean and 90th percentile intakes were obtained for combined representative approved food uses of BCD (see Appendix for lists of food codes used). The intakes were reported for the following population groups: • infants, age 0 to 1 year • toddlers, age 1 to 2 years, • children, ages 2 to 5 years, . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
Results and Discussion
Food Usage The individual food uses for BCD are synopsized in Appendix 1. Food codes representative of each approved use were selected from the Food and Nutrition Database for Dietary Studies (FNDDS) for the appropriate biennial NHANES survey. In FNDDS, the primary (usually generic) description of a given food is assigned a unique 8-digit food code (USDA, 2012) that appears in Appendix 1.
Food Survey Results
The estimated "all-user" total intakes of BCD from all approved food uses of BCD in the U.S. by population group is summarized in Table 2. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101 However, every food category in which BCD is approved for use does not necessarily incorporate BCD into every product in the category at the maximum approved use level. As a result, the values in Figure 1, Figure 2, and Table 2 are corrected using the total amount of BCD consumed in food in the United States during the period of the survey. The correction was derived from US population numbers and market research on the global β cyclodextrin industry (Global and Chinese Β Cyclodextrin Industry, 2016). is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101/2020.07.24.20161547 doi: medRxiv preprint 1012 tons of BCD were shipped into the US and presumably consumed in US foods in 2016 (Global and Chinese Β Cyclodextrin Industry, 2016). This number was converted to grams and divided by the number of BCD consumers in the United States to give: 1012000000 g / 223377598 consumers = 4.53 g/consumer/yr. Dividing the grams per consumer per year by 365 gives grams per consumer per day, or 0.0124 g. To derive the correction factor the maximum g/consumer/day for ages 2+ in the NHANES table is divided by the actual g/day, 2.0237/0.0124 = 163.2. The estimated maximum exposures provided by the 2015-2016 NHANES were divided by 163.2 to get estimated actual exposures in Table 2.
Development
Scale-up of a novel pharmaceutical may prove difficult during a pandemic, especially with common inactive ingredients such as cyclodextrin, which are used in a wide variety of industries as mentioned (Astray et al., 2009;Hahn, 2020;Loftsson et al., 2014;Loftsson and Duchêne, 2007). -Cyclodextrin, like many active and inactive pharmaceutical ingredients, is now supplied through a global supply chain (GSC) system. Cyclodextrin is made in less than 100 manufacturing sites worldwide, with the majority of these manufacturing sites located overseas in Western Europe and Japan. With a weakened workforce and broken links for importing and exporting goods during the global pandemic, shortages in pharmaceutical products have been reported (Hahn, 2020). Additional research will investigate the feasibility of scale-up of a novel pharmaceutical during periods of material and workforce shortages. This work will construct a model able to identify and mitigate potential supply chain risks and ultimately allow scale-up to occur. The model will need to incorporate country-level reserves and trade data similar to that of the work done by Korniejczuk or Marchand (Korniejczuk, 2019;Marchand et al., 2016). Additionally, the model should capture the actions and interactions of those that will be supplying the material and those that would be further harmed by the shifting inventory adjustments required for producing the new product. This aspect could be feasibly done by integrating social dilemma models such as in Hauser's work (Hauser et al., 2019).
Conclusion
BCD use is increasing in terms of the number of foods approved for BCD incorporation, and in 2016 69.17% of the total U.S. population of 2+ years were established as consumers of BCD based on the approved food uses. However, the mean intakes of BCD by the 2016 BCD consumers from all approved food uses were estimated to be 12.4 mg/person/day or 0.2 mg/kg body weight/day, a slight decrease from 2014 (Lodder, 2017). The heavy consumer (90th percentile all-user) intakes of BCD from all approved food-uses were estimated to be 30.3 mg/person/day or 0.5 mg/kg body weight/day. The initial human clinical studies of BSN389 for COVID-19 will use 1.5 μg of BCD. This is over a factor of 1,000 smaller than the expected daily . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 27, 2020. . https://doi.org/10.1101/2020.07.24.20161547 doi: medRxiv preprint intake from the food uses, and much less than the amount required to move the average consumer from the 50th to the 90th percentile of consumption. For these reasons, the use of BCD in the BSN389 formulation is an negligible inclusion to daily intake and should be safe for subjects in the COVID-19 clinical trial. | 2020-07-27T19:01:43.561Z | 2020-07-27T00:00:00.000 | {
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139774528 | pes2o/s2orc | v3-fos-license | Finite Element Analysis on a Newly-Modified Method for the Taylor Impact Test to Measure the Stress-Strain Curve by the Only Single Test Using Pure Aluminum
In this study, finite element analyses are performed to obtain a stress-strain curve for ductile materials by a combination between the distributions of axial stress and strain at a certain time as a result of one single Taylor impact test. In the modified Taylor impact test proposed here, a measurement of the external impact force by the Hopkinson pressure bar placed instead of the rigid wall, and an assumption of bi-linear distribution of an axial internal force, are introduced as well as a measurement of deformed profiles at certain time. In order to obtain the realistic results by computations, at first, the parameters in a nonlinear rate sensitive hardening law are identified from the quasi-static and impact tests of pure aluminum at various strain rates and temperature conducted. In the impact test, a miniaturized testing apparatus based on the split Hopkinson pressure bar (SHPB) technique is introduced to achieve a similar level of strain rate as 104 s−1, to the Taylor test. Then, a finite element simulation of the modified test is performed using a commercial software by using the user-subroutine for the hardening law with the identified parameters. By comparing the stress-strain curves obtained by the proposed method and direct calculation of the hardening law, the validity is discussed. Finally, the feasibility of the proposed method is studied.
Introduction
The Taylor impact test, established by Taylor [1], is a quite simple impact compressive test.In this test, a cylindrical slender specimen is shot to, and strikes the surface of a rigid body wall.After striking, the length of the deformed specimen is measured to determine the mechanical properties of the materials.As a result of the test, maximum strain rates as high as 10 3 -10 5 s −1 can be easily obtained.The test is applicable to a wide variety of materials such as metals [2], polymers [3,4], foams [5,6] etc.The test was originally used for measuring the impact yield strength, and the fairly reliable value of the strength can be obtained.
It is actively discussed that modifications to the theoretical formulae in the original method are needed to calculate the strength, because only constant deceleration at the free end of specimens is considered.For example, Wilkins and Guinan [7] experimentally derive the conclusion that the ratio between length in the region due to plastic deformation and the total length of the deformed specimen is almost constant, with respect to the impact velocity, and they propose a simplification method of the formulae by Taylor [1].In order to reduce the effect of friction and to avoid the indentation of the wall by specimens, the symmetric Taylor impact test [8,9] is also proposed.
In conjunction with the above discussions in terms of the Taylor impact test, a further modification is made to measure a stress-strain curve of ductile materials.Jones et al. [10,11] extend the theoretical formulae by including not only the yield stress, but also work hardening effect.Julien et al. [12] made a big challenge to measure stress-strain curve in brass, based on the test, by using various kinds of formulae, as well as formulae by Jones et al. [10,11] to compare between them.However, according to their method, numerous repetitions of the experiments are necessary to obtain one single stress-strain curve.In addition, these formulae only considering the deformation of the specimen, the external force is not measured and questions about the accuracy of the measurement results arise.From this point of view, Lopatnikov et al. [5] and Yahaya et al. [6] attempted to use measuring devices such as the Hopkinson pressure bar and a load cell made of quartz, with high accuracy and band width, by replacing the rigid wall.However, this was not applied to measuring the stress-strain curve by just one single number of the test, although the time history of the external force could be obtained.
The goal of this study is to modify the testing method for obtaining the stress-strain curve as a result of one single Taylor impact test.A combination between the distributions of axial stress and strain at a certain time is employed.In the modification, two following items are introduced into the test.One is a measurement of the external impact force by the Hopkinson pressure bar placed instead of the rigid wall, and the other is an assumption of bi-linear distribution of an axial internal force calculated by a measurement of deformed profiles at certain time.In order to confirm the feasibility and the validity of the modifications, finite element analyses on the basis of the proposed testing method are performed for pure aluminum.In order to simulate the tests, at first, the quasi-static and impact tests of pure aluminum at various strain rate and temperature are conducted.In the impact test, a miniaturized testing apparatus based on the split Hopkinson pressure bar (SHPB) technique is introduced to achieve the similar level of strain rate as 10 4 s −1 and to avoid the punching or indentation displacement [13].Here, the nonlinear rate-sensitive hardening model proposed by Allen et al. [14] is chosen, because it has been reported that the stress becomes nonlinear with the respect to strain rate at high strain rate [15].Then, the parameters in the model are identified from the experimental results after correcting the stress value to remove the friction effect [16,17].Next, the finite element simulation of the modified Taylor impact test is performed using a commercial software, MSC Marc (2014), with the user-subroutine for the model by Allen et al. [14] and the identified parameters.The feasibility and the validity of the proposed method is studied by comparing the stress-strain curve obtained by the proposed method and direct calculation of the model by Allen et al. [14].
It must be emphasized that the selection of the hardening model is out of the main scope of this paper.A modification method of the Taylor impact test is proposed to obtain a stress-strain curve by one single number from the test.In order to confirm the feasibility and validity of the proposed method as introduced above, this study is based on the finite element simulation by using arbitrary hardening law.However, it is required that the model can express nonlinearity with respect to the strain rate, and it differs from the model by Johnson and Cook [18] in as realistic situation as possible.Similar to the work done by Iwamoto and Yokoyama [16], it should be confirmed that the result obtained through the simulation as an output is similar to the hardening law used as an input.
A Proposition of a Modified Taylor Impact Test
As explained in the Introduction, the Hopkinson pressure bar is introduced to capture the impact force by a specimen.At the end of the bar, the cylindrical specimen is hit.The internal force at an interface between elastic and plastic deformation F s (t) can be calculated from stress σ 0 (t) at the interface by Taylor [1] as: F s (t) = σ 0 (t)A 0 = ρA 0 l e (t) ..
s denote the initial cross-sectional area of the specimen, the density, length of an elastically-deformed region, and the acceleration at the free end of the specimen, respectively.Here, it is assumed that the cross-sectional area in the elastically-deformed region can be the same as the initial value, even though it is slightly changed.
Jones et al.
[10] have proposed that the deformation behavior in the Taylor impact test can be divided into three phases, depending on the position of the plastic wave front in the specimen.The details of the three phases proposed by Jones et al. [10] are expressed below. Figure 1 shows the schematic drawing of the three phases during deformation.In this figure, l p (t) is length of a plastically-deformed region.Here, the period from the start of deformation to the time t 1 is referred to as the phase 1, the period from the time t 1 to t 2 is referred to as phase 2, and the period after the time t 2 is referred to as phase 3.As shown in Figure 1, Jones et al. [10] assumed that the phase 1 was nonlinear with respect to the time immediately after the impact.In addition, phase 2 represents a time period in which the plastic wave front is moving linearly with respect to time.The moving velocity of the plastic wave front is constant in this phase.After that, the particle velocity of the specimen becomes 0. Therefore, the region where the moving velocity of the plastic wave front approaches null can be considered to be phase 3.However, Jones et al.
[10] provides no clear descriptions of the behavior of the specimen at each of these phases, the transition times t 1 and t 2 , as well as its conditions.Focusing on phase 2, it is assumed that Here, 0 , e () and ̈ denote the initial cross-sectional area of the specimen, the density, length of an elastically-deformed region, and the acceleration at the free end of the specimen, respectively.Here, it is assumed that the cross-sectional area in the elastically-deformed region can be the same as the initial value, even though it is slightly changed.
Jones et al.
[10] have proposed that the deformation behavior in the Taylor impact test can be divided into three phases, depending on the position of the plastic wave front in the specimen.The details of the three phases proposed by Jones et al. [10] are expressed below. Figure 1 shows the schematic drawing of the three phases during deformation.In this figure, () is length of a plastically-deformed region.Here, the period from the start of deformation to the time t1 is referred to as the phase 1, the period from the time t1 to t2 is referred to as phase 2, and the period after the time t2 is referred to as phase 3.As shown in Figure 1, Jones et al. [10] assumed that the phase 1 was nonlinear with respect to the time immediately after the impact.In addition, phase 2 represents a time period in which the plastic wave front is moving linearly with respect to time.The moving velocity of the plastic wave front is constant in this phase.After that, the particle velocity of the specimen becomes 0. Therefore, the region where the moving velocity of the plastic wave front approaches null can be considered to be phase 3.However, Jones et al.
[10] provides no clear descriptions of the behavior of the specimen at each of these phases, the transition times t1 and t2, as well as its conditions.Focusing on phase 2, it is assumed that ̈ becomes almost constant.Additionally, ̈ can be measured by the difference in the outline of the deformed specimen at a different elapsed time.According to an inertia effect in the axial direction of the specimen, it is be realized that the internal force is distributed along with the axial direction of a specimen.Figure 2 shows the schematic drawing of the deformed specimen, and the axial distribution of the internal force.As shown in this figure, the linear distribution of the internal force in each region is assumed here.Hereafter, both regions are called the elastic part and the plastic part.Both and can be measured from the deformed profile of the specimen at an arbitrary time.
Based on the above assumptions, the distribution of the internal force can be calculated.The distribution of the axial internal force in the plastically-deformed and elastically-deformed regions of the specimen can be obtained through the following equations, which are expressed as a ratio between the internal force that is computed by the above assumption, and the current cross-sectional area: According to an inertia effect in the axial direction of the specimen, it is be realized that the internal force is distributed along with the axial direction of a specimen.Figure 2 shows the schematic drawing of the deformed specimen, and the axial distribution of the internal force.As shown in this figure, the linear distribution of the internal force in each region is assumed here.Hereafter, both regions are called the elastic part and the plastic part.Both l e and l p can be measured from the deformed profile of the specimen at an arbitrary time.
Based on the above assumptions, the distribution of the internal force can be calculated.The distribution of the axial internal force in the plastically-deformed and elastically-deformed regions of the specimen can be obtained through the following equations, which are expressed as a ratio between the internal force that is computed by the above assumption, and the current cross-sectional area: x 0 < x < l p and l e (t) x − l p (t) l p < x < l . ( Here, F 0 (t) and x denote the external force at an impact face of the specimen and the position from an origin at the impact end of the specimen.The true normal stress distribution of the specimen in the plastically-and elastically-deformed regions can be obtained through the following equations, which are expressed as a ratio between the internal force, computed by the above equation and the current cross-sectional area: (3) ( Here, 0 () and denote the external force at an impact face of the specimen and the position from an origin at the impact end of the specimen.The true normal stress distribution of the specimen in the plastically-and elastically-deformed regions can be obtained through the following equations, which are expressed as a ratio between the internal force, computed by the above equation and the current cross-sectional area: ( Figure 2. A schematic drawing of deformed specimen and axial distribution of internal force.
House et al. [19] introduces a high-speed camera for the first time, and conducts the Taylor impact test using oxygen-free copper to measure the shape of the specimen during deformation.In this study, the axial strain is calculated at each position in the axial direction of the specimen from the specimen shape under deformation.According to their method with assumptions of constant volume during plastic deformation and the dominance of plastic deformation, the axial strain can be calculated as: Here, (, ) denotes the current cross-sectional area of the specimen at a position .
The Hardening Law to Nonlinear Strain Rate Sensitivity for the Finite Element Simulation
In order to simulate realistic deformation behavior, a selection of the hardening law, which indicates a relationship between the uniaxial stress, temperature, strain rate, and strain is quite important.Among the various hardening laws proposed in the past, there are three representative models.Firstly, the power-law model can be expressed as: House et al. [19] introduces a high-speed camera for the first time, and conducts the Taylor impact test using oxygen-free copper to measure the shape of the specimen during deformation.In this study, the axial strain is calculated at each position in the axial direction of the specimen from the specimen shape under deformation.According to their method with assumptions of constant volume during plastic deformation and the dominance of plastic deformation, the axial strain can be calculated as: Here, A(x, t) denotes the current cross-sectional area of the specimen at a position x.
The Hardening Law to Nonlinear Strain Rate Sensitivity for the Finite Element Simulation
In order to simulate realistic deformation behavior, a selection of the hardening law, which indicates a relationship between the uniaxial stress, temperature, strain rate, and strain is quite important.Among the various hardening laws proposed in the past, there are three representative models.Firstly, the power-law model can be expressed as: Here, σ, ε p , E, n, and K respectively mean the equivalent stress, the equivalent plastic strain, Young's modulus, work-hardening exponent, and a material constant.The Ramberg-Osgood model [20] is applicable by adding the term of elastic strain into Equation (5), and is widely used for simulating the deformation behavior of major metallic materials.However, expressing the strain rate sensitivity as well as temperature dependency is quite tough, because strain rate and temperature are not included into the model.Several efforts on its extension are made; however, it can be said that a reliable and unified model is not found up to now.
Secondary, the hardening law proposed by Johnson and Cook [18] is frequently used as expressed in: .
ε 0 , T, T r , and T m denote the equivalent plastic strain rate, the reference strain rate, the temperature of the material, the room temperature, and the melting temperature of the material, respectively.The parameters A and B are respectively the yield stress and the work hardening coefficient at the strain rate of .ε 0 and the temperature of T r .The parameter m expresses the effect of thermal softening behaviour.The parameter C indicates the coefficient of strain rate sensitivity.This model has already implemented into a lot of commercial FE codes because of its usability, as well as the availability of identified parameters in the model for various kinds of materials.
However, it is common that Equations ( 5) and ( 6) are empirical and phenomenological.In addition, it is not based on any underlying physics such as a thermal activation process, based on the dislocation theory.Thirdly, more physically-based models, for example, the model proposed by Zerilli and Armstrong [21] for fcc metals, can more legitimately be extrapolated, as expressed in: Here, d is the average grain size, and C 1 to C 5 are parameters.The second term inside the exponential function indicates the rate sensitivity.This term also depends on the temperature.However, it is quite hard to say that all three representative model can express the nonlinear rate sensitivity of materials, which appears especially at higher strain rate.
On the other hand, Allen et al. [14] proposed a nonlinear hardening model with respect to the strain rate, expressed as the following equation: Here, the parameter C indicates the exponent of strain rate sensitivity.This model is quite similar to the Johnson-Cook model expressed in Equation ( 6).The difference is the second bracket on the right-hand side to show the rate sensitivity.The rate sensitivity of the stress becomes nonlinear with respect to the logarithmic strain rate.
Like the work done by Iwamoto and Yokoyama [16], the selection of the hardening model was not a major target of this paper.In this research work, the nonlinear hardening model proposed by Allen et al. [14] expressed in Equation ( 8) is adopted for the finite element simulation of the modified Taylor impact test, because it can be used at the high strain rate to express nonlinear behavior with Metals 2018, 8, 642 6 of 16 respect to the strain rate.In addition, the capability of the model is quite higher since only small changes from frequently-used Johnson-Cook model [18] in Equation ( 6) are sufficient.
A Removing Method of the Frictional Effect in Impact Compressive Tests Based on the SHPB Technique
In the past, it has been reported that the friction and radial inertia in the impact test based on SHPB method have an effect on the stress during the deformation [16].Therefore, it is necessary to consider the relationship between the friction coefficient and slenderness ratio: Here, σ m , σ, ∆σ f , ν, µ, and λ denote modified value of stress, measured stress, increment value of stress due to friction, Poisson's ratio, friction coefficient, and the slenderness ratio, respectively.Kii et al. [17] attempt to reduce the friction effect without any corrections of stress value by using a hollow specimen.In order to perform finite element analyses to show an effectiveness of hollow specimens, they also propose a method to correct the value of stress based on the above Equation ( 9) to identify the parameters in Johnson-Cook hardening law.In their method, σ m , which means the true value of stress in the material, should be constant with respect to λ at constant values of ν and µ.In order to remove the frictional effect based on Equation ( 9), compressive tests using specimens with various λ are carried out.Then, σ m calculated by Equation ( 9) with respect to µ is plotted against λ, and the linear approximate curve at the various values of µ are drawn.When the slope of the linear curve becomes almost zero, both µ and σ m can be identified at the same time.As a result of both quasi-static and impact tests, the slope between the slenderness ratio and stress at a certain level of strain can be obtained.
Material and Specimen
The specimen was made of A1070, which is a pure aluminum under the Japan Industrial Standard (JIS).The shape of the specimen for the quasi-static test and impact test at 10 −3 s −1 was a cylinder 14 mm in diameter.A circular plate with 3.6 mm in diameter for the impact test at 10 4 s −1 was used.From Equation ( 9), it can be seen that the slenderness ratio would also have an effect on the stress; therefore, the same value of slenderness ratio in specimens was used in both the quasi-static and impact tests.In order to remove the frictional effect based on Equation ( 9), the length or the thickness of the specimens was controlled to obtain the various slenderness ratios of 0.5, 1.0, 1.5, and 2.0 in the case of the quasi-static tests, and 0.4, 0.5, 0.7, and 1.0 in the case of the impact tests.All the specimens were annealed in a vacuum at 623 K for 1 h.
Quasi-Static and Impact Compressive Tests at Different Strain Rates and Temperatures
Quasi-static compressive tests were conducted by a material testing machine (Shimadzu AG-250 kN, Shimadzu corporation, Kyoto, Japan) with lubricant of MoS 2 .In order to determine the friction coefficient for quasi-static, the tests were conducted at room temperature for 10 −3 s −1 of the strain rate.Next, the strain rate to measure the rate sensitivity in the stress-strain curves was set to 10 −1 , 10 −2 , and 10 −3 s −1 , and the test temperature was the room temperature.Then, it is carried out at 373, 473, and 573 K under 10 −3 s −1 of the strain rate to obtain the temperature dependency in the stress-strain curves.Because the thermal energy was released to the outside faster than the speed of deformation during the quasi-static test, the temperature rise of the specimen could be vanished.After the quasi-static test to measure the elastic properties by using two rosette gauges, Young's modulus was 67 GPa and Poisson's ratio was 0.35.
It was shown that the stress-strain curve for the strain rate higher than 10 2 -10 4 s −1 could be measured by using the SHPB technique with a thin cylindrical specimen [22].The conventional size of the impact testing apparatus based on the SHPB method with 16 mm in diameter of the bars [23] was used to measure not only the stress-strain curve, but also the friction coefficient at the strain rate of 10 3 s −1 .However, with the decrease in the diameter ratio of the specimen and the pressure bar to achieve the strain rate of 10 4 s −1 , the punching or the indentation displacement [13] was increased.Consequently, it was difficult to measure the stress-strain curve accurately because of the effect of the punching displacement [13].In order to obtain the stress-strain curve at a higher strain rate over 10 4 s −1 , the impact compressive test was conducted by using the miniature testing apparatus, based on the SHPB method.Figure 3 shows the schematic illustration on the outline of used apparatus.The material of the striker bar and the pressure bars was SUJ2, which was bearing steel in the JIS standard, the lengths of the striker, input, and output bars were 150 mm, 400 mm, and 400 mm respectively.The diameter of the bars is 4 mm.The validity of the compressive tests obtained from the miniaturized testing apparatus, based on the SHPB method was discussed by comparing with the result that was obtained from the conventional size of the testing apparatus, based on the SHPB method [23] at a similar strain rate.
Finite Element Simulation of the Modified Taylor Impact Test
By using a commercial finite element software MSC Marc 2014 (Version 2014, MSC Software corporation, Los Angels, CA, USA), the finite element analyses of the modified Taylor impact test were performed at the initial velocities of the specimen, at 154 m/s.The finite element model is shown as Figure 4.As shown in Figure 4a, the specimen and the pressure bar were modelled as axisymmetric bodies.The shape of specimen was a cylinder 8 mm in diameter and 40 mm in length.The shape and dimensions were typical [12].To measure the external force, the Hopkinson pressure bar was used in the apparatus of the Taylor impact test to replace the rigid wall.On the interface between the specimen and the stress bar, a hard contact condition with the kinematic friction was only considered, and its coefficient was set to the values that were determined later, based on Equation (9).The finite element used here was a four nodes bi-linear axisymmetric element.The numbers of elements and nodes were 4000 and 4221 respectively for the specimen, and 100,000 and 102,051 respectively for the pressure bar.Julien et al. [12] chose the Arbitrary Lagrangian-Eulerian (ALE) formulation to simulate the Taylor impact test of brass material in a three-dimensional space because the finite element (FE) meshes were extremely distorted around the contact region.The FE discretization in the present study was uniform, but the mesh size of 200 μm was small, as shown in Figure 4b, and the remeshing option was used to reduce such difficulties related to the distortion.It must be noted that, as explained above in Figure 1, excessive deformation of the specimen in the Phase 3 reduced the applicability of this method.Thus, the extremely huge deformed region induced errors, even though the stress and the strain in the region was precisely predicted.Like the compressive test, the specimen was assumed to be made of A1070.The pressure bar of 200 mm in diameter and 400 mm in length was assumed to be made of SUJ2.On the assumption of the actual experiment, the incident stress wave of the Hopkinson bar by generating an impacted specimen was captured.The pressure bar was assumed to be an isotropic linear elastic body with 7900 kg/m 3 of the density, 209 GPa of Young's modulus and 0.3 of the Poisson's ratio.The density of the specimen was set to 2700 kg/m 3 from the nominal value, based on a catalogue of pure aluminum.Unfortunately, the nonlinear hardening model in Equation ( 8) has not been implemented into any major commercial software, including MSC Marc 2014.Thus, it was implemented by using the usersubroutine named "WKLDP".
