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+# Artifact Removal From Semg Signals Recorded During Fully Unsupervised Daily Activities
+
+Digital Health
+
+![0_image_0.png](0_image_0.png) Volume 9: 1–16 © The Author(s) 2023 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/20552076231164239 journals.sagepub.com/home/dhj Álvaro Costa-García1 , Shotaro Okajima1, Ningjia Yang1 and Shingo Shimoda1
+
+## Abstract
+
+Objective: In this study, we propose a method for removing artifacts from superficial electromyography (sEMG) data, which have been widely proposed for health monitoring because they encompass the basic neuromuscular processes underlying human motion.
+
+Methods: Our method is based on a spectral source decomposition from single-channel data using a non-negative matrix factorization. The algorithm is validated with two data sets: the first contained muscle activity coupled to artificially generated noises and the second comprised signals recorded under fully unsupervised conditions. Algorithm performance was further assessed by comparison with other state-of-the-art approaches for noise removal using a single channel.
+
+Results: The comparison of methods shows that the proposed algorithm achieves the highest performance on the noiseremoval process in terms of signal-to-noise ratio reconstruction, root means square error, and correlation coefficient with the original muscle activity. Moreover, the spectral distribution of the extracted sources shows high correlation with the noise sources traditionally associated to sEMG recordings.
+
+Conclusion: This research shows the ability of spectral source separation to detect and remove noise sources coupled to sEMG signals recorded during unsupervised daily activities which opens the door to the implementation of sEMG recording during daily activities for motor and health monitoring. Keywords Electromyography, daily logging, muscle activity, health monitoring, artifact removal Submission date: 10 October 2022; Acceptance date: 1 March 2023
+
+## Introduction
+
+The adoption of new technologies by large population groups has paved the way for the emergence of what is known as the "digital health era", in which people are encouraged to take up new digital media technologies to engage in health self-monitoring.1,2 This trend has been further accentuated since 2020, when the COVID-19 pandemic triggered a situation in which people were requested to socially distance and reduce visits to hospitals and other health centers.3,4 Governments and health organizations have promoted the development and implementation of self-monitoring networks to reduce health care system burden and improve societal health.5–7 Feasible self-monitoring is usually based on cheap and easy-to-use technologies that actually target societal needs. Some examples are self-measurement of blood glucose by people with diabetes8 and heart beat monitoring for athletes and people with heart disease.9,10 It is likely that new self-monitoring technologies will be adopted by the general populace in the coming years. 1Intelligent Behavior Control Unit, RIKEN Institute, Nagoya, Japan Corresponding author: Álvaro Costa-García, Intelligent Behavior Control Unit, RIKEN Institute, Nagoya, Japan. Email: alvaro.costagarcia@riken.jp Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/
+open-access-at-sage).
+
+![1_image_0.png](1_image_0.png)
+
+![1_image_1.png](1_image_1.png)
+
+The monitoring of muscle activity during daily life has been proposed for the early diagnosis and treatment of the age-related loss of muscle mass known as sarcopenia,11 head, neck, and back pain,12 and anxiety.13 Moreover, superficial electromyography (sEMG) has been extensively studied and correlated to the basic neuromuscular processes underlying human motion.14 For these reasons, daily logging of sEMG signals will likely play an important role in the future of health monitoring.
+
+Although the recording of muscle activity during daily life presents several challenges,15 sEMG can now be acquired using wearable and increasingly inexpensive devices.16–19 In general, sEMG signals present a high signal-to-noise ratio (SNR) compared with other physiological signals such as electroencephalography and magnetic resonance images.
+
+In the laboratory, it is relatively simple to create experimental conditions in which artifacts affecting sEMG are
+
+![2_image_0.png](2_image_0.png)
+
+suppressed or controlled. However, it becomes more challenging in the analysis of large amounts of data recorded during unsupervised daily tasks. The wide range of environmental and behavioral changes during daily activities generates huge variability in signal quality because the noises affecting muscle recordings during these conditions present heterogeneous distributions and are difficult to predict. Therefore, the identification and removal of noises during unsupervised recordings are one of the main challenges facing the implementation of daily health logging systems based on this technology.
+
+Noise sources for sEMG signals are mainly divided into three groups: white Gaussian noise (WGN), which has an equally distributed spectrum; power line interference (PLI), which mainly affects 50/60 Hz (depending on the country);
+and low-frequency artifacts (LFAs), which include electrocardiography (for electrodes around the chest) and a range of disturbances produced by motion (e.g. electrode vibration, hanging wires, and wireless data loss).20,21 Raw sEMG signals have a bandwidth in the approximate range of 10–200 Hz.22,23 Regular bandpass filtering is normally accepted to reduce LFAs and WGN while a
+
+![3_image_0.png](3_image_0.png)
+
+common technique to remove PLI is the application of a 50/60-Hz Notch filter.24,25 However, this approach is not time discriminant; that is, it requires a priori knowledge of the noises affecting the signals and also assumes that the noises have a homogeneous distribution and temporal modulation. These assumptions can be true under controlled conditions but should not be made during unsupervised recordings.
+
+Previous studies with high-density sEMG (HD-EMG)
+arrays have proposed noise removal methods based on source separation approaches such as canonical correlation analysis21,26 and independent component analysis (ICA).27 Their aim was to separate sEMG from noise components by using redundant information recorded from several electrodes placed on the same muscle. These approaches are strongly dependent on the amount of spatial information available.
+
+For daily logging of sEMG data, the use of HD arrays is not always a feasible option. Apart from the increased cost associated with HD-EMG arrays, not all muscles are large enough to allow multiple simultaneous sEMG recordings. Aware of this issue, Mijovic et al. proposed a generalized noise source separation method from single-channel biological signal recordings that combines empirical-mode decomposition (EMD) (to decompose a signal into spectrally separated sources) and ICA (to extract statistically independent sources).28 This technique was successfully validated with experimental data recorded under laboratory conditions and compared with other similar approaches. However, EMD decomposition assumes that the spectral components extracted are present throughout the signal under analysis. In this case, the limitations of EMD analysis during fully unsupervised conditions are unclear.
+
+However, it is clear that the development and validation of a method for sEMG artifact removal during fully unsupervised activities is currently lacking a state-of-the-art application of this technology. For this reason, the current work proposes a novel algorithm for the analysis of sEMG data with the two following main goals: (a) the effective detection of noise sources from data recorded during fully unsupervised conditions, and (b) the development of a noise removal technique that allows the discrimination of valid sEMG data from large and highly contaminated signals.
+
+Our approach targets the separation of muscle activity from noise sources by using a non-negative matrix factorization (NMF) over the signal spectrum. To perform such a decomposition, the NMF algorithm extracts common spectral patterns from a number of signals. Redundancy is achieved by the temporal segmentation of the signal into overlapping epochs. The data used for source extraction are the normalized spectral magnitude of those epochs.
+
+This article describes the proposed methodology and compares its performance with that of regular bandpass filtering and of the method proposed by Mijovic et al.28 Two different data sets were used to validate methodology performance. A
+set of sEMG signals combined with artificially created noises was used to compare the signal reconstruction performance
+
+![4_image_0.png](4_image_0.png)
+
+among methods. In addition, to show the effects of each method on real data, a second set of signals recorded during fully unsupervised conditions was used.
+
+## Materials And Methods Conceptual Source Decomposition
+
+In general, most of the power of raw sEMG signals is contained in a frequency range between 10 and 200 Hz,22,23 with an approximate energy distribution as presented in Figure 1(A). Although the noise sources might overlap the spectral distribution of sEMG data, they present independent modulation. This means that, with enough information, the spectrum of sEMG signals contaminated with different noise sources (Figure 1(B)) can be decomposed into signal and noise components. Figure 1(C) shows an approximate graphical representation of the spectral distribution associated with the three main noise sources affecting sEMG signals
+(LFAs, PLI, and WGN).21 The present work focuses on the detection and removal of these sources by using redundant information extracted from temporally shifted signal epochs.
+
+## General Workflow
+
+Figure 2 shows the general workflow of the methodology presented in this work. First, single-/multi-channel signals
+
+![5_image_0.png](5_image_0.png)
+
+are segmented (Figure 2—segmentation) and the spectrum of each epoch is computed and normalized (Figure 2—spectral computation). Standardized spectrums undergo a NMF
+where the noise sources are decoupled from the sEMG spectrum (Figure 2—source separation). Then, noise source contributions are removed from each epoch, leaving only the contribution related to sEMG activation (Figure 2—filter design/noise removal). Clean epochs are shifted back to the time domain and the final signal is reconstructed by the temporal arrangement of all of the clean epochs. The technical details of each stage are provided in Appendix 1.
+
+## Noise Removal
+
+After source decomposition, the spectrum of each epoch
+(Epe(f)) can be reconstructed (REpe(f)) as the linear combination of one sEMG source and three noise sources, as in:
+REpe(f) = sEMGSourcee(f) + NoiseSourcese(f); (1)
+where
+
+$$s E M G S o u r c e_{e}(f)=s E M G(f)\cdot W_{e}^{s E M G};$$
+e ; (2)
+$$N o i s e S o u r c e s_{e}(f)=W G N(f)\cdot W_{e}^{W G N}+L F A(f)$$
+$$\cdot W_{\epsilon}^{L F A}+P L I(f)\cdot W_{\epsilon}^{s E M G},$$
+e ; (3)
+where sEMG(f), WGN(f), LFA(f), and PLI(f) are the sources associated with sEMG and each noise, respectively, and WsEMG
+e , WWGN
+e , WLFA
+e , and WPLI
+e are the weights of each source associated with the epoch e. From this source-based reconstruction, it is possible to define an epoch-specific filter that removes the noise contributions from each frequency of the original epoch (Epe(f)). The filter is described by the following equation:
+
+$$S F_{e}(f)=1-{\frac{N o i s e S a u r c e s_{e}(f)}{R E p_{e}(f)}}$$
+$$(4)$$
+
+Finally, the clean spectrum of each epoch e is computed as the element-by-element multiplication of the original epoch spectrum Epe(f) by the epoch-specific filter SFpe(f), as in:
+
+$$C l e a n E p_{e}(f)=E p_{e}(f)\cdot S F_{e}(f)$$
+CleanEpe(f) = Epe(f) · SFe(f) (5)
+Method comparison
+To test the proposed methodology, the extraction and removal of noise sources was compared among three different methods. The first comprised a regular bandpass and notch filtering (hereinafter, RFilt), the second was based on the approach introduced in this work (hereinafter, FFT-NMF),
+and the third used the EMD-ICA approach previously introduced in the literature.28 The technical details of the parameter tuning for each method can be found in Appendix 2.
+
+The NMF and ICA source separation techniques require a priori selection of the number of sources from which data will be decomposed. Four sources were selected in both methods (FFT-NMF and EMD-ICA) (one source representing the sEMG data and three sources representing WGN, PLI, and LFA noises). Both source separation algorithms return
+
+![6_image_0.png](6_image_0.png)
+
+randomly arranged components. Therefore, to avoid biases generated by a visual component arrangement, the sources extracted were rearranged according to their similarity to the model spectrums shown in Figure 3. These signals are a simplifi cation of the spectral magnitudes associated with each expected spectral source. Similarity was tested by computing
+
+![7_image_0.png](7_image_0.png)
+
+the coefficient correlation matrix between the sources extracted from each session and the modeled spectral distributions. The highest coefficient values were used to rearrange the data.
+
+Two data sets were used to validate and compare methodology performance. The first data set included single-channel sessions in which sEMG data recorded under laboratory conditions were combined with different distributions of artificially generated noise sources (including LFAs, PLI, and WGN). The second data set comprised sessions of sEMG data recorded during unsupervised daily activities.
+
+## Data Set 1: Artificial Noises Artificial Noise Data Generation
+
+Figure 4 describes the process followed to include artificial noises into sEMG data recorded under laboratory conditions. A 10-min session of sEMG recordings was combined with three types of noises with spectral distributions corresponding to those associated with LFA, PLI, and WGN
+noises.
+
+Noise source modulation and their temporal features were randomized to achieve the unstable behavior expected from unsupervised recordings. Figure 5 describes the details for noise generation and sEMG data acquisition. Figure 5(A) shows a 10-min sEMG signal recorded from the sternocleidomastoid muscle of a healthy subject under laboratory conditions. The subject was instructed to sit in a chair and wait for 10 min. During the waiting period, the subject was allowed to perform any desired postural changes and head motions. The fast-Fourier transform
+(FFT) of the data presented in the right graph of Figure 5(A) shows the spectral distribution of the power of the data.
+
+Figure 5(B) shows the elements required for noise generation. Each noise source was described by two vectors.
+
+The first was a 10-min normalized noise source (NNS) in which the spectral distribution corresponded with one of the three main noises affecting sEMG data. The second was a 10-min vector defining the noise appearance periods and modulating its amplitude (NAV) during the 10-min session. The element-by-element multiplication of both vectors defined the features and behavior of a single noise source during a single session. Figure 5(C) shows the process followed to generate the NNS. First, a random 10-min vector was generated with values between 1 and
+−1 (top-left graph—Figure 5(C)). The spectrum of this vector was computed through a FFT and divided into its magnitude and phase (note that its magnitude behaves as WGN with an equally distributed spectrum). The noise source spectral distributions modeled in Figure 3 were used to fit the computed magnitude into the spectral distribution expected for LFAs, PLI, or WGN. The modified magnitude and its original phase were used to reconstruct the signal in the temporal domain by an inverse FFT (IFFT). Finally, the temporal signal was normalized between 1 and −1.
+
+For NAV generation (Figure 5(D)), several parameters were considered. Each noise source was set to be active for 80% of the total session duration (8 out of 10 min).
+
+Moreover, to decide the length and amplitude of each activation period, two vectors were used. These vectors,
+
+![8_image_0.png](8_image_0.png)
+
+amplitude vector (AV), and length vector (LV), were defined as:
+
+$$(6)$$
+
+REpe(f) = [MaxAmp, MaxAmp − incA, ... , MinAmp];
+(6)
+
+$AV=[MaxAmp,\ MaxAmp-incA,\ \ldots,\ MinAmp];$ (6)
+$$M i n A m p=\frac{3}{4}\cdot s E M G_{p95};$$ $$M a x A m p=1.5\cdot s E M G_{p95};$$
+incA = MaxAmp − MinAmp 99 ; (9)
+LV = [MaxLen, MaxLen − incL, ... , MinLen]; (6)
+$$\begin{array}{l}\mbox{MinAmp}=\frac{3}{4}\cdot\mbox{sEMG}_{p95};\\ \mbox{MaxAmp}=1.5\cdot\mbox{sEMG}_{p95};\\ \mbox{incL}=\frac{\mbox{MaxLen}-\mbox{MinLen}}{99};\end{array}\tag{9}$$
+(7)  $\frac{1}{2}$  . 
+$${\mathrm{(8)}}$$
+$$(9)$$
+where sEMGp95 is the 95th percentile of the rectified amplitude from the sEMG signal and EMGLen is the length of
+
+![9_image_0.png](9_image_0.png)
+
+the session (10 min). Both vectors contain 100 values arranged in a descending order for AV and an ascending order for LV. Figure 5(D) summarizes the algorithm work-flow to generate the NAV. First, a random amplitude and length are chosen from AV and LV, respectively. In addition, a random starting sample is chosen from the number of samples within the total length of the data. If these randomly chosen values allow the creation of an activation period that fits within the session time and that do not overlap with any other activation period, it gets included in the NAV. The algorithm keeps repeating this loop, adding new activation periods, until the total time of noise activation reaches 80% of the signal length. In addition, a stability index (SI) was defined as a number between 1 and 100. The SI was used to tune the range of available values in AV and LV as:
+
+$$A V_{\small{\small{available}}}=A V[S I{\small{:}}e n d];$$ $$L V_{\small{\small{available}}}=L V[S I{\small{:}}e n d];$$
+
+Therefore, for an SI=100, there will be a single value available for both vectors (AVavailable = AV[100] = MinAmp and LVavailable = LV[100] = MaxLen) and for SI=1, all amplitude and length values defined in equations (6) and
+(10) will be available. Modification of this parameter allows tuning of the noise variability both in amplitude and active time.
+
+## Data Set Generation
+
+The process described in Figure 4 was repeated 100 times for each SI = [10, 20, 30, 40, 50, 60, 70, 80, 90, 100],
+giving a total amount of 1000 10-min sessions in which different distributions of LFA, PLI, and WGN sources were added to the sEMG signal.
+
+## Method Performance Validation
+
+$$(10)$$
+$$(11)$$
+
+The results obtained from this data set were used to compare the performances of each method regarding the separation of sEMG signals from noise sources. For that purpose, the original SNR data were compared to the SNR predicted from each method after source decomposition. Efficiency in signal and noise reconstruction was quantified by two parameters. The first parameter was the root mean square error (RMSE), computed as:
+
+$$R M S E={\sqrt{a v g((S_{0}-S_{r})^{2})}};$$
+$$(12)^{\frac{1}{2}}$$
+; (12)
+
+![10_image_0.png](10_image_0.png)
+
+where avg((S0 − Sr)
+2) is the averaged value of the square difference between the original signal So and the reconstructed signal Sr. This parameter quantifies the energy difference between two signals. The second parameter was the correlation coefficient between the original and reconstructed recordings. This coefficient quantifies, with a value between
+−1 and 1, the similarity in the shapes of the two vectors.
+
+## Data Set 2: Unsupervised Semg Recordings Experimental Set-Up
+
+Unsupervised sEMG data were acquired using a medium density band recording 8 channels from 16 bipolar electrodes (Figure 6(B)) with a sampling frequency of 2000 Hz. A single electrode located in the area of the sternocleidomastoid muscle was selected for analysis. The system included a belt pouch with a battery providing autonomy of around 10 h. Every 10 min, a data file was stored in a SD card (Figure 6(C)).
+
+## Participants
+
+Five patients with neck pain were requested to wear the sEMG band shown in Figure 6(A). They were instructed on how to set up the recording device and all of them provided signed informed consent according to the Declaration of Helsinki. No further instructions were given, and the participants were asked to continue with their normal daily activities.
+
+## Data Set
+
+Each patient recorded around 5 h of muscle activity. Thirty 10-min recordings were extracted from each patient, giving a total of 150 10-min sessions (30 sessions × 5 patients).
+
+## Data Evaluation
+
+This data set was used to visualize the spectral distribution of sEMG and the noise sources extracted by the RFilt, FFT-NMF, and EMD-ICA methods during real unsupervised sEMG recordings. Moreover, the separability between spectral component pairs was quantified for each method as their Bhattacharyya distance (Bdist), first introduced by Bhattacharyya et al.29 This parameter is closely related to the Bhattacharyya coefficient, which measures the amount of overlap between two statistical samples or populations.
+
+Bdist can provide any value in the range 0 < Bdist < ∞,
+with larger values associated with higher class separability.
+
+## Details Of The Study
+
+Patients' sEMG data was fully recorded by them during their daily lives in Saitama and Tokyo Prefectures in Japan during periods that varied between 4 and 17 days.
+
+The conceptualization of the experiment, generation of artificial noises, and recordings of clean sEMG signals were performed at the same time in the facilities of Riken in Nagoya. Data analysis, results extraction, and interpretation were done during the following months also in Riken's laboratory in Nagoya.
+
+## Results Semg Reconstruction Performance
+
+The signal/noise decomposition performance for each method was assessed by using the data set including real sEMG data coupled to artificially created noises (data set 1) in which the original sources and SNR were known. Figure 7(A) compares the SNR of the original data with that predicted from source decomposition using the RFilt, FFT-NMF, and EMD-ICA methods. Each boxplot represents the SNRs of 100 10-min sessions associated with different values of the SI, as shown on the x-axis (original SNR,
+blue; FFT-NMF-predicted SNR, green; EMD-ICA-predicted SNR, red; RFilt-predicted SNR, orange). As designed, the real SNR (blue) decreases with an increasing variability of noise appearance. The predicted SNRs presented significant differences for the RFilt, FFT-NMF, and EMD-ICA methods, both compared with original SNR and among each other. However, the SNR predicted by the FFT-NMF algorithm exhibited higher accuracy and less variability that predicted by the RFilt and EMD-ICA methods, which showed a significantly higher standard deviation for all values of the SI. Moreover, the RFilt method presented significantly lower performance in SNR prediction and was also more affected by noise stability than the methods based on source decomposition.
+
+Figure 7(B) and (C) uses the same boxplot format to show the RMSE and correlation coefficients between the original sEMG data and the sEMG data reconstructed by each method (FFT-NMF, green; EMD-ICA, red; and RFilt, orange). For each SI value, the FFT-NMF method showed less RMSE (error recovering the energy of the original data) and a higher correlation coefficient (shape similarity to original data) than the RFilt and EMD-ICA
+methods. The increase in the noise variability (a decrease in the SI) produced, in the three methods, a decrease in the energy and shape reconstruction performance. This phenomenon was stronger in the sEMG data extracted using the RFilt method. Moreover, the FFT-NMF algorithm showed a more stable reconstruction (lower standard deviation)
+for lower stability values (0.7≥SI≥0.1).
+
+## Real Data Analysis
+
+Extracted sources. Data set 2 was used to compare the signal decomposition results from the FFT-NMF and EMD-ICA algorithms applied to data recorded from the sternocleidomastoid muscle during unsupervised tasks. In Figure 8(A) and (B), the four spectral components extracted using each method are presented according to their median
+(red), 25th and 75th percentiles (blue), and maximum and minimum values (back) computed from the set of 150 sessions (30 10-min session×5 subjects). Spectral components were arranged according to their correlation coefficient with the sources modeled in Figure 3 that represent a simplification of the spectral distributions expected for the sEMG
+and LFA, WGN, and PLI sources. Figure 8(C) shows the Bhattacharyya distances computed between each pair of components extracted using each method. The spectral distributions of sources extracted using the FFT-NMF method showed higher similarity with the conceptual decomposition of the data into four sources representing the sEMG data and WGN, PLI, and LFA sources. Moreover, Bhattacharyya distances exhibited higher class separability for component pairs extracted using the FFT-NMF method. Figure 8(D) shows an example of the FFT-NMF algorithm reconstructing the original sEMG signals recorded under a controlled environment from versus their version where with the addition of artificial noises were added.
+
+Clean data and sEMG estimation. Figure 9 shows the results of the use of the FFT-NMF algorithm to remove the noise contributions from two 10-min recordings from the sternocleidomastoid muscle during fully unsupervised tasks. The graphs on the left show the raw data (top graph), the clean sEMG signal (blue) +original signal reconstruction (OSR) (orange) (middle graph), and the epoch-wise predicted SNR (bottom graph). The right graphs show a zoom-in of selected periods that allows a clear visualization of the effects of the noise removal technique. Both for raw signals (two upper graphs) and clean data (two lower graphs), a time/amplitude and spectrogram representation are shown. Three signal periods labeled as 1∗, 2∗, and 3∗ are highlighted from both examples to discuss algorithm effects. 1∗ shows an example in which the main contribution of the algorithm is the removal of WGN (affecting mainly the higher frequency bands), allowing a clear visualization of the muscle activity after cleaning. 2∗ shows an example in which the strong coupling of PLI is removed by the algorithm, leaving only the part in which a small sEMG activation happened. 3∗ shows an example of the ability of the FFT-NMF algorithm to removing the appearance of a LFA that produced low-frequency variations in the raw data. The OSR ratio and SNR provide extra information about the relative power between the predicted sEMG signals and noises. Both ratios can be used for a time-wise estimation of the physiological meaning of cleaned signals.
+
+Finally, Figure 10 shows an example of the RFilt, FFT-NMF, and EMD-ICA methods applied to a highly contaminated session where most of the recorded data was noise.
+
+The blue data represent the raw recorded data while the
+
+## Discussion
+
+orange data represent the signal after noise removal for each method. The effects on the spectral domain can be seen in the respective FFTs presented in the right graphs.
+
+should carefully check the origins of the LFA noises coupled to their signals. Given the multi-origin nature of these noises, the fine-tuning of their reference spectral distribution could be vital for the proper detection and removal of these artifacts.
+
+## Conclusion
+
+This research shows the ability of spectral source separation to detect and remove noise sources coupled to sEMG signals recorded during unsupervised daily activities. The algorithm presented showed a better performance than traditional and other state-of-the-art methodologies for noise removal, which opens the door to the implementation of sEMG recording during daily activities for motor and health monitoring.
+
+As mentioned in the Introduction, social trends toward subject engagement with self-health monitoring and the development of increasingly cheap and wearable sEMG devices put this technology in the early mass adoption stage. Under this scenario, the first big challenge of people in charge of sEMG big data analysis will be artifact removal and discrimination of valid muscle activity. In this regard, the current methodology was developed as a tool for the treatment of large sets of sEMG data recorded during fully unsupervised daily activities. Our method provides a robust basis for the preliminary treatment and classification of data recorded under highly variable conditions.
+
+Nevertheless, there are still several challenges that remain to be addressed to further improve this implementation. One of the outstanding challenges is in the development and inclusion of hardware-specific modules in the amplification stage of sEMG sensors that apply the proposed algorithm during recording. Moreover, future researches must address the characterization of the spectral distribution of other potential noises affecting sEMG signals.
+
+Finally, in future works, this methodology will be used to treat sEMG signals recorded during the daily lives of people in age ranges where muscle conditions such as back and neck pain, and sarcopenia present their first symptoms, potentially enabling for their early detection and treatment. The effects of artifact reduction on the later stage of signal processing and analysis of sEMG signals must also be discussed according to the results of future works. Acknowledgments: The authors would like to thank the Toyota Motor Corporation for their promotion of this work and the Saitama Neuropsychiatric Institute for assisting with recruitment of participants for this research.
+
+Authors' Note: Álvaro Costa-García, National Institute of Advance Industrial Science and Technology, Chiba, Kashiwa, Japan; Shotaro To the best of our knowledge, the current work presents the first method for the effective detection and removal of noise sources from sEMG recordings during fully unsupervised daily activities. The method can be fully automated after establishment of the number of sources (four in the case of this article) and their spectral distributions (Figure 3). Depending on the data set under analysis, the number of sources and the model of their spectral distribution can be tuned to increase method performance.
+
+The method was compared to traditional signal filtering and to a state-of-the-art approach (EMD-ICA) previously introduced in the literature.28 The FFT-NMF method showed higher performance both when reconstructing the original sEMG data and when predicting the SNR
+(Figure 7). The segmentation of the original data into overlapping epochs generates sufficient signal redundancy for the extraction of common spectral sources, which allows its application to both single- and multi-channel recordings. Moreover, epoching decouples time and frequency domains, which improves the detection of sources whose contributions are not stable in time. The results also show that traditional noise removal (RFilt) is more affected by higher noise variabilities than FFT-NMF and EMD-ICA methods (Figure 7). The sources extracted with the FFT-NMF algorithm present a spectral distribution that correlates with the main noises affecting sEMG data
+(Figure 8(A)).20 Such a correlation was not found in the sources extracted using the EMD-ICA method, at least when applied to fully unsupervised recordings
+(Figure 8(B)). The epoch-specific filters generated by the FFT-NMF method allow fine-tuning of the spectral bands and power reduction in accordance with the noises affecting the data in each time period (Figure 9).
+
+In addition, OSR and SNR indices allow the detection of temporal periods of interest that can be used to estimate the physiological meaning of the cleaned signals. Finally, the FFT-NMF algorithm allows a signal reconstruction for illustrating which spectral features present a similar distribution to the one expected from sEMG signals (Figure 10).
+
+The effectiveness of the current algorithm is strongly related to the number of noises coupled to the sEMG signals because the redundancy in their spectral distributions over time allows the extraction of the spectral pattern associated with each noise. The algorithm might present limitations if applied to large data sets that present a very low amount of coupled noises because there will not be enough redundant noise information to extract common spectral patterns between epochs. Moreover, researchers interested in applying the current methodology Okajima, Nagoya University, Aichi, Nagoya, Japan; Shingo Shimoda, Nagoya University, Aichi, Nagoya, Japan.
+
+Contributorship: ACG and SS researched the literature and conceived the study. ACG, NY, and SO were involved in methodology development, data recording, and analysis. SO and SS contributed to the recruitment of participants. ACG wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.
+
+Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
+
+Ethical approval: The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Riken (Reference Wako3 28-13). Informed consent was obtained from all participants involved in the study.
+
+Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Toyota Motor Corporation and a JSPS KAKENHI grant (number 18K18431).
+
+Guarantor: ACG accepts responsibility for the overall integrity of the manuscript (including ethics, data handling, reporting of results, and study conduct).
+
+ORCID iDs: Álvaro Costa-García https://orcid.org/0000-00030097-2793 References 1. Lupton D. The digitally engaged patient: self-monitoring and self-care in the digital health era. Soc Theory Health 2013; 11:
+256–270.
+
+2. Lancaster K, Abuzour A, Khaira M, et al. The use and effects of electronic health tools for patient self-monitoring and reporting of outcomes following medication use: systematic review. J Med Internet Res 2018; 20: e294.
+
+3. Chakraborty I and Maity P. COVID-19 outbreak: migration, effects on society, global environment and prevention. Sci Total Environ 2020; 728: 138882.
+
+4. Bayoumy AB, Veugen CC, van Rijssen LB, et al.
+
+Self-monitoring of the tympanic membrane: an opportunity for telemedicine during times of COVID-19 and beyond. J Otol 2021; 16: 120–122.
+
+5. Wahlqvist ML. Self-monitoring networks for personal and societal health: dietary patterns, activities, blood pressure and Covid-19. Asia Pac J Clin Nutr 2020; 29: 446–449.
+
+6. Lim HM, Teo CH, Ng CJ, et al. An automated patient selfmonitoring system to reduce health care system burden during the COVID-19 pandemic in Malaysia: development and implementation study. JMIR Med Inform 2021; 9:
+e23427.
+
+7. Whiting P and Elwenspoek M. Accuracy of self-monitoring heart rate, respiratory rate and oxygen saturation in patients with symptoms suggestive of covid infection.
+
+8. Benjamin EM. Self-monitoring of blood glucose: the basics.
+
+Clin Diabetes 2002; 20: 45–47.
+
+9. Dooley EE, Golaszewski NM and Bartholomew JB.
+
+Estimating accuracy at exercise intensities: a comparative study of self-monitoring heart rate and physical activity wearable devices. JMIR Mhealth Uhealth 2017; 5: e7043.
+
+10. Forerunner G and Fuse M. Comparison of wearables for selfmonitoring of heart rate in coronary rehabilitation patients.
+
+Georgian Medical 2021; 315: 78–85.
+
+11. Caroppo A, Rescio G, Leone A, et al. A surface electromyographybased platform for the evaluation of sarcopenia.
+
+12. Hart JD and Cichanski KA. A comparison of frontal EMG biofeedback and neck EMG biofeedback in the treatment of musclecontraction headache. Biofeedback Self Regul 1981; 6: 63–74.
+
+13. Viens DA. The effects of self-monitoring, emg biofeedback, and relaxation tapes in the treatment of tension headaches.
+
+14. Merletti R and Farina D (eds). Surface electromyography:
+physiology, engineering, and applications. Hoboken, New Jersey, USA: John Wiley & Sons, 2016.
+
+15. Milosevic B, Benatti S and Farella E. Design challenges for wearable EMG applications. InDesign, Automation & Test in Europe Conference & Exhibition (DATE), 2017 2017 Mar 27 (pp. 1432-1437). IEEE.
+
+16. Cerone GL, Botter A and Gazzoni M. A modular, smart, and wearable system for high density sEMG detection. IEEE Trans Biomed Eng 2019; 66: 3371–3380.
+
+17. Pino EJ, Arias Y and Aqueveque P. Wearable EMG shirt for upper limb training. In2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) 2018 Jul 18 (pp. 4406-4409). IEEE.
+
+18. Yamaguchi T, Mikami S, Maeda M, et al. Portable and wearable electromyographic devices for the assessment of sleep bruxism and awake bruxism: a literature review. CRANIO®
+2020; 41(1): 69–77.
+
+19. Michelsen JS, Lund MC, Alkjær T, et al. Wearable electromyography recordings during daily life activities in children with cerebral palsy. Dev Med Child Neurol 2020; 62: 714–722.
+
+20. Clancy EA, Morin EL and Merletti R. Sampling, noisereduction and amplitude estimation issues in surface electromyography. J Electromyogr Kinesiol 2002; 12: 1–6.
+
+21. Al Harrach M, Boudaoud S, Hassan M, et al. Denoising of HD-sEMG signals using canonical correlation analysis. Med Biol Eng Comput 2017; 55: 375–388.
+
+22. Wakeling JM and Rozitis AI. Spectral properties of myoelectric signals from different motor units in the leg extensor muscles. J Exp Biol 2004; 207: 2519–2528.
+
+23. Von Tscharner V and Goepfert B. Estimation of the interplay between groups of fast and slow muscle fibers of the tibialis anterior and gastrocnemius muscle while running. J
+Electromyogr Kinesiol 2006; 16: 188–197.
+
+24. De Luca CJ, Gilmore LD, Kuznetsov M, et al. Filtering the surface EMG signal: movement artifact and baseline noise contamination. J Biomech 2010; 43: 1573–1579.
+
+25. Wang J, Tang L and Bronlund JE. Surface EMG signal amplification and filtering. Int J Comput Appl 2013; 82: 15–22.
+
+26. Anand D, Bhateja V, Srivastava A, et al. An approach for the preprocessing of EMG signals using canonical correlation analysis. InSmart Computing and Informatics: Proceedings of the First International Conference on SCI 2016, Volume 2 2018 (pp. 201-208). Springer Singapore.
+
+27. Azzerboni B, Carpentieri M, La Foresta F, et al. Neural-ICA
+and wavelet transform for artifacts removal in surface EMG. In2004 IEEE International Joint Conference on Neural Networks (IEEE Cat. No. 04CH37541) 2004 Jul 25 (Vol. 4, pp. 3223-3228). IEEE.
+
+28. Mijović B, De Vos M, Gligorijević I, et al. Source separation from single-channel recordings by combining empirical-mode decomposition and independent component analysis. IEEE Trans Biomed Eng 2010; 57: 2188–2196.
+
+29. Bhattacharyya A. On a measure of divergence between two multinomial populations. Sankhyā: The Indian J Stat 1946; 7(4): 401–406.
+
+30. Zhang Y and Fang Y. A NMF algorithm for blind separation of uncorrelated signals. In2007 International Conference on Wavelet Analysis and Pattern Recognition 2007 Nov 2 (Vol. 3, pp. 999-1003). IEEE.
+
+## Appendix 1 Fft-Nmf Method Stages
+
+Segmentation. Signals are divided into L sample epochs with O sample overlap. The length of the window (L) can be modified depending on the bandwidth of interest and the sampling frequency and following the Nyquist criteria
+(Fmax < Fsamp/2). The selection of the overlap size (O)
+will depend on the desired temporal accuracy. Wider overlaps will result in a larger number of epochs, which leads to an increase in the amount of redundant information for the source separation stage by also increasing the computational demands.
+
+Spectrum computation. The spectrum of each epoch is computed through the FFT and divided into its magnitude
+(MAG) and phase (PHA). Phases are kept for the temporal reconstruction of the data (see the "Signal reconstruction" section) and the magnitudes are normalized as:
+
+$$N M a g_{e}={\frac{M a g_{e}}{\sum_{i=1}^{n}M a g_{e}^{i}}}$$
+$$(17)$$
+
+where Mage is a vector containing the spectral magnitude of the epoch e, NMage is a vector containing i normalized frequencies, and n is the total number of frequencies under analysis (i = 1, 2, 3, ... , n Hz). This process normalizes the NMage area to 1. The goal is to remove the amplitude variations and emphasize the shape of the spectrum. While the amplitude of noises might experience large variations during unsupervised recordings, their spectral shape will present higher stability. Therefore, it is expected that the extraction of components by the source separation algorithm should perform better when applied to normalized spectral shapes. The amplitude information associated with each epoch e (ae = n i=1 Magie) will be used for filter design during the noise removal stage (see the
+"Noise removal" section).
+
+Source separation. To decompose the data into x = 1 + N
+sources (one sEMG component and the N noise components),
+the set of normalized spectral epochs was described as:
+
+$$E=W\cdot S\tag{18}$$  $E\in R^{\rm c\!xf},\ \ W\in R^{\rm c\!cx},\ \ S\in R^{\rm x\!cf}$ (19)
+where E is an e × f matrix of normalized spectrums (with e the number of epochs and f the frequencies under analysis), W
+is an e × x matrix containing the weights used to reduce the e epochs to an x-dimensional space, and S is an x × f matrix containing the shape of the x spectral sources. Matrices W and S can be calculated from E through the NMF algorithm by fixing the x-dimensionality. The NMF algorithm extracts the S component matrix by minimizing the correlation between them.30 Signal reconstruction. Signal reconstruction is divided into two stages. In the first stage, the clean spectral magnitude
+(CMage) and the original phase (PHAe) of each epoch e are used to reconstruct their temporal information through an IFFT. This inverse transformation applied to discrete data results in the appearance of amplitude spikes at the beginning and end of each epoch that were not present in the original signal (Figure 11(A)). In the second stage, the time series of the clean epochs are recombined to reconstruct the sEMG signal. To remove the spikes appearing in the temporal reconstruction of each epoch, two recombination strategies are proposed (Figure 11(B) and (C)).
+
+The first strategy (Figure 11(B)) requires that, during the segmentation process, the overlap length is selected as half of the window length (O =L/2). This allows two reconstructions of the original signal, either using the even epochs or using the odd epochs. The spikes of each reconstructed signal will be shifted O =L/2 samples. The final sEMG
+data are reconstructed by substituting the spike samples of the signal reconstructed from even epochs with the same temporal samples of the signal reconstructed from odd epochs (which do not include the spikes). The second reconstruction strategy requires larger overlaps between segments
+(O > L/2) so that the sEMG signal can be reconstructed an X
+number of times, as presented in Figure 11(C). Once all of the signals are aligned in time, the final sEMG reconstruction is obtained by computing the sample-by-sample median value across all of the extracted vectors. Although this second strategy uses more information for an accurate signal reconstruction, the increased overlap produces an increased number of epochs, which leads to higher computational demands during the source separation stage.
+
+Estimation of the sEMG contribution. To perform a time-wise identification of the SNR, the following index was computed:
+
+## Snre = Cmage Nmage(20)
+
+where SNRe is the ratio of the epoch e between the nonnormalized spectral magnitude CMage after noise removal and the summation of the non-normalized spectral magnitudes of the noise sources (see NoiseSourcese(f) in equation
+(3)). Moreover, the ratio between the signal before and after denoising was computed as:
+
+$$O S R_{e}={\frac{C M a g_{e}}{M a g_{e}}}$$
+Mage(21)
+where OSRe is the ratio of the epoch e between the nonnormalized spectral magnitudes CMage (after noise removal) and Mage (before noise removal). This index represents the percentage of the contribution of the predicted muscle activity to the original signal.
+
+## Appendix 2 Method Parameters
+
+Rfilt method. Each 10-min session was bandpass-filtered between 10 and 200 Hz to isolate the frequency range commonly associated with sEMG activity.22,23 In addition, a Notch filter was applied at 50, 100, and 150 Hz to remove the main contribution and harmonics of PLI.
+
+FFT-NMF method. The parameters for the application of the FFT-NMF method are the following:
+
+$$(21)$$
+
+Segmentation stage. Window size (L) of 2000 samples
+(1 s) and overlap (O) of 1000 samples (0.5 s).
+
+Spectral computation stage. Evaluation of the spectral range between 1 and 1000 Hz to meet Nyquist criteria.
+
+Normalized spectral magnitudes were submitted to the source separation stage.
+
+Source separation stage. Signals were decomposed into four different sources (x=4). One source represented muscle activity while the other three sources represented the main types of noise affecting sEMG signals (LFAs, PLI, and WGN).
+
+Reconstruction stage. To keep the computational demands low during the source separation stage, the first reconstruction strategy (see Appendix 1) was applied.
+
+sEMG identification. SNR and OSR (see Appendix 2)
+were computed by comparing the original data and the clean data to provide a quantifiable threshold for the discrimination of valid sEMG signals.
+
+EMD-ICA method. Temporal data from each 10-min session was decomposed into physically significant components based on EMD. ICA was applied over these modes to obtain four statistically significant sources (one representing the sEMG data and three representing WGN, PLI, and LFA noises). The spectral magnitude of the four sources extracted using the ICA were computed through a FFT with a resolution of one sample per frequency (from 1 to 1000 Hz) to compare the extracted sources in the same domain as the ones extracted using the FFT-NMF method.

medical/md/PMC10099623.md

@@ -0,0 +1,286 @@
+DOI: 10.1111/ene.15629 
+
+# Sex Differences In Imaging And Clinical Characteristics Of Patients From The Wake-Up Trial
+
+Anke Wouters1,2,3 | Lauranne Scheldeman2,3,4 | Hannelore **Liessens4** | Patrick Dupont5,6 | Florent Boutitie7 | Bastian Cheng8 | Martin **Ebinger9,10** | Matthias Endres9,11,12,13,14 | Jochen B. Fiebach9 | Christian Gerloff8 | Keith W. **Muir15** | Norbert Nighoghossian16 | Salvador Pedraza17 | Claus Z. **Simonsen18** | Vincent Thijs19,20 | Götz Thomalla8 | Robin **Lemmens2,3,4**
+1Neurology, Amsterdam University Medical Centers location AMC, Amsterdam, the Netherlands 2Department of Neurosciences, Experimental Neurology, KU Leuven - University of Leuven, Leuven, Belgium 3Center for Brain & Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium 4Department of Neurology, University Hospitals Leuven, Leuven, Belgium 5Department of Neurosciences, Laboratory for Cognitive Neurology, KU Leuven - University of Leuven, Leuven, Belgium 6Leuven Brain Institute, Leuven, Belgium 7Hospices Civils de Lyon, Service de Biostatistique, Université Lyon, Lyon, France 8Klinik und Poliklinik für Neurologie, Kopf- und Neurozentrum, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 9Center for Stroke Research Berlin (CSB), Charité - Universitätsmedizin Berlin, Berlin, Germany 10Klinik für Neurologie, Medical Park Berlin Humboldtmühle, Berlin, Germany 11Klinik und Hochschulambulanz für Neurologie, Charité– Universitätsmedizin Berlin, Berlin, Germany 12German Center for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany 13German Center for Neurodegenerative Diseases (DZNE), partner site Berlin, Berlin, Germany 14ExcellenceCluster NeuroCure, Berlin, Germany 15Institute of Neuroscience & Psychology, University of Glasgow, Glasgow, UK 16Department of Stroke Medicine, Université Claude Bernard Lyon 1; Hospices Civils de Lyon, Lyon, France 17Department of Radiology, Institut de Diagnostic per la Image (IDI), Hospital Dr Josep Trueta, Institut d'Investigació Biomedica de Girona (IDIBGI), Parc Hospitalari Marti i Julia de Salt - Edifici M2, Girona, Spain 18Department of Neurology, Aarhus University Hospital, Aarhus, Denmark 19Stroke Theme, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Heidelberg, Victoria, Australia 20Department of Neurology, Austin Health, Heidelberg, Victoria, Australia Correspondence Anke Wouters, Herestraat 49, 3000 Leuven, Belgium. Email: anke.wouters@kuleuven.be
+
+## Abstract
+
+Background and purpose: Sex-based differences in acute ischemic stroke are a wellknown phenomenon. We aimed to explore these differences between women and men in the Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke (WAKE-UP) trial. Methods: We compared baseline demographic and imaging characteristics (visual fluidattenuated inversion recovery [FLAIR] positivity, relative FLAIR signal intensity, collateral status) between women and men in all screened patients. In randomized patients (i.e., those with Anke Wouters and Lauranne Scheldeman contributed equally. 
+
+This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. *European Journal of Neurology* published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.
+
+diffusion-weighted imaging (DWI)–FLAIR mismatch), we evaluated a modifying role of sex on the treatment effect of alteplase in multivariable logistic regression, with treatment adjusted for National Institute of Health Stroke Scale (NIHSS) score and age. Dependent variables were modified Rankin Scale (mRS) score of 0–1 at 90 days and distribution of mRS scores at 90 days. Results: Of 1362 screened patients, 529 (38.8%) were women. Women were older than men, had higher baseline NIHSS scores and smoked less frequently. FLAIR positivity of the DWI lesion was equally present in women (174/529, 33.1%) and men (273/833, 33.3%; 
+p = 1.00) and other imaging variables also did not differ between the sexes. In a total of 503 randomized patients, of whom 178 were women (35.4%), sex did not modify the treatment effect of alteplase on mRS score 0–1 or on the total distribution of mRS scores.
+
+Conclusion: As in many other stroke trials, more men than women were included in the WAKE-UP trial, but the presence of a visual DWI–FLAIR mismatch and the relative FLAIR 
+signal intensity did not differ between the sexes. The treatment effect of alteplase was not modified by sex.
+
+KEYWORDS
+ischemic stroke, sex, MRI, wake-up
+
+## Introduction
+
+Sex-based differences in the acute stroke setting have been described. Women more often present with non-typical and more severe stroke symptoms and with stroke mimics [1–4]. Furthermore, they may have longer delays from stroke onset to emergency department arrival as well as longer door-to-imaging times, and receive treatment with alteplase less often, despite having no difference in overall treatment eligibility [5–15]. Visual fluid-attenuated inversion recovery (FLAIR) positivity and the relative FLAIR (rFLAIR) signal intensity within the area of a diffusion-weighted imaging (DWI) lesion are related to the time from symptom onset [16, 17]. Therefore differences between men and women with regard to these imaging variables in unknown stroke onset patients could be assumed but are unknown. Furthermore, men and women have similar outcomes after treatment with intravenous recombinant tissue plasminogen activator, but a stronger treatment effect may exist in women and they might have worse outcomes if acute stoke is untreated [18–21]. However, there is substantial between-study variability and more recent studies suggest that the gap between men and women has narrowed over recent years [6, 10]. In this post hoc analysis of the Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke (WAKE-UP) trial [22], we explored sex-based differences in baseline imaging (FLAIR lesion, DWI lesion, rFLAIR signal intensity and quality of collateral circulation) and clinical characteristics in screened patients, and outcome in randomized women versus men.
+
+## Patients And Methods Study Design And Patient Population
+
+In this post hoc analysis, we studied sex-based differences in demographic, clinical, and imaging baseline characteristics and clinical outcome in patients screened and randomized in the WAKE-UP trial. The WAKE-UP trial was a multicenter, randomized, doubleblind, placebo-controlled trial, designed to investigate if patients with an unknown time of stroke onset benefit from thrombolysis in the presence of a DWI–FLAIR mismatch [22]. Patients or their legal representatives provided written informed consent according to national and local regulations. The trial was approved for each study site by the competent authorities and the corresponding ethics committee. Patients were screened for the presence of a DWI– FLAIR mismatch with magnetic resonance imaging (MRI; screened patients) and randomized to placebo or alteplase if this mismatch was present (randomized patients). Only randomized patients received follow-up with imaging at 24–36 hours and documentation of clinical outcome.
+
+## Analysis Of Imaging Characteristics
+
+As in the original WAKE-UP trial, acute MRI images of the screened patients were visually rated for the presence of a DWI-positive lesion on the B1000 images and for a FLAIR hyperintense signal (i.e., FLAIR-positive) within the region of the acute stroke. The DWI– FLAIR mismatch was defined as a DWI-positive lesion with no signal alterations on the FLAIR [22]. Furthermore, we investigated the maturation of the FLAIR lesion in men versus women in screened patients in the WAKE-UP trial who had FLAIR of sufficient quality at baseline available. The rFLAIR signal intensity was calculated as described previously [23]. To summarize, the rFLAIR signal intensity was calculated as the ratio of the FLAIR signal intensity in one voxel and median FLAIR signal intensity of all voxels within a sphere with radius of 15 mm around a homologue voxel in the contralateral hemisphere. We defined the ischemic core lesion and its volume using RAPID software, which calculates the ischemic core lesion based on the apparent diffusion coefficient threshold <620 × 10−6mm2/s. 
+
+Only patients with a core lesion volume >10 ml were included for further analysis.
+
+To investigate sex-based differences in the quality of the collateral circulation, we studied the hypoperfusion intensity ratio (HIR) 
+in patients screened in the WAKE-UP trial who also underwent perfusion-weighted imaging [24, 25]. We used RAPID software (iSchemaView) to calculate the perfusion lesion, defined as tissue with a time to the maximum of the residue function (Tmax) of >6 s. 
+
+For patients with a minimal perfusion lesion of 10 ml, we calculated the HIR as the ratio of the lesion volume with Tmax >10 s and the lesion volume with a Tmax >6 s. We dichotomized the HIR at a threshold of 0.5 to define collateral status: <0.5 as an indicator of good and 
+≥0.5 of poor collaterals [25].
+
+## Statistical Analysis
+
+The baseline characteristics were compared between men and women in the screened and randomized population using twosample t-tests for parametric data, Wilcoxon rank-sum tests for nonparametric data, and chi-squared tests for categorical data.
+
+In randomized patients (i.e., those with the DWI–FLAIR mismatch), we evaluated the influence of sex on treatment effect of alteplase in multivariable logistic regression with treatment, sex and their interaction term as independent variables (adjusted for NIHSS 
+score >10 and age >60 years, as in the original trial) [22] to predict good outcome, defined as a score on the modified Rankin Scale (mRS) of 0–1 at 90 days, and mortality at 90 days. The categorical shift in distribution of the mRS scores was studied with proportional odds logistic regression analysis, adjusted for the same variables. p values < 0.05 were taken to indicate statistical significance. All the analyses were conducted in R.
+
+## Results Baseline Demographic, Clinical And Imaging Characteristics In Screened Patients
+
+In the population of 1,362 screened patients, more men (833; 61.2%) were screened than women (p< 0.01). Women were older, had higher baseline NIHSS score, higher baseline mRS score and smoked less frequently (Table 1). Furthermore, they presented more often with aphasia compared to men (9.3% vs. 5.3%; p = 0.01). 
+
+A trend towards a lower proportion of women being randomized compared to men was not statistically significant (178/529 women, 33.6% vs. 325/833 men, 39.0%; p = 0.05). The presence of a hyperintense FLAIR signal within the DWI lesion did not differ between women (174/529, 33.1%) and men (273/833, 33.3%; p = 1.00). Also, the proportion of screened patients without a DWI-positive lesion was similar between the sexes (27.6% of women vs. 24.6% of men; p = 0.52; Table 1).
+
+TABLE 1 Baseline characteristics of the screened patients.
+
+Women 
+
+(n = **529)**
+
+Men 
+
+(n = 833) p **value**
+
+Age, years 70 (61–76) 67 (57–74) <0.01
+
+Arterial hypertension 288 (54.9%) 421 (50.9%) 0.30 Atrial fibrillation 47 (9.0%) 60 (7.3%) 0.53 Hypercholesterolemia 157 (29.9%) 272 (32.9%) 0.52 Type 2 diabetes 75 (14.3%) 149 (18.3%) 0.20
+
+Smoking 89 (18.4%) 223 (29.6%) <0.01
+
+Pre-stroke mRS score 0.04
+
+0 422 (80.1%) 698 (84.9%) 1 99 (18.8%) 112 (13.6%) 2 2 (0.4%) 7 (0.9%) 3 3 (0.6%) 1 (0.1%)
+
+4 1 (0.2%) 4 (0.5%)
+
+Median NIHSS score 7 (4–12) 5 (3–9) <0.01
+
+Reason for unknown 
+
+time of symptom onset
+
+Night-sleep 445 (85.1%) 734 (88.9%) Day-sleep 20 (3.8%) 24 (2.9%) Aphasia 49 (9.3%) 44 (5.3%) Other 5 (1.0%) 16 (1.9%)
+
+Randomization rate 178 (33.6%) 325 (39.0%) 0.05 FLAIR positivity 174 (33.1%) 273 (33.3%) 1.00 DWI negativity 97 (27.6%) 125 (24.6%) 0.52
+
+Note: Data are median (IQR) or n (%).
+
+Abbreviations: DWI, diffusion-weighted imaging; FLAIR, fluidattenuated inversion recovery; IQR, interquartile range; mRS, Modified Rankin Scale; NIHSS, National Institute Health Stroke Scale.
+
+Analysis of the rFLAIR signal intensity (as a proxy of time from symptom onset to imaging) was successful in 307 patients with min-
+
+| 0.03   |
+|--------|
+
+imal core lesion of 10 ml. The mean rFLAIR signal intensity did not differ in women (ratio 1.08, interquartile range [IQR] 1.05–1.13) versus men (ratio 1.07, IQR 1.03–1.12; p = 0.31).