Finite Element Simulation of the Modified Taylor Impact Test
By using a commercial finite element software MSC Marc 2014 (Version 2014, MSC Software corporation, Los Angels, CA, USA), the finite element analyses of the modified Taylor impact test were performed at the initial velocities of the specimen, at 154 m/s.The finite element model is shown as Figure 4.As shown in Figure 4a, the specimen and the pressure bar were modelled as axisymmetric bodies.The shape of specimen was a cylinder 8 mm in diameter and 40 mm in length.The shape and dimensions were typical [12].To measure the external force, the Hopkinson pressure bar was used in the apparatus of the Taylor impact test to replace the rigid wall.On the interface between the specimen and the stress bar, a hard contact condition with the kinematic friction was only considered, and its coefficient was set to the values that were determined later, based on Equation (9).The finite element used here was a four nodes bi-linear axisymmetric element.The numbers of elements and nodes were 4000 and 4221 respectively for the specimen, and 100,000 and 102,051 respectively for the pressure bar.Julien et al. [12] chose the Arbitrary Lagrangian-Eulerian (ALE) formulation to simulate the Taylor impact test of brass material in a three-dimensional space because the finite element (FE) meshes were extremely distorted around the contact region.The FE discretization in the present study was uniform, but the mesh size of 200 µm was small, as shown in Figure 4b, and the remeshing option was used to reduce such difficulties related to the distortion.It must be noted that, as explained above in Figure 1, excessive deformation of the specimen in the Phase 3 reduced the applicability of this method.Thus, the extremely huge deformed region induced errors, even though the stress and the strain in the region was precisely predicted.Like the compressive test, the specimen was assumed to be made of A1070.The pressure bar of 200 mm in diameter and 400 mm in length was assumed to be made of SUJ2.On the assumption of the actual experiment, the incident stress wave of the Hopkinson bar by generating an impacted specimen was captured.The pressure bar was assumed to Metals 2018, 8, 642 8 of 16 be an isotropic linear elastic body with 7900 kg/m 3 of the density, 209 GPa of Young's modulus and 0.3 of the Poisson's ratio.The density of the specimen was set to 2700 kg/m 3 from the nominal value, based on a catalogue of pure aluminum.Unfortunately, the nonlinear hardening model in Equation ( 8) has not been implemented into any major commercial software, including MSC Marc 2014.Thus, it was implemented by using the user-subroutine named "WKLDP".related to the distortion.It must be noted that, as explained above in Figure 1, excessive deformation of the specimen in the Phase 3 reduced the applicability of this method.Thus, the extremely huge deformed region induced errors, even though the stress and the strain in the region was precisely predicted.Like the compressive test, the specimen was assumed to be made of A1070.The pressure bar of 200 mm in diameter and 400 mm in length was assumed to be made of SUJ2.On the assumption of the actual experiment, the incident stress wave of the Hopkinson bar by generating an impacted specimen was captured.The pressure bar was assumed to be an isotropic linear elastic body with 7900 kg/m 3 of the density, 209 GPa of Young's modulus and 0.3 of the Poisson's ratio.The density of the specimen was set to 2700 kg/m 3 from the nominal value, based on a catalogue of pure aluminum.Unfortunately, the nonlinear hardening model in Equation ( 8) has not been implemented into any major commercial software, including MSC Marc 2014.Thus, it was implemented by using the usersubroutine named "WKLDP".On MSC Marc 2014, the analysis class chosen was Thermal/Structural, and the large strain option was checked.For the post-processes, the deformed profiles of the specimen were output at several points in elapsed time to obtain (, ) , e () , and () in Equations ( 1), (3), and (4).The "historyplot" option was chosen to calculate ̈ in Equation (1).By combining the axial stress and strain at a certain time, the stress-strain curve could be obtained.Additionally, for a comparison with the axial stress and strain calculated by the deformed profiles, the "pathplot" option was also used to draw the axial stress and strain, along with the central axis of the specimen from their contour data.
Compressive Test from Quasi-Static to Impact Range
First of all, the validity of the impact test by the miniature impact testing apparatus was confirmed.Figure 5 shows the stress-strain curve obtained by setting the almost similar value of strain rate with the testing apparatus based on the SHPB method, with the bars being 16mm in diameter.From this figure, it can be found that the stress-strain curve obtained from the miniature apparatus almost coincided with the result of the conventional testing apparatus in the range of the target strain rate for this experiment.The strain rate obtained in both testing apparatuses was similar.
From the above, it can be estimated that the miniaturized testing apparatus was valid.Additionally, it can also be seen that the size of the specimen had almost no effect on the stress-strain curve in the range of the strain rate, as 10 3 s −1 .On MSC Marc 2014, the analysis class chosen was Thermal/Structural, and the large strain option was checked.For the post-processes, the deformed profiles of the specimen were output at several points in elapsed time to obtain A(x, t), l e (t), and l p (t) in Equations ( 1), (3), and (4).The "historyplot" option was chosen to calculate .. s in Equation (1).By combining the axial stress and strain at a certain time, the stress-strain curve could be obtained.Additionally, for a comparison with the axial stress and strain calculated by the deformed profiles, the "pathplot" option was also used to draw the axial stress and strain, along with the central axis of the specimen from their contour data.
Compressive Test from Quasi-Static to Impact Range
First of all, the validity of the impact test by the miniature impact testing apparatus was confirmed.Figure 5 shows the stress-strain curve obtained by setting the almost similar value of strain rate with the testing apparatus based on the SHPB method, with the bars being 16mm in diameter.From this figure, it can be found that the stress-strain curve obtained from the miniature apparatus almost coincided with the result of the conventional testing apparatus in the range of the target strain rate for this experiment.The strain rate obtained in both testing apparatuses was similar.From the above, it can be estimated that the miniaturized testing apparatus was valid.Additionally, it can also be seen that the size of the specimen had almost no effect on the stress-strain curve in the range of the strain rate, as 10 3 s −1 .
Figure 6 shows the relationship between modified stress σ m , calculated by Equation ( 9) for each friction coefficient and slenderness ratio obtained by (a) quasi-static and (b) impact tests.As performed by Kii et al. [17], the approximated linear curve was drawn for each of the plots of the conditions, based on Equation ( 9), and the slope of the curve was plotted against the friction coefficient.As per the results of an extrapolation when the slope becomes zero, the friction coefficient was determined as 0.18 for the quasi-static test, and 0.03 for the impact test, respectively.At the same time, the modified stress σ m could be also determined, so that modified values were used to identify the parameter in the model.
strain rate with the testing apparatus based on the SHPB method, with the bars being 16mm in diameter.From this figure, it can be found that the stress-strain curve obtained from the miniature apparatus almost coincided with the result of the conventional testing apparatus in the range of the target strain rate for this experiment.The strain rate obtained in both testing apparatuses was similar.
From the above, it can be estimated that the miniaturized testing apparatus was valid.Additionally, it can also be seen that the size of the specimen had almost no effect on the stress-strain curve in the range of the strain rate, as 10 3 s −1 .
Figure 5.
A comparison between the stress-strain curves obtained by the conventional and miniature testing apparatuses.
Figure 6 shows the relationship between modified stress , calculated by Equation ( 9) for each friction coefficient and slenderness ratio obtained by (a) quasi-static and (b) impact tests.As performed by Kii et al. [17], the approximated linear curve was drawn for each of the plots of the In order to identify the parameters in the hardening laws, the least-square method or some other optimization technique is employed [2,24].Here, the following identification method by a manual-like procedure was chosen.At first, .ε 0 in Equation ( 8) is set to the lowest strain rate as 10 −3 .By fitting the experimentally-obtained curve at .ε 0 and T r , A, B, and n could be identified.Then, m was determined from the experimental data at .ε 0 by changing T. Finally, C could be obtained from the experimental data of the stress for 0.1 of the strain at various strain rates.As a result of the procedure, all the identified parameters in Equation ( 8) are shown in Table 1.
Metals 2018, 8, x FOR PEER REVIEW 9 of 16 conditions, based on Equation ( 9), and the slope of the curve was plotted against the friction coefficient.As per the results of an extrapolation when the slope becomes zero, the friction coefficient was determined as 0.18 for the quasi-static test, and 0.03 for the impact test, respectively.At the same time, the modified stress could be also determined, so that modified values were used to identify the parameter in the model.In order to identify the parameters in the hardening laws, the least-square method or some other optimization technique is employed [2,24].Here, the following identification method by a manuallike procedure was chosen.At first, ̇0 in Equation ( 8) is set to the lowest strain rate as 10 −3 .By fitting the experimentally-obtained curve at ̇0 and , , , and could be identified.Then, was determined from the experimental data at ̇0 by changing .Finally, could be obtained from the experimental data of the stress for 0.1 of the strain at various strain rates.As a result of the procedure, all the identified parameters in Equation ( 8) are shown in Table 1. Figure 7 shows the stress-strain relationship at various (a) strain rate and (b) temperature.In the figure, the experiment results with the modified stress are shown by dashed lines, and the results drawn by calculating the model in Equation ( 8) are shown by solid lines.From this figure, it can be understood that the experimental results and the results by model by Allen et al. [14] were in good agreement in the small strain range of less than 0.1.From this result, it was found that the model could express the rate-sensitive and temperature-dependent deformation behavior accurately at a strain rate range of around 10 4 s −1 .Figure 7 shows the stress-strain relationship at various (a) strain rate and (b) temperature.In the figure, the experiment results with the modified stress are shown by dashed lines, and the results drawn by calculating the model in Equation ( 8) are shown by solid lines.From this figure, it can be understood that the experimental results and the results by model by Allen et al. [14] were in good agreement in the small strain range of less than 0.1.From this result, it was found that the model could express the rate-sensitive and temperature-dependent deformation behavior accurately at a strain rate range of around 10 4 s −1 .Figure 8 shows the relationship between the stress and strain rate for true axial strain of 0.1 and 0.15, obtained by experimental methods, and the model proposed by Allen et al. [14].It has been reported that pure aluminum, which is the face-centered cubic structure, shows a steep rise in stress at the strain rate of about 5000 s −1 from the past study [15].Furthermore, the stress becomes nonlinear with respect to the strain rate.As shown in this figure, it was understood that the stress value obtained from the miniaturized testing apparatus showed a sharp rise around the strain rate of 10 3 s −1 , and the nonlinearity could be also confirmed, as in the previous studies [15].From these results, the nonlinear strain rate sensitivity of the material could be measured correctly by using the miniature testing apparatus.In addition, from the results obtained by the model proposed by Allen et al. [14], the stress value rose sharply at a strain rate of 10 2 /s.This fairly good agreement could be confirmed with the results that were obtained by experimental methods, at a strain rate over 10 4 s −1 .Overall, the model proposed by Allen et al. [14] could be used at a super high strain rate.Figure 8 shows the relationship between the stress and strain rate for true axial strain of 0.1 and 0.15, obtained by experimental methods, and the model proposed by Allen et al. [14].It has been reported that pure aluminum, which is the face-centered cubic structure, shows a steep rise in stress at the strain rate of about 5000 s −1 from the past study [15].Furthermore, the stress becomes nonlinear with respect to the strain rate.As shown in this figure, it was understood that the stress value obtained from the miniaturized testing apparatus showed a sharp rise around the strain rate of 10 3 s −1 , and the nonlinearity could be also confirmed, as in the previous studies [15].From these results, the nonlinear strain rate sensitivity of the material could be measured correctly by using the miniature testing apparatus.In addition, from the results obtained by the model proposed by Allen et al. [14], the stress value rose sharply at a strain rate of 10 2 s −1 .This fairly good agreement could be confirmed with the results that were obtained by experimental methods, at a strain rate over 10 4 s −1 .Overall, the model proposed by Allen et al. [14] could be used at a super high strain rate.Figure 8 shows the relationship between the stress and strain rate for true axial strain of 0.1 and 0.15, obtained by experimental methods, and the model proposed by Allen et al. [14].It has been reported that pure aluminum, which is the face-centered cubic structure, shows a steep rise in stress at the strain rate of about 5000 s −1 from the past study [15].Furthermore, the stress becomes nonlinear with respect to the strain rate.As shown in this figure, it was understood that the stress value obtained from the miniaturized testing apparatus showed a sharp rise around the strain rate of 10 3 s −1 , and the nonlinearity could be also confirmed, as in the previous studies [15].From these results, the nonlinear strain rate sensitivity of the material could be measured correctly by using the miniature testing apparatus.In addition, from the results obtained by the model proposed by Allen et al. [14], the stress value rose sharply at a strain rate of 10 2 /s.This fairly good agreement could be confirmed with the results that were obtained by experimental methods, at a strain rate over 10 4 s −1 .Overall, the model proposed by Allen et al. [14] could be used at a super high strain rate.
Computation of the Modified Taylor Impact Test by FEM
Using finite element analysis, the modified Taylor impact test proposed here was performed with an initial impact speed of the specimen at 154 m/s.As a result, the above-mentioned calculation method of a stress-strain curve explained in Section 2.1 was studied.Using the time history of the external force obtained from the pressure bar, the velocity at the free end of the specimen, the time history of the deformed profile in the specimen, at each time, the stress-strain curve was calculated by the method shown previously.The stress-strain curve obtained in the analysis was compared with the curve that was calculated by the model expressed in Equation ( 8), and the validity of the obtained stress-strain curve by the above-mentioned method was investigated.
Figure 9 shows (a) the time history of the external force obtained by finite element analysis (FEA) at the positions of 10, 20, and 30 mm away from the impact surface as the origin, and (b) a contour plot of axial stress during the impact of the specimen at 20 µs of time elapsed.In Figure 9a, the black line shows the time history of the reaction force by a contact acting on the impact surface of the specimen, and the green, blue, and red lines represent the time histories of the external force as measured on the surface of the pressure bar at 10, 20, and 30 mm from the impact surface, respectively.From this figure, it was found that the time history of the external force at the position 20 mm or more away from the impact surface could be accurately measured, excluding a spike generated immediately after the impact.In addition, as shown in Figure 9b, the one-dimensional stress wave propagation has not been achieved in the region within 10 mm from the impact surface.From the above results, it was considered that an accurate measurement was difficult when the stress wave was measured near the impact surface.In the finite element analysis, it was assumed that the specimen completely impacted, with the center of the pressure bar.However, when actually conducting this proposed method based on the Taylor impact test, a statistical variation in a position on the impact surface, where the specimen collides with the pressure bar easily occurs.Therefore, it is sufficiently conceivable that the region in the three-dimensional stress wave propagation in the pressure bar becomes larger than the region obtained by the FEA, as shown in Figure 9b.Therefore, in order to reliably measure the time history of the external force, the measurement position is determined to be 30 mm from the impact surface.
Computation of the Modified Taylor Impact Test by FEM
Using finite element analysis, the modified Taylor impact test proposed here was performed with an initial impact speed of the specimen at 154 m/s.As a result, the above-mentioned calculation method of a stress-strain curve explained in Section 2.1 was studied.Using the time history of the external force obtained from the pressure bar, the velocity at the free end of the specimen, the time history of the deformed profile in the specimen, at each time, the stress-strain curve was calculated by the method shown previously.The stress-strain curve obtained in the analysis was compared with the curve that was calculated by the model expressed in Equation ( 8), and the validity of the obtained stress-strain curve by the above-mentioned method was investigated.
Figure 9 shows (a) the time history of the external force obtained by finite element analysis (FEA) at the positions of 10, 20, and 30 mm away from the impact surface as the origin, and (b) a contour plot of axial stress during the impact of the specimen at 20 μs of time elapsed.In Figure 9a, the black line shows the time history of the reaction force by a contact acting on the impact surface of the specimen, and the green, blue, and red lines represent the time histories of the external force as measured on the surface of the pressure bar at 10, 20, and 30 mm from the impact surface, respectively.From this figure, it was found that the time history of the external force at the position 20 mm or more away from the impact surface could be accurately measured, excluding a spike generated immediately after the impact.In addition, as shown in Figure 9b, the one-dimensional stress wave propagation has not been achieved in the region within 10 mm from the impact surface.From the above results, it was considered that an accurate measurement was difficult when the stress wave was measured near the impact surface.In the finite element analysis, it was assumed that the specimen completely impacted, with the center of the pressure bar.However, when actually conducting this proposed method based on the Taylor impact test, a statistical variation in a position on the impact surface, where the specimen collides with the pressure bar easily occurs.Therefore, it is sufficiently conceivable that the region in the three-dimensional stress wave propagation in the pressure bar becomes larger than the region obtained by the FEA, as shown in Figure 9b.Therefore, in order to reliably measure the time history of the external force, the measurement position is determined to be 30 mm from the impact surface.
(a) Figure 10 shows the time histories of (a) the external force obtained from the pressure bar and the velocity at the free end of the specimen, and (b) the length of the specimen and the distance of the plastic wave front from the impact surface.In Figure 10a, the black and red lines represent the external force and the velocity at the free end of the specimen.In Figure 10b, the black and red lines represent the length of the specimen and the position of the plastic wave front, respectively.As shown in Figure 10a, the duration of the external force was about 92 μs, and the time to decrease to 0 m/s for the velocity at the free end of the specimen is about 78 μs.From this result, it was found that the impact state continued for about 14 μs after the velocity at the free end of the specimen became 0 m/s.Just after the velocity at the free end of the specimen achieves 0 m/s, the length of the specimen becomes about 33 mm, as shown in Figure 10b.From this length, the time period to reciprocate the elastic stress wave once in the specimen was calculated as 13.2 μs, which was the time to decrease the external force rapidly after the velocity at the end of the specimen to become 0 m/s.This almost coincided with the time that was required for the history to decrease to zero.Therefore, it can be inferred that this phenomenon was due to the fact that it takes time for the unloading wave to reciprocate in the test piece after the particle velocity at the end of the specimen becomes zero.Since the duration of the time history of external force is the time during which the pressure bar and the specimen are in contact, the duration of the time history of the external force was the duration of this test.Moreover, as shown in Figure 10b, the position of the plastic wave front changed nonlinearly from the start of the impact to about 14 μs, then it showed a linear change, and its change became nonlinear again after about 50 μs.This represented the transition time from phase 1 to 2 and from 2 to 3, respectively, as described before.In this case, 1 and 2 were about 14 and 50 μs.̈ in Equation ( 1) could be captured by the slope of the red line in Figure 10a, from 1 to 2 .Figure 10 shows the time histories of (a) the external force obtained from the pressure bar and the velocity at the free end of the specimen, and (b) the length of the specimen and the distance of the plastic wave front from the impact surface.In Figure 10a, the black and red lines represent the external force and the velocity at the free end of the specimen.In Figure 10b, the black and red lines represent the length of the specimen and the position of the plastic wave front, respectively.As shown in Figure 10a, the duration of the external force was about 92 µs, and the time to decrease to 0 m/s for the velocity at the free end of the specimen is about 78 µs.From this result, it was found that the impact state continued for about 14 µs after the velocity at the free end of the specimen became 0 m/s.Just after the velocity at the free end of the specimen achieves 0 m/s, the length of the specimen becomes about 33 mm, as shown in Figure 10b.From this length, the time period to reciprocate the elastic stress wave once in the specimen was calculated as 13.2 µs, which was the time to decrease the external force rapidly after the velocity at the end of the specimen to become 0 m/s.This almost coincided with the time that was required for the history to decrease to zero.Therefore, it can be inferred that this phenomenon was due to the fact that it takes time for the unloading wave to reciprocate in the test piece after the particle velocity at the end of the specimen becomes zero.Since the duration of the time history of external force is the time during which the pressure bar and the specimen are in contact, the duration of the time history of the external force was the duration of this test.Moreover, as shown in Figure 10b, the position of the plastic wave front changed nonlinearly from the start of the impact to about 14 µs, then it showed a linear change, and its change became nonlinear again after about 50 µs.This represented the transition time from phase 1 to 2 and from 2 to 3, respectively, as described before.In this case, t 1 and t 2 were about 14 and 50 µs.Figure 11 shows the distribution of axial strain calculated from various methods at (a) 20 and (b) 50 μs of elapsed time since the impact.In the figure, the black line shows the results calculated by Equation ( 4) with the outline of the deformed specimen, the blue line shows the results obtained from the contour plot of FEA, the red line shows the average strain of the plastic part, and the dashed line shows the position of the plastic wave front.From this figure, the distribution of axial strain calculated by Equation ( 4) was in good agreement with that obtained from the contour plot, and it was evaluated that the calculated distribution by Equation ( 4) was appropriate.In addition, an increase in a quite highly deformed region, as similar to the buckling at 50 μs, could be observed locally.
Figure 12 shows the distribution of axial stress in the specimen at (a) 20 and (b) 50 μs of elapsed time since the impact.In this figure, the black line is the calculated result from the bi-linear approximation by Equation (3), the red line is the obtained result from a contour map by FEA, the blue line is the stress at the interface calculated by Equation (1), and the dashed line is the position of the plastic wave front, respectively.From this figure, the stress at the interface calculated by Equation (1) and the stress value at the plastic wave front obtained by the FEA agreed well.This means that in phase 2, the calculation of stress distribution by bi-linear approximation was applicable.Also, in the time after 20 μs, the stress value decreased in the region with the higher value of axial strain near the impact surface.The precision for predicting the distribution was much higher at 20 μs.Therefore, a good measurement could be obtained at a time close to 1 .Figure 11 shows the distribution of axial strain calculated from various methods at (a) 20 and (b) 50 µs of elapsed time since the impact.In the figure, the black line shows the results calculated by Equation ( 4) with the outline of the deformed specimen, the blue line shows the results obtained from the contour plot of FEA, the red line shows the average strain of the plastic part, and the dashed line shows the position of the plastic wave front.From this figure, the distribution of axial strain calculated by Equation ( 4) was in good agreement with that obtained from the contour plot, and it was evaluated that the calculated distribution by Equation (4) was appropriate.In addition, an increase in a quite highly deformed region, as similar to the buckling at 50 µs, could be observed locally.
Figure 12 shows the distribution of axial stress in the specimen at (a) 20 and (b) 50 µs of elapsed time since the impact.In this figure, the black line is the calculated result from the bi-linear approximation by Equation (3), the red line is the obtained result from a contour map by FEA, the blue line is the stress at the interface calculated by Equation (1), and the dashed line is the position of the plastic wave front, respectively.From this figure, the stress at the interface calculated by Equation (1) and the stress value at the plastic wave front obtained by the FEA agreed well.This means that in phase 2, the calculation of stress distribution by bi-linear approximation was applicable.Also, in the time after 20 µs, the stress value decreased in the region with the higher value of axial strain near the impact surface.The precision for predicting the distribution was much higher at 20 µs.Therefore, a good measurement could be obtained at a time close to t 1 .Figure 11 shows the distribution of axial strain calculated from various methods at (a) 20 and (b) 50 μs of elapsed time since the impact.In the figure, the black line shows the results calculated by Equation ( 4) with the outline of the deformed specimen, the blue line shows the results obtained from the contour plot of FEA, the red line shows the average strain of the plastic part, and the dashed line shows the position of the plastic wave front.From this figure, the distribution of axial strain calculated by Equation ( 4) was in good agreement with that obtained from the contour plot, and it was evaluated that the calculated distribution by Equation (4) was appropriate.In addition, an increase in a quite highly deformed region, as similar to the buckling at 50 μs, could be observed locally.
Figure 12 shows the distribution of axial stress in the specimen at (a) 20 and (b) 50 μs of elapsed time since the impact.In this figure, the black line is the calculated result from the bi-linear approximation by Equation (3), the red line is the obtained result from a contour map by FEA, the blue line is the stress at the interface calculated by Equation (1), and the dashed line is the position of the plastic wave front, respectively.From this figure, the stress at the interface calculated by Equation (1) and the stress value at the plastic wave front obtained by the FEA agreed well.This means that in phase 2, the calculation of stress distribution by bi-linear approximation was applicable.Also, in the time after 20 μs, the stress value decreased in the region with the higher value of axial strain near the impact surface.The precision for predicting the distribution was much higher at 20 μs.Therefore, a good measurement could be obtained at a time close to 1 .Figure 13 shows the stress-strain curves at each elapsed time.In the figure, the black line is the calculated curve from the bi-linear approximation, the red line is the obtained curve from FEA, and the blue line is directly calculated from the model by Allen et al. [14] in Equation (8).From this figure (a), the stress-strain curves obtained by the bi-linear approximation and the contour plot showed good agreement at 20 μs, when it could be seen that the approximation of the stress distribution was possible, as shown in Figure 13.However, it is understood that the difference from the curve calculated by the model gets larger as the strain increases.Since it is conceivable that the starting time of plastic deformation everywhere in the specimen is also different, it is possible that the stress value becomes low in the higher strain region located around the impact surface.On the other hand, in the smaller region of strain less than 0.05, the curve obtained by these three methods showed fairly good agreement at 50 μs.From this figure, it was found that better measurements of the curve could be realized at a time close to 2 , when the plastic wave sufficiently propagated at a constant velocity.Figure 13 shows the stress-strain curves at each elapsed time.In the figure, the black line is the calculated curve from the bi-linear approximation, the red line is the obtained curve from FEA, and the blue line is directly calculated from the model by Allen et al. [14] in Equation (8).From this figure (a), the stress-strain curves obtained by the bi-linear approximation and the contour plot showed good agreement at 20 µs, when it could be seen that the approximation of the stress distribution was possible, as shown in Figure 13.However, it is understood that the difference from the curve calculated by the model gets larger as the strain increases.Since it is conceivable that the starting time of plastic deformation everywhere in the specimen is also different, it is possible that the stress value becomes low in the higher strain region located around the impact surface.On the other hand, in the smaller region of strain less than 0.05, the curve obtained by these three methods showed fairly good agreement at 50 µs.From this figure, it was found that better measurements of the curve could be realized at a time close to t 2 , when the plastic wave sufficiently propagated at a constant velocity.Figure 13 shows the stress-strain curves at each elapsed time.In the figure, the black line is the calculated curve from the bi-linear approximation, the red line is the obtained curve from FEA, and the blue line is directly calculated from the model by Allen et al. [14] in Equation (8).From this figure (a), the stress-strain curves obtained by the bi-linear approximation and the contour plot showed good agreement at 20 μs, when it could be seen that the approximation of the stress distribution was possible, as shown in Figure 13.However, it is understood that the difference from the curve calculated by the model gets larger as the strain increases.Since it is conceivable that the starting time of plastic deformation everywhere in the specimen is also different, it is possible that the stress value becomes low in the higher strain region located around the impact surface.On the other hand, in the smaller region of strain less than 0.05, the curve obtained by these three methods showed fairly good agreement at 50 μs.From this figure, it was found that better measurements of the curve could be realized at a time close to 2 , when the plastic wave sufficiently propagated at a constant velocity.Of course, this method was applicable when the loading only occurred in the entire region of specimens.The deformation history at different material points must be considered carefully if the distribution of stress and strain is linked together.On the other hand, it is difficult to establish the method of the calculation of the strain rate.Therefore, it was necessary to establish the calculation method with a higher precision of the stress-strain curve and strain rate, by using not only the theory of stress wave propagation, but also the laws of physics from the deformation of the specimens, as well as the time history of the external force.
Concluding Remarks
In this study, to measure the stress-strain curve at only one single trial of the Taylor impact test, its modification method was newly proposed.In the method, a measurement of the external force by the Hopkinson pressure bar, an assumption of bi-linear distribution of an internal force and a combination between the distributions of the axial stress and strain at a certain time, were employed.Before the feasibility of the proposed method was studied, at first, the quasi-static and the impact tests at various strain rates and temperatures were conducted to identify the parameters in a nonlinear rate sensitive hardening law.Then, the hardening law with the identified parameters was implemented into the commercial code through the user-subroutine.Next, a series of finite element simulations was performed by using the implemented model to confirm the feasibility.The following results were obtained.
1.
It is possible to obtain a valid stress-strain curve at only one single trial of the Taylor impact test.2.
In the Phase 2, the distribution of the axial internal force can be approximated bi-linearly with respect to the axial position of the specimen.
3.
It can be observed that the axial stress decreases mainly in the region of higher strain.4.
The choice of elapsed time during Phase 2 is quite important, in order to obtain the correct stress-strain curve.
At present, the strain rate cannot be calculated and stress-strain curve can be obtained at smaller strains of less than 0.1.Further modifications of this method are necessary to solve the above issues.
Figure 1 .
Figure 1.A schematic drawing of specimen at 1 , 2 , 3 , and the time history of the wave front of the plastic region, to explain the partition of phase in the deformation behaviour of the specimen during the Taylor impact test.
Figure 1 .
Figure1.A schematic drawing of specimen at t 1 , t 2 , t 3 , and the time history of the wave front of the plastic region, to explain the partition of phase in the deformation behaviour of the specimen during the Taylor impact test.
Figure 2 .
Figure 2. A schematic drawing of deformed specimen and axial distribution of internal force.
Metals 2018, 8, x FOR PEER REVIEW 7 of 16the lengths of the striker, input, and output bars were 150 mm, 400 mm, and 400 mm respectively.The diameter of the bars is 4 mm.The validity of the compressive tests obtained from the miniaturized testing apparatus, based on the SHPB method was discussed by comparing with the result that was obtained from the conventional size of the testing apparatus, based on the SHPB method[23] at a similar strain rate.
Figure 3 .
Figure 3.A schematic illustration of established miniature split Hopkinson pressure bar (SHPB) testing apparatus.
Figure 3 .
Figure 3.A schematic illustration of established miniature split Hopkinson pressure bar (SHPB) testing apparatus.
Figure 4 .
Figure 4.The finite element model of the modified Taylor impact test using a pressure bar.(a) The whole view and (b) the magnified view around an interface between the specimen and the pressure bar, as shown in the red circle of the figure (a) (dimensions in mm).
Figure 4 .
Figure 4.The finite element model of the modified Taylor impact test using a pressure bar.(a) The whole view and (b) the magnified view around an interface between the specimen and the pressure bar, as shown in the red circle of the figure (a) (dimensions in mm).
Figure 5 .
Figure 5.A comparison between the stress-strain curves obtained by the conventional and miniature testing apparatuses.
Figure 6 .
Figure 6.The relationship between modified stress calculated by Equation (9) for each friction coefficient and slenderness ratio obtained by (a) quasi-static and (b) impact tests using an annealed specimen.
Figure 6 .
Figure 6.The relationship between modified stress calculated by Equation (9) for each friction coefficient and slenderness ratio obtained by (a) quasi-static and (b) impact tests using an annealed specimen.
Figure 7 .
Figure 7. Stress-strain curves obtained by a quasi-static and impact test at various temperatures and directly calculated by Equation (8).(a) At various strain rates; (b) At various temperatures.
Figure 8 .
Figure 8. Stress-strain rate relationship obtained by quasi-static to impact tests using the established miniature testing apparatus.
Figure 7 .
Figure 7. Stress-strain curves obtained by a quasi-static and impact test at various temperatures and directly calculated by Equation (8).(a) At various strain rates; (b) At various temperatures.
Figure 7 .
Figure 7. Stress-strain curves obtained by a quasi-static and impact test at various temperatures and directly calculated by Equation (8).(a) At various strain rates; (b) At various temperatures.
Figure 8 .
Figure 8. Stress-strain rate relationship obtained by quasi-static to impact tests using the established miniature testing apparatus.
Figure 8 .
Figure 8. Stress-strain rate relationship obtained by quasi-static to impact tests using the established miniature testing apparatus.
Figure 9 .
Figure 9. Determination of a measuring position of external force from the impact surface.(a) Time history of the external force.(b) Contour of axial stress at 20 µs.
sFigure 10 .
Figure 10.Time histories of some important parameters in the specimen.(a) External force and velocity in the elastic part.(b) Length and position of the plastic wave front.
Figure 10 .
Figure 10.Time histories of some important parameters in the specimen.(a) External force and velocity in the elastic part.(b) Length and position of the plastic wave front.
Figure 10 .
Figure 10.Time histories of some important parameters in the specimen.(a) External force and velocity in the elastic part.(b) Length and position of the plastic wave front.
Figure 11 .
Figure 11.Distribution of the true strain obtained by Equation (4), a contour as a result of FEA and average of strain at each elapsed time.(a) 20 µs; (b) 50 µs.
Figure 11 .Figure 12 .
Figure 11.Distribution of the true strain obtained by Equation (4), a contour as a result of FEA and average of strain at each elapsed time.(a) 20 μs; (b) 50 μs.
Figure 12 .
Figure 12.Distribution of axial stress obtained by a bi-linear approximation using Equation (3), the FEA, and the calculated yield stress by Equation (1) at each elapsed time since the impact.(a) 20 µs, (b) 50 µs.
Table 1 .
[14]material parameters of the models by Allen et al.[14], determined by experimental results.
Table 1 .
[14]material parameters of the models by Allen et al.[14], determined by experimental results. | 2019-03-11T02:39:48.342Z | 2018-08-15T00:00:00.000 | {
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797130 | pes2o/s2orc | v3-fos-license | Viscoelastic and elastomeric active matter: Linear instability and nonlinear dynamics
We consider a continuum model of active viscoelastic matter, whereby an active nematic liquid-crystal is coupled to a minimal model of polymer dynamics with a viscoelastic relaxation time $\tau_C$. To explore the resulting interplay between active and polymeric dynamics, we first generalise a linear stability analysis (from earlier studies without polymer) to derive criteria for the onset of spontaneous heterogeneous flows (strain rate) and/or deformations (strain). We find two modes of instability. The first is a viscous mode, associated with strain rate perturbations. It dominates for relatively small values of $\tau_C$ and is a simple generalisation of the instability known previously without polymer. The second is an elastomeric mode, associated with strain perturbations, which dominates at large $\tau_C$ and persists even as $\tau_C\to\infty$. We explore the novel dynamical states to which these instabilities lead by means of direct numerical simulations. These reveal oscillatory shear-banded states in 1D, and activity-driven turbulence in 2D even in the elastomeric limit $\tau_C\to\infty$. Adding polymer can also have calming effects, increasing the net throughput of spontaneous flow along a channel in a new type of"drag-reduction". Finally the effect of including strong, antagonistic coupling between nematic and polymer is examined numerically, revealing a rich array of spontaneously flowing states.
We consider a continuum model of active viscoelastic matter, whereby an active nematic liquidcrystal is coupled to a minimal model of polymer dynamics with a viscoelastic relaxation time τC. To explore the resulting interplay between active and polymeric dynamics, we first generalise a linear stability analysis (from earlier studies without polymer) to derive criteria for the onset of spontaneous heterogeneous flows (strain rate) and/or deformations (strain). We find two modes of instability. The first is a viscous mode, associated with strain rate perturbations. It dominates for relatively small values of τC and is a simple generalisation of the instability known previously without polymer. The second is an elastomeric mode, associated with strain perturbations, which dominates at large τC and persists even as τC → ∞. We explore the dynamical states to which these instabilities lead by means of direct numerical simulations. These reveal oscillatory shear-banded states in 1D, and activity-driven turbulence in 2D even in the elastomeric limit τC → ∞. Adding polymer can also have calming effects, increasing the net throughput of spontaneous flow along a channel in a type of "drag-reduction". Finally the effect of including strong, antagonistic coupling between nematic and polymer is examined numerically, revealing a rich array of spontaneously flowing states.
I. INTRODUCTION
Examples of active matter include bacterial swarms, the cellular cytoskeleton, and in vitro 'cell extracts' that comprise only polymeric filaments, molecular motors and a fuel supply [1][2][3][4]. Such materials are not only of direct biophysical significance, but also represent a wider class of systems in statistical physics in which strong deviations from thermal equilibrium emerge at the collective macroscopic scale due to the underlying active dynamics of the system's microscopic subunits (rotating bacterial flagella, marching molecular motors that bridge cytoskeletal filaments, etc.). Arising at the level of the microscopic subunits, this driving is distinct from the macroscopic boundary driving of, for example, an imposed shear flow.
For an active particle subject to no externally imposed force, the simplest perturbation it can exert on the local fluid environment is that of a force dipole. Depending on the sign of this dipole the activity is classified as extensile (where the two forces act from the centre of mass of the dipole outwards towards the fluid) or contractile (where the opposite applies). Collectively, a fluid of these active particles can exhibit non-equilibrium emergent phenomena on macroscopic lengthscales that greatly exceed the particle size or spacing. These include activity-induced ordering, and bulk fluid flows that arise spontaneously even in the absence of external driving. Depending on the strength of the activity, these flows may remain steady and laminar at the scale of the system; show oscillatory limit cycles at that scale or below; or exhibit spatiotemporal chaos. The latter effect closely resembles conventional inertial turbulence in a passive Newtonian fluid and is accordingly often termed "active turbulence" or "bacterial turbulence" [2,[5][6][7][8][9]. However its mechanism is distinct from that of inertial turbulence: it stems from a balance between active stress and orientational relaxation, rather than between inertia and viscosity.
Depending on their symmetry, ordered phases of active fluids can be described by either a polar [10,11] or nematic order parameter [1,12,13]. In this work we consider the latter case, and throughout denote the nematic order parameter by Q. Indeed, many theoretical descriptions of active matter are based on simple continuum models for the hydrodynamics of a suspension of rod-like objects, originally developed to describe a passive liquid crystal [14,15]. To describe an active material, such a model is then augmented by the leading-order terms characterising the violations of time-reversal symmetry that arise from activity. In particular, this gives an additional active contribution Σ A = −ζQ to the fluid's stress tensor, where the activity parameter ζ is positive for extensile systems and negative for contractile ones.
Even without detailed knowledge of the value of ζ, the approach just described is capable of robust predictions. For example, beyond a critical threshold level of activity an initially quiescent fluid is generically predicted to become unstable to the formation of a spontaneously flowing state [12]. This threshold, for a finite system size, depends on whether the system is extensile or contractile. However in both cases it decreases with increasing system size [10], tending to zero in the limit of a bulk sample. Any level of activity, however small, can then trigger the formation of spontaneous flows. Consistent with these analytical predictions, numerical solutions of active nematic continuum models [5,[7][8][9] have indeed revealed a host of spontaneously flowing states resembling experimental observations in bacterial swarms [2] and microtubule-based cell extracts [4]. Both of these are extensile nematics, to which we restrict ourselves in our numerical studies below [11].
Active nematic fluids are often referred to informally as 'active gels' [5,10], particularly in a biological context. But while all liquid crystals are somewhat viscoelastic, for example due to the slow motion of topological defects, most existing models of active matter assume fast local relaxations and so fail to model "gels" in the conventional sense of the term, as understood by polymer or colloid physicists [16,17]. Certainly the standard continuum models of active matter [1,13] do not capture the types of slow viscoelastic dynamics that one might expect in the cytoskeleton, which contains long-chain flexible polymers and other cytoplasmic components that are expected to have long intrinsic relaxation times (or even divergent ones in the case of a cross-linked network). This is a major shortcoming, because this slow viscoelastic dynamics would be expected to couple strongly to the active liquid crystalline dynamics and thereby potentially radically modify the effects of activity.
The effect of a viscoelastic polymeric background is also likely to play an important role in modifying active flows and diffusion [18] at a supra-cellular level. Indeed, long-chain molecules are present in mucus, saliva, and many other viscoelastic fluids both inside and outside the body that are susceptible to colonization by swarms of motile bacteria. Moreover, many bacteria secrete their own polymers [19], particularly during biofilm formation (which is however not the topic of this paper). This suggests an evolutionary advantage for bacteria in controlling the viscoelasticity of their surroundings, supporting the view that viscoelasticity and active motion are coupled in a nontrivial way.
With this motivation, the aim of this paper is to study in greater depth the predictions of a model first presented in Ref. [20], which addresses at a continuum level the interplay between active liquid-crystalline dynamics and slow polymeric modes of relaxation. This approach is distinct from, but complements, recent studies of individual swimmers in viscoelastic fluids [21][22][23][24][25], which show that swimming speeds can be either enhanced or suppressed relative to those in a Newtonian solvent, depending on details of the swimming mechanism and particle geometry. Such details do not enter our continuum picture, however, which focuses on emergent and potentially universal many-body behavior at larger length scales.
In a biological context, active matter is often found in confined geometries such as the interior of a cell. Indeed it has been argued that the confinement of subcellular active matter may in part be responsible for cytoplasmic streaming [26,27], an important process whereby coherent fluid flows facilitate the circulation of nutrients and organelles within the cell [28]. At a larger scale, a recent study of cell migration in artificial channels observed increases in mean cell velocity and flow coherence as the channel was narrowed [29]. Also, suspensions of B. subtilis were observed to form stable spiral structures when confined in a droplet [30]. In view of these observations, it is important that any numerical study should consider carefully the effects of system size. In what follows we choose a rectangular channel geometry of fixed aspect ratio bounded by a pair of parallel hard walls. The size of this channel is notionally fixed in simulation units, but we study finite size effects by then varying instead the microscopic length scales in the problem.
As we shall explore in detail below, the interplay of active and polymeric dynamics leads to a host of exotic spontaneous flow states. These include oscillatory shear-banded states in 1D, while in 2D we find activitydriven turbulence even in the limit of infinite viscoelastic relaxation time in which our model describes an active elastomer. In other regimes we find that adding polymer can have calming effects, increasing the net throughput of spontaneous flow along a channel in a type of "dragreduction".
The paper is structured as follows. In Sec. II we review the equations of motion that describe the coupled dynamics of an active nematic with a viscoelastic polymer, as derived in Ref. [20]. In Sec. III we discuss the simulated sample geometry and give details of our numerical methods. In Sec. IV we perform a linear stability analysis to derive the threshold of instability to spontaneous flow. The results of this linear calculation then provide a routemap for performing full nonlinear simulations in Secs. V and VI. In Sec. V we restrict those simulations to one spatial dimension for simplicity, while in Sec. VI we perform full 2D simulations. Sec. VII contains a summary of our results, and the outlook for future work.
II. MODEL EQUATIONS
In this section we remind the reader of the model equations, as developed originally in Ref. [20]. The state of liquid crystal (nematic) ordering is denoted by a traceless symmetric tensor Q = q nn − 1 3 I , wheren is the nematic director and q is the degree of ordering. The polymeric conformation is similarly denoted by the symmetric tensor C = rr , where r is the end-to-end vector of a chain (or subchain, depending on the level of description used), normalized so that C = I in equilibrium. We adopt a single-fluid description in which the concentration fields of polymer and liquid crystal are assumed to remain uniform (in contrast to a two-fluid approach as taken by, for example, Ref. [31]). Accordingly, all three components -liquid crystal, polymer and solvent -share the same centre-of-mass velocity, v. The symmetric and antisymmetric parts of the velocity-gradient tensor (∇v) ij ≡ ∂ i v j are denoted D and Ω respectively. For any other tensors the symmetric, antisymmetric, and traceless parts carry superscripts S, A and T.
We introduce a free energy density f = f Q (Q, ∇Q) + f C (C) + f QC (Q, C), where f Q and f C are the standard forms for nematics [14] and dumb-bell polymers [32] respectively. Accordingly we have in which G Q sets the scale of the bulk free energy density, K is the nematic elastic constant, and γ is a control parameter for the isotropic-nematic transition. Likewise where G C is the polymer elastic modulus. The lowest order passive coupling between Q and C is where both terms vanish for undeformed polymers (C = I).
Here κ controls how the polymer pressure shifts the isotropic-nematic transition and for simplicity we set κ = 0 throughout. The second term makes it energetically preferable for Q and C to align with major axes parallel for χ < 0, and perpendicular for χ > 0. From the volume-integrated free energy F = f dV , the nematic molecular field H ≡ −[δF/δQ] ST follows as The corresponding molecular field B ≡ −[δF/δC] S for the polymer obeys Using these molecular fields we follow Ref. [20] in developing minimally coupled equations of motion for Q and C that respectively reduce to the Beris-Edwards liquid crystal theory and the Johnson-Segalman (JS) polymer model in appropriate limits [14]. We then allow for conformational diffusion in the polymer sector [33], which adds a gradient term in C of kinetic origin [20]. (Alternatively one can incorporate a non-local term in the polymer free energy, though this then produces a more complex form for the polymer stress [34].) To this is added a minimal set of active terms [12], supposing for simplicity that the polymers are not themselves active, but that the origin of the activity resides entirely in Q. This is enough to capture, for example, the effect of adding polymer to a cell extract, or the collective dynamics of bacterial suspensions in mucus. (A contrasting approach is to build a system of polymers directly from active elements [35]). There remain two active terms linear in Q. One of these can be absorbed into f Q , and the other is the familiar active deviatoric stress Σ A = −ζQ [12].
The resulting equations of motion for Q and C are: Here ξ is the flow-alignment parameter of the nematic [36] and a is the slip parameter of the JS model. Setting a = 1 recovers the Oldroyd B model [37]. Each controls the relative tendency of molecules to align with streamlines versus rotating with the local vorticity. Parameters τ Q , τ C are intrinsic local relaxation times for nematic and polymer, while ℓ C governs conformational diffusion in the JS sector [33]. The velocity field v obeys the Navier Stokes equation for an incompressible fluid Here the total stress Σ = −P I + 2ηD + Σ A + Σ Q + Σ C combines an isotropic pressure P , a contribution from a Newtonian solvent of viscosity η, two reactive stresses and the active stress Σ A = −ζQ. The colon in Eq. 10 denotes contraction over the second and third Cartesian indices. In what follows we shall assume inertialess (creeping) flow, and set ρ = 0 in Eq. 8.
Having set out this model in which the dynamics of the polymer and liquid crystal are fully coupled, we now consider certain limits in which the coupling between them is diminished. Clearly, setting the constants χ and κ to zero in Eqn. 3 eliminates any thermodynamic coupling between Q and C. It is important to understand, however, that even in this case of zero thermodynamic coupling, Q and C remain non-trivially coupled in a purely kinematic way: any changes in C or Q perturb the stress Σ, which in turn perturbs the flow field v, which in turn drives both Q and C. Accordingly, even without any thermodynamic coupling we shall find in what follows a rich array of regimes in which the dynamics of Q and C show strong coupling. We therefore defer the case of true thermodynamic coupling to Sec. VI C.
As already noted, in the limit in which we simply remove the polymeric and active components (G C = κ = χ = ζ = 0), the model reduces to the Beris-Edwards theory of liquid crystals. Removing instead the liquid crystalline component (G Q = K = κ = χ = 0) recovers the Johnson-Segalman model of polymeric fluids. These separate models have been studied comprehensively in the earlier literature and we do not consider them further here.
Note finally, and importantly, that in the limit τ C → ∞ our polymer behaves as an elastomer, without any local mechanism for stress relaxation. At zero activity, our model then describes a passive nematic elastomer [38].
A. Geometry and boundary conditions
We consider a two-dimensional (2D) slab of active viscoelastic matter confined between parallel plates separated by a distance L y in the y-direction, and of length L x in the x-direction. At the plates we choose boundary conditions of no-slip and no-permeation for the fluid velocity v: and zero-gradient boundary conditions for Q and C: In the direction x parallel to the plates we adopt periodic boundary conditions for all variables.
The adoption of a zero-gradient boundary condition for the polymeric conformation tensor C is standard practice in the literature on shear banding, though it remains to be justified microscopically. A zero-gradient boundary condition on the nematic order parameter, allowing free rotation of Q at the wall, has been used in some previous studies of active materials [39,40], whereas other studies have adopted anchoring boundary conditions [41]. Recent work comparing both these boundary conditions has shown the essential physics to be qualitatively unchanged by the choice made [5], particularly at large activities where flowing states develop structure on a length-scale much smaller than the system size.
Despite this, we attach the following note of caution. In Sec. IV below we calculate the critical threshold activity required for the onset spontaneous flow, starting from an initial base state of no flow and a perfectly ordered director field. Crucially, we will find this threshold to depend strongly on the orientation of the director in that initial state: states where the director is initially aligned in the x-and y-directions become separately unstable at activities an order of magnitude apart. That observation in turn has important implications for the boundary condition on Q, because a boundary condition that favors anchoring of the director parallel (resp. perpendicular) to the walls would be likely to favor an initial condition in which the director is aligned in the x (resp. y) direction, leading to different threshold activities in each case. This suggests that, in some instances, the choice of boundary conditions can have an important effect on the system's behavior. Indeed recent experiments demonstrate that 'living liquid-crystals', in which conventional liquid crystals are made active by the addition of swimming bacteria, can be influenced by anchoring boundary conditions [42].
On the other hand, the adoption of a zero gradient boundary condition considerably simplifies our linear stability analysis below, because it permits a spatially homogeneous base state. This in turn means that heterogeneous perturbations to that base state can easily be expressed in a cosine basis. In contrast, anchoring boundary conditions could lead to inhomogeneous base states, complicating the analysis. Accordingly, we work with zero gradient boundary conditions throughout.
We restrict the fluid velocities to lie in the x − y plane, setting v z = 0, and assume translational invariance of all variables along z. In some calculations we further assume translational invariance along x, restricting our states to vary only in 1D, along y. However we work throughout with 3D tensors for both Q and C, allowing non-zero components Q αβ and C αβ for α, β = x, y, z. In principle this could allow the principal axis (director) of Q and/or C to point out of the xy plane of the simulation. Analysing our numerical results, however, we find in practice that in most of our simulations Q and C do remain confined to the xy plane [43,44]. We therefore expect the key physics to be robust to the dimensionality of the order parameter tensors [45].
B. Units and Parameters
We work in units of length such that the gap size L y = 1, of time such that the liquid crystal's local relaxation time τ Q = 1, and of mass such that its characteristic modulus G Q = 1. The value of the parameter η Q = G Q τ Q that controls the passive nematic viscosity is then also equal to unity. We work in the creeping flow limit of zero Reynolds number, setting ρ → 0. We use a cell aspect ratio L x /L y = 4.
To enable a direct comparison with previous numerical studies of active matter without polymer present [5], we fix the solvent viscosity η = 0.567 throughout. In the Q sector of the dynamics we set the isotropic-nematic control parameter γ = 3 so that the passive liquid crystal lies well within its nematic phase [46]. We set the flowalignment parameter at ξ = 0.7, which lies within the flow-aligning regime.
In the polymer sector we set the slip parameter a = 1, such that the Johnson-Segalman model reduces to the Oldroyd B model. This eliminates any possibility of a shearbanding instability originating purely from the polymer dynamics. Theoretical studies suggest that this value, which is widely adopted for flexible polymers, may also be reasonable for dense cross-linked filaments [47,48].
With many of the model's parameters having been fixed as just described, there remain only four to be explored numerically: the activity ζ, a diffusivity parameter ∆ (defined below) that governs relaxation of spatial gradients in the system, the polymer relaxation time τ C and Symbol values explored parameter its modulus G C , and (when non zero) the coupling constant χ. In our chosen units, these are adimensionalised as summarised in Table I. The tildes denote adimensional quantities, but our units have been chosen to make the tildes redundant, and we now drop them. Table I also shows the representative ranges that we shall explore numerically for these parameters. While these are chosen on the basis of order-of-magnitude estimates at best, they might reasonably describe the recent experiments of Sanchez et al. on an extensile kinesinmicrotubule mixture akin to a cytoskeletal gel [4], where the level of activity was varied via the concentration of ATP. In Ref. [49], the modulus of a (contractile) actin gel was found to increase by a factor of ten compared to the passive case in the presence of myosin motor activity, suggesting an upper bound to the dimensionless activity parameter of ζ/G Q ≈ 10.
In our model, the polymer could in principle describe a range of viscoelastic behaviors within the cell, including the effects of the cytosol, which comprises entangled protein filaments and organelles [50]. To encompass this diversity, we vary the polymer relaxation time from τ C = 10 −2 , for which the polymeric dynamics are rapid and only contribute extra viscosity to the fluid as a whole, to τ C → ∞, where the polymer effectively acts as an elastomeric solid.
Throughout most of what follows we assume a constant value of η C ≡ G C τ C , which controls the polymer viscosity, setting η C = 1. To maintain this between runs in which the polymer relaxation time τ C changes, the polymer elastic modulus G C must change in inverse proportion to τ C . This assumption of a constant polymer viscosity is however relaxed in those sections where we consider the true elastomeric limit τ C → ∞ (at a fixed value of G C ) of an active nematic in a crosslinked polymeric background.
The crossover length-scale at which elastic distortions in a passive nematic compete with its bulk free energy is ℓ Q ≡ K/G Q . Recasting the Q constitutive equation (Eq. 6) in this notation, the diffusive term in the molecular field then becomes ℓ 2 Q τQ ∇ 2 Q. The diffusive JS model prescribes an analogous term in the polymer sector, with coefficient ℓ 2 C /τ C [33]. In most of our numerical runs we assume these to be equal, setting: Note that taking the elastomeric limit τ C → ∞ at a fixed value of the diffusivity parameter ∆ means taking l 2 C → ∞ in tandem. In some places below we instead consider fixed l C = l Q . Taking the elastomeric limit in that case then eliminates the diffusive term ∆ C = l 2 C /τ C from the polymer dynamics; setting l C = l Q is then the same as setting l C = 0 in this limit. However many of our 2D simulations then show an unphysical instability that leads to structure on the scale of the numerical grid. The retention of finite ∆ C = ∆ as τ C → ∞ avoids this problem; we discuss the issue further in Section VI B.
Values for ∆ are then selected so that the associated lengthscale l Q obeys ∆x < ℓ Q ≪ L y , where ∆x is the numerical grid size (for which L x /1024 or L x /2048 is a reasonable lower bound, constrained by a maximum feasible number of grid points N x = 1024 or 2048). In this way, the spatial structures predicted by the model are well resolved on the scale of the numerical mesh, and also fit comfortably within the simulation box. The velocity correlation length for kinesin microtubule mixtures has been estimated at l v ≈ 100µm [4], and if we assume that our lengthscale ℓ Q is an order of magnitude smaller than this [45], the range of values of l Q that we explore numerically would imply a channel width in the range 400µm → 3mm, as might describe a typical microfluidic device.
As noted above, throughout most of what follows we disable explicit thermodynamic coupling between Q and C by setting χ = 0 and κ = 0. The only remaining coupling between Q and C is then purely kinematic, caused by the two components sharing a common fluid velocity v. In Sec. VI C, we move finally to address the case of explicit thermodynamic coupling with χ = 0.
C. Numerical Details
In our numerical scheme we evaluate spatially local terms in both constitutive equations using an explicit Euler time-stepping scheme. To solve the Stokes equation we use a stream function formulation, with a hybrid numerical scheme in which gradient terms in the x and y directions are computed using Fourier and Crank-Nicolson methods respectively [51,52]. Spatially diffusive terms in the constitutive equations are likewise solved using this hybrid approach. Advective terms are computed using third order upwind differencing.
We performed the following checks of our numerics. For each run we verified the results to be robust against halving either the spatial mesh size or the time step. Note however that because of the erratic (and quite possibly chaotic) nature of many of the dynamical states explored, converging individual trajectories is not a realistic prospect. We instead performed convergence tests on time-averaged quantities, and on the overall characteristics of states in the phase diagram. Finally, in our nonlinear runs we have carefully checked the behavior in the early stages of instability for consistency with the growth rates found from our linear stability analysis.
IV. LINEAR STABILITY ANALYSIS
In this section we perform a linear stability analysis of an initially homogeneous, non-flowing base state to determine the regions of parameter space in which such a state is linearly unstable to the formation of a heterogeneous state with spontaneous flows. Doing so enables us to understand the key instabilities present in the model, and to form a helpful roadmap in selecting parameter values for the much more computationally expensive nonlinear runs in later sections. We set the explicit thermodynamic couplings to zero throughout this section: κ = χ = 0.