+
+Of 1,362 screened patients, 186 had successful RAPID analysis of perfusion-weighted imaging and a minimal Tmax >6 s lesion of 10 ml. An HIR ≥0.5 suggestive of poorer collateral status did not differ significantly between men and women (40/110 women, 36.3%, vs. 
+
+22/76 men, 28.9%; p = 0.37).
+
+## Patient Characteristics, Outcome And Treatment Effect In Randomized Patients
+
+Of 503 randomized patients, 178 were female (35.4%). Baseline and outcome characteristics for men and women are presented in Table 2. Similarly to the screened population, women were older, had higher baseline NIHSS scores and smoked less frequently. Of all TABLE 2 Baseline and outcome characteristics of the randomized patients.
+
+Women 
+
+(n = 178)
+
+Men 
+
+(n = 325) p **value**
+
+Age, years 70 (63–76) 67 (58–73) 0.02 Arterial hypertension 101 (56.7%) 165 (50.8%) 0.20 Atrial fibrillation 29 (16.3%) 30 (9.2%) 0.06 Hypercholesterolemia 67 (37.6%) 111 (34.2%) 0.38 Type 2 diabetes 24 (13.5%) 58 (17.8%) 0.13 Smoking 42 (24.1%) 91 (29.2%) 0.01 Median NIHSS score 7 (4–11) 5 (3–9) 0.01 Reason for unknown 
+
+time of symptom onset
+
+| randomized patients. Reason for unknown  time of symptom  onset Core volume at  baseline, mla Hemorrhage type PH1c Hemorrhage type PH2c   |
+|-------------------------------------------------------------------------------------------------------------------------------------------|
+
+Night-sleep 156 (87.6%) 293 (90.2%) Day-sleep 10 (5.6%) 13 (4.0%) Aphasia 9 (5.1%) 12 (3.7%) Other 3 (1.7%) 7 (2.1%)
+
+Treatment with IV rtPA 89 (50.0%) 165 (50.8%) 0.94 Core volume at 
+
+baseline, mla
+
+2.5 (0.96–11) 2.1 (0.73–7.5) 0.14
+
+mRS scoreb 2 (1–3) 2 (1–3) 0.69 mRS score 0–1b 76 (44.2%) 157 (49.4%) 0.32
+
+Mortalityb 5 (2.9%) 8 (2.5%) 1.00
+
+Overall 6 (3.4%) 7 (2.2%) 0.36 Group with IV rtPA 3 (3.4%) 4 (2.4%) 0.83 Overall 2 (1.1%) 8 (2.5%) 0.29 Group with IV rtPA 1 (1.1%) 8 (4.8%) 0.28
+
+Note: Data are median (IQR) or n (%). Abbreviations: IQR, interquartile range; IV rtPA, intravenous recombinant tissue plasminogen activator; mRS, modified Rankin Scale; NIHSS, National Institute Health Stroke Scale.
+
+aFourteen patients had missing data.
+
+bThirteen patients were lost to follow-up. Outcome parameters were assessed at Day 90.
+
+cSeven patients had missing data.
+
+| 0.67   |
+|--------|
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+randomized patients, 249 received placebo (35.7% women, 64.3% men) and 254 received recombinant tissue plasminogen activator (35.0% women, 65.0% men). No day 90 outcome was available for 13 patients lost to follow-up. There was no difference between women and men in the rate of good outcome (44.2% vs. 49.4%; p = 0.32), in mortality (2.9% vs. 2.5%; p = 1.00) or in the distribution of mRS 
+scores at 90 days (adjusted odds ratio [aOR] 1.14, 95% confidence interval [CI] 0.82–1.60; Figure 1). Although the effect of thrombolysis on mRS score 0–1 was significant in men (aOR 1.79 95% CI 
+1.10–2.91; p = 0.02) but not in women (aOR 1.36, 95% CI 0.74–2.59; p = 0.35), sex did not modify the treatment effect of alteplase on mRS score 0–1 (p = 0.57 for interaction) or on the total distribution of the mRS scores (p = 0.75 for interaction; Figure 2).
+
+## Discussion
+
+In this post hoc analysis of the WAKE-UP trial, we found that the proportion of women screened for inclusion in the trial was lower compared to men. Moreover, women were older, had increased stroke severity at admission and smoked less often than men. Presence of DWI–FLAIR mismatch was similar in women and men and there were similar proportions of patients that had no DWI lesion on baseline imaging. Also, we found no difference in other imaging variables, such as rFLAIR signal intensity and quality of the collateral circulation. Outcomes were similar between men and women, and sex did not modify the treatment effect of alteplase on clinical outcome.
+
+The literature consistently reports that women are older at stroke onset [2, 4, 26]. The most obvious explanation is that women live longer than men, in concert with a higher incidence of stroke with aging. The higher severity of stroke on admission has been attributed to women more often presenting with proximal occlusions, anterior circulation stroke and cardio-embolic strokes, which are more frequently severe compared to other stroke mechanisms [13, 27, 28]. Another explanation might be older age and women more often having worse premorbid functional status [8, 28]. In our population of screened patients, women were indeed older and presented with a higher pre-stroke mRS score. Furthermore, women presented more often with aphasia, possibly reflected in the higher NIHSS score. This sex disparity regarding language disturbance at presentation has been described previously [29, 30]. A smaller stroke lesion volume threshold to cause aphasia for women as compared to men has been postulated as a potential cause [31]. The lower incidence of smoking in female WAKE-UP patients is also consistent with previous literature [32]. Despite the non-significant results in our study, there was also a trend towards a higher frequency of hypertension and atrial fibrillation in women, as described in the literature [5, 8].
+
+Collateral circulation as assessed by the HIR was not different between women and men. A recent study showed better collaterals in women, associated with better functional outcome. Women had smaller perfusion lesions, less ischemic growth, and smaller final infarct volumes [33]. Similar results of better collateral circulation, smaller baseline core volumes, and slower ischemic core growth among women were reported in DEFUSE 3 [34]. In WAKE-UP, we included a more heterogeneous population. Many patients had small DWI and perfusion lesions and only approximately one-third of patients presented with large vessel occlusions, hampering a robust analysis of collateral status by sex [22].
+
+Fewer women than men were screened with MRI for the presence of a DWI–FLAIR mismatch. One explanation for this could be that women are older when presenting with stroke, exceeding the upper age limit of 80 years as defined by the inclusion criteria in the original WAKE-UP trial. Furthermore, poorer premorbid functionality similarly could have led to exclusion of more women than men because WAKE-UP excluded patients with an mRS score >1 [28]. 
+
+Also the number of women presenting with a wake-up stroke could have been lower compared to men due to different stroke mechanisms. However, because data are lacking on the total number of women presenting to the sites during the trial, this remains speculative. Since sites did not document reasons for not screening patients with unknown stroke onset, the possibility of more atypical symptoms and more risk averseness in women compared to men, although reported by others, could not be assessed [26, 35]. A higher frequency of stroke mimics could be a possible explanation for fewer women being randomized in an MRI-based stroke study. However, in our analysis, lack of DWI lesion as a reason for exclusion was equal between the sexes, which suggests similar presentation of stroke mimics (although some of these patients could have had transient ischemic attacks without a DWI lesion). An overview of sex representation in different fields of stroke research found a similar under-representation of women [36]. The average percentage of female participants is approximately 40% but the reasons for this under-representation in randomized controlled stroke trials has not been extensively investigated [32]. More equal sex representation in randomized controlled trials is desirable but in order to achieve this, better understanding of the factors that limit participation of women is needed. Our data indicate no difference in eligibility based on imaging features.
+
+The presence of a DWI–FLAIR mismatch was equal in women and men. As reports exist on longer times from stroke onset to emergency department arrival in women, we explored the visual FLAIR positivity and rFLAIR signal intensity within the DWI lesion because this is known to increase with time after symptom onset [9, 17]. However, we did not identify more FLAIR-positive lesions, neither did we identify an increase in the rFLAIR signal intensity in women, arguing against a delay between stroke onset to time of imaging in women with wake-up stroke.
+
+The treatment effect of alteplase on clinical outcome in patients with unknown onset of stroke and a DWI–FLAIR mismatch was not modified by sex. The rate of excellent functional outcome and mortality was comparable between men and women. There is heterogeneity in the literature about the differences in outcome or effect of alteplase between men and women. Some authors report similar outcomes at hospital discharge and 90-day survival [4, 19], whereas others report worse functional outcome [27] and higher mortality rate in women [5], or even a more favourable in-hospital recovery in women compared to men and higher likeliness of favourable functional outcome at discharge [13]. Different pathophysiological and hormonal-based theories are described to explain the sex differences [19, 20, 27]. Interestingly, sex differences were no longer present in many studies after correction for age, stroke severity and premorbid function [28]. In the WAKE-UP trial, an upper age limit of 80 years and minimal premorbid functionality state were predefined inclusion criteria, possibly reducing the confounding effect of these variables. Furthermore, the analysis of the interaction between sex and treatment effect was adjusted for age and NIHSS score. This revealed the lack of a modifying effect of sex on clinical outcomes in this population.
+
+Our study has some limitations. First of all, we had no insights into the rationale of each local investigator to decide not to screen patients with unknown stroke onset time with MRI, nor on the total number of female stroke patients presenting to the different study sites. Furthermore, we only had outcome data from the population of randomized patients because screened patients had no follow-up after documentation of the absence of a DWI–FLAIR mismatch. Therefore, we cannot exclude sex-based variation in outcome for wake-up stroke patients without the DWI–FLAIR mismatch at admission. In the population of randomized patients, subgroups may have been too small to study the treatment effect of alteplase in men and women separately.
+
+In conclusion, in the WAKE-UP trial, fewer women than men were included in MRI screening. Women were older, had higher prestroke mRS scores, higher NIHSS scores at admission, and smoked less frequently. Imaging characteristics did not differ between the sexes. In this post hoc analysis, there was no effect of sex on the clinical outcome after treatment with alteplase.
+
+## Author Contributions
+
+lecturer from Acandis, Alexion, Amarin, Bayer, Boehringer Ingelheim, BristolMyersSquibb, Daichii Sankyo, Portola and Stryker. K.W.M. reports fees as a consultant from Boehringer Ingelheim, Abbvie and Biogen, and institutional support from Boehringer Ingelheim for the ATTEST-2 trial, all outside the submitted work. V.T. reports personal fees and non-financial support from Boehringer Ingelheim, Pfizer/ BMS, Bayer, Sygnis, Amgen and Allergan outside the submitted work. All remaining authors report nothing to disclose.
+
+## Data Availability Statement
+
+The data that support the findings of this study are available from the corresponding author upon reasonable request.
+
+## Orcid
+
+Anke Wouters https://orcid.org/0000-0001-5229-2699 Lauranne Scheldeman https://orcid.org/0000-0002-5263-3550 Keith W. Muir https://orcid.org/0000-0001-9535-022X
+Norbert Nighoghossian https://orcid.org/0000-0003-0594-4409 Anke Wouters: Conceptualization; Investigation; Writing—original draft; Methodology; Visualization. Lauranne Scheldeman: Investigation; Writing—original draft; Methodology; Conceptualization; Visualization. Hannelore Liessens: Writing—original draft; Conceptualization. Patrick Dupont: Writing—review and editing; Methodology. Florent Boutitie: Writing—review and editing; Methodology. Bastian Cheng: Writingreview and editing; Data curation. Martin Ebinger: Writing—review and editing. Matthias Endres: Writing—review and editing. Jochen B Fiebach: Writing—review and editing. Christian Gerloff: Writingreview and editing. Keith W Muir: Writing—review and editing. Norbert Nighoghossian: Writing—review and editing. Salvador Pedraza: Writing—review and editing. Claus Z Simonsen: Writing—review and editing. Vincent Thijs: Writing—review and editing. Götz Thomalla: Writing—review and editing. Robin Lemmens: Writing—review and editing; Supervision; Methodology; Conceptualization; Investigation.
+
+## Funding Information
+
+The WAKE-UP trial was supported by a grant (278276) from the European Union Seventh Framework Program. M.E. received funding from DFG under Germany´s Excellence Strategy—EXC- 2049—390688087, BMBF, DZNE, DZHK, EU, Corona Foundation and Fondation Leducq. R.L. is a senior clinical investigator for Research foundation Flanders.
+
+## Conflicts Of Interest
+
+L.S. reports grants from Research Foundation Flanders during the conduct of the study (L.S. is supported by Research Foundation Flanders, PhD fellowship fundamental research 1193620N) and other (congress participation) from Daiichi Sankyo outside the submitted work. R.L. has no personal disclosures, but reports consultancy fees paid to KU Leuven from Ischemaview and Boehringer Ingelheim. J.B.F. reports outside the submitted work personal fees from Abbvie, AC Immune, Artemida, Bioclinica/Clario, Biogen, BMS, Brainomix, Cerevast, Daiichi-Sankyo, Eisai, F.Hoffmann-La Roche AG, Eli Lilly, Guerbet, Ionis Pharmaceuticals, IQVIA, Janssen, Julius clinical, jung diagnostics, Lysogene, Merck, Nicolab, Premier Research and Tau Rx. C.Z.S. reports research grants from Novo Nordisk Foundation and Health Research Foundation of Central Denmark Region. M.End reports grants from Bayer and fees paid to the Charité from AstraZeneca, Bayer, Boehringer Ingelheim, BMS, Daiichi Sankyo, Amgen, GSK, Sanofi, Covidien, Novartis and Pfizer, all outside the submitted work. G.T. reports fees as a consultant or 
+
+## References
+
+1. Stuart-Shor EM, Wellenius GA, Dellolacono DM, Mittleman MA. 
+
+Gender differences in presenting and prodromal stroke symptoms. Stroke. 2009;40:1121-1126.
+
+2. Gall SL, Donnan G, Dewey HM, et al. Sex differences in presentation, severity, and management of stroke in a population-based study. *Neurology*. 2010;74:975-981.
+
+3. Artto V, Putaala J, Strbian D, et al. Stroke mimics and intravenous thrombolysis. *Ann Emerg Med*. 2012;59:27-32.
+
+4. Fraticelli L, Freyssenge J, Claustre C, et al. Sex-related differences in management and outcome of acute ischemic stroke in eligible patients to thrombolysis. *Cerebrovasc Dis*. 2019;47:196-203.
+
+5. Park SJ, Do SS, Ro YS, Song KJ, Oh J. Gender differences in emergency stroke care and hospital outcome in acute ischemic stroke: a multicenter observational study. *Am J Emerg Med*. 2013;31:178-184.
+
+6. Reeves M, Bhatt A, Jajou P, Brown M, Lisabeth L. Sex differences in the use of intravenous rt-PA thrombolysis treatment for acute ischemic stroke: a meta-analysis. *Stroke*. 2009;40:1743-1749.
+
+7. Burton KR, Kapral MK, Li S, et al. Predictors of diagnostic neuroimaging delays among adults presenting with symptoms suggestive of acute stroke in Ontario: a prospective cohort study. *Can Med Assoc* Open Access J. 2016;4:E331-E337.
+
+8. Asdaghi N, Romano JG, Wang K, et al. Sex disparities in ischemic stroke care: FL-PR CReSD study (Florida-Puerto Rico Collaboration to Reduce Stroke Disparities). *Stroke*. 2016;47:2618-2626.
+
+9. Mainz J, Andersen G, Valentin JB, Gude MF, Johnsen SP. 
+
+Disentangling sex differences in use of reperfusion therapy in patients with acute ischemic stroke. *Stroke*. 2020;51:2332-2338.
+
+10. Strong B, Lisabeth LD, Reeves M. Sex differences in IV thrombolysis treatment for acute ischemic stroke: a systematic review and meta-analysis. *Neurology*. 2020;95:e11-e22.
+
+11. Fredwall M, Sternberg S, Blackhurst D, Lee A, Leacock R, 
+Nathaniel TI. Gender differences in exclusion criteria for recombinant tissue-type plasminogen activator. *J stroke Cerebrovasc Dis*. 
+
+2016;25:2569-2574.
+
+12. Nagaraja N, Olasoji EB, Patel UK. Sex and racial disparity in utilization and outcomes of t-PA and thrombectomy in acute ischemic stroke. *J stroke Cerebrovasc Dis*. 2020;29:104954.
+
+13. Bonkhoff AK, Karch A, Weber R, Wellmann J, Berger K. Female stroke: sex differences in acute treatment and early outcomes of acute ischemic stroke. *Stroke*. 2021;52:406-415.
+
+27. Reeves MJ, Bushnell CD, Howard G, et al. Sex differences in stroke: 
+epidemiology, clinical presentation, medical care, and outcomes. Lancet Neurol. 2008;7:915-926.
+
+28. Manwani B, McCullough LD. On the basis of sex. *Stroke*. 
+
+2019;50:2285-2287.
+
+29. Hier DB, Yoon WB, Mohr JP, Price TR, Wolf PA. Gender and aphasia in the stroke data bank. *Brain and Language*. 1994;47:155-167.
+
+30. Di Carlo A, Lamassa M, Baldereschi M, et al. Sex differences in the clinical presentation, resource use, and 3-month outcome of acute stroke in Europe: Data from a multicenter multinational hospitalbased registry. *Stroke*. 2003;34:1114-1119.
+
+31. Bonkhoff AK, Schirmer MD, Bretzner M, et al. Outcome after acute ischemic stroke is linked to sex-specific lesion patterns. *Nat* Commun. 2021;12:1-14.
+
+32. Carcel C, Woodward M, Wang X, Bushnell C, Sandset EC. Sex matters in stroke: a review of recent evidence on the differences be-tween women and men. *Front Neuroendocrinol*. 2020;59:100870.
+
+33. Demeestere J, Christensen S, Mlynash M, et al. Effect of sex on clinical outcome and imaging after endovascular treatment of largevessel ischemic stroke. *J Stroke Cerebrovasc Dis*. 2021;30:105468.
+
+34. Dula AN, Mlynash M, Zuck ND, Albers GW, Warach SJ, DEFUSE 3 Investigators. Neuroimaging in ischemic stroke is different between men and women in the DEFUSE 3 cohort. *Stroke*. 2020;51:481-488.
+
+35. Kapral MK, Devon J, Winter A-L, Wang J, Peters A, Bondy SJ. Gender differences in stroke care decision-making. *Med Care*. 2006;44:70-80.
+
+36. Tsivgoulis G, Katsanos AH, Caso V. Under-representation of women in stroke randomized controlled trials: Inadvertent selection bias leading to suboptimal conclusions. *Ther Adv Neurol Disord*. 
+
+2017;10:241-244.
+
+14. De Silva DA, Ebinger M, Davis SM. Gender issues in acute stroke thrombolysis. *J Clin Neurosci*. 2009;16:501-504.
+
+15. Tsivgoulis G, Katsanos AH, Malhotra K, et al. Thrombolysis for acute ischemic stroke in the unwitnessed or extended therapeutic time window. *Neurology*. 2020;94:e1241-e1248.
+
+16. Thomalla G, Cheng B, Ebinger M, et al. DWI-FLAIR mismatch for the identification of patients with acute ischaemic stroke within 4·5 h of symptom onset (PRE-FLAIR): a multicentre observational study. *Lancet Neurol*. 2011;10:978-986.
+
+17. Cheng B, Brinkmann M, Forkert ND, et al. Quantitative measurements of relative fluid-attenuated inversion recovery (FLAIR) signal intensities in acute stroke for the prediction of time from symptom onset. *J Cereb Blood Flow Metab*. 2013;33:76-84.
+
+18. Lindley RI, Wardlaw JM, Whiteley WN, et al. Alteplase for acute ischemic stroke: outcomes by clinically important subgroups in the Third International Stroke Trial. *Stroke*. 2015;46:746-756.
+
+19. Hametner C, Macisaac RL, Kellert L, Abdul-Rahim AH, Ringleb PA, 
+Lees KR. Sex and stroke in thrombolyzed patients and controls. Stroke. 2017;48:367-374.
+
+20. Kent DM, Price LL, Ringleb P, Hill MD, Selker HP. Sex-based differences in response to recombinant tissue plasminogen activator in acute ischemic stroke: a pooled analysis of randomized clinical trials. *Stroke*. 2005;36:62-65.
+
+21. Shobha N, Sylaja PN, Kapral MK, Fang J, Hill MD. Differences in stroke outcome based on sex. *Neurology*. 2010;74:767-771.
+
+22. Thomalla G, Simonsen CZ, Boutitie F, et al. MRI-guided thrombolysis for stroke with unknown time of onset. *N Engl J Med*. 
+
+2018;379:611-622.
+
+23. Scheldeman L, Wouters A, Dupont P, et al. Reversible edema in the penumbra correlates with severity of hypoperfusion. *Stroke*. 2021;52:2338-2346.
+
+24. Olivot JM, Mlynash M, Inoue M, et al. Hypoperfusion intensity ratio predicts infarct progression and functional outcome in the DEFUSE 2 Cohort. *Stroke*. 2014;45:1018-1023.
+
+25. Guenego A, Mlynash M, Christensen S, et al. Hypoperfusion ratio predicts infarct growth during transfer for thrombectomy. *Ann* Neurol. 2018;84:616-620.
+
+26. Berglund A, Schenck-Gustafsson K, von Euler M. Sex differences in the presentation of stroke. *Maturitas*. 2017;99:47-50.
+
+How to cite this article: Wouters A, Scheldeman L, 
+Liessens H, et al. Sex differences in imaging and clinical characteristics of patients from the WAKE-UP trial. *Eur* J *Neurol*. 2023;30:641-647. doi:10.1111/ene.15629

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+# Optimizing Point-Of-Care Testing Strategies For Diagnosis And Treatment Of Hepatitis C Virus Infection In Australia: A Modelbased Cost-Effectiveness Analysis
+
+Sophy T. F. Shih,a,∗ Qinglu Cheng,a Joanne Carson,a Heather Valerio,a Yumi Sheehan,a Richard T. Gray,a Evan B. Cunningham,a Jisoo A. Kwon,a Andrew R. Lloyd,a Gregory J. Dore,a Virginia Wiseman,a,b and Jason Grebelya aThe Kirby Institute, University of New South Wales, Level 6, Wallace Wurth Building, Sydney 2052, Australia bDepartment of Global Health and Development, London School of Hygiene and Tropical Medicine, London, UK
+Summary Background Timely diagnosis and treatment of hepatitis C virus (HCV) is critical to achieve elimination goals. This study evaluated the cost-effectiveness of point-of-care testing strategies for HCV compared to laboratory-based testing in standard-of-care. Methods Cost-effectiveness analyses were undertaken from the perspective of Australian Governments as funders by modelling point-of-care testing strategies compared to standard-of-care in needle and syringe programs, drug treatment clinics, and prisons. Point-of-care testing strategies included immediate point-of-care HCV RNA testing and combined point-of-care HCV antibody and reflex RNA testing for HCV antibody positive people (with and without consideration of previous treatment). Sensitivity analyses were performed to investigate the cost per treatment initiation with different testing strategies at different HCV antibody prevalence levels.
+
+Findings The average costs per HCV treatment initiation by point-of-care testing, from A$890 to A$1406, were up to 35% lower compared to standard-of-care ranging from A$1248 to A$1632 depending on settings. The average costs per treatment initiation by point-of-care testing for three settings ranged from A$1080 to A$1406 for RNA, A$960–
+A$1310 for combined antibody/RNA without treatment history consideration, and A$890–A$1189 for combined antibody/RNA with treatment history consideration. When HCV antibody prevalence was <74%, combined pointof-care HCV antibody and point-of-care RNA testing were the most cost-effective strategies. Modest increases in treatment uptake by 8%–31% were required for immediate point-of-care HCV RNA testing to achieve equivalent cost per treatment initiation compared to standard-of-care.
+
+Interpretation Point-of-care testing is more cost-effective than standard of care for populations at risk of HCV. Testing strategies combining point-of-care HCV antibody and RNA testing are likely to be cost-effective in most settings.
+
+Funding National Health and Medical Research Council. Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: HCV; Health economics; Modelling; Antibody testing; RNA testing Introduction The ability to meet the World Health Organization's hepatitis C virus (HCV) elimination targets is threatened by low diagnosis (23%)1 and treatment (5% of people diagnosed) globally.2 Increasing HCV testing uptake is hampered by current diagnostic pathways requiring multiple visits, contributing to loss to follow-up.3 In Australia, modelling suggests that HCV RNA testing must increase by 50% annually to achieve elimination by 2030.4 Traditional testing pathways involve an HCV antibody test to confirm exposure and an HCV RNA test to detect active infection. Point-of-care HCV RNA testing for detection of current HCV infection in 1 h has changed HCV clinical management,5,6 enabling diagnosis and treatment in a single-visit,7–11 increasing testing acceptability,12,13 reducing loss to follow-up7–11 and increasing treatment uptake.7–11 Point-of-care HCV
+RNA tests have good technical accuracy5,6,14 and clinical utility, with high treatment uptake following testing in needle and syringe programs (NSPs) (81%),7 supervised consumption sites (89%),9 mobile outreach models (74%),8 and prisons (control: 22%; intervention 93%).11
+*Corresponding author. University of New South Wales, Level 6, Wallace Wurth Building, Sydney 2052, Australia.
+
+E-mail address: sshih@kirby.unsw.edu.au (S.T.F. Shih).
+
+The Lancet Regional Health - Western Pacific 2023;36: 100750 Published Online 4 April 2023 https://doi.org/10.
+
+1016/j.lanwpc.2023. 100750
+
+## Research In Context
+
+Evidence before this study We searched several databases, including MEDLINE, Scopus, Web of Science, Cochrane Central Register of Controlled Trials, Embase, Cost-Effectiveness Analysis Registry, International HTA database, Econlit, and PsycINFO, up to 7 April 2022 by the search terms "Hepatitis C" OR "HCV" OR "hepacivirus", and "screen* OR "test" OR "tested" OR "testing" OR "treat*" OR "diagnos*", and "costs and cost analysis" OR "cost*" OR "healthcare resource*" OR "economic*" OR "QALY* OR "ICER* OR "life year*" OR "LY" OR "DALY". Collectively, four protocols of randomized controlled trials, feasibility and cohort studies involving point-of-care HCV testing were identified. Before the empirical evidence and trial-based cost-effectiveness were available, many modelling cost-effectiveness analyses suggested that HCV point-of-care testing could identify more cases, achieve more cures, lead to greater life expectancy with QALY gains. Most modelling studies indicated point-of-care testing was more expensive in the short-term but has the potential to lower the long-term lifetime costs by reducing HCV incidence and prevalence. Two papers reported results of HCV point-of-care testing combined with HIV testing or offered concomitantly to COVID-19 vaccination showing the prospect of point-of-care testing to identify undiagnosed infections at a reasonable cost and a feasible way to implement in practice. Added value of this study This study evaluated the cost-effectiveness of point-of-care testing strategies compared to standard of care in key Australian service settings for HCV elimination, including prisons, needle and syringe programs, and drug treatment clinics. Our analyses determined the cost-effective point-ofcare testing strategies for different HCV antibody prevalence However, point-of-care HCV RNA testing is more expensive than point-of-care antibody testing and has a longer time to result (60 min for an RNA result vs. 1–20 min for a negative antibody result and 1–15 min for a positive antibody result). Combining point-of-care HCV antibody and RNA testing could optimise testing outcomes, enhance cost-effectiveness and acceptability, without impact on effectiveness.
+
+Many modelling cost-effectiveness studies suggested that HCV point-of-care testing could identify more cases, achieve more cures, lead to greater life expectancy with QALY gains.15–24 However, studies exploring the cost-effectiveness of HCV point-of-care testing strategies are limited to particular populations (mostly low- and middle-income countries),18,19,24 have lacked detailed costing information,17–20 and have not included realworld epidemiological and clinical data to inform analyses.17–20 Understanding the HCV prevalence and settings where different point-of-care testing strategies should be used is critical to inform implementation, levels and the treatment uptake required for point-of-care testing to be more cost-effective than standard of care. This study identifies which point-of-care testing strategies are more cost-effective to implement across service settings and HCV prevalence and provides economic evidence to guide point-of-care testing to optimize treatment outcomes. This information is critical for the implementation and scale-up of point-of-care testing for HCV infection globally and will therefore inform clinical guidelines, clinical practice, and health policy, including national and international guidelines. Implications of all the available evidence Our findings suggested that all point-of-care HCV testing strategies had a lower average cost per treatment initiation compared to standard of care across all settings evaluated and HCV antibody prevalence. Based on HCV prevalence, different testing strategies should be used to optimize the testing and treatment outcomes. When HCV antibody prevalence was
+<74%, combined point-of-care HCV antibody and point-ofcare RNA testing (with and without consideration of treatment history) is shown to be the most cost-effective strategies. Modest increases in treatment uptake by 8%–31%
+were required for immediate point-of-care HCV RNA testing to achieve equivalent cost per treatment initiation compared to standard of care. Combining point-of-care HCV antibody and RNA testing could optimise testing outcomes, enhance cost-effectiveness by linking more people with current HCV to treatment, and improve patient acceptability by reducing time to receipt of results. This study is important for informing the future implementation and evaluation of point-of-care testing strategies to achieve HCV elimination targets.
+
+funding, and integration into policy/practice. There is a lack of economic evidence to guide implementation of different testing strategies to optimize the outcomes.
+
+This study evaluated the cost-effectiveness of pointof-care testing strategies including immediate point-ofcare HCV RNA testing and combined point-of-care HCV antibody and RNA testing for HCV antibody positive people (with and without consideration of previous treatment) compared to standard of care in key service settings for HCV elimination. This study determined the most cost-effective point-of-care testing strategies for different HCV antibody prevalence levels and the treatment uptake required for point-of-care testing to be more cost-effective than standard of care.
+
+## Methods Economic Evaluation
+
+This economic evaluation followed the Consolidated Health Economic Evaluation Reporting Standards 2022
+(CHEERS 2022) Statement.25 The PICO (Population, Intervention, Comparator, Outcomes) statement and evaluation parameters are presented in Supplementary Table S1.
+
+## Study Perspective
+
+Cost-effectiveness analysis was undertaken from the perspective of the Australian Governments as the funders responsible for the delivery and funding of HCV
+testing and treatment, recognising settings where healthcare provision is segregated (e.g., testing in prison is paid by the State government only and testing in the community is paid by the State and Federal governments).
+
+Study population and setting The target population was people at risk of HCV infection. Study settings included prisons, NSPs, and drug treatment clinics.
+
+Diagnostic strategies A. Point-of-care HCV RNA testing.
+
+B. Combined point-of-care HCV antibody and reflex point-of-care RNA testing (for antibody positive people).
+
+C. Combined point-of-care HCV antibody and reflex point-of-care RNA testing (for antibody positive people) in those self-reporting no previous HCV
+treatment and immediate point-of-care RNA testing in people self-reporting previous HCV treatment.
+
+D. Standard of care (on-site sample collection by venepuncture and laboratory-based HCV antibody followed by RNA testing if antibody positive).
+We assumed that people with detectable HCV RNA
+following point-of-care testing were offered same-visit treatment and people receiving standard of care required 2–3 visits for testing and treatment.26
+
+## Outcome Measured
+
+Outcomes included: 1) current HCV infection (detectable RNA); and 2) direct-acting antiviral (DAA) treatment initiation in people with current HCV infection.
+
+The time horizon of analysis was one year, thus discounting was not applied.
+
+## Analytic Methods
+
+Decision analytic models were constructed to assess the four HCV diagnostic strategies using the TreeAge software (Fig. 1).27 Further modelling details are provided in the Supplementary Materials. Costs and outcomes for point-of-care HCV testing strategies were compared to standard of care, considering HCV prevalence, treatment history, and test accuracy. Costs and outcomes among the three point-of-care testing strategies were also compared to investigate the most efficient way to conduct point-of-care HCV testing in each setting.
+
+Cost-effectiveness analyses estimated the average costs of diagnosis and treatment initiation by the four testing strategies in prisons, NSPs, and drug treatment clinics.
+
+Model parameters for outcomes Model parameters and estimate sources are presented in Table 1. HCV antibody and RNA prevalence, and selfreported treatment history were estimated based on Australian studies evaluating HCV testing, diagnosis and treatment in prisons, NSPs, and drug treatment clinics.28,29 Health outcomes were assessed under each testing strategy, including true positives identified and people with current infection linked to treatment. Assay diagnostic performance was sourced from peerreviewed literature and manufacturer specifications.
+
+## Costs
+
+An ingredient approach of costing was used to identify all resources required to perform testing and clinical assessment prior to treatment initiation. Costs of pointof-care RNA testing were estimated at three levels: 1)
+variable costs only (device, testing cartridge, consumables, and staff time); 2) direct costs including variable costs and fixed costs of IT/connectivity, quality assurance, and training; 3) total program costs including direct variable and fixed costs, and indirect program coordination/communication costs (Table 2 and Supplementary Table S2). Direct costs (variable and fixed costs for the operation of HCV detection, diagnosis, assessment and reporting as per clinical and public health requirements) were used in the base case analysis and sensitivity analysis were performed considering costs ranging from variable costs only (testing) to total program costs.
+
+Variable costs (tests and labour costs involved in testing/clinical assessment) were measured from clinical studies.7,11,28 Labour costs included nursing staff time (at different seniority levels according to tasks performed),30 infectious disease specialists, and correctional officers (in prisons). Capital cost for point-of-care testing device was proportionated to unit cost per test (annuitization discounted 3% per annum with 10 years effective life). Testing kit, testing cartridge, and consumables for point-of-care testing and IT/connectivity, quality assurance, training, and program coordination were estimated based on the financial information from the National Australian Hepatitis C Point-of-Care Testing Program (Supplementary Table S2).
+
+Costs associated with tests and clinical assessments prior to treatment initiation were considered, thus DAA pharmaceutical costs were excluded (Table 2). Clinical assessments costs included labour costs and costs of serology tests for hepatitis B virus and human immunodeficiency virus, liver function tests, full blood count, urea, electrolytes and creatine, pregnancy test (women only) and FibroScan. The Australian Medical Benefit Schedule was used for testing costs and services
+
+![3_image_0.png](3_image_0.png)
+
+| Parameter                                                                                                                                                                                                                                                                                                                                                                                                     | Setting              | Range for sensitivity Distribution in probabilistic   | Source            |             |            |                      |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|-------------------------------------------------------|-------------------|-------------|------------|----------------------|
+| analyses                                                                                                                                                                                                                                                                                                                                                                                                      | sensitivity analyses |                                                       |                   |             |            |                      |
+| Prison (95% CI)                                                                                                                                                                                                                                                                                                                                                                                               | Needle Syringe       | Drug treatment clinic                                 |                   |             |            |                      |
+| Program (95% CI)                                                                                                                                                                                                                                                                                                                                                                                              | (95% CI)             |                                                       |                   |             |            |                      |
+| Proportion of people tested                                                                                                                                                                                                                                                                                                                                                                                   | 1                    | 1                                                     | 1                 | 0.50–1.00   | Triangular | Assumptiona          |
+| HCV Antibody prevalence                                                                                                                                                                                                                                                                                                                                                                                       | 0.37 (0.33, 0.40)    | 0.61 (0.54, 0.68)                                     | 0.69 (0.66, 0.72) | 0.10–0.90   | Beta       | 28,29                |
+| HCV RNA (+) in HCV Antibody (+)                                                                                                                                                                                                                                                                                                                                                                               | 0.32 (0.26, 0.38)    | 0.30 (0.22, 0.38)                                     | 0.20 (0.17, 0.23) | 0.20–0.40   | Beta       | 28,29                |
+| HCV RNA prevalence                                                                                                                                                                                                                                                                                                                                                                                            | 0.12 (0.09, 0.14)    | 0.18 (0.13, 0.24)                                     | 0.14 (0.12, 0.16) | 0.10–0.20   | Beta       | 28,29                |
+| HCV Antibody (+) in HCV RNA (−)                                                                                                                                                                                                                                                                                                                                                                               | 0.28 (0.25, 0.32)    | 0.53 (0.45, 0.60)                                     | 0.64 (0.61, 0.68) | 0.20–0.70   | Beta       | 28,29                |
+| Point-of-care RNA invalid test due to                                                                                                                                                                                                                                                                                                                                                                         | 0.03                 | 0.03                                                  | 0.03              | 0.01–0.1    | Triangular | Assumption           |
+| operation error Sensitivity of test - Point-of-care HCV antibody                                                                                                                                                                                                                                                                                                                                              | 0.993                | 0.993                                                 | 0.993             | 0.961–0.999 | Beta       | Bioline HCV, Abbottb |
+| - Point-of-care HCV RNA                                                                                                                                                                                                                                                                                                                                                                                       | 1.00                 | 1.00                                                  | 1.00              | 0.84–1.00   | Beta       | 5,6                  |
+| - Laboratory HCV antibody                                                                                                                                                                                                                                                                                                                                                                                     | 1.00                 | 1.00                                                  | 1.00              |             |            |                      |
+| - Laboratory HCV RNA                                                                                                                                                                                                                                                                                                                                                                                          | 1.00                 | 1.00                                                  | 1.00              |             |            |                      |
+| Specificity of test - Point-of-care HCV antibody                                                                                                                                                                                                                                                                                                                                                              | 0.981                | 0.981                                                 | 0.981             | 0.945–0.994 | Beta       | Bioline HCV, Abbottb |
+| - Point-of-care HCV RNA                                                                                                                                                                                                                                                                                                                                                                                       | 1.00                 | 1.00                                                  | 1.00              | 0.81–1.00   | Beta       | 5,6                  |
+| - Laboratory HCV antibody                                                                                                                                                                                                                                                                                                                                                                                     | 1.00                 | 1.00                                                  | 1.00              | Assumptionc |            |                      |
+| - Laboratory HCV RNA                                                                                                                                                                                                                                                                                                                                                                                          | 1.00                 | 1.00                                                  | 1.00              | Assumptionc |            |                      |
+| Proportion lost to follow-up - Point-of-care RNA re-test                                                                                                                                                                                                                                                                                                                                                      | 0.25                 | 0.25                                                  | 0.25              | 0.20–0.30   | Triangular | Assumption           |
+| - Standard of care RNA test                                                                                                                                                                                                                                                                                                                                                                                   | 0.31                 | 0.31                                                  | 0.31              | 0.30–0.32   | Beta       | 3,11                 |
+| Treatment uptake - Point-of-care                                                                                                                                                                                                                                                                                                                                                                              | 0.93 (0.78, 0.99)    | 0.81 (0.62, 0.94)                                     | 0.81 (0.62, 0.94) | 0.70–0.99   | Beta       | 7,11                 |
+| - Standard of care                                                                                                                                                                                                                                                                                                                                                                                            | 0.60 (0.57, 0.63)    | 0.59 (0.56, 0.61)                                     | 0.59 (0.56, 0.61) | 0.20–0.65   | Beta       | 3,11                 |
+| Self-report history of HCV treatment                                                                                                                                                                                                                                                                                                                                                                          | 0.14 (0.11, 0.17)    | 0.18 (0.07, 0.33)                                     | 0.26 (0.19, 0.34) | 0.10–0.30   | Beta       | 28,29                |
+| history in RNA (+)                                                                                                                                                                                                                                                                                                                                                                                            | 0.09 (0.08, 0.10)    | 0.39 (0.31, 0.46)                                     | 0.43 (0.39, 0.46) | 0.05–0.50   | Beta       | 28,29                |
+| Self-report history of HCV treatment history in RNA (−) Treatment-naïve in HCV Antibody (+)                                                                                                                                                                                                                                                                                                                   | 0.74 (0.72, 0.77)    | 0.43 (0.35, 0.52)                                     | 0.42 (0.38, 0.46) | 0.30–0.80   | Beta       | 28,29                |
+| Treatment-naïve in HCV RNA (+)                                                                                                                                                                                                                                                                                                                                                                                | 0.86 (0.83, 0.88)    | 0.83 (0.67, 0.93)                                     | 0.74 (0.66, 0.81) | 0.60–0.90   | Beta       | 28,29                |
+| Self-report history of HCV treatment                                                                                                                                                                                                                                                                                                                                                                          | 0.10 (0.09, 0.11)    | 0.35 (0.28, 0.41)                                     | 0.40 (0.37, 0.43) | 0.10–0.40   | Beta       | 28,29                |
+| HCV Antibody (+) and RNA (−) in TxNaïve                                                                                                                                                                                                                                                                                                                                                                                                               | 0.17 (0.15, 0.18)    | 0.23 (0.15, 0.32)                                     | 0.38 (0.33, 0.42) | 0.10–0.40   | Beta       | 28,29                |
+| a Proportion of people tested: Assume to be 100% in the base case and tested in the ranges 0.5–1.0 in sensitivity analysis. b Sensitivity and specificity of POC HCV-Antibody test: by Bioline HCV, Abbott, product specification insert. c Sensitivity and specificity of lab HCV Antibody and RNA tests: assume 100% as gold standard tests. Table 1: Decision analytical model epidemiological parameters. |                      |                                                       |                   |             |            |                      |
+
+rendered by private healthcare providers, and government employment pay rates were used for public healthcare providers.31,32 Costs were valued in 2021 Australian Dollar (A$).
+
+## Cost-Effectiveness Analysis Incremental Analysis
+
+Costs and outcomes were compared to standard of care laboratory-based HCV diagnosis for each point-of-care testing strategy. Pairwise comparisons (testing strategy A vs. B, A vs. C, B vs. C) were carried out for the three point-of-care testing strategies in three settings.
+
+Incremental costs were compared to incremental outcomes to generate incremental cost-effectiveness ratios
+(ICERs).
+
+## Sensitivity Analysis
+
+Deterministic univariate sensitivity analysis was performed to investigate the cost per treatment initiation for each testing strategy by HCV antibody prevalence and treatment uptake. The most efficient and costeffective testing strategies by HCV antibody prevalence were identified for each setting. Threshold analyses were undertaken to identify the minimum treatment uptake for point-of-care testing strategies to achieve equivalent average cost per treatment initiation compared to standard of care. One-way sensitivity analysis was performed with all model parameters with value ranges presented in Table 1 (effectiveness variables) and Supplementary Table S2 (cost variables).
+
+Probabilistic sensitivity analysis with 1000 iterations was
+
+![5_image_0.png](5_image_0.png)
+
+| Test                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Costs                 | Source                 |                                                            |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------|------------------------|------------------------------------------------------------|
+| Base case estimate (low, high)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                       |                        |                                                            |
+| Point-of-care RNA test                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | Testing variable cost | Direct cost (including | Total cost                                                 |
+| variable and fixed costs)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                       |                        |                                                            |
+| $96 ($77, $165)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | $129 ($109, $199)     | $153 ($133, $222)      | Cepheid 2019, 2020a Cepheid 2019b National Programc TEMPOd |
+| Point-of-care combined HCV Ab and RNA tests                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Anti-HCV only         | Anti-HCV Reflex RNA    | RNA onlye                                                  |
+| $34 ($30, $86)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | $163 ($139, $285)     | $129 ($109, $199)      | 7                                                          |
+| Standard of care laboratory HCV tests                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Anti-HCV only         | Anti-HCV & RNA         | RNA onlye                                                  |
+| $38 ($34, $90)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | $153 ($117, $191)     | $115 ($103, $174)      | 31                                                         |
+| Treatment initiation assessment                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | $180 ($162, $198)     | 28,31                  |                                                            |
+| a GeneXpert IV 4sites Laptop model with lifespan 10 years for 375 screened annually per site, including annual service and maintenance fees. Discount 20%∼50% applied for sensitivity analysis. b Costs include cartridge, reagent, delivery fee, and consumables. c National Point-of-care Testing Program budget 2021. d Ranges are based on TEMPO pilot data - 2 patients per hour by CNS, additional 15 min for reflex RNA after positive HCV antibody. e Immediate RNA tests with treatment history. Table 2: Cost estimates for point-of-care HCV RNA, point-of-care anti-HCV and standard of care laboratory HCV tests. (Costs in 2021 A$). |                       |                        |                                                            |
+
+conducted to illustrate the impact of parameter variability on costs and outcomes.
+
+## Role Of The Funding Source
+
+The study was supported by the Point of Care Research Consortium for Infectious Disease in the Asia Pacific
+(RAPID) which is funded through a National Health and Medical Research Council Centre for Research Excellence Grant in Australia. The funders did not have any role in study design, data collection, data analysis, interpretation, writing of the report.
+
+## Results Average Cost-Effectiveness For Diagnosis Of Hcv Infection
+
+With standard of care HCV testing, the average costs per HCV-infected person detected were $748 in prisons, $630 in NSPs, and $857 in drug treatment clinics (base case) (Table 3). The expected average cost of HCV diagnosis by point-of-care testing varied by setting and testing strategy, A$689–A$1140 per person detected in prisons, A$575–A$729 in NSPs, and A$817–A$957 in drug treatment clinics, depending on testing strategies
+(Table 3). Across all settings, diagnosis by immediate point-of-care HCV RNA testing was the most expensive testing strategy (A$729–A$1140), while combining point-of-care HCV antibody and reflex point-of-care RNA testing with consideration of treatment history achieved the lowest average cost per HCV-infected person detected (A$575–A$817).
+
+## Average Cost-Effectiveness For Hcv Treatment Initiation
+
+With standard of care HCV testing, the average cost per treatment initiation was $1426 in prisons, $1248 in NSPs, and $1632 in drug treatment clinics (base case)
+(Table 3). Across all settings, the average cost per treatment initiation with point-of-care testing was up to 35% less than the average cost in standard of care. The expected average cost per treatment initiation by pointof-care testing ranged from A$921–A$1406 in prisons, A$890–A$1080 in NSPs, to A$1189–A$1362 in drug treatment clinics, depending on testing strategies
+(Table 3). The average cost of immediate point-of-care HCV RNA testing per treatment initiation in the base case analysis was A$1406 in prison, A$1080 in NSP, and A$1362 in drug treatment clinics. The average cost per treatment initiation with combined point-of-care HCV
+antibody and reflex RNA testing was lower than immediate point-of-care RNA testing, with the lowest average cost per treatment initiation when considering previous treatment history (prisons A$921 vs. A$960, NSP A$890 vs. A$970, and drug treatment clinics A$1189 vs. A$1310).
+
+## Incremental Cost-Effectiveness Ratios
+
+Compared to standard of care, point-of-care HCV testing strategies detected more infections and increased treatment uptake with higher cost per person tested, resulting in ICERs from A$408 to A$2409 per detectable RNA and A$424–A$1386 per treatment initiation. Among the three point-of-care testing strategies, combined antibody and reflex RNA testing with consideration of treatment history was always the most efficient (lowest average cost per treatment initiation).
+
+Immediate RNA testing was more expensive but detected more people with HCV and associated with higher treatment uptake, resulting in ICERs from A$33,764 to A$89,757 for each additional person initiating HCV
+treatment, compared to the most efficient combined point-of-care testing strategy considering treatment history (strategy A vs. C in Table 4). When compared to the combined point-of-care antibody and reflex RNA
+tests irrespective of treatment history, immediate RNA
+testing produced ICERs from A$8698 in drug treatment
+
+| Number to test for one person diagnosed or treated                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |      |     |        |        |             |       |      |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|-----|--------|--------|-------------|-------|------|
+| Detection of active HCV infection Prison setting POC HCV RNA for all                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | $132 | $66 | 0.1159 | 0.0274 | $2409 $1140 | 8.6   |      |
+| POC HCV-Antibody for all with reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | $83  | $17 | 0.1151 | 0.0266 | $650        | $725  | 8.7  |
+| POC HCV Antibody for treatment-naive with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | $79  | $13 | 0.1153 | 0.0268 | $493 $689   | 8.7   |      |
+| Standard of Care                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | $66  | -   | 0.0885 | -      | -           | $748  | 11.3 |
+| Needle Syringe Program Setting POC HCV RNA for all                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | $132 | $47 | 0.1813 | 0.0453 | $1027       | $729  | 5.5  |
+| POC HCV-Antibody for all with reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | $115 | $29 | 0.1800 | 0.0441 | $669        | $640  | 5.6  |
+| POC HCV Antibody for treatment-naive with reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | $104 | $18 | 0.1802 | 0.0443 | $408        | $575  | 5.5  |
+| Standard of Care                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | $86  | -   | 0.1359 | -      | -           | $630  | 7.4  |
+| Drug Treatment Setting POC HCV RNA for all                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | $132 | $40 | 0.1381 | 0.0308 | $1308       | $957  | 7.2  |
+| POC HCV-Antibody for all with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | $126 | $34 | 0.1371 | 0.0299 | $1127       | $916  | 7.3  |
+| POC HCV Antibody for Tx-naive with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | $112 | $20 | 0.1740 | 0.0301 | $676        | $817  | 7.3  |
+| Standard of Care                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | $92  | -   | 0.1072 | -      | -           | $857  | 9.3  |
+| HCV detected and linked to treatment Prison setting POC HCV RNA for all                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | $152 | $76 | 0.1078 | 0.0547 | $1386 $1406 | 9.3   |      |
+| POC HCV-Antibody for all with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | $103 | $27 | 0.1071 | 0.0539 | $500 $960   | 9.3   |      |
+| POC HCV Antibody for Tx-naive with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | $99  | $23 | 0.1072 | 0.0541 | $424        | $921  | 9.3  |
+| Standard of Care                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | $76  | -   | 0.0531 | -      | -           | $1426 | 18.8 |
+| Needle Syringe Program Setting POC HCV RNA for all                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | $159 | $59 | 0.1468 | 0.0666 | $879 $1080  | 6.8   |      |
+| POC HCV-Antibody for all with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | $141 | $41 | 0.1458 | 0.0656 | $630        | $970  | 6.9  |
+| POC HCV Antibody for Tx-naive with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | $130 | $30 | 0.1460 | 0.0658 | $455 $890   | 6.9   |      |
+| Standard of Care                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | $100 | -   | 0.0802 | -      | -           | $1248 | 12.5 |
+| Drug Treatment Setting POC HCV RNA for all                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | $152 | $49 | 0.1118 | 0.0486 | $1010 $1362 | 8.9   |      |
+| POC HCV-Antibody for all with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | $146 | $42 | 0.1111 | 0.0478 | $884 $1310  | 9.0   |      |
+| POC HCV Antibody for Tx-naive with Reflex HCV-RNA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | $132 | $29 | 0.1113 | 0.0480 | $605 $1189  | 9.0   |      |
+| Standard of Care                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | $103 | -   | 0.0633 | -      | $1632       | 15.8  |      |
+| HCV: hepatitis C, ICER: incremental cost-effective ratio, POC: point-of-care, Tx: treatment. - The comparator to generate incremental cost/effectiveness. Table 3: Average cost per person tested, HCV detected, and people treated by three point-of-care testing strategies and incremental cost-effectiveness ratio compared to standard of care by laboratory testing. Strategy Average cost per person tested Incremental Effectiveness cost (HCV diagnosed or treated) Incremental ICER Average cost per effectiveness person diagnosed or treated |      |     |        |        |             |       |      |
+
+clinics to A$64,706 in prisons (strategy A vs. B in Table 4). Pairwise comparisons of the two combined point-of-care antibody and reflex RNA testing showed that, in all settings, the testing strategy without consideration of treatment history was dominated (more expensive and less people treated) by the combined tests with consideration of treatment history. Costeffectiveness frontier presented in a cost-effectiveness plane in Fig. 2 illustrates the points of base case corresponding to four testing strategies with the gradient of the line representing the ICER of the testing strategy comparison between two alternatives. The testing strategy of point-of-care antibody testing for everyone and reflex RNA test for antibody positives sit on the left side of frontier lines, meaning this strategy was dominated by the expansion of intervention undominated lines and would not be a choice for consideration from costeffectiveness point of view.
+
+## Sensitivity Analysis
+
+To assess the impact of HCV antibody prevalence on cost-effectiveness of different testing strategies, one-way sensitivity analysis was performed (Fig. 3). Combined point-of-care HCV antibody testing with reflex point-ofcare RNA testing with consideration of previous treatment was the most cost-effective testing strategy across all settings up to an HCV antibody prevalence of 79% in prisons and 86% in NSPs and drug treatment clinics.