A. General procedure
We consider an initially homogeneous, quiescent, uniaxial base state with Here q is the magnitude of the nematic order parameter, and n α its director. We shall consider two alternative choices for the initial director field:n = (1, 0, 0) and (0, 1, 0), oriented parallel and perpendicular to the walls respectively. Note that, although we have specified the Q αβ of this initial base state to be uniaxial, our calculations for departures from the base state do not impose that restriction. The polymer is initially undeformed, consistent with the absence of velocity gradients; with our boundary conditions this absence also implies v α = 0 in the base state such that the fluid is initially at rest.
We now examine the linear stability of this base state to perturbations that are heterogeneous in one spatial dimension (1D), with wavevector in the flow gradient direction, y. Within this 1D assumption, the condition of fluid incompressibility further demands that the only element of the velocity gradient tensor that might become nonzero isγ ≡ ∂ y v x . To simplify the notation, we collect all the relevant variables into the quantity φ = (Q, C,γ). To the base state φ = Q, C,γ prescribed in Eq. 13 above, we then add Fourier-mode perturbations with small amplitudes φ k = Q k , C k ,γ k . Our boundary conditions at y = 0, L y require that the wavevector k takes discrete values, k = πm/L y , with integer m.
Substituting Eq. 14 into the model equations derived in Sec. II and expanding in powers of the mode amplitudes gives, at first order, a linearised equation set for the dynamics of the perturbations: where Note that p k contains only a subset of the full list of variables originally specified in φ k . The reasons for this are twofold. First, the Stokes equation requires ∇·Σ = 0, with Σ the total stress tensor. This enables us to expresṡ γ k directly in terms of Q k and C k aṡ in an obvious notation. This eliminatesγ k as a dynamical variable: physically, the shear rate is enslaved to the viscoelastic stress components by the requirement of instantaneous force balance in the limit of creeping flow. Secondly, we find that the set of components Q k αz and C k αz evolve independently from the xx, xy, yy components contained in p; this z-related set furthermore always have negative eigenvalues, rendering them stable. Accordingly, the only dynamical variables relevant to us are those listed in p above.
The eigenvalues of the matrix M k then determine whether the initial base state is stable or unstable to the growth of perturbations. Any eigenvalue with a positive real part corresponds to an unstable mode that grows exponentially (in this linear regime), taking the system away from the initial homogeneous, non-flowing base state, and towards a heterogeneous state with a spontaneous flow.
In what follows we seek a threshold value of the activity at which the base state first becomes linearly unstable, with the values of the other model parameters held fixed. Sweeping the activity level upwards, the critical threshold ζ c at which instability first sets in is the value at which where ω + is the eigenvalue with the largest real part. It is possible to show that the only non-trivial (potentially unstable) modes of M k have eigenvalues ω ± given by and For each value of Λ, then, two different modes of instability are possible as determined by Eq. 17. We term these the viscous and elastomeric instabilities, and explore them in the next two subsections respectively. For each such mode of instability, the activity threshold depends on Λ. This in turn depends on whether the initial director obeysn = (0, 1, 0), for which the growing mode involves a bend deformation, or obeysn = (1, 0, 0), for which it involves splay.
B. Viscous instability
For the first mode of instability, the discriminant b 2 −4c in Eq. 17 remains positive and the eigenvalues of M k are purely real. The condition Re(ω + ) > 0 for the onset of instability then simplifies to the condition c < 0. Predividing c by the final term on the right hand side of Eq. 19 and multiplying by η then gives the conditions for instability as We identify the four terms on the left hand side as the wavevector-dependent zero-shear viscosities associated with the polymer, the passive nematic stress, the active nematic stress and the solvent respectively. We can therefore rewrite this criterion as where in fact η k Q,passive and η k are independent of k. Each term in Eq. 22 is positive, apart from the term η k Q,active . A simplified analysis, that casts the full tensorial nematic order parameter Q in a simplified form in terms of a directorn with a fixed degree of nematic ordering [53], then suggests that where the multiplication symbols serve to emphasize the correspondence of terms between the two expressions. In this equation, an initial base state director orientation alongx is linked to a director perturbation δn in the y direction, corresponding to a splay instability; whereas an initial base state director alongŷ is linked to a perturbation δn in thex direction, corresponding to a bend instability. Recall that the perturbation wavevector k is in theŷ direction in all these 1D calculations. (Note that all other analytical and numerical results employ the full Q formulation, with the exception of Eq. 27 where we make the analogous argument for the elastomeric instability.) Within Eqs. 22 and 23 we recognise the mechanism that drives this instability as follows: a perturbation in the shear rate causes a perturbation in the director orientation, which in turn causes a perturbation in the active contribution to the stress field, which must provide a counterbalancing contribution from the other stress components to maintain force balance. The two shear rate perturbations in this loop are in the same sense, signifying positive feedback. Rearranging Eq. 21, we find the critical activity for the onset of instability to be where we recognise r Q ≡ ℓ 2 Q k 2 /τ Q as the on-diagonal rate of relaxation of a perturbation to the director field at wavelength k.
For values of the activity just above threshold, the perturbations grow very slowly. They furthermore do so without any oscillatory component because the eigenvalues are real in this case. Relative to this slow growth, the fluid's intrinsic relaxation timescales τ C and τ Q become infinitely fast as one approaches the threshold. Accordingly the fluid's resistance to this instability appears only via its zero-frequency viscosities, η C and η Q : the relaxation times τ C and τ Q cannot appear in Eq. 22 independently of these viscosities. The effect of the polymer is therefore simply to increase the fluid's overall viscosity, delaying the onset of instability. The dependence of the threshold on η C = G C τ C is thus independent of whether τ C is varied at fixed G C , or G C at fixed τ C . Accordingly, we call this mode the 'viscous instability' in what follows.
In view of the relatively trivial role of the polymer in determining the onset of this viscous instability, we expect the result just obtained to match onto the one in the earlier literature for a purely nematic active fluid, without polymer. Indeed, substituting K ≡ ℓ 2 Q G Q recovers the criterion in the form originally derived in Refs. [10,40], with an additional term to account for the extra viscosity contributed by the polymer.
In the limit of infinite system size, k → 0 in Eq. 24, we further recover the prediction ζ visc c → 0, implying that a bulk active nematic will be unstable for any level of activity, however small [10,12]. One important prediction of the present work is that this generic instability of a bulk active nematic persists even with polymer present.
C. Elastomeric instability
An alternative mode of instability arises when the discriminant b 2 − 4c in Eq. 17 is negative and the eigenvalues of M k have an imaginary part, suggesting a Hopf bifurcation with oscillatory dynamics [54]. The criterion Re(ω + ) = 0 for the onset of instability then simplifies to the condition (25) At a given wavevector k, we identity the five terms in b as being respectively proportional to (i) the rate of change of polymer stress during an impulsive strain; (ii) the rate of change of passive nematic stress during an impulsive strain; (iii) the rate of change of active nematic stress during an impulsive strain; (iv) the on-diagonal rate of relaxation r c ≡ (1 + ℓ 2 C k 2 )/τ C for a perturbation in the polymer conformation, and (v) the on-diagonal rate of relaxation r Q for a perturbation in the director field.
Therefore we write the criterion for the onset of instability as Among the terms on the left hand side, each is always positive apart from the third. Following the approach described previously for the viscous instability, in which we rewrite the derivative in terms of the director field n, the third term reads We recognise the mechanism that drives this instability as follows: a perturbation in the shear strain field causes a perturbation in the director orientation, which in turn causes a perturbation in the shear stress field, which in turn must cause a perturbation in the strain field to maintain force balance. The two shear strain perturbations in this loop are in the same sense, signifying positive feedback. Indeed this is essentially the same mechanism as for the viscous instability above, but with the strain rate replaced by the strain. Accordingly, we call this mode the 'elastomeric instability'. Rearranging Eq. 25 we get an expression for the critical activity for the onset of instability as In contrast to the viscous instability discussed above, at the threshold of this new instability the perturbations grow subject to a superposed oscillatory component that has a time period prescribed by the imaginary part of the eigenvalue, τ ω = 2π/Im(ω + ). The presence of this time scale means that the relative value of the polymer's relaxation time is relevant to this instability, in contrast to its unimportance to the viscous one: τ C enters Eq. 28 directly rather than merely via the viscosity τ C G C . Put differently, the polymer's viscoelasticity is important to this new instability, in contrast to the viscous instability, which depends only on its viscosity.
Two distinct thresholds again arise for the elastomeric instability according to whether the deformation corresponds to a bend or a splay in the director field, which is in turn determined by whether the initial director lies in the y or x direction as set by Λ (see Eq. 20).
D. Viscous -elastomeric crossover
As described in the previous two subsections, viscoelastic active matter is predicted to exhibit two distinct modes of instability: a viscous mode and an elastomeric one. The activity thresholds for these two modes are compared in the phase diagrams of Figs. 1 and 2.
As can be seen, the thresholds for the two instabilities display qualitatively different dependences on the polymer relaxation time τ C and on the system size. Indeed, taking the elastomeric limit τ C → ∞ for any fixed G C and fixed ℓ C = ℓ Q raises indefinitely the threshold for the viscous instability: ζ visc c → ∞. The fluid's viscosity approaches infinity in this limit, switching off the viscous instability entirely. In contrast the threshold for the onset of the elastomeric instability remains finite even in the purely elastomeric limit τ C → ∞: (where we have also taken the infinite system size limit for clarity of expression). Crucially, then, this second instability can arise even in the limit of an elastomeric solid, because it involves perturbations in strain rather than strain rate. Now consider instead fixing τ C , G C , so that the polymer viscosity remains finite, and taking the limit ℓ C = ℓ Q → 0. This is equivalent to taking the limit of infinite system size L y → ∞, for which the threshold for the viscous instability tends to zero: for a bulk system, any level of activity, however small, triggers this instability. In contrast the activity threshold for the elastic instability remains finite even in this bulk limit. These two facts can be seen in Fig. 1.
The physical origin of this important difference in system-size dependence can be understood by comparing the driving terms for the two modes. The term ∂n/∂γ in the viscous criterion (recall Eq. 23) scales as 1/k 2 , because an infinitesimal shear rate perturbation can cause finite rotation of the director field in the limit of k → 0. (This stems from the Goldstone mode associated with the spontaneously broken rotational symmetry. A nematic phase exerts no restoring force against global rotations of a state of uniform Q, implying by continuity that the force likewise vanishes in the limit k → 0 [55].) In contrast, the term ∂n/∂γ in the elastic criterion (recall Eq. 27) is finite at low k, because small strain perturbations cause only small changes in director, even in the limit of zero wavevector.
With these remarks in mind, we now see that dominance of the viscous instability over the elastomeric one (or vice versa) will depend on the ratio τ C /τ Q of the polymer and nematic relaxation times, and also on the system size, measured in relation to the lengths l Q , l C (as set by the gradient terms in the equations of motion). For any fixed system size, below a critical value of the polymer relaxation time τ C = τ * C the viscous instability threshold ζ visc c lies below the elastic one: the viscous instability arises first on increasing the activity, and ζ visc c sets the stability criterion for our base state. In contrast, for τ C > τ * C the elastomeric instability arises before the viscous one, and ζ elast c sets the relevant criterion. This crossover value τ * C can easily be shown to obey which for large system sizes simplifies to τ * C ≈ τ Q /ℓ 2 Q k 2 . This form can be understood as follows. The quantity τ Q /ℓ 2 Q k 2 is the orientational relaxation time for longwavelength distortions in the nematic phase, which diverges as k → 0. (Recall that this is a Goldstone mode, with no penalty for global rotations in the director field.) For polymer relaxation times faster than this, τ C < τ * C , the polymer behaves in a viscous way relative to the nematic mode, and the viscous instability dominates. In the opposite case the polymer behaves elastically, and the elastomeric instability dominates.
In the limit of infinite system size the crossover value diverges: τ * C → ∞. In this bulk limit the viscous instability is triggered first (at zero activity threshold) for all values of τ C , whereas the threshold of the elastomeric instability remains finite even for a bulk sample. As noted in the introduction, however, many biological environments are strongly confined. This motivates the use of a finite box size in our simulations, and a correspondingly careful exploration of both the viscous and elastomeric instabilities. For our typical choice of parameter values given in Table I, the crossover between the two instabilities occurs at τ * C ≈ η Q /(Kk 2 ) = O(10 3 τ Q ).
V. NONLINEAR DYNAMICS (1D)
In the previous section we derived the activity threshold for linear instability to a state of spontaneous flow in an active nematic with added polymer. We now use those results as a roadmap to explore the model's full nonlinear dynamics. We shall restrict ourselves in this section to 1D calculations, with heterogeneity in the y direction only, deferring a study of 2D effects to Sec. VI below. In each run we initialise the system in the homogeneous base state given by Eq. 13, with the directorn aligned in the x-direction. Accordingly, any instabilities that arise below originate from splay deformations. We subject this base state to small added perturbations at the start of the run of the form δγ = Our model has three competing local relaxation times: the liquid-crystal relaxation time τ Q , the polymer relaxation time τ C , and the active forcing timescale τ a = η/ζ, which was identified in Ref. [56] by balancing the active modulus ζ against the solvent viscosity η. As discussed previously, the first of these, τ Q , is our unit of time. We now examine the competition between the second and third timescales by producing phase diagrams in the (ζ, τ C ) plane. Related to τ a is the active timescalē τ a = η/(ζ − ζ c ), with the denominator shifted to account for the fact that in a finite system the critical threshold for the onset of activity is non-zero. (Note thatτ a → τ a as L y → ∞ or ζ → ∞.) Whenever the active timescale is shorter than the two relaxation times, we can expect oscillatory dynamics [56]. We will confirm this expectation: first (in Sec. V A) for a material of fixed polymer viscosity η C = 1, where we find the dominant period of oscillation τ osc to be set by τ C , and then (in Sec. V B) for an elastomeric material with τ C → ∞, where instead we find τ osc ∝τ a . In a biological context, oscillatory states have been observed in fibroblast cells [57] with a period of oscillation proportional to the myosin motor activity. Similar states have also been studied in Drosophila embryos [58], where oscillations are thought to play a key role in cell shape formation (morphogenesis).
A. Phase diagram: fixed viscosity
We begin by considering the case of a fixed polymer viscosity η C = G C τ C = 1. (Recall that the passive nematic viscosity η Q = 1 by our choice of units, and the solvent viscosity η = 0.567 for consistency with Ref. [5].) Between runs we vary the polymer relaxation time τ C , and in tandem therefore also vary G C = 1/τ C . This will allow us to confirm the direct role of the polymer relaxation time τ C in the dynamics, while removing any potential complications arising from variations in the overall viscosity. In varying τ C in this section we choose to keep ℓ C = ℓ Q fixed. Note that this effectively eliminates the diffusivity ∆ C = ℓ 2 C /τ C from the polymer dynamics in the elastomeric limit τ C → ∞. The predictions of our linear stability analysis in this case are shown in the (ζ, τ C ) plane in Fig. 3, with four sets of curves corresponding to different values of ℓ C = ℓ Q .
The results of our nonlinear simulations at fixed ℓ Q = ℓ C = 0.004 are shown in Fig. 4, with the linear stability thresholds also repeated on the same axes for comparison. The green shaded region indicates when oscillatory instabilities are expected from the linear stability analysis at any point in the discrete spectrum of modes k = mπ/L y (cf. Fig. 3 where oscillatory instabilities are also marked with shaded regions, but where we instead fix k = π/L y and plot multiple values of ℓ Q = ℓ C ). As can be seen, crosses) in our nonlinear simulations, consistent with the analytical prediction of a stable base state in this regime. For values of the activity above ζ c we observe spontaneously flowing shear banded states (black circles).
These spontaneously flowing states may be categorised according to whether they approach a steady state at long times, or instead oscillate. In particular, for activity values exceeding the threshold for the viscous instability, but below that for the elastomeric instability, we observe steady shear banded states (open circles), as seen in earlier studies of the model in the absence of polymer [5]. In contrast, for values of the activity that also exceed the threshold for the elastomeric instability, and roughly correspond to τ C /τ a > 200 (blue line in Fig. 4), we find time-dependent states in which the shear bands oscillate with a period set by the viscoelastic relaxation timescale τ C . No counterpart to these oscillatory states was observed in the earlier study without polymer [5]. This confirms our view that the polymer relaxation time is integral to their dynamics.
The separation of timescales (large τ C /τ a ) required to see these oscillations is reminiscent of earlier work addressing rheological chaos [59]. Intuitively, if the active timescale τ a = η/ζ is sufficiently long, then both polymer and liquid crystal can relax any activity-driven deformation and the resulting shear-banded steady state is time-independent. Conversely, if the polymer cannot relax the activity-induced stress quickly enough, the polymer dynamics lag behind, resulting in oscillations. This is also somewhat analogous to the mechanism described in Ref. [56], although in that case the coupling was to a slowly diffusing concentration field rather than a slowly relaxing polymer.
To gain further insight into these oscillatory shear banded states, we now explore in detail two particular examples denoted respectively by the blue triangle and red square in the phase diagram of Fig. 4.
Oscillatory interface, fixed flow direction. The first example, denoted by the blue triangle in Fig. 4, corresponds to a time-dependent state in which the position of the interface between two shear bands oscillates as a function of time. See Fig. 5 for a space-time plot of the shear rateγ (top left) and of the polymer shear stress, Σ C xy (bottom left). The time period is set by the polymer relaxation time τ C , making it clear that these oscillations are governed by the viscoelastic dynamics of the polymer. As the interface position deviates from the centre, large polymeric stresses are seen to develop in the narrower band until eventually the interface turns round and returns in the opposite direction, relaxing the polymer somewhat. The total shear stress and integrated throughput, Ψ = Ly 0 v x dy also have time-dependent signals (not shown) with a period set by τ C .
In this example the sign of the throughputi.e., the overall direction of the flow -remains constant in time. This can be seen by noting that the region of negative shear rate remains always below the one of positive shear rate in Fig. 5 (top left), meaning that the velocity profile is always overall nose-shaped and pointing to the left. (Note that a state with sustained throughput to the right could equally well have developed, had we made small changes in the initial conditions.) Oscillatory interface, switching flow direction. The second example is denoted by the red square in Fig. 4, with space-time plots ofγ and Σ C xy in Fig. 5 (right). Here two distinct kinds of oscillation are seen in the course of any run. On a slow timescale ≈ 2τ C the flow periodically reverses direction, as evidenced by the up-down switching between the bands of positive and negative shear rate. Another, more rapid, oscillation is evident: the through-put direction remains the same but the position of the interface fluctuates with a mechanism resembling the one discussed in the previous example above.
As already noted above, in this section we have kept the values of ℓ C = ℓ Q fixed as the polymer relaxation time changes. Had we instead chosen to keep ℓ 2 Q /τ Q = ℓ 2 C /τ C ≡ ∆ fixed as τ C varied, the initial linear instability would not be oscillatory. While a oscillatory linear instability doesn't necessarily imply oscillatory nonlinear dynamics, our results in this section demonstrate a reasonable correlation between the two (compare green region in Fig. 4 with oscillatory banded states (shaded and filled circles)).
B. Spontaneous flow in elastomeric solids
The case of an active elastomeric solid can be approached within our formalism by more than one route, yielding slightly different limiting descriptions that may be appropriate to different physical cases. One choice concerns the way in which the infinite viscosity limit is taken; a second and independent choice concerns the behavior of the polymer stress diffusivity ∆ C . In the previous section we studied the dependence of the model's behavior on the polymer relaxation time τ C at fixed polymer viscosity η C = G C τ C = 1. We also fixed the values of the microscopic lengthscales ℓ C = ℓ Q , in relation to the macroscopic system size. In the regime of large polymer relaxation times (τ C ≃ 10 6 ) we found sustained oscillations in the system's long time response. Such large values of τ C correspond for all practical purposes to an elastomeric solid, but with a tiny modulus G C to maintain the overall constraint of fixed polymer viscosity η C = G C τ C = 1.
In this section we consider instead taking the "true" limit of an elastomeric solid τ C → ∞ at a fixed value of the polymer modulus G C , so that the polymer viscosity η C = G C τ C now also diverges as τ C → ∞. We also fix the value of the diffusivity parameter ℓ 2 Q /τ Q = ℓ 2 C /τ C = ∆. In this way, taking the limit τ C → ∞ means taking the limit l C → ∞ also.
Our choice of nonzero polymer stress diffusivity ∆ C = ∆ > 0 in the elastomeric limit is not obvious, since physically there is no reason for l C to diverge in that limit. However, we have found that in our 2D nonlinear work setting ∆ C = 0 generally leads to a short-scale numerical instability unless prohibitively small time-and spatial-stepsizes are used. We explore this issue further in Sec. VI B, but even there set ∆ C = ∆ to retain direct comparability of parameters between the 1D and 2D numerics.
The predictions of our linear stability analysis in this case are shown in Fig. 6 in the (ζ, τ C ) plane, for fixed G C = 0.1 and various fixed values of ∆. In this figure, τ C → ∞ is approached by taking the ordinate to infinity. Note how Fig. 6, which has fixed G C and ∆ C , differs from Fig. 1, which had fixed G C and ℓ C . In particular, the viscous instability is not eliminated when the elastomeric limit is taken at fixed ∆ C in Fig. 6, apparently because the polymer stress can still redistribute itself spatially by diffusion despite having a divergent local viscoelastic relaxation time τ C . (The latter ensures that the volumeaveraged polymer stress cannot decay; see Sec. VI B for further discussion.) Furthermore, the regime in which an oscillatory instability is predicted has been eliminated in Fig. 6 compared to its strong presence in Fig. 1. Linear stability results calculated in the limit τ C = ∞, again at various fixed ∆ Q = ∆ C ≡ ∆, are shown in the plane of polymer modulus and activity (ζ, G C ) in Fig. 7. With these linear stability results in mind, we now perform nonlinear runs in the elastomeric limit τ C = ∞ for the particular case of G C = 0.1 and ∆ = 10 −4 . For these parameters the critical activity threshold is ζ c = 2.41. Accordingly, we simulate a sequence of activity values ζ = 2 → 3.2 straddling this threshold. The results are shown in Fig. 8. The upper panel shows the time evolution of the (largest mode of the) perturbation to the base state, clearly showing exponential decay (linear stability) for activity values ζ < ζ c and early time exponential growth (linear instability) for ζ > ζ c , giving way to saturation at long times due to nonlinear effects. We have checked that the slopes of the lines in Fig. 8a) correspond to the eigenvalues predicted by the linear stability analysis.
For the particular run ζ = 3.2 we now examine the system's behavior at long times, once it has settled to the ultimate nonlinear attractor of the dynamics. As before we find states that are shear banded. Note, however, that conventional static shear bands are forbidden by the elastomeric nature of the polymer: a static band with a fixed non-zero shear rate would indefinitely load the polymer, leading to divergent polymer stresses. Indeed, inspecting the shear-rate profilesγ(y, t) in Fig. 8b, we see instead travelling bands of local shear-rateγ = ±0.15, such that the fluid at any location in the flow cell is alternately sheared forward then backward. Corresponding to this, the shear component C xy of the polymer stress (Fig. 8b) also alternates in sign. These results are reminiscent of traveling density bands seen experimentally in cytoskeletal extracts [60]. Note that the initial linear instability is not oscillatory, consistent with the prediction in our linear stability analysis that eigenvalues are purely real for ℓ 2 Q /τ Q = ℓ 2 C /τ C = ∆; recall Figs. 6 and 7. Because the polymer relaxation timescale τ C is infinite in these runs, the period of oscillation τ osc must now be set by a separate timescale. To identify this timescale we performed simulations for a range of values of the activity ζ and solvent viscosity η. In each, we measured τ osc via the largest peak in the Fourier series of the throughput as a function of time. See Fig. 9 inset. Collecting the data from all these runs in Fig. 9 (main), we find reasonable evidence for the scaling Physicallyτ a is related to the timescale of active forcing identified in Ref. [56]. As discussed in that study, if the active forcing is faster than the relaxation timescales, τ a < τ Q ≪ τ C , both nematic and polymer lag behind, resulting in oscillatory behavior. Note, however, that for large values of the activity the flow becomes increasingly aperiodic and τ osc becomes less clearly defined, resulting in minor deviations from the suggested scaling law. To summarise, we have shown both by linear stability analysis and also by full nonlinear simulations (so far in one spatial dimension) that an elastomeric active nematic generically undergoes instability towards a spontaneously flowing state which must be oscillatory, otherwise the polymers would suffer infinite loading.
VI. NONLINEAR DYNAMICS (2D)
In the previous section we saw a rich array of dynamical states with heterogeneity in one spatial dimension (1D). These would clearly have been forbidden in any description that constrained the system to remain homogeneous in all directions (0D). We now increase the dimensionality further, to consider spatio-temporal dynamics in 2D. Two new features are immediately anticipated that were forbidden in 1D. One is the presence of ± 1 2 point defects in the director field [8,9,45]. Another is the possibility of extensional flow, which was forbidden by the constraint of fluid incompressibility in a 1D flow field of the form v = v(y)x.
Consistent with these expectations, an earlier study of active nematics (without polymer) indeed found a much richer spectrum of phase behavior in 2D than in 1D [5]. We now briefly remind the reader of those earlier findings, as a starting point from which to understand the effects of polymer below. Accordingly, the model's phase behaviour without polymer is summarised in Fig. 10 (top) in the plane of (ζ, ∆), where ∆ ≡ ℓ 2 Q /τ Q . The solid line shows the linear instability threshold (Eq. 22) for the onset of the 1D viscous bending instability, given a homogeneous initial state with the directorn along y. The dotted line is the counterpart for the 1D viscous splay mode, given an initial director along x. As expected the instability manifests itself at high ζ and low ∆, consistent with the fact that smaller system sizes (large ∆) tend to be stabilising.
In the same diagram, the results of numerical runs performed without polymer in 2D are shown by symbols.
Runs are initialised withn along y, with a perturbation δQ αβ = 32 n 32 m δA mn αβ cos (πmy/L y ) cos (2πnx/L x + θ n αβ ), where δ = 10 −10 , A mn αβ is a randomly selected magnitude drawn from a uniform distribution on [−1, 1] and θ n αβ ∈ [0, 2π) is a randomly selected phase (analogous perturbations are also added to C αβ when present). In the region where no linear instability is predicted the system remains homogeneous and quiescent (no spontaneous flow), even in these 2D runs. In contrast, beyond the threshold of the (bend) instability we find spontaneously flowing states, as expected. For activities only just above threshold, the ultimate solution corresponds to a steady 1D shear-banded state (triangular symbols), even though these runs in principle allow heterogeneity in 2D. In contrast, further into the unstable regime we find fully 2D spontaneous flow states. At intermediate activities we observe a range of oscillatory states, including states in which defect pairs undulate along the channel; these we denote by squares in Fig. 10. Spatially, these states exhibit repeating structures at a lengthscale roughly set by the channel width L y . As the activity is increased (or ∆ decreased), these oscillatory states gain additional frequency components when plotting a given scalar observable (e.g., throughput) against time. When there is no longer any discernible periodicity in this signal, we term the state aperiodic. In this limit the characteristic lengthscale of the resulting nematic texture decreases [45,61] and the order parameter fields lose any obvious spatial periodicity. The numerically observed crossover line ζ 2D c separating 1D from 2D states is shown as a dashed line.
In each run, once the system has attained its ultimate attractor we measure the net throughput of fluid along the channel in the main flow direction x. The criterion that we adopt to represent significant throughput is discussed in the Appendix. States meeting this throughput criterion are represented in Fig. 10 by solid symbols, and those without significant throughput are shown by open symbols. As might be expected, the 1D laminar banded states show significant throughput. In contrast most of the 2D states, with just a few exceptions, lack any coherent throughput: the more complicated velocity rolls associated with them have no overall flow direction.
With the above discussion in mind, we now consider the effects of adding polymer. We start in Sec. VI A by considering a viscoelastic polymer with a fixed viscos- ity η C = G C τ C . We then turn to the elastomeric limit τ C → ∞ in Sec. VI B. In Secs. VI A and VI B we restrict ourselves for simplicity to the case of zero thermodynamic coupling between the nematic and polymer, χ = κ = 0. (As discussed previously, a strong kinematic coupling is however still present.) Finally in Sec. VI C we consider the effects of including explicit thermodynamic coupling.
A. Viscoelastic active matter
In this section we consider a polymer with a finite viscoelastic relaxation timescale τ C . To avoid any complications associated with changing the overall viscosity, we consider fixed η C = G C τ C . (Accordingly, between runs in which τ C progressively increases, G C is progressively decreased in tandem.) We first explore the effects of polymer on the 2D nonlinear dynamics by revisiting the (ζ, ∆) phase diagram discussed above.
Long-time behavior: active drag reduction
The lower panel of Fig. 10 shows the phase diagram of the model, with polymer now included, for a polymer relaxation time τ C = 4.0. Compared to the polymerfree case (top panel) a dramatic difference is immediately apparent. Whereas without polymer the regime of significant throughput is confined predominantly to that of laminar 1D banded states, with added polymer even the majority of the more complicated 2D time-dependent flow states show a significant throughput. Three representative snapshot states with a significant throughput are shown in Fig. 11.
To explore this further, we pick a single set of parameters ζ = 3.2, ∆ = 2 × 10 −5 representative of the regime of fully developed active turbulence in Fig. 10 (for both τ C = 0.0 and τ C = 4.0) and perform a separate series of runs across which we vary the value of the polymeric relaxation time τ C , exploring the full range from the Newtonian limit (for the polymer) τ C → 0 to the elastomeric limit τ C → ∞. (Recall that the polymer modulus G C must decrease in inverse proportion to τ C , given our fixed value of η C = G C τ C = 1; likewise ℓ 2 C must increase in proportion to τ C to keep a fixed value of ∆.) For this series of runs, we plot in Fig. 12 the rootmean-square fluid velocity v rms ≡ |v| 2 x , the mean throughput, and the mean defect density [62]. (Each such quantity is time-averaged across a run, after discarding the initial transient.) As expected, in the regime of small τ C where the polymer acts simply as an additional solvent, none of these quantities change with τ C .
In contrast, once τ C > τ Q the active stress has to work against an increasingly elastic fluid, and v rms decreases with increasing polymer relaxation time. Indeed, for τ C → ∞ the polymer effectively arrests the flow altogether. (This holds only for the final behavior, after discarding the initial transient; we show below that this transient can in fact be long-lived, and have its own rich dynamics.) Despite this monotonic decrease of the root-meansquare fluid velocity, the mean throughput has a nonmonotonic dependence on τ C . For small τ C , the throughput is essentially zero, consistent with the model's limiting behavior in the absence of polymer discussed above. As τ C increases to become comparable with τ Q the throughput increases dramatically, suggesting a more coherent flow state capable of sustaining a net flow in one direction. (Indeed, not only does the mean throughput increase but the fluctuations of the throughput about this mean decrease.) Finally, for large τ C the throughput again decreases as the overall flow arrests.
This gradual transition with increasing τ C to a state of significant throughput, followed by arrest, suggests a progressive increase in the spatial coherence of the flow with increasing τ C . Consistent with this, we see in Fig. 12b that the defect density n in the nematic's director field markedly falls with increasing τ C , with the onset of this decline roughly coinciding with the peak in throughput. A strong correlation between v rms and defect density has been noted previously in Refs. [45,61,63].
We have demonstrated, then, that adding polymer to a fluid showing fully developed active turbulence calms the short scale structure of the spontaneous active flow, decreasing the nematic defect density, and thereby increasing the flow correlation length to give a more organized flow state, often with a sustained net throughput [20,64] For a passive Newtonian fluid displaying conventional inertial turbulence in a pressure-driven pipe flow at high Reynolds number [65], the addition of a small amount of long-chain polymer is known to calm the flow and reduce the ratio of imposed pressure gradient to throughput [66], in an effect known as 'polymer drag reduction'. The phenomenon just described for our active fluid, whereby (zero Reynolds number) active turbulence is calmed by the addition of polymer is strongly reminiscent of that effect, and we term it 'active drag reduction' accordingly. The persistent, coherent motion to which it leads may have a biophysical analogue in cytoplasmic streaming, where it is believed that active cytoskeletal materials such as actomyosin play a role in generating coherent flows, facilitating the transport of nutrients and organelles within the cell [26,27].
Transient dynamics: extensional catastrophe
In the previous subsection we discussed the long-time behavior of viscoelastic active matter in 2D, ignoring the initial transient en route to the ultimate attractor of the dynamics. We now turn to consider that initial transient.
Again we pick a single set of parameters (ζ = 2.2, ∆ = 4 × 10 −5 ), representative of the regime of fully developed active turbulence in the phase diagram of Fig. 10. We then perform a series of runs, varying the polymer relaxation time τ C across the full range from τ C → 0 to the elastomeric limit τ C → ∞. We again fix both the overall polymer viscosity (η C = 1) and the diffusivity ∆.
In each run we initialise the system as usual in a state that is homogeneous and quiescent, with the polymer undeformed, C = I. For the given parameters we expect perturbations to grow exponentially until nonlinear effects become important; this is confirmed in the early time growth dynamics in Fig. 13. (Note that the time Fig. 13 don't appear chaotic until the turbulent state becomes fully developed, i.e., at later times in the run.)
signals in
The degree to which the polymer becomes deformed by this instability, as measured by the growth in Tr(C − I), increases with increasing τ C . This is to be expected: polymeric strains build up at a rate set by the velocity gradients in the fluid, and relax at a rate set by 1/τ C . Indeed a polymer obeying the Oldroyd B equation will, if subject to an indefinitely sustained simple shear flow of rateγ, acquire a shear strainγτ C ; and, if subject to an indefinitely sustained extensional flow of rateǫ, acquire an extensional strainǫτ C /(1 − 2ǫτ C ) [67]. The latter is an infamous result, predicting the polymers to suffer unbounded deformation for any sustained extensional flow of rateǫ > 1/2τ C . (In reality this growth would ultimately be cut off by finite chain extensibility or chain rupture, neither of which is captured by the Oldroyd-B model.) Although the actively turbulent flow states that develop here do not subject any given fluid element to an indefinitely sustained extensional flow, they do nonetheless include regions with significant extension rates. To characterise these, we define a local extensional Deborah number De = τ C 1 2 (D : D), and take its spatial average De . In view of our above discussion of the Oldroyd B model's extensional catastrophe, we might then reasonably expect a dramatic growth of polymer strain in any regime where De > De c ≈ 1 2 .
In confirmation of this, the time-evolution of De is plotted in Fig. 13a for each in our series of runs. For small τ C (warm colors), De remains small and the spatially average polymer strain Tr[C − I] likewise remains modest. In contrast, for larger τ C (cold colors) De exceeds De c ≈ 1 2 leading to a dramatic effect on the polymer conformation: following the initial linear instability, we observe a second period of exponential chain stretching that generates huge polymer strains. See Fig. 13b.
Any growth in the polymer strain gives a corresponding growth in the polymeric contribution Σ C = G C (C − I) to the stress. Recalling that G C = 1/τ C for this series of runs, the large values of τ C for which dramatic chain stretching occurs have correspondingly small values of G C . Accordingly, as long as the polymer strain remains O(1), the polymeric contribution to the stress will remain small. In contrast, in the regime of dramatic chain stretch the polymer strains become huge and the polymer stress does then become comparable with the nematic and Newtonian ones, despite the small polymer modulus G C . This in turn feeds back on the flow field, mitigating the extensional flows and halting the divergence of the polymer stress.
Limit cycle
In the previous two subsections we considered state points (ζ, ∆) deep in the active-turbulent region of the phase diagram in Fig. 10 (which has τ C = 4.0). While the transient dynamics of corresponding runs at much larger τ C revealed that polymer deformation proliferates due to the appreciable extensional component of the turbulent activity-driven flow field, it is unclear how this phenomenon manifests in the long time dynamics. Therefore we consider a point (ζ, ∆) in Fig. 10 with a more moderate activity (which is less deeply turbulent) and again perform a corresponding run at a much larger τ C = 1000 (once again at fixed η C = 1), where we now run the simulation until long times t max ∼ O(10τ C ).
The results are shown in Fig. 14. In this case the system ultimately settles to an oscillatory state whose period is set by the polymer relaxation time τ C . Over the course of one cycle, the system switches from a quasi-1D banded state dominated by bending in the director field (see the snapshot at time t 1 ), to a different quasi-1D banded state dominated by splay (at time t 2 ), via a fully 2D intermediate state with velocity rolls. A representative snapshot of such a 2D intermediate state is shown at time t 3 .
Closer study of the full run reveals that the transition from bend to splay initiates via the director, at a single value of x midway between the plates, rotating by π/2. This disturbance then propagates along the interface until the whole system is splayed. (Destabilisation of the bend state to form the splay state has been seen previously in Ref. [68].) The splay-banded state then apparently remains stable for a time of order τ C , before developing a roll-like instability around t 3 = 4400 char-acterised by alternative pairs of ± 1 2 topological defects [55] in the nematic director field.
The degree of inhomogeneity during these transitions can be monitored quantitatively through the power spectrum P (k x , t) of the spatial Fourier transform of the nonzero order parameters φ = (Q xx , Q xy , Q yy , C xx , C xy , C yy ): Any purely 1D state would have P (k x , t) = 0 for k x > 0. As can be seen, the quasi-1D bend and splay states indeed have small P . The 2D roll-like states that arise during the transition between these quasi-1D states have, in contrast, a much larger P = O(1). See Fig. 14a. This oscillating effective dimensionality of the flow state directly influences the polymeric stretching, which we monitor as before with a scalar extension measure Tr[C − I] (see Fig. 14b). The constraint of fluid incompressibility means that an extensional component to the flow field can only arise in the 2D states. Because of this, during times when the system occupies a quasi-1D bend or splay state the polymer extension relaxes on a timescale τ C . In contrast, the intervening 2D roll state contains regions of extensional flow leading to significant polymer stretching.
It is worth noting that the nonlinear oscillatory state reported here would not have been predicted from our 1D linear stability analysis, for which the corresponding linear instability is not oscillatory. However a 2D linear stability calculation that linearises about one of the inhomogeneous banded states (shown in Fig. 14) may well predict this behavior. Note that oscillatory dynamics in active systems is widely seen in a biophysical context. For example, shape oscillations in developing cells are believed to be driven by actomyosin networks [69], which have been theoretically described using an elastic model [58].
B. Elastomeric active matter
In the previous subsection we saw that adding a polymer of even modest relaxation time has a dramatic effect on the phase behavior of confined active nematics. For example, it gives rise to an unusual drag reduction effect with strong enhancements in the flow field's coherence and net throughput, for a fixed level of activity. We also explored the effect of changing the polymeric relaxation time τ C , moving towards the elastomeric limit of large τ C . We did so at a fixed overall polymer viscosity η C = 1, with G C decreasing in inverse proportion to τ C .
In this section we consider the "true" elastomeric limit, in which τ C → ∞ is taken at finite G C so that the polymeric viscosity diverges. Physically, this could be realised by increasing the crosslink density and/or chain length in an entangled polymer of fixed concentration: our model might then describe an actomyosin cell extract in a background of lightly cross-linked polymer gel. To implement this limit in our simulations we simply remove the local relaxation term prefactored by 1/τ C in the polymer equation of motion. As mentioned already for the 1D case in Sec. V B, we do however retain finite stress diffusivity, ∆ C = ℓ 2 C /τ C , meaning that ℓ C → ∞ as the elastomeric limit is taken. We discuss this choice further at the end of this subsection.
The predictions of our linear instability analysis in this limit were shown in Fig. 7. We now explore the instability more fully by performing nonlinear 2D simulations, at infinite τ C , for a range of polymer moduli G C = 10 −8 → 10 −1 . We choose values of the activity (ζ = 3.2) and diffusivity (∆ = 8 × 10 −5 ) for which the initially homogeneous state is predicted to be unstable. In each run, we find that the initial homogeneous state indeed destabilises to form a heterogeneous, complex liquid crystalline texture with a high density n of defects in the director field; see Fig. 15a and Fig. 16 (top). Associated with this buildup of defects is a complex 'elastically turbulent' deformation field with regions of (transient) extensional flow, which result in exponential stretching of the polymer; see Fig. 15b. Because in these runs the polymer modulus obeys G C < G Q , appreciable polymer strain can develop (inset of Fig. 15b) during this early time regime before the resulting polymeric contribution to the stress becomes comparable with solvent and nematic stresses, which are O(1) in our units. (A comparable phenomenon is seen in some models of polymeric glasses [70].) Once the polymer stress does become comparable with the other stresses, the 'elastic turbulence' arrests into a complex but almost frozen defect pattern. Thereafter the defect density shown in Fig. 15a slowly coarsens over time, roughly as t −1 , which is the same as the classical result for coarsening in a passive nematic [71]. See Fig. 16 for snapshots of the nematic texture during this process. Although a defect-free state n → 0 might arise in the true steady state limit t → ∞, the coarsening process that leads to it is sufficiently slow that the strain pattern created by the arrested active turbulence might easily be mistaken for a final steady state. Note that the mean polymer extension continues to grow in this coarsening regime, albeit slowly (roughly logarithmically at intermediate times) with some suggestion of eventual saturation seen in the numerics. This elastomeric arrest of the active turbulent flow field, where strong polymer stretching in extensional flow regions creates strong stresses that oppose the flow, is mechanistically reminiscent of the drag reduction effects reported earlier. Note that the quasihomogeneous state that develops at long times is drastically different from the base state in our earlier linear stability calculation (which assumes that the polymer is undeformed), and is therefore not subject to the same linear instability as found there.
We now return to an issue raised previously in this section and earlier in Sec. V B, which is our choice to retain finite polymer stress diffusivity ∆ C = ∆ > 0 even in the elastomeric limit of τ C → ∞. Since ∆ C = l 2 C /τ C , this requires matching divergence of the length l C . (The physical meaning of this length is ambiguous, but it is often assumed to be of order the polymer coil size.) Our reason for this choice is ultimately pragmatic: we have found that our 2D numerical simulations become highly unstable, at the discretization scale, if performed with ∆ C = 0. A credible explanation of this behavior is as follows. We have seen that polymers encounter regions of strong elongation in 2D active flows. These convert 'globular' initial stress fluctuations into 'threadlike' ones, creating fine structure in the transverse direction as set by the local compression axis. Once elongation is strong, numerical instability is inevitable unless there is a restoring term to iron out these short-scale fluctuations. However, in the absence of thermodynamic couplings (see next section), the only term in the polymer dynamics that can achieve this is ∆ C . Thus we retain finite ∆ C in order to capture the short lengthscale physics of local stress redistribution. This mechanism, whatever its details, should not disappear for lightly cross-linked elastomers, since their physics essentially merges with that of free chains at short lengthscales (below the cross-link separation).
Note that in an elastomer of finite ∆ C , although the mean stress caused by an applied deformation can never relax (which is the defining feature of a solid), any inhomogeneous polymer stress will relax to a uniform one eventually, via the stress diffusion term. This may seem a bit artificial, although a similar effect can arise in many elastomeric systems by other pathways. One of these, although absent from the present model, is solvent permeation flow [32]. Interestingly, one might expect that thermodynamic coupling to the Q field, whose stress diffusivity does not vanish as τ C → ∞, could also provide enough stress redistribution to stabilize the numerics. Indeed we find this to be the case, as we now discuss.
C. Explicit thermodynamic coupling
Throughout the preceding results sections we set κ = χ = 0 in Eq. 3, so that there is no direct thermodynamic coupling between Q and C. In that way, the only coupling between Q and C was purely kinematic, arising because they share a common fluid velocity v which is influenced by both sources of stress. In this subsection, we study the effects of a direct coupling.
The κ term in Eq. 3 controls how the polymer pressure shifts the isotropic-nematic transition. Our interest in this paper lies far from the transition region, deep within the nematic phase, so for simplicity we set κ = 0. The remaining coupling parameter, χ, governs the energetics of relative orientations of Q and C. For χ < 0, it is favorable for Q and C to align. Indeed experiments (in the passive limit) suggest that single semi-flexible polymers can couple to the nematic director field in this fashion [72]. However, such co-alignment tends to arise kinematically even when χ = 0, since Q and C both have similar alignment tendencies in relation to the spontaneous velocity field (at least for our choices of the flow-alignment and slip parameters ξ and a). We therefore do not expect substantially new physics to arise in this case. Therefore we focus here on antagonistic coupling χ > 0, where Q and C now prefer to orient with their major axes perpendicular. We do not perform an exhaustive search of parameter space but instead present a selection of the intriguing states observed.
Persistent elastomeric turbulence
We have shown previously (in 1D) that persistent oscillatory states can develop in the elastomeric limit τ C → ∞, provided that we keep ℓ C fixed. Our 2D simulations produce significantly more complex flow fields characterised by strong extensional components which, as previously discussed, can be numerically problematic. Without any polymer diffusion we found that our numerics required impractically severe convergence criteria; with polymer diffusion retained (by imposing ∆ C = ℓ 2 C /τ C = ∆ Q ) the simulations are stabilised but the initial "turbulent solid" state slowly coarsens away.
However we might anticipate a similar stabilisation to result also from a small thermodynamic coupling between C and Q, the latter of which retains spatial gradients even in the limit τ C → ∞. Indeed, with this coupling we have successfully performed runs at zero polymer diffusivity, i.e., ∆ C = 0, albeit using a significantly smaller space-and time-steps [73]. Intriguingly, we find that the transient elastomeric turbulence discussed previously now continues indefinitely, producing an unusual oscillatory state. This can be readily visualised by plotting the paths of massless tracer particles as they are advected by the flow, as shown in Fig. 17. Without polymer present, these tracers perform a random walk as they are advected by the turbulent flow. However in the elastomeric limit, the tracer trajectories remain confined to periodic orbits for the duration of the simulation. In this limit, the activity-driven, turbulent flow field stretches and distorts the elastic polymer background which, at large enough strains, reacts by producing a restoring force that "undoes" any displacement, eventually returning displaced material points to their initial location (or nearby). We also plot the defect density n, throughput Ψ and rms velocity in Fig. 18. These all apparently reach quasi-steady state values after a short time, and show no signs of the coarsening discussed previously for ∆ C = 0 in Fig. 15. Also note that the throughput oscillates about zero, consistent with our expectation that an elastic solid should undergo no net displacement. The severe numerical requirements when ∆ C = 0 restrict our discussion to the qualitative aspects of the state; a more sophisticated numerical implementation would be required to fully quantify the elastomeric turbulence seen here.
Shear Bands with interfacial defects
For the remaining examples we restore ∆ C = ∆ Q = ∆ and fix a finite value of τ C comparable to τ Q . The first of these (which has χ = 0.002) demonstrates an intriguing, polymer-driven disorder-order transition. Choosing parameters characteristic of the chaotic state (when χ = 0), at early stages of the run we find the defect-rich disordered state observed without polymer (Fig. 19c). However as the simulation progresses, ordered regions of nearly uniform directorn spread in from the walls towards the centre of the channel, forming an increasingly shear-banded like state (Fig. 19d). Eventually, the only remaining evidence of the earlier chaotic state are pairs of defects embedded in the interface (Fig. 19e).
This transition can be seen quantitatively by examining the power spectrum P (k x , t) in Fig. 19a. During the initial chaotic phase, the first 20 wavevectors (plotted) contribute significantly to P (k x , t), indicating significant spatial structure in the x-direction, with dynamics aperiodic in time. At long times, once the banded state forms, all amplitudes P (k x , t) are attenuated, particularly at large k x . This (admittedly extreme) example is consistent with our drag reduction argument, whereby polymer calms short-scale structure. Snapshots of the evolution of this state in Fig. 19c/d/e demonstrate a clear transition from a disordered to an almost ordered state.
Correlated with this suppression of short scale structure is a dramatic increase in the throughput (Fig. 19b). While the chaotic state at early times has zero mean throughput, the latter banded state develops a strong net flow in a spontaneously chosen direction, in this example towards the right.
Interestingly, during the intermediate phase between chaotic and banded states, we occasionally find transient, rotating spiral structures, which when viewed macroscopically [74] possess an integer topological charge +1. (See Fig. 19d.) Such structures are commonly found in polar active materials [60,[75][76][77], but not in apolar nematics which much more commonly display ± 1 2 defects [4], as were found above. In general, integer defects in passive nematics tend to dissociate [55], a tendency maintained in the active case without polymer. (One exception is in highly confined cylindrical geometries, as studied in Ref. [26] where the authors found a single +1 defect at low activities, which split into a pair of + 1 2 defects only at higher activity values.) It appears that antagonistic coupling can help promote integer defects, but the detailed mechanism for this remains unclear.
Shuffling state
By increasing the activity with other parameters fixed, we can disrupt the above shear-banded state. The result, Fig. 20 (upper snapshot), is a state that shuffles back and forth as a whole, with defects travelling along regions in which q, the largest eigenvalue of Q, is small. This mechanism is similar to that reported in Ref. [68] (without polymer), where defect motion in 'walls' (regions of high local distortion) was observed. The throughput time series in Fig. 20 reveals that the flow switches direction periodically on a timescale of order the viscoelastic relaxation timescale τ C , once again confirming the direct influence of polymer on the dynamics.
Order-disorder coexistence
With a larger coupling constant (χ = 0.004), even more exotic states can develop. In Fig. 20 (lower snapshot) we show a state exhibiting coexistence between chaotic/oscillatory regions (where the director lies in the xy plane shown) and pseudo-quiescent regions (where the director points along z). The active regions travel back and forth, trapped between the bounding walls. Repeating the simulations a number of times with different seeds for the 2D perturbation, we find that these pseudoquiescent regions sometimes grow to envelop the whole system, which then remains quiescent indefinitely. Such quiescent regions are likely made possible by our assumption of translational invariance along the vorticity axis z. (Recall that in our simulations we consider 3D order parameter tensors Q and C residing in a 2D space). It may be that fully 3D simulations are required to fully resolve the dynamical behavior of this state, which could easily then show structure also in the z direction. We leave such 3D investigations to future work.
VII. CONCLUSIONS
We have studied in depth the linear stability and nonlinear dynamics of the minimally coupled model for viscoelastic active matter derived in Ref. [20]. While treating 3D structural tensors for both the active nematic and the polymer sectors, our studies assumed either 2D flow states (translationally invariant along the vorticity axis z) or 1D states (invariant also along the periodic axis x).
For appreciable activities, the phase diagram without polymer is dominated by chaotic states with no net throughput [5]. Adding polymer results in a 'active dragreduction effect' whereby the short scale flow structure is calmed, resulting in a reduced density of nematic point defects. The increased flow correlation length is associated with net material transport around the periodic boundary conditions of our simulation. In fully confined geometries this would likely also help promote long-lived steady circulation as opposed to a mixing flow [27].
While adding polymer can delay the onset of spontaneous flow, our results predict a critical activity which does not diverge in the elastomeric limit τ C → ∞, and indeed vanishes for large enough systems just as it does without polymer [10]. Thus activity-driven flows can occur within active materials that are ultimately solid: these might be called "true active gels". Numerical simulation of such materials in 1D indeed reveals oscillatory shear-banded states where the flow direction switches periodically as the polymer stress is loaded. A range of complex states involving nontrivial interplay of viscoelasticity and activity were seen in systems of large but finite polymer relaxation time τ C .
As the relaxation time of the polymer was increased, the effect of activity-driven extensional flows was shown to be important. When the spatially averaged Deborah number (which describes the ratio of polymer and extensional timescales) exceeds a critical value, we found that the initial linear instability is followed by a period of rapid, exponential deformation of the polymer. For sufficiently large τ C , this resulted in oscillatory states which cycled between rapid extensional deformation of the polymer and slow stress relaxation on a timescale τ C .
In the elastomeric limit, τ C → ∞ the ultimate fate of the oscillatory and/or turbulent states that we discovered remains sensitive to numerical details of the model. Retention of finite stress diffusivity (which greatly improve numerical stability) in this limit causes ultimate relaxation to a state of uniform stress; this precludes any permanent state of oscillation or elastic turbulence. However, numerically we were able to switch off the stress diffusivity once thermodynamic coupling between nematic and polymeric degrees of freedom was included. In this case we found that the system could be truly a "turbu-lent solid" even in steady state. That is, the coupled system shows chaotic, activity-induced velocity fields that persist indefinitely but that reverse sign often enough locally that the elastic strain remains bounded, as a solid requires.
More generally, we found a wide range of complex and interesting spontaneous flow states when Q and C are antagonistically coupled at the free energy level. Depending on the strength of the coupling, our simulations show that polymer can drive a transition from active turbulence to near-laminar banded flow, result in states with oscillatory dynamics on a timescale τ C , and create regions where director is oriented out of the plane. Fully 3D simulations should provide useful insight, particularly for the last of these.
Our prediction of active turbulence in soft solid materials (τ Q → ∞) arises when G C /G Q 0.1. This looks experimentally feasible for subcellular active matter (though probably not swarms of bacteria) within a lightly cross-linked polymer gel. The symmetry breaking characterised by net throughputs, which we observe at moderate polymer relaxation times (τ C /τ Q ≈ 10), could have particular relevance for studies of cytoplasmic streaming, where coherent flow is crucial for material transport within the cell. The same physics may also apply, albeit at larger scales, to cell migration in confined geometries [29]. Our results with explicit coupling that show oscillatory, 'shuffling' states may also be relevant in describing the cell shape oscillations of Ref. [57].