+
+When HCV antibody prevalence was <74%, combined point-of-care HCV antibody and point-of-care RNA
+testing, both with and without consideration of treatment history, were more cost-effective than immediate
+
+| Pairwise                                                         | Strategy                                          | Average                                                              |      |        |         |            |
+|------------------------------------------------------------------|---------------------------------------------------|----------------------------------------------------------------------|------|--------|---------|------------|
+| comparison                                                       | cost per person tested                            | Incremental Effectiveness cost (HCV detected or treated) Incremental | ICER |        |         |            |
+| effectiveness                                                    |                                                   |                                                                      |      |        |         |            |
+| Prison setting Detection of active HCV infection                 |                                                   |                                                                      |      |        |         |            |
+| A                                                                | POC HCV RNA for all                               | $132                                                                 | $49  | 0.1159 | 0.0008  | $60,000    |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $83                                                                  | -    | 0.1151 | -       | -          |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $83                                                                  | 4    | 0.1151 | −0.0002 | dominateda |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $79                                                                  | -    | 0.1153 | -       | -          |
+| A                                                                | POC HCV RNA for all                               | $132                                                                 | $53  | 0.1159 | 0.0006  | $83,310    |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $79                                                                  | -    | 0.1153 | -       | -          |
+| HCV detected & linked to treatment                               |                                                   |                                                                      |      |        |         |            |
+| A                                                                | POC HCV RNA for all                               | $152                                                                 | $49  | 0.1078 | 0.0008  | $64,700    |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $103                                                                 | -    | 0.1071 | -       | -          |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $103                                                                 | $4   | 0.1071 | −0.0002 | dominated  |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $99                                                                  | -    | 0.1072 | -       | -          |
+| A                                                                | POC HCV RNA for all                               | $152                                                                 | $53  | 0.1078 | 0.0006  | $89,800    |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $99                                                                  | -    | 0.1072 | -       | -          |
+| Needle Syringe Program Setting Detection of active HCV infection |                                                   |                                                                      |      |        |         |            |
+| A                                                                | POC HCV RNA for all                               | $132                                                                 | $17  | 0.1813 | 0.0013  | $13,400    |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $115                                                                 | -    | 0.1800 | -       | -          |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $115                                                                 | $11  | 0.1800 | −0.0002 | dominated  |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $104                                                                 | -    | 0.1802 | -       | -          |
+| A                                                                | POC HCV RNA for all                               | $132                                                                 | $28  | 0.1813 | 0.0010  | $27,200    |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $104                                                                 | -    | 0.1802 | -       | -          |
+| HCV detected & linked to treatment                               |                                                   |                                                                      |      |        |         |            |
+| A                                                                | POC HCV RNA for all                               | $159                                                                 | $17  | 0.1468 | 0.0010  | $16,800    |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $141                                                                 | -    | 0.1458 | -       | -          |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $141                                                                 | $11  | 0.1458 | −0.0002 | dominated  |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $130                                                                 | -    | 0.1460 | -       | -          |
+| A                                                                | POC HCV RNA for all                               | $159                                                                 | $29  | 0.1468 | 0.0008  | $33,800    |
+| C                                                                | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $130                                                                 | -    | 0.1460 | -       | -          |
+| Drug Treatment Setting Detection of active HCV infection         |                                                   |                                                                      |      |        |         |            |
+| A                                                                | POC HCV RNA for all                               | $132                                                                 | $7   | 0.1381 | 0.0010  | $6900      |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $126                                                                 | -    | 0.1371 | -       | -          |
+| B                                                                | POC HCV-Antibody for all with Reflex HCV-RNA      | $126                                                                 | $13  | 0.1371 | −0.0003 | dominated  |
+| C                                                                | POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $112                                                                 | -    | 0.1374 | -       | -          |
+| A                                                                | POC HCV RNA for all                               | $132                                                                 | $20  | 0.1381 | 0.0007  | $27,900    |
+| C                                                                | POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $112                                                                 | -    | 0.1374 | -       | -          |
+| (Table 4 continues on next page)                                 |                                                   |                                                                      |      |        |         |            |
+
+![8_image_0.png](8_image_0.png)
+
+![8_image_1.png](8_image_1.png)
+
+A POC HCV RNA for all $152 $7 0.1118 0.0008 $8700 B POC HCV-Antibody for all with Reflex HCV-RNA $146 - 0.1111 - –
+B POC HCV-Antibody for all with Reflex HCV-RNA $146 $13 0.1111 −0.0002 dominated C POC HCV Antibody for Tx-naive with Reflex HCV-RNA $132 - 0.1113 - –
+A POC HCV RNA for all $152 $20 0.1118 0.0006 $34,600 C POC HCV Antibody for Tx-naive with Reflex HCV-RNA $132 - 0.1113 - –
+ICER: incremental cost-effective ratio. - The comparator to generate incremental cost/effectiveness. aDominated: meaning more costly but less effective.
+
+Table 4: Pairwise comparison between point-of-care testing strategies for HCV detection and treatment initiation for the base cases in three settings.
+point-of-care RNA testing and lab-based standard of care testing. At an HCV antibody prevalence <35% in prison,
+<45% in NSP, and <49% in drug treatment clinics, immediate point-of-care RNA testing was the least costeffective strategy; while at an antibody prevalence >79%
+in prisons and >86% in NSPs and drug treatment clinics, immediate point-of-care RNA testing became the most cost-effective testing strategy because the cost of the HCV antibody test is not wasted on people who are highly likely to be HCV antibody positive.
+
+The key factor determining the cost-effectiveness per person treated was the difference in treatment uptake between point-of-care and standard of care testing.
+
+Threshold analyses demonstrated that for immediate point-of-care RNA testing, treatment uptake levels at 67%–91% would achieve an equivalent average cost per treatment initiation as those in standard of care (59%–
+60%, depending on setting) (Table 5 and Fig. 4). For combined point-of-care HCV antibody and reflex RNA
+testing, treatment uptake of 54%–57% with consideration of prior treatment and 58%–64% without consideration of prior treatment, were required to achieve an equivalent average cost per treatment initiation as in standard of care.
+
+One-way sensitivity analyses suggested that point-ofcare RNA test costs, number of annual tests per site, and number of samples collected in 1 h played a role in determining the ICERs of point-of-care testing compared to standard of care (Supplementary Fig. S1). Standard of care treatment uptake also impacted ICERs by determining the magnitude of improvements in treatment uptake with point-of-care testing.
+
+Accounting for model input variability by simultaneously changing all parameters with plausible ranges
+(including effectiveness and costs variables) in the probabilistic sensitivity analysis, the three point-of-care testing strategies showed an increased treatment initiation compared to standard of care at a higher cost, albeit with great uncertainly (scatterplots of cost and effectiveness in Supplementary Fig. S2). Additionally, cost-effectiveness planes of incremental cost and incremental effectiveness of pairwise comparisons between the three point-of-care testing strategies are presented in Supplementary Fig. S3, which further validates the robustness of our analyses and strengthen the base case results presented in Table 4 and Fig. 2. That is, strategy B was dominated by strategy C, i.e., POC antibody testing for everyone without treatment history consideration and reflex with RNA testing (strategy B) less effective and more costly than the same testing with treatment history consideration (strategy C).
+
+## Discussion
+
+This study demonstrated that all point-of-care HCV
+testing strategies had a lower average cost per treatment initiation compared to standard of care across settings and HCV antibody prevalence. When HCV antibody prevalence was below 74%, combining point-of-care HCV antibody and reflex point-of-care RNA testing
+(with or without consideration of previous treatment experience) is most cost-effective. Only modest improvements in treatment uptake are required for pointof-care RNA testing to be cost-effective compared to standard of care. Our study findings are important to inform optimal strategies for the scale-up of point-ofcare testing in settings critical to support HCV
+elimination.
+
+Point-of-care RNA testing was more cost-effective than standard of care, consistent with studies in the US, Canada, and Africa.15–21 Limitations of previous studies included limited cost inclusions,15,17 underestimated point-of-care RNA testing costs,20 and simplified testing pathways assuming no loss to followup.19 The strengths of this study are the availability of detailed costing data, the inclusion of costs for critical aspects of point-of-care testing (e.g., operator training,
+
+![9_image_0.png](9_image_0.png)
+
+quality assurance, and IT/connectivity), and real-world data for key parameters related to HCV prevalence and treatment. The superior cost-effectiveness of point-ofcare testing was primarily driven by a higher treatment uptake compared to standard of care (despite the higher costs related to point-of-care testing). Most costs related to standard of care were attributed to wasted testing costs due to loss of follow-up in the care cascade compared to point-of-care testing.
+
+When the HCV antibody prevalence was below 74%, combined point-of-care HCV antibody with reflex point-of-care HCV RNA testing was most cost-effective, with consideration of previous treatment history further improving cost-effectiveness. This testing strategy was more cost-effective because people who were HCV antibody negative received a less expensive antibody test rather than an RNA test. The improved cost-effectiveness of this strategy is primarily driven by the decreased costs of point-of-care antibody testing (A$34 including test kit, consumables, labour)
+compared to immediate point-of-care RNA testing
+(A$96 including GeneXpert machine, test cartridge, consumables, labour). Point-of-care HCV antibody testing also has a shorter time to result compared to point-of-care HCV RNA testing (60 min). Combined point-of-care HCV antibody and RNA testing strategies are likely to improve patient and staff acceptability by reducing time to result for those who are HCV antibody negative (1–20 min vs. 60 min), while not considerably increasing the total time to result for those who are positive (61–65 min in total). It is unclear whether a combined point-of-care and RNA
+Fig. 3: Average cost per HCV treatment initiation in relation to HCV antibody prevalence in three settings (a) Prison (b) Needle Syringe
+
+![10_image_0.png](10_image_0.png)
+
+Program and (c) Drug treatment clinics.
+
+testing strategy has any impact on treatment uptake or response to therapy. Further research is needed to understand the effectiveness, acceptability, implementation challenges, and long-term cost-effectiveness to inform implementation, funding, and integration into policy and practice.
+
+This study suggested that only modest increases in treatment uptake from 59% to 60% in standard of care
+
+| Setting                                                                                                                                                                                                                                                                                                                                                                                                                    | Treatment uptake in the base case   | Threshold of point-of-care treatment uptake for equal average cost per person treated compared to standard of care   |                                                                                                     |                                                  |                   |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------|----------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------|--------------------------------------------------|-------------------|
+| SOC (95% CI)                                                                                                                                                                                                                                                                                                                                                                                                               | POC (95% CI)                        | Combined POC HCV antibody and reflex RNA with treatment history consideration, base case (range)                     | Combined POC HCV antibody and reflex RNA without treatment history consideration, base case (range) | Immediate POC HCV RNA for all, base case (range) |                   |
+| Prison                                                                                                                                                                                                                                                                                                                                                                                                                     | 0.60 (0.57, 0.63)                   | 0.93 (0.78, 0.99)                                                                                                    | 0.55 (0.52, 0.59)                                                                                   | 0.58 (0.55, 0.59)                                | 0.91 (0.88, 0.95) |
+| Needle Syringe Program                                                                                                                                                                                                                                                                                                                                                                                                     | 0.59 (0.56, 0.61)                   | 0.81 (0.62, 0.94)                                                                                                    | 0.54 (0.52, 0.56)                                                                                   | 0.60 (0.57, 0.62)                                | 0.68 (0.66, 0.70) |
+| Drug treatment clinic                                                                                                                                                                                                                                                                                                                                                                                                      | 0.59 (0.56, 0.61)                   | 0.81 (0.62, 0.94)                                                                                                    | 0.57 (0.53, 0.59)                                                                                   | 0.64 (0.60, 0.66)                                | 0.67 (0.62, 0.68) |
+| POC: Point-of-Care, SOC: Standard of Care. Difference in treatment linkage between POC and SOC: base case 0.93–0.60, worst case 0.78–0.63, best case 0.99–0.57 in prison; base case 0.81–0.59, worst case 0.62–0.61, best case 0.94–0.56 in NSP and drug treatment clinics. Table 5: Threshold of point-of-care treatment uptake for equal average cost per person treated compared to standard of care in three settings. |                                     |                                                                                                                      |                                                                                                     |                                                  |                   |
+
+![11_image_0.png](11_image_0.png)
+
+to 67%–91% with immediate point-of-care HCV RNA
+testing is needed for the higher-cost point-of-care HCV RNA testing to become a preferred strategy compared to standard of care. Clinical studies have demonstrated that high treatment uptake (81%–93%) following pointof-care HCV RNA testing can be achieved in various settings.7–11 In our analyses, treatment uptake in the standard of care was based on the proportion of diagnosed people in 2016–2017 in New South Wales, providing an accurate estimate of HCV treatment uptake.3 The benefit of point-of-care testing with same-visit treatment is the ability to detect current infection and initiate treatment in a timely manner.7,11 This study demonstrates that if real-world treatment uptake following point-of-care RNA testing is lower than pilot studies to date, point-of-care testing is still likely to be more cost-effective than standard of care. Lower treatment uptake is required by the point-of-care combined testing with treatment history consideration to achieve equivalent average costs per treatment initiation in standard of care, suggesting the relative advantage of point-of-care HCV antibody testing in cost terms.
+
+This study has several limitations. Our models are based on empirical data from clinical trials and studies in Australian service settings. Thus, diagnosis outcomes for the base cases in each setting are mainly built on the epidemiological profiles of the population in these Australian settings.28,29 The results are driven by the attributes of these studies, particularly the HCV antibody and RNA prevalence and treatment history among the study population. The findings may not be comparable to other populations with different epidemiological characteristics from these Australian studies. Interpretation and application of our study results for other populations and settings should be undertaken cautiously as HCV prevalence and testing costs would vary widely (e.g., homeless people compared to patients of mental health services or attendees of general practice).
+
+Treatment costs for people diagnosed with HCV
+were excluded as the end of testing pathway for this cost-effectiveness analysis was initiation, rather than completion, of DAA treatment. The DAA reimbursement agreement between the Australian government and the pharmaceutical manufacturers stipulates the prices by a fixed budget regardless of number of patients treated.33,34 Thus, the additional benefits of increasing treatment did not incur additional costs; therefore, the DAA pharmaceutical cost was regarded as a sunk cost and was excluded from this analysis. Lastly, lack of modelling the long-term outcomes for people living with HCV is a key limitation of the current analysis. The one-year time horizon means that potential long-term costs and benefits of treatment with respect to prevention of HCV transmission and reductions in morbidity/mortality are not captured.
+
+Our work in the near future will include the measurement of quality-adjusted life-years (QALYs) and mathematical modelling to estimate expected transmission and long-term disease burden benefits to guide implementation and enable comparison with other public health interventions and programs. In the interim, this study lays the foundations for future modelling by providing useful information on costs and benefits that can be expected in the short term and by identifying uncertain estimates requiring more detailed investigation for future extrapolation beyond the trial follow-up period. This study also provides a model that could be used to assess scale-up of point-ofcare testing strategies in other countries that could be parametrised to the local epidemiology, including the Western Pacific. This is important given the recent inclusion of point-of-care testing in the new recommendations for simplified service delivery and diagnostic testing by the World Health Organization.35
+
+## Conclusion
+
+Point-of-care HCV testing enables diagnosis and treatment in a single-visit, increases testing acceptability, and reduces loss to follow-up, addressing the drop-off in the HCV care cascade. Combining point-of-care HCV
+antibody and RNA testing could optimise testing outcomes, enhancing cost-effectiveness and patient acceptability. This study is important for informing the future implementation of point-of-care testing strategies to achieve HCV elimination targets.
+
+Contributors S.T.F.S., V.W., and J.G. conceived the study. S.T.F.S., R.T.G., A.R.L., V.W., and J.G. obtained funding. S.T.F.S., Q.C., and R.T.G. contributed to model development. S.T.F.S. and J.G. designed the analyses, interpreted the results, and drafted the manuscript. J.C., H.V., Y.S., E.B.C.,
+J.A.K., A.R.L., G.J.D., and J.G. provided data and validated model inputs. V.W. and J.G. supervised the project. All authors reviewed, edited, and approved the manuscript. Data sharing statement Models parameters are presented in Table 1 and Supplementary Table S2. Data sources for model parameters will be made available on request.
+
+Declaration of interests R.T.G. has received funding for his research from WHO and has provided non-funded project advice to Gilead and ViiV. Y.S. is a coinvestigator on investigator-initiated research grants from AbbVie and Gilead Sciences. A.R.L. has received investigator-initiated research grants from AbbVie, Gilead Sciences, and Sequiris. G.J.D. is a consultant/advisor and has received research grants from Abbvie, Abbot Diagnostics, Gilead Sciences, Bristol Myers Squibb, Cepheid, GlaxoSmithKline, Merck, Janssen and Roche. J.G. is a consultant/
+advisor and has received research grants from AbbVie, Biolytical, Camurus, Cepheid, Gilead Sciences, Hologic, Indivor, and Merck/MSD
+and has received honoraria from AbbVie, Cepheid, Gilead Sciences, and Merck. No input into this work was provided by any of the above listed organisations or institutions.
+
+All other authors have no conflicts of interest to declare.
+
+Acknowledgement We would like to acknowledge the contributors to the studies which provided data that informed the analyses presented in this paper.
+
+Funding: This work was supported by the Point of Care Research Consortium for Infectious Disease in the Asia Pacific (RAPID)
+which is funded through a National Health and Medical Research Council Centre for Research Excellence Grant [grant number 1153647]. The funding body has no role in the writing of the manuscript or the decision to submit the manuscript for publication. The Kirby Institute is funded by the Australian Government Department of Health and Ageing. The views expressed in this publication do not necessarily represent the position of the Australian Government. G.J.D. is supported through a National Health and Medical Research Council Investigator Grant [2008276]. J.G. is supported through a National Health and Medical Research Council Investigator Grant [1176131]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.
+
+org/10.1016/j.lanwpc.2023.100750. References
+
+![12_image_0.png](12_image_0.png)
+
+1 WHO. Global health sector strategy on viral hepatitis 2016-2021.
+
+Geneva: WHO; 2017.
+
+2 Polaris Observatory HCV Collaborators. Global change in hepatitis C virus prevalence and cascade of care between 2015 and 2020: a modelling study. Lancet Gastroenterol Hepatol. 2022;7(5):396–415.
+
+3 Yousafzai MT, Alavi M, Valerio H, Hajarizadeh B, Grebely J,
+Dore GJ. Timely hepatitis C RNA testing and treatment in the era of direct-acting antiviral therapy among people with hepatitis C in New South Wales, Australia. Viruses. 2022;14(7):1496.
+
+4 Scott N, Sacks-Davis R, Wade AJ, et al. Australia needs to increase testing to achieve hepatitis C elimination. Med J Aust. 2020;212(8):365–370.
+
+5 Grebely J, Lamoury FMJ, Hajarizadeh B, et al. Evaluation of the Xpert HCV Viral Load point-of-care assay from venepuncturecollected and finger-stick capillary whole-blood samples: a cohort study. Lancet Gastroenterol Hepatol. 2017;2(7):514–520.
+
+6 Lamoury FMJ, Bajis S, Hajarizadeh B, et al. Evaluation of the Xpert HCV Viral Load finger-stick point-of-care assay. J Infect Dis.
+
+2018;217(12):1889–1896.
+
+7 Grebely J, Gilliver R, McNaughton T, et al. Single-visit hepatitis C
+point-of-care testing, linkage to nursing care, and peer-supported treatment among people with recent injecting drug use at a peer-led needle and syringe program: The TEMPO Pilot Study. Int J Drug Pol. 2023;114:103982.
+
+8 MacIsaac M, Whitton B, Anderson J, et al. Rapid point of care HCV
+testing allows high throughout HCV screening and rapid treatment uptake among PWID attending a medically supervised injecting room. International Conference on Hepatitis Care in Substance Users. 2021.
+
+9 O'Loan J, Young M, Mooney M, O'Flynn M. Same day delivery!
+
+HCV point of care testing in South East Queensland mar-ginalised communities simplifies diagnosis and ensures rapid access to treatment. International Conference on Hepatitis Care in Substance Users. 2021.
+
+10 Ralton L, McCartney E, Ferguson C, et al. Prompt - point of care testing for hepatitis C in the priority settings of mental health, prisons and drug & alcohol facilities. International Conference on Hepatitis Care in Substance Users. 2021.
+
+11 Sheehan Y, Cunningham E, Cochrane A, et al. A 'one-stop-shop' point-of-care hepatitis C RNA testing intervention to enhance treatment uptake in a reception prison: the PIVOT study. J Hepatol.
+
+(Submitted 27 September 2022, Manuscript number JHEPAT-D22-01849).
+
+12 Bajis S, Maher L, Treloar C, et al. Acceptability and preferences of point-of-care finger-stick whole-blood and venepuncture hepatitis C
+virus testing among people who inject drugs in Australia. Int J Drug Policy. 2018;61:23–30.
+
+13 Lafferty L, Cochrane A, Sheehan Y, Treloar C, Grebely J, Lloyd AR.
+
+"That was quick, simple, and easy": patient perceptions of acceptability of point-of-care hepatitis C RNA testing at a reception prison.
+
+Int J Drug Policy. 2022;99:103456.
+
+14 Catlett B, Hajarizadeh B, Cunningham E, et al. Diagnostic accuracy of assays using point-of-care testing or dried blood spot samples for the determination of HCV RNA: a systematic review. J Infect Dis. 2022;226:1005–1021.
+
+15 Assoumou SA, Nolen S, Hagan L, et al. Hepatitis C management at federally qualified health centers during the opioid epidemic: a cost-effectiveness study. Am J Med. 2020;133(11):e641–e658.
+
+16 Assoumou SA, Tasillo A, Leff JA, et al. Cost-effectiveness of onetime hepatitis C screening strategies among adolescents and young adults in primary care settings. Clin Infect Dis. 2018; 66(3):376–384.
+
+17 Duchesne L, Dussault C, Godin A, Maheu-Giroux M, Kronfli N.
+
+Implementing opt-out hepatitis C virus (HCV) screening in Canadian provincial prisons: a model-based cost-effectiveness analysis.
+
+Int J Drug Policy. 2021;96:103345.
+
+18 Duchesne L, Hejblum G, Njouom R, et al. Model-based costeffectiveness estimates of testing strategies for diagnosing hepatitis C virus infection in Central and Western Africa. PLoS One.
+
+2020;15(8):e0238035.
+
+19 Duchesne L, Hejblum G, Toure Kane NC, et al. Model-based costeffectiveness estimates of testing strategies for diagnosing hepatitis C virus infection in people who use injecting drugs in Senegal. Int J Drug Policy. 2020;75:102613.
+
+20 Koo V, Tian F, Wong WWL. Cost-effectiveness analysis of hepatitis C virus (HCV) point-of-care assay for HCV screening. Liver Int.
+
+2022;42(4):787–795.
+
+21 Malaisamy M, Nagarajan K, Kirti T, et al. Economic evaluation of implementing a rapid point-of-care screening test for the identification of hepatitis C virus under National Viral Hepatitis Control Programme in Tamil Nadu, South India. J Glob Infect Dis. 2021;13(3):126–132.
+
+22 Mohamed Z, Scott N, Nayagam S, et al. Cost effectiveness of simplified HCV screening-and-treatment interventions for people who inject drugs in Dar-es-Salaam, Tanzania. Int J Drug Policy. 2022;99:103458.
+
+23 Scott N, Doyle JS, Wilson DP, et al. Reaching hepatitis C virus elimination targets requires health system interventions to enhance the care cascade. Int J Drug Policy. 2017;47:107–116.
+
+24 Chhatwal J, Chen Q, Wang X, et al. Assessment of the feasibility and cost of hepatitis C elimination in Pakistan. JAMA Netw Open. 2019;2(5):e193613.
+
+25 Husereau D, Drummond M, Augustovski F, et al. Consolidated Health Economic Evaluation Reporting Standards 2022 (CHEERS 2022) statement: updated reporting guidance for health economic evaluations. Value Health. 2022;25(1):3–9.
+
+26 Palmer AY, Wade AJ, Draper B, et al. A cost-effectiveness analysis of primary versus hospital-based specialist care for direct acting antiviral hepatitis C treatment. Int J Drug Policy. 2020;76:102633.
+
+27 TreeAge Pro Healthcare. In: © 2021 TreeAge Software LLC. 2022. 28 Hajarizadeh B, Grebely J, Byrne M, et al. Evaluation of hepatitis C
+treatment-as-prevention within Australian prisons (SToP-C): a prospective cohort study. Lancet Gastroenterol Hepatol. 2021;6(7):533–546.
+
+29 Valerio H, Alavi M, Silk D, et al. Progress towards elimination of hepatitis C infection among people who inject drugs in Australia:
+the ETHOS Engage Study. Clin Infect Dis. 2020;73(1):e69–e78.
+
+30 Palmer A, Papaluca T, Stoove M, et al. A costing analysis of a statewide, nurse-led hepatitis C treatment model in prison. Int J Drug Policy. 2021;94:103203.
+
+31 Medicare benefit schedule book [internet] [cited Oct 2020]. Available from: http://www.mbsonline.gov.au/internet/mbsonline/
+publishing.nsf/Content/Home; 2018.
+
+32 Public health system nurses' & midwives' (state) award 2021 [internet].
+
+2021. [cited Nov 15, 2021].
+
+33 Drug Utilisation Sub-Committee PBAC. Direct acting antiviral medicines for the treatment of chronic hepatitis C. In: Department of Health. Canberra, Australia: Australian Commonwealth Governement; 2018.
+
+34 Moon S, Erickson E. Universal medicine access through lump-sum remuneration - Australia's approach to hepatitis C. N Engl J Med.
+
+2019;380(7):607–610.
+
+35 World Health Organization. Updated recommendations on HCV
+simplified service delivery and HCV diagnostics: policy brief. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO. Geneva, Switzerland.

medical/md/PMC10523130.md

@@ -0,0 +1,360 @@
+
+## Review Article
+
+Prostate cancer
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_1.png](0_image_1.png)
+
+pISSN: 2287-4208 / eISSN: 2287-4690 World J Mens Health 2023 Oct 41(4): 759-768 https://doi.org/10.5534/wjmh.220202
+
+# Emerging Relationship Between The Gut Microbiome And Prostate Cancer
+
+Makoto Matsushita1 , Kazutoshi Fujita1,2 , Koji Hatano1 , Marco A. De Velasco2,3 , Akira Tsujimura4 , Hirotsugu Uemura2 , Norio Nonomura1 1 Department of Urology, Osaka University Graduate School of Medicine, Suita, Departments of 2Urology and 3Genome Biology, Kindai University Faculty of Medicine, Osakasayama, 4Department of Urology, Juntendo University Urayasu Hospital, Urayasu, Japan The human gut microbiota changes under the influence of environmental and genetic factors, affecting human health. Extensive studies have revealed that the gut microbiome is closely associated with many non-intestinal diseases. Among these, the influence of the gut microbiome on cancer biology and the efficacy of cancer therapy has attracted much attention. Prostate cancer cells are affected by direct contact with the microbiota of local tissues and urine, and a relationship between prostate cancer cells and the gut microbiota has been suggested. In the human gut microbiota, bacterial composition differs depending on prostate cancer characteristics, such as histological grade and castration resistance. Moreover, the involvement of several intestinal bacteria in testosterone metabolism has been demonstrated, suggesting that they may affect prostate cancer progression and treatment through this mechanism. Basic research indicates that the gut microbiome also plays an important role in the underlying biology of prostate cancer through multiple mechanisms owing to the activity of microbial-derived metabolites and components. In this review, we describe the evidence surrounding the emerging relationship between the gut microbiome and prostate cancer, termed the "gut-prostate axis." Keywords: Bacteria; Gut microbiome; Host microbial interactions; Prostate cancer; Testosterone This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
+
+## Introduction
+
+Various bacterial species are present on every surface of the human body and form unique flora, depending on their location. In particular, there are approximately 1013–1014 total bacteria in the colon, making the gut microbiota the largest flora in the body [1]. It is now known that the bacteria that make up the gut microbiota interact with us through various factors. Lifestyle factors such as diet and exercise are the primary influencers of the composition of the gut microbiota [2]. In turn, intestinal bacterial components and their metabolites can influence human biological systems such as the immune system [3].
+
+Some bacteria-induced diseases have long been known and studied; however, very few commensal bacteria have been targeted for the treatment and prevention of these diseases [4]. Recent advances in bacterial analysis have revealed that numerous diseases are affected by local or distant microbiomes. The study of the relationship between humans and their microbiota first focused on intestinal diseases, such as inflammatory bowel disease (IBD), because the local environment is rich in bacteria [5-8]. In addition, intervention with the gut microbiota by fecal microbiota transplantation and probiotics is becoming a therapeutic approach for these intestinal diseases [9]. More recently, the systemic effects of intestinal bacteria have been analyzed, and these symbiotic bacteria have been found to influence the state of diseases, including diabetes, dementia, hepatic steatosis, and non-intestinal cancers in distant organs [10-12]. Because the interactions between the intestinal tract and other distant organs are connected via their microbiota and believed to play an important role in human health, these major relationships have been termed, for example, the "gut-liver axis, gut-brain axis, and gut-kidney axis" [11,13,14].
+
+Diseases involving the prostate have also been reported in several bacterial studies. Prostate cancer is strongly associated with lifestyle, factors, including diet, and and genetic factors, including race; in particular, animal fat intake and African-American race are respective risk factors for prostate cancer [15,16]. These factors modulate the risk of prostate cancer by altering the gut microbiome. In this review, we investigate the relationship between the gut microbiota and prostate cancer, using findings from previous studies.
+
+## Clinical Research On Gut Microbiota And Prostate Cancer
+
+The gut microbiota can affect the incidence and progression of various types of cancers, but fewer reports consider its relationship with prostate cancer than other cancers. Recent studies in the USA have reported that the composition of the gut microbiota differs depending on the status of prostate cancer. In 2018, Liss et al [17] analyzed the gut microbiota of 133 American men who underwent prostate biopsy and reported a relationship between human prostate cancer and the gut microbiome for the first time. Their cohort primarily included African-Americans and Caucasians. The composition of intestinal bacteria was significantly different between men with positive and those with negative biopsy results (p<0.01), and patients with prostate cancer had a higher ratio of *Bacteroides* and Streptococcus species in their gut microbiota (p<0.04). 
+
+The researchers also performed a gene-based functional analysis of gut microbiota, revealing that the folate and arginine metabolism pathways were enriched in patients with prostate cancer. Because of the increased dependency on folate in prostate cancer cells, intensive research has been conducted on the relationship between them, and it has been shown that blood folate levels are correlated with prostate cancer risk and proliferation rate [18,19]. Furthermore, in a mouse model of prostate cancer, oral folate intake affected both the blood and prostate folate levels, and dietary folate deficiency improved cancer pathology [20]. Risk scoring based on 10 microbiome-derived metabolic pathways can detect prostate cancer with higher accuracy than the standard prostate-specific antigen (PSA) test. These results suggest that specific intestinal bacteria and their metabolic functions could play a role in the risk of prostate cancer, similar to that observed in several other diseases.
+
+In another report, *Bacteroides massiliensis* was more common in the gut microbiota of Caucasian males with prostate cancer than in those with benign prostatic hyperplasia (BPH), whereas *Faecalibacterium prausnitzii* and *Eubacterium rectalis* were less common [21]. F. 
+
+prausnitzii is a common anti-inflammatory bacterium, and its decrease is associated with a higher risk of Crohn's disease recurrence [22]. In addition, culture supernatants of this bacterium exhibit anti-inflammatory effects by reducing NF-κB activity in intestinal epithelial cells, both *in vitro* and *in vivo* [22]. These results suggest that some metabolites secreted by *F. prausnitzii* may contribute to the prevention of prostate cancer and IBD; therefore, this species is potentially beneficial as a probiotic. The composition of the gut microbiota of non-Hispanic Caucasians did not differ significantly between patients with and without prostate cancer [23]. 
+
+In Caucasian-centric communities, these results suggest that there may be no major specificity in the gut microbiota of patients with prostate cancer, but some Bacteroides species may predominate.
+
+A few reports on the gut microbiome profile of patients with prostate cancer living outside the USA 
+show different results. Our group has previously analyzed the gut microbiota composition of 152 Japanese patients who underwent prostate biopsy [24]. In this study, patients with high-grade prostate cancer (grade group >2) showed a significantly higher abundance of Alistipes and Lachnospira. *Alistipes* was enriched in the gut microbiota of patients with colorectal cancer, and *Alistepes* administration induced colitis in rodents by activating IL-6 signaling and promoting tumorigenesis [25,26]. The same mechanism may be responsible for the progression of prostate cancer. On the other hand, *Lachnospira* belongs to the family Lachnospiraceae, which is considered to contain beneficial bacteria that have anti-inflammatory effects and locally inhibit colon cancer [27,28]. However, as discussed later, short-chain fatty acids (SCFAs) produced by this family promote prostate cancer growth [29]. Moreover, a new index calculated from the abundance of 18 intestinal bacteria characteristic of patients with highgrade prostate cancer could discriminate the cancer status with greater accuracy than the PSA test (area under the curve [AUC]=0.81 vs. 0.67). Despite the different bacteria identified in studies in the USA, our functional analysis of the gut microbiome of patients with high-grade prostate cancer showed high starch and sucrose metabolism pathways, which are also common pathways seen in Americans. These results support previous reports that the profile of gut microbiota varies by region and race [30,31]. It also suggests that although different intestinal bacteria are involved in prostate cancer risk by region, their functions may be common across regions.
+
+Since the microbiota, which consists of foreign elements, interacts with the immune system, it is often associated with inflammation. Urinary and prostatic tissue-residing microbiota can directly cause prostatitis and prostatic hyperplasia through inflammatory processes [32,33]. Gut microbiota are involved locally in the pathogenesis of these inflammatory diseases, but may also play a role in more distant organs via immune and inflammatory processes [34]. In a previous study, we found that men with enlarged prostates had significantly higher Firmicutes/Bacteroidetes ratios in the gut microbiota than those without enlarged prostates [35]. 
+
+An imbalance between Firmicutes and Bacteroidetes in the human gut microbiota is considered an indicator of dysbiosis, and has also been observed in patients with obesity, IBD, nonalcoholic steatohepatitis, and myocarditis, suggesting a link to systemic inflammation [3640]. Thus, our results indicate that inflammation caused by intestinal bacteria may also affect distant prostate cancers. Furthermore, gut microbiota may be associated not only with hypertrophy but also with inflammatory carcinogenesis in the prostate. In the future, there is a need to investigate the independent impact of these bacterial factors on prostate cancer risk, as both gut microbiota and prostate cancer development are affected by various lifestyle factors, such as diet and exercise.
+
+## The Regulation Of Testosterone By Intestinal Bacteria
+
+Some bacteria reportedly affect testosterone levels in humans through deglucuronidation of testosterone in the gut, wherein the biologically active form is subsequently reabsorbed into the host [41]. Conversely, testosterone can alter the composition of gut microbiota 
+[42,43]. We have previously shown that the fraction of the Firmicutes phylum in the gut microbiota of elderly Japanese men (median age, 71.0 y) was significantly correlated with serum testosterone levels (r=0.3323, p=0.01) [44]. A similar trend was observed in younger cohorts. Shin et al. also found that in 31 men with a median age of 37.45 years, serum testosterone levels were positively correlated with several bacteria belonging to the phylum Firmicutes, including Megamonas
+(r=0.4161, p=0.02) and *Ruminococcus* (r=0.4589, p=0.01) 
+[45]. Similar to our study, in a group of adolescents 
+(median age, 10.99 y), testosterone levels were positively correlated with *Ruminococcus* (r=0.371, p=0.040) and Dorea (r=0.471, p=0.007), both of which belong to Firmicutes [46].
+
+Despite age-related changes in the gut microbiota composition, bacteria belonging to the phylum Firmicutes may be responsible for testosterone regulation in hosts. Fecal transplantation from adult male mice into immature female mice elevated their testosterone levels, indicating that the gut microbiome of males produces testosterone [47]. Oral administration of *Lactobacillus*, which belongs to Firmicutes, also increased testosterone levels in mice [48,49]. These results suggest that probiotics may improve testosterone-related health conditions and diseases in men.
+
+The gut microbiota may also influence the development of breast cancer because of its ability to deconjugate conjugated estrogen excreted in the intestinal tract [50]. In addition, intestinal bacteria can metabolize estrogen-like compounds such as enterodiol [51]. 
+
+Similarly, bacterial-derived testosterone analogs may be relevant in prostate cancer, and further studies are needed in this category.
+
+The gut microbiota has recently been described as an androgen-producing "organ." Accordingly, androgen deprivation therapy administered as a systemic treatment for prostate cancer can alter the gut microbiota. 
+
+Sfanos et al [52] analyzed the gut microbiota of 30 American men with various stages of prostate cancer and healthy controls. The results showed that patients with prostate cancer had a significantly different microbial composition from healthy controls (p=0.02). 
+
+In addition, treatment with anti-androgen therapy reduced the diversity of the gut microbiota in these patients and increased the proportion of *Akkermansia muciniphila* (0.002 vs. 0.055, p=0.002), which is also known to increase responses to immunotherapy [53]. 
+
+In an *in vivo* study using a mouse model, Akkermansia muciniphila decreased in the gut microbiota after castration, and testosterone propionate supplementation increased this bacterium [54]. These conflicting results may be due to the underlying differences in the composition of the gut microbiota in humans and rodents. 
+
+Further studies are needed to clarify the interaction between testosterone and *Akkermansia muciniphila*. 
+
+Thus, during treatment selection, we should consider the possibility that androgen deprivation therapy influences the efficacy of other types of cancer treatments through gut microbiome-mediated mechanisms.
+
+## The Role Of The Gut Microbiome In Prostate Cancer Pathogenesis
+
+Although human microbiome studies suggest that the gut microbiota plays a role in prostate cancer progression, few basic studies have elucidated the mechanisms by which this can occur. In other diseases, including non-intestinal diseases, basic research has revealed that intestinal immunity or bacterial-derived metabolites influenced by the gut microbiota can have augmenting or suppressive effects. Thus, the two-sided mechanisms by which the intestinal microbiota affects human health are complex, but have become clearer in recent studies.
+
+Previous findings have shown that animal fat intake promotes cancer growth in a prostate cancer mouse model, specifically in prostate-specific *Pten* knockout mice, through various mechanisms [55-57]. In our previous study, oral administration of antibiotics significantly reduced prostate cancer growth in mice fed a high-fat diet (HFD) (p=0.015) [29]. This study provides the first firm evidence that the gut microbiome is involved in prostate cancer progression. We found that this inhibitory effect involved a reduction in SCFAs produced by intestinal bacteria. SCFAs, major metabolites of the gut microbiota, stimulate systemic as well as local prostatic insulin-like growth factor 1 (IGF-1) 
+production and activate the IGF-1 signaling pathway in prostate cancer cells, which in turn promotes cancer growth [29]. The results of our study suggest that influencing the gut microbiota by diet and medications may play a role in the pathogenesis of prostate cancer.
+
+In contrast, butyrate, an SCFA, exerts anti-inflammatory effects through the activation of Gpr109a, inhibits carcinogenesis in the gut, and is a beneficial metabolite in humans [58]. Interestingly, this metabolite has opposing functions in the prostate and gut, indicating that the profile of healthy and beneficial gut microbiota may differ for each disease. For example, cohort studies have shown that some dairy products increase the risk of prostate cancer, whereas the opposite is observed in colorectal cancer [59-61]. The cause has not been identified, although the high butyrate content in milk suggests that this compound may be responsible for this discrepancy [62]. Since obese persons are at an increased risk of high-grade prostate cancer and have been found to have high levels of SCFAs in their stool, SCFAs are likely to be involved in prostate cancer risk 
+[63,64].
+
+Other mechanisms also suggest the involvement of gut microbiota in prostate cancer. We found that HFD-induced dysbiosis of the gut microbiota in mice elevated intestinal permeability and promoted lipopolysaccharide (LPS) leakage from this tract [65]. Serum LPS levels were significantly higher in prostate cancer model mice fed HFD than in those fed the control diet 
+(p=0.005). Prostate local histamine signaling activated by LPS can promote inflammatory prostate cancer growth by stimulating the interleukin-6 (IL6)-signal transducer and activator of transcription 3 (STAT3) 
+axis and increasing myeloid-derived suppressor cells 
+(MDSCs); however, the LPS/toll-like receptor 4 (TLR4) signaling block inhibited HFD-induced prostate cancer growth [65]. In another report, it was found that gut dysbiosis and increased intratumoral LPS promoted prostate cancer progression and docetaxel resistance via the IL6-STAT3 axis *in vivo* and *in vitro* [66]. Intestinal bacterial components have also been reported to cause a variety of diseases in distant organs via inflammatory and immune mechanisms. The influx of gut microbiota-derived endotoxins in the portal circulation promotes TLR4 expression and is associated with hepatic inflammation and steatosis [67,68]. Obesityinduced hepatic translocation of lipoteichoic acid, one of the components of the cell wall of gram-positive bacteria, accelerates the senescence of hepatic stellate cells, promoting HCC progression via prostaglandin E2-mediated suppression of antitumor immunity [69]. 
+
+Endotoxins and amyloids from gram-negative bacteria can penetrate the blood-brain barrier and induce amyloid β aggregation and neuroinflammation in the central nervous system, suggesting that bacterial molecules and metabolites may be involved in the onset and progression of Alzheimer's disease [70]. Bacterial components other than LPS may also play key roles in the inflammatory and immune-related development and progression of prostate cancer.
+
+Modulation of the immune system and other factors by intestinal bacteria is associated with the efficacy of various prostate cancer treatments. In an in vitro gut microbiota-simulated model, Daisley et al [71] 
+demonstrated that abiraterone acetate, a hormonebased chemotherapeutic used to treat prostate cancer, increased *Akkamansia muciniphila* and suggested that the species enhanced the supply of bacteria-derived vitamin K2, which in turn increased the efficacy of this medication. *Akkamansia* in the gut microbiota can also improve the efficacy of immunotherapeutic checkpoint inhibitors in several cancer types [53]. In prostate cancer, extracellular vesicles from this species activate antitumor immunity by interacting with CD8-positive T 
+cells and macrophages [72]. Gut microbiota can also affect immunity in the prostate, a distant organ, through bacterial components that circulate in the body.
+
+Other mechanisms of prostate cancer regulation by androgens in the gut microbiome have also been demonstrated. Pernigoni et al [73] found that the gut microbiota of patients and murine models with castration-resistant prostate cancer (CRPC) was enriched with Ruminococcus and *Bacteroides* genera. These intestinal bacteria transform androgens into their active forms and are involved in prostate cancer progression by supplying the active androgens. Indeed, elimination of the microbiota by antibiotics delayed the acquisition of castration resistance in prostate cancer, but transplantation with stools of patients with CRPC in mice accelerated their prostate cancer progression. Conversely, transplantation of stools of patients with castrationsensitive prostate cancer or the particular species *Prevotella stercorea* inhibited cancer progression.
+
+The reliable presence of multiple mechanisms, acting via metabolites, immunity, and androgens, suggests that a complex signaling network mediated by the microbiome is formed between the gut and prostate (Fig. 
+
+![4_image_0.png](4_image_0.png)
+
+Fig. 1. Overview of the "Gut-Prostate Axis". Specific taxa and dysbiosis in the gut microbiome regulate crucial features of prostate cancer through multiple mechanisms.
+
+1). Future studies can help provide a more complete profile of this "gut-prostate axis." Understanding this axis may lead to improved prevention and treatment of prostate cancer using specialized probiotics or prebiotics. In addition, fecal microbiota transplantation, as a more direct treatment method, has been in clinical trials for the treatment of some intestinal diseases [74], 
+and its application in prostate cancer is expected soon.
+
+## Prostate Cancer And The Local Microbiota
+
+In addition to the gut microbiome, the relationship between prostate cancer risk and the local microbiota has been studied. In the microbiota of human prostate tissue, *Cutibacterium acnes*, previously classified as Propionibacterium acnes, which commonly colonizes the skin, is more abundant in patients with prostate cancer. 
+
+Chen and Wei [75] investigated the genomic sequence of *C. acnes* in three sets of RNA-seq data from prostatic tissues of Australian, Caucasian, and Chinese patients and detected *C. acnes* in both cancer and adjacent tissue samples, but not in non-cancerous samples. Cavarretta 
+[76] also showed by bacterial genome sequencing of 16 prostatectomy specimens that *Propionibacterium* was more common in cancerous and peri-cancerous areas than in non-cancerous areas (60% vs. 49%).
+
+In a study of 100 samples from patients with prostate cancer and 50 samples from men without cancer, C. acnes was found to be significantly more prevalent in patients with prostate cancer (60% vs. 26%, p=0.001) 
+[77]. This is important, given that *C. acnes* can colonize prostate tissue and cause chronic inflammation in a rat model [78]. In addition, *Escherichia* has been detected in prostate cancer tissues in several studies, and is a genus of interest [79,80]. This genus mainly colonizes the gut, and the colibactin produced by some strains promotes carcinogenesis through DNA damage caused by double-strand breaks [81]. *Escherichia*-colonized prostate tissue may also have a similar oncogenic effect.
+
+The association of prostate cancer with the microbiota present in urine and prostatic fluid that comes in close contact with the prostate gland has also been investigated, and several small-scale studies using metagenomic analysis have shown a common abundance of *Streptococcus* in patients with prostate cancer 
+[23,82,83]. Although the mechanism underlying this **Table 1.** List of bacteria-related factors with potential impacts on prostate cancer**Bacterial factor** 
+
+![5_image_0.png](5_image_0.png)
+
+remains unclear, these results suggest that the microbiome present in urine and prostatic fluid may be more directly involved in prostate cancer than in the gut. 
+
+However, urine and prostate tissue contain far fewer numbers of bacteria than stool, and sequencing-based methods for detecting microbes in samples with low microbial concentrations are susceptible to contamination. Further verification, including contamination-free sample collection methods and appropriate sequencing controls, is required to obtain reliable results.
+
+The gut microbiome may be indirectly involved in the carcinogenesis and progression of prostate cancer, but the local microbiome also appears to be directly involved. The composition of the microbiota in the prostate is different from that seen in stool, and it is likely that different bacterial associations and activities exist locally compared to indirect actions from the gut microbiota.
+
+## Conclusions
+
+In addition to the importance of the local microbiome, the relationship between the gut microbiota and prostate cancer has been extensively investigated in both basic and clinical studies, and these suggest that the gut-prostate axis houses various bacterial associations through different mechanisms (Table 1). Furthermore, since the gut microbiome affects androgen production, it may be associated with the pathogenesis of several predominant male health conditions, such as the late onset of hypogonadism, erectile dysfunction, and BPH. However, the overall complexity of the axes remains unclear. Both genetic predisposition and environmental factors play a role in the pathogenesis of prostate cancer [84,85]. The gut microbiota is known to be susceptible to the same host factors [86,87]. Further studies should examine the gut-prostate axis as well as relevant genetic and environmental factors to determine the true impact of the gut microbiome on prostate cancer development and progression. These studies are expected to provide further evidence for the importance of the gut microbiome and support its modification for the prevention, diagnosis, and treatment of prostate cancer.
+
+## Conflict Of Interest
+
+The authors have nothing to disclose.
+
+## Funding
+
+This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant No. JP21K09421).
+
+## Author Contribution
+
+Conceptualization: MM, KF, KH, MADV, AT, HU, NN. Data curation: MM. Formal analysis: MM. Funding acquisition: MM. 
+
+Investigation: MM. Methodology: MM. Project administration: 
+MM, KF, KH, MADV, AT, HU, NN. Resources: MM. Software: MM. Supervision: AT, HU, NN. Validation: MM, KF. Visualization: MM. Writing - original draft: MM. Writing - review & 
+editing: KF, KH, MADV, AT, HU, NN.
+
+## References
+
+1. Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, et al. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355-9.
+
+2. Strasser B, Wolters M, Weyh C, Krüger K, Ticinesi A. The effects of lifestyle and diet on gut microbiota composition, inflammation and muscle performance in our aging society. Nutrients 2021;13:2045.
+
+3. Rooks MG, Garrett WS. Gut microbiota, metabolites and host immunity. Nat Rev Immunol 2016;16:341-52.
+
+4. de Brito BB, da Silva FAF, Soares AS, Pereira VA, Santos MLC, 
+Sampaio MM, et al. Pathogenesis and clinical management of *Helicobacter pylori* gastric infection. World J Gastroenterol 2019;25:5578-89.
+
+5. Tilg H, Adolph TE, Gerner RR, Moschen AR. The intestinal microbiota in colorectal cancer. Cancer Cell 2018;33:954-64.
+
+6. Nishida A, Inoue R, Inatomi O, Bamba S, Naito Y, Andoh A. 
+
+Gut microbiota in the pathogenesis of inflammatory bowel disease. Clin J Gastroenterol 2018;11:1-10.
+
+7. Ohkusa T, Sato N, Ogihara T, Morita K, Ogawa M, Okayasu I. Fusobacterium varium localized in the colonic mucosa of patients with ulcerative colitis stimulates species-specific antibody. J Gastroenterol Hepatol 2002;17:849-53.
+
+8. Lupp C, Robertson ML, Wickham ME, Sekirov I, Champion OL, Gaynor EC, et al. Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host Microbe 2007;2:204.
+
+9. Imdad A, Nicholson MR, Tanner-Smith EE, Zackular JP, Gomez-Duarte OG, Beaulieu DB, et al. Fecal transplantation for treatment of inflammatory bowel disease. Cochrane Database Syst Rev 2018;11:CD012774.
+
+10. Teng NMY, Price CA, McKee AM, Hall LJ, Robinson SD. 
+
+Exploring the impact of gut microbiota and diet on breast cancer risk and progression. Int J Cancer 2021;149:494-504.
+
+11. Cryan JF, O'Riordan KJ, Cowan CSM, Sandhu KV, Bastiaanssen TFS, Boehme M, et al. The microbiota-gut-brain axis. Physiol Rev 2019;99:1877-2013.
+
+12. Gurung M, Li Z, You H, Rodrigues R, Jump DB, Morgun A, 
+et al. Role of gut microbiota in type 2 diabetes pathophysiology. EBioMedicine 2020;51:102590.
+
+13. Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: pathophysiological basis for therapy. J Hepatol 2020;72:558-77.
+
+14. Stavropoulou E, Kantartzi K, Tsigalou C, Konstantinidis T, 
+Romanidou G, Voidarou C, et al. Focus on the gut-kidney axis in health and disease. Front Med (Lausanne) 2021;7:620102.
+
+15. Matsushita M, Fujita K, Nonomura N. Influence of diet and nutrition on prostate cancer. Int J Mol Sci 2020;21:1447.
+
+16. Gann PH. Risk factors for prostate cancer. Rev Urol 2002;4(Suppl 5):S3-10.
+
+17. Liss MA, White JR, Goros M, Gelfond J, Leach R, Johnson-
+Pais T, et al. Metabolic biosynthesis pathways identified from fecal microbiome associated with prostate cancer. Eur Urol 2018;74:575-82.
+
+18. Price AJ, Travis RC, Appleby PN, Albanes D, Barricarte Gurrea A, Bjørge T, et al. Circulating folate and vitamin B12 and risk of prostate cancer: a collaborative analysis of individual participant data from six cohorts including 6875 cases and 8104 controls. Eur Urol 2016;70:941-51.
+
+19. Tomaszewski JJ, Cummings JL, Parwani AV, Dhir R, Mason JB, Nelson JB, et al. Increased cancer cell proliferation in prostate cancer patients with high levels of serum folate. Prostate 2011;71:1287-93.
+
+20. Bistulfi G, Foster BA, Karasik E, Gillard B, Miecznikowski J, 
+Dhiman VK, et al. Dietary folate deficiency blocks prostate cancer progression in the TRAMP model. Cancer Prev Res (Phila) 2011;4:1825-34.
+
+21. Golombos DM, Ayangbesan A, O'Malley P, Lewicki P, Barlow L, Barbieri CE, et al. The role of gut microbiome in the pathogenesis of prostate cancer: a prospective, pilot study. Urology 2018;111:122-8.
+
+22. Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermúdez-Humarán LG, Gratadoux JJ, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A 2008;105:16731-6.
+
+23. Alanee S, El-Zawahry A, Dynda D, Dabaja A, McVary K, Karr M, et al. A prospective study to examine the association of the urinary and fecal microbiota with prostate cancer diagnosis after transrectal biopsy of the prostate using 16sRNA gene analysis. Prostate 2019;79:81-7.
+
+![7_image_0.png](7_image_0.png)
+
+24. Matsushita M, Fujita K, Motooka D, Hatano K, Fukae S, 
+Kawamura N, et al. The gut microbiota associated with high- Gleason prostate cancer. Cancer Sci 2021;112:3125-35.