We hope our work will promote experiments on these and other forms of active viscoelastic matter; while the effects of viscoelasticity on individual swimmers have been studied previously [78,79], we are not aware of an equivalent study for bulk, orientationally ordered phases. One related study did however consider the linear stability of the bulk isotropic phase in an Oldroyd-B fluid [18]. While the specifics of that model differ from the one presented here [80], both studies find (i) that increasing η C at fixed τ C will always eventually suppress the spontaneous flow instability, (ii) that at small τ C the polymer simply renormalises the solvent viscosity (this is our viscous limit) and (iii) a region at large τ C where unstable oscillatory behaviour is predicted (this is our elastomeric limit). which is independent of x because of fluid incompressibility. Because this quantity generally exhibits significant fluctuations in time, particularly in the chaotic regime, we define our criterion for significant or 'net' throughput as being when the mean of the throughput time-series µ Ψ exceeds the standard deviation σ Ψ : and which converge to constant values as t → ∞. Note that under this definition, states that have non-zero mean throughput will fail the criterion if this mean is less than the standard deviation of the time-series. In practice, we find that the flow direction can intermittently switch. See, for example, Fig. 21 left. If naively averaged as above, this would clearly produce zero mean throughput, at least in the limit t → ∞. Therefore we instead perform a least-squares fit, fitting the throughput histogram with two Gaussians of width σ Ψ , centred at ±µ Ψ . We have explicitly checked that our results are robust to the number of histogram bins used. Examples of both throughput and non-throughput states are shown in Fig. 21. We mark states satisfying criterion Eq. A.2 in the phase diagrams with solid symbols, and those failing it with empty symbols. | 2016-03-29T11:29:44.000Z | 2015-12-14T00:00:00.000 | {
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236405646 | pes2o/s2orc | v3-fos-license | An Analysis of the Students’ Difficulties in TOEFL Prediction Test of Listening Section
This study is to know the students’ difficulty in doing the TOEFL prediction test, especially the listening section in ITB AAS Indonesia. This study uses descriptive qualitative research by using a questionnaire. The result shows that the students’ difficulty in doing TOEFL listening both external and internal factors. In external factors, include: 1) the speaker's accent (75.5 %), 2) the speakers’ speed (75.9 %), 3) the speakers’ intonation/emphasis (73 %), 4) the speakers’ pause in pronouncing a sentence (70.3 %), 5) the choice of words and foreign terms conveyed by the speaker (71 %), 6) the sentence structure conveyed by the speaker is too complex (54.8 %), 7) audio interruption causes the audio sounds less/unclear (54.8 %). In internal factors, include: 1) 64.3 % of students do not have previous experience doing TOEFL test, 2) 58.5 % of students have lack of practice in TOEFL listening, 3) 78.8 % of students have limited time in doing TOEFL listening test, 4) 62.2 % of students feel a lot of listening questions which consist of 50 questions, 5) 76.2 % of students do not have hearing impairment in listening, 6) 51 % of students have memory limitations when listening to TOEFL, 7) 48.1 % of students lack of motivation and enthusiasm, 8) 52.7 % of students lack of concentration or focus, 9) 53.5 % of students have limited mastery of foreign/unfamiliar vocabularies, 10) 47.3 % of students feel boredom when listening, 11) 56 % of students feel easily distracted by sounds or other things, 12) 51 % of students tend to translate any foreign vocabularies when listening, 13) 52.7 % of students have trouble catching or finding keywords, 14) 44.4 % of students are busy along with other activities when listening, such as playing writing instruments, taking notes or doing other things.
INTRODUCTION
TOEFL stands for Test of English as a Foreign Language. It is a standardized academic English test primarily taken by students applying to universities in the United States. The TOEFL is also accepted as proof of English skill by some universities outside the United States. More than seven thousand colleges and universities in one hundred and thirty countries, including the best universities in the world, such as the U.S., Canada, UK, Australia, and New Zealand. This test is not only used to register at the university but is also required if you want to work in government agencies, take care of licensing, companies, and also for scholarship registration. Setiawan (2013) states that In many nations, TOEFL was used as a criterion for university admission or for obtaining a scholarship. Several Indonesian universities need the TOEFL examination as part of their graduation criteria (Soali & Pujiani, 2020). TOEFL also becomes one of the standard requirements to enter Indonesian universities (Rahmah, 2019).
TOEFL examines language skills that are tailored to the academic and professional fields. These fields are packaged and divided into sections. TOEFL test usually consists of three sections with 140 questions. TOEFL includes three aspects, namely (1) Listening and missing detailed information, lack of concentration, lack of understanding of English accents, interpret word for word, lack of knowledge of vocabulary and pronunciation, unable to distinguish certain words and sounds. Analyzing students' difficulties toward listening comprehension helped the students in listening test as well as TOEFL test (Darti & Asmawati, 2017).
The listening comprehension section aims to test our abilities in understanding spoken language. However, to be able to understand spoken language in addition to having to get used to listening to English spoken, we must know English grammar (grammar) adequately. All sentences in this section use the same sentence grammatical (grammatically correct) and usually in the form of a complete sentence. All these sentences are pronounced in conversational English. According to Ang-zie (2020, p. 10), to answer listening comprehension questions well, we are required to have the skill to understand the stress and tone, understand the difference in sound, understand idioms, understand conversational phrases, understanding groups or verb phrases, find implied information (not clearly stated), understand comparisons, and understand the meaning of the conversation or conversation. The listening section is divided into separately timed parts. They are short conversations, long conversations, and talks/monologues. In each part, we will listen to the audio-only one time (Putlack et al., 2020). Kasmini & Kadarmo (2014, p. 12) states that all questions in the listening section will usually be played through a headset on the computer or tape using speakers (loudspeakers) that have been prepared for the TOEFL test. Speakers in the tape using American English pronunciations and expressions whose speech and expression are somewhat different from British English (British English). We need to understand the difference especially the way it's pronounced.
Some previous studies were conducted related to TOEFL listening comprehension. In the first research entitled "Investigating Medical Students' Difficulties in TOEFL Listening Test" written by (Rahmi et al., 2020). This research examined the reactions of the students to listening problems in taking the TOEFL test listening portion and their attempts to resolve the difficulties. Most respondents were found to have difficulties recognizing the pronunciation of the native speaker (64%). As the top rank of students' attempts to solve listening difficulties of the TOEFL exam when viewing English TV programs or movies (65.10 percent) and listening to English songs (60 percent). In the second research entitled "Students Difficulties in Passing Listening Section in TOEFL Prediction Test" written by (Chairuddin & Ulfa, 2018).
This research is to recognize difficulties for learners in passing the TOEFL prediction test listening portion. It is found that there are many facets of student issues, and the most difficult items are: 1) capture the idea of the speaker, 2) recognize the idiomatic language, 3) define the coherent marker, 4) give the utterance a literal meaning, 5) retain the important details. The third research entitled "An Analysis of the Difficulties Encountered by Non-English Department Students in TOEFL Test of Listening Section" written by (Pratiwi, 2017). This research was largely aimed at exploring the difficulties of students listening to the TOEFL exam. The outcome of this study reveals that in the listening portion of the TOEFL exam, the recapitulation score of the students was weak, by percentage 60 percent of students include 9 students of fifteen participants are very low understanding, and 40 percent of students include 6 students are low understanding. This suggests that the students are not comfortable with the TOEFL exam. In the fourth research entitled "Analyzing Students' TOEFL Listening Comprehension Test Performance" written by (Yuliandani et al., 2014). The goal of this analysis is to figure out the most challenging part of the TOEFL listening interpretation and to find out the problematic aspects. Double negative expressions (23 percent) are the most problematic aspect, while the most perfected aspect is passive (77 percent). The most troublesome aspect in discussions and talks is the subject matter and accurate knowledge is the mastered aspect. In the fifth research entitled "Students' Difficulties and Strategies in Doing Listening Section On TOEFL-Like Test" written by (Pujiana, 2015). This analysis is to discover the problems encountered by the students and the approach they used in the TOEFL test listening segment. The effect is that the listening section is not simple for them and that part C (lecture) is the most difficult part of the listening section. The result reveals that students interpret attention, keywords, time, and emphasis as issues. The students usually used some techniques to solve the challenge.
From five previous studies, those have similarities and differences between these studies. Those studies have a similar focus on TOEFL listening comprehension and its difficulties. But there are differences between those previous studies and this study is the objective. Previous studies above focus on the question items related to listening comprehensions such as the pronunciation of the native speaker, the idea of the speaker, idiomatic language, coherent marker, literal meaning, important details, double negative expressions, passive form, attention, keywords, time, and emphasis as issues. It shows only in the external factor. While this research focuses on the difficulties in doing the TOEFL listening test based on internal and external factors related to it.
Based on the explanation above, the researcher is interested to research the TOEFL Prediction Test of Reading Comprehension. Finally, the researcher takes the title "The Students' Opinion Toward TOEFL Prediction Test of Reading Comprehension Skill". The researcher formulates the objective of this study is to know the students' difficulty in doing the TOEFL prediction test of listening comprehension skill in ITB AAS Indonesia.
THEORETICAL FRAMEWORK
In TOEFL, the listening comprehension section is to measure the skill to understand spoken English. It contains listening passages and questions about each passage (Gear & Gear, 2002, p. 73). Listening questions are divided into 3 parts, namely Part A, Part B, and Part C.
Part A consists of 30 short conversations between two people, each of which will end with one question that you must answer in the correct answer choices (A, B, C, D). This part of Part A consists of 30 questions. Part B consists of two longer conversations between two people and each conversation is followed by several questions. Generally, the part in Part B consists of seven to eight questions. In part, C consists of 3 long speeches (talks) and only involves one person (monologue). And each speech will be followed by a few questions. Generally for Part C, this consists of seven to eleven questions.
In each of these sections, there will be directions or directions for working on the questions that will be read out by native speakers in audio. The words spoken by native speakers in this direction are almost the same in every TOEFL test. So, it's good if we already know and are familiar with the direction before taking the TOEFL test so that when the direction is being read, we can use this opportunity to read as many answer choices as possible and guess about what questions for the answer choices.
In this section or session, our listening skills will be thoroughly tested. This section is used to test the test participant's ability to listen to a speech or spoken language in English. The participants are expected to be able to listen to every conversation well that comes from a tape recorder or other media. Listening Comprehension on the TOEFL test is always a frightening specter for test-takers, not infrequently many participants fail to pass the test in this session. The time to answer each question in the listening comprehension is very short. We only have no more than 8 seconds per problem. For listening questions, it is very important to read all the answer choices.
RESEARCH METHODOLOGY
This study used descriptive qualitative research. Qualitative research is defined as a study that focuses on understanding in the naturalistic setting or everyday life, or of a certain phenomenon or person (Neergaard & Ulhøi, 2007, p. 383). They were studies that include the context in which the study's phenomenon is embedded. Taylor et al. (2015, p. 8) stated that qualitative researchers developed concepts, ideas, thoughts, and understanding from patterns in the data rather than collecting data to analyze models, hypotheses, or theories.
The method of collecting data in this study used a questionnaire. Data collection techniques were the most important step in research because the main purpose of the research was to get the data. McNabb (2004, p. 109) stated that a questionnaire could be used to gather information about a large number of respondents (populations) or small groups (samples). In this research, the respondents were fifth-semester students of ITB AAS Indonesia. The researcher uses a closed question. It required the respondent to choose from several predetermined responses (Andrew et al., 2011, p. 82). In this research, the researcher used media for sharing questionnaires by using Google Forms. Google forms provided a simple interface to construct interactive forms that contain a variety of question types (multiple-choice, short answer, long answer, dropdown, etc. (Talbert, 2017). Here, the questions were in the form of multiple-choice with four answer options A, B, C, or D.
In this study, the researcher used the technique of data analysis based on Miles and Huberman (1994) which is involving three steps: data reduction, data display, and conclusion drawing/verification. The results of questionnaire data processing were often presented in descriptive form.
FINDINGS AND DISCUSSION FINDINGS
The objective of this study is to know the students' difficulty in doing the TOEFL prediction test of listening comprehension skills. Two factors cause the students are difficulty in doing TOEFL listening in both external and internal factors. Below the findings of the research as follow: The first statement about "The speaker's accent affects your TOEFL Listening skill". There are 182 students or 75.5 % who said 'yes, 56 students or 23.2 % said 'sometimes, and only 3 students or 1.2 % said 'no'. Based on those answers, it shows that most students feel that the speaker's accent affects their TOEFL Listening skill". The second statement about "Speakers' speed affects your TOEFL Listening skill". There are 183 students or 75.9 % who said 'yes, 52 students or 21.6 % said 'sometimes, and only 6 students or 2.5 % said 'no'. Based on those answers, it shows that most students feel that the speakers' speed affects their TOEFL Listening skills. The third statement about "Speakers' intonation/emphasis affects your TOEFL Listening skill". There are 176 students or 73 % who said 'yes, 59 students or 24.5 % said 'sometimes, and only 6 students or 2.5 % said 'no'. Based on those answers, it shows that most students feel that the speakers' intonation/emphasis affects their TOEFL Listening skills. The fourth statement about "Speakers' pause in pronouncing a sentence affects your TOEFL Listening skill". There are 144 students or 59.8 % who said 'yes' 73 students or 70.3 % said sometimes only 24 students or 10 % said 'no'. Based on those answers, it shows that most students feel that the speakers' pause in pronouncing a sentence affects their TOEFL Listening skills. The fifth statement about "The choice of words (diction) and foreign terms conveyed by the speaker affects your TOEFL listening skill". There are 171 students or 71 % said yes, 60 students or 24.9 % said 'sometimes only 10 students or 4.1 % said 'no'. Based on those answers, it shows that most students feel that the choice of words (diction) and foreign terms conveyed by the speaker affects their TOEFL listening skills. The sixth statement about "The sentence structure conveyed by the speaker is too complex then affects your TOEFL Listening skill". There are 132 students or 54.8 % who said 'yes', 95 students, or 39.4 % said 'sometimes only 14 students or 5.8 % said 'no'. Based on those answers, it shows that most students feel that the sentence structure conveyed by the speaker is too complex then affects their TOEFL Listening skills. The seventh statement about "There is audio interruption so that the audio sounds less/unclear in Listening TOEFL." There are 132 students or 54.8 % said 'yes', 72 students or 29.8 % said 'sometimes', and 37 students or 15.4 % said 'no'. Based on those answers, it shows that most students feel that audio interruption makes the audio sounds less/unclear in Listening TOEFL". Form the table above shows that some internal factors caused difficulty in doing the TOEFL prediction test of the listening section. The 1 st statement about "Have previous experience in doing TOEFL test before". There are 86 students or 35.7 % who said 'yes' and 155 students or 64.3 % said 'no'. Based on those answers, it shows that most students do not have previous experience in listening to the TOEFL. The 2 nd statement about "Lack of practice in listening to TOEFL". There are 100 students or 41.5 % who said 'yes' and 141 students or 58.5 % said 'no'. Based on those answers, it shows that most students do not lack practice in listening to TOEFL before. The 3 rd statement about "Limited time in doing TOEFL listening test". There are 190 students or 78.8 % who said 'yes' and 51 students or 21.2 % said 'no'. Based on those answers, it shows that most students have limited time in doing the TOEFL listening test.
The 4 th statement about "A lot of listening questions which consist of 50 questions". There are 150 students or 62.2 % who said 'many', 90 students or 37.3 % said 'enough', and 51 students or 21.2 % said 'less'. Based on those answers, it shows that most students feel that many listening questions because it consists of 50 questions. The 5 th statement about "Have hearing impairment in listening to TOEFL". There are 45 students or 18.7 % who said 'yes', 31 students or 12.9 % said 'sometimes' and 162 students or 76.2 % said 'no'. Based on those answers, it shows that most students do not have hearing impairment in listening to TOEFL. The 6 th statement about "Have memory limitations when listening to TOEFL". There are 123 students or 51 % who said 'yes', 87 students or 36.1 % said 'sometimes' and 31 students or 12.9 % said 'no'. Based on those answers, it shows that most students feel that they have memory limitations when listening to TOEFL. The 7 th statement about "Lack of motivation and enthusiasm when listening to TOEFL". There are 51 students or 21.2 % who said 'yes', 116 students, or 48.1 % said 'sometimes' and 74 students, or 30.7 % said 'no'. Based on those answers, it shows that most students sometimes feel a lack of motivation and enthusiasm when listening to TOEFL. The 8 th statement about "Lack of concentration or focus when listening to TOEFL". There are 82 students or 34 % who said 'yes', 127 students or 52.7 % said 'sometimes' and 32 students or 13.3 % said 'no'. Based on those answers, it shows that most students sometimes feel a lack of concentration or focus when listening to TOEFL. The 9 th statement about "Have a limited mastery of foreign or unfamiliar vocabulary when listening to TOEFL". There are 129 students or 53.5 % who said 'yes', 103 students or 42.7 % said 'sometimes' and 9 students or 3.7 % said 'no'. Based on those answers, it shows that most students feel to have limited mastery of foreign or unfamiliar vocabulary when listening to TOEFL. The 10 th statement about "The appearance of boredom when listening to TOEFL". There are 100 students or 41.5 % who said 'yes', 114 students or 47.3 % said 'sometimes' and 27 students or 11.2 % said 'no'. Based on those answers, it shows that most students have boredom when listening to TOEFL. The 11 th statement about "Easily distracted by sounds or other things when listening to TOEFL". There are 135 students or 56 % who said 'yes', 89 students or 36.9 % said 'sometimes' and 17 students or 7.1 % said 'no'. Based on those answers, it shows that most students feel that they are easily distracted by sounds or other things when listening to TOEFL". The 12 th statement about "Tend to interpret any foreign vocabulary when listening to TOEFL". There are 123 students or 51 % who said 'yes', 105 students or 43.6 % said 'sometimes' and 13 students or 5.4 % said 'no'. Based on those answers, it shows that most students tend to interpret any foreign vocabulary when listening to TOEFL. The 13 th statement about "Have trouble catching or finding keywords when listening to TOEFL". There are 108 students or 44.8 % who said 'yes', 127 students, or 52.7 % said 'sometimes' and only 6 students, or 2.5 % said 'no'. Based on those answers, it shows that most students sometimes have trouble catching or finding keywords when listening to TOEFL. The 14 th statement about "Busy alone with other activities when listening to TOEFL, for example playing writing instruments, taking notes or doing other things". There are 38 students or 15.8 % who said 'yes', 107 students or 44.4 % said 'sometimes' and 96 students or 39.8 % said 'no'. Based on those answers, it shows that most students sometimes busy along with other activities when listening to TOEFL, for example playing writing instruments, taking notes, or doing other things.
DISCUSSION
The first session of the TOEFL test is a listening comprehension test (Listening Comprehension). This listening session generally consists of 50 questions. The students/learners will hear the recording in English and we have to respond or answer questions related to the recording. The students/learners have to listen to the recording as much as possible because The students/learners will only hear the recording once and the recorded material is not written in the test book. There are three sessions in this listening test: PART A which consists of 30 short conversations, each conversation is followed by several questions, PART B which consists of 2 long conversations followed by several questions, and PART C which consists of 3 talks/lectures (like a lecturer explaining a certain topic).
In the TOEFL test, The students/learners will meet several question sessions. One of them is a listening session. In this session, The students/learners will hear English native speakers talk, have dialogues, or even listen to short speeches or short speeches. After listening to the audio, we must answer the questions related to the audio earlier. In dealing with this listening question, several strategies are needed in doing the listening test. The listening Comprehension Section aims to test the skill to understand spoken language. However, to be able to understand the spoken language, besides having to get used to listening to spoken English, we must have a knowledge of the structure of adequate English (grammar). All sentences in this section use a grammatically correct sentence (correct according to grammar) and usually in the form of a complete sentence (Purnaning et al., 2014, p. 10). All these sentences are pronounced in conversational English.
Listening is the most difficult part of the TOEFL test for most people. For many people listen is the most difficult language skill (Sudarmono, 2018, p. 35). According to Marwan (2020), Students experience three categories of difficulties: those related to the subject, those related to the listener, and those related to the physical environment. For the structure section and reading we can anticipate by understanding grammar material and master a lot of basic vocabulary, then practice the TOEFL test. Difficulty in doing TOEFL listening questions is a fairly complex activity, meaning that there are many aspects and many factors that influence it. These factors are interrelated, in other words, that reading difficulty is influenced by one another. Students need to implement more strategies, and teachers or educators are recommended to inspire them and develop their awareness to use more strategies when taking a TOEFL (Razmalia & Gani, 2017).
Two factors cause the students are difficulty in doing TOEFL listening in both external and internal factors. In external factors, they are: 1) The speaker's accent affects your TOEFL Listening skill. 2) Speakers' speed affects your TOEFL Listening skill. 3) Speakers' intonation/emphasis affects your TOEFL Listening skill. 4) The speakers' pause in pronouncing a sentence affects your TOEFL Listening skill. 5) The choice of words (diction) and foreign terms conveyed by the speaker affects your TOEFL listening skill. 6) The sentence structure conveyed by the speaker is too complex so it affects the TOEFL Listening skill. 7) Audio interruption makes the audio sounds less/unclear when Listening TOEFL.
In internal factors, they are: 1) Do not have previous experience doing TOEFL test before. 2) Have a lack of practice in listening to TOEFL.
3) Have limited time in doing the TOEFL listening test. 4) A lot of listening questions which consist of 50 questions. 5) Have hearing impairment in listening to TOEFL. 6) Have memory limitations when listening to TOEFL. 7) Lack of motivation and enthusiasm when listening to TOEFL. 8) Lack of concentration or focus when listening to TOEFL. 9) Have limited mastery of foreign or unfamiliar vocabulary when listening. 10) The appearance of boredom when listening to TOEFL. 11) Easily distracted by sounds or other things when listening to TOEFL. 12) Tend to translate any foreign vocabulary when listening to TOEFL. 13) Have trouble catching or finding keywords when listening to TOEFL. 14) Busy alone with other activities when listening to TOEFL, for example playing writing instruments, taking notes, or doing other things.
There are a variety of instructor techniques for teaching TOEFL Listening Preparation (Khobir & Qonaatun, 2020). First, teacher strategies will help students become more confident speakers and listeners. Second, instructor tactics will help students feel more at ease when taking the TOEFL test. Third, the student's ability to understand native speakers can be improved by listening to them often. Fourth, the student should take a more involved role in taking the TOEFL. Finally, the student understands how to answer type questions on the TOEFL, especially in the listening section of the strategies.
CONCLUSION
TOEFL Listening section is arguably a little more difficult than the other section. It is not surprising that many people find it difficult to improve their English Listening skills. Unlike the other TOEFL sections, listening skills cannot be improved in just a short time. It takes stages and processes that are gradual until someone can sort out the words that are heard well. Listening is the most difficult part of the TOEFL test in most people's view. For the structure section and reading, the students/learners can anticipate by understanding grammar material and master a lot of basic vocabulary, then practice a lot do the TOEFL standard questions. Difficulty in doing TOEFL listening questions is a fairly complex activity, meaning that there are many aspects and many factors that influence it both external or internal. Internal factors include the listener's physical condition and the listener's psychological condition. The physical condition of a listener is an important factor that determines the success and quality of listening. These factors are interrelated, in other words, that reading difficulty is influenced by one another. External factors include environmental conditions (physical environment and social environment). Environmental factors have a big influence on the success of the listening process. Environmental factors in the form of the physical environment and social environment. As good listeners, The students/learners should know and understand what factors influence the listening process and try to minimize them, so that listening can run smoothly and optimally. Listening is an important activity in everyday life, not only in school but wherever we are. Be a good listener so that we can increase our knowledge and knowledge, and strengthen the brotherly relationship between humans, because one of the objectives of listening is to communicate and to get information. | 2021-07-27T00:05:21.863Z | 2021-05-28T00:00:00.000 | {
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267648787 | pes2o/s2orc | v3-fos-license | The Absurdity of the Ordinary: The Fragile Affinity Between Imagination and Materiality in the Finnish Urban Periphery
This anthropological study focuses on spatially ordered dimensions of sociocultural life in Kontula, a suburban housing estate located at the urban margins of Helsinki, Finland. With a notorious reputation since its construction in the 1960s, it has come to represent the numerous ills of contemporary urbanity, from poverty and substance abuse to failed immigration policies. Its urban transformation is explored as the entanglement of imagination and materiality, a make-believe space that privileges neither the social constructionist nor the purely materialist perspective. I study the everyday life of its inhabitants as recurring and routinized episodes, occasionally interrupted by events that disturb its embodied flow and force inhabitants to reflect upon their spatially situated practices. I argue that the everyday encounters in rapidly transforming Kontula are simultaneously experienced as absurd and ordinary, and constitute the ordering principles of its affective geography.
1
Please note that the given corresponding author address is a personal address and not an academic address.
Please check.
2
As per style, citations are not allowed in the abstract.Hence the citation "Navaro-Yashin, 2012" has been deleted from the abstract.Please check and confirm.
3
As per APA, sections should use at least two subsections or use none.In the "The Absurd Heart of Kontula: Encounters at the Open-Air Shopping Centre" section, there is only one subsection, "Everyone Has a Story."Please indicate where a second subsection could be added within "The Absurd Heart of Kontula: Encounters at the Open-Air Shopping Centre," whether "Everyone Has a Story" can be deleted (just the heading), or whether the "Everyone Has a Story" can be formatted similar to "The Absurd Heart of Kontula: Encounters at the Open-Air Shopping Centre" (i.e., two separate sections).
4
As per APA, sections should use at least two subsections or use none.In the "Ordering the Diversity" section, there is only one subsection, "The Normalization of Absurdity."Please indicate where a second subsection could be added within "Ordering the Diversity," whether "The Normalization of Absurdity" can be deleted (just the heading), or whether the "The Normalization of Absurdity" can be formatted similar to "Ordering the Diversity" (i.e., two separate sections).
Introduction
The metro map of the capital region of Finland is extremely simple.One line connects the fringes of the city of Espoo in the west with the urban peripheries of Helsinki in the east.The otherwise solitary metro line forks into two for the last easternmost stops.The 15-min passage from the city center toward the east carries a specific significance in the vernacular geography of Helsinki: According to the dominant conception, reproduced vividly in the mainstream media, most of the eastern districts of Helsinki have an aura of a whole range of social problems.The specific character of these problems is both embodied and reflected upon in the course of daily life, with various logics of inclusion and exclusion, relative to their location.Kontula, my field site, is the second to last stop on the line.
I was traveling from the city center to Kontula with Jukka 1 -a former bus driver who had retired early because of mental health problems and substance abuse-and we were only three stops away.We had been on a cinema excursion, organized by one of the active non-governmental organizations (NGOs) in Kontula.Jukka participated regularly in various kinds of voluntary work and had lived in Kontula for more than 30 years.
It's really quite rare that I leave Kontula; sometimes I go to meet my parents who live far away and occasionally my brother takes me to see a band somewhere in Helsinki.I do not really fit into the city centre.
We overheard a discussion by two women, around 50 years of age, who spoke in a very selfconfident manner: "Ok, we are now heading into the lawless east.It doesn't look like Finland anymore with all these immigrants around," the one right behind me started."I wonder what it would feel like to live here.I think I wouldn't have the guts, especially not with my children," the other added.Jukka grew visibly agitated but did not say anything and the women got off at the next stop, Itäkeskus, known for its large shopping mall.