+
+25. Dai Z, Coker OO, Nakatsu G, Wu WKK, Zhao L, Chen Z, et al. Multi-cohort analysis of colorectal cancer metagenome identified altered bacteria across populations and universal bacterial markers. Microbiome 2018;6:70.
+
+26. Moschen AR, Gerner RR, Wang J, Klepsch V, Adolph TE, 
+Reider SJ, et al. Lipocalin 2 protects from inflammation and tumorigenesis associated with gut microbiota alterations. Cell Host Microbe 2016;19:455-69.
+
+27. Meehan CJ, Beiko RG. A phylogenomic view of ecological specialization in the Lachnospiraceae, a family of digestive tract-associated bacteria. Genome Biol Evol 2014;6:703-13.
+
+28. Yachida S, Mizutani S, Shiroma H, Shiba S, Nakajima T, Sakamoto T, et al. Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer. Nat Med 2019;25:968-76.
+
+29. Matsushita M, Fujita K, Hayashi T, Kayama H, Motooka D, 
+Hase H, et al. Gut microbiota-derived short-chain fatty acids promote prostate cancer growth via IGF1 signaling. Cancer Res 2021;81:4014-26.
+
+30. Nishijima S, Suda W, Oshima K, Kim SW, Hirose Y, Morita H, 
+et al. The gut microbiome of healthy Japanese and its microbial and functional uniqueness. DNA Res 2016;23:125-33.
+
+31. Chen L, Zhang YH, Huang T, Cai YD. Gene expression profiling gut microbiota in different races of humans. Sci Rep 2016;6:23075.
+
+32. Lee HY, Wang JW, Juan YS, Li CC, Liu CJ, Cho SY, et al. The impact of urine microbiota in patients with lower urinary tract symptoms. Ann Clin Microbiol Antimicrob 2021;20:23.
+
+33. Miyake M, Tatsumi Y, Ohnishi K, Fujii T, Nakai Y, Tanaka N, 
+et al. Prostate diseases and microbiome in the prostate, gut, and urine. Prostate Int 2022;10:96-107.
+
+34. Al Bander Z, Nitert MD, Mousa A, Naderpoor N. The gut microbiota and inflammation: an overview. Int J Environ Res Public Health 2020;17:7618.
+
+35. Takezawa K, Fujita K, Matsushita M, Motooka D, Hatano K, 
+Banno E, et al. The Firmicutes/Bacteroidetes ratio of the human gut microbiota is associated with prostate enlargement. Prostate 2021;81:1287-93.
+
+36. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022-3.
+
+37. Stojanov S, Berlec A, Štrukelj B. The influence of probiotics on the Firmicutes/Bacteroidetes ratio in the treatment of obesity and inflammatory bowel disease. Microorganisms 
+
+## 2020;8:1715.
+
+38. Vallianou N, Christodoulatos GS, Karampela I, Tsilingiris D, 
+Magkos F, Stratigou T, et al. Understanding the role of the gut microbiome and microbial metabolites in non-alcoholic fatty liver disease: current evidence and perspectives. Biomolecules 2021;12:56.
+
+39. Sawicka-Śmiarowska E, Moniuszko-Malinowska A, Kamiński KA. Which microbes like my diet and what does it mean for my heart? Nutrients 2021;13:4146.
+
+40. Malesza IJ, Malesza M, Walkowiak J, Mussin N, Walkowiak D, Aringazina R, et al. High-fat, Western-style diet, systemic inflammation, and gut microbiota: a narrative review. Cells 2021;10:3164.
+
+41. Cross TL, Kasahara K, Rey FE. Sexual dimorphism of cardiometabolic dysfunction: gut microbiome in the play? Mol Metab 2018;15:70-81.
+
+42. Barroso A, Santos-Marcos JA, Perdices-Lopez C, Vega-Rojas A, Sanchez-Garrido MA, Krylova Y, et al. Neonatal exposure to androgens dynamically alters gut microbiota architecture. J Endocrinol 2020;247:69-85.
+
+43. Moreno-Indias I, Sánchez-Alcoholado L, Sánchez-Garrido MÁ, Martín-Núñez GM, Pérez-Jiménez F, Tena-Sempere M, et al. Neonatal androgen exposure causes persistent gut microbiota dysbiosis related to metabolic disease in adult female rats. Endocrinology 2016;157:4888-98.
+
+44. Matsushita M, Fujita K, Motooka D, Hatano K, Hata J, Nishimoto M, et al. Firmicutes in gut microbiota correlate with blood testosterone levels in elderly men. World J Mens Health 2022;40:517-25.
+
+45. Shin JH, Park YH, Sim M, Kim SA, Joung H, Shin DM. 
+
+Serum level of sex steroid hormone is associated with diversity and profiles of human gut microbiome. Res Microbiol 2019;170:192-201.
+
+46. Yuan X, Chen R, Zhang Y, Lin X, Yang X. Gut microbiota: effect of pubertal status. BMC Microbiol 2020;20:334.
+
+47. Markle JG, Frank DN, Mortin-Toth S, Robertson CE, Feazel LM, Rolle-Kampczyk U, et al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science 2013;339:1084-8.
+
+48. Lee J, Yang W, Hostetler A, Schultz N, Suckow MA, Stewart KL, et al. Characterization of the anti-inflammatory Lactobacillus reuteri BM36301 and its probiotic benefits on aged mice. BMC Microbiol 2016;16:69.
+
+49. Dardmeh F, Alipour H, Gazerani P, van der Horst G, Brandsborg E, Nielsen HI. Lactobacillus rhamnosus PB01 (DSM 14870) supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model. PLoS One 2017;12:e0185964.
+
+50. Yang J, Tan Q, Fu Q, Zhou Y, Hu Y, Tang S, et al. Gastrointestinal microbiome and breast cancer: correlations, mechanisms and potential clinical implications. Breast Cancer 2017;24:220-8.
+
+51. Parida S, Sharma D. The microbiome-estrogen connection and breast cancer risk. Cells 2019;8:1642.
+
+52. Sfanos KS, Markowski MC, Peiffer LB, Ernst SE, White JR, 
+Pienta KJ, et al. Compositional differences in gastrointestinal microbiota in prostate cancer patients treated with androgen axis-targeted therapies. Prostate Cancer Prostatic Dis 2018;21:539-48.
+
+53. Cani PD, Depommier C, Derrien M, Everard A, de Vos WM. 
+
+Akkermansia muciniphila: paradigm for next-generation beneficial microorganisms. Nat Rev Gastroenterol Hepatol 2022;19:625-37. Erratum in: Nat Rev Gastroenterol Hepatol 2022;19:682 54. Song CH, Kim N, Nam RH, Choi SI, Jang JY, Lee HN. Changes in gut microbiome upon orchiectomy and testosterone administration in AOM/DSS-induced colon cancer mouse model. Cancer Res Treat 2022. doi: 10.4143/crt.2022.080 [Epub].
+
+55. Hayashi T, Fujita K, Matsushita M, Hayashi Y, Uemura M, 
+Nonomura N. Metformin inhibits prostate cancer growth induced by a high-fat diet in Pten-deficient model mice. Int J Urol 2019;26:307-9.
+
+56. Hayashi T, Fujita K, Nojima S, Hayashi Y, Nakano K, Ishizuya Y, et al. High-fat diet-induced inflammation accelerates prostate cancer growth via IL6 signaling. Clin Cancer Res 2018;24:4309-18.
+
+57. Narita S, Nara T, Sato H, Koizumi A, Huang M, Inoue T, et al. 
+
+Research evidence on high-fat diet-induced prostate cancer development and progression. J Clin Med 2019;8:597.
+
+58. Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, 
+et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 2014;40:128-39.
+
+59. Orlich MJ, Mashchak AD, Jaceldo-Siegl K, Utt JT, Knutsen SF, 
+Sveen LE, et al. Dairy foods, calcium intakes, and risk of incident prostate cancer in adventist health study-2. Am J Clin Nutr 2022;116:314-24.
+
+60. Aune D, Navarro Rosenblatt DA, Chan DS, Vieira AR, Vieira R, Greenwood DC, et al. Dairy products, calcium, and prostate cancer risk: a systematic review and meta-analysis of cohort studies. Am J Clin Nutr 2015;101:87-117.
+
+61. Collatuzzo G, Seyyedsalehi MS, Rezaeianzadeh A, Marzban M, Rashidian H, Hadji M, et al. Consumption of yoghurt and other dairy products and risk of colorectal cancer in Iran: the IROPICAN study. Nutrients 2022;14:2506.
+
+62. Harrison S, Lennon R, Holly J, Higgins JPT, Gardner M, 
+Perks C, et al. Does milk intake promote prostate cancer initiation or progression via effects on insulin-like growth factors (IGFs)? A systematic review and meta-analysis. Cancer Causes Control 2017;28:497-528.
+
+63. Fujita K, Hayashi T, Matsushita M, Uemura M, Nonomura N. Obesity, inflammation, and prostate cancer. J Clin Med 2019;8:201.
+
+64. Schwiertz A, Taras D, Schäfer K, Beijer S, Bos NA, Donus C, 
+et al. Microbiota and SCFA in lean and overweight healthy subjects. Obesity (Silver Spring) 2010;18:190-5.
+
+65. Matsushita M, Fujita K, Hatano K, Hayashi T, Kayama H, Motooka D, et al. High-fat diet promotes prostate cancer growth through histamine signaling. Int J Cancer 2022;151:623-36.
+
+66. Zhong W, Wu K, Long Z, Zhou X, Zhong C, Wang S, et al. 
+
+Gut dysbiosis promotes prostate cancer progression and docetaxel resistance via activating NF-κB-IL6-STAT3 axis. Microbiome 2022;10:94.
+
+67. Henao-Mejia J, Elinav E, Jin C, Hao L, Mehal WZ, Strowig T, 
+et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 2012;482:179-85.
+
+68. Rivera CA, Adegboyega P, van Rooijen N, Tagalicud A, Allman M, Wallace M. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol 2007;47:571-9.
+
+69. Loo TM, Kamachi F, Watanabe Y, Yoshimoto S, Kanda H, 
+Arai Y, et al. Gut microbiota promotes obesity-associated liver cancer through PGE2-mediated suppression of antitumor immunity. Cancer Discov 2017;7:522-38.
+
+70. Kim HS, Kim S, Shin SJ, Park YH, Nam Y, Kim CW, et al. 
+
+Gram-negative bacteria and their lipopolysaccharides in Alzheimer's disease: pathologic roles and therapeutic implications. Transl Neurodegener 2021;10:49.
+
+71. Daisley BA, Chanyi RM, Abdur-Rashid K, Al KF, Gibbons S, 
+Chmiel JA, et al. Abiraterone acetate preferentially enriches for the gut commensal Akkermansia muciniphila in castrateresistant prostate cancer patients. Nat Commun 2020;11:4822. Erratum in: Nat Commun 2020;11:6394.
+
+72. Luo ZW, Xia K, Liu YW, Liu JH, Rao SS, Hu XK, et al. Extracellular vesicles from *Akkermansia muciniphila* elicit antitumor immunity against prostate cancer via modulation of CD8+ T cells and macrophages. Int J Nanomedicine 2021;16:2949-63.
+
+73. Pernigoni N, Zagato E, Calcinotto A, Troiani M, Mestre RP, 
+Calì B, et al. Commensal bacteria promote endocrine resistance in prostate cancer through androgen biosynthesis. Science 2021;374:216-24.
+
+74. Wortelboer K, Nieuwdorp M, Herrema H. Fecal microbiota transplantation beyond Clostridioides difficile infections. EBioMedicine 2019;44:716-29.
+
+75. Chen Y, Wei J. Identification of pathogen signatures in prostate cancer using RNA-seq. PLoS One 2015;10:e0128955.
+
+76. Cavarretta I, Ferrarese R, Cazzaniga W, Saita D, Lucianò R, 
+Ceresola ER, et al. The microbiome of the prostate tumor microenvironment. Eur Urol 2017;72:625-31.
+
+77. Davidsson S, Mölling P, Rider JR, Unemo M, Karlsson MG, 
+Carlsson J, et al. Frequency and typing of Propionibacterium acnes in prostate tissue obtained from men with and without prostate cancer. Infect Agent Cancer 2016;11:26.
+
+78. Olsson J, Drott JB, Laurantzon L, Laurantzon O, Bergh A, 
+Elgh F. Chronic prostatic infection and inflammation by Propionibacterium acnes in a rat prostate infection model. PLoS One 2012;7:e51434. Erratum in: PLoS One 2013;8:10.1371/ annotation/2160e616-aa79-4097-96ab-e143d2a4d136.
+
+79. Feng Y, Jaratlerdsiri W, Patrick SM, Lyons RJ, Haynes AM, 
+Collins CC, et al. Metagenomic analysis reveals a rich bacterial content in high-risk prostate tumors from African men. Prostate 2019;79:1731-8.
+
+80. Feng Y, Ramnarine VR, Bell R, Volik S, Davicioni E, Hayes VM, et al. Metagenomic and metatranscriptomic analysis of human prostate microbiota from patients with prostate cancer. BMC Genomics 2019;20:146.
+
+81. Nougayrède JP, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, Gottschalk G, et al. Escherichia coli induces DNA doublestrand breaks in eukaryotic cells. Science 2006;313:848-51.
+
+82. Ma X, Chi C, Fan L, Dong B, Shao X, Xie S, et al. The microbiome of prostate fluid is associated with prostate cancer. Front Microbiol 2019;10:1664.
+
+83. Shrestha E, White JR, Yu SH, Kulac I, Ertunc O, De Marzo AM, et al. Profiling the urinary microbiome in men with positive versus negative biopsies for prostate cancer. J Urol 2018;199:161-71.
+
+84. Demichelis F, Stanford JL. Genetic predisposition to prostate cancer: update and future perspectives. Urol Oncol 2015;33:75-84.
+
+85. Bostwick DG, Burke HB, Djakiew D, Euling S, Ho SM, 
+Landolph J, et al. Human prostate cancer risk factors. Cancer 2004;101(10 Suppl):2371-490.
+
+86. Wang J, Tang M, Jiang H, Wu B, Cai W, Hu C, et al. The role of adrenergic activation on murine luteal cell viability and progesterone production. Theriogenology 2016;86:1182-8.
+
+87. Claesson MJ, Jeffery IB, Conde S, Power SE, O'Connor EM, 
+Cusack S, et al. Gut microbiota composition correlates with diet and health in the elderly. Nature 2012;488:178-84.

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+Contents lists available at ScienceDirect
+
+![0_image_3.png](0_image_3.png)
+
+# Critical Care And Resuscitation
+
+![0_Image_4.Png](0_Image_4.Png)
+
+journal homepage: www.elsevier.com/locate/ccrj Original Article
+
+![0_image_2.png](0_image_2.png)
+
+The impact of body mass index on long-term survival after ICU admission due to COVID-19: A retrospective multicentre study Ashwin Subramaniam, MBBS MMed FRACP FCICM a, b, c, d, *, Ryan Ruiyang Ling, MBBS e, Emma J. Ridley, PhD d, f, David V. Pilcher, MBBS MRCP(UK) FRACP FCICM d, g, h a Department of Intensive Care, Peninsula Health, Frankston, Victoria, Australia; b Department of Intensive Care, Dandenong Hospital, Monash Health, Dandenong, Victoria, Australia; c Peninsula Clinical School, Monash University, Frankston, Victoria, Australia; d Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), School of Public Health and Preventive Medicine, Monash University, Melbourne, Victoria, Australia; e Yong Loo Lin School of Medicine, National University of Singapore, Singapore; f Department of Nutrition and Dietetics, Alfred Hospital, Melbourne, Victoria, Australia; g Department of Intensive Care, Alfred Hospital, Melbourne, Victoria, Australia; h Centre for Outcome and Resource Evaluation, Australian and New Zealand Intensive Care Society, Melbourne, Victoria, Australia article information
+
+## Abstract
+
+| Article history: Received 16 October 2023 Accepted 18 October 2023   |
+|----------------------------------------------------------------------|
+
+| Keywords: Body mass index Obesity Obesity paradox COVID-19 ANZICS adult patient database Long-term survival   |
+|---------------------------------------------------------------------------------------------------------------|
+
+Objective: The impact of obesity on long-term survival after intensive care unit (ICU) admission with severe coronavirus disease 2019 (COVID-19) is unclear. We aimed to quantify the impact of obesity on time to death up to two years in patients admitted to Australian and New Zealand ICUs.
+
+Design: Retrospective multicentre study. Setting: 92 ICUs between 1st January 2020 through to 31st December 2020 in New Zealand and 31st March 2022 in Australia with COVID-19, reported in the Australian and New Zealand Intensive Care Society adult patient database.
+
+Participants: All patients with documented height and weight to estimate the body mass index (BMI)
+were included. Obesity was classified patients according to the World Health Organization recommendations.
+
+Interventions and main outcome measures: The primary outcome was survival time up to two years after ICU admission. The effect of obesity on time to death was assessed using a Cox proportional hazards model. Confounders were acute illness severity, sex, frailty, hospital type and jurisdiction for all patients.
+
+Results: We examined 2,931 patients; the median BMI was 30.2 (IQR 25.6e36.0) kg/m2. Patients with a BMI 30 kg/m2 were younger (median [IQR] age 57.7 [46.2e69.0] vs. 63.0 [50.0e73.6]; p < 0.001) than those with a BMI <30 kg/m2. Most patients (76.6%; 2,244/2,931) were discharged alive after ICU admission. The mortality at two years was highest for BMI categories <18.5 kg/m2 (35.4%) and 18.5e24.9 kg/m2 (31.1%), while lowest for BMI 40 kg/m2 (14.5%). After adjusting for confounders and with BMI
+18.5e24.9 kg/m2 category as a reference, only the BMI 40 kg/m2 category patients had improved survival up to 2 years (hazard ratio ¼ 0.51; 95%CI: 0.34e0.76).
+
+Conclusions: The obesity paradox appears to exist beyond hospital discharge in critically ill patients with COVID-19 admitted in Australian and New Zealand ICUs. A BMI 40 kg/m2 was associated with a higher survival time of up to two years.
+
+© 2023 The Authors. Published by Elsevier B.V. on behalf of College of Intensive Care Medicine of Australia and New Zealand. This is an open access article under the CC BY-NC-ND license (http://
+creativecommons.org/licenses/by-nc-nd/4.0/).
+
+Abbreviations: ANZ, Australia and New Zealand; ANZICS, Australia and New Zealand Intensive Care Society; ANZROD, Australia and New Zealand risk of death; APACHE,
+Acute Physiology and Chronic Health Evaluation; APD, Adult Patient Database; BMI, body mass index; COPD, chronic obstructive pulmonary disease; COVID-19, coronavirus disease 2019; CFS, Clinical Frailty Scale; ECMO, extracorporeal membrane oxygenation; ED, emergency department; HR, hazard ratio; ICU, intensive care unit; IQR, interquartile range; LOS, length of stay; MET, medical emergency team; MI, myocardial infarction; NIV, non-invasive ventilation; n, number; OR, odds ratio; RRT, renal replacement therapy; SD, standard deviation.
+
+* Corresponding author at: Intensive care specialist, Department of Intensive Care, Frankston Hospital, 2 Hastings Road, Frankston, Victoria, 3199, Australia. Tel.: þ61 3 9784 7422.
+
+E-mail address: ashwin.subramaniam@monash.edu (A. Subramaniam).
+
+@catchdrash (A. Subramaniam)
+https://doi.org/10.1016/j.ccrj.2023.10.004 1441-2772/© 2023 The Authors. Published by Elsevier B.V. on behalf of College of Intensive Care Medicine of Australia and New Zealand. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+## Key Points
+
+Question: What is the association between BMI and survival time up to 2 years after ICU admission due to COVID-19?
+
+Findings: In this bi-national retrospective study, we found that only patients with a BMI 40 kg/m2 with severe COVID19 were independently associated with better survival time up to two years following an ICU admission in Australia and New Zealand. The obesity paradox appears to exist beyond hospital discharge in critically ill patients with COVID-19.
+
+Meaning: Our findings suggest that future studies should explore the reasons why there is a survival advantage for morbidly obese patients with COVID-19. Tweet: A retrospective multicentre study from ANZ found that only patients with a BMI  40 kg/m2 with severe COVID19 were independently associated with better survival time up to two years following an ICU admission. The obesity paradox appears to exist beyond hospital discharge in critically ill patients with COVID-19.
+
+## 1. Introduction
+
+The prevalence of obesity is increasing globally, with approximately 20 % of patients admitted to the intensive care unit (ICU)
+obese.1e3 While obesity is typically associated with morbidity and mortality and poses additional care challenges in managing the critically ill, emerging literature has paradoxically reported higher short-term survival for patients with critical illness who are obese, also known as the obesity paradox.2,4e7 A large epidemiological study reporting on patients in ICUs from Australia and New Zealand observed that 35 % of the patients were obese and confirmed that some level of obesity was associated with overall lower in-hospital mortality.8 A recent study observed the obesity paradox in survivors of critical illness up to 4 years after their ICU
+admission.9 There are however no accepted physiological mechanistic models to explain why morbid obesity could be protective.
+
+Although the causal mechanisms are unknown, some pathophysiologic mechanisms such as higher energy reserves, anti-inflammatory immune profile, the role of adipose tissue, prevention of muscle wasting and an association between increased BMI and lower risk of hypoglycaemia may all provide explanations for this obesity paradox.4,8e10 Apart from chronic disease, sarcopenia, malnutrition and smoking status, other non-physiological methodological explanations have been postulated.8,11,12 Furthermore, many biases such as selection bias, confounder bias, collider stratification bias, reporting bias, treatment bias and publication bias may be added to explain this phenomenon.1,8 A large retrospective study demonstrated that the obesity paradox is more than just a simple association between body mass index (BMI) and mortality and emphasized the importance of acute illness severity.13 Several studies have found an association between coronavirus disease 2019 (COVID-19) severity and obesity, 14e21 such that obesity was labelled a risk factor for severe COVID-19, ICU
+admission and mechanical ventilation.18 As excess body fat mass results in various hormonal, metabolic, and inflammatory changes,22 it was hypothesized that adipose tissue may play a vital role in the mechanism of progression of COVID-19.23,24 In patients with severe COVID-19, there is a linear association between body mass index (BMI) and hospitalization, ICU admission and the need for mechanical ventilation and death among patients with COVID19.14,17e20,25e39 Furthermore, the Health Outcome Predictive Evaluation for COVID-19 Registry revealed no evidence of obesity paradox in patients with COVID-19.40 However, a recent study found that obese patients with COVID-19 admitted to hospital had lower survival, but amongst those who have been admitted to ICU,
+there is a higher survival associated with obesity.25 These discrepancies in findings may be due to differences in study population characteristics, and most meta-analyses showed significant heterogeneity.39 To our knowledge, the impact of obesity on long-term survival after intensive care unit (ICU) admission with severe COVID-19 has never been investigated. Therefore, our primary aim was to investigate the association between BMI and long-term survival after ICU
+admission due to COVID-19. We hypothesized that after adjusting for confounders, increasing levels of obesity would be associated with higher survival for up to two years.
+
+## 2. Methods: 2.1. Study Design And Participants
+
+We performed a retrospective multicentre study of all critically ill adult (age 16 years) patients admitted to Australian and New Zealand ICUs for their index ICU admission with a diagnosis of COVID-19. The patients were identified as suspected or confirmed COVID-19 based on the diagnostic code allocated as the cause of ICU
+admission (Supplementary Table 1). We only included the first ICU
+admission per hospitalization. Patients transferred to another ICU
+were excluded. We also excluded patients if there were no documented height and weight to estimate BMI, if the outcome was missing or if admitted for organ donation purposes.
+
+## 2.2. Data Sources And Measurement
+
+Data were extracted from the Australian and New Zealand Intensive Care Society (ANZICS) adult patient database, a binational clinical quality registry dataset, collected by the ANZICS
+Centre for Outcomes and Resources Evaluation, that contains information on all admissions to 98 % of adult ICUs in Australia and 67 % of ICUs in New Zealand. ICU admission records between 1st January 2020 through 31st December 2020 in New Zealand and 31st March 2022 in Australia, were linked to the date of death recorded in the national death registers in each country using an encoded linkage key. This provided a maximum follow-up of 27 months. Data collectors receive regular training and quality assurance reviews and data is collected using a standardized data dictionary. In addition, regular automated data checks further ensure the validity of recorded data.41 Apart from each patient's demographic details, the registry also captures their diagnostic, biochemical, physiological, and chronic health parameters from the first 24 h of ICU admission as required to calculate illness severity 184 A. Subramaniam et al. / Critical Care and Resuscitation 25 (2023) 182e192 scores. The definitions are described in the ANZICS adult patient database data dictionary.42
+
+## 2.3. Variables
+
+We extracted data on patient demographics (age, sex, comorbidities, ethnicity, ICU admission source, smoking status), frailty status using the clinical frailty scale (CFS), BMI (based on patient's weight and height which could have been estimated at ICU admission), ICU organ supports (receipt of invasivemechanical ventilation, non-invasive ventilation, vasopressors, extracorporeal membrane oxygenation, and/or renal replacement therapy), ICU and hospital mortality, ICU and hospital length of stay and discharge destinations (home, chronic care facility or rehabilitation).
+
+## 2.4. Bmi Definition
+
+BMI was classified according to the World Health Organization recommendations:43 BMI <18.5 kg/m2 'underweight'; BMI between 18.5  24.9 kg/m2 'healthy weight'; BMI 25.0 and  29.9 kg/m2
+'overweight' and BMI30 kg/m2 'obese'. Obesity was further classified into three severity categories: class-I (BMI 30.0e34.9 kg/m2),
+class-II (BMI 35.0e39.9 kg/m2) and class-III (BMI 40 kg/m2).44 BMI
+was calculated based on the available weight and height in the ANZICS adult patient database (body weight (kg) divided by height squared (m2)).
+
+## 2.5. Confounding Variables
+
+Based on a previous study, the confounding variables included in the model pertain to the ICU (location, type, and size), patient (frailty, age, gender, and comorbidities), ICU admission (care type, source, treatment limitations and pre-ICU length of stay) and patient acute illness severity.45 Illness severity was determined using the ANZROD which is a highly predictive mortality prediction model used for benchmarking ICU performance in Australia and New Zealand.46,47 ANZROD includes components of the APACHE IV
+scoring system, such as age, chronic illnesses, acute physiological disturbance and diagnosis, and the presence of treatment limitation on admission to ICU and provides an accurate estimate of the severity of illness in the first 24 h of ICU admission. Recent studies demonstrated that ANZROD was predictive of longer-term outcomes.48,49 Frailty was included as a confounder as 20 % of patients with COVID-19 admitted to ICU were frail.49e51
+
+## 2.6. Outcomes
+
+The primary outcome was a survival time of up to two years and is reported as observed mortality at one and two years after ICU
+admission. Secondary outcomes included ICU and hospital mortality, ICU and hospital length of stays, and discharge destinations.
+
+## 2.7. Subgroup Analysis
+
+Predefined subgroup analyses based on those who survived hospitalisation, and those receiving mechanical ventilation were performed.
+
+## 2.8. Statistical Analysis
+
+For categorical variables, we reported counts with percentage (n
+(%)) and made comparisons between BMI categories using Chisquared tests. For continuous data, we report normally distributed data using means (standard deviation) and non-parametric data using median (interquartile range [IQR]). We made comparisons using the student's t-test for normally distributed data and the ManneWhitney U test otherwise. All analyses were performed with the BMI 18.5e24.9 kg/m2 category as the reference.
+
+We treated BMI both as a continuous and categorical variable in separate regression models. As estimates can cluster around centres, we used a robust-variance sandwich-type estimator to derive the standard errors and account for this clustering.52 Overall survival estimates are displayed using KaplaneMeier curve plots. The effect of BMI on time to death was assessed using a Cox proportional hazards model, adjusting for ANZROD, male sex, CFS, hospital type (tertiary, metropolitan, rural/regional, and private) and jurisdiction (Australian states and New Zealand), clustered by site, and site treated as a random effect. The results were reported using hazard ratios (HR, 95%CI). We assigned CFS and ANZROD as nonlinear predictors in the regression analysis where the estimation algorithm arranges the predictors based on significance to determine the best fitting fractional-polynomial function for each predictor while assuming all other predictors are linear using the mfp package on R. We conducted an additional post hoc analysis looking at an interaction effect of age*obesity treated both as continuous and categorical variables while adjusting for illness severity, hospital type, jurisdiction, and frailty. We performed data analysis using R4.2.2 (The R Foundation).53 We used a two-sided p-value of
+<0.05 to indicate statistical significance.
+
+## 2.9. Ethics Approval
+
+All experimental protocols were approved by The Alfred Hospital Ethics Committee (local reference 413/19).
+
+## 3. Results
+
+During the study period, the ANZICS adult patient database listed 5834 patients with COVID-19 hospitalizations which were linked with the National Death Index. After excluding: 64 patients from New Zealand admitted in 2021; 413 patients who were transferred from another ICU; and 2423 patients who had insufficient information to estimate BMI, the final study population comprised 2931 patients from 87 Australian and 5 New Zealand ICUs (Supplementary Fig. 1). Compared to patients with estimated BMI, those without BMI were of similar age, frailty status and illness severity, less commonly were mechanically ventilated and had lower 2-year mortality after ICU admission (Supplementary Table 2).
+
+Of the total 2931 patients, 50.9 % (n ¼ 1493) had a BMI of 30 kg/m2 (median [IQR] BMI 30.2 [25.6e36.0]) kg/m2). Patients with obesity were younger (median [IQR] age 57.7 [46.2e69.0] vs.
+
+63.0 [50.0e73.6]; p < 0.001), a higher proportion had diabetes mellitus and a lower proportion of solid organ or haematological malignancies or immunosuppressive conditions or treatment limitations at ICU admission. Patients with obesity were more likely to receive invasive and non-invasive ventilatory and vasopressor supports than those without obesity. No difference in the receipt of other organ supports was observed. The baseline characteristics of patients with COVID-19 based on BMI categories are summarized in Table 1, while Supplementary Table 3 summarizes the comparison based on obesity status.
+
+## 3.1. Primary Outcome
+
+The mortality at two years progressively decreased with increasing BMI, highest for BMI <18.5 kg/m2 (35.4 %) and lowest for BMI 40 kg/m2 (14.5 %; p < 0.001) (Table 2, Fig. 1, Supplementary Table 4). The median [IQR] survival time for patients with obesity was 6.4 [4.5e16.8] months when compared to 7.1 [4.0e20.6] months for patients with a BMI <30 kg/m2 (p < 0.001). After adjusting for
+
+| Table 1 Baseline characteristics, illness severity and ICU management of patients with COVID-19 based on BMI categories. BMI <18.5 kg/m2 18.5e24.9 kg/m2 25.0e29.9 kg/m2                                                                                              | 30.0e34.9 kg/m2                                                            | 35.0e39.9 kg/m2   | 40.0 kg/m2                   | p-value           |                   |                   |         |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------|-------------------|-------------------|-------------------|-------------------|-------------------|---------|
+| (Underweight)                                                                                                                                                                                                                                                         | (Healthy)                                                                  | (Overweight)      | (Class 1 obesity) | (Class 2 obesity) | (Class 3 obesity) |                   |         |
+| Number                                                                                                                                                                                                                                                                | 48 (1.6 %)                                                                 | 595 (20.3 %)      | 793 (27.1 %)      | 659 (22.5 %)      | 375 (12.8 %)      | 459 (15.7 %)      | e       |
+| Male sex                                                                                                                                                                                                                                                              | 20 (41.7 %)                                                                | 370 (62.2 %)      | 534 (67.3 %)      | 390 (59.2 %)      | 206 (54.9 %)      | 212 (46.2 %)      | < 0.001 |
+| Indigenous status                                                                                                                                                                                                                                                     | 4 (8.9 %)                                                                  | 34 (5.9 %)        | 27 (3.6 %)        | 18 (2.8 %)        | 9 (2.5 %)         | 20 (4.7 %)        | 0.030   |
+| CFS score                                                                                                                                                                                                                                                             | 5 (4, 6)                                                                   | 3 (2, 4)          | 3 (2, 4)          | 3 (2, 4)          | 3 (2, 4)          | 3 (3, 4)          | < 0.001 |
+| Age (years)                                                                                                                                                                                                                                                           | 63.5 (50.1, 76.4)                                                          | 63.0 (49.0, 73.6) | 62.7 (50.7, 73.5) | 60.0 (49.7, 70.1) | 58.0 (45.9, 70.0) | 52.7 (41.7, 64.4) | < 0.001 |
+| Hospital classification - Public Tertiary                                                                                                                                                                                                                             | 16 (33.3 %)                                                                | 312 (52.4 %)      | 430 (54.2 %)      | 347 (52.7 %)      | 205 (54.7 %)      | 216 (47.1 %)      | < 0.001 |
+| - Public Metropolitan                                                                                                                                                                                                                                                 | 17 (35.4 %)                                                                | 167 (28.1 %)      | 250 (31.5 %)      | 231 (35.1 %)      | 118 (31.5 %)      | 177 (38.6 %)      |         |
+| - Public rural/regional                                                                                                                                                                                                                                               | 13 (27.1 %)                                                                | 100 (16.8 %)      | 100 (12.6 %)      | 77 (11.7 %)       | 48 (12.8 %)       | 63 (13.7 %)       |         |
+| - Private                                                                                                                                                                                                                                                             | 2 (4.2 %)                                                                  | 16 (2.7 %)        | 13 (1.6 %)        | 4 (0.6 %)         | 4 (1.1 %)         | 5 (1.1 %)         |         |
+| ICU admission source - Emergency department                                                                                                                                                                                                                           | 29 (60.4 %)                                                                | 372 (62.5 %)      | 420 (53.0 %)      | 341 (51.7 %)      | 186 (49.6 %)      | 250 (54.5 %)      | < 0.001 |
+| - Ward                                                                                                                                                                                                                                                                | 12 (25.0 %)                                                                | 187 (31.4 %)      | 329 (41.5 %)      | 282 (42.8 %)      | 164 (43.7 %)      | 189 (41.2 %)      |         |
+| - Other hospital                                                                                                                                                                                                                                                      | 4 (8.3 %)                                                                  | 29 (4.9 %)        | 38 (4.8 %)        | 33 (5.0 %)        | 22 (5.6 %)        | 16 (3.5 %)        |         |
+| - Operating theatre/ Recovery                                                                                                                                                                                                                                         | 0 (0 %)                                                                    | 1 (0.2 %)         | 1 (0.1 %)         | 0 (0 %)           | 1 (0.3 %)         | 1 (0.2 %)         |         |
+| - Direct admit                                                                                                                                                                                                                                                        | 3 (6.3 %)                                                                  | 6 (1.0 %)         | 5 (0.6 %)         | 3 (0.5 %)         | 2 (0.5 %)         | 4 (0.9 %)         |         |
+| Documented co-morbidities - Chronic respiratory condition                                                                                                                                                                                                             | 24 (50.0 %)                                                                | 95 (16.0 %)       | 88 (11.1 %)       | 82 (12.4 %)       | 46 (12.3 %)       | 80 (17.4 %)       | < 0.001 |
+| - Chronic cardiovascular condition                                                                                                                                                                                                                                    | 5 (10.4 %)                                                                 | 55 (9.2 %)        | 57 (7.2 %)        | 57 (8.6 %)        | 29 (7.7 %)        | 40 (8.7 %)        | 0.83    |
+| - Chronic renal failure                                                                                                                                                                                                                                               | 1 (2.1 %)                                                                  | 19 (3.2 %)        | 24 (3.0 %)        | 18 (2.7 %)        | 13 (3.5 %)        | 14 (3.1 %)        | 0.017   |
+| - Chronic liver disease                                                                                                                                                                                                                                               | 4 (8.3 %)                                                                  | 4 (0.7 %)         | 2 (0.3 %)         | 8 (1.2 %)         | 5 (1.3 %)         | 6 (1.3 %)         | < 0.001 |
+| - Diabetes mellitus                                                                                                                                                                                                                                                   | 8 (16.7 %)                                                                 | 126 (21.2 %)      | 199 (25.1 %)      | 228 (34.6 %)      | 120 (32.0 %)      | 148 (32.2 %)      | < 0.001 |
+| - Cancer                                                                                                                                                                                                                                                              | 2 (4.2 %)                                                                  | 33 (5.5 %)        | 30 (3.8 %)        | 14 (2.1 %)        | 8 (2.1 %)         | 7 (1.5 %)         | 0.003   |
+| - Immunosuppression                                                                                                                                                                                                                                                   | 5 (10.0 %)                                                                 | 40 (7.1 %)        | 51 (6.1 %)        | 35 (5.3 %)        | 14 (3.6 %)        | 9 (1.8 %)         | < 0.001 |
+| - Lymphoma                                                                                                                                                                                                                                                            | 0 (0 %)                                                                    | 7 (1.2 %)         | 5 (0.6 %)         | 2 (0.3 %)         | 3 (0.8 %)         | 1 (0.2 %)         | 0.44    |
+| - Leukaemia                                                                                                                                                                                                                                                           | 0 (0 %)                                                                    | 11 (1.8 %)        | 15 (1.9 %)        | 8 (1.2 %)         | 2 (0.5 %)         | 2 (0.4 %)         | 0.19    |
+| Miscellaneous - ICU admission post-MET call                                                                                                                                                                                                                           | 8 (16.7 %)                                                                 | 129 (21.9 %)      | 238 (30.1 %)      | 198 (30.3 %)      | 127 (34.0 %)      | 133 (29.2 %)      | < 0.001 |
+| - Treatment limitations                                                                                                                                                                                                                                               | 16 (33.3 %)                                                                | 103 (17.3 %)      | 102 (12.9 %)      | 70 (10.6 %)       | 42 (11.2 %)       | 44 (9.6 %)        | < 0.001 |
+| - Cardiac arrest 24 h prior                                                                                                                                                                                                                                           | 2 (4.2 %)                                                                  | 14 (2.4 %)        | 11 (1.4 %)        | 6 (0.9 %)         | 4 (1.1 %)         | 2 (0.4 %)         | 0.004   |
+| - Pre-ICU (hours)                                                                                                                                                                                                                                                     | 9.0 (3.7, 19.4)                                                            | 7.3 (4.0, 24.0)   | 9.8 (4.5, 40.4)   | 10.6 (4.5, 39.3)  | 10.0 (4.1, 46.2)  | 8.6 (4.4, 42.4)   | 0.033   |
+| Organ failure scores - APACHE-II                                                                                                                                                                                                                                      | 17.7 [6.1]                                                                 | 16.5 [7.7]        | 15.9 [6.7]        | 15.8 [6.4]        | 15.2 [6.6]        | 15.0 [6.3]        | < 0.001 |
+| - APACHE-III                                                                                                                                                                                                                                                          | 56.4 [21.0]                                                                | 53.7 [15.7]       | 53.2 [21.8]       | 51.9 [20.1]       | 50.0 [20.0]       | 48.3 [19.0]       | < 0.001 |
+| - ANZROD (%)                                                                                                                                                                                                                                                          | 8.6 (4.4, 18.4)                                                            | 6.3 (2.5, 14.3)   | 5.8 (2.5, 13.2)   | 5.0 (2.6, 11.9)   | 4.8 (2.3, 9.7)    | 3.9 (2.0, 8.7)    | < 0.001 |
+| Organ supports - Mechanical ventilation                                                                                                                                                                                                                               | 6 (12.5 %)                                                                 | 158 (27.0 %)      | 277 (35.4 %)      | 239 (36.5 %)      | 144 (38.6 %)      | 190 (42.4 %)      | < 0.001 |
+| - MV duration (hours)                                                                                                                                                                                                                                                 | 326 (85, 548)                                                              | 105 (26, 226)     | 128 (27, 308)     | 152 (49, 315)     | 167 (42, 290)     | 142 (42, 256)     | 0.09    |
+| - Non-invasive ventilation                                                                                                                                                                                                                                            | 15 (31.3 %)                                                                | 162 (28.0 %)      | 284 (36.4 %)      | 289 (44.4 %)      | 152 (41.0 %)      | 235 (52.6 %)      | < 0.001 |
+| - Vasopressor and inotropes                                                                                                                                                                                                                                           | 13 (27.1 %)                                                                | 209 (35.7 %)      | 307 (39.1 %)      | 270 (41.3 %)      | 158 (42.2 %)      | 187 (41.6 %)      | 0.047   |
+| - Renal replacement therapy                                                                                                                                                                                                                                           | 2 (4.3 %)                                                                  | 23 (4.0 %)        | 52 (6.7 %)        | 38 (5.9 %)        | 20 (5.5 %)        | 17 (3.9 %)        | 0.018   |
+| - ECMO                                                                                                                                                                                                                                                                | 0 (0 %)                                                                    | 5 (0.9 %)         | 10 (1.3 %)        | 12 (1.9 %)        | 3 (0.8 %)         | 8 (1.8 %)         | 0.61    |
+| - Tracheostomy                                                                                                                                                                                                                                                        | 2 (4.3 %)                                                                  | 14 (2.4 %)        | 35 (4.6 %)        | 26 (4.0 %)        | 19 (5.2 %)        | 15 (3.4 %)        | 0.40    |
+| Data are n (%), mean [SD] or median (IQR). CFS e clinical frailty scale, SD e standard deviation, IQR e interquartile range, BMI e body mass index, MET e medical emergency team, APACHE e Acute Physiology and Chronic Health Evaluation, ICU e intensive care unit, | A. Subramaniam et al. / Critical Care and Resuscitation 25 (2023) 182 e192 |                   |                   |                   |                   |                   |         |
+
+186 
+
+![4_image_0.png](4_image_0.png)
+
+This retrospective study examined the impact of obesity
+(defined via BMI) on long-term survival time up to two years after ICU admission among patients with severe COVID-19 in Australia and New Zealand. We found that more than half the patients had obesity, which was higher than pre-pandemic. Patients with Table 2Raw outcomes of patients with COVID-19 based on BMI categories.obesity were younger and had lower illness severity scores than
+confounders, the Cox proportional hazards regression demonstrated higher survival time up to two years only for patients with BMI 40 kg/m2 (HR ¼ 0.51; 95%CI: 0.34e0.76) (Table 3). When BMI
+was modelled as a continuous variable, a non-linear relationship with time to death was noted. Patients with BMIs between BMI 32 and 60 kg/m2 had the best survival (Fig. 2).
+
+## 3.2. Subgroup Analyses
+
+Patients who survived hospitalization: 2570 patients were discharged alive from the hospital; 52.2 % (n ¼ 1342) were obese.
+
+Kaplan Meier survival curves estimated that patients with BMI 
+40 kg/m2 had the highest survival compared to other BMI categories (p < 0.001; Supplementary Fig. 2, Supplementary Table 5).
+
+Similar findings of higher survival time up to two years only for patients with BMI 40 kg/m2 were observed in the adjusted Cox proportional hazards regression (Supplementary Table 6). The log hazards 2-year survival demonstrated a non-linear survival advantage for patients with BMI >25 kg/m2 (Supplementary Fig. 3).
+
+Patients receiving mechanical ventilation: 1015 patients received mechanical ventilation, of which 56.6 % (n ¼ 574) were obese. The duration of mechanical ventilation was also similar across BMI
+categories (Supplementary Table 7). Kaplan Meier survival curves estimated that patients with BMI category <18.5 kg/m2 had the lowest 2-year survival, while BMI 40 kg/m2 had the highest
+(Supplementary Fig. 2). The adjusted Cox proportional hazards regression demonstrated a higher survival time of up to 2 years for all patients with BMI 25 kg/m2 (Supplementary Table 8). The log hazards' 2-year survival demonstrated a non-linear survival advantage beyond BMI 25 kg/m2 (Supplementary Fig. 3).
+
+## 3.3. Secondary Outcomes
+
+The hospital mortality was highest for BMI 18.5e24.9 kg/m2
+(15 %, n ¼ 89), and lowest for patients with BMI 40 kg/m2 (8.0 %,
+n ¼ 37). Compared to patients with BMI <18.5 and 18.5e24.9 kg/
+m2, patients with BMI 25.0e29.9 kg/m2 and patients with all three obesity classes had longer ICU and hospital length of stays
+(p < 0.001 and p ¼ 0.015, respectively). There was no difference in the discharge destinations between BMI categories among patients discharged to their usual residence, to rehabilitation or a nursing home, respectively. The raw secondary outcomes are summarized in Table 2.
+
+## 3.4. Post Hoc Analysis
+
+When the mortality was standardised by age and obesity in the population, there was no interaction effect between age and obesity (pinteraction ¼ 0.32), when both were treated as continuous variables, adjusted for confounders (Supplementary Table 9).
+
+However, when BMI was categorised, age significantly interacted with only BMI<18.5 kg/m2 (pinteraction ¼ 0.004) and BMI
+35.0e35.9 kg/m2 (pinteraction ¼ 0.026) categories, when adjusted for the same confounders (Supplementary Table 10).
+
+## 4. Discussion 4.1. Executive Summary
+
+![5_image_0.png](5_image_0.png)
+
+those who were within the normal or underweight range. Secondly, in the adjusted analysis, only patients with BMI 40 kg/m2 were associated with higher survival times up to two years. Third, most of the patients who survived hospitalization were alive at 2 years, the highest for patients with BMI 40 kg/m2. Fourth, all three obesity classes had higher two-year survival among those needing mechanical ventilation. Finally, our findings suggest that the obesity paradox exists beyond hospital discharge and up to 2 years in critically ill patients with COVID-19 admitted to Australian and New Zealand ICUs.
+
+| ANZROD, male sex, frailty (CFS), hospital type and jurisdiction for all patients with COVID-19. BMI was treated as a categorical variable. Predictor HR (95%CI) p-value BMI categories  <18.5 kg/m2 0.69 (0.41e1.19) 0.18  18.5e24.9 kg/m2 Reference  25.0e29.9 kg/m2 0.90 (0.67e1.20) 0.46  30.0e34.9 kg/m2 0.84 (0.66e1.05) 0.13  35.0e39.9 kg/m2 0.95 (0.65e1.36) 0.78  40.0 kg/m2 0.51 (0.34e0.76) 0.001 Sex  Male sex 1.17 (0.99e1.39) 0.06 Patient factors  log (ANZROD/10) 2.02 (1.79e2.27) < 0.001  log (CFS/10) 6.30 (5.19e7.26) < 0.001 Hospital classification  Metropolitan Reference  Private 1.12 (0.67e1.88) 0.65  Rural/Regional 0.86 (0.68e1.09) 0.21  Tertiary 0.90 (0.73e1.12) 0.39 Jurisdiction  New South Wales Reference  Northern Territory 0.88 (0.07e2.09) 0.27  New Zealand 1.55 (0.58e4.15) 0.39  Queensland 0.60 (0.37e0.98) 0.041  South Australia 0.26 (0.19e0.34) < 0.001  Tasmania 0.00 (0.00e0.00) < 0.001  Victoria 0.81 (0.67e0.99) 0.035  Western Australia 0.98 (0.30e3.20) 0.98 CFS e Clinical Frailty Scale, BMI e body mass index, ANZROD e Australia New Zealand risk of death.   |
+|---|
+
+## 4.2. Relationship To Previous Studies
+
+The in-hospital mortality rates for patients with COVID-19 in Australia and New Zealand were lower than in other countries.50,54 Recent studies from Australia have found that in-hospital mortality rates in patients with severe COVID-19 reached nearly 15 %51,54 and that this was highest in the third wave of COVID-19 (26th June to 1st November 2021).54 While one study reported that 6-month survival was less than 50 % following ICU admission,55 others have found that more than 50 % of patients receiving mechanical ventilation for COVID-19 survived to 180 days.56 However, no studies reported long-term outcomes while taking BMI into account.
+
+Previous evidence suggests that being overweight and moderately obese was protective with lower mortality when compared with underweight BMI, normal BMI, or more severe obesity.25 Our
+
+![6_image_0.png](6_image_0.png)
+
+study observed that patients with obesity were younger, had fewer comorbidities, and had lower CFS and acute illness severity scores when compared to the underweight group who were relatively older patients, had more comorbidities, frailer with higher acute illness severity scores. A recent study found that ICU survivors demonstrated greater annual increases in lean and fat mass.57 Contrarily, although the point estimates for the other obese groups were also in the same direction, raising the possibility that this an overall trend to greater survival with progressively increasing BMI, our study found that survival time up to 2 years was independently associated only for patients with BMI 40 kg/m2.
+
+Our findings, therefore, suggest that an obesity paradox exists among survivors of COVID-19 beyond hospital discharge up to two years. Furthermore, a study that looked at the mortality trends between three COVID-19 surges among 1868 patients in the USA found that low BMI was associated with higher hospital mortality in patients admitted to ICU.58 In contrast, our study did not identify any not independent association with long-term survival.
+
+Previous evidence suggests that COVID-19 patients with a BMI of 30 kg/m2 were independently associated with needing mechanical ventilation.19,59 In contrast to the findings of a pre-pandemic large multicentre study which found that patients with class-II
+and class-III obesity had longer durations of mechanical ventilation and ICU care, we found similar durations of mechanical ventilation across all BMI categories.
+
+## 4.3. Study Implications
+
+We found an over-representation of patients with severe COVID-19 who were obese in Australian and New Zealand ICUs when compared to the large Australian epidemiological study pre-
+COVID-19.8 Our findings suggest the existence of obesity paradox existed beyond hospitalization for up to two years in critically ill patients. These findings will help determine the mortality risk associated with obesity in patients with severe COVID-19 to individualize patient intervention, such as healthy eating, minimizing ultra-processed foods and lifestyle modification.60 Furthermore, the COVID-19 vaccine's effectiveness in inducing protective humoral immunity is possibly reduced among obese individuals, therefore will require timely booster doses to improve their neutralizing immunity.61 Furthermore, priority should be given to exploring the reasons why there is a survival advantage for morbidly obese patients with COVID-19.
+
+## 4.4. Strengths
+
+Our study has several notable strengths. Firstly, our study spans numerous ICUs that enrolled patients across Australia and New Zealand. Secondly, the relatively larger sample of high-quality data increased the precision of our estimates. Thirdly, we incorporated several pre-specified secondary analyses to assess the association of obesity with survival of up to two years.
+
+## 4.5. Limitations
+
+There are a few limitations to this study. First, the retrospective study design meant that data collection was reliant on existing datasets and medical records. As a result, patients who did not have a recorded height or weight were excluded, reducing the sample size of our study. However, despite some group differences compared to those without BMI, the illness severity was similar, suggesting that our study is representative. In addition, there is a 189 possibility of data coding inaccuracy, and without site-based auditing of diagnostic codes, we cannot be certain about the degree of misclassification if any, and what its effects are on our findings. While we believe that our cohort is broadly representative of the larger population, this cannot be confirmed. Moreover, as a retrospective registry-based study, it is only possible to highlight associations and no causal inferences can be drawn. Furthermore, pre-ICU factors used to estimate illness severity may be colliders or confounders. This is an inherent limitation of the registry which does not capture pre-ICU factors that may influence ICU admission.
+
+Second, we did not have any information regarding the number of patients that were referred for and denied ICU admission. Third, the Australian and New Zealand healthcare system has been very fortunate with the magnitude of COVID-19 infections being largely under control, therefore the results may not be generalizable in resource-constrained healthcare systems. Fourth, the BMI was estimated only once at ICU admission. Although the median time spent outside the ICU was 7e10 h, fluid management acutely and the loss of lean mass (over days to weeks in the hospital) could have affected the weight and BMI. Fifth, although there is evidence that patients with obesity were more prone to have pathological pulmonary limitation and pulmonary gas exchange impairment to exercise compared with nonobese COVID-19 patients,62 we did not have the functional outcome following hospitalisation. Sixth, after discharge, the database did not record any ongoing healthcare needs following discharge. As a result, it is challenging to determine the precise impact of BMI after discharge on long-term survival. Seventh, COVID-19, its treatments, and in many places the composition of patients admitted to the ICU with COVID-19 rapidly evolved during the pandemic. Although this could have accounted for era effects in their analyses, a recent study from Australia did not show any difference based on the year of admission.50 Eighth, although many biases are related to the obesity paradox,1,8 collider stratification bias could be highly likely to underlie our study's findings and the decreased risk of mortality associated with obesity when in fact there is no biological basis for this relationship.63 Finally, the results from this dataset cannot be translated to a non-critically ill population. The existing public health message that the healthy-weight BMI is 18.5e24.9 kg/m2 has evidential support, including an association with a range of chronic diseases and early death, and the over-representation of patients with obesity and above in this dataset may speak to the increased burden of disease associated with obesity.8
+
+## 5. Conclusion
+
+In a large bi-national retrospective study, we confirmed the existence of the obesity paradox existed beyond hospital discharge in critically ill patients with COVID-19 admitted in Australian and New Zealand ICUs. Patients with BMI 40 kg/m2 were associated with a higher survival time of up to two years. Future studies should explore reasons why there is a survival advantage for morbidly obese patients with COVID-19.
+
+## Disclaimer
+
+Ethics approval and consent to participate:
+
+- All experimental protocols were approved by The Alfred Hospital Ethics Committee (Project No: 413/19, Project Title BMI and frailty in Critical Illness, approved 11/01/2022) approved this study with a waiver of informed consent. This was a sub-study of the larger study titled "BMI and frailty in Critical Illness".
+- ANZICS Centre for Outcome and Resource Evaluation Management Committee granted access to the ANZICS-APD following standing protocols on 11/16/2022.
+
+- All methods were carried out following the relevant guidelines and regulations of the Declaration of Helsinki.
+
+- Consent for publication e Not applicable Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available as these are linked from two registries (ANZICS, and the National Death Index), but are available from the corresponding author upon reasonable request.
+
+## Funding
+
+No financial support, including any institutional departmental funds, was used for the study.
+
+## Statement Of Authors' Contributions To The Manuscript
+
+1. Designed Research:
+ Study conception: AS
+ Study design: AS, RRL, ER, DP
+ Development of overall research plan: AS, RRL, ER, DP
+ Study oversight: AS
+
+## 2. Conducted Research: N/A. 3. Provided Essential Reagents/Materials: Dp Is The Custodian Of The Anzics Dataset. 4. Analysis Of Data:
+
+ Data analysis, performed statistical analysis: AS, RRL Long-term survival statistical analysis and interpretation: DP
+ Tables and figures: RRL, AS
+
+## 5. Wrote Paper:
+
+ Original drafting of the manuscript: AS Critical revision of the manuscript for intellectually important content: AS, RRL, ER, DP
+
+## 6. Primary Responsibility For Final Content: As.
+
+7. Other: AS and DP were responsible for the decision to submit the manuscript.
+
+8. All authors provided critical conceptual input, interpreted the data analysis, and read, and approved the final draft.
+
+## Conflict Of Interest
+
+The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: n/a If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
+
+## Acknowledgement
+
+The authors and the ANZICS CORE management committee would like to thank clinicians, data collectors and researchers at the following contributing sites:
+
+## Appendix A. Supplementary Data
+
+Supplementary data to this article can be found online at https://doi.org/10.1016/j.ccrj.2023.10.004.
+
+| Australian Capital Territory Calvary Hospital (Canberra) ICU            | Canberra Hospital ICU                         |                                               |
+|-------------------------------------------------------------------------|-----------------------------------------------|-----------------------------------------------|
+| New South Wales Bankstown-Lidcombe Hospital ICU                         | Hornsby Ku-ring-gai Hospital ICU              | Shoalhaven Hospital ICU                       |
+| Bathurst Base Hospital ICU                                              | John Hunter Hospital ICU                      | St George Hospital (Sydney) ICU               |
+| Blacktown Hospital ICU                                                  | Lismore Base Hospital ICU                     | St Vincent's Hospital (Sydney) ICU            |
+| Calvary Mater Newcastle ICU                                             | Maitland Hospital ICU                         | Goulburn Base Hospital ICU                    |
+| Campbelltown Hospital ICU                                               | Nepean Hospital ICU                           | Sydney Adventist Hospital ICU                 |
+| Coffs Harbour Health Campus ICU                                         | Northern Beaches Hospital ICU                 | Tamworth Base Hospital ICU                    |
+| Concord Hospital (Sydney) ICU                                           | Orange Base Hospital ICU                      | The Chris O'Brien Lifehouse ICU               |
+| Dubbo Base Hospital ICU                                                 | Port Macquarie Base Hospital ICU              | Tweed Heads District Hospital ICU             |
+| Fairfield Hospital ICU                                                  | Prince of Wales Hospital ICU                  | Westmead Hospital ICU                         |
+| Gosford Hospital ICU                                                    | Royal North Shore Hospital ICU                | Wyong Hospital ICU                            |
+| Sutherland Hospital & Community Health Services ICU                     | Royal Prince Alfred Hospital ICU              | Wollongong Hospital ICU                       |
+| Northern Territory Alice Springs Hospital ICU                           | Royal Darwin Hospital ICU                     |                                               |
+| Queensland Caboolture Hospital ICU                                      | Mater Adults Hospital (Brisbane) ICU          | Redcliffe Hospital ICU                        |
+| Cairns Hospital ICU                                                     | Mater Health Services North Queensland ICU    | Royal Brisbane and Women's Hospital ICU       |
+| Gold Coast University Hospital ICU                                      | The Prince Charles Hospital ICU               | St Vincent's Private Hospital Northside ICU   |
+| Hervey Bay Hospital ICU                                                 | Mater Private Hospital (Brisbane) ICU         | St Vincent's Private Hospital (Toowoomba) ICU |
+| Logan Hospital ICU                                                      | Noosa Hospital ICU                            | Sunshine Coast University Hospital ICU        |
+| Mackay Base Hospital ICU                                                | Princess Alexandra Hospital ICU               | Toowoomba Hospital ICU                        |
+| South Australia Calvary Adelaide Hospital ICU                           | Flinders Medical Centre ICU                   | Royal Adelaide Hospital ICU                   |
+| Tasmania Launceston General Hospital ICU                                | Royal Hobart Hospital ICU                     |                                               |
+| Victoria Alfred Hospital ICU                                            | Epworth Freemasons Hospital ICU               | Mulgrave Private Hospital ICU                 |
+| Austin Hospital ICU                                                     | Epworth Geelong ICU                           | Royal Melbourne Hospital ICU                  |
+| Box Hill Hospital ICU                                                   | Footscray Hospital ICU                        | St John of God Hospital (Bendigo) ICU         |
+| Cabrini Hospital ICU                                                    | Frankston Hospital ICU                        | St Vincent's Hospital (Melbourne) ICU         |
+| Casey Hospital ICU                                                      | Goulburn Valley Health ICU                    | Sunshine Hospital ICU                         |
+| Central Gippsland Health Service ICU                                    | Holmesglen Private Hospital ICU               | The Northern Hospital ICU                     |
+| Dandenong Hospital ICU                                                  | Knox Private Hospital ICU                     | University Hospital Geelong ICU               |
+| Bendigo Health Care Group Hospital ICU                                  | Monash Medical CentreeClayton Campus ICU      |                                               |
+| Western Australia Fiona Stanley Hospital ICU                            | Joondalup Health Campus ICU                   | Royal Perth Hospital ICU                      |
+| Sir Charles Gairdner Hospital ICU New Zealand Christchurch Hospital ICU | Nelson Hospital ICU                           | Waikato Hospital ICU                          |
+| Middlemore Hospital ICU                                                 | Whangarei Area Hospital ICU, Northland Health |                                               |
+
+## References
+
+[1] Decruyenaere A, Steen J, Colpaert K, Benoit DD, Decruyenaere J,
+Vansteelandt S. The obesity paradox in critically ill patients: a causal learning approach to a casual finding. Crit Care 2020;24(1):485. https://doi.org/
+10.1186/s13054-020-03199-5.
+
+[2] Schetz M, De Jong A, Deane AM, Druml W, Hemelaar P, Pelosi P, et al. Obesity in the critically ill: a narrative review. Intensive Care Med 2019;45(6):757e69.
+
+https://doi.org/10.1007/s00134-019-05594-1.
+
+[3] Sakr Y, Alhussami I, Nanchal R, Wunderink RG, Pellis T, Wittebole X, et al.
+
+Being overweight is associated with greater survival in ICU patients: results from the intensive care over nations audit. Crit Care Med 2015;43(12):
+2623e32. https://doi.org/10.1097/ccm.0000000000001310 (In eng).
+
+[4] Karampela I, Chrysanthopoulou E, Christodoulatos GS, Dalamaga M. Is there an obesity paradox in critical illness? Epidemiologic and metabolic considerations. Curr Obes Rep 2020;9(3):231e44. https://doi.org/10.1007/s13679020-00394-x.
+
+[5] Pepper DJ, Sun J, Welsh J, Cui X, Suffredini AF, Eichacker PQ. Increased body mass index and adjusted mortality in ICU patients with sepsis or septic shock: a systematic review and meta-analysis. Crit Care 2016;20(1):181. https:// doi.org/10.1186/s13054-016-1360-z.
+
+[6] Hutagalung R, Marques J, Kobylka K, Zeidan M, Kabisch B, Brunkhorst F,
+et al. The obesity paradox in surgical intensive care unit patients.
+
+Intensive Care Med 2011;37(11):1793. https://doi.org/10.1007/s00134011-2321-2.
+
+[7] Acharya P, Upadhyay L, Qavi A, Naaraayan A, Jesmajian S, Acharya S, et al. The paradox prevails: outcomes are better in critically ill obese patients regardless of the comorbidity burden. J Crit Care 2019;53:25e31. https://doi.org/
+10.1016/j.jcrc.2019.05.004 (In eng).
+
+[8] Secombe P, Woodman R, Chan S, Pilcher D, van Haren F. Epidemiology and outcomes of obese critically ill patients in Australia and New Zealand. Crit Care Resusc 2020;22(1):35e44. https://www.ncbi.nlm.nih.gov/pubmed/
+32102641.
+
+[9] Zhou D, Wang C, Lin Q, Li T. The obesity paradox for survivors of critically ill patients. Crit Care 2022;26(1):198. https://doi.org/10.1186/s13054-02204074-1.
+
+[10] Plecko D, Bennett N, Martensson J, Bellomo R. The obesity paradox and hypoglycemia in critically ill patients. Crit Care 2021;25(1):378. https://doi.org/
+10.1186/s13054-021-03795-z.
+
+[11] Tobias DK, Hu FB. Does being overweight really reduce mortality? Obesity 2013;21(9):1746e9. https://doi.org/10.1002/oby.20602.
+
+[12] Schooling CM, Au Yeung SL. "Selection bias by death" and other ways collider bias may cause the obesity paradox. Epidemiology 2017;28(2):e16e7. https:// doi.org/10.1097/EDE.0000000000000591.
+
+[13] Jagan N, Morrow LE, Walters RW, Plambeck RW, Wallen TJ, Patel TM, et al.
+
+Sepsis and the obesity paradox: size matters in more than one way. Crit Care Med 2020;48(9):e776e82. https://doi.org/10.1097/ccm.0000000000004459
+(In eng).
+
+[14] Williamson EJ, Walker AJ, Bhaskaran K, Bacon S, Bates C, Morton CE, et al.
+
+Factors associated with COVID-19-related death using OpenSAFELY. Nature 2020;584(7821):430e6. https://doi.org/10.1038/s41586-020-2521-4 (In eng).
+
+[15] Caussy C, Pattou F, Wallet F, Simon C, Chalopin S, Telliam C, et al. Prevalence of obesity among adult inpatients with COVID-19 in France. Lancet Diabetes 191 Endocrinol 2020;8(7):562e4. https://doi.org/10.1016/s2213-8587(20)
+30160-1 (In eng).
+
+[16] Petrilli CM, Jones SA, Yang J, Rajagopalan H, O'Donnell L, Chernyak Y, et al.
+
+Factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in New York City: prospective cohort study. BMJ 2020;369:m1966. https://doi.org/10.1136/bmj.m1966 (In eng).
+
+[17] Goyal P, Ringel JB, Rajan M, Choi JJ, Pinheiro LC, Li HA, et al. Obesity and COVID-19 in New York city: a retrospective cohort study. Ann Intern Med 2020;173(10):855e8. https://doi.org/10.7326/m20-2730 (In eng).
+
+[18] Simonnet A, Chetboun M, Poissy J, Raverdy V, Noulette J, Duhamel A, et al.
+
+High prevalence of obesity in severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) requiring invasive mechanical ventilation. Obesity 2020;28(7):1195e9. https://doi.org/10.1002/oby.22831 (In eng).
+
+[19] Kalligeros M, Shehadeh F, Mylona EK, Benitez G, Beckwith CG, Chan PA, et al.
+
+Association of obesity with disease severity among patients with coronavirus disease 2019. Obesity 2020;28(7):1200e4. https://doi.org/10.1002/oby.22859
+(In eng).
+
+[20] Palaiodimos L, Kokkinidis DG, Li W, Karamanis D, Ognibene J, Arora S, et al.
+
+Severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher in-hospital mortality, in a cohort of patients with COVID-19 in the Bronx, New York. Metabolism 2020;108: 154262. https://doi.org/10.1016/j.metabol.2020.154262 (In eng).
+
+[21] Rebelos E, Moriconi D, Virdis A, Taddei S, Foschi D, Nannipieri M. Letter to the Editor: importance of metabolic health in the era of COVID-19. Metabolism 2020;108:154247. https://doi.org/10.1016/j.metabol.2020.154247 (In eng).
+
+[22] Huttunen R, Syrjanen J. Obesity and the risk and outcome of infection. Int J
+Obes 2013;37(3):333e40. https://doi.org/10.1038/ijo.2012.62.
+
+[23] Luzi L, Radaelli MG. Influenza and obesity: its odd relationship and the lessons for COVID-19 pandemic. Acta Diabetol 2020;57(6):759e64. https://doi.org/
+10.1007/s00592-020-01522-8.
+
+[24] Malavazos AE, Corsi Romanelli MM, Bandera F, Iacobellis G. Targeting the adipose tissue in COVID-19. Obesity 2020;28(7):1178e9. https://doi.org/
+10.1002/oby.22844.
+
+[25] Chetboun M, Raverdy V, Labreuche J, Simonnet A, Wallet F, Caussy C, et al. BMI
+and pneumonia outcomes in critically ill COVID-19 patients: an international multicenter study. Obesity 2021;29(9):1477e86. https://doi.org/10.1002/
+oby.23223.
+
+[26] Bello-Chavolla OY, Bahena-Lopez JP, Antonio-Villa NE, Vargas-V  azquez A,
+Gonzalez-Díaz A, M  arquez-Salinas A, et al. Predicting mortality due to SARSCoV-2: a mechanistic score relating obesity and diabetes to COVID-19 outcomes in Mexico. J Clin Endocrinol Metab 2020;105(8). https://doi.org/
+10.1210/clinem/dgaa346 (In eng).
+
+[27] Busetto L, Bettini S, Fabris R, Serra R, Dal Pra C, Maffei P, et al. Obesity and COVID-19: an Italian snapshot. Obesity 2020;28(9):1600e5. https://doi.org/
+10.1002/oby.22918 (In eng).
+
+[28] Cai Q, Chen F, Wang T, Luo F, Liu X, Wu Q, et al. Obesity and COVID-19 severity in a designated hospital in shenzhen, China. Diabetes Care 2020;43(7):
+1392e8. https://doi.org/10.2337/dc20-0576 (In eng).
+
+[29] Gao M, Piernas C, Astbury NM, Hippisley-Cox J, O'Rahilly S, Aveyard P, et al.
+
+Associations between body-mass index and COVID-19 severity in 6.9 million people in England: a prospective, community-based, cohort study. Lancet Diabetes Endocrinol 2021;9(6):350e9. https://doi.org/10.1016/S22138587(21)00089-9.
+
+[30] Sanchis-Gomar F, Lavie CJ, Mehra MR, Henry BM, Lippi G. Obesity and outcomes in COVID-19: when an epidemic and pandemic collide. Mayo Clin Proc 2020;95(7):1445e53. https://doi.org/10.1016/j.mayocp.2020.05.006 (In eng).
+
+[31] Anderson MR, Geleris J, Anderson DR, Zucker J, Nobel YR, Freedberg D, et al.
+
+Body mass index and risk for intubation or death in SARS-CoV-2 infection : a retrospective cohort study. Ann Intern Med 2020;173(10):782e90. https://
+doi.org/10.7326/m20-3214 (In eng).
+
+[32] Tartof SY, Qian L, Hong V, Wei R, Nadjafi RF, Fischer H, et al. Obesity and mortality among patients diagnosed with COVID-19: results from an integrated health care organization. Ann Intern Med 2020;173(10):773e81.
+
+https://doi.org/10.7326/m20-3742 (In eng).
+
+[33] Sharma A, Garg A, Rout A, Lavie CJ. Association of obesity with more critical illness in COVID-19. Mayo Clin Proc 2020;95(9):2040e2. https://doi.org/
+10.1016/j.mayocp.2020.06.046 (In eng).
+
+[34] Lavie CJ, Coursin DB, Long MT. The obesity paradox in infections and implications for COVID-19. Mayo Clin Proc 2021;96(3):518e20. https://doi.org/
+10.1016/j.mayocp.2021.01.014.
+
+[35] Docherty AB, Harrison EM, Green CA, Hardwick HE, Pius P, Norman L, et al.
+
+Features of 20 133 UK patients in hospital with covid-19 using the ISARIC
+WHO Clinical Characterisation Protocol: prospective observational cohort study. BMJ 2020;369:m1985. https://doi.org/10.1136/bmj.m1985 (In eng).
+
+[36] Foldi M, Farkas N, Kiss S, Z € adori N, V  ancsa S, Szak  o L, et al. Obesity is a risk factor for developing critical condition in COVID-19 patients: a systematic review and meta-analysis. Obes Rev 2020;21(10):e13095. https://doi.org/
+10.1111/obr.13095.
+
+[37] Huang Y, Lu Y, Huang YM, Wang M, Ling W, Sui Y, et al. Obesity in patients with COVID-19: a systematic review and meta-analysis. Metabolism 2020;113:154378. https://doi.org/10.1016/j.metabol.2020.154378 (In eng).
+
+[38] Liu M, Deng C, Yuan P, Ma J, Yu P, Chen J, et al. Is there an exposure-effect relationship between body mass index and invasive mechanical ventilation, severity, and death in patients with COVID-19? Evidence from an updated meta-analysis. Obes Rev 2020;21(11):e13149. https://doi.org/10.1111/ obr.13149.
+
+[39] Soeroto AY, Soetedjo NN, Purwiga A, Santoso P, Kulsum ID, Suryadinata H,
+et al. Effect of increased BMI and obesity on the outcome of COVID-19 adult patients: a systematic review and meta-analysis. Diabetes Metabol Syndr 2020;14(6):1897e904. https://doi.org/10.1016/j.dsx.2020.09.029 (In eng).
+
+[40] Abumayyaleh M, Núnez Gil IJ, El-Battrawy I, Estrada V, Becerra-Mu ~ noz VM, El- ~
+Aparisi A, et al. Does there exist an obesity paradox in COVID-19? Insights of the international HOPE-COVID-19-registry. Obes Res Clin Pract 2021;15(3):
+275e80. https://doi.org/10.1016/j.orcp.2021.02.008 (In eng).
+
+[41] ANZICS Centre for Outcomes and Resource Evaluation. Centre for resource and outcomes evaluation 2020 report. 2021. https://www.anzics.com.au/wpcontent/uploads/2020/11/2019-CORE-Report.pdf.
+
+[42] ANZCIS Centre for Outcomes and Resource Evaluation. Adult Patient Database Data Dictionary. (https://www.anzics.com.au/wp-content/uploads/2021/03/ ANZICS-APD-Dictionary.pdf).
+
+[43] Obesity: preventing and managing the global epidemic. Report of a WHO
+consultation. World Health Organ Tech Rep Ser 2000;894(i-xii):1e253 [In eng)].
+
+[44] Poirier P, Giles TD, Bray GA, Hong Y, Stern JS, Pi-Sunyer FX, et al. Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss. Arterioscler Thromb Vasc Biol 2006;26(5):968e76. https://doi.org/10.1161/
+01.ATV.0000216787.85457.f3
+(In eng).
+
+[45] Darvall JN, Bellomo R, Bailey M, Young PJ, Rockwood K, Pilcher D. Impact of frailty on persistent critical illness: a population-based cohort study.
+
+Intensive Care Med 2022;48(3):343e51. https://doi.org/10.1007/s00134022-06617-0.
+
+[46] Pilcher D, Paul E, Bailey M, Huckson S. The Australian and New Zealand Risk of Death (ANZROD) model: getting mortality prediction right for intensive care units. Crit Care Resusc 2014;16(1):3e4 [In eng)].
+
+[47] Paul E, Bailey M, Kasza J, Pilcher D. The ANZROD model: better benchmarking of ICU outcomes and detection of outliers. Crit Care Resusc 2016;18(1):25e36
+[In eng)].
+
+[48] Subramaniam A, Ueno R, Tiruvoipati R, Srikanth V, Bailey M, Pilcher D.
+
+Comparison of the predictive ability of clinical frailty scale and hospital frailty risk score to determine long-term survival in critically ill patients: a multicentre retrospective cohort study. Crit Care 2022;26(1):121. https://doi.org/
+10.1186/s13054-022-03987-1.
+
+[49] Subramaniam A, Ueno R, Tiruvoipati R, Darvall J, Srikanth V, Bailey M, et al.
+
+Comparing the clinical frailty scale and an international classification of diseases-10 modified frailty index in predicting long-term survival in critically ill patients. Crit Care Explor 2022;4(10):e0777. https://doi.org/10.1097/
+CCE.0000000000000777.
+
+[50] Subramaniam A, Shekar K, Anstey C, Tiruvoipati R, Pilcher D. Impact of frailty on clinical outcomes in patients with and without COVID-19 pneumonitis admitted to intensive care units in Australia and New Zealand: a retrospective registry data analysis. Crit Care 2022;26(1):301. https://doi.org/10.1186/
+s13054-022-04177-9.
+
+[51] Subramaniam A, Anstey C, Curtis JR, Ashwin S, Ponnapa Reddy M,
+Aliberti MJR, et al. Characteristics and outcomes of patients with frailty admitted to ICU with coronavirus disease 2019: an individual patient data meta-analysis. Crit Care Explor 2022;4(1):e0616. https://doi.org/10.1097/
+CCE.0000000000000616.
+
+[52] Wei LJ, Lin DY, Weissfeld L. Regression analysis of multivariate incomplete failure time data by modeling marginal distributions. J Am Stat Assoc 1989;84(408):1065e73. https://doi.org/10.2307/2290084.
+
+[53] Team RC. R: A language and environment for statistical computing. . R
+Foundation for Statistical Computing. (https://www.R-project.org/).
+
+[54] Begum H, Neto AS, Alliegro P, Broadley T, Trapani T, Campbell LT, et al. People in intensive care with COVID-19: demographic and clinical features during the first, second, and third pandemic waves in Australia. Med J Aust 2022;217(7):352e60. https://doi.org/10.5694/mja2.51590 (In eng).
+
+[55] Taniguchi LU, Avelino-Silva TJ, Dias MB, Jacob-Filho W, Aliberti MJR. Association of frailty, organ support, and long-term survival in critically ill patients with COVID-19. Crit Care Explor 2022;4(6):e0712. https://doi.org/10.1097/
+CCE.0000000000000712.
+
+[56] Penuelas O, del Campo-Albendea L, de Aledo ALG, A ~ n~on JM, Rodríguez- 
+Solís C, Mancebo J, et al. Long-term survival of mechanically ventilated patients with severe COVID-19: an observational cohort study. Ann Intensive Care 2021;11(1):143. https://doi.org/10.1186/s13613-02100929-y.
+
+[57] Thackeray M, Kotowicz MA, Pasco JA, Mohebbi M, Orford N. Changes in body composition in the year following critical illness: a case-control study. J Crit Care 2022;71:154043. https://doi.org/10.1016/j.jcrc.2022.154043.
+
+[58] Auld SC, Harrington KRV, Adelman MW, Robichaux CJ, Overton EC, CaridiScheible M, et al. Trends in ICU mortality from coronavirus disease 2019: a tale of three surges. Crit Care Med 2022;50(2):245e55. https://doi.org/
+10.1097/ccm.0000000000005185 (In eng).
+
+[59] Martino JL, Stapleton RD, Wang M, Day AG, Cahill NE, Dixon AE, et al. Extreme obesity and outcomes in critically ill patients. Chest 2011;140(5):1198e206.
+
+https://doi.org/10.1378/chest.10-3023 (In eng).
+
+[60] Popkin BM, Du S, Green WD, Beck MA, Algaith T, Herbst CH, et al. Individuals with obesity and COVID-19: a global perspective on the epidemiology and 192 
+
+biological relationships. Obes Rev 2020;21(11):e13128. https://doi.org/ 10.1111/obr.13128.
+
+[61] Faizo AA, Qashqari FS, El-Kafrawy SA, Barasheed O, Almashjary MN,
+Alfelali M, et al. A potential association between obesity and reduced effectiveness of COVID-19 vaccine-induced neutralizing humoral immunity. J Med Virol 2023;95(1):e28130. https://doi.org/10.1002/jmv.28130
+(In eng).
+
+[62] Lacavalerie MR, Pierre-Francois S, Agossou M, Inamo J, Cabie A, Barnay JL, et al.
+
+Obese patients with long COVID-19 display abnormal hyperventilatory response and impaired gas exchange at peak exercise. Future Cardiol 2022;18(7):577e84. https://doi.org/10.2217/fca-2022-0017 (In eng).
+
+[63] Banack HR, Kaufman JS. Does selection bias explain the obesity paradox among individuals with cardiovascular disease? Ann Epidemiol 2015;25(5):
+342e9. https://doi.org/10.1016/j.annepidem.2015.02.008.

medical/md/PMC10882963.md

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+pubs.acs.org/est Article
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_1.png](0_image_1.png)
+
+# A U.S. Lead Exposure Hotspots **Analysis**
+
+Valerie G. Zartarian,* Jianping Xue, Antonios G. Poulakos, Rogelio Tornero-Velez, Lindsay W. Stanek, Emily Snyder, Veronica Helms Garrison, Kathryn Egan, and Joseph G. Courtney Cite **This:** Environ. Sci. *Technol.* 2024, 58, 3311−3321 Read **Online**
+ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: To identify U.S. lead exposure risk hotspots, we expanded upon geospatial statistical methods from a published Michigan case study. The evaluation of identified hotspots using five lead indices, based on housing age and sociodemographic data, showed moderate-to-substantial agreement with state-identified higher-risk locations from nine public health department reports
+(45−78%) and with hotspots of children's blood lead data from Michigan and Ohio (e.g., Cohen's kappa scores of 0.49−0.63). Applying geospatial cluster analysis and 80th−100th percentile methods to the lead indices, the number of U.S. census tracts ranged from ∼8% (intersection of indices) to ∼41% (combination of indices). Analyses of the number of children <6 years old living in those census tracts revealed the states (e.g., Illinois, Michigan, New Jersey, New York, Ohio, Pennsylvania, Massachusetts, California, Texas) and counties with highest potential lead exposure risk. Results support use of available lead indices as surrogates to identify locations in the absence of consistent, complete blood lead level (BLL) data across the United States. Ground-truthing with local knowledge, additional BLL data, and environmental data is needed to improve identification and analysis of lead exposure and BLL hotspots for interventions. While the science evolves, these screening results can inform "deeper dive" analyses for targeting lead actions. KEYWORDS: children's health, environmental health, metals, indices, mapping, *biomonitoring*
+- **INTRODUCTION**
+While average blood lead levels in the U.S. have declined, millions of children and adults are still exposed to various sources of lead.1−3 Although there are known sources of lead exposure (e.g., paint in older homes, drinking water from lead pipes, soil, and consumer products),4 it is difficult to identify communities that may have disproportionate exposures because of limitations in children's blood lead surveillance data and gaps in environmental and other exposure data.5 There is no known level of lead exposure to be without risk,6−8 and many communities are disproportionately impacted.9,10 Identifying and addressing remaining lead exposure risk hotspots are priorities in the United States. The Federal Lead Action Plan10 and the U.S. Environmental Protection Agency
+(EPA) Lead Strategy (e.g., Goal 2, "Identify Communities with High Lead Exposures and Improve Their Health Outcomes")9 highlight the need for lead mapping as part of whole-ofgovernment efforts to address high exposure risk locations and disparities. Data mapping can inform screening and prioritization efforts to guide interventions and "deeper dive" analyses
+(such as enhancing children's blood lead level (BLL) surveillance data analyses and lead source apportionment analyses). These analyses can assist in efforts around primary prevention; lead-based paint mitigation; lead remediation, enforcement, education, and outreach.11 Federal agencies are collaborating to identify geographic locations and populations at risk for lead exposure so that they can be addressed proactively. Examples include targeting HUD remediation grants, EPA environmental cleanup actions, and CDC primary prevention and enhanced blood lead testing programs for children.5 The EPA, U.S. Department of Housing and Urban Development (HUD), Centers for Disease Control and Prevention (CDC), and other organizations are applying geospatial statistical methods with available data (blood lead surveillance data, lead indices, and environmental data) to identify locations that are disproportionately at risk. However, there are remaining gaps and challenges such as cross-agency data collection and integration.5 The interagency lead mapping state-of-the-science paper5 presented a data integration roadmap to identify places for public health action:
+
+| Received:                   | September 22, 2023   |
+|-----------------------------|----------------------|
+| Revised:                    | December 7, 2023     |
+| Accepted:                   | December 8, 2023     |
+| Published: February 9, 2024 |                      |
+
+![0_image_2.png](0_image_2.png)
+
+"For national scale, one could use lead indices and *evaluate* identified geographic locations by available BLL data and local knowledge...While the science is evolving to *compare* available lead indices and models against each other and evaluate or ground-truth them against states'-measured surveillance BLL data...an analyst or decision-maker may want to use them collectively to identify high-risk locations to cast a wider net or to use their intersecting locations for a more focused *list...".*
+This paper provides a next step in the research to identify U.S. lead exposure hotspots using existing data.
+
+- MATERIALS AND **METHODS**
+The general approach taken for this analysis consists of the following four steps:
+1. Statistically evaluated hotspots identified with lead indices against children's BLL surveillance data:
+(a) from Michigan (MI) and Ohio (OH), using the BLL
+data and statistical methods described in Xue et al.12 and Stanek et al.,13 respectively, and an expanded set of national lead indices;
+(b) from matching hotspots identified using lead indices with community hotspots identified in 9 state health department public reports (listed in Zartarian et al.5)
+and quantifying the percent; a match is defined here as a community with at least one census tract identified by the lead indices in our analyses; 2. Compared existing national indices against each other and against available BLL surveillance data using sensitivity, specificity, and Cohen's kappa score to determine which indices are the statistically strongest predictors of hotspots for the national-scale analysis; 3. Produced census tract-level maps for the United States that visualize the intersection and collective combination of hotspots based on the two methods discussed in Xue et al.,12 top 20 (i.e., 80th−100th) percentiles and GetisOrd Gi*14 geospatial cluster hotspots analysis methods; 4. Conducted national-scale analyses to identify states and counties with the highest potential lead exposure risk, based on the considered indices and the number of children younger than six years old in the identified 2010 census tracts (n = 73,086 census tracts containing at least one child <6 years old in the 50 states).
+
+In this paper, hotspots refer to geographic locations with a higher prevalence of children's lead exposures, based on the percentage of higher BLLs among children and/or identification by lead exposure indices using two statistical methods:
+top 20 (80th−100th) percentile and Getis-Ord Gi* geospatial cluster hotspot analysis (ESRI ArcGIS Desktop v.10.8.2 and Pro 3.1 Hot Spot Analysis tool15). The top 20 (80th−100th)
+percentile method uses a direct percentile calculation conducted with traditional statistical software (i.e., Microsoft Excel). The Getis-Ord Gi* geospatial cluster hotspot analysis method uses the ArcGIS Hot Spot tool15 which is a spatial interpolation function that calculates the Getis-Ord Gi* statistic for the exposure value of each census tract within the context of neighboring ones. To be identified as a geographic location with a higher prevalence of children's lead exposures, a given census tract will have higher BLL values and/or high lead exposure index values that are also adjacent to other census tracts with higher BLL values and/or high lead
+exposure index values. More detail on the application of this method can be found in Xue et al.12 In our analysis, "higher BLLs" (referred to previously as elevated BLLs or EBLLs) are defined as greater than or equal to CDC's previous blood lead reference value of 5 μg/dL.16 We elected to use CDC's 2012 blood lead reference value
+(BLRV)16 of 5 μg/dL for this analysis to remain consistent with the 2010 census data, MI and OH BLLs acquired through EPA's data use agreements, and the public state health department reports used in this analysis (all of which contain data predating 2020). The BLRV was updated to 3.5 μg/dL in 2021.17 The five lead exposure indices and models (referred to collectively as lead indices) used in this national-scale analysis are based on publicly available data on older housing (prior to 1980) and sociodemographic variables. We included the following indices: (1) the EPA EJSCREEN Lead Paint EJ
+Index ("EJSCREEN Index");18 (2) Schultz et al., 2017-based BLL model ("Schultz Model") developed by the EPA Office of Research and Development (ORD);19 (3) U.S. Department of Housing and Urban Development Deteriorated Paint Index
+(HUD DPI);20 (4) the Vox U.S. Lead Risk Exposure score
+("Vox");21 and Random Forest (RF) Regression EBLL
+Prediction Model ("RF Model"). The first four were summarized by Zartarian et al.5 Two versions of the new Random Forest (RF) Regression EBLL Prediction Model developed by EPA ORD22,23 were also included: RF Model version 1 (v1) and RF Model version 2 (v2). "RF Model v1" refers to version 1 of the regression model built using Ohio 2007−2011 BLL data and the 2013 OH DH report model.24 It includes the following five variables: percent of homes built prior to 1940, percent of homes built prior to 1950, percent of families whose income-to-poverty ratio was greater than 2, percent of population with either high school or higher education, and percent of non-Hispanic African Americans in each census tract. Demographic data originate from the American Community Survey 2013−2017 5-year summary file.25 "RF Model v2" refers to a modified version that includes the following variables: percent of homes built prior to 1940, percent of homes built prior to 1950, and percent of families whose income-to-poverty ratio was greater than 2. Note that RF Model v2, which is a reduced form of v1, is included in a subset of tables and figures in the Results and Discussion, as it was developed after the initial analyses were conducted. Please see the Supporting Information for more details.
+
+We used BLL data from Michigan (∼1.9 million BLL data points; 2006−2016 among children <6 years old) and Ohio
+(∼2.3 million BLL data points; 2005−2018 among children <6 years old) acquired through data use agreements between the state health departments and the EPA/ORD. More detail on these data can be found in Xue et al. (MI data)12 and Stanek et al. (OH data).13 For steps 1 and 2, indices were compared together and with children's BLL data from MI and OH and state-identified locations in public health department reports. The identified lead exposure indices hotspots were evaluated by applying Cohen's kappa26 agreement statistic (<0.4 = low; >0.4−0.6 =
+moderate; >0.6−0.8 = substantial; >0.8−0.99 = near perfect agreement) and sensitivity and specificity analyses. Note that some reports refer to these locations as "high risk communities" while others refer to them differently, e.g., "selected community". In the context of this application, sensitivity is the percentage of true positives (i.e., the rate at
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+which a model or index correctly predicts a hotspot for BLLs) and specificity is the percentage of true negatives (i.e., the rate at which a model or index correctly predicts a nonhotspot for BLLs).
+
+To compare hotspots identified by the lead indices with
+"high risk locations" identified by state health departments based on their BLL surveillance data, we assessed the summary of available data and reports listed in Zartarian et al. figure B
+(left panel) and table D.5 Note that 9 of 32 public reports identified hotspots at the community/city/town level; the rest reported them at other geographic scales (i.e., state, county, parish, health jurisdiction, zip code). The 9 states used for evaluation were: Connecticut (city/town), Maine (city/town), Massachusetts (city/town, census tracts), Michigan (city/ town), New Hampshire (city/town, census tracts), New Jersey (municipality), Ohio (city/town, county), Pennsylvania (municipality), and Rhode Island (city). As Table D in Zartarian et al.5 shows, states used different criteria, geographic scales, time periods, and blood lead reference values for identifying higher risk locations.
+
+After comparing and evaluating the indices using the statistical methods (i.e., top 20th percentile and Getis-Ord Gi* geospatial clustering) described in Xue et al.,12 the intersection and collective combinations were mapped with ESRI ArcGIS Desktop version 10.8.2. This data intersection and combination was executed in ArcGIS by performing spatial data joining using census tract geographic identification numbers (GEOIDs).
+
+We summarized the results into tables by U.S. states and
+
+![2_image_2.png](2_image_2.png)
+
+counties, ranking them by total number of children <6 years old (per 2010 Census27) in identified census tracts based on the lead exposure indices results. Corresponding county and state ranking tables are displayed by index or model identification (1 = identified as a lead exposure risk hotspot by index or model; 0 = not identified by index or model as a lead exposure risk hotspot; Total = total number of indices and models that identified the respective location). For Tables S1−S8 identifying states and counties with the highest potential lead exposure risk (as defined by total number of children <6 years old identified by the indices based on housing age and sociodemographics), we featured the Schultz Model, Vox, and RF Model v1 based on the evaluation results. Our intent of identifying states and counties is to inform further tailored analyses for lead mitigation efforts.
+
+- RESULTS AND DISCUSSION
+National-Scale Lead Exposure Hotspot Maps **Based**
+on 5 Indices and **Models.** Figure 1 shows national analyses of high lead exposure locations with five available indices/
+models, using the Getis-Ord Gi* geospatial cluster method and the top 20 percentile (80th−100th) method. The top panels show the intersection of the five indices, and the bottom shows the combination. Figures S-1 and S-2 show the individual hotspot maps for the five indices using the two statistical methods. The maps look different due to the nuances in underlying science and data as discussed in Zartarian et al.,5 i.e., national-scale lead indices constructed with different
+
+![3_image_0.png](3_image_0.png)
+
+Figure 2. National scale convergence analyses of the highest potential lead (Pb) exposure risk locations identified using 5 available indices and models with the Getis-Ord Gi* geospatial cluster and top 20 percentile (80th−100th) methods.
+
+| state                                                                                                                                                  | EJSCREEN Index   | Schultz Model   | HUD Index   | Random Forest Model v1   | Vox   | totala   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|-----------------|-------------|--------------------------|-------|----------|
+| Illinois                                                                                                                                               | 1                | 1               | 1           | 1                        | 1     | 5        |
+| Michigan                                                                                                                                               | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New Jersey                                                                                                                                             | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New York                                                                                                                                               | 1                | 1               | 1           | 1                        | 1     | 5        |
+| Ohio                                                                                                                                                   | 1                | 1               | 1           | 1                        | 1     | 5        |
+| Pennsylvania                                                                                                                                           | 1                | 1               | 1           | 1                        | 1     | 5        |
+| Massachusetts                                                                                                                                          | 0                | 1               | 1           | 1                        | 1     | 4        |
+| California                                                                                                                                             | 1                | 1               | 0           | 1                        | 1     | 4        |
+| Indiana                                                                                                                                                | 0                | 0               | 1           | 1                        | 1     | 3        |
+| Texas                                                                                                                                                  | 1                | 1               | 1           | 0                        | 1     | 4        |
+| Connecticut                                                                                                                                            | 0                | 0               | 1           | 0                        | 0     | 1        |
+| Florida                                                                                                                                                | 1                | 1               | 0           | 0                        | 0     | 2        |
+| Maryland                                                                                                                                               | 1                | 0               | 0           | 0                        | 0     | 1        |
+| Wisconsin                                                                                                                                              | 0                | 0               | 0           | 1                        | 0     | 1        |
+| a Total = total number of indices and/or models that identified the respective location. 1 = identified by index or model. 0 = not identified by index |                  |                 |             |                          |       |          |
+
+methods, variables, and geographic scales. For example, different years of housing age (% pre-1940 vs pre-1960 vs pre-1980 homes) or different sociodemographic variables (e.g., % people of color vs % non-Hispanic African American) can produce different looking maps with differences in hotspots identified, depending on the location and population.
+
+Figure 2 shows the intersection and combination of the two statistical methods on the same maps, along with the number of U.S. census tracts identified. Considering the intersection of the five indices, the number of census tracts is 562 (0.8%)
+based on Getis-Ord Gi* identified only, 1869 (2.6%) based on top 20 percentiles identified only, and 3430 (4.7%) identified by both analyses (for a total of ∼8%). Considering the combination of the five indices, there is much more coverage over the entire United States, with the number of census tracts as 2107 (2.9%) based on Getis-Ord Gi* identified only, 9234
+(12.6%) based on top 20 percentiles identified only, and 18 867 (25.8%) identified by both analyses (for a total of
+∼41%).
+
+States and Counties with the Most Children **Living** in Hotspot Census Tracts Based on Lead Exposure **Indices.** Utilizing five indices and the two geospatial statistical methods
+(Getis-Ord Gi* and top 20 percentiles), states and counties with the most children <6 years old residing in hotspot census tracts include:
+- IL, MI, NJ, NY, OH, PA (identified by 5 indices); MA,
+CA, TX (identified by 4 indices) (Getis-Ord Gi* geospatial cluster method) as shown in Table 1 along with other results (i.e., states identified by 1, 2, or 3 indices);
+- IL, MI, NJ, NY, OH, PA, TX (identified by 5 indices);
+CA (identified by 4 indices) (top 20, 80th−100th, percentile method) as shown in Table S-1 along with other results (i.e., states identified by 1, 2, or 3 indices);
+
+| state                                                                                                                                                  | county                | EJSCREEN Index   | Schultz Model   | HUD Index   | Random Forest Model v1   | Vox   | totala   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------|------------------|-----------------|-------------|--------------------------|-------|----------|
+| Illinois                                                                                                                                               | Cook County           | 1                | 1               | 1           | 1                        | 1     | 5        |
+| Michigan                                                                                                                                               | Wayne County          | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New York                                                                                                                                               | Bronx County          | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New York                                                                                                                                               | Kings County          | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New York                                                                                                                                               | New York County       | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New York                                                                                                                                               | Queens County         | 1                | 1               | 1           | 1                        | 1     | 5        |
+| Pennsylvania                                                                                                                                           | Philadelphia County   | 1                | 1               | 1           | 1                        | 1     | 5        |
+| New Jersey                                                                                                                                             | Essex County          | 0                | 1               | 1           | 1                        | 1     | 4        |
+| California                                                                                                                                             | Los Angeles County    | 1                | 1               | 0           | 1                        | 1     | 4        |
+| Maryland                                                                                                                                               | Baltimore City        | 0                | 1               | 0           | 1                        | 1     | 3        |
+| Massachusetts                                                                                                                                          | Suffolk County        | 0                | 0               | 1           | 1                        | 1     | 3        |
+| Ohio                                                                                                                                                   | Cuyahoga County       | 0                | 1               | 0           | 1                        | 1     | 3        |
+| Pennsylvania                                                                                                                                           | Allegheny County      | 0                | 0               | 1           | 1                        | 1     | 3        |
+| Texas                                                                                                                                                  | Harris County         | 1                | 1               | 1           | 0                        | 1     | 4        |
+| Wisconsin                                                                                                                                              | Milwaukee County      | 0                | 1               | 1           | 1                        | 1     | 4        |
+| Arizona                                                                                                                                                | Maricopa County       | 1                | 1               | 0           | 0                        | 1     | 3        |
+| California                                                                                                                                             | Alameda County        | 1                | 0               | 0           | 1                        | 1     | 3        |
+| Florida                                                                                                                                                | Miami-Dade County     | 1                | 1               | 0           | 0                        | 1     | 3        |
+| Indiana                                                                                                                                                | Marion County         | 0                | 1               | 1           | 0                        | 0     | 2        |
+| New Jersey                                                                                                                                             | Hudson County         | 0                | 0               | 1           | 1                        | 0     | 2        |
+| New York                                                                                                                                               | Erie County           | 0                | 0               | 1           | 1                        | 0     | 2        |
+| Connecticut                                                                                                                                            | New Haven County      | 0                | 0               | 1           | 0                        | 0     | 1        |
+| Massachusetts                                                                                                                                          | Middlesex County      | 0                | 0               | 1           | 0                        | 1     | 2        |
+| New York                                                                                                                                               | Westchester County    | 0                | 0               | 1           | 0                        | 0     | 1        |
+| Ohio                                                                                                                                                   | Hamilton County       | 0                | 1               | 0           | 1                        | 0     | 2        |
+| Rhode Island                                                                                                                                           | Providence County     | 0                | 0               | 0           | 1                        | 0     | 1        |
+| Tennessee                                                                                                                                              | Shelby County         | 0                | 1               | 0           | 0                        | 0     | 1        |
+| Texas                                                                                                                                                  | Dallas County         | 1                | 1               | 0           | 0                        | 0     | 2        |
+| California                                                                                                                                             | Fresno County         | 1                | 0               | 0           | 0                        | 0     | 1        |
+| California                                                                                                                                             | Kern County           | 1                | 0               | 0           | 0                        | 0     | 1        |
+| California                                                                                                                                             | Orange County         | 1                | 0               | 0           | 0                        | 0     | 1        |
+| California                                                                                                                                             | Riverside County      | 1                | 0               | 0           | 0                        | 0     | 1        |
+| California                                                                                                                                             | San Bernardino County | 1                | 0               | 0           | 0                        | 0     | 1        |
+| California                                                                                                                                             | San Diego County      | 1                | 0               | 0           | 0                        | 0     | 1        |
+| California                                                                                                                                             | San Francisco County  | 0                | 0               | 0           | 0                        | 1     | 1        |
+| District of Columbia                                                                                                                                   | District of Columbia  | 0                | 1               | 0           | 0                        | 0     | 1        |
+| Missouri                                                                                                                                               | St. Louis city        | 0                | 0               | 0           | 1                        | 0     | 1        |
+| New Jersey                                                                                                                                             | Middlesex County      | 0                | 0               | 1           | 0                        | 0     | 1        |
+| New York                                                                                                                                               | Suffolk County        | 0                | 0               | 1           | 0                        | 0     | 1        |
+| Texas                                                                                                                                                  | Bexar County          | 1                | 0               | 0           | 0                        | 0     | 1        |
+| a Total = total number of indices and/or models that identified the respective location. 1 = identified by index or model. 0 = not identified by index |                       |                  |                 |             |                          |       |          |
+
+- Cook County, IL, Wayne County, MI, Bronx, Kings, New York, Queens counties, NY, Philadelphia County, PA (identified by 5 indices); Essex County, NJ, Los Angeles County, CA, Harris County, TX, Milwaukee County, WI (identified by 4 indices); others shown in Table 2 (Getis-Ord Gi* geospatial cluster method);
+- Cook County, IL, Wayne County, MI, Bronx, Kings, Queens counties, NY, Philadelphia County, PA, Los Angeles County, CA, Harris County, TX, Milwaukee County, WI (identified by 5 indices); Essex County, NJ, New York County, NY, Dallas County, TX (identified by 4 indices); others shown in Table S-2 (top 20, 80th−
+100th, percentile method).
+
+Details of states and counties identified based on the number of children in lead indices hotspot census tracts are
+shown in Tables S-3, S-4, and S-5 and S-6, S-7, and S-8, respectively. Of the three featured indices (RF Model v1, Schultz Model, and Vox), the Vox score yielded the highest number of children living in identified lead exposure risk hotspots in New York state (627 045; Table S-4) and in Los Angeles County (279 113) (Table S-7).
+
+Evaluation of Lead Indices and Hotspots **Identified.**
+Statistical evaluation of hotspots identified using five lead indices showed good agreement with OH and MI BLL data hotspots. Comparing the indices vs BLLs, Cohen's kappa scores demonstrate moderate to substantial statistical agreement (0.49−0.63) between hotspots (Table 3). Generally, the RF Model v1 and v2 had the strongest agreement results (0.63 and 0.54/0.52 for MI and OH, respectively). Tables S-9 and S10 further illustrate strong statistical agreement with sensitivity Table 3. Statistical Agreement Analyses (Cohen's **Kappa)** of the Highest Potential Lead (Pb) Exposure Risk **Hotspots** Identified: Michigan and Ohio Blood Lead Levels **(BLLs)** vs Pb Exposure Indices and **Models**a
+
+| Michigan BLLs                                                                                                             | Ohio BLLs   |             |      |      |             |      |
+|---------------------------------------------------------------------------------------------------------------------------|-------------|-------------|------|------|-------------|------|
+| GetisOrd                                                                                                                           | GetisOrd             |             |      |      |             |      |
+| top 20                                                                                                                    | top 20      |             |      |      |             |      |
+| N                                                                                                                         | Gi*         | percentiles | N    | Gi*  | percentiles |      |
+| kappa                                                                                                                     | kappa       |             |      |      |             |      |
+| EJSCREEN                                                                                                                  | 2401        | 0.56        | 0.53 | 2850 | 0.59        | 0.50 |
+| Index HUD DPI                                                                                                             | 2401        | 0.50        | 0.43 | 2850 | 0.49        | 0.42 |
+| Random Forest                                                                                                             | 2401        | 0.63        | 0.52 | 2850 | 0.54        | 0.51 |
+| Model version 1 Schultz Model                                                                                             | 2401        | 0.49        | 0.49 | 2850 | 0.46        | 0.51 |
+| Vox                                                                                                                       | 2401        | 0.50        | 0.55 | 2850 | 0.44        | 0.51 |
+| Random Forest                                                                                                             | 2401        | 0.63        | 0.52 | 2850 | 0.52        | 0.51 |
+| Model version 2                                                                                                           |             |             |      |      |             |      |
+| a Cohen's kappa agreement statistic: <0.4 low; >0.4−0.6 moderate; >0.6−0.8 substantial; >0.8−0.99 near perfect agreement. |             |             |      |      |             |      |
+
+and specificity analyses: sensitivity of 91−96% for three of the indices (RF Model v1, Schultz Model, and Vox − Getis-Ord Gi*); specificity of 78−94% for all indices (top 20 percentiles and Getis-Ord Gi*). Compared against each other, the lead indices demonstrate low to near-perfect statistical agreement
+(e.g., kappa scores of 0.37−0.87 as shown in Table S-11).
+
+The hotspots identified with the five considered lead indices match well with community hotspots identified in the available state health department reports (Tables 4 and 5). For the U.S., the results were 45% (EJSCREEN Index), 62% (Schultz Model), 78% (HUD DPI), 58% (RF Model v1), 72% (Vox), and 61% (RF Model v2). For OH and MI, the results were 74% (EJSCREEN Index), 95% (Schultz Model), 89% (HUD DPI), 89% (RF Model v1), 100% (Vox), and 95% (RF Model v2). Since the convergence analyses of RF Model v1, Schultz Model, and Vox show better statistical agreement with each other (Cohen's kappa of ∼0.7) than with EJSCREEN Index and HUD DPI (kappa ranged from 0.47 to 0.59 with the GetisOrd Gi* method) for the national scale analysis (Table S-11),
+we focused on these three indices for identifying states and counties with the highest potential lead exposure risk (as mentioned in Materials and Methods).
+
+- DISCUSSION
+Strengths. This paper presents an innovative, state-of-thescience geospatial data analysis and screening approach to help identify and address U.S. lead exposure hotspot locations.
+
+Current limitations and gaps in environmental, sociodemographic, and blood lead data make it challenging to develop a single U.S. blood lead map or blood lead prediction model accounting for all sources of lead exposure. Thus, our methodology is to utilize a convergence and collective set of currently available information, considering uncertainties and different data sets and approaches. The results are displayed visually with U.S. hotspot map figures showing the intersection and combination of five available lead indices and corresponding tables with locations they identified. The accompanying statistical analyses in the tables presented show the strength of the indices used and verification of locations they identified.
+
+Our results support the use of available lead indices in the absence of consistent, complete BLL surveillance data across Environmental Science & Technology **pubs.acs.org/est** Article the United States. This analysis utilizes peer-reviewed, published lead exposure hotspot analysis methods in EPA's Michigan lead analysis paper12 and builds off an interagency
+(EPA/HUD/CDC) lead mapping state-of-the-science paper.5 Statistical evaluation of the available lead indices was conducted in multiple ways: with MI and OH children's BLL surveillance data and locations identified from 9 state health department reports.