After the metro doors closed, Jukka said: If I'd have heard those words in Kontula, I would have challenged them immediately.The East might have its share of problems but I'm tired of hearing about them.There are lots of good people around but everyone thinks that we're all junkies and criminals-why is no one telling the truth about this?I tell you, I don't understand why so many immigrants are placed in Kontula, I can't stand all the noise they make where I live, but, he struggled to find words, "I still prefer them to these idiots.Year after year I understand less what is happening in Kontula-it's all getting mixed up to the point of madness, but, still, we have to stick together over here." This vignette raises several questions.I will explore here the relational qualities of urban space: How movement in the city does not occur in a neutral grid, but is shaped in an uneven manner, through the entanglements of material, affective and sensorial realms.For Jukka, the material aspects blended with memories and immediate sensations throughout the passage.The borders between the districts did not follow the official designations but were experienced as fluid and subject to change.How is it that some urban spaces are endowed with distinct qualities that make them stand apart?In the case above, the passage toward Kontula and, on a larger scale, the gradual movement toward the east, signified a sense of homeliness for Jukka, not an entry into a lawless urban zone.At the same time, he was aware of the reputation of the area but felt that it was something he could not control, and that the two women, ignorant and coming from elsewhere, were reinforcing it.However, it was easier to find solidarity in the lifeworld Jukka found increasingly absurd and incomprehensible than to follow the mainstream views.My focus is on these ambivalent and rapidly shifting positions in everyday life: An urban space like Kontula is recognized as inferior and stigmatized, but, often, in a paradoxical manner, is also a source of pride, or even a safe haven for its marginalized inhabitants.
Jukka's encounter, based on immediate and embodied experience rather than on analytical reflection about the spatial divisions of the city, was one of the many demonstrating the affective geography of Helsinki in action.My aim here is to illustrate, supported by ethnographic data, how the deviations from what is experienced as normal and encounters with incommensurable lifeworlds have become part and parcel of the everyday neighboring practices of an urban periphery.I argue that explorations of urban futures, especially in rapidly diversifying margins, should acknowledge in a more detailed manner the embeddedness of the material and the imagined.In place like Kontula, questions of diversity and belonging do not fit into neat categorizations of class, origin, or ethnicity: They are in a state of flux, often simultaneously incomprehensible and ordinary.Everyday life revolves around "the capacity to live with difference" (Hall, 1993, p. 361).
Analytically, I will focus on two broad themes that can be summed up under the rubrics of affective geography (Navaro-Yashin, 2012) and accelerated urban change (Hall, 2012).Together, they will help us understand in a more comprehensive manner how the residents of Kontula become aware of their lifeworlds, establish a sense of belonging in a marginalized area, and invest into the everyday rituals in the surroundings experienced as increasingly absurd while, puzzlingly, perpetuating powerful associations of home.I use absurd here as a catch-all term for exaggeratedly unusual, unconventional, and unexpected characteristics, a sensation that there is too much of everything and that reality is becoming increasingly difficult to grasp.This glimpse into the current realities of the urban margins in Finland explores how the material and affective realms of urban space relate to sociocultural expressions of conviviality and confusion.
My curiosity was aroused already at the early stages of my research by the vividness with which the residents described their surroundings, commonly associated with mass-produced concrete structures and the lack of features to identify with.The gray walls of the central open-air shopping center with its dilapidated squares and the adjoining metro station had powerful affective qualities that connected with the embodied experiences, memories, and spatiality of the city.Yael Navaro-Yashin (2012) defines affective geography as "cartography, at one and the same time, of the affects of an outer environment and those of interior human selves, as they are interrelated" (p.24).In this sense, the inner and the outer merge and become indistinguishable.From a slightly different angle, emphasizing the historical formation of affective atmospheres, Laurent Berlant (2011) points out how affective responses are anchored in history and "may be said significantly to exemplify shared historical time" (p.15).Kontula's urban history is short in years but extremely dense; it is shared by its inhabitants as an incommensurable fit between the demonizing accounts of the mainstream media and the lived realities of the everyday, realized through powerful affective responses.
Methodologically, the straightforward opposition between the self and the environment, the social constructionist and new materialist approaches, can be challenged by focusing on spatiality.This perspective emphasizes the notion of make-believe in the ongoing creation of spaces, the interplay between making and believing, "a play on the notion of the de facto: something that exists, but not really; an entity that has been crafted and erected phantasmatically, that has been believed through the making or materialized in the imagining" (Navaro-Yashin, 2012, pp. 5, 28).The aim is to bring the phantasmatic and the tangible together without privileging one or the other.However, while Navaro-Yashin studies how the uncanny affective geography of Northern Cyprus haunts the normal life of people, in my argument the positions are reversed: I look at how the absurdity of the accelerated urban change becomes normalized in Kontula.In my study, the making and believing are situated in an urban setting that is plagued by notorious reputation and celebrated for its diversity.Kontula is, paradoxically, simultaneously filled with significance and associated with uniform suburban landscape, consisting of nondescript human containers and minimal municipal services.The phantasmatic and tangible are entangled in often surprising ways that result in interesting considerations for better understanding of diversity and urban futures-both in Helsinki and globally.
This study offers a critical perspective on super-diversity, a summary term for moving beyond society divided into simple ethnic groups (Vertovec, 2007), into lifeworlds with more complex classifications, some of them ephemeral and others more lasting.I follow the view that the everyday has become filled "with new emergent modes of coexistence" (Back & Sinha, 2018, p. 25) and "the everyday multiculture that emerges through the senses has outpaced both cultural theory and many city dwellers' own accounts of their lives" (Rhys- Taylor, 2013, p. 405).I argue here that ethnicity or cultural background are not the primary identifiers in the everyday encounters in Kontula.There are moments when they are foregrounded but, generally, the affective geography directs the attention toward more amorphous sense of difference, not to be reduced to crude generalizations of race or origin.The local sense of affective geography is in stark contrast with the sensationalist news accounts of the area, portraying it as a case of "failed integration" or "multicultural ghetto." I begin with a discussion on urban geography and argue that the perspective based on affective geography bridges the division between the material and sensory in a fruitful way.I move on to analyze how the complex bordering practices based on the relative location of Kontula provide ways to establish connections and separations, as well as comparisons and evaluations, between urban spaces.After discussing the advantages of studying these issues ethnographically, I then focus on the most significant site of my study, Kontula's dilapidated open-air shopping center.I discuss how the neighborhoods of Kontula have been situated in relation to different concerns throughout their history and how their inhabitants relate and react to the accelerated urban change.
Awareness of the Qualities of Urban Space
The neighborhoods of Kontula have occupied a special place in the vernacular geography of Helsinki since their construction in the mid-1960s.Unlike most Finnish suburban housing estates, Kontula has had a strong identity, with the historically shifting qualities of a problem zone that have also been shared by those who have never visited the place (see, e.g., Kokkonen, 2002;Tuominen, 2020Tuominen, , 2022)).Alongside its reputation on the municipal and national level, it is also situated in the global hierarchy of value (Herzfeld, 2004), a shared recognition of classifications that enable their comparison in different contexts.Globally, Kontula's position corresponds to a small set of urban spaces in every country that have become renowned and reviled in "the discourses of journalism, politics, and scholarship, as well as in ordinary conversation as synonyms for social hell" (Wacquant et al., 2014(Wacquant et al., , p. 1273)).
Interestingly, Kontula occupies a paradoxical position in the classification of urban areas: It is both a bleak place with no significant sense of the past, a surface that forces the residents to live in its dreary present (cf. Reed, 2002, p. 137), and a place with too much meaning, impossible to order and categorize into a coherent whole.As I will demonstrate later, there are several ways to deal with the material and sensory overload of a place that for many residents has rapidly changed beyond recognition.While most of the daily life lacks variety the routinized episodes are heightened by occasional moments of increased awareness, reflection, and occasionally incomprehension (Taylor, 1989(Taylor, , 1992)).My study illustrates the dynamic of making and believing in the practices of classification and bordering, especially in relation to the assumed normality elsewhere.
At the core of the bordering practices of Kontula is the concept of neighborhood, powerful as contested territory and meaning, resisting attempts of standardization (Martin, 2003, p. 362).Instead of a clearly bounded whole, I concentrate on "nested zones that subdivide the environment around one's home into sections of distinct spatial, social, and emotional nearness" (Kusenbach, 2008, p. 231), an affective geography resulting from the mix of people and the built environment.In Kontula, the open-air shopping center signifies the core, bringing together the residents of the surrounding neighborhoods.
Much of my ethnographic fieldwork was characterized by encounters with the unexpected, often amusing or even absurd, that had become central to the residents' awareness of the place.Their most richly lived and felt relationships to the immediate environment were both embodied and reflected upon.In these situations, the quotidian flow of life was interrupted by a complex sensation that forced the individual to step back and concentrate on the qualities of the place, as a way to challenge the previous understanding (Basso, 1996, p. 54;Taylor, 1992Taylor, , 1995)).In addition to self-reflection, the sensory experience of places-familiar or surprising-inspired thinking about "other places, other people, other times, whole networks of associations that ramify unaccountably within the expanding spheres of awareness that they themselves engender.The experience of sensing places, then, is thus both roundly reciprocal and incorrigibly dynamic" (Basso, 1996, p. 55).At the same time, the residents' sensory experience was closely tied into the habitual life of Kontula, realized in encounters reproducing its particular affective atmosphere (Berlant, 2011) and geography, entangling the material and the sensorial that was shared by its residents but not immune to change (Navaro-Yashin, 2012).
Obviously, a comprehensive review of the vast literature on how affect has been studied in different disciplines, from experimental psychology to cultural geography, is outside the scope of this article.
Instead, for my argument, I follow Navaro-Yashin's (2012) characterization of affect as "a charge that has a part to play in the sociality of the human beings who inhabit a space" (p.20), as something that reaches beyond the realms of subjectivity but may be experienced by human beings (Navaro-Yashin, 2012).In my analysis, I combine the intricate ways make-believe operates in urban space with a study of historically situated bordering practices that shape the modes of belonging and exclusion.In the beginning vignette, Jukka wanted to defend Kontula but did not because we were not on his home ground.At the same time, he claimed that he understands less and less what is happening in Kontula but still insisted on the need for solidarity among its residents.
Bordering Practices and Absurd Urban Life
In an approach that emphasizes the make-believe qualities of space, the conception of borders as tangible objects severely limits the analysis.It is more appropriate to see them as processes, a form of technique.Rather than borders themselves, my focus is on bordering, "the process of classifying and ordering space and relations between here and elsewhere in the world" (Green, 2013, p. 350).The location of Kontula should be seen as relative, subject to a range of different ways of measuring and comparison, its meaning and value depending upon "its relations with, and separations from, other places" (Green, 2012, p. 6).Borders conceptualized in this manner are not fixed on the present but contain all the previous ways of how they have been thought and performed-this is an ongoing process of generating new connections and disconnections (Green, 2010, p. 272).
It is also important to note that the comparison does not only occur between units of similar size and standing.In addition to comparing different neighborhoods with one another, within a city or even globally, it is common to reflect upon distinctions at different scales: city and the suburbs, city and the provinces, city and the nation, etc. (Koefoed & Simonsen, 2012, p. 634).In this vein, Kontula is simultaneously bordered as an exceptional suburban housing estate, an exemplary case of global developments, the future of Helsinki, and a village-like collection of neighborhoods.
One of the recurring themes in my ethnographic data concerned accelerated urbanity, an ordered sense of neighborhood and community disappearing and becoming harder to comprehend (see Hall, 2012 for a similar argument about London).Many of my informants experienced this sense of confusion and lack of comprehension as a permanent condition in their lives.This was not about dealing with economic uncertainty or a precarious work situation, in their own right very common among them, but, rather, about the unpredictability and absurdity of social encounters and the very nature of the urban environment."Anything can happen around here!" was a very common way to describe the area, and "See, this is Kontula!," was a typical reaction to something unexpected.According to these statements, everyday life in Kontula did not follow the same rules as elsewhere in Helsinki, exemplified by the assumed normality of the central districts.This embodiment of rapid change is a crucial dimension of the make-believe processes in Kontula.
My approach to diversity in Kontula focuses on its ordinariness.Theoretically, I follow Paul Gilroy's (2005) use of the term conviviality but emphasize its connection to living with all kinds of diversity.Everyday encounters between people who are familiar with one another as they frequent the same places, are, for the most of the time, rather unremarkable.For young people, they signify normality for they represent the only sociocultural realities they know.Les Back and Shamser Sinha (2018) summarize conviviality as "an unruly, spontaneous social pattern produced by metropolitan social groups living in close proximity with each other" (p.134).I consider a place-specific sense of conviviality, embodied and routinized by the inhabitants, as an important bordering practice that operates on both material (referring to the physical boundaries, however porous) and affective (referring to the affective geography of relative location) realms.Life is considered to be different elsewhere.
Methods
My research is based on ethnographic fieldwork conducted in Kontula in 2017 and 2018 (12 months/3 months).However, my participant observation has continued after this period in the form of participating in voluntary work for different NGOs, discussing my research and writings with the residents, and just spending time in the area.While the focus has been on long-term participant observation, the fieldwork has been supported with semistructured interviews, studying media accounts, historical studies of the area, and statistical data.More than 30 interviews, lasting from half an hour to several hours, have been used mostly to confirm and study further insights and questions arising from participant observation.Following Alpa Shah (2017), my ethnographic work emphasizes "production of knowledge through being and action; it is praxis, the process by which theory is dialectically produced and realised in action" (p.45).I will refer to this aspect throughout the article.
The advantages of long-term fieldwork already started to show during my first weeks in Kontula.The residents began to share their reflections in a more contemplative manner, and discussions became more relaxed.Living in a place that had for a long time been a symbol for the ills and evils of urban marginality had made most of them wary.There was a steady flow of journalists, researchers, NGO professionals, and students with questionnaires, almost all of them concentrating on social problems, reproducing the predefined classifications, and mostly disappearing after the forms were filled in.I was once approached to provide contact information for three "unemployed" and five "immigrants" for research purposes.Long-term engagement with a holistic approach to sociocultural diversity and my detailed study of relations between different groups gave my research access to views that were very different to those expressed during the first encounters.Initial comments mostly confirmed the expectations of the mainstream society and sensationalist media accounts, reproducing the image of a problem area (see Back & Sinha, 2018).
In contrast to the rehearsed reactions to preformulated questions, everyday life is based on an embodied understanding of sociality, a way to act in different situations in an appropriate way, rather than following a ready map inside our heads (Taylor, 1992, p. 217).In Kontula, this principle was acknowledged in colloquial speech, with assertions of "senselessness," "outrageousness," and "madness," and expectations that just about anything might happen.Trying to understand how people experienced and reacted to these situations became a crucial part of my research.The stereotype of a gloomy suburban housing estate, a landscape of uniform gray concrete, was brought together with urban diversity unlike anywhere else in Helsinki.
My own position as a white university-educated male researcher differed from many, but by no means all, of the people in my field site.There was a lot of good-natured banter about having a "Doctor" to observe how the marginalized people were living, and I had an unexpected advantage of having lived my childhood and youth in another stigmatized district in Helsinki-a fact that many in Kontula saw as bringing us together.The fluctuation of intimacy and estrangement, discussions of personal feelings, and views on complex global developments are presented here as lived engagement, vignettes, and comments that illuminate themes and contradictions that were repeatedly brought up in the everyday encounters between people in Kontula.The interactions were at their most dense at the run-down open-air shopping center-the heart of Kontula where people from its neighborhoods congregated and where material, sensory, and imaginary clashed in often surprising ways.
The Absurd Heart of Kontula: Encounters at the Open-Air Shopping Center
Building suburban housing estates was Finland's response to the large-scale migration from the countryside to the cities in the 1950s and 1960s (Lento, 2006).The Finnish word for them, lähiö, has connotations with this specific type of housing, uninviting but affordable blocks of flats with large green areas between them, but it also refers to a distinctive style of living, even a particular mentality of the dwellers (see Ilmonen, 2016;Kokkonen, 2002;Stjernberg, 2019;Tuominen, 2020Tuominen, , 2022 for detailed discussions on definitions and history).
For the purposes of this article, the history of Kontula can be summarized broadly into three phases.Its first decades, since construction began in 1963, are characterized by the promise of modernization, especially for people who had moved in from traditional working-class districts or the countryside to the new and spacious housing with modern amenities.However, the rowdiness of the place, its relatively large proportion of social housing, remoteness, and the rootlessness of its residents became early on part of its image through media accounts (see especially Kokkonen, 2002;Roivainen, 1999).Its notorious reputation and territorial stigma became allencompassing in the 1990s with the deep economic recession and quick rise in unemployment, mostly as a result of the loss of traditional working-class jobs.From the 2000s on, the emphasis has shifted largely into supposed failures in immigration and integration (Tuominen, 2020).
In statistical terms, 36.2% of the 15,000 residents of Kontula do not speak the official languages of Finland (Finnish, Swedish, Sámi) as their native tongue (City of Helsinki, 2019).The figure is among the highest in Finland and has grown rapidly in the past 30 years, but is quite low compared with many other European localities associated with immigration.The percentage of inhabitants receiving social benefits is 20% (compared with 11.9% in the Helsinki region), almost double the average but not exceptional in Helsinki (City of Helsinki, 2018).There are many suburban housing estates that match these numbers but have not developed a similar sense of territorial stigma.I suggest that the sense of absurdity and accelerated change has to do with the distinct make-believe character of Kontula's neighborhoods, especially the affective geography of its notorious open-air shopping center.
Thirty-eight open-air shopping centers were built in Helsinki and its surrounding municipalities in the 1960s to act as their effective centers.In addition to shops, they housed the municipal services and acted as local hubs for public transport.After the 1960s, a new type, the enclosed shopping mall, started to emerge in Finland and spread into the central areas of the Finnish cities in the 1980s (Lahti, 2017).The open-air shopping center in Kontula was built in 1967 and expanded in 1986 together with the extension of the metro line.Currently, it is the biggest of its kind in Finland with more than 22,000 square meters of retail space and more than 30,000 visitors in a day.However, it is vastly different from the spectacular, fully enclosed indoor malls, associated with almost magical qualities (see Goss, 1993 andMiller &Laketa, 2019 for affective sensibilities of the malls)-the shopping experience in Kontula is mostly limited to the bare necessities.
Despite that, for many of the residents, it is the center of their public lives, with grocery stores, pubs, and restaurants, a youth center, a municipal library, a health center, and other integral services.For the vast majority of those who do not live in Kontula, it has become a powerful symbol of urban failure, expressed by a friend of mine, and conveying a familiar view of the whole district: "If the shopping centre area is the public face of Kontula, I don't want to know what happens outside it, in the private." Public life at the shopping center is public to the degree that people might refer to it jokingly as a theater or even a colosseum.Its three smallish squares, connected by narrow passages lined with shops, restaurants, and bars make it very different from the sleepy residential neighborhoods surrounding it.It is composed of relatively uniform structures with two floors; the central square has a circular building resembling a tower that houses a nightclub; the Kontula square is surrounded by the concentration of municipal services, and the children's square hosts a dilapidated playground.These are all familiar signs of the centers of suburban housing estates, recognized even by Finns who have never visited one.Many of the open-air shopping centers have already been demolished, and the few remaining ones will probably follow soon.The architecture brings one back in time to the 1960s but what the local refer to as the atmosphere or feeling of the place is nowadays very different.
The overall appearance is dreary and unkempt but, at the same time, extremely lively around the clock.People pass by on their way home from the metro, the residents use the services provided as a part of their daily routines and, for a large group of people, it is the center of their social lives, a place to see and be seen.For them, in the words of Suzanne Hall (2012), "local worlds are places in which they are not simply dependent, but also highly invested" (p.96).In the environment of accelerated urban change, the seemingly insignificant everyday rituals host countless encounters: "They potentially provide recognisable rhythms, thereby offering diverse individuals a format of time, space and etiquette in which to engage with one another" (Hall, 2012, pp. 59-60).There were, of course, also residents who avoided the shopping center area for its restlessness or they felt indifferent about it.However, I concentrate here on the loose community of people who had embraced the shopping center's bounded social world.
"Everyone Has a Story"
My initial contacts in Kontula all emphasized the same thing: It would not be difficult to find "material" for my study, for everyone would have a story to tell.This was in unison with claims that Kontula is full of characters who do not fit into the uniform expectations of the central districts.The stories of differences reinforced the sense of absurdity and set the border between two kinds of normality.There was, for example, a woman with a briefcase and the look of an office clerk about her who would erupt into shouting obscenities at any minute, a man of scruffy appearance who supposedly had millions in his bank account, a woman who started giving hugs to strangers at the beginning of the coronavirus 2019 (COVID-19) pandemic, an old lady who would come to feed the rats in the middle of the night, a man who would drink around the clock throughout the winter months but would spend summers abroad sober, and many more.They were presented as ordinary features of the place that invited different responses.
Abdi, 25, of Somali descent, had settled in Kontula after migrating to Finland 10 years ago and living in other parts of Helsinki for 7 years.He had just found a job filling shelves in a supermarket that specializes in products from the Middle East.It was located nearby, just two metro stops away, in another run-down open-air shopping center.Abdi's everyday life was largely restricted to these two places.We were having a routine discussion at his local pub.
Everything I need is close by, I belong here.I tend to forget how long it was since I visited the centre.A couple of weeks ago I had to go to the centre.The Eid celebrations were coming and I needed a new stylish suit-that's very important in my community-and I couldn't find one that I liked, he began and took a sip of his beer.
I went through every shop in East Helsinki but still no luck.I had to go to the centre.The big airconditioned shopping mall made me feel nervous and I wanted to get back here quick.I don't know what it is but I felt completely out of place.It's the atmosphere of those places, not just the people.
Abdi did not want to be misunderstood: Don't think that I would be scared of those places.It's true that there is more racism in the centre but I don't mind.Here, it is a crazy mix of cultures but for me it represents a balance.I can go to a pub and I can celebrate religious holidays.Some of my friends living elsewhere say that anything can happen in Kontula.I tell them that I enjoy it, nothing is happening in the boring parts of the city.
As if to amplify his message, Abdi took a can of beer from his backpack and filled his pint glass in full view of the bartender.Noticing my amazement, he grinned, "I can drink my own beer here because I don't like the brand they have on tap.Sometimes they make me buy something.This is Kontula!" The distance of Kontula from elsewhere and the borders separating it from other places are measured not just in physical terms but in different interrelated hierarchies of value.According to the global hierarchy of value, it is peripheral and stigmatized, as opposed to the central and the most valued districts (Herzfeld, 2004).Its difference from the mainstream society is coupled with the assumed behavioral distance of its residents with regard to the mainstream norms, values, and behaviors (Hastings, 2004, p. 236).The residential stigma makes it difficult for some people to leave their familiar areas (see, e.g., August, 2014;Bourdieu & Accardo, 1999;Hall, 2012;Tuominen, 2016Tuominen, , 2020;;Wacquant, 2010).As Abdi's discomfort indicates, the difference between places was based on not just analytical reflection but also affective and embodied sensory experience.It is true that Kontula shopping center is loud and often rowdy; there is a lot of substance abuse happening and more than 10 small bars, among the cheapest in Helsinki, make it very different from the shiny shopping malls of Helsinki.
The affective geography of the place thrives on public display that has become the cornerstone of its sociality: What is considered disorder consists of doing things like drinking and hanging out in public-things that are done legitimately in private in the more reputable areas (Sampson, 2009, p. 7).Perhaps, this is where the playful comparisons to a theater (or colosseum) come from.In the countless encounters that I participated in, Kontula was referred to as energetic, diverse, and eager for new expressions.At the same time, it was absurd, out of control, and "too much."The hierarchy of places and the careful drawing of borders connecting and separating them played a major role in how these qualities were evaluated.At the same time, it was possible to reorder this sense of chaos and marginality.[AQ:3]
Ordering the Diversity
The make-believe space in Kontula is a result of historical transformations and their reactions.The vernacular history of Kontula, modeled as an accelerating urban transformation toward diversity, is related to developments in other areas.Jukka, Abdi, and many other residents often referred to feeling out of place or not fitting in elsewhere-Kontula had a diversity that they had access to.This was a way of situating oneself into the affective geography of Helsinki, for the cosmopolitan diversity of the city center was very different from the cosmopolitan diversity in the periphery.Neil Smith (2005) summarizes the difference thus: The pursuit of difference, diversity, and distinction forms the basis of the new urban ideology but it is not without contradiction.It embodies a search for diversity as long as it is highly ordered, and a glorification of the past as along as it is safely brought into the present.(p. 114) On the basis of a short visit or the sensationalist media accounts, the everyday reality of Kontula's shopping center area might feel out of control.Usually, this was vehemently denied by many of the residents, who blamed the media for stigmatization (see Tuominen, 2020).It was occasionally "too much" but nevertheless associated with an affective sense of homeliness and belonging.The responses emphasized the embodied sense of attachment and solidarity: I have never felt welcome in other parts of Helsinki.Here in the East I have room to breathe.I don't feel like a Finn-I will never be one.But I sure am from Kontula.I know everyone and everything here!(Ali, 25, moved to Finland from Iraq 10 years ago) I was born here twenty years ago but my family moved to Birmingham when I was one.Now I'm back here to do my military service [compulsory for Finnish males].I don't know that much about Finland but every time I'm back here I stay in Kontula.Other people say that this is a shithole but it feels like home to me.Perhaps I'm a little bit crazy to keep a smiling face here.I was born like that.Born to survive.(Suldaan, 20, Finn of Somali origin) We have always welcomed new people here.Some people say that all the shit trickles to the East, but we are proud of what we have.Posh people might not like our way of life but we stick together and survive no matter what happens.We're not easily scared.I'm the type who can laugh in the middle of hardship.(Esko, 60, Finn of Finnish origin, born in Kontula) These were general affirmations of belonging and community, and variations of them circulated widely throughout my fieldwork.They were expressions of territorial solidarity by people who had been denied the sense of belonging elsewhere.Their engagement with diversity was the result of limited mobility, both socially and physically.The affective geography of the city center of Helsinki created an uncomfortable feeling of being in a wrong place outside the familiar surroundings.I also met many residents who had no problems leaving Kontula, who, nevertheless, joked about the outside world being sterile and bland, with twice as expensive pints and kebabs.These quotidian exchanges constituted important bordering practices, reproduced the makebelieve space of Kontula, and established crucial senses of belonging, often unrecognized and ignored in urban studies.Understanding of urban diversity is highly localized and often polarized: Especially, the recently gentrified former working-class districts closer to the city center portray diversity, vibrancy, and nonconformist lifestyles but in highly ordered sense.
The Normalization of Absurdity
The residents of Kontula often referred to what I interpret as accelerated urban change and the increasing absurdity of their everyday lives but their reactions for this were varied.I came across moments of longing for a more comprehensible lifeworld, but they were usually contrasted with claims of resilience, of "pulling through" under any circumstances, possessing moral strength to endure (cf.Hochschild, 2016).In a slightly gloomy way, the optimism itself, referred to as an inherent characteristic, was turned into an attribute that makes life bearable (Berlant, 2011, p.14).