+
+We used census tract level analyses to identify, on a national scale, the states and counties with the highest potential lead exposure risk based on the number of children living in lead exposure hotspots. These results can inform further tailored analyses for lead mitigation efforts. For example, these screening analysis results could include identifying the most disproportionately impacted communities that might be eligible for federal or state lead mitigation programs; determining where resources should be focused to reduce lead-based paint exposures, replace lead service lines, and address lead-contaminated soils and other sources; assessing additional data needs and priority locations for collecting more environmental and biomonitoring data to identify lead exposure hotspots and their drivers. The analysis presented in this paper to advance the science for identifying high lead exposure locations supports U.S. efforts including the Federal Lead Action Plan,10 EPA Lead Strategy,9 Biden-Harris Lead Pipe and Paint Action Plan,28 and Biden-Harris "Get the Lead Out" Partnership.29 This science can guide other lead hotspot identification and verification efforts and inform lead reduction actions through collaborations with state and local health departments.30 Not only are these national-scale maps and results informative to help target U.S. efforts, but the approaches of this paper could also inform lead discussions and efforts in other countries. For example, a report to G7 ministers31 on the outcomes of the November 2022 workshop entitled, "Lead as a Major Threat for Human Health and the Environment: An Integrated Approach Strengthening Cooperation toward Solutions," discussed options for future work to reduce sources of lead and minimize lead exposure in low- and middle-income countries (LMICs) that included: "helping LMICs conduct initial diagnostic assessments about the prevalence of lead poisoning and identification and ranking of sources of lead exposure." In the U.S. and other countries, while data collection and hotspot mapping science evolves to help pinpoint the highest risk locations, short-term national and community actions can be informed by existing best practices and toolkits (e.g., EPA's Local Lead Action Plan Guide;32 United Nations Environment Programme, Toolkit for establishing laws to eliminate lead paint33).
+
+Limitations. There are a number of limitations of this analysis and opportunities to enhance data and methods to identify lead exposure risk hotspots. The lead indices used in this analysis include 2010 census data, geographic identifiers, and boundaries. The 2020 census data, geographic identifiers, and boundaries cannot be fully incorporated at this time due to incomplete census data or index incompatibility. As mentioned in the Materials and Methods, this analysis uses 5 μg/dL rather than the current CDC blood lead reference value of 3.5 μg/dL
+to be consistent with the underlying data in the lead indices and the data used to evaluate them (public reports and BLL data from MI and OH). Although this may be considered a limitation of the analysis, Xue et al.12 found that, when comparing MI census tract hotspots using 5 μg/dL vs
+
+Table 4. State-Identified Locations in 9 Public Health Department Reports (Based on Blood Lead Levels ≥5 μg/dL) **Across**
+
+the United States That Were Also Identified by the Getis-Ord Gi* Analysis of Lead Exposure Indices and **Models**a
+
+| the United States That Were Also Identified by the Getis-Ord Gi* Analysis of Lead Exposure Indices and Modelsa state health department community hotspotb EJSCREEN Schultz HUD Random Forest Model community index-model identification count Index Model Index v1 Vox Akron, OH 1 1 1 1 1 5 Atlantic City, NJ 1 1 1 1 1 5 Bridgeport, CT 1 1 1 1 1 5 Brockton, MA 1 1 1 1 1 5 Canton, OH 1 1 1 1 1 5 Central Falls, RI 1 1 1 1 1 5 Cincinnati, OH 1 1 1 1 1 5 Cleveland, OH 1 1 1 1 1 5 Columbus, OH 1 1 1 1 1 5 Dayton, OH 1 1 1 1 1 5 Detroit, MI 1 1 1 1 1 5 East Cleveland, OH 1 1 1 1 1 5 East Orange, NJ 1 1 1 1 1 5 Flint, MI 1 1 1 1 1 5 Hamtramck, MI 1 1 1 1 1 5 Harrisburg, PA 1 1 1 1 1 5 Hartford, CT 1 1 1 1 1 5 Highland Park, MI 1 1 1 1 1 5 Irvington, NJ 1 1 1 1 1 5 Lancaster, PA 1 1 1 1 1 5 Meriden, CT 1 1 1 1 1 5 Muskegon, MI 1 1 1 1 1 5 New Haven, CT 1 1 1 1 1 5 Paterson, NJ 1 1 1 1 1 5 Plainfield, NJ 1 1 1 1 1 5 Reading, PA 1 1 1 1 1 5 Toledo, OH 1 1 1 1 1 5 Trenton, NJ 1 1 1 1 1 5 Waterbury, CT 1 1 1 1 1 5 York, PA 1 1 1 1 1 5 Youngstown, OH 1 1 1 1 1 5 Jackson, MI 0 1 1 1 1 4 Lewiston, ME 0 1 1 1 1 4 New Bedford, MA 0 1 1 1 1 4 Portland, ME 0 1 1 1 1 4 Providence, RI 0 1 1 1 1 4 Scranton, PA 0 1 1 1 1 4 Springfield, OH 0 1 1 1 1 4 Woonsocket, RI 0 1 1 1 1 4 Grand Rapids, MI 0 1 1 1 1 4 Bangor, ME 0 1 1 0 1 3 Adams, MA 0 0 1 0 1 2 Auburn, ME 0 0 1 0 1 2 Gloucester, MA 0 0 1 0 1 2 Newark, NJ 0 1 1 0 0 2 Newport, RI 0 0 1 0 1 2 North Adams, MA 0 0 1 0 1 2 Total 31 43 54 40 50 Percentc 45 62 78 58 72   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+a1 = identified by index or model; 0 = not identified by index or model. bState health department reports and their respective community hotspots are sourced from Table D in Zartarian, V.; Poulakos, A.; Garrison, V. H.; Spalt, N.; Tornero-Velez, R.; Xue, J.; Egan, K.; Courtney, J. Lead Data Mapping to Prioritize US Locations for Whole-of-Government Exposure Prevention Efforts: State of the Science, Federal Collaborations, and Remaining Challenges. Am. J. Public *Health*. 2022, 112 (S7), S658−S669. DOI: 10.2105/AJPH.2022.307051 (ref 5). cPercent refers to the percentage of community hotspots identified by each respective index or model. Percentages were calculated by taking the values in the "Total" row and dividing them by 69 (i.e., the total number of state health department community hotspots in the aforementioned 9 public health department reports). 57 community hotspots were identified by at least one index/model. 47 of the 69 are shown in the table as these are the communities identified by two or more indices/models (this was also done due to table formatting and spacing limitations).
+
+Table 5. State-Identified Locations in Michigan and Ohio Public Health Department Reports (Based on Blood Lead **Levels** ≥5 μg/dL) That Were Also Identified by the Getis-Ord Gi* Analysis of Lead Exposure Indices and **Models**a
+
+| Pb exposure indices and models                                                                                                                      |          |         |     |                     |                                            |    |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------|----------|---------|-----|---------------------|--------------------------------------------|----|
+| state health dept. community hotspotb                                                                                                               | EJSCREEN | Schultz | HUD | Random Forest Model | community index-model identification count |    |
+| Index                                                                                                                                               | Model    | Index   | v1  | Vox                 |                                            |    |
+| Akron, OH                                                                                                                                           | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Canton, OH                                                                                                                                          | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Cincinnati, OH                                                                                                                                      | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Cleveland, OH                                                                                                                                       | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Columbus, OH                                                                                                                                        | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Dayton, OH                                                                                                                                          | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Detroit, MI                                                                                                                                         | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| East Cleveland, OH                                                                                                                                  | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Flint, MI                                                                                                                                           | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Hamtramck, MI                                                                                                                                       | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Highland Park, MI                                                                                                                                   | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Muskegon, MI                                                                                                                                        | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Toledo, OH                                                                                                                                          | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Youngstown, OH                                                                                                                                      | 1        | 1       | 1   | 1                   | 1                                          | 5  |
+| Jackson, MI                                                                                                                                         | 0        | 1       | 1   | 1                   | 1                                          | 4  |
+| Springfield, OH                                                                                                                                     | 0        | 1       | 1   | 1                   | 1                                          | 4  |
+| Grand Rapids, MI                                                                                                                                    | 0        | 1       | 1   | 1                   | 1                                          | 4  |
+| Lansing, MI                                                                                                                                         | 0        | 1       | 0   | 0                   | 1                                          | 2  |
+| Adrian, MI                                                                                                                                          | 0        | 0       | 0   | 0                   | 1                                          | 1  |
+| Total                                                                                                                                               | 14       | 18      | 17  | 17                  | 19                                         |    |
+| Percentc                                                                                                                                            | 74       | 95      | 89  | 89                  | 100                                        |    |
+| a 1 = identified by index or model; 0 = not identified by index or model. b State health department reports and their respective community hotspots |          |         |     |                     |                                            |    |
+
+3.5 μg/dL, a Cohen's kappa value of ∼0.94 was calculated, demonstrating near perfect statistical agreement. Furthermore, the use of 3.5 μg/dL as a blood lead reference value has yet to be adopted by all state health departments, which presently limits and complicates its use in a national scale analysis.
+
+This research focuses on children because of available blood lead data, but reducing lead exposure is also important for adults.1 We note that some researchers have questioned the interpretability of agreement statistics such as Cohen's kappa.34,35 Only nine state health department reports identified community-level locations; this, along with most of them not reporting at the census tract level, limited the extent to which we could evaluate the indices across the country.
+
+Ideally, more blood lead data would be available for groundtruthing of index-identified hotspots.
+
+The indices utilized are moderate to substantial predictors of higher BLLs and do not include environmental data and sources that could likely improve the identification of lead exposure hotspots (e.g., prevalence of lead service lines; drinking water and soil lead concentration data in residences, schools, and daycare centers; other data discussed in Zartarian et al.5). We emphasize that, because the indices and models used in this paper are based on publicly available housing age and sociodemographic data, the lead exposure risk hotspots identified in this national-scale analysis are most relevant to old housing-related sources of lead (e.g., lead-based paint, residential soil and dust, and lead in drinking water in old homes with lead pipes). Identification of high-risk locations and their underlying sources may change as environmental data, and ideally also food and consumer products data, are incorporated into the indices and finer scales are considered. Understanding which lead sources and pathways are contributing the most to BLLs within lead exposure hotspots identified is important for prioritizing exposure reduction actions and resources. However, source apportionment can vary by location and scenarios,36,37 and quantification is challenging without an integrated database of multimedia environmental lead and BLL data.5 Table 1 results, based on Pb indices constructed with old housing and sociodemographic data from the census, may potentially be skewed toward states with higher population densities because of significantly more old housing stock (i.e., in northeastern and north-central U.S. in comparison to the south and other parts of the U.S). Rural counties may appear less than urban areas in this analysis because of smaller populations and potentially lower quantity of census data from those counties used in the indices. Prior research with Michigan data showed that the HUD DPI, EJSCREEN Index, and Schultz Model predominantly identify urban hot spots, particularly with the geospatial cluster analysis (Xue et al.12). This U.S. analysis paper and the ones it builds upon focus on potential lead exposure risk from old housing-related sources, based on population level (census tract, county, and state results) not at the individual or household levels.
+
+As a screening approach to identify locations with an increased potential of high lead exposure or higher BLLs, this national-scale analysis cannot identify sources at particular addresses or risk at an individual level. Our methodology focuses on the census tract scale for reasons discussed in Xue et al..12 To target resources for local-scale source and risk mitigation, further investigation of hotspots identified should be conducted to identify individuals who may have been exposed.30 More verification or "ground-truthing" of lead hotspots is needed using more blood lead surveillance data, as it becomes available, and information from local partners with on-the-ground knowledge of their communities.30 We also note that counties with high population densities were identified by most or all of the indices used, which may raise the question of whether population density alone may be sufficient in determining high lead exposure risk. As described in the U.S. EPA Integrated Science Assessment for Lead,4 old housing�a key environmental variable included in all lead exposure indices considered in this paper�is a widely known and acknowledged indicator of potential lead exposures. This reinforces its use over simple population-based metrics.
+
+Furthermore, lead exposure risk has been observed to disproportionately impact specific communities.9,10 From a social and environmental justice perspective, population density data alone cannot account for this.
+
+Research **Needs.** More research is needed to understand the differences in high potential lead exposure risk locations identified and the choice of the most appropriate index or indices for different geographic locations. In addition, while some indices statistically agree more with higher BLL hotspots for MI and OH (as shown in Table 3), the requisite BLL data to be able to preferentially select one over another are not available across the entire U.S. Thus, our national approach is to intersect or combine indices (as shown in Figure 2) to narrow or broaden the set of locations for lead-related efforts, depending on the intended purpose and available resources.
+
+Future research can also include enhancing and expanding the current lead indices to address the various limitations as more data become available. Research is underway to enhance the Random Forest Model by incorporating environmental data in EPA/OECA's POST (EPA Office of Enforcement and Compliance Assurance Pb Occurrence and Source Tool described in Zartarian et al.5). For example, analyses have been conducted to determine the relative impact of lead service line (LSL) prevalence vs old housing in two cities where LSL data are available22 and to geospatially analyze linkages between commercial sources of lead and childhood BLLs.38 CDC is currently developing a web-based Lead Exposure Risk Index (LERI) that will incorporate multiple demographic, geographic, and environmental risk factors at the census tract level. This tool is intended for use by health agencies and providers to identify high-risk areas where children should be tested for lead and to target lead-reduction interventions.39 In the next two years, HUD plans to geocode the historic database of HUD grants' production of lead-safe housing units and update the Deteriorated Paint Index in 2024 after the 2023 American Housing Survey, Healthy Homes supplement is fielded.
+
+Due to the current data limitations discussed in Zartarian et al.,5 development of lead indices as surrogates in the absence of complete and accurate population-level BLL surveillance data requires a whole-of-government approach. EPA, HUD, and CDC continue to collaborate and evolve the research and address data gaps. Various groups are working to collect more representative BLL surveillance data and detailed environmental lead sources and measurement data that could help evaluate and improve future indices.30 Collaborative interagency case studies applying the EPA Lead Strategy Goal 2
+"blueprint"9,30 to identify and prioritize potential places for action are further developing and applying these methods to determine drivers of lead exposure hotspots. While science in this area evolves, these U.S. screening results identifying states, counties, and census tracts with potential high lead exposure risk can inform further efforts to target lead actions (such as those described in Breysse et al.11 and Zartarian et al.5) for public health protection.
+
+The overarching public health questions this research is addressing to help prioritize actions are as follows: Where are exposure risk hotspots and what sources are driving them? The lead-focused methodologies in this paper and previous research it expands upon, using available blood lead surveillance data and surrogate exposure indices, could potentially be applied to other chemicals (and countries) depending on available data.
+
+- ASSOCIATED **CONTENT**
+
+## Data Availability **Statement**
+
+Data from this paper are available in EPA's Environmental Dataset Gateway at https://doi.org/10.23719/1529907.
+
+*sı Supporting **Information**
+The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.3c07881.
+
+Additional maps and analyses of high lead exposure risk locations based on available lead indices and blood lead data (PDF) Detailed background information and method details on Random Forest Model v1 and v2; table showing variables used; figure showing model validation and performance (PDF)
+- AUTHOR INFORMATION
+
+## Corresponding **Author**
+
+Valerie G. **Zartarian** − U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States;* orcid.org/00090003-7788-7033; Phone: 617-620-3148; Email: Zartarian.valerie@epa.gov
+
+## Authors
+
+Jianping Xue − U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States* Antonios G. **Poulakos** − U.S. Environmental *Protection* Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United States; LinTech Global, Inc., Boston, Massachusetts 02109, United States; orcid.org/0000-0001-5755-3595 Rogelio **Tornero-Velez** − U.S. Environmental *Protection* Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States;*
+orcid.org/0000-0003-2102-0548 Lindsay W. **Stanek** − U.S. Environmental Protection *Agency,*
+Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States;* orcid.org/00000002-9921-7458 Emily **Snyder** − U.S. Environmental Protection Agency, *Office* of Research and Development, Research Triangle Park, *North* Carolina 27711, United *States*
+
+Veronica Helms **Garrison** − U.S. Department of Housing and Urban Development, Office of Policy Development and Research, Washington, D.C. 20410, United *States* Kathryn **Egan** − Centers for Disease Control and Prevention, National Center for Environmental Health, Agency for Toxic Substances and Disease Registry, Office of Science, *Atlanta,*
+Georgia 30329, United *States* Joseph G. **Courtney** − Centers for Disease Control and Prevention, National Center for Environmental *Health,* Division of Environmental Health Science and *Practice,* Atlanta, Georgia 30329, United *States* Complete contact information is available at: https://pubs.acs.org/10.1021/acs.est.3c07881
+
+## Notes
+
+The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency, U.S. Department of Housing and Urban Development, Centers for Disease Control and Prevention, or state partners. Any mention of trade names, products, or services does not imply an endorsement by the U.S. Government or EPA, HUD, or CDC. EPA, HUD, and CDC do not endorse any commercial products, services, or enterprises. In addition, the contractor's role did not include establishing agency policy. Michigan Department of Health and Human Services (MDHHS) provided blood lead data used in this paper, pursuant to Data Use Agreement 201909-157. EPA assumes full responsibility for the analysis and interpretation of the data. This paper also includes analyses with blood lead data provided by the Ohio Department of Health (ODH), through the Ohio Public Health Information Warehouse under an approved Data Use Agreement. The use of these data should not be considered an endorsement of this study or its conclusions by ODH. Research included in this analysis was approved under Institutional Review Boards (IRBs) through University of North Carolina at Chapel Hill (UNC; 16-2302), MDHHS (201703-12-EA), and ODH (2019-41). The authors declare no competing financial interest. J.X. is now retired from the EPA.
+
+- **ACKNOWLEDGMENTS**
+We gratefully acknowledge the technical reviewers; EPA/ORD lead mapping team, managers, and support staff; EPA ORD and Region 5 coauthors of the MI paper; EPA, HUD, and CDC collaborators; coauthors of state-of-the-science lead mapping paper; partners in Michigan MDHHS, Michigan EGLE, and Ohio DOH. This article has been reviewed by EPA, HUD, and CDC and approved for publication. The coauthors appreciate technical reviewers and managers from our agencies who supported this article and related research. We gratefully acknowledge the EPA's Office of Research and Development; the CDC Lead Exposure Risk Index Working Group, Agency for Toxic Substances and Disease Registry, Office of Community Health and Hazard Assessment and the National Center for Environmental Health, Division of Environmental Health Science and Practice; the HUD Office of Policy Development and Research. The mapping and analysis work has been funded in part by the U.S. EPA under the East Coast Help Desk Operations (ECHO)−Multi Region Information Technology (IT) Services Support Contract and the U.S. EPA Region 1 IT Services Support Task Order; specifically, LinTech Global, Inc.'s ECHO Contract No. GS35F-0343W/68HE0319F0020 with the U.S. Environmental Protection Agency, Regions 1, 2, & 3.
+
+- **REFERENCES**
+(1) Lamas, G. A.; Bhatnagar, A.; Jones, M. R.; Mann, K. K.; Nasir, K.; Tellez-Plaza, M.; Ujueta, F.; Navas-Acien, A. the American Heart Association Council on Epidemiology and Prevention; Council on Cardiovascular and Stroke Nursing; Council on Lifestyle and Cardiometabolic Health; Council on Peripheral Vascular Disease; Council on the Kidney in Cardiovascular Disease. Contaminant Metals as Cardiovascular Risk Factors: A Scientific Statement from the American Heart Association. J. Am. Heart *Assoc.* **2023**, 12, No. e029852.
+
+(2) Egan, K. B.; Cornwell, C. R.; Courtney, J. G.; Ettinger, A. S.
+
+Blood lead levels in US children ages 1−11 years, 1976−2016.
+
+Environ. Health *Perspect.* **2021**, 129 (3), 37003.
+
+(3) Hauptman, M.; Niles, J. K.; Gudin, J.; Kaufman, H. W.
+
+Individual- and community-level factors associated with detectable and elevated blood lead levels in US children: results from a national clinical laboratory. JAMA *Pediatr* **2021**, 175 (12), 1252−1260.
+
+(4) Integrated Science Assessment (ISA) for Lead (Final Report, *July* 2013); EPA-600-R-10-075F; U.S. Environmental Protection Agency: Washington, DC, 2013.
+
+(5) Zartarian, V.; Poulakos, A.; Garrison, V. H.; Spalt, N.; TorneroVelez, R.; Xue, J.; Egan, K.; Courtney, J. Lead Data Mapping to Prioritize US Locations for Whole-of-Government Exposure Prevention Efforts: State of the Science, Federal Collaborations, and Remaining Challenges. Am. J. Public *Health.* **2022**, 112 (S7), S658−
+S669.
+
+(6) Centers for Disease Control and Prevention. Childhood Lead Poisoning Prevention Program: Lead Poisoning *Prevention*; https://
+www.cdc.gov/nceh/lead/prevention/default.htm (updated September 2, 2022; accessed 2023-09-01).
+
+(7) Agency for Toxic Substances and Disease Registry. Toxicological profile for *lead*; U.S. Department of Health and Human Services, Public Health Service: Atlanta, GA, 2020;https://wwwn.cdc.gov/ TSP/ToxProfiles/ToxProfiles.aspx?id=96&tid=2web2 (accessed 2023-09-01).
+
+(8) U.S. Environmental Protection Agency. EPA Lead (Pb) *Research*;
+https://www.epa.gov/land-research/epa-lead-pb-research (updated November 7, 2022; accessed 2023-09-01).
+
+(9) U.S. Environmental Protection Agency. EPA strategy to *reduce* lead exposures and disparities in US *communities*; November 16, 2021; https://www.epa.gov/system/files/documents/2021-11/updatedpublic-comment-draft-lead-strategy-11-16-2021.pdf (accessed 202309-01).
+
+(10) President's Task Force on Environmental Health Risks and Safety Risks to Children. Federal Action Plan to Reduce Childhood *Lead* Exposures and Associated Health *Impacts*; December 2018; https:// www.epa.gov/sites/default/files/2018-12/documents/fedactionplan_ lead_final.pdf (accessed 2023-09-01).
+
+(11) Breysse, P. N.; Cascio, W. E.; Geller, A. M.; Choiniere, C. J.;
+Ammon, M. Targeting Coordinated Federal Efforts to Address Persistent Hazardous Exposures to Lead. Am. J. Public *Health.* **2022**,
+112 (S7), S640−S646.
+
+(12) Xue, J.; Zartarian, V.; Tornero-Velez, R.; Stanek, L. W.;
+Poulakos, A.; Walts, A.; Triantafillou, K.; Suero, M.; Grokhowsky, N. A generalizable evaluated approach, applying advanced geospatial statistical methods, to identify high lead (Pb) exposure locations at census tract scale: Michigan case study. Environ. Health *Perspect.* **2022**, 130 (7), 77004.
+
+(13) Stanek, L. W.; Xue, J.; Zartarian, V. M.; Poulakos, A.; TorneroVelez, R.; Snyder, E.; Walts, A.; Triantafillou, K. Identification of *High* Lead Exposure Locations in Ohio at the Census Tract Scale *using* a Generalizable Geospatial Hotspot *Approach*. **2024**, in journal review.
+
+(14) Ord, J. K.; Getis, A. Local spatial autocorrelation statistics:
+distributional issues and an application. Geogr. *Anal.* **1995**, 27 (4),
+286−306.
+
+(15) ESRI ArcGIS Pro. How Hot Spot Analysis (Getis-Ord Gi*)
+works; 2023; https://pro.arcgis.com/en/pro-app/latest/toolreference/spatial-statistics/h-how-hot-spot-analysis-getis-ord-gispatial-stati.htm (accessed 2023-09-01).
+
+(16) Centers for Disease Control and Prevention. Childhood Lead Poisoning Prevention Program: Blood Lead Reference *Value*; https://
+www.cdc.gov/nceh/lead/data/blood-lead-reference-value.htm (updated December 2, 2022; accessed 2023-09-01).
+
+(17) Ruckart, P. Z.; Jones, R. L.; Courtney, J. G.; LeBlanc, T. T.;
+Jackson, W.; Karwowski, M. P.; Cheng, P. Y.; Allwood, P.; Svendsen, E. R.; Breysse, P. N. Update of the blood lead reference value − United States, 2021. Morb. Mortal. Wkly. *Rep.* **2021**, 70 (43), 1509−
+1512.
+
+(18) U.S. Environmental Protection Agency; Office of Environmental Justice EJScreen: Environmental Justice Screening and *Mapping* Tool; https://www.epa.gov/ejscreen (accessed 2023-09-01).
+
+(19) Schultz, B. D.; Morara, M.; Buxton, B. E.; Weintraub, M.
+
+Predicting blood-lead levels among US children at the census tract level. Environ. *Justice.* **2017**, 10 (5), 129−136.
+
+(20) Garrison, V. E.; Ashley, P. J. Identifying jurisdictions at risk of containing housing units with deteriorated paint: results and targeting implications for the U.S. Department of Housing and Urban Development. J. Public Health Manag. *Pract.* **2021**, 27 (6), 546−557.
+
+(21) Vox. The risk of lead poisoning isn't just in Flint. So we mapped the risk in every neighborhood in *America*; 2016; https://www.vox.com/ a/lead-exposure-risk-map (accessed 2023-09-01).
+
+(22) Tornero-Velez, R.; Zartarian, V.; Poulakos, A.; Shao, N.;
+Stanek, L.; Buckley, T.; Goldberg, M. Tapping into Lead Service Line Information: Two City Case Study. In National Environmental *Health* Association (NEHA) Conference Lead Mapping Session. Lead Data Mapping: Methods and Tools for Lead Prioritization, Prevention, and Mitigation, New Orleans, LA, August 1, 2023.
+
+(23) Zartarian, V.; Xue, J.; Poulakos, A.; Tornero-Velez, R.; Stanek, L.; Geller, A. Identifying High Lead (Pb) Exposure Locations in the US With an Environmental Justice Focus. Oral presentation at the Society of Toxicology (SOT) conference, Session on Translating *Lead* Toxicology; Engaging Stakeholders and *Communities*, San Diego, CA, March 26, 2022.
+
+(24) Ohio Department of Health. Ohio Healthy Homes and Lead Poisoning Prevention Program prepared by the Ohio State University Statistical Consulting Service. Final Report on Targeted Testing Plan for Childhood Lead *Poisoning*, April 30, 2013.
+
+(25) U.S. Census Bureau. Detailed Tables, 2013−2017 American Community Survey 5-year *estimates*; https://data.census.gov/all?g= 010XX00US$1400000=2017=ACS+5- Year+Estimates+Detailed+Tables (accessed 2023-09-01).
+
+(26) Cohen, J. A coefficient of agreement for nominal scales. *Educ.*
+Psychol. *Meas.* **1960**, 20 (1), 37−46.
+
+(27) U.S. Census Bureau. 2010 Decennial *Census*; https://data.
+
+census.gov/advanced?y=2010 (accessed 2023-09-01).
+
+(28) The White House. Biden-Harris Lead Pipe and Paint Action Plan; December 16, 2021; https://www.whitehouse.gov/briefingroom/statements-releases/2021/12/16/fact-sheet-the-biden-harrislead-pipe-and-paint-action-plan/ (accessed 2023-09-01).
+
+(29) The White House. Biden-Harris "Get the Lead Out" *Partnership*;
+January 26, 2023; https://www.whitehouse.gov/build/briefing-room/ 2023/01/26/biden-harris-get-the-lead-out-partnership/ (accessed 2023-09-01).
+
+(30) Zartarian, V.; Oliveri, A.; Gross-Davis, C.; Triantafillou, K.;
+Walts, A.; Spalt, N.; Poulakos, A.; Tornero-Velez, R.; Lytle, D.;
+Christian, M.; Stanek, L.; Snyder, E. Work-in-Progress Whole-ofGovernment Lead (Pb) "Blueprint" Examples for Michigan and West Virginia. In National Environmental Health Association (NEHA) Conference Lead Mapping Session. Lead Data Mapping: Methods and Tools for Lead Prioritization, Prevention, and *Mitigation*, New Orleans LA, August 1, 2023.
+
+Environmental Science & Technology **pubs.acs.org/est** Article
+(31) German Federal Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection. G7 Workshop. Lead as a Major Threat for Human Health and the *Environment:*
+An Integrated Approach Strengthening Cooperation toward *Solutions*;
+Report to G7 ministers on Key Workshop Outcomes; November 9−
+10, 2023; https://www.bmuv.de/fileadmin/Daten_BMU/ Download_PDF/Europa___International/outcome_g7_workshop_ lead_2022_bf.pdf (accessed 2023-09-01).
+
+(32) U.S. Environmental Protection Agency. Build Your Local *Lead* Action Plan: A Guide for Local Leaders, Local Lead Action Plan *Guide*;
+https://www.epa.gov/lead/llap-guide (updated July 25, 2023; accessed 2023-09-01).
+
+(33) United Nations Environment Programme. Toolkit for establishing laws to eliminate lead *paint*; https://www.unep.org/toolkitestablishing-laws-eliminate-lead-paint (accessed 2023-09-01).
+
+(34) Pontius, R. G., Jr.; Millones, M. Death to Kappa: birth of quantity disagreement and allocation disagreement for accuracy assessment. Int. J. Remote *Sens.* **2011**, 32 (15), 4407−29.
+
+(35) McHugh, M. L. Interrater reliability: the kappa statistic.
+
+Biochem. Med. *(Zagreb)* **2012**, 22 (3), 276−282.
+
+(36) Stanek, L. W.; Xue, J.; Lay, C. R.; Helm, E. C.; Schock, M.;
+Lytle, D. A.; Speth, T. F.; Zartarian, V. G. Modeled impacts of drinking water Pb reduction scenarios on children's exposures and blood lead levels. Environ. Sci. *Technol.* **2020**, 54 (15), 9474−9482.
+
+(37) Zartarian, V.; Xue, J.; Tornero-Velez, R.; Brown, J. Children's lead exposure: a multimedia modeling analysis to guide public health decision-making. Environ. Health *Perspect.* **2017**, 125 (9), 097009.
+
+(38) Kulikowski, N.; Tornero-Velez, R.; Lamichhane, A.; Kim, H.;
+Gohari, Z.; Hall, A.; Spalt, N.; DeFlorio-Barker, S. Geospatial Analysis to Assess Linkage between Commercial Sources of Lead (Pb) and Childhood Blood Lead Levels. Get the Lead Out, Beyond Gas and Paint: Databases, Crowdsourcing, and Other Approaches to Understand Modern Day Lead Exposures. In International Society of Exposure Science (ISES) Conference. Symposium: Track *Vulnerabilities* in Environmental Exposure and *Health*, August 30, 2023.
+
+(39) Egan, K.; Courtney, J. CDC-ATSDR Lead Exposure Risk Index
+(LERI). In Lead Exposure and Prevention Advisory *Committee* (LEPAC) *meeting*; December 3, 2021; https://www.cdc.gov/nceh/ lead/advisory/lepac-meeting-12-3-21.html (accessed 2023-09-01).

medical/md/PMC10954656.md

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+# A Tale Of Three Chambers: Cor Triatriatum Sinistrum
+
+Maliha Butt, MD, Husban Halim, MD, Ataul Qureshi, MD, Ryan Koene, MD, Su Aung, MD,
+Tara Karamlou, MD, and Ashish Saini, MD, Canton, Cleveland, and Akron, Ohio INTRODUCTION
+Cor triatriatum sinistrum (CTS) is one of the rarest congenital abnormalities of the heart (0.1%-0.4%) that is defined by the presence of an abnormal fibromuscular septum dividing the left atrium (LA) into two chambers.1 CASE PRESENTATION
+A 40-year-old patient with a history of sinus bradycardia who had undergone dual chamber pacemaker implantation at 37 years of age, with morbid obesity, type 2 diabetes mellitus, and hyperlipidemia, presented with palpitations, exertional dyspnea, and chest pain. The patient also reported having ''three chambers in the heart'' found at the age of 18 years during evaluation of a syncopal episode. Interestingly, the patient did not follow up with any cardiologist, although they did undergo two transthoracic echocardiograms
+(TTE) during hospital admissions for noncardiac reasons that showed possible cor triatriatum and a left ventricular ejection fraction (EF) of 50% in 2014 (Figure 1). On presentation to our hospital, the patient was diagnosed with heart failure with reduced EF of 30% and started on guideline-directed medical therapy, which resulted in improved EF (Figure 2). The patient was morbidly obese and referred for bariatric surgery, resulting in significant reduction in weight. They also had recurrent episodes of symptomatic paroxysmal atrial fibrillation and were referred to an electrophysiologist. The patient underwent direct-current cardioversion two times as well as radiofrequency pulmonary vein isolation ablation. Transesophageal echocardiography
+(TEE) was done to further evaluate cardiac anatomy and revealed CTS with a small opening of 0.8 cm2 (Figures 3-5, Videos 1 and 2).
+
+The interatrial septum was normal. There was no anomalous pulmonary venous return. There was concern for mitral stenosis–like physiology caused by the CTS membrane, so the patient was referred to an adult congenital heart disease clinic. Right heart catheterization showed pulmonary artery pressure of 50/30 mm Hg, pulmonary capillary wedge pressure of 30.0 mm Hg, and pulmonary venous resistance of 0.8 Wood unit. The cardiac index was 3.5 L/min/m2.
+
+Coronary angiography showed no significant coronary artery disease.
+
+The patient underwent surgical correction of CTS. Intraoperative findings included a small patent foramen ovale and a large LA membrane with a tiny aperture near the septum. Surgical LA membrane resection, patent foramen ovale closure, and biatrial maze and LA
+appendage ligation was performed. Antiarrhythmics were continued, and anticoagulation was stopped after 3 months. The patient continued to do well at 6-month follow-up, with no further atrial fibrillation or symptoms of congestive heart failure.
+
+DISCUSSION CTS is a congenital heart disease in which the LA is divided into two compartments by a fold of tissue, a membrane, or a fibromuscular band. The exact pathophysiology is unknown, but several theories have been proposed, including malseptation (abnormal growth of septum primum that bisects the atrium), entrapment (embryonic sinus venosus traps the common pulmonary vein and prevents its incorporation into the LA), and the most widely accepted theory, malincorporation, in which the pulmonary vein fails to incorporate into the LA,
+forming a septumlike appendage that divides the LA into two chambers. Classically, the true atrial chamber is in contact with the atrioventricular valve and contains the atrial appendage as well as the true atrial septum that bears the fossa ovalis, while the accessory portion of the atrium receives venous blood. The membrane that separates the affected atrium into two parts has been documented to be of various sizes and shapes and may be with or without any openings.
+
+This determines the severity of symptoms and complications of the disease. The Loeffler classification divides CTS into three groups:
+group 1, absence of connection between the superior and inferior (true atrium) chambers; group 2, one or a few small openings in the intra-atrial membrane; and group 3, wide opening between superior and inferior chamber.
+
+Although the first two groups cause severe symptoms in childhood and result in high mortality, group 3 usually presents in adulthood, as it may remain asymptomatic for years. The spectrum of symptoms largely depends on the severity of obstruction caused by the membrane. Patients are likely to be asymptomatic when the aperture or fenestration is larger than 1 cm.2 In a systematic review of literature, Rudiene_ et al.
+
+3 found only 170 reported cases of CTS in adults, with a mean age at diagnosis of 43 years. Forty-one percent of the patients had restrictive physiology and resulting complications of CTS,
+including atrial fibrillation (32.8%), congestive heart failure (44.3%),
+and pulmonary hypertension (27.1%). Pulmonary edema during labor has also been reported in young adult women.4 It is unclear why patients with Loeffler group 3 CTS who are asymptomatic for years later develop complications. It is thought that there might be electrical and mechanical cardiac remodeling of the tissue predisposing to atrial fibrillation or mitral stenosis–like physiology. Another explanation is the degenerative changes and calcification that can occur over years to create a restrictive physiology of the From the Department of Cardiology, Aultman Hospital, Canton, Ohio (M.B., H.H., A.Q., R.K., S.A.); the Department of Cardiothoracic Surgery, Cleveland Clinic Main Campus, Cleveland, Ohio (T.K.); and the Adult Congenital Heart Service, Akron Children's Hospital Heart Center, Akron, Ohio (A.S.).
+
+Keywords: Cor triatriatum sinistrum, Atrial fibrillation, Systolic heart failure, Congenital heart disease, Surgical membrane resection Correspondence: Maliha Butt, MD, Department of Cardiology, Aultman Hospital, 2600 6th Street SW, Canton, OH, 44710. (E-mail: *maliha.butt@aultman.com*).
+
+Published by Elsevier Inc. on behalf of the American Society of Echocardiography. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 2468-6441 https://doi.org/10.1016/j.case.2023.12.019 221 VIDEO HIGHLIGHTS
+Video 1: Two-dimensional TEE, midesophageal view, without
+(left) and with (right) color flow Doppler, demonstrates flow acceleration across the opening in the cor triatriatum membrane.
+
+Video 2: Three-dimensional TEE, midesophageal view, demonstrates the CTS membrane.
+
+View thevideo contentonlineatwww.cvcasejournal.com.
+
+membrane. However, this theory was disputed by a retrospective study of patients with CTS who were followed for 6 years and showed no significant increase in membrane gradient in patients who were managed conservatively.5 Regrettably, there are no available studies following patients with CTS for a longer duration to confirm the findings of Fuchs et al.
+
+5 Presenting symptoms of complicated CTS may include syncope, neurologic deficits, chest pain, and shortness of breath. Subtle cardiac auscultatory findings suggestive of CTS may be present on physical examination. Cardiac murmur is typically diastolic with a loud P2 when pulmonary hypertension is present. The absence of an opening snap or a loud S1 marks the difference between CTS and mitral stenosis.
+
+Signs of right heart failure, pulmonary hypertension, and pulmonary congestion may also be present.
+
+Diagnostic evaluation begins with TTE. On the four-chamber view, the membrane of CTS is seen as a thin linear structure bisecting the atria distally to the mitral valve and the LA appendage. In the long-axis view, the superior part of the membrane is usually parallel to the aortic wall, while its inferior part connects to the LA posterior wall. One or more foramina may be seen within the membrane, with an increased velocity on color flow Doppler. TTE is also useful in identifying other associated congenital cardiac anomalies, such as atrial septal defects, ventricular septal defects, and tricuspid or mitral stenosis. Spectral Doppler interrogation is used to measure the mean and maximal transmembrane pressure gradients and maximal velocity as well as the pulmonary artery pressure. It has been reported that severe obstruction is indicated by a maximum Doppler velocity >2 m/sec. Although three-dimensional TTE may be useful to delineate the size and anatomy of the orifice, TEE is often required for this purpose. TEE also helps determine the relation of the membrane to other cardiac structures, especially to differentiate CTS from supravalvular ring, the latter being located below the LA appendage. Three-dimensional matrix-array TEE can also provide detailed visualization of the orifice or fenestrations of CTS. A retrospective analysis showed a 50% higher detection rate of CTS on preoperative computed tomographic imaging compared with echocardiography. In 80% of patients, complex cardiac anomalies were either missed or misdiagnosed on echocardiography.6 Thus, echocardiography alone may not be sufficient to describe the anatomic presentation of the defect and an integrative diagnosis may be helpful. Cardiovascular magnetic resonance (CMR) can be helpful in the assessment of hemodynamics, valve disease, and chamber size and function. Sakamoto et al.
+
+7 described two cases of CTS in adults in which the use of CMR provided additional anatomic information that was not evident on TTE, particularly regarding the location of atrial communication and anatomy of the pulmonary veins.
+
+Figure 1 Two-dimensional TTE, apical four-chamber view, systolic phase performed 8 years before the current presentation,
+
+![1_image_0.png](1_image_0.png)
+
+demonstrates the cor triatriatum membrane (CTM) dividing the left atrium (LA) into true and accessory chambers. LV, Left ventricle; MV, mitral valve; RA, right atrium; RV, right ventricle.
+
+According to the updated 2018 American Heart Association and American College of Cardiology guidelines for the management of adult congenital heart diseases, adults presenting with CTS should be evaluated for other congenital abnormalities, particularly atrial septal defect, ventricular septal defect, and anomalous pulmonary venous connection (Class 1). In adults with previous repair of CTS and recurrent symptoms, they recommend evaluating for pulmonary vein stenosis (Class 2a).8 The management of CTS requires a multidisciplinary approach.
+
+Early involvement of the congenital heart disease team, electrophysiologists, and the cardiac surgery team will help accurately diagnose and understand the extent of the condition, tailor treatment plan on the basis of age, overall health, and severity of disease, as well as provide ongoing follow-up and monitoring for complications.
+
+Cardiopulmonary exercise testing can be useful for baseline functional assessment and serial testing in adult congenital heart diseases
+(2018 American Heart Association/American College of Cardiology Class 2a recommendation), but there is scarce evidence of its use in patients with CTS.9 Exercise stress echocardiography can also be useful to determine the functional severity of the membrane.10 An incidental echocardiographic finding of LA extra membrane with no pressure gradient in an asymptomatic patient requires monitoring only. When exertional dyspnea and signs of pulmonary congestion occur, diuretics and preload reduction are the mainstays of medical therapy.
+
+Atrial arrhythmias should be adequately treated with ventricular rate control and appropriate anticoagulation, as they may quickly lead to cardiac decompensation. As with other congenital heart diseases, it is not uncommon for patients with CTS to have other complex interatrial arrhythmias aside from atrial fibrillation.11 When performing catheter ablation for atrial fibrillation, additional electrophysiologic testing (e.g., rapid atrial pacing) should be considered to assess for atrial flutters and atrial tachycardias. Successful management of atrial fibrillation in patients with CTS with catheter ablation is reported, but long-term outcomes have not been reported.12 However, catheter ablation can be challenging in these patients with an altered LA anatomy. First, when performing transseptal access to the LA, there needs to be specific focus on entering the ''accessory atrium'' receiving blood from the pulmonary veins. As CTS can
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+involve the interatrial septum, there may be less interatrial septum available for transeptal access. Second, once access into the ''accessory atrium'' is achieved, catheter manipulation may be constrained because there is a smaller than usual space in which to perform catheter ablation of the pulmonary veins.13 In our patient, the CTS membrane was noted to originate from the posterolateral ridge but was mostly out of the way.
+
+According to the 2018 American Heart Association and American College of Cardiology guidelines, surgical repair is indicated for adults with CTS for symptoms attributable to the obstruction or a substantial gradient across the membrane $8 mm Hg (mean, 17 mm Hg; range, 8-40 mm Hg; Class 1).8 Complete resection of the membrane and closure of the atrial septum with a pericardial patch is a common approach. Associated congenital defects need to be corrected at the same time. Surgical outcomes are usually favorable at well-experienced centers, with nearly all patients becoming asymptomatic at follow-up and an overall reported survival 10 years of 83%.14 Percutaneous strategies have also been explored as a treatment modality. There are no guidelines for selecting patients for percutaneous treatment. However, Li et al.
+
+15 suggested that balloon
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+angioplasty can be considered as a bridge to surgery or as definitive therapy in patients with acute heart failure who are unfit for surgery at the time or symptomatic pregnant patients who have compatible anatomic variants of CTS. They concluded that anatomic characteristics, determined using advanced imaging such as CMR, should be considered to predict the technical success of catheter-based interventional strategies and that percutaneous strategies can be recommended only for isolated forms of CTS in which all pulmonary veins ultimately drain into the LA. Given that the long-term efficacy of percutaneous strategies remains to be confirmed, surgery is still the mainstay of treatment. CONCLUSION
+CTS is an important differential diagnosis in young patients with systolic heart failure, even if they have multiple comorbidities. Early multidisciplinary involvement is important for timely diagnosis and intervention.
+
+ETHICS STATEMENT The authors declare that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.
+
+CONSENT STATEMENT
+The authors declare that since this was a non-interventional, retrospective, observational study utilizing de-identified data, informed consent was not required from the patient under an IRB exemption status.
+
+FUNDING STATEMENT The authors declare that this report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
+
+DISCLOSURE STATEMENT
+The authors report no conflict of interest. ACKNOWLEDGMENT We thank sonographers Sylvia V. McAninch and Nicole Miller for their assistance in the acquisition of images.
+
+## Supplementary Data
+
+Supplementary data related to this article can be found at https://doi. org/10.1016/j.case.2023.12.019.
+
+REFERENCES
+1. Al Sabri AR, Joshi N, Al Kindi H, Al Senaidi KS. Cor Triatriatum Sinistrum: a rare disease with a common presentation. Sultan Qaboos Univ Med J 2021;21:e324-6.
+
+2. Nassar PN, Hamdan RH. Cor triatriatum sinistrum: classification and imaging modalities. Eur J Cardiovasc Med 2011;1:84-7.
+
+3. Rudiene V, Hjortshøj CMS, Glaveckait _ e S, Zakarkait _ e D, Petrulionien _ e_ Z, -
+Gumbiene L, et al. Cor triatriatum sinistrum diagnosed in the adulthood: a _ systematic review. Heart 2019;105:1197-202.
+
+4. Davlouros PA, Koutsogiannis N, Karatza A, Alexopoulos D. An unusual case of cor triatriatum sinister presenting as pulmonary edema during labor. Int J Cardiol 2011;150:e92-3.
+
+5. Fuchs M, Connolly H, Said S, Egbe A. Outcomes in patients with cor triatriatum sinister. Congenit Heart Dis 2018;13:628-32. 6. Goel A, Viswamitra S, Reddy BN, Gaduputi J. Computed tomography features of cor triatriatum: an institutional review. Br J Radiol 2021; 94:20201252.
+
+7. Sakamoto I, Matsunaga N, Hayashi K, Ogawa Y, Fukui J. Cine-magnetic resonance imaging of cor triatriatum. Chest 1994;106:1586-9.
+
+8. Stout KK, Daniels CJ, Aboulhosn JA, Bozkurt B, Broberg CS, Colman JM,
+et al. 2018 AHA/ACC guideline for the management of adults with congenital heart disease: a report of the American College of Cardiology/American Heart Association task force on clinical practice guidelines
+[published correction appears in J Am Coll Cardiol. 2019 may 14;73(18):2361-2362]. J Am Coll Cardiol 2019;73:e81-192.
+
+9. Tuan SH, Chen GB, Chen CH, Liou I, Sun SF, Wu SY, et al. Serial cardiopulmonary exercise testing in an asymptomatic young female receiving percutaneous balloon dilatation for cor triatriatum sinistrum at an early age. J Formos Med Assoc 2021;120:1143-7.
+
+10. Cotrim CA, Cafe H, Jo~ao I, Cotrim N, Guardado J, Cordeiro P, et al. Exercise stress echocardiography: where are we now? World J Cardiol 2022;14:
+64-82.
+
+11. Okada A, Kato K, Shoda M, Tabata H, Yoshie K, Kuwahara K. Successful catheter ablation of atrial tachycardia in cor triatriatum sinister: a figureof-8 reentry in the left atrial membrane. HeartRhythm Case Rep 2020; 7:109-11.
+
+12. Minciuna IA, Cismaru G, Puiu M, Roșu R, Amet D, Anghelina D, et al.
+
+Atrial fibrillation ablation in a patient with cor triatriatum sinister and left common pulmonary vein: impact of left atrium anatomy on ablation approach. Life (Basel) 2022;12:992.
+
+13. Borne RT, Gonzalez J, Khanna A, Sauer WH, Thai Nguyen D. Getting to the right left atrium: catheter ablation of atrial fibrillation and mitral annular flutter in cor triatriatum. HeartRhythm Case Rep 2016;2:502-5.
+
+14. Saxena P, Burkhart HM, Schaff HV, Daly R, Joyce LD, Dearani JA. Surgical repair of cor triatriatum sinister: the Mayo Clinic 50-year experience. Ann Thorac Surg 2014;97:1659.
+
+15. Li WW, Koolbergen DR, Bouma BJ, Hazekamp MG, de Mol BA, de Winter RJ. Cathether-based interventional strategies for cor triatriatum in the adult - feasibility study through a hybrid approach. BMC Cardiovasc Disord 2015;15:68.

medical/md/PMC2196312.md

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+# A Novel Inhibitory Receptor (Ilt3) Expressed On
+
+## Monocytes, Macrophages, And Dendritic Cells Involved In Antigen Processing
+
+By Marina Cella,* Christian Döhring,* Jacqueline Samaridis,* Mark Dessing,* Manfred Brockhaus,‡ Antonio Lanzavecchia,* and Marco Colonna* From the *Basel Institute for Immunology, CH-4005 Basel, Switzerland; and ‡*Hoffmann-La Roche* AG, CH-4002 Basel, Switzerland
+
+## Summary
+
+Immunoglobulin-like transcript (ILT) 3 is a novel cell surface molecule of the immunoglobulin superfamily, which is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells. The cytoplasmic region of ILT3 contains putative immunoreceptor tyrosine-based inhibitory motifs that suggest an inhibitory function of ILT3. Indeed, co-ligation of ILT3 to stimulatory receptors expressed by APCs results in a dramatic blunting of the increased [Ca21]i and tyrosine phosphorylation triggered by these receptors. Signal extinction involves SH2-containing protein tyrosine phosphatase 1, which is recruited by ILT3 upon cross-linking. ILT3 can also function in antigen capture and presentation.
+
+It is efficiently internalized upon cross-linking, and delivers its ligand to an intracellular compartment where it is processed and presented to T cells. Thus, ILT3 is a novel inhibitory receptor that can negatively regulate activation of APCs and can be used by APCs for antigen uptake.
+
+Engagement of B cell antigen receptor, TCR, and several Fc receptor (FcR) complexes by cognate ligands results in tyrosine phosphorylation of cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs)1 (1–6).
+
+Phosphorylated ITAMs recruit and activate protein tyrosine kinases, which trigger a cascade of intracellular phosphorylation events leading to cellular activation. Immune cell responses are negatively regulated when stimulatory receptors are co-ligated to inhibitory receptors. These include the low affinity FcR for IgG, FcgRIIB, and CD22 in B
+cells (7–10), the Ly-49 molecules, the killer cell inhibitory receptors (KIRs), the NKG2A/CD94 heterodimer in NK cells (11–13), and glycoprotein (gp)49B1 in mast cells and NK cells (14–16). All of these receptors are characterized by the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs; 17). Upon receptor engagement, cytoplasmic ITIMs are tyrosine phosphorylated, and subsequently bind the SH2 domain(s) of SHP-1 protein tyrosine phosphatase (18–23) and/or SHIP inositol phosphatase (24), which mediate downregulation of cell activation.
+
+Recently, we have described two new members of the immunoglobulin superfamily (Ig-SF), called immunoglobulin-like transcript 1 (ILT1) and ILT2 (25). These are homologous to bovine Fcg2 receptor (Fcg2R; 26), murine cell surface antigen gp49 (27, 14), human KIRs (28–30), human Fca receptor (FcaR; 31) and map to human chromosome 19, as do the FcaR and the KIRs. ILT1 and ILT2 are characterized by similar extracellular regions consisting of four Ig-SF domains. Their transmembrane and cytoplasmic domains, however, differ substantially. ILT1 has a charged residue of arginine within the transmembrane region, followed by a short cytoplasmic tail with no ITAMs or ITIMs. Similar characteristics are found in human FcaRI and bovine Fcg2R, which trigger cell responses via associated signaltransducing, ITAM-containing subunits. Conversely, ILT2 has a long cytoplasmic tail, which contains putative ITIMs similar to those known to bind SHP-1 in KIRs. ILT1 and ILT2 also display different tissue distributions. Although ILT1 is predominantly expressed by myeloid cells, ILT2 is expressed by B cell lines. Thus, ILT1 and ILT2 may mediate activation of myeloid cell responses and inhibition of B cell activation, respectively.
+
+Here we characterize a third member of the ILT family, called ILT3, which, similarly to ILT2, displays a long cytoplasmic tail containing putative ITIMs. By using an antiILT3 mAb, we have found that ILT3 is selectively expressed on myeloid APCs. Functional studies show that ILT3 behaves as an inhibitory receptor when cross-linked to a stimulatory receptor. A cytoplasmic component of the ILT3-mediated negative signaling pathway is the SH2containing phosphatase SHP-1, which becomes associated with ILT3 upon cross-linking. Furthermore, ILT3 is internalized and ILT3 ligands are efficiently presented to specific T cells, suggesting that ILT3 may be involved in antigen uptake and presentation.
+
+## Materials And Methods
+
+Cells. THP1 is a human myeloid cell line. C1R and 721.221 are MHC class I–deficient, EBV-transformed human B cell lines. Jurkat is a human T cell line. All of these cells were grown in RPMI, 10% FCS. Monocytes were prepared from PBMCs by adhesion to plastic. In brief, PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient, washed with RPMI,
+resuspended at 5 3 106/ml in RPMI, 10% FCS and cultured in six-well plates for 1 h at 378C. Nonadherent cells were removed by washing twice with RPMI. Adherent cells were cultured overnight in RPMI, 10% FCS supplemented with 50 ng/ml GM-CSF and 1,000 U/ml IL-4. Dendritic cells (DCs) were obtained from monocytes as previously described (32). Macrophages were obtained by culturing monocytes for 10 d in six-well plates at a concentration of 106 cells/ml in RPMI, 20% human serum supplemented with 1 ng/ml macrophage CSF (M-CSF) (33, 34).