This pride in survival in an increasingly heterogeneous world was a key component of social relations.My ethnographic vignettes demonstrate the different ways of dealing with the absurd and the unexpected.In the beginning of the article, Jukka claimed that he does not understand what is happening in the place he calls home, but he would still prefer it to the pretentious lives of the mainstream.For Abdi, things were happening in Kontula while most of Helsinki was boring.The affective geography of Kontula allowed Ali and Suldaan to be themselves, to breathe and to smile.They embraced it wholeheartedly, felt safe and at home despite the territorial stigma.Others used irony as a tool to negotiate the contradictions between the official narratives and lived experiences (Juntunen & Laakkonen, 2019) or resorted to forms of cultural intimacy, taking features of external embarrassment-untidiness, noise, and cheap beer-as a source of pride and common sociality at the inhabitants' collective expense (Herzfeld, 2016, p. 7).
As someone born in Kontula, Esko emphasized the ease of access and tolerance of diversity across racial, class, and other boundaries.However, this did not mean that discrimination did not exist in the make-believe space of Kontula.While different aspects of diversity were valued in Kontula than in the central districts and the desired futures of the areas followed divergent trajectories, the hierarchies were not a matter of individuals' decisions.Many who did not conform to the expectations of mainstream society because of their cultural background or lifestyle found Kontula welcoming and open-minded, but this did not extend to everyone.The Bulgarian and Romanian Roma were generally shunned, just like elsewhere in Helsinki, and among the migrants, there was a local hierarchy of belonging, based on time of arrival into the country (see Back & Sinha, 2018, p. 72).However, even for the newcomers, the sense of belonging was easier to attain here than elsewhere in Helsinki.The relationships were convivial in Gilroy's (2005) sense: People spending time in pubs and attending NGO-organized activities did not necessarily like each other.However, "strange others" became "familiars others" often through banal exchanges that increased attachment to the social environment (Koefoed & Simonsen, 2011, p. 355).These encounters made Kontula distinctive in the affective geography of Helsinki.[AQ:4]
Conclusion
A cynic would argue that people who prefer Kontula above other districts, despite its stigma and deprivation, are just revealing simple defense mechanisms that have taken hold amid the squalor.Certainly, Kontula's residents had their moments of doubt: Conversations about enjoying the bleak surroundings of the dilapidated concrete structures in the company of undesirables were often accompanied with hesitation and hearty laughter.At the same time, they expressed choices by people who could have chosen otherwise and presented a prime example of everyday universalism-identifying and valorizing something that is relatively common and accessible, independent of resources (Lamont, 2019, p. 686).
The group I have focused on included both long-term residents and newcomers, ethnic Finns and migrants.For them, the everyday encounters had become "an unremarkable fact of life even with all of its irreducible kaleidoscopic complexity" (Back & Sinha, 2018, p. 164).This form of accelerated urbanity had created a shared experience of incomprehension and absurdity, shaping some neighborhoods and districts more than others, with varying responses.This is a sign of cultural vitality, a powerful experience that brings together the material and sensory qualities of urban neighborhoods.
For anthropological studies of space, ethnographic analysis of affective geography opens up intriguing ways to understand urban transformation.The mundane details of everyday life grow in significance and reveal complex dynamics of borders and belonging in contemporary societies.Focus on affective geographies also opens up new possibilities for social inclusion, which are much more spontaneous than the official narratives on integration.An ethnographic study of a place like Kontula reveals its affective geography at different scales, a make-believe space that is subject to historical transformations of spatial hierarchies.As scholars in social sciences have increasingly pointed out (see, e.g., Back & Sinha, 2018;Glick Schiller, 2012;Hall, 2012), these everyday socialities have been theorized insufficiently and the multiple spontaneous connections that create new possibilities for group life have been insufficiently recognized.For many, it is easier to embrace the absurd urbanity of Kontula than to relate to Finnish mainstream society.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. | 2024-06-21T13:20:50.755Z | 0001-01-01T00:00:00.000 | {
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54015753 | pes2o/s2orc | v3-fos-license | Efficiency Enhanced Colloidal Mn-Doped Type II Core / Shell ZnSe / CdS Quantum Dot Sensitized Hybrid Solar Cells
1Department of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology (KTH), 106 91 Stockholm, Sweden 2Department of Applied Physics/Bimolecular Physics, School of Science, Royal Institute of Technology (KTH), 106 91 Stockholm, Sweden 3Organic Chemistry, Centre of Molecular Devices, Department of Chemistry, School of Chemical Science and Engineering, Royal Institute of Technology (KTH), 100 44 Stockholm, Sweden
Introduction
Nanotechnology has led to huge progress in the use of semiconductor nanocrystals for applications in diverse areas like organic light emitting diodes [1], biosensing, biolabeling [2,3], solar cells [4][5][6], and imaging and detection [7], to mention a few examples.The world energy demands for renewable and cheap resources of solar energy have generated a large interest in sensitized solar cell technology due to high power conversion efficiency with low cost of production [8,9].Among various kinds of sensitizers employed in sensitized solar cells, quantum dots (QDs) are regarded as promising candidates by virtue of their size-dependent optical and electronic properties, high light-absorption ability, photostability, and multiple exciton generation [10].In particular, type II core/shell quantum dots are promising for efficient sensitization due to their long-time charge separation and possibility for electron confinement in the conduction band of the shell when their band structure is carefully designed [7,11].These nanoscale crystals are capable of integrating multistructures with different functionalization into a single nanoscale particle with controllable electronic structure for development of photovoltaic cells.In this heterostructure, the core and shell are made up of two different semiconductors, with a higher conduction (valence) band of the core than the conduction (valence) band of the shell (Scheme 1).
This offers charge carrier localization in two separate materials so that electrons and holes are confined in the shell and core, respectively.Moreover, the use of type II nanocrystals in solar cell applications leads to better power conversion efficiency compared to the corresponding nanocrystals made up entirely from the core or shell materials [12,13].Apart from the wide photon absorption range for type II QDs, the improvement also refers to an effective charge separation of electron-hole pairs in the type II nanostructures that facilitates electron abstraction from QDs, suppresses recombination, and therefore leads to better electron transportation [14][15][16].It follows that much recent research efforts have been devoted to the synthesis of different type II core/shell structured QDs, like TiO 2 /CdS [17], CdSe/ZnSe [18], CdTe/CdSe [19][20][21][22], CdTe/CdS [23], CdSe/ZnTe [24], CdSe/ZnTe [25], ZnTe/CdSe [26,27], CdS/CdSe [28], CdS/ZnSe [16,18], and ZnSe/CdS [29], as well as to the use of such QDs in emerging technology for solar cell applications.As an example, theoretical calculations from density-functional and many-body perturbation theory show the conduction and valence band offsets of 0.66 and 0.32 eV for ZnSe/CdS, respectively [30].Since the effective mass of the electron is lower than the hole in ZnSe [31,32], one can expect efficient conductivity in the ZnSe/CdS interface particularly where the electron accumulation is made by doping a paramagnetic material like Mn with semifilled d orbital.
It has been shown that use of CdTe/CdSe core/shell nanocrystals prepared by the one-pot synthesis method without core seed purification could make structural and optical properties of nanocrystals comparable to the nanocrystals synthesized using purified core seed, which can give higher absorption and better crystallinity [33].Neo et al. have investigated the effect of shell thickness and surface passivation as another strategy to improve the efficiency of type II PbS/CdSbased solar cells [34].Metal ion doping as a band gap engineering tool has also been employed for improvement of type II-based solar cell performance because metal ions could make changes in the Fermi level, band gap, and conductance [35].Particularly, Mn-doping was recently used in CdS/CdSe sensitized cells as a strategy to boost solar cell efficiency due to very long lifetime of Mn d−d transitions ( 4 T 1 -6 A 1 ) [36].Mn-doping in semiconductors like ZnS, ZnSe, CdS, and CdSe shows dramatic increase in lifetime due to the spin forbidden 4 T 1 -6 A 1 transition of the Mn [37][38][39][40][41][42].In case of Mn-doped CdS, through doping with Mn 2+ , a pair of d bands ( 4 T 1 and 6 A 1 ) is inserted between the conduction and valence bands of CdS QDs, which alters the electronhole separation and recombination dynamics, allowing the generation of long-lived charge carriers with microsecondscale lifetime more slowly than in the case of undoped QDs [37].Utilization and transfer (transfer of electron and hole toward TiO 2 substrate and electrolyte, resp.) of these long-lived and special-separated carriers by band alignment engineering in core/shell structures should be a superior opportunity to design a QDSSCs device with higher power conversion efficiency.To the best of our knowledge, Mndoped ZnSe/CdS has not been synthesized and investigated in quantum dots sensitized solar cells (QDSSCs).Since the lattice constant of the CdS shell is larger than that of the ZnSe core (5.82Å and 5.67 Å for shell and core, resp.),CdS will become strained if it is carefully grown on ZnSe with a smaller lattice constant.In case of strain, both the direct and indirect band gaps (Γ-and L-band, resp.) in the CdS shell are affected by the strain amount and type (compressive or tensile).As the direct band gap shrinks faster than the indirect one in a strained material, -type doping like Mn 2+ -doping could make Γ-band transitions suitable for detecting the light [43][44][45].
Here we present a colloidal synthesis of a novel type II Mn-doped ZnSe/CdS core/shell QD system as sensitizer with different Mn concentration (0-3%) and test its utilization in QD sensitized solar cells.This is the first time that Mn-doped ZnSe/CdS core/shell QDs are successfully synthesized by the method of hot injection and used as a strategy to boost solar cell efficiency.As explained in what is to follow it was found that a proper balance of Mn concentration could tailor the band gap and core/shell conduction band edge, causing a better electron transfer from QDs to the TiO 2 photoelectrode, a broader absorption, and, consequently, higher solar cell efficiency.
Results and Discussions
Novel Mn-doped ZnSe/CdS QDs with different Mn concentration were synthesized by hot injection method.To investigate the effect of doping, all QDs were synthesized at the same conditions and with the same amount of ZnSe cores.To get precise amount of ZnSe core, they were synthesized in one batch and after washing by methanol and acetone, they were divided into several parts to be utilized as ZnSe core in ZnSe/CdS and Mn-doped ZnSe/CdS.To grow shell material onto ZnSe core, the mixture of S and (Mn-doped) Cd precursors was slowly injected into core solution at 240 ∘ C, followed by keeping 240 ∘ C for 20 min.Afterwards the reaction was cooled down to room temperature for purification and characterization.Transmission electron microscopy, operating in 100 KV, was employed to evaluate quality and size distribution of the nanocrystals.TEM images of ZnSe/CdS core/shell (Figure 1) demonstrate the size of core/shell to be 6-7 nm.
In order to measure the actual Mn concentration incorporated into the nanocrystals, inductively coupled plasma atomic emission spectroscopy (ICP-AES, Thermo Scientific ICAP 6500) was employed.Nanocrystals were digested completely with nitric acid (67%, 0.1 mM) and diluted with DI water to obtain 10 mL of clear solution for ICP-AES measurement [46][47][48].The results with details are listed in Table 1 where the real Mn/Cd molar ratio is confirmed.
After being successful in synthesizing the QDs, we prepared different photoanodes sensitized with these QDs: (F) 2, and (G) 3%.Figure 2 shows the absorption of QDsdeposited photoelectrodes and fluorescence of QDs dispersed in chloroform.It was seen that pure ZnSe/CdS give broader absorption with a band edge up to 488 nm compared to ZnSe with 437 nm.It shows that both the absorption and emission peak wavelengths in type II ZnSe/CdS core/shell QDs are much longer than those of the pure core or shell materials, resulting from the fact that the energy states (conduction and valence band) for the photogenerated electrons and holes are located in CdS shell and ZnSe core, respectively.In order to calculate the band gap of the nanocrystals, the absorption coefficient () was applied for near-edge optical absorption using the following equation [49,50]: where is a constant and is the optical band gap.
According to the graph in Figure 3 plotted for (ℎ]) 2 in terms of (ℎ]), the band gap values of the nanocrystals were obtained from extrapolating the straight portion of the curve to zero absorption coefficients and they are listed in Table 2. Therefore, the heterostructure band gap corresponds to the energy separation between the conduction band (CB) edge of the shell and the valence band (VB) edge of the core [11,16], which results in a smaller band gap and a broader absorption spectrum compared to the ZnSe or CdS nanocrystals.Although there is a red shift for low concentration of Mn (0.25-1%), a blue shift is obtained for high concentration (2-3%).The red and blue shifts are attributed to the decrease and increase of the band gap (Table 2), respectively.The band gap decreased for these samples of (C)-(E) and increased for these samples of (F)-(G) due to the contribution of Mn as a metal dopant in CdS and the low solid solubility of Mn (<1%) in the CdS nanocrystals [51][52][53][54].It means that, below 1%, introduction of Mn could lead to decrease of the band gap but, after 1%, it leads to increase of the band gap because of the MnS phase.
To examine the performance of the devices, they were tested under AM 1.5 G simulated solar irradiation with intensity of 100 mW⋅cm −2 .Figure 4 shows the - characteristics of solar cell devices sensitized with various QDs.The device parameters including the short-circuit current ( SC ), opencircuit voltage ( OC ), fill factor (ff), and power conversion efficiency () of all samples are summarized in Table 3.
The ZnSe/CdS core/shell shows much higher current than the ZnSe core which may be attributed to special carrier extraction and extension of the light-absorption range in type II heterostructures.Compared to previous work in which the CdS shell of the investigated ZnSe/CdS QDs was synthesized by deposition of Cd and S separately [11], in this study the colloidal ZnSe/CdS core/shell, of which the CdS shell was synthesized by direct deposition of mixture of Cd and S solution, shows higher current and voltage and more than 2 times higher efficiency of 0.47 V and 2.23 mA and 0.56%, respectively, indicating the better crystallization quality of CdS shell synthesized by direct deposition.The strain is another issue in heterostructures that can increase electron mobility [55,56].Since the crystal size of CdS bulk (5.82) is bigger than the ZnSe bulk (5.67 Å) [57], one can expect strain in the CdS crystals.Since the strain can lead to enhanced electron transport as reported, for instance, in [58], it can be expected that the electrons in the strained CdS shell are transported faster than in unstrained CdS.
A device sensitized with low Mn-doped ZnSe/CdS (0.25%) shows a dramatic increase in all device parameters with about 10 times and 3 times higher efficiency compared to ZnSe and ZnSe/CdS, respectively.Since all samples are coated with 2 cycles of a ZnS SILAR layer, which blocks the electron recombination with the electrolyte [59] and passivates defect states that can trap electron-hole pairs [60], we expect a stabilizing effect by this coating.In case of the Mn-doped ZnSe/CdS sensitized device, the open-circuit voltage, short-circuit current, fill factor, and consequently device efficiency decrease with increase of Mn concentration due to the decrease of absorption range and the increase in nanocrystal band gap (Figures 2 and 3).New energy states in the band gap of the nanocrystal can make the carriers better separated and decrease recombination [12][13][14].
The best values of device parameters are found for the lowest Mn concentration, possibly due to lower carrier recombination induced by new states generated by proper concentrations of Mn.It should also be mentioned that, like Mn-doped GaAs [61], Mn atoms can occupy three types of sites in the Cd 1-2 Mn S lattice matrix.They can occupy the substitutional or interstitial Cd lattice sites to form Cd 1-2 Mn S with the wurtzite lattice structure.Mn atoms can also precipitate out to form different phases, for example, MnS nanocrystals.Since the solid solubility of Mn in CdS is too low [51][52][53] (<1%), less than 1% induces substitutional or interstitial sites and more than 1% induces other phases like MnS that can act as impurity and therefore decrease the device performance.
The Incident Photon to Current Efficiency (IPCE) of all devices was measured to evaluate the photocurrent response to incident light (shown in Figure 5).IPCE, sometimes referred to as external quantum efficiency (EQE), is expressed by the following equation [62]: where inj is the quantum yield, coll is the electron collection efficiency, and LHE() is the light harvesting efficiency at the wavelength of the incident light and is derived via The power conversion efficiency is comparatively low for devices sensitized by ZnSe due to limited light absorption but increases in ZnSe/CdS.This is related to the broader absorption and the special carrier separation in this type II heterostructure.The observed rather dramatic increase in IPCE spectra of Mn-doped ZnSe/CdS compared to undoped ZnSe/CdS may be due to a more efficient electron collection and electron injection efficiency.It is worthwhile to stress that band structure and CB manipulation via band gap engineering (Figures 2 and 3) by proper Mn concentration doped into the nanocrystals not only give superior ability to enhance light absorption but also can facilitate carrier transfer, which may be responsible for the improvement of the device efficiency.However, higher doping ratios may generate impurities or defects like MnS, which can increase charge recombination and inhibit electron injection into the TiO 2 substrate.The excited states dynamics of QDs were further investigated by time-resolved fluorescence lifetime measurements (experimental details in the supporting information in Supplementary Material available online at http://dx.doi.org/10.1155/2015/921903).Figures 6(a S1 (supporting information).The average lifetime of QDs was estimated based on [63] where is the th exponential component.In our procedure, a triexponential decay model ( = 3) was found to be necessary to properly fit the experimental fluorescence decay curves [64,65], and no substantial improvement in the goodness-of-fit was obtained for higher .(See fitting residuals in Figures S1 and S2 in the supporting information.)It is found that the average fluorescence lifetime of ZnSe/CdS QDs (49 ns) is much longer than that of ZnSe (2 ns) when they are attached to the insulator.This is due to special charge separation in type II nanostructures that can decrease their wave function overlap and delay radiative recombination, resulting in a long fluorescence lifetime [11].It was mentioned that photogenerated holes can be moved to the valence band of ZnSe and that photogenerated electrons will be localized in CdS.When the QDs are attached to the insulator, there is no electron injection, but if deposited to the TiO 2 , another electron deactivation route is created that causes electrons to be injected from the QDs into the TiO 2 with lower conduction band energy, which results in a decrease of the lifetime.The corresponding electron injection rate constant can be estimated from [64,65] where (QD) and (QD+TiO 2 ) are the fluorescence lifetimes of the QDs attached to the TiO 2 and insulator glass, respectively.As it is shown in Table S1, a higher injection rate constant of the QDs is ascribed to the lower Mn-doped type II ZnSe/CdS QDs that are responsible for the efficiency of the device.
To get more understanding of the charge transport in type II ZnSe/CdS QDs with different Mn concentration, a physical model like Scheme 1 is considered.The ZnSe as core is covered by the CdS shell and the conduction bands and valence bands edge of TiO 2 , ZnSe core, and CdS shell are formed while considering the quantum confinement effect in the type II heterostructure.Coupling of the ZnSe and CdS moieties leads to special carrier separation and interfacial wave function interaction increased by increasing the Mn concentration.The results indicate that by increasing Mn content until 1% not only is the band gap decreased, which results in special indirect band gap with some red shift of the absorption band edge, but also the device parameters are increased because of lower recombination and enhancement of the electron injection rate.It is notable that the Mn 4 T 1 mid-state between CdS CB and TiO 2 CB can facilitate electron transfer from QDs into TiO 2 .Further amount of doping can reverse the results (not shown here).
Conclusions
Novel colloidal Mn-doped ZnSe/CdS core/shell QDs with various Mn concentrations were successfully synthesized and applied to sensitized solar cells.It was demonstrated that QDs with proper Mn-doping could cause an increase in the absorption spectra and red shift in the absorption band edge and in the photoluminescence emission peak.The mid-states generated by Mn can facilitate electron transfer from the QDs to the TiO 2 substrate.With superior light absorption, better carrier separation, and efficient electron injection rate a power conversion efficiency of 1.27% is presented, which is about 2 and 3 times larger than those of core and undoped QDs sensitized solar cells, respectively.The present work suggests that band structure manipulation in type II core/shell nanostructure offers an effective way to improve light harvesting and control of charge transfer via efficient charge separation in sensitized solar cells and that Mn-doping opens a new window to increase device efficiency.
There is a multitude of lines of research to embark upon for future improvement of QDs sensitized solar cell of the kind proposed here.One such line of research concerns surface passivation.In fact, the organic ligands used can act as dangling bonds on the QDs surface and trap carriers, which calls for surface passivation of the QDs as a way to improve device performance, for instance, by using hydrophilic materials or ions.These and other measures will be pursued in our future studies.
Experimental
4.1.Quantum Dots Synthesis.Zinc stearate, gray selenium, cadmium oxide, sulfide, and manganese nitrate tetrahydrate were bought from Aldrich Company as precursors for Zn, Se, Cd, S, and Mn sources, respectively.The targeted heteronanocrystals are fabricated by a two-step synthesis composed of the fabrication of ZnSe core nanoparticle followed by a deposition of pure or Mn-doped CdS shell.Synthesis of both ZnSe core and pure or Mn-doped ZnSe/CdS core/shell under different Mn concentrations is based on previously published procedures [16,29] with some modifications briefly explained as follows.
Firstly, in order to synthesize ZnSe core nanocrystals from organic solution, specific amounts of selenium 0.0118 g and 0.4 mL of trioctylphosphine (TOP) were placed in a one-necked flask while stirring to make selenium dissolved in TOP.The reaction was conducted under nitrogen atmosphere.When the mixture became clear, the solution was kept in a clean syringe to be used in the next step.0.0950 g zinc stearate, 0.1874 g stearic acid, and 2 mL of octadecane (ODA) were mixed together in a 25 mL three-necked flask.The mixture was slowly heated to 120 ∘ C (about 2 ∘ C/min) while stirring and pumping to remove additional elements from solution.The mixture was then heated to 240 ∘ C under nitrogen flow to make zinc dissolved in ODE where the solution appeared colorless and clear.Then a selenium stock solution prepared in the last step was swiftly injected into the reaction flask.The solution temperature was controlled and monitored to be kept at about 280 ∘ C.After 20 min, the reaction solution was cooled down to 60 ∘ C and 5 mL of chloroform was added to the solution to allow the quantum dots to be dissolved and suspended.The product of this step was ZnSe core, which contains byproducts and free ligands.To purify, it was washed 4 times by acetone and methanol.Typically, 5 mL of chloroform, 10 mL of acetone, and 2 mL of methanol were slowly added to the QDs solution followed by centrifugation for 3 min at 12400 rpm.The upper colorless layer was removed and QDs on the bottom were dissolved in chloroform for the next washing.Monodisperse ZnSe core produced in this step was sealed and kept in a cool and dark area to be used for shell deposition.
For preparing the CdS shell, three steps were considered.First, totally 0.0050 g of sulfur was dissolved in 3 mL of ODE while pumping and heating slowly to 80 ∘ C and then cooled to room temperature.Second, totally 0.0190 g of cadmium oxide (in case of Mn-doping, a proper amount of Mn was added to Cd precursor) was mixed in 0.4 mL of oleic acid (OA) and 3.5 mL of ODE in a 25 mL flask while stirring and heating slowly to 100 ∘ C.After degassing process, the temperature was increased to 280 ∘ C to make Cd dissolved completely in the solution.When the solution became clear, it was cooled down to 60 ∘ C. Third, mix these two precursor solutions (S and Cd) together as a source for shell growth.The final mixture was slowly injected (3 mL/h) into the reaction vessel, which contained 375 g ODA and ZnSe core dispersed in 2 mL of ODE at 240 ∘ C.After 20 min, the reaction was terminated and cooled down to room temperature for purification by chloroform and acetone.4.2.Photoanode Preparation.FTO (fluorine tin oxide) conductive glasses (Sigma Aldrich, sheet resistance of 7 Ω/sq) were used as photoelectrode substrates.FTO glasses were cut to targeted size (1 × 2 cm) and sequentially washed by soap, KOH dissolved in 2-propanol, acetone, ethanol, and DI water via sonication for 30 min for each washing step, followed by immersion in 40 mM TiCl 4 solution at 80 ∘ C for 40 minutes to give a TiO 2 blocking layer.Mesoporous TiO 2 layers were prepared by deposition of three transparent layers and one scattering layer with commercial TiO 2 pastes (Ti-Nanoxide T/SP with 20 nm and Ti-Nanoxide R/SP with > 100 nm particle size for transparent and scattering layer, resp.) by screen-printing technique concluding by heating at 70 ∘ C for each layer deposition.The samples were postannealed at 500 ∘ C for 30 min to make the layers porous by removing the organic part at high temperature.
QDs dissolved in chloroform were deposited drop by drop on TiO 2 films and left to be dried in the air.Excess QDs not adsorbed on TiO 2 were washed by chloroform.To passivate QDs, ZnS block layers were deposited by two cycles of SILAR method.Each cycle contains dipping the samples into sulfur solution (Na 2 S dissolved in DI) for 1 min followed by washing with DI water and zinc solution (zinc nitrate hydrate dissolved in DI) for 1 min followed by washing with DI water.
Fluorescence Lifetime
Measurements by Time-Correlated Single-Photon Counting.Time-correlated single-photon (TCSPC) measurements were performed on a spectrofluorometer with a TCSPC option (FluoroMax3, Horiba Jobin Yvon).A NanoLED (Horiba Jobin Yvon) emitting at 488 nm with a repetition rate of 1 MHz and pulse width of 1,4 ns was used as an excitation.Measurements were stopped when 3000 photon counts were collected in the peak channel using 2048 channels with 0,4 ns/channel.The instrument response function was recorded using a 2% Ludox (Sigma Aldrich) solution.To avoid reabsorption and reemission effects and also not to saturate the detectors, the sample concentration was kept strictly below 5 M.Recorded curves were fitted with a three-exponential decay using the software Decay Analysis Software v.6 (IHB).
) and 6(b) show the fluorescence emission decay of different QDs deposited on insulator glass and conductive glass/TiO 2 , respectively.The fluorescence intensity decay curves recorded from QDs were successfully fitted to a three-exponential decay model.The fitting parameters are summarized in Table Photon intensity Photon intensity
4. 3 .
Counter Electrode Preparation.Cu 2 S counter electrode was prepared by dipping brass sheet (Sigma Aldrich, resistivity of 1.673 Ω⋅cm) in hydrochloric acid at 80 ∘ C for 10 minutes and then washing with DI water and methanol.4.4.Device Assembly.Both photoelectrode and Cu 2 S counter electrode prepared in the last steps were stocked together by cell spacer while the electrodes were heating at 80 ∘ C. The S 2− /S 2− electrolyte (2 M S, 2 M Na 2 S, and 0.2 M KCl in a methanol-water (v/v, 3/7) solution) was finally injected into the cell through the hole preconstructed on top of the brass sheet.After electrolyte injection, the hole was sealed by sealing tape for the next electrical characterization.
Table 1 :
Summary of Cd and Mn concentration of nanocrystals.
Table 2 :
Band gap values of nanocrystals derived from Figure3.
Table 3 :
Summary of device parameters. | 2018-11-22T13:03:26.264Z | 2015-01-01T00:00:00.000 | {
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