+
+cDNA Synthesis, Oligonucleotide Primers, PCR, and Cloning.
+
+The 39 end of ILT3 was cloned by rapid amplification of cDNA 39 end (35). In brief, RNA was prepared from 721.221 and C1R
+cells as described (36) and single-strand cDNA was synthesized by reverse transcription of 1 mg of total RNA using Moloney murine leukemia virus reverse transcriptase and a (dT)17 adaptor in a reaction volume of 20 ml. cDNA was amplified by two nested PCRs. The 59 oligonucleotides were CCCCCAGCGACCCCCTGGA (nucleotide 926–944 of ILT2) and ACGGTGGCCTCAGGAGAGA (nucleotide 1003–1021 of ILT2) (25). In both PCRs, the 39 primer corresponded to the adaptor primer.
+
+PCRs (50 ml volume) contained 1 ml of total cDNA sample, PCR primers (0.5 mM each), 29-deoxynucleoside 59-triphosphates (125 mM each), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM
+MgCl2, 0.01% gelatin, and 0.25 units of Taq polymerase. PCR
+was carried out for 35 cycles, each consisting of 1-min steps at 94, 55, and 728C. Amplified products were gel purified in 1.2% agarose gel, cloned into pCRII (Invitrogen, San Diego, CA), and sequenced. The 59 end of ILT3 was cloned by two consecutive cycles of 59 rapid amplification of cDNA ends (35).
+
+Production of ILT3 Human IgG1 Fusion Protein. To produce ILT3 as a soluble fusion protein, we constructed a chimeric gene consisting of ILT3 extracellular domains and human IgG1 constant regions. The cDNA fragment encoding the ILT3 extracellular region was amplified by PCR from cloned plasmid DNA. The 59 primer (GATC*GAATTC*ATGATCCCCACCTTCACGGCT) contained an EcoRI restriction site (*italic*) and the ILT3 start codon. The 39 primer (CTGATC*AAGCTT*ATACTTACCCTCCCAGTGCCTTCTCAGACC) provided a HindIII restriction site (*italic*), a splice donor sequence, and seven ILT3 codons preceding the transmembrane domain. The z800-bp PCR product was cut with EcoRI and HindIII, and ligated into an expression vector containing the exons for hinge, CH2 and CH3 region of human IgG1, the guanosine phosphotransferase gene conferring resistance to mycophenolic acid, and the k promoter for the expression in mouse myeloma (37). Transfections, selection of secreting transfectants, and purification of fusion protein from culture supernatants were performed as described (38).
+
+Production of anti-ILT3 mAbs. 10-wk-old, female BALB/c mice
+(Iffa-Credo, L'Arbresle, France) received an initial injection of 100 mg of ILT3 human IgG1 fusion protein (ILT3-HuIgG1),
+mixed 1:1 (vol/vol) with Alu-Gel-S (Serva Biochemicals, Paramus, NJ), behind the neck. 4 wk later, they were given a booster immunization with the same immunogen, followed after 2 wk by a final injection of 100 mg of purified ILT3-HuIgG1. 3 d later, mice were killed and draining lymph node cells were isolated and fused with the myeloma fusion partner, Ag8.653, using polyethylene glycol 4000. Hybridoma supernatants were screened in two steps. First, an ELISA was performed using ILT3-HuIgG1 in the coating step and human-adsorbed alkaline phosphatase–labeled goat anti–mouse IgG as secondary antibody. Supernatants from clones that were positive in ELISA were then tested by FACS
+analysis for staining C1R cells by flow cytometry.
+
+Transfections. ILT1, ILT2, and ILT3 cDNAs were subcloned into pCDNA3 (Invitrogen) and transfected into COS7 cells by lipofectin (GIBCO BRL, Gaithersburg, MD). Cell surface expression of ILTs was assessed 48 h after transfection by FACS analysis. A stable transfectant of ILT3 in Jurkat T cells was obtained as previously described (28).
+
+Antibodies and FACS *Staining.* Before staining, all the cells were preincubated with PBS, 20% human serum for 30 min on ice, to block Fc receptors. In two- and three-color staining experiments, cells were stained with the following primary mAbs: OKT3
+(anti-CD3, IgG2a; American Type Culture Collection, Rockville, MD), CD16 (IgG2a; Milan Analytica AG, La Roche, Switzerland), 3C10 (anti-CD14, IgG2b; American Type Culture Collection), 1F5.4 (anti-CD20, IgG2a; American Type Culture Collection), M-T102 (anti-CD1a, IgG2b; PharMingen, San Diego, CA), L243 (anti-HLA-DR, IgG2a; American Type Culture Collection), and HB15a (anti-CD83, IgG2b; provided by Dr. T. Tedder, Duke University, Durham, NC). As secondary antibodies, we used human-adsorbed FITC- or PE-conjugated goat anti–mouse IgG1, IgG2a, and IgG2b (Southern Biotechnology Assoc., Inc., Birmingham, AL). In three-color stainings, biotin-labeled goat anti–mouse IgG2a was followed by streptavidin-allophycocyanin (SBA). Stained cells were analyzed by flow cytometry on a FACStar Plus (Becton Dickinson, Mountain View, CA) using the LYSYS II software.
+
+Immunoprecipitations. Cells were surface labeled with 1 mCi of Na125I using the sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate method (39). For metabolic labeling with [32P]orthophosphate, cells were incubated in phosphate-free DMEM (Sigma Chemical Co., St. Louis, MO) containing 10% Tris-buffered saline–dialyzed FCS for 1 h. [32P]orthophosphate (Amersham Corp., Arlington Heights, IL) was then added at 0.5 mCi/ml and incubation was continued for 5 h. After surface or metabolic labeling, cells were lysed in 1% Triton X-100, 100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. After overnight preclearing with protein G–sepharose, lysates were incubated with culture supernatants of either ZM3.8 (anti-ILT3, IgG1) or control IgG1 mAbs
+(5.133, anti-NKAT4 [40] and 1B7.11, anti-2,4,6 TNP, American Type Culture Collection) at 148C for 4 h, and immune complexes were precipitated by addition of protein G–Sepharose for 1 h at 148C. Precipitates were washed 3 times with lysis buffer, followed by a final wash with 10 mM Tris-HCl, pH 7.4, and resuspended in nonreducing or reducing sample buffer. SDS-PAGE
+analysis was performed according to a standard procedure. After the run, gels were dried and exposed to autoradiography film
+(Amersham Corp.) for 2–5 d.
+
+Measurement of Cytosolic Calcium in Macrophages and Monocytes. Monocytes and macrophages were loaded with Indo-1 AM (Sigma Chemical Co.) as described (41). In stimulation experiments, anti-CD11b mAb (44aabc; American Type Culture Collection) and anti-CD16 mAb (B73.1; American Type Culture Collection) were added to monocytes and macrophages, respectively, followed by a F(ab9)2 goat anti–mouse IgG (Milan Analytica)
+as cross-linker. Cells were then analyzed on a flow cytofluorimeter (FACS Vantage or Coulter Elite Enhanced Sort Performance
+[Coulter Corp., Hialeah, FL]) to detect Ca21 fluxes. Stimulation of monocytes with the anti-HLA-DR mAb 3.8B1 (Cella, M., and A. Lanzavecchia unpublished data) was performed without a cross-linker. In inhibition experiments, cells were preincubated for 10 min at room temperature either with ZM3.8 mAb, or with the anti–MHC class I W6/32 mAb. After washing with PBS, either anti-CD11b, anti-HLA-DR, or anti-CD16 mAbs were added followed by the cross-linking antibody, and cells were analyzed by flow cytometry to detect Ca21 fluxes. Only live (based on forward scatter criteria) and Indo-1–loaded cells (based on 405 nM
+versus 525 nM emission spectra) were included in the analysis.
+
+Immunoblotting. For antiphosphotyrosine blots, monocytes (2 3 106) were incubated for 2 min at 378C with medium, ZM3.8 mAb, 3.8B1 mAb, or with both mAbs in the absence or in the presence of a secondary cross-linker. Cells were washed in icecold PBS and lysed as previously described (20). Cell lysates were boiled, separated on SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase (HRP)-coupled 4G10
+(500 ng/ml; Upstate Biotechnology Inc., Lake Placid, NY). Immunoblotted proteins were visualized by chemiluminescence using the enhanced chemiluminescence detection reagents (Amersham Corp.). For anti-SHP blots, 107 monocytes were plated on six-well tissue culture plates previously coated with purified ZM3.8 or 5.133 mAbs at 50 mg/ml, centrifuged for 2 min, shifted to 378C for 2 min, collected in cold PBS, 0.5 mM EDTA,
+and washed twice. Identical number of cells stimulated with ZM3.8 or 5.133 mAbs were lysed and immunoprecipitated with ZM3.8 and protein G as described above. Precipitated samples were boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose (Hybond-C extra; Amersham Corp.). Membranes were blocked with PBS, 3% nonfat, dry milk and probed anti-SHP-1 rabbit antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) followed by HRP-labeled goat anti–rabbit Ig (SBA). Immunoblotted proteins were visualized by chemiluminescence. Membranes were stripped of antibody by incubating for 30 min at 508C in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM TrisHCL, pH 6.7, according to the protocol supplied by Amersham Corp. Stripped blots were reprobed after blocking with antiSHP-2 rabbit antiserum (Santa Cruz Biotechnology).
+
+Internalization Assay. Monocytes were cultured for 24–48 h in culture medium containing GM-CSF and IL-4, as described above. One aliquot of monocytes was fixed for 1 min at room temperature in RPMI, 0.05% glutaraldehyde. Another aliquot was used without fixation. Both samples were stained with ZM3.8 mAb for 40 min on ice, washed twice with ice-cold PBS,
+1% FCS, and subsequently stained with biotin-labeled F(ab9)2 goat anti–mouse IgG for 1 h on ice. After washing with ice-cold PBS, 1% FCS, cells were incubated at 378C in prewarmed RPMI, 10% FCS for various times to allow internalization. Cells were then cooled on ice, washed once with PBS, 0.1% NaN3, stained with PE-conjugated streptavidin for 30 min on ice, washed, and analyzed by FACS. As a measure of internalization in nonfixed cells, we used the percental decrease of the median fluorescence intensity (MFI) as compared to control samples kept at 48C. The percentage decrease of MFI observed in fixed cells was taken as a measure of the off-rate of the antibody at 378C.
+
+Antigen Presentation Assay. 4 3 104 irradiated monocytes (3,000 rad) were co-cultured with 8 3 104 cells of the B13 T cell clone
+(42) in 96-well flat-bottom microplates in the presence of serial dilutions of IgG1 mAbs. mAbs used in the assay were the following: ZM3.8 (anti-ILT3, IgG1) or 5.133 (anti-NKAT4 KIR,
+IgG1), 1B7.11 (anti-2,4,6 TNP, IgG1; American Type Culture Collection), and 32.2 (anti-FcgRI, IgG1; American Type Culture Collection). After 48 h, the cultures were pulsed with [3H]thymidine (1 mCi/well, specific activity 5 Ci/mmol), and the radioactivity incorporated was measured after additional 16 h. The data were plotted against the concentration of mAbs determined by ELISA using a purified mouse IgG1 (anti-CD68, Dako Corp.,
+Carpinteria, CA) as a standard.
+
+Figure 1. Amino acid sequence of human ILT3, aligned with ILT1 and ILT2. Since ILT1 and ILT2 consist of four Ig-SF
+domains, only the two domains with higher homology to ILT3 were included in the alignment.
+
+The alignment was generated by the Clustal method. Gaps (*dashes*)
+were introduced to maximize homologies. Amino acids identical to the consensus are indicated by dots. Conserved cysteines involved in disulfide bonds in the extracellular domains are identified by asterisks. Cytoplasmic tyrosine-based motifs potentially involved in signal transduction and/or endocytosis are boxed.
+
+ILT3 has no N-linked glycosylation sites (N-X-S/T). Amino acid residues are numbered on the right side, beginning with the first residue of each of the predicted domains. ss, signal sequence; *Ig1–4*, Ig-SF extracellular domains; cp, connecting peptide; tm, transmembrane domain; cy, cytoplasmic domain.
+
+ILT3 cDNA sequence is available from EMBL/GenBank/DDBJ under accession number U82979.
+
+## Results
+
+![3_Image_0.Png](3_Image_0.Png)
+
+ILT3 Is a Member of the ILT Multigene Family Located on Human Chromosome 19. During the process of cloning the 39 end of ILT2 cDNA from EBV-B cell lines (25), we obtained an additional cDNA sequence that differed from ILT2 by several amino acid residues. Amplification of the 59 end of this fragment yielded a new cDNA, called ILT3, which contains a single open reading frame encoding a transmembrane protein of 447 amino acids with a predicted molecular mass of z47 kD (Fig. 1). The amino acid sequence begins with a hydrophobic signal peptide of 23 amino acids followed by an extracellular region composed of two C2 type Ig-SF domains (43). Each domain shows two characteristic cysteines that are 49 and 50 residues apart, flanked by conserved residues (Val-x-Leu/Ile-x-Cys and His/Tyr-x-Gly-x-Tyr-x-Cys-Tyr/Phe, respectively, where x is any amino acid). The putative transmembrane domain of ILT3 consists of 21 amino acids, followed by a long cytoplasmic region of 167 amino acids, which is characterized by the presence of one Tyr-x-x-Val motif followed by two Tyr-x-x-Leu motifs spaced by 26 amino acid residues. These Tyr-x-x-Leu pairs and their spacing are reminiscent of the Tyr-x-x-Leu motifs identified in KIRs as binding sites for protein tyrosine phosphatase SHP-1
+(19–23).
+
+Comparison of the predicted amino acid sequence of ILT3 with Ig-SF protein sequences, revealed that ILT3 is closely related to ILTs (64% similarity with ILT2), bovine Fcg2R (45%), murine cell surface antigen gp49 isoforms
+(39–43%), and, to a lesser extent, to KIRs (23–32%) and human FcaR (28%). Genomic DNA analysis of human–
+hamster hybrid cell lines, each with a different partial complement of human chromosomes, showed hybridizing bands only in samples containing human chromosome 19 (data not shown). Thus, ILT3 belongs to the ILT family which maps to human chromosome 19, as do the FcaR
+and the KIR genes. Expression of ILT3 by RNA blot analysis revealed a major transcript of z1.6 kb (data not shown). Interestingly, this transcript was weakly expressed in the B cell lines C1R or 721.221, from which ILT3 was initially cloned, whereas it was predominantly expressed in monocytes purified from PBMCs and in the myeloid cell line THP1. No transcripts were detected in the T cell line Jurkat.
+
+ILT3 Is Expressed on Monocytes, Macrophages, and Dendritic Cells. To characterize the ILT3-encoded cell surface mol-
+
+![4_image_0.png](4_image_0.png)
+
+ecule, we produced anti-ILT3 mAbs using a soluble ILT3-
+HuIgG1 fusion protein as an immunogen. The ZM3.8 mAb specifically recognized ILT3 on transiently transfected COS cells (data not shown). In PBMCs, ZM3.8 mAb stained only CD141 monocytes, whereas no staining was observed in CD31 T cells, CD161 NK cells, or CD201 B cells (Fig. 2 A). Interestingly, in monocyte-enriched populations derived from peripheral blood, ZM3.8 also stained CD831 cells (Fig. 2 A), as well as CD14-/HLA-DRhigh cells (Fig. 2 B). Both of these cell populations correspond to circulating primary DCs (44–46). In addition, ILT3 was expressed by both DCs and macrophages derived from monocytes cultured with GM-CSF plus IL-4 or M-CSF, respectively
+(Fig. 2, C and D; 32–34).
+
+We also tested ILT3 expression in several cell lines.
+
+Among four myelo-monocytic cell lines (U937, Monomac6, THP1, and HL-60), only THP1 reacted with ZM3.8. All tested EBV-B cell lines (n 5 10) were negative, with the exception of C1R. No cell surface expression was detected either on purified fresh B cells or on B cells activated by CD40L
+in the presence of IL-4, IL-2, and IL-10 (data not shown). Finally, ZM3.8 did not stain neutrophils (data not shown). Thus, ILT3 is selectively expressed on myeloid APCs.
+
+To define the biochemical characteristics of ILT3, we carried out immunoprecipitations from ILT3-transfected Jurkat T cells and monocytes (Fig. 3). A prominent band of z55 kD in ILT3-transfected T cells was detected under nonreducing and reducing conditions, while a z58–60 kD
+band was detected in monocytes (Fig. 3 A) and in THP1
+(data not shown). N- and O-deglycosylation of immunoprecipitates did not produce any change in the size of the bands. After labeling cells with [32P]orthophosphate, ILT3 was immunoprecipitated as a constitutively phosphorylated molecule (Fig. 3 B). The discrepancy in apparent molecular mass of ILT3 between ILT3-transfected T cells and monocytes may be due to a different degree of phosphorylation in distinct cell types.
+
+Intracellular Ca21 *Mobilization Responses Triggered by* CD11b, MHC Class II, and CD16 in APCs Are Negatively Regulated by Co-ligation with ILT3. The presence of putative ITIMs suggested an inhibitory role of ILT3 on APC
+activation. To test this hypothesis, we triggered monocytes and macrophages with mAbs that have been shown to induce Ca21 mobilization, such as anti-CD11b (47, 48), antiHLA-DR (49–51), and anti-FcgRIII (CD16; 52) mAbs, and tested whether recruitment of ILT3 to the triggering receptors inhibits Ca21 mobilization. As shown in Fig. 4 A,
+ligation of surface CD11b with a specific mAb followed by a secondary cross-linking antibody elicited a rapid and transient rise in intracellular calcium concentration ([Ca21]i).
+
+On the contrary, preincubation of monocytes with ZM3.8 mAb, followed by addition of anti-CD11b and co-crosslinking of CD11b and ILT3 by a secondary antibody, resulted in complete inhibition of the [Ca21]i increase (Fig. 4 D). In control experiments, the [Ca21]i increase induced by CD11b was not affected by co-ligation of CD11b with MHC class I. Also, cross-linking of ILT3 alone did not evoke Ca21 mobilization, indicating that ILT3 is not a stimulatory receptor (data not shown).
+
+It has been reported that some anti–MHC class II mAbs can trigger functional responses in B lymphocytes (49–51). We have recently developed an anti-HLA-DR mAb, called 3.8B1, which triggers a rapid rise in [Ca21]i, even in the absence of a secondary cross-linking antibody (Fig. 4 B). Co-ligation of ILT3 and HLA-DR resulted in significant inhibition of [Ca21]i increase (Fig. 4 E). Interestingly, HLA-DR–mediated stimulation of monocytes was not affected by preincubation of monocytes with anti-ILT3 anti-
+
+![4_image_1.png](4_image_1.png)
+
+Figure 5. (A) Protein tyrosine
+
+![5_image_0.png](5_image_0.png)
+
+phosphorylation stimulated by treatment of monocytes with the anti-HLA-DR mAb 3.8B1 is inhibited by co-ligation of HLADR with ILT3. Monocytes were incubated with medium alone
+(lane 1), with ZM3.8 mAb (antiILT3; lane 2), with 3.8B1 mAb
+(anti-HLA-DR; lane 3) or with both mAbs in the absence (lane 4) or in the presence (lane 5) of a secondary cross-linking antibody. Cell lysates were separated on SDS-PAGE, transferred to nitrocellulose, and probed with HRP-coupled antiphosphotyrosine mAb 4G10.
+
+(B) Association of SHP-1 with ILT3 is increased upon ILT3 cross-linking. Monocytes were stimulated with ZM3.8 (lane 2)
+or with a control IgG (5.133 mAb) (lane 1) coated on plastic plates. Cells were kept at 378C for 2 min, harvested, and lysed. ILT3 was immunoprecipitated with ZM3.8. Proteins were separated on SDSPAGE, transferred to nitrocellulose, and probed with anti-SHP-1, followed by HRP-conjugated goat anti–rabbit Ig. The migration of SHP-1 is marked by the arrow.
+body in the absence of the secondary cross-linking antibody, indicating that recruitment of ILT3 to the stimulatory receptor is necessary to block the [Ca21]i increase (Fig. 4 G).
+
+The low affinity receptor for IgG, FcgRIII, is expressed on NK cells and macrophages and has been shown to trigger Ca21 mobilization in NK cells (52, 53). Similarly, we showed that cross-linking of FcgRIII in macrophages evokes a strong increase of [Ca21]i (Fig. 4 C). Despite the low expression of ILT3 on macrophages (see Fig. 2 D), co-ligation of ILT3 and FcgRIII inhibited the [Ca21]i increase, especially at early time points (Fig. 4 F). Together, these results demonstrate that co-ligation of ILT3 with a triggering receptor results in signal extinction.
+
+SHP-1 Is Recruited to ILT3 upon Cross-linking. To test whether inhibition of Ca21 mobilization responses was paralleled by inhibition of tyrosine phosphorylation, we performed antiphosphotyrosine immunoblotting on whole cell lysates of monocytes stimulated via HLA-DR in the absence or in the presence of co-ligation with ILT3. As shown in Fig. 5 A, monocytes triggered via HLA-DR displayed a substantial increase of tyrosine phosphorylation (lane 3), as compared to monocytes incubated with medium alone (lane 1)
+or with anti-ILT3 mAb (lane 2). Co-ligation of ILT3 with HLA-DR resulted in a dramatic reduction of the intensity and number of tyrosine phosphorylated species (lane 5). In the absence of secondary cross-linking antibody, ILT3 did not block HLA-DR–triggered tyrosine phosphorylation increase (lane 4).
+
+Since in NK cells, KIRs downregulate calcium mobilization and tyrosine phosphorylation by recruiting SHP-1 and SHP-2 (19–23), we tested whether these phosphatases could be also involved in the negative signaling pathway Figure 6. ZM3.8 mAb is internalized upon cross-linking.
+
+![5_image_1.png](5_image_1.png)
+
+ZM3.8 bound to monocytes on ice did not disappear from the cell surface when the cells were shifted at 378C, in the absence of a cross-linking antibody (m). After cross-linking with a F(ab9)2 biotin-labeled secondary antibody, ZM3.8 was rapidly internalized, becoming undetectable by FACS analysis in 30–60 min
+(h). In fixed cells, only a minimal decrease of the median fluorescence intensity was detected over time (j). Cell surface–
+bound ZM3.8 was revealed by PE-conjugated goat anti–mouse IgG1 (m) or by PE-conjugated streptavidin (h, j). The MFI
+was determined by FACS analysis. The percentage decrease of MFI as compared to a control sample kept at 48C was used as a measure of internalization.
+
+mediated by ILT3. ILT3 was cross-linked on monocytes by ZM3.8 mAb attached to the plastic surface of tissue culture plates and subsequently immunoprecipitated from cell lysates. In control experiments, ILT3 was immunoprecipitated from unstimulated cells. Immunoblotting of immunoprecipitates with anti-SHP-1 antibodies demonstrated association of SHP-1 with ILT3 that significantly increases upon ILT3 cross-linking (Fig. 5 B). On the contrary, no association with SHP-2 was detected (data not shown).
+
+These results implicate SHP-1 as a cytosolic component of the signal extinction mediated by ILT3.
+
+Cross-linking of ILT3 on Monocytes Results in Receptor Internalization and Delivery into an Antigen-processing Compartment. Since ILT3 is selectively expressed on APCs and displays putative tyrosine-x-x-valine/leucine internalization motifs in the cytoplasmic tail (54–56), we analyzed the ability of ILT3 to endocytose and deliver its ligand to an antigen-processing and loading compartment. As shown in Fig. 6, ZM3.8 mAb bound at 08C to monocytes was not internalized when the cells were shifted to 378C. We therefore examined whether internalization occurs in the presence of a secondary cross-linker. ZM3.8 mAb was bound on ice to monocytes and cross-linked by a biotinylated F(ab9)2 secondary antibody. Cells were subsequently warmed to 378C to allow internalization. After various time points, cells were returned to ice and the amount of ZM3.8 mAb remaining on the cell surface was determined by flow cytometry using PE-conjugated streptavidin. Freshly isolated monocytes efficiently internalized bound ZM3.8 mAb, as demonstrated by a 60–80% reduction after 30 min at 378C and by a complete disappearance after 60–90 min
+(Fig. 6). In control experiments, no significant decrease of surface-bound ZM3.8 was observed using fixed monocytes, ruling out a detachment of the primary or secondary antibodies from the cell surface at 378C (Fig. 6). In addi1748 An Inhibitory Receptor Expressed in Myeloid Antigen-presenting Cells
+
+![6_image_0.png](6_image_0.png)
+
+tion, when 125I-labeled streptavidin was added as a third step reagent before incubation at 378C, no significant decrease of cell-bound radioactivity was observed over time, thus excluding shedding of ILT3 from the cell surface (data not shown).
+
+To further investigate a possible role of ILT3 in antigen capture, we evaluated the ability of monocytes to present ZM3.8 mAb to a CD41 class II–restricted T cell clone specific for a mouse IgG1 peptide epitope (42). The presentation of the ZM3.8 mAb was compared to that of IgG1 mAbs that bind to other receptors (anti-FcgRI mAb) or do not bind to surface molecules and are taken up in the fluid phase [anti-TNP and anti-NKAT4 KIR mAbs]. As shown in Fig. 7, monocytes presented ZM3.8 mAb to T cells 50–
+100-fold more efficiently than the anti-FcgRI mAb, and 400–500-fold more efficiently than anti-TNP and antiNKAT4 KIR mAbs. Together, these experiments indicate that ILT3 can efficiently deliver its ligand to an intracellular compartment where class II loading can occur.
+
+## Discussion
+
+Previous work has extensively shown that inhibitory receptors regulate cell activation in B, NK, mast cells, and subsets of T cells when co-ligated with ITAM-containing stimulatory receptor complexes. Our data extend this concept to professional APCs, showing that ILT3 negatively regulates APC functional responses triggered via stimulatory receptors, such as CD11b, CD16, and even MHC
+class II. In addition to its inhibitory function, ILT3 is involved in antigen uptake. It is noteworthy that ILT3 is expressed on DCs. These are a unique type of leukocytes whose primary function is to efficiently capture antigens, process them, and present them to naive T cells (57). Although the uptake and processing of antigen is a major function of DCs, only a few mechanisms and receptors specialized for antigen capture have been identified on DCs
+(58–60). Our data indicate that ILT3 is a unique receptor expressed in DCs, able to target the ligand into processing and peptide-loading compartments. Thus, ILT3 displays a dual function of inhibitory receptor and antigen-capturing molecule. A similar dual function has been previously shown for the low affinity receptor for IgG, FcgRIIB (7, 8).
+
+The ligand of ILT3 is still unknown. ILT3 is closely related to bovine Fcg2R and, to a lesser extent, to human FcaR, suggesting that ILT3 may be an Fc receptor. However, we did not detect any significant binding of human monomeric and heat-aggregated IgM, IgG, IgA, and IgE to ILT3-transfected Jurkat T cells (data not shown). ILT3 also shows homology to KIRs, which suggests that it may be a receptor for MHC class I molecules. We have investigated this possibility by analyzing binding of soluble ILT3-
+HuIgG1 fusion protein to MHC class I transfectants in the class I–deficient mutant 721.221. No significant binding could be detected by FACS analysis under the same experimental conditions in which KIR-HuIgG fusion proteins bind to MHC class I molecules (data not shown). We are investigating the possibility that ILT3 is a receptor for cell surface molecules related to MHC class I molecules, such as nonclassical class I molecules (CD1 [61], MR1 [62],
+MIC [63]), for MHC class II molecules, or for serum factors such as complement components. ILT3 is a member of the ILT subfamily of Ig-SF molecules, which is located on chromosome 19, most likely in close linkage with KIRs. Another member of this family, ILT2, is characterized by similar cytoplasmic tyrosine-based motifs such as ILT3, and is expressed in B cell lines, suggesting that this may be a putative inhibitory resceptor in B cells. We have also cloned additional cDNAs which encode novel molecules homologous to ILT2 and ILT3
+(ILT4 and ILT5, GenBank accession numbers AF000574 and AF000575). Thus, the number of Ig-SF inhibitory receptors with different tissue distribution and specificity is likely to increase.
+
+Received for publication 30 January 1997.
+
+## References
+
+1. Cambier, J.C., C.M. Pleiman, and M.R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. *Annu. Rev. Immunol.* 12:457–486.
+
+2. Weiss, A., and D.R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. *Cell.* 76:263–274.
+
+3. Letourneur, F., and R.D. Klausner. 1991. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor z family proteins. *Proc. Natl. Acad. Sci. USA.* 88:8905–8909.
+
+4. Letourneur, O., I.C. Kennedy, A.T. Brini, J.R. Ortaldo, J.J.
+
+O'Shea, and J.P. Kinet. 1991. Characterization of the family of dimers associated with Fc receptors (Fce RI and Fcg RIII).
+
+J. Immunol. 147:2652–2656.
+
+5. Bonnerot, C., S. Amigorena, D. Choquet, R. Pavlovich, V.
+
+Choukroun, and W.H. Fridman. 1992. Role of associated g-chain in tyrosine kinase activation via murine FcgRIII.
+
+EMBO (Eur. Mol. Biol. Organ.) J. 11:2747–2757.
+
+6. Daeron, M., O. Malbec, C. Bonnerot, S. Latour, D.M. Segal, and W.H. Fridman. 1994. Tyrosine-containing activation motifdependent phagocytosis in mast cells. *J. Immunol.* 152:783–792.
+
+7. Amigorena, S., C. Bonnerot, J.R. Drake, D. Choquet, W.
+
+Hunziker, J.G. Guillet, P. Webster, C. Sautes, I. Mellman, and W.H. Fridman. 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. *Science (Wash. DC).* 256:1808–1812.
+
+8. Muta, T., T. Kurosaki, Z. Misulovin, M. Sanchez, M.C.
+
+Nussenzweig, and J.V. Ravetch. 1994. A 13-amino-acid motif in the cytoplasmic domain of FcgRIIB modulates B-cell receptor signalling. *Nature (Lond.).* 368:70–73.
+
+9. Daeron, M., S. Latour, O. Malbec, E. Espinosa, P. Pina, S.
+
+Pasmans, and W.H. Fridman. 1995. The same tyrosine-based inhibition motif, in the intracytoplasmic domain of FcgRIIB,
+regulates negatively BCR-, TCR-, and FcR-dependent cell activation. *Immunity.* 3:635–646.
+
+10. Doody, G.M., L.B. Justement, C.C. Delibrias, R.J. Matthews, J. Lin, M.L. Thomas, and D.T. Fearon. 1995. A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. *Science (Wash. DC).* 269:242–244.
+
+11. Yokoyama, W.M., and W.E. Seaman. 1993. The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. *Annu. Rev. Immunol.*
+11:613–635.
+
+12. Lanier, L.L., and J.H. Phillips. 1996. Inhibitory MHC class I
+receptor on NK cells and T cells. *Immunol. Today.* 17:86–91.
+
+13. Lazetic, S., C. Chang, J.P. Houchins, L.L. Lanier, and J.H.
+
+Phillips. 1996. Human natural killer cell receptors involved in MHC class I recognition are disulfide linked heterodimers of CD94 and NKG2 subunits. *J. Immunol.* 157:4741–4745.
+
+14. Katz, H.R., E. Vivier, M.C. Castells, M.J. McCormick, J.M.
+
+Chambers, and F.K. Austen. 1996. Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high-affinity Fc receptor for IgE. Proc. Natl. Acad. Sci. USA. 93:10809–10814.
+
+15. Wang, L.L., I.K. Mehta, P.A. LeBlanc, and W.M. Yokoyama.
+
+1997. Mouse natural killer cells express gp49B1, a structural homologue of human killer inhibitory receptors. *J. Immunol.*
+158:13–17.
+
+16. Rojo, S., D.N. Burshtyn, E.O. Long, and N. Wagtmann.
+
+1997. Type I transmembrane receptor with inhibitory function in mouse mast cells and NK cells. *J. Immunol.* 158:9–12.
+
+17. Thomas, M.L. 1995. Of ITAMs and ITIMs: turning on and off the B cell antigen receptor. *J. Exp. Med.* 181:1953–1956.
+
+18. D'Ambrosio, D., K.L. Hippen, S.A. Minskoff, I. Mellman, G. Pani, K.A. Siminovitch, and J.C. Cambier. 1995. Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by FcgRIIB1. *Science (Wash. DC).*
+268:293–297.
+
+19. Burshtyn, D.N., A.M. Scharenberg, N. Wagtmann, S. Rajagopalan, K. Berrada, T. Yi, J.P. Kinet, and E.O. Long. 1996.
+
+Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor. *Immunity.* 4:77–85.
+
+20. Campbell, K.S., M. Dessing, M. Lopez Botet, M. Cella, and M. Colonna. 1996. Tyrosine phosphorylation of a human killer inhibitory receptor recruits protein tyrosine phosphatase 1C. *J. Exp. Med.* 184:93–100.
+
+21. Olcese, L., P. Lang, F. Vely, A. Cambiaggi, D. Marguet, M.
+
+Blery, K.L. Hippen, R. Biassoni, A. Moretta, L. Moretta, J.C. Cambier, and E. Vivier. 1996. Human and mouse killercell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. *J. Immunol.* 156:4531–4534.
+
+22. Fry, A.M., L.L. Lanier, and A. Weiss. 1996. Phosphotyrosines in the killer cell inhibitory receptor motif of NKB1 are required for negative signaling and for association with protein tyrosine phosphatase 1C. *J. Exp. Med.* 184:295–300.
+
+23. Binstadt, B.A., K.M. Brumbaugh, C.J. Dick, A.M. Scharenberg, B.L. Williams, M. Colonna, L.L. Lanier, J.-P. Kinet, R.T. Abraham, and P.J. Leibson. 1996. Sequential involvement of Lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activation. Immunity. 5:629–638.
+
+24. Ono, M., S. Bolland, P. Tempst, and J.V. Ravetch. 1996.
+
+Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor FcgRIIB. *Nature*
+(Lond.). 383:263–266.
+
+25. Samaridis, J., and M. Colonna. 1997. Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. *Eur. J. Immunol.* 27:660–665.
+
+26. Zhang, G., J.R. Young, C.A. Tregaskes, P. Sopp, and C.J.
+
+Howard. 1995. Identification of a novel class of mammalian Fcg receptor. *J. Immunol.* 155:1534–1541.
+
+27. Arm, J.P., M.F. Gurish, D.S. Reynolds, H.C. Scott, C.S.
+
+Gartner, K.F. Austen, and H.R. Katz. 1991. Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily. *J. Biol. Chem.* 266:15966–15973.
+
+28. Colonna, M., and J. Samaridis. 1995. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. *Science* (Wash. DC). 268:405–408.
+
+1750 An Inhibitory Receptor Expressed in Myeloid Antigen-presenting Cells 29. Wagtmann, N., R. Biassoni, C. Cantoni, S. Verdiani, M.S.
+
+Malnati, M. Vitale, C. Bottino, L. Moretta, A. Moretta, and E.O. Long. 1995. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains. *Immunity.* 2:439–449.
+
+30. D'Andrea, A., C. Chang, K. Franz-Bacon, T. McClanahan, J.H. Phillips, and L.L. Lanier. 1995. Molecular cloning of NKB1. A natural killer cell receptor for HLA-B allotypes. J. Immunol. 155:2306–2310.
+
+31. Maliszewski, C.R., C.J. March, M.A. Schoenborn, S. Gimpel, and L. Shen. 1990. Expression cloning of a human Fc receptor for IgA. *J. Exp. Med.* 172:1665–1672.
+
+32. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. *J. Exp. Med.* 179:1109–1118.
+
+33. Bender, A., M. Sapp, G. Schuler, R.M. Steinman, and N.
+
+Bhardwaj. 1996. Improved methods for the generation of dendritic cells from non proliferating progenitors in human blood. *J. Immunol. Methods.* 196:121–135.
+
+34. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, and G. Schuler. 1996. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. *J. Immunol. Methods.* 196:137–151.
+
+35. Frohman, M.A., M.K. Dush, and G.R. Martin. 1988. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.
+
+Proc. Natl. Acad. Sci. USA. 85:8998–9002.
+
+36. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 8.11.
+
+37. Traunecker, A., F. Oliveri, and K. Karjalainen. 1991. Myeloma based expression system for production of large mammalian proteins. *Trends Biotechnol.* 9:109–113.
+
+38. Dohring, C., and M. Colonna. 1996. Human natural killer cell inhibitory receptors bind to HLA class I molecules. *Eur.* J. Immunol. 26:365–369.
+
+39. Reid, P.A., and C. Watts. 1990. Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments. *Nature (Lond.).* 346:655–657.
+
+40. Dohring, C., D. Scheidegger, J. Samaridis, M. Cella, and M.
+
+Colonna. 1996. A human killer inhibitory receptor specific for HLA-A. *J. Immunol.* 156:3098–3101.
+
+41. Valitutti, S., M. Dessing, and A. Lanzavecchia. 1993. Role of cAMP in regulating cytotoxic T lymphocyte adhesion and motility. *Eur. J. Immunol.* 23:790–795.
+
+42. Lanzavecchia, A., S. Abrignani, D. Scheidegger, R. Obrist, B. Dorken, and G. Moldenhauer. 1988. Antibodies as antigens. The use of mouse monoclonal antibodies to focus human T cells against selected targets. *J. Exp. Med.* 167:345–352.
+
+43. Williams, A.F., and N.A. Barclay. 1988. The immunoglobulin superfamily-domains for cell surface recognition. Ann.
+
+Rev. Immunol. 6:381–405.
+
+44. Zhou, L.J., and T.F. Tedder. 1995. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. *J. Immunol.* 154:3821–3835.
+
+45. Zhou, L.J., and T.F. Tedder. 1996. CD141 blood monocytes can differentiate into functionally mature CD831 dendritic cells. *Proc. Natl. Acad. Sci. USA.* 93:2588–2592.
+
+46. O'Doherty, U., R.M. Steinman, M. Peng, P.U. Cameron, S.
+
+Gezelter, I. Kopeloff, W.J. Swiggard, M. Pope, and N.
+
+Bhardwaj. 1993. Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. *J. Exp. Med.* 178:1067–1076.
+
+47. Altieri, D.C., S.J. Stamnes, and C.G. Gahmberg. 1992. Regulated Ca21 signalling through leukocyte CD11b/CD18 integrin. *Biochem. J.* 288:465–473.
+
+48. Crockett-Torabi, E., B. Sulenbarger, C.W. Smith, and J.C. Fantone. 1995. Activation of human neutrophils through L-selectin and Mac-1 molecules. *J. Immunol.* 154:2291–2302.
+
+49. Cambier, J.C., D.C. Morrison, M.M. Chien, and K.R. Lehmann. 1991. Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia crosslinking. J. Immunol. 146:2075–2082.
+
+50. Cambier, J.C., and K.R. Lehmann. 1989. Ia-mediated signal transduction leads to proliferation of primed B lymphocytes. J. Exp. Med. 170:877–886.
+
+51. Charron, D., S. Brick-Ghannam, R. Ramirez, and N.
+
+Mooney. 1991. HLA class-II-mediated B-lymphocyte activation: signal transduction and physiologic consequences. Res.
+
+Immunol. 142:467–474.
+
+52. Cassatella, M.A., I. Anegon, M.C. Cuturi, P. Griskey, G.
+
+Trinchieri, and B. Perussia. 1989. FcgR(CD16) interaction with ligand induces Ca21 mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca21 in FcgR
+(CD16)-induced transcription and expression of lymphokine genes. *J. Exp. Med.* 169:549–567.
+
+53. Ravetch, J.V., and J.P. Kinet. 1991. Fc receptors. *Annu. Rev.*
+Immunol. 9:457–492.
+
+54. Trowbridge, I.S., J.F. Collawn, and C.R. Hopkins. 1993.
+
+Signal-dependent membrane protein trafficking in the endocytic pathway. *Annu. Rev. Cell. Biol.* 9:129–161.
+
+55. Letourneur, F., and R.D. Klausner. 1992. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. *Cell.* 69:1143–1157.
+
+56. Sandoval, I.V., and O. Bakke. 1994. Targeting of membrane proteins to endosomes and lysosomes. *Trends Cell Biol.* 4: 292–297.
+
+57. Steinman, R.M. 1991. The dendritic cell system and its role in immunogenicity. *Annu. Rev. Immunol.* 9:271–296.
+
+58. Jiang, W., W.J. Swiggard, C. Heufler, M. Peng, A. Mirza, R.M. Steinman, and M.C. Nussenzweig. 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. *Nature (Lond.).* 375:151–155.
+
+59. Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995.
+
+Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. *J. Exp. Med.* 182:389–400.
+
+60. Bieber, T., H. de la Salle, A. Wollenberg, J. Hakimi, R.
+
+Chizzonite, J. Ring, D. Hanau, and C. de la Salle. 1992. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FceRI). *J. Exp. Med.* 175:1285–1290.
+
+61. Porcelli, S.A. 1995. The CD1 family: a third lineage of antigen-presenting molecules. *Adv. Immunol.* 59:1–98.
+
+62. Hashimoto, K., M. Hirai, and Y. Kurosawa. 1995. A gene outside the human MHC related to classical HLA class I genes. *Science (Wash. DC).* 269:693–695.
+
+63. Bahram, S., M. Bresnahan, D.E. Geraghty, and T. Spies.
+
+1994. A second lineage of mammalian major histocompatibility complex class I genes. *Proc. Natl. Acad. Sci. USA.* 91:
+6259–6263.

medical/md/PMC2529247.md

@@ -0,0 +1,506 @@
+Journal of Experimental Botany, Vol. 59, No. 12, pp. 3485–3499, 2008 doi:10.1093/jxb/ern201 Advance Access publication 06 August, 2008 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
+
+# Research Paper Arabidopsis Sterol Carrier Protein-2 Is Required For Normal Development Of Seeds And Seedlings
+
+Bing Song Zheng1,2, Elin Ro¨nnberg1, Lenita Viitanen3, Tiina A. Salminen3, Krister Lundgren4, Thomas Moritz4 and Johan Edqvist5,* 1 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7080, 750 07 Uppsala, Sweden 2 School of Forestry and Biotechnology, Zhejiang Forestry University, 311300, Lin An, China 3 Department of Biochemistry and Pharmacy, A˚bo Akademi University, Artillerigatan 6 A III, FIN-20520 Turku, Finland 4 Umea˚ Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umea˚, Sweden 5 IFM Biology, Linko¨ping University, 581 83 Linko¨ping, Sweden Received 25 April 2008; Revised 27 June 2008; Accepted 4 July 2008
+
+## Abstract
+
+The Arabidopsis thaliana sterol carrier protein-2 (AtSCP2) is a small, basic and peroxisomal protein that in vitro enhances the transfer of lipids between membranes. AtSCP2 and all other plant SCP-2 that have been identified are single-domain polypeptides, whereas in many other eukaryotes SCP-2 domains are expressed in the terminus of multidomain polypeptides. The AtSCP2 transcript is expressed in all analysed tissues and developmental stages, with the highest levels in floral tissues and in maturing seeds.
+
+The expression of AtSCP2 is highly correlated with the multifunctional protein-2 (MFP2) involved in b-oxidation.
+
+A. thaliana Atscp2-1 plants deficient in AtSCP2 show altered seed morphology, a delayed germination, and are dependent on an exogenous carbon source to avoid a delayed seedling establishment. Metabolomic investigations revealed 110 variables (putative metabolites) that differed in relative concentration between Atscp2-1 and normal A. thaliana wild-type seedlings.
+
+Microarray analysis revealed that many genes whose expression is altered in mutants with a deficiency in the glyoxylate pathway, also have a changed expression level in Atscp2-1.
+
+Key words: Arabidopsis, b-oxidation, germination, glyoxylate
+
+![0_image_0.png](0_image_0.png) cycle, lipid, lipid transport, metabolomics, microarray, peroxisomes, SCP-2.
+
+## Introduction
+
+Sterol carrier protein-2 (SCP-2) is an intracellular, small, basic protein domain that in vitro stimulates the transfer of lipids between membranes (Ritter et al., 1971; Bloj et al., 1978). In mammals, SCP-2 is implicated to have role in peroxisomal b-oxidation. The exact function of SCP-2 in b-oxidation is unclear, but it might facilitate the presentation and solubilization of the substrates or stabilizing the enzymes involved in catalysing the reaction cycles
+(Seedorf et al., 2000). Such suggestions are mainly based on studies of the mammalian peroxisomal proteins sterol carrier protein-X (SCP-X) and D-bifunctional protein
+(DBP), which both contain C-terminal SCP-2 domains.
+
+The human gene SCPX (also known as SCP2) encodes SCP-X, which consists of a 3-ketoacyl-CoA thiolase domain connected to a C-terminal SCP-2 domain (Ohba et al., 1994). Due to the existence of dual promoters, as well as proteolytic cleavage sites the SCPX encoded SCP-2 domain is also expressed as a single-domain protein
+(Ohba et al., 1995). SCP-X and the single-domain SCP-2 are both predominantly located to peroxisomes. Gene
+
+## 3486 Zheng Et Al.
+
+targeting in mice revealed that complete deficiency of SCPX resulted in an impaired catabolism of 2-methyl branched-chain fatty acyl CoAs as shown by a 10-fold accumulation of phytanic acid in SCPX(–/–) mice (Seedorf et al., 1998). Further, it has been demonstrated with FRET microscopy that the single-domain SCP-2 interacts in peroxisomes with enzymes involved in b-oxidation such as acyl-CoA oxidase, bifunctional protein, and 3-ketoacylCoA thiolase (Wouters et al., 1998). It may be noteworthy that plants are not encoding any gene orthologous to the 3-ketoacyl-CoA thiolase in SCP-X (Edqvist and Blomqvist, 2006).
+
+A SCP-2 domain is also present in the C-terminus of the mammalian DBP (also referred to as MFE-2), which in the human genome is encoded from HSD17B4. DBP has domains for D-3 (equivalent to 3R)-hydroxyacyl-CoA
+dehydrogenase, 2-enoyl-CoA hydratase, and SCP-2
+(Adamski et al., 1995; Leenders et al., 1998) and catalyses the second and third steps of the b-oxidation pathway. Reactions catalysed by the mammalian DBP
+proceed through D-3-hydroxyl-CoA esters. DBP is suggested to be required for the peroxisomal b-oxidation of the enoyl-CoA esters of very long chain fatty acids, pristanic acid, and of dihydroxycholestanoic acid (DHCA)
+(Wanders, 2004). The role of the SCP-2 domain in DBP is unclear, but it contains a peroxisomal targeting signal (PTS1) that locates DBP to the peroxisomes. The structural and functional conservation may indicate that the SCP-2 domain in DBP also has additional functions, such as to interact with enzymatic domains of DBP to form an extended hydrophobic cavity for the hydrophobic tails of some b-oxidation substrates (Haapalainen et al., 2001).
+
+A. thaliana do not encode DBP, and there are no plant genes identified orthologous to the D-3-hydroxyacyl-CoA
+dehydrogenase domain of mammalian DBP (Edqvist and Blomqvist, 2006). Rather, the A. thaliana multifunctional proteins AIM1 and MFP2 each share domain structure and approximately 50% amino acid sequence similarity to the human peroxisomal L-bifunctional protein (LBP) (also referred to as MFE-1) (Kiema et al., 2002). LBP and DBP
+lack significant sequence similarity and LBP, as well as AIM1 and MFP2, are not carrying any C-terminal SCP-2 domain. Furthermore, reactions catalysed by LBP, AIM1, and MFP2 proceeds through L-3-hydroxyl-CoA esters.
+
+Thus, plants do not encode DBP or SCP-X, but it has recently been shown that plants also encode and express SCP-2 (Eklund and Edqvist, 2003; Edqvist et al., 2004; Edqvist and Blomqvist, 2006). The gene AtSCP2 (At5g42890) on chromosome 5 encodes the sole SCP-2 domain in the A. thaliana genome. AtSCP2 is a 13.6 kDa protein with a pI of 9.2, which localizes to peroxisomes through its C-terminal PST1 targeting signal. It has lipid transfer activity in vitro (Edqvist et al., 2004). The known crystal structures of SCP-2 from rabbit (Choinowski et al., 2000), of the SCP-2 domain of the human DBP
+(Haapalainen et al., 2001) and of yellow fever mosquito SCP-2 (Dyer et al., 2003) have an a/b-fold consisting of a five stranded b-sheet and four or five a-helices. A
+C-terminal segment, together with part of the b-sheet and four a-helices form a hydrophobic tunnel, which is very suitable for binding of lipids or other hydrophobic ligands.
+
+According to generated models, AtSCP2 has a similar a/b-fold forming a hydrophobic tunnel (Edqvist et al.,
+2004). AtSCP2 and also all other plant SCP-2 that have been identified are single-domain polypeptides (Edqvist and Blomqvist, 2006; Viitanen et al., 2006), whereas, as indicated above, SCP-2 domains in animals and many other eukaryotes are often present in the terminal of polypeptides which carry multiple protein domains.
+
+The only SCP-2 domain encoded in A. thaliana is the single-domain protein AtSCP-2. As described above and in Edqvist and Blomqvist (2006), the situation is more complex in animals, with larger SCP-2 gene families and often quite complicated arrays of protein domain fusions.
+
+We reason that this turns A. thaliana into a very suitable model organism for studying the function of the still enigmatic SCP-2 domain. Here, an initial investigation on the biological function of AtSCP2 is presented. It is shown that the activity of the peroxisomal protein AtSCP2 is important for the metabolism in A. thaliana seeds and seedlings.
+
+## Materials And Methods
+
+Plant materials and growth conditions A. thaliana ecotype Columbia (Col-0) was used as the wild-type plant. Seeds of the T-DNA insertion lines Sail_1231_F11 were purchased from the European Arabidopsis Stock Centre (NASC)
+(Loughborough, UK). The Sail_1231_F11 line is referred to as Atscp2-1. Seeds were surface-sterilized (washed in 70% ethanol for 1 min and in 15% chlorine and 0.5% SDS for 10 min followed by at least four washes in sterile distilled water) and sown on halfstrength Murashige and Skoog medium (1/2 MS). Before cultivation, seed dormancy was broken by 72 h of cold treatment (4 C).
+
+The synthetic auxin 2,4-dichlorophenoxybutyric acid (2,4-DB)
+(0.1 lM, 4 lM) and indole-3-butyric acid (IBA) (3 lM, 30 lM)
+were added to the autoclaved medium where indicated. Ten-day-old plants grown under sterile conditions were transplanted on soil mixed with vermiculite (2:1 v/v). The plants were cultivated under controlled conditions in environmental chambers at 20–22 C under long day (16/8 h light/dark) conditions. The Atscp2-1 mutant was back-crossed to wild-type A. thaliana Col-0.
+
+For expression of AtSCP2 in Atscp2-1 under the control of its own promoter, a DNA fragment carrying the AtSCP2 gene including the promoter was obtained through amplification of A. thaliana genomic DNA with primers ATSCP2promattB1F (5\#-
+GGGGACAAGTTTGTACAAAAAAGCAGGCTCACACCTCCTATTTATCGGACAT-3\#) and AtSCP2attB2R (5\#-GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAACTTTGAAGGTTTACGGAAGAT-3\#). The PCR fragment was recombined into the destination vector pMDC99 (Curtis and Grossniklaus, 2003)
+resulting in the plasmid pJE602. For expression of AtSCP2 cDNA
+under control of the cauliflower mosaic virus (CaMV) 35S promoter, a fragment carrying a cDNA copy of AtSCP2 was amplified from A. thaliana cDNA with ATSCP2attB1F (5\#-
+GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCGAATACCCAACTCAAATC-3\#) and ATSCP2attB2R. The PCR fragment was recombined into destination vector pMDC32 (Curtis and Grossniklaus, 2003) yielding plasmid pJE601. Recombination events were done with the Gateway technology from Invitrogen
+(Carlsbad, CA, USA). pJE601 and pJE602 were transformed into Agrobacterium tumefaciens C58. The floral dip method (Clough and Bent, 1998) was used to transform A. thaliana Atscp2-1 with A. tumefaciens C58 carrying pJE601 or pJE602. Transformations and selection of transformants were done at the Uppsala Transgenic Arabidopsis Facility. The transformants obtained were denoted Atscp2-1(35S::AtSCP2) for transformation with pJE601 and Atscp21(AtSCP2::AtSCP2) for transformation with pJE602.
+
+## Phenotypic Assays
+
+The hypocotyl and root lengths were measured on 3-d-old and 7-dold seedlings grown on ½ MS medium with 0% or 1% sucrose. At least 20 seedlings were measured from each growth condition and time point. All experiments were performed in triplicate. Rosette diameter was measured at the widest point of the plant without disturbing any leaves under light. The developing seeds were counted after self-pollination. The percentage of seed germination was scored every 12 h after transferring stratified seeds on the MS media to a growth chamber. Germination was defined as an obvious protrusion of the radicle through the seed coat. Several seed lots were tested with similar results.
+
+## Histochemical And Quantitative Gus Activity Assays
+
+A DNA fragment carrying the AtSCP2 promoter was amplified from the A. thaliana Col-0 genome by the use of primers SCPPrU2
+(5\#-CACACCTCCTATTTATCGGACAT-3\#) and SCPPrN2 (5\#-
+GATTTTTGTTAGAGACTGGCACG-3\#). The PCR primers were designed such that a fragment was amplified stretching from the untranslated region of the nearest gene upstream of AtSCP2 to the 5' untranslated region of AtSCP2. The obtained 1.4 kb AtSCP2 promoter fragment was inserted into vector PCR2.1-TOPO (Invitrogen) to yield the plasmid pER2. The AtSCP2 promoter fragment was released from pER2 by restriction enzymes XbaI+BamH1, and subsequently fused to the b-glucuronidase (GUS) reporter gene by ligation to the XbaI+BamH1 sites of vector pBI101 resulting in plasmid pER1. The plasmid pER1 was transformed to A. tumefaciens C58.
+
+Histochemical GUS-assays were performed as described by Jefferson et al. (1987). Plant tissues were incubated in a substrate solution containing 50 mM Na-phosphate buffer (pH 7.0), 1 mM
+5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid cyclohexyl ammonium salt (X-GlcA CHA) (Duchefa Biochemie, Haarlem, The Netherlands), 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, and 0.01%
+(w/v) Triton X-100 at 37 C overnight. Stained samples were incubated in 95% ethanol at room temperature to extract the chlorophyll.
+
+## Quantitative Real-Time Reverse Transcriptase-Pcr, Reverse Transcriptase-Pcr And Genomic Pcr
+
+RNA was extracted from A. thaliana using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Five lg RNA was used for cDNA synthesis using oligo dT-primer and Superscript II
+Rnase-Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Amplification of the cDNA was performed in the presence of gene-specific primers and the SYBR Green PCR
+master mix (Applied Biosystems, Foster City, CA, USA) in MicroAmp Optical 96-well reaction plates with optical covers using an ABI Prism 7000 Sequence Detector (Applied Biosystems).
+
+Reaction conditions were 50 C for 2 min, 94 C for 10 min, followed by 40 cycles of 94 C for 15 s and 60 C for 1 min. All cDNA-samples were included in triplicate in all assays. Primers were designed using Primer express software (Applied Biosystems).
+
+Relative quantification of gene expression data was carried out with the 2–DDCT or comparative CT method (Livak and Schmittgen, 2001). The threshold cycle (CT) indicates the cycle number at which the amount of amplified transcript reaches a fixed threshold.
+
+Expression levels were normalized with the CT values obtained for the A. thaliana tubulin b-2/b-3 chain (At5g62690). The following gene-specific primers were used: AtSCP2: SCPF1 (5\#-GCCGGGAAAGAGGTCACA-3\#) and SCPR1 (5\#-TGTAAGTAACCTCTTCAAACCCTAATTTCT-3); A. thaliana tubulin b-2/b-3 chain
+(At5g62690): Tub1 (5\#-ACACCAGACATAGTAGCAGAAATCAAG-3\#) and Tub2 (5\#-GAGCCTTACAACGCTACTCTGTCTGTC-3\#); At3g21720: 3g21720F (5\#-TTGCACAGATCGCAGACATCA-3\#) and 3g21720R (5\#-GAATTGGGTGCATTCATTGAGA-3\#); At1g06570: 1g06570F (5\#-CTCCGCCAAATCCGATCTT-3\#) and 1g06570R (5\#-GGAGGTCACCGGAGGTGAGT-3\#);
+At3g47340: 3g47340F (5\#-AACGTCTTGCCGTCATCGAT-3\#) and 3g47340R (5\#-CAATGGTCTTGTCCTCGTTGAA-3\#); At1g08630:
+1g08630F (5\#-CGATGCGAGAAGCAATGTGTA-3\#) and 1g08630R
+(5\#-CGTCTAGCCGTTGGGTCATATC-3\#); At1g21400: 1g21400F
+(5\#-AGACACAGGCTGATCATTTGGTT-3\#) and 1g21400R (5\#-
+TTTGCCTCCTGGGAAATCC-3\#); At3g45140: 3g45140F (5\#-AGCCCCAATGGAAACAAGTCT-3\#) and 3g45140R (5\#-AGCAAGATTCCATAGCCAGCA-3\#); At5g55730: 5g55730F (5\#-GGCACCAGAGGATGGTGATG-3\#) and 5g55730R (5\#-TCTTTCCTTTCGCTTTCCCTTT-3\#); At5g04960: 5g04960F (5\#-AAACGCGAGCCGATCAAG-3\#) and 5g04960R (5\#-AGAGCTCCGTGAT-GGTGACTTT3\#); At3g14210: 3g14210F (5\#-CAGGAGGAAATGGCTCATCTTCTA-3\#) and 3g14210R (5\#-AGCTCACGGACC-GTCATTG-3\#);
+At1g52050: 1g52050F (5\#-TTCCTAAGCTACAGAAGTTTGTTCATGCATG-3\#) and 1g52050R (5\#-CATAGTTGACAACATCGGAATCG-3\#).
+
+Reverse transcriptase-PCR (RT-PCR) was done as described previously (Edqvist et al., 2004). The oligonucleotides ATSCPRT1 and ATSCPRT2 were used for expression analysis of AtSCP2 cDNA, while UBL1 and UBL2 were used for amplification of cDNA for the ubiquitin-conjugating enzyme E2-21 kD (At5g41340) (Edqvist et al., 2004).
+
+For isolation of A. thaliana genomic DNA, 500 ll of an extraction buffer consisting of 200 mM TRIS, 250 mM NaCl, and 25 mM EDTA was added to plant tissues. After addition of six glass beads (3 mm in diameter), the tissues were disrupted by incubation in a FAST-prep instrument (MP Biomedicals, Irvine, CA, USA) for 30 s. 25 ll of 10% SDS was added, and the samples were incubated at 65 C for 10 min and then centrifuged for 5 min.
+
+450 ll of the supernatant was removed and precipitated with an equal volume of cold isopropanol. After incubation for 2 min at room temperature, samples were centrifuged for 5 min. The supernatant was discarded, and the DNA pellet obtained was dried, dissolved in water, and used for PCR. The following primers were used for analysing genomic DNA from Atscp2-1: SAIL_1231_F11_RP
+(5\#-AACATTGCTCCAAAGGTTGGT-3\#), SAIL_1231_F11_LP
+(5\#-GGACCAAATCCAAGTCACACA-3\#) and SAIL_LB1 (5\#-
+GCCTTTTCAGAAATGGATAAATAGCCTTGC-3\#).
+
+## Expression Profiling Through Microarray Analysis
+
+Two-day-old seedlings of the Atscp2-1 and wild-type plants grown under light conditions on 1/2 MS without sucrose were harvested and frozen quickly in liquid nitrogen. Two-day-old seedlings were selected for the expression analysis due to the high AtSCP-2 expression levels and the manifested phenotype of young Atscp2-1
+
+## 3488 Zheng Et Al.
+
+seedlings when grown on 1/2 MS without exogenous carbon source. RNA samples were extracted using RNeasy Plant Mini Kit
+(Qiagen). Five lg of total RNA was converted to double-stranded cDNA using the SuperScript polymerase II (Invitrogen) with a T7-dT primer incorporating a T7 RNA polymerase promoter
+(Ambion, Austin, TX, USA). The double-stranded cDNA was amplified to cRNA through in vitro transcription using Megascript T7 kit (Ambion). To obtain aminoallyl-labelled cDNA the amplified cRNA was used as template for cDNA synthesis performed with SuperScript III (Invitrogen) in the presence of dATP, dCTP, dGTP,
+and aminoallyl-dUTP (aa-dUTP). After completion of cDNA
+synthesis unincorporated aa-dUTP was removed by using Qiagen QIAquick PCR purification kit. The purified aminoallyl-labelled cDNA (aa-cDNA) was lyophilized and subsequently dissolved in 4.5 ll of 0.1 M Na2CO3-buffer, pH 9.0. To label the aa-cDNA,
+4.5 ll of the NHS-ester Cy3 or Cy5 dye, prepared in DMSO, was added and the mixture was incubated at room temperature for 1 h in darkness. The two resulting dye-labelled cDNA pools were mixed and then co-hybridized to the same A. thaliana CATMA microarray slide (Allemeersch et al., 2005) for 2 d at 42 C in a water bath.
+
+After that, the arrays were washed and then scanned using a GenePix 4000B array scanner (Molecular Devices, Sunnyvale, CA, USA). The raw data were stored and analysed using BASE
+(https://base.lcb.uu.se) and the Linnaeus Centre for Bioinformatics
+(LCB) Data Warehouse (https://dw.lcb.uu.se) at LCB and the WCN
+Expression Array Facility at Uppsala University and The Swedish University of Agricultural Sciences, Uppsala, Sweden. Four replicates were made. The empirical Bayes methods B-statistics (Filtering Bstat1 >7.21) (Lo¨nnstedt and Speed, 2002; Smyth et al., 2003; Lo¨nnstedt and Britton, 2005) was applied to rank the genes in order of evidence for differential expression, from the strongest to the weakest evidence.
+
+## Metabolomics
+
+Metabolites were extracted from 3-d-old seedlings of A. thaliana wild-type and Atscp2-1. Seedlings were grown on 1/2 MS without sucrose. The young seedlings were selected for metabolome profiling due to the high AtSCP-2 expression levels in wild-type and the manifested phenotype in young Atscp2-1 seedlings when grown on 1/2 MS without sucrose. From each genotype, 10 samples of 20 mg fresh weight were used for the analysis. Extraction of metabolites, GC/MS analysis, and data processing were done as described previously (Gullberg et al., 2004; Jonsson et al., 2005). All multivariate statistical investigations (PCA, PLS-DA) were performed using Simca software 10.5.0.0 (Umetrics, Umea˚, Sweden). The following statistics for the PLS-DA models are discussed throughout this paper: R2X is the cumulative modelled variation in X, R2Y is the cumulative modelled variation in Y and Q2Y is the cumulative predicted variation in Y, according to cross-validation. The range of these parameters is 0–1, where 1 indicates a perfect fit.
+
+## Docking Of Auxin Precursors Into The Homology Model Of Arabidopsis Thaliana Scp-2
+
+The docking program GOLD 3.2 (Jones et al., 1995, 1997) was used to dock the auxin precursors IBA and 2,4-DB into the previously created homology model of AtSCP-2 (Edqvist et al., 2004), which is based on the crystal structure of the SCP-2-like domain of human DBP (Haapalainen et al., 2001). The IBA and 2,4-DB structures were generated using the program SYBYL 8.0
+(Tripos International, St Louis, MO, USA). Ten independent genetic algorithm runs with the default docking parameters were made in GOLD for the ligands. The binding site was restricted within a 15 A˚ radius of the side-chain hydrogen (HZ) of Phe112 in the AtSCP2 model. The docking was stopped if the three best scoring solutions were within 1.5 A˚ rmsd of each other. The cavity in the AtSCP2 model was identified using the program SURFNET
+(Laskowski, 1995). The docking results were visualized and examined in the BODIL modelling environment (Lehtonen et al.,
+2004).
+
+## Results Expression Of Atscp2 During Development
+
+To gain insight on the expression pattern of AtSCP2, we analyzed large amount of data accessible in public databases (i.e. www.weigelworld.org, www.arabidopsis.org, and www.genevestigator.ethz.ch) from microarray analysis of gene expression during A. thaliana development. Figure 1 shows the expression of AtSCP2 in 63 samples from different tissues or stages of development.
+
+The data are from the AtGenExpress expression atlas
+(www.weigelworld.org) (Schmid et al., 2005) and shows that the AtSCP2 mRNA is present in all tissues and at all stages of the life of the plant. The highest levels of the AtSCP2 transcript was found in seeds, such as in green cotyledons of maturating seeds (Fig. 1, sample 63), and in floral tissues, such as the stamens of stage 12 flowers (Fig. 1, sample 51) and petals (Fig. 1, sample 50) and stamens of stage 15 flowers (Fig. 1, sample 52). The accumulation of the AtSCP2 transcript in roots, stems, buds, siliques, inflorescences, and leaves of mature A. thaliana plants was analysed with quantitative real-time RT-PCR
+(data not shown). The results obtained confirmed that the AtSCP2 mRNA is abundant in most A. thaliana tissues.
+
+The A. thaliana AtSCP2 promoter was fused to the b-glucuronidase (GUS) reporter gene and the temporal and spatial patterns of expression were assessed during plant growth and development (Fig. 2). Endosperm (Fig. 2B),
+embryo (Fig. 2C), and 2-d-old seedlings (Fig. 2D) showed very high GUS activity. Staining was also detected in vascular tissues and hydathodes of cotyledons (Fig. 2E),
+in trichomes of the rosette leaves (Fig. 2F), in the receptacles (Fig. 2G, H), in vascular tissues of sepals and petals (Fig. 2G, I, J), in the style and stigma of the carpels (Fig. 2G, K), and in anthers, filaments, and pollen
+(Fig. 2G, L). The funiculi of the siliques were also shown to contain GUS activity (Fig. 2M).
+
+The performance of the AtSCP2 promoter-GUS fusion was also assayed in response to light, darkness, and sucrose in 1–7-d-old seedlings (Fig. 3). The 1-d-old seedlings showed high levels of GUS-activity, indicating high levels of activity of the AtSCP2 promoter early during germination. There was a decrease in expression from the AtSCP2 promoter with time, as 7-d-old seedlings showed lower GUS activity compared with 1-d-old and 3-d-old seedlings. Dark-grown seedlings showed a more intense staining, indicating a higher activity of the AtSCP2 promoter. Interestingly, the GUS activity increased with
+
+![4_image_0.png](4_image_0.png)
+
+increasing amounts of sucrose when grown under constant illumination as well as in darkness. The microarray data compiled in Fig. 1 also revealed a slight increase in the accumulation of the AtSCP2 transcript in seedlings in response to sucrose (compare samples 4 and 5, and samples 27 and 28).
+
+## Correlated Expression Patterns Of Atscp2 To Other Genes
+
+To establish a functional context of the expression pattern of AtSCP2, it was analysed whether its expression is correlated to that of other genes. To calculate Pearson correlation coefficients (R) from microarray analysis of gene expression, the gene correlator function of Genevestigator (www.genevestigator.ethz.ch) (Zimmermann et al.,
+2004, 2005) and the ExpressionAngler function of the Botany Array Resource in the University of Toronto
+(bbc.botany.utoronto.ca) (Toufighi et al., 2005) were used. Results from such investigations using ExpressionAngler on data from the AtGenExpress experiments are shown in Fig. 4. At the ExpressionAngler, the data are divided into four groups: tissue, hormone, stress, and pathogen for samples collected from various tissues and developmental stages, after hormone treatments, after abiotic stresses or biotic stresses, respectively. The 100 genes from each group giving the highest R to AtSCP2 were listed. The R cut-off values required for genes to appear on the TOP100-lists were R¼0.923 (hormone), R¼0.800 (stress), R¼0.656 (pathogen), and R¼0.686 (tissue). R >0.7 is generally considered a rule-of-thumb threshold for true correlation and used in various analysis (Lee et al., 2004; Ren et al., 2005). Figure 4 shows the R of the 26 genes that were identified in the lists from at least two of the four groups. Firstly, it can be noted that no gene was found in all the lists, and that only one gene (At4g07390 encoding a PQ-loop repeat family protein) appeared in three of the lists. Of the 25 remaining genes there are three genes (MFP2, PED1, and PMDH1) which encode proteins involved in the b-oxidation cycle (Hayashi et al.,
+1998; Rylott et al., 2006; Pracharoenwattana et al., 2007). The correlation of expression between MFP2 and AtSCP2 is particularly strong, with R¼0.82 for tissue samples and R¼0.99 for the hormone samples.
+
+## Characterization Of An Atscp2 T-Dna Insertion Mutant
+
+To elucidate the biological role of AtSCP2 further, the TDNA insertion line Sail_1231_F11 was obtained from NASC (Fig. 5A). The Sail_1231_F11 line is referred to as Atscp2-1. In Atscp2-1 the T-DNA insertion is in the second intron of AtSCP2. A homozygous insertion line was obtained as confirmed by PCR analysis (Fig. 5B). Transcript analysis using RT-PCR showed that no AtSCP2 transcript was detected in Atscp2-1 seedlings (Fig. 5C).
+
+Adult plants of Atscp2-1 were normal in appearance (data not shown). Since the expression analysis revealed the highest levels of the AtSCP2 transcript in floral tissues and during seed maturation, seed development in Atscp2-1 was investigated. Thirty-three siliques were removed from wild-type and Atscp2-1 plants, and the developing seeds were examined under a microscope and counted. For
+
+![5_image_0.png](5_image_0.png)
+
+wild-type plants, the average silique contained 56 green seeds, whereas the average Atscp2-1 silique contained 49 green seeds. According to the performed t test, the seed per silique numbers of Atscp2-1 were significantly different from wild-type at a P-value of 0.000155. When the morphology of the seeds was examined after harvest, it was revealed that seeds from the Atscp2-1 plants had an imperfect appearance (Fig. 6). Seeds from Atscp2-1 plants complemented with wild-type AtSCP-2 from either pJE602 containing a genomic fragment carrying AtSCP2 including the AtSCP2 promoter [Atscp2-1(AtSCP2::AtSCP2)] or pJE601 carrying a cDNA copy of AtSCP2 connected to the CaMV 35S promoter [Atscp2-1(35S::AtSCP2)] showed wild-type morphology (data not shown).
+
+The germination of Atscp2-1 seeds was tested by scoring the radicle emergence every 12 h after transferring stratified seeds on 1/2 MS media to a growth chamber. The germination kinetics was significantly slower for Atscp2-1 seeds compared to wild-type seeds (Fig. 7). 75% of the Atscp2-1 seeds were germinated after 72 h
+
+![6_image_0.png](6_image_0.png)
+
+incubation showing that the mutant seeds also had a lower germination frequency than the wild-type. Neither the lowered frequency nor the slower kinetics of germination could be rescued by the addition of 1% sucrose to the media. Germination kinetics resembling A. thaliana wildtype were obtained when Atscp2-1 was complemented with wild-type AtSCP-2 (Fig. 7).
+
+In order to investigate whether the lack of SCP-2 had an effect on seedling establishment, seeds from Atscp2-1 and wild-type were placed on agar media with or without sucrose and germinated under light or in darkness (Fig. 8).
+
+After 3 d on media without sucrose, the Atscp2-1 seedlings were clearly distinguishable from the wild-type.
+
+The insertion line showed smaller cotyledon rosettes and shorter hypocotyls than wild-type and the root elongation was also retarded (Fig. 8A–F). Seven days after sowing the root length was still retarded compared with the wildtype when grown without a carbohydrate supplement
+(Fig. 8E, F). Also in the dark, root elongation was inhibited on media lacking sucrose as the length of the primary root in Atscp2-1 was reduced on average to 67% and 48% of the wild-type length when assayed after 3 d and 7 d, respectively. The length of the mutant hypocotyls were slightly reduced in darkness on media lacking sucrose, as they showed a 20% and 14% reduction in length after 3 d and 7 d, respectively. Growth and development of Atscp2-1 on 1% sucrose closely matched the wild-type control, in light (Fig. 8A–F) as well as in darkness (data not shown).
+
+## Effect Of 2,4-Db And Iba On Germination Of Atscp2 Mutants
+
+2,4-DB is b-oxidized to 2,4-dichlorophenoxyacetic acid, which inhibits root elongation. Several A. thaliana mutants with deficiencies in b-oxidation have been identified in screens for plants that elongated roots on normally inhibitory concentrations of 2,4-DB (Hayashi et al., 1998).
+
+To investigate whether the AtSCP2 protein may have a role in b-oxidation of auxin precursors and analogues, seeds of Atscp2-1 were germinated on media containing 0.1 lM and 4 lM 2,4-DB and root elongation was followed. The growth inhibition of root elongation of Atscp2-1 was indistinguishable from the control wild-type plants on any tested concentration of 2,4-DB indicating that the AtSCP2 is not involved in catabolism of 2,4-DB (data not shown). Several b-oxidation mutants also show resistance to the growth inhibition caused by IBA (Zolman et al., 2001; Adham et al., 2005). However, Atscp2-1 also showed wild-type sensitivity to IBA (data not shown).
+
+The binding mode of IBA and 2,4-DB into the homology model of AtSCP2 was studied by the automated docking program GOLD, which gave four docking results for IBA and three results for 2,4-DB. The ligands were positioned quite similarly in all the dockings and only the results with the highest fitness are presented in Fig. 9. Both IBA and 2,4-DB were positioned with their carboxyl groups in one of the cavity openings (near Gln92, Gln110, Ile102, and Leu106). The indole of IBA
+
+![7_image_0.png](7_image_0.png)
+
+and the dichlorophenyl group of 2,4-DB were positioned towards the hydrophobic cavity (near Ile37, Phe74, Phe76, Phe81, Met100, and Phe112). The SURFNET plot shows that the AtSCP2 model has a very large tunnel-like cavity. Based on the docking results, the IBA and 2,4-DB molecules take up only a part of the cavity volume and, thus, do not extensively interact with the large hydrophobic cavity, indicating that they are not optimal AtSCP2 ligands (Fig. 9).
+
+## Expression Profiling Of Atscp2-1
+
+To reveal the molecular mechanisms underlying the delayed root elongation of Atscp2-1 seedlings, expression profiling was performed using the CATMA microarray slides (Allemeersch et al., 2005). RNA was isolated from 2-d-old seedlings of wild-type and Atscp2-1 germinated in the light on media lacking sucrose. The RNA was used as the template for the synthesis of cDNA, which subsequently was labelled with Cy5 and Cy3 and hybridized to the gene-specific tags of 150–500 bp on the microarray slide. The empirical Bayes methods B-statistics (Lo¨nnstedt and Speed, 2002; Smyth et al., 2003; Lo¨nnstedt and Britton, 2005) was applied to identify and rank the 100 reporters that, among about 25 000 reporters on the slide, showed the most significant changes in expression pattern in Atscp2-1. These 100 reporters corresponded to 94 different genes from the A. thaliana genome of which 54 were up-regulated and 40 were down-regulated (including AtSCP2) in Atscp2-1 (see Supplementary Table S1 at JXB
+online). The expression pattern of 10 genes identified as up- or down-regulated in the microarray analysis was tested with quantitative real-time RT-PCR using gene specific primers. The results confirmed that these genes had a modified expression in Atscp2-1 (see Supplementary Table S2 at JXB online).
+
+Those genes with a significantly altered expression represent a large range of functional categories, such as carbohydrate and amino acid metabolism, transport, and stress response. Interestingly, several genes that previously were shown to have an altered expression in seedlings of A. thaliana mutants icl-2 and mls-2 with mutations in genes encoding the key enzymes in the glyoxylate cycle, isocitrate lyase (ICL), and malate synthase (MLS) (Cornah et al., 2004), also showed an altered expression in Atscp2-1
+
+![8_image_0.png](8_image_0.png)
+
+![8_image_1.png](8_image_1.png)
+
+(Table 1). In fact, of the 20 genes listed showing the most significant overexpression in icl-2 mutants, six were among the 54 genes that were up-regulated on our Top 100 list.
+
+Two out of 12 genes that showed a 2-fold repression in mls-2, and 2 out of 10 genes showing a 2-fold overexpression in mls-2 were also identified in our Top 100 list.
+
+Three genes, ASN1 (At3g47340), At2g05540, and THA1
+(At1g08630), are overexpressed in Atscp2-1, icl-2, and mls2. ASN1 is a glutamine-dependent asparagine synthase
+(Lam et al., 1994), At2g055490 is a Gly-rich protein, and THA1 is a Thr aldolase, which catalyse the formation of Gly from Thr (Joshi et al., 2006).
+
+## Metabolome Analysis Of Atscp2-1
+
+We were interested in identifying metabolites that showed either significantly increased or decreased levels in Atscp2-1. Metabolome analyses were performed on 3-dold seedlings of Atscp2-1 and A. thaliana wild-type. The samples were extracted, derivatized, and analysed by GCMS according to Gullberg et al. (2004). The GC-MS data were analysed using hierarchical multivariate curve resolution (H-MCR; (Jonsson et al., 2005)). The data were centred and scaled to unit variance prior to partial least squares discriminant analysis (PLS-DA) classification of the genotypes. The obtained PLS-DA model (three components; R2X¼0.48; R2Y¼0.99; Q2Y¼0.93) shows a clear separation of the genotypes for the first two components (data not shown). The identification of differences was performed by interpretation of the loadings (as described in Trygg and Wold, 2002) from the PLS-DA
+model together with the 99% confidence intervals calculated using jack-knifing. In seedlings, 421 variables were detected, 110 of those showed a significant difference between wild-type and Atscp2-1 according to PLS-DA
+analysis (one component; R2X¼0.20; R2Y¼0.91; Q2Y¼0.81) and interpretation of first loading vector as described above. The significant metabolites were identified by comparison of retention index and mass spectra with retention index and mass spectra libraries (Schauer et al., 2005). The 20 metabolites that showed the most significant difference in accumulation between Atscp2-1 and wild-type were ranked. Twelve different metabolites
+
+![9_image_0.png](9_image_0.png)
+
+![9_image_1.png](9_image_1.png)
+
+were identified on the ranking list (Table 2). There were higher levels of Gln, pyroglutamic acid (a putative derivatization artefact from glutamate), Asp, b-Ala, fumaric acid, glyceraldehyde, and ribose in wild-type seedlings compared to Atscp2-1 (Table 2). The Atscp2-1 seedlings contained enhanced levels of Ser, Gly, Asn, 3cyanoalanine, and 5-methylthiopentanitrile (tentative identification). Comparison with the previously published metabolome analyses of icl-2 and mls-2 seedlings (Cornah et al., 2004) revealed several similarities as these mutants were also shown to have lowered amounts of Gln, and that mls-2 and Atscp2-1 seedlings both contained increased levels of Ser and Gly. The increased levels of Asn in Atscp2-1 coincide with increased expression of the asparagine synthase ASN1, as shown by the microarray analysis of Atscp2-1 seedlings. Elevated levels of Gly correlated with increased expression of the Thr aldolase THA1 in Atscp2-1.
+
+## Discussion
+
+It has been shown that AtSCP2 is ubiquitously expressed in maturing seeds, young seedlings, and floral tissues, although the transcript is present throughout all developmental stages of A. thaliana. The expression of AtSCP2 is correlated to the expression of enzymes of the b-oxidation machinery, such as MFP2, PED1, and PMDH1. The AtSCP2-deficient Atscp2-1 show altered seed morphology, compromised germination, and require exogenous carbohydrates to avoid delayed seedling establishment. Transcriptome and metabolome analysis of AtSCP2-1 revealed similarities to the glyoxylate cycle mutants icl-2 and mls-2, such as increased expression of the genes ASN1 and THA1, decreased levels of Gln, and elevated levels of Gly and Ser.
+
+According to current knowledge on b-oxidation and the glyoxylate cycle in A. thaliana (reviewed in Baker et al.,
+2006), fatty acids are delivered into the peroxisomes by the peroxisomal ABC-transporter CTS (also referred to as PED3 or PXA1) (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002). The delivered fatty acids are activated to acyl-CoA esters through the activity of peroxisomal acyl-CoA synthetases such as the long-chain acyl-CoA synthetases LACS6 and LACS7 (Fulda et al., 2004). The acyl-CoA esters then go through the repeated cleavage of acetate units from the thiol end through the activities of the b-oxidation enzymes: acyl-CoA oxidases ACX1-6, the multifunctional proteins MFP2 or AIM1, and the 3-ketoacyl-CoA thiolases (PED1, KAT1 and
+
+![10_image_0.png](10_image_0.png)
+
+PKT2) (Hayashi et al, 1998, 1999; Richmond and Bleecker, 1999; Germain et al., 2001; Adham et al., 2005; Rylott et al., 2006). To allow for conversion of lipids into sugar via gluconeogenesis, the acetate obtained
+
+## Functional Analysis Of Atscp2 3495
+
+may be fed into the glyoxylate cycle for the synthesis of succinate, malate, and oxaloacetate. The reactions in the glyoxylate cycle are catalysed by citrate synthase (CSY), aconitase, ICL, MLS, and malate dehydrogenase.
+
+Seedling establishment of the Atscp2-1 mutant is not dependent on an exogenous supply of sucrose, but sucrose is required to avoid a delay. Sucrose-dependent seedling establishment has been shown for many A. thaliana mutants deficient in proteins with roles in b-oxidation or the glyoxylate cycle, such as the acx1acx2 mutant deficient in the ACX1 long chain acyl-CoA oxidase and the ACX2 very long chain acyl CoA oxidase (Adham et al., 2005), the aim1 mutant (Richmond and Bleecker, 1999), the mfp2 mutant (Rylott et al., 2006), the pex6 mutant deficient in a putative peroxisomal ATPase (Zolman and Bartel, 2004), the pxa1/ped3/cts mutant (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002), the lacs6lacs7 mutant
+(Fulda et al., 2004), the ped1/kat2 mutant (Hayashi et al., 1998; Germain et al., 2001), the csy2csy3 mutant (Pracharoenwattana et al., 2005), the mls-2 mutant (Cornah et al., 2004), and the sdp2 mutant deficient in peroxisomal monohydroascorbate reductase (Eastmond, 2007). The sucrose dependency of these mutants is considered to reflect a lack of gluconeogenesis as a consequence of reduced fatty acid b-oxidation or a blocked glyoxylate cycle.
+
+Many of the above sucrose-dependent mutants with defects in b-oxidation are able to grow in toxic levels of 2,4-DB or IBA. The phenotype of the Atscp2-1 mutant resembles that of the mfp2, lacs6lacs7, acx1acx2, and sdp2 mutants, which are sucrose-dependent but still sensitive to 2,4-DB and IBA (Fulda et al., 2004; PinfieldWells et al., 2005; Rylott et al., 2006; Eastmond, 2007).
+
+The phenotype of the lacs6lacs7 mutant may be explained by the fact that probably another CoA-synthetase is capable of activating 2,4-DB and IBA to the
+
+| to icl-2 and mls-2. NA, not applicable (see Cornah et al., 2004). Gene Descriptiona   | Atscp2-1b                      | icl-2bc   | mls-2bc   |    |
+|---------------------------------------------------------------------------------------|--------------------------------|-----------|-----------|----|
+| THA1                                                                                  | Threonine aldolase             | +         | +         | +  |
+| (At1g08630) At1g21400                                                                 | 2-Oxoisovalerate dehydrogenase | +         | +         | NA |
+| At2g05540                                                                             | Gly-rich protein               | +         | +         | +  |
+| ASN1                                                                                  | Gln-dependent Asn-synthetase   | +         | +         | +  |
+| (At3g47340) At5g20230                                                                 | Blue-copper binding protein    | +         | +         | NA |
+| At5g50600                                                                             | Hydroxysteroid dehydrogenase 1 | +         | +         | NA |
+| At3g14210                                                                             | Myrosinase-associated protein  | -         | NA        | -  |
+| At1g52060                                                                             | Jasmonate-inducible protein    | -         | NA        | -  |
+
+| d-old seedlings of wild-type and Atscp2-1 Compound Wild-type Relativea mean area 699% confidence interval   | Atscp2-1 Relativea mean area 699% confidence interval   |               |
+|-------------------------------------------------------------------------------------------------------------|---------------------------------------------------------|---------------|
+| Asp                                                                                                         | 14 12263572                                             | 880862170     |
+| b-Ala                                                                                                       | 7986374                                                 | 396665        |
+| Fumaric acid                                                                                                | 15826412                                                | 8876163       |
+| Gln                                                                                                         | 174 860637561                                           | 89 282610191  |
+| Glyceraldehyde                                                                                              | 8156266                                                 | 323671        |
+| Pyroglutamic acid                                                                                           | 312 447655162                                           | 182 199618855 |
+| Ribose                                                                                                      | 8846265                                                 | 458654        |
+| Asn                                                                                                         | 647761874                                               | 11 38061560   |
+| 3-Cyanoalanine                                                                                              | 7526100                                                 | 1171697       |
+| Gly                                                                                                         | 22 28963940                                             | 50 760611217  |
+| 5-Methylthiopentanenitrileb                                                                                 | 060                                                     | 8766136       |
+| Ser                                                                                                         | 27 87969060                                             | 58 18965825   |
+| a Corrected for internal standards and weight. b Tentative identification.                                  |                                                         |               |
+
+corresponding CoA-thioester before b-oxidation. Moreover, IBA and 2,4-DB sensitivity shows that the deficiency of the two long chain acyl-CoA synthetases does not cause a general block of the b-oxidation cycle in the lacs6lacs7 mutant. The 2,4-DB sensitivity of mfp2 mutants is probably due to that the other A. thaliana multifunctional protein AIM1 is active on 2,4-DB in a mfp2 background (Rylott et al., 2006). In the case of the acx1acx2 mutants, the 2,4-DB sensitivity could be due to ACX1 and ACX2 showing specificity for medium to very long chain acyl-CoA species, whereas 2,4-DB is metabolized by ACXs with short-chain specificity as suggested by Pinfield-Wells et al. (2005). The 2,4-DB sensitivity shown for sdp2 was hypothesized to be due to many peroxisomal matrix and membrane proteins remaining functional in the mutant (Eastmond, 2007). The IBA and 2,4-DB sensitivity of the Atscp2-1 mutant show that AtSCP2 is not required for b-oxidation of those compounds, and that the b-oxidation cycle is not blocked in the mutant. Furthermore, the docking analysis of IBA and 2,4-DB shows that they are not the most favourable ligands for AtSCP2, indicating that AtSCP2 would not be involved in the binding or transfer of the auxin precursors.
+
+The Atscp2-1 mutant shows a compromised germination, with slower germination kinetics as well as a lower germination frequency. Unlike the post-germinative growth phenotype, the germination phenotype of Atscp2-1 is not rescued by the addition of exogenous sugar. The slow germination phenotype of Atscp2-1 resembles the phenotype of the lac6lac7 double mutant which reached 50%
+germination in 4.5 d compared to 1.5 d for wild-type (Footitt et al., 2006). Sucrose-independent germination phenotypes have also been shown for acx1acx2, ped1/ kat2, and cts mutants (Pinfield-Wells et al., 2005). Seeds from these mutants showed germination frequencies below 30%, indicating a more severe block in germination compared to Atscp2-1. Seeds from ped1/kat2 and cts mutants contain significant amounts of sucrose (Footitt et al., 2002; Pritchard et al., 2002) suggesting that the germination phenotypes are not due to a limited supply of soluble sugars. Rather, as suggested recently (Pinfield-Wells et al., 2005) the b-oxidation pathway may, during germination, be utilized for the synthesis of a specific signal molecule promoting germination, or to degrade a molecule that inhibits germination. This capacity may then be compromised in Atscp2-1, lac6lac7, acx1acx2, ped1/kat2, and cts mutants leading to an inhibition of germination.
+
+During development and maturation of A. thaliana seeds, the total fatty acid levels in the seeds increase, until the later stages of seed maturation when the levels drop to close to 30% relative to the peak levels (Baud et al, 2002). Metabolic studies of developing embryos of Brassica napus showed that at least 10% of the fatty acids stored as triacylglycerol was lost during the desiccation phase of seed development. Interestingly, metabolic labelling of the embryos revealed that b-oxidation was not associated with net gluconeogenic activity (Chia et al., 2005). The function of fatty acid breakdown during seed development remains unclear, nevertheless, the activity during seed maturation may explain the altered morphology of the Atscp2-1 seeds if AtSCP2 is involved in peroxisomal lipid utilization. We were somewhat surprised to find that sucrose stimulated the expression of AtSCP2. Rather, it had been expected that sucrose would repress the expression, as increased levels of sucrose could be signalling a limited need for lipid utilization. Further investigations of the function of AtSCP2 may possibly reveal the significance of this observation.
+
+The Atscp2-1 seedlings had elevated levels of Asn, Ser, and Gly, and decreased levels of Asp, Glu, and Gln.
+
+Changed levels of Gln, Ser, and Gly were previously reported for icl-2 and mls-2 seedlings (Cornah et al., 2004). Asn accumulates in response to sugar starvation, probably due to an increase in expression of Asn synthase
+(Lam et al., 1994; Azevedo et al., 2006), which catalyses the formation of Asn through the transfer of an amide group from Gln to Asp. Our microarray experiments revealed overaccumulation of the ASN1 transcript in Atscp2-1 seedlings (see Supplementary Table S1 at JXB
+online) which suggests that the modified levels of Asn, Asp, Gln, and Glu in the Atscp2-1 mutant are consequences of increased accumulation of Asn synthase. Interestingly, ASN1 had elevated expression levels also in icl-2 and mls-2 seedlings (Cornah et al., 2004). Why are deficiencies in AtSCP2, ICL, and MLS leading to increased expression of ASN1? Possibly, when an exogenous carbon source is lacking, these mutant seedlings are in a condition suggestive of sugar starvation. Sugar starvation triggers ASN1 expression and leads to increased levels of Asn. Atscp2-1 also had increased levels of Gly and Ser. This was also seen for the mls-2 seedlings. In the case of mls-2, Cornah et al. (2004) hypothesized that when the glyoxylate cycle is blocked at MLS, glyoxylate feeds into the photorespiratory pathway. The increased activity of the photorespiratory pathway then leads to elevated levels of Gly and Ser. The elevated levels of Gly and Ser in Atscp2-1 possibly indicate that AtSCP2 also functions in the glyoxylate cycle. This hypothesis may also be supported by the decreased levels of fumarate in Atscp2-1. An underperforming glyoxylate cycle in Atscp2-1 may lead to lowered levels of fumarate, as fumarate is produced in the citric acid cycle from succinate, which is a product of the glyoxylate cycle. The elevated Gly levels may also, at least partially, be due to the demonstrated overexpression of THA1 in Atscp2-1. THA1, which is mainly expressed in seeds and young seedlings, encodes a Thr aldolase functioning in Thr catabolism where it is catalysing the conversion of Thr to Gly (Joshi et al.,
+2006). A tha1 mutant has a 50% decrease in Gly content (Joshi et al., 2006), which indicates that it is not unlikely that the overexpression of THA1 in Atscp2-1 would result in elevated Gly levels. It is interesting that the expression of THA1 was also induced in mls-2 and icl-2 seedlings
+(Cornah et al, 2004).Thus, it can not be excluded that the elevated Gly levels in mls-2 seedlings is, to some extent, related to increased THA1 activity.
+
+We have presented data here showing that the activity of the peroxisomal protein AtSCP2 is important for metabolism in A. thaliana. A function in b-oxidation is supported from the co-expression with MFP2, PED1, and PMDH1. The common pattern seen in animals, fungi, and protists with fusions of SCP-2 domains to catalytic domains involved in b-oxidation could also support a direct involvement in b-oxidation for AtSCP2. However, the 2,4-DB-sensitivity shown for Atscp2-1 may suggest that AtSCP2 is not required for b-oxidation, at least not for all compounds. A function related to the glyoxylate cycle is supported by the similarities between Atscp2-1 and the glyoxylate cycle mutants icl-2 and mls-2, as revealed by transcriptome and metabolome analyses. Another possibility that cannot be excluded is that the function of AtSCP2 is not directly connected to a specific metabolic pathway. Rather, AtSCP2 could be involved in the transport, solubilization, and presentation of hydrophobic reaction intermediates from several metabolic pathways.
+
+Further detailed investigations, including lipid profiling of Atscp2-1 seedlings, as well as AtSCP2-protein and AtSCP2-ligand interactions may provide us with additional clues to the biological function of AtSCP2.
+
+## Supplementary Data
+
+Supplementary data are available at JXB online.
+
+Table S1. Ranking of genes with significantly altered expression in Atscp2-1 relative to wild-type.
+
+Table S2. Comparison of results from microarray and quantitative real-time reverse transcriptase PCR (qRTPCR) analysis of gene expression in wild-type and Atscp2-1 seedlings.
+
+## Acknowledgements
+
+We thank Maria Andersson, Gunilla Swa¨rd, and Gun Ro¨nnqvist for technical assistance, Kristina Blomqvist for comments on the manuscript, and Jens Sundstro¨m for tubulin primers and advice on real-time PCR. We thank Anette Hagberg for help with designing and analysing microarray experiments. This work was supported by The Swedish Research Council (JE), Carl Trygger Foundation (JE),
+China Scholarship Council (BSZ), and the Natural Science Foundation of Zhejiang Province (Y305314) (BSZ).
+
+## References
+
+Adamski J, Normand T, Leenders F, Monte D, Begue A,
+Stehelin D, Jungblut PW, de Launoit Y. 1995. Molecular cloning of a novel widely expressed human 80 kDa 17 b-hydroxysteroid dehydrogenase IV. Biochemical Journal 311, 437–443.
+
+Adham AR, Zolman BK, Millius A, Bartel B. 2005. Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in beta-oxidation. The Plant Journal 41, 859–874.
+
+Allemeersch J, Durinck S, Vanderhaeghen R, et al. 2005.
+
+Benchmarking the CATMA microarray. A novel tool for Arabidopsis transcriptome analysis. Plant Physiology 137, 588–601.
+
+Azevedo RA, Lancien M, Lea PJ. 2006. The aspartic acid metabolic pathway, an exciting and essential pathway in plants.
+
+Amino Acids 30, 143–162.
+
+Baker A, Graham IA, Holdsworth M, Smith SM,
+Theodoulou FL. 2006. Chewing the fat: b-oxidation in signalling and development. Trends in Plant Science 11, 124–132.
+
+Baud S, Boutin JP, Miquel M, Lepiniec L, Rochat C. 2002. An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiology and Biochemistry 40, 151–160.
+
+Bloj B, Hughes ME, Wilson DB, Zilversmit DB. 1978. Isolation and amino acid analysis of a nonspecific phospholipid transfer protein from rat liver. FEBS Letters 96, 87–89.
+
+Chia TY, Pike MJ, Rawsthorne S. 2005. Storage oil breakdown during embryo development of Brassica napus (L.). Journal of Experimental Botany 56, 1285–1296.
+
+Choinowski T, Hauser H, Piontek K. 2000. Structure of sterol carrier protein 2 at 1.8 A˚ resolution reveals a hydrophobic tunnel suitable for lipid binding. Biochemistry 39, 1897–1902.
+
+Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743.
+
+Cornah JE, Germain V, Ward JL, Beale MH, Smith SM. 2004.
+
+Lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis mutants lacking the glyoxylate cycle enzyme malate synthase. Journal of Biological Chemistry 279, 42916–42923.
+
+Curtis MD, Grossniklaus U. 2003. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology 133, 462–469.
+
+Dyer DH, Lovell S, Thoden JB, Holden HM, Rayment I,
+Lan Q. 2003. The structural determination of an insect sterol carrier protein-2 with a ligand-bound C16 fatty acid at 1.35A
+resolution. Journal of Biological Chemistry 278, 39085–39091.
+
+Eastmond PJ. 2007. MONODEHYROASCORBATE
+REDUCTASE4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis. The Plant Cell 19, 1376–1387.
+
+Edqvist J, Blomqvist K. 2006. Fusion and fission, the evolution of sterol carrier protein-2. Journal of Molecular Evolution 62, 292–306.
+
+Edqvist J, Ro¨nnberg E, Rosenquist S, Blomqvist K, Viitanen L,
+Salminen TA, Nylund M, Tuuf J, Mattjus P. 2004. Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2. Journal of Biological Chemistry 279, 53544–53553.
+
+Eklund DM, Edqvist J. 2003. Localization of non-specific lipid transfer proteins correlate with programmed cell death responses during endosperm degradation in Euphorbia lagascae seedlings.
+
+Plant Physiology 132, 1249–1259.
+
+Footitt S, Slocombe SP, Larner V, Kurup S, Wu Y, Larson T,
+Graham I, Baker A, Holdsworth M. 2002. Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP. EMBO Journal 21, 2912–2922.
+
+Footitt S, Marquez J, Schmuths H, Baker A, Theodoulou FL,
+Holdsworth M. 2006. Analysis of the role of COMATOSE and peroxisomal b-oxidation in the determination of germination potential in Arabidopsis. Journal of Experimental Botany 57, 2805–2814.
+
+Fulda M, Schnurr J, Abbadi A, Heinz E, Browse J. 2004.
+
+Peroxisomal acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana. The Plant Cell 16, 394–405.
+
+Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N,
+Carde JP, Bryce JH, Graham IA, Smith SM. 2001. Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid b-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. The Plant Journal 28, 1–12.
+
+Gullberg J, Jonsson P, Nordstro¨m A, Sjo¨stro¨m M, Moritz T.
+
+2004. A strategy for identifying differences in large series of metabolomic samples analysed by GC/MS. Analytical Chemistry 76, 1738–1745.
+
+Haapalainen AM, van Aalten DM, Merilainen G, Jalonen JE,
+Pirila P, Wierenga RK, Hiltunen JK, Glumoff T. 2001. Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A˚ resolution.
+
+Journal of Molecular Biology 313, 1127–1138.
+
+Hayashi H, De Bellis L, Ciurli A, Kondo M, Hayashi M,
+Nishimura M. 1999. A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes. Journal of Biological Chemistry 274, 12715–12721.
+
+Hayashi M, Nito K, Takei-Hoshi R, Yagi M, Kondo M,
+Suenaga A, Yamaya T, Nishimura M. 2002. Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid b-oxidation. Plant and Cell Physiology 43, 1–11.
+
+Hayashi M, Toriyama K, Kondo M, Nishimura M. 1998. 2,4-
+Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid b-oxidation. The Plant Cell 10, 183–195.
+
+Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions: bglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6, 3901–3907.
+
+Jones G, Willett P, Glen RC. 1995. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. Journal of Molecular Biology 245, 43–53.
+
+Jones G, Willett P, Glen RC, Leach AR, Taylor R. 1997.
+
+Development and validation of a genetic algorithm for flexible docking. Journal of Molecular Biology 267, 727–748.
+
+Joshi V, Laubengayer KM, Schauer N, Fernie AR, Jander G.
+
+2006. Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool.
+
+The Plant Cell 18, 3564–3575.
+
+Jonsson P, Johansson A, Gullberg J, Trygg J, Jiye A, Grung B,
+Marklund S, Sjo¨stro¨m M, Antti H, Moritz T. 2005. High through-put data analysis for detecting and identifying differences between samples in GC/MS-based metabolomic analyses. Analytical Chemistry 77, 5635–5642.
+
+Kiema TR, Taskinen JP, Pirila PL, Koivuranta KT,
+Wierenga RK, Hiltunen JK. 2002. Organization of the multifunctional enzyme type 1: interaction between N- and Cterminal domains is required for the hydratase-1/isomerase activity. Biochemical Journal 367, 433–441.
+
+Lam HM, Peng SS, Coruzzi GM. 1994. Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana. Plant Physiology 106, 1347–1357.
+
+Laskowski RA. 1995. SURFNET: a program for visualizing molecular surfaces, cavities, and intermolecular interactions.
+
+Journal of Molecular Graphics 13, 323–330.
+
+Lee HK, Hsu AK, Sajdak J, Qin J, Pavlidis P. 2004. Coexpression analysis of human genes across many micro array data sets. Genome Research 14, 1085–1094.
+
+Leenders F, Dolez V, Begue A, Moller G, Gloeckner JC, de Launoit Y, Adamski J. 1998. Structure of the gene for the human 17b-hydroxysteroid dehydrogenase type IV. Mammalian Genome 9, 1036–1041.
+
+Lehtonen JV, Still DJ, Rantanen VV, et al. 2004. BODIL:
+a molecular modelling environment for structure-function analysis and drug design. Journal of Computer-Aided Molecular Design 18, 401–419.
+
+Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCT
+method. Methods 25, 402–408.
+
+Lo¨nnstedt I, Britton T. 2005. Hierarchical Bayes models for cDNA microarray gene expression. Biostatistics 6, 279–291.
+
+Lo¨nnstedt I, Speed TP. 2002. Replicated microarray data.
+
+Statistica Sinica 12, 31–46.
+
+Ohba T, Holt JA, Billheimer JT, Strauss 3rd JF. 1995. Human sterol carrier protein x/sterol carrier protein 2 gene has two promoters. Biochemistry 34, 10660–10668.
+
+Ohba T, Rennert H, Pfeifer SM, He Z, Yamamoto R, Holt JA,
+Billheimer JT, Strauss 3rd JF. 1994. The structure of the human sterol carrier protein X/sterol carrier protein 2 gene
+(SCP2). Genomics 24, 370–374.
+
+Pinfield-Wells H, Rylott EL, Gilday AD, Graham S, Job K,
+Larson TR, Graham IA. 2005. Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism. The Plant Journal 43, 861–872.
+
+Pracharoenwattana I, Cornah JE, Smith SM. 2005. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. The Plant Cell 17, 2037–2048.
+
+Pracharoenwattana I, Cornah JE, Smith SM. 2007. Arabidopsis peroxisomal malate dehydrogenase functions in b-oxidation but not in the glyoxylate cycle. The Plant Journal 50, 381–390.
+
+Pritchard SL, Charlton WL, Baker A, Graham IA. 2002.
+
+Germination and storage reserve mobilization are regulated independently in Arabidopsis. The Plant Journal 31, 639–647.
+
+Ren XY, Fiers MW, Stiekema WJ, Nap JP. 2005. Local coexpression domains of two to four genes in the genome of Arabidopsis. Plant Physiology 138, 923–934.
+
+Richmond TA, Bleecker AB. 1999. A defect in b-oxidation causes abnormal inflorescence development in Arabidopsis. The Plant Cell 11, 1911–1924.
+
+Ritter MC, Dempsey ME. 1971. Specificity and role in cholesterol biosynthesis of a squalene and sterol carrier protein. Journal of Biological Chemistry 246, 1536–1539.
+
+Rylott EL, Eastmond PJ, Gilday AD, Slocombe SP, Larson TR,
+Baker A, Graham IA. 2006. The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal b-oxidation is essential for seedling establishment. The Plant Journal 45, 930–941.
+
+Schauer N, Steinhauser D, Strelkov S, et al. 2005. GC-MS
+libraries for the rapid identification of metabolites in complex biological samples. FEBS Letters 579, 1332–1337.
+
+Schmid M, Davison TS, Henz SR, Pape UJ, Demar M,
+Vingron M, Scholkopf B, Weigel D, Lohmann JU. 2005. A gene expression map of Arabidopsis thaliana development.
+
+Nature Genetics 37, 501–506.
+
+Seedorf U, Ellinghaus P, Nofer JR. 2000. Sterol carrier protein-2.
+
+Biochimica et Biophysica Acta 1486, 45–54.
+
+Seedorf U, Raabe M, Ellinghaus P, et al. 1998. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-X
+gene function. Genes and Development 12, 1189–1201.
+
+Smyth GK, Yang YH, Speed T. 2003. Statistical issues in cDNA
+microarray data analysis. Methods in Molecular Biology 224, 111–136.
+
+Toufighi K, Brady SM, Austin R, Ly E, Provart NJ. 2005. The Botany Array Resource: e-Northerns, Expression Angling, and promoter analyses. The Plant Journal 43, 153–163.
+
+Trygg J, Wold S. 2002. Orthogonal projections to latent structures
+(O-PLS). Journal of Chemometrics 16, 119–128.
+
+Viitanen L, Nylund M, Eklund DM, Alm C, Eriksson AK,
+Tuuf J, Salminen TA, Mattjus P, Edqvist J. 2006. Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity. FEBS
+Journal 273, 5641–5655.
+
+Wanders RJ. 2004. Peroxisomes, lipid metabolism, and peroxisomal disorders. Molecular Genetics and Metabolism 83, 16–27.
+
+Wouters FS, Bastiaens PIH, Wirtz KWA, Jovin TM. 1998.
+
+FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO Journal 17, 7179–
+7189.
+
+Zimmermann P, Hennig L, Gruissem W. 2005. Gene-expression analysis and network discovery using Genevestigator. Trends in Plant Science 10, 407–409.
+
+Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W.
+
+2004. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiology 136, 2621–2632.
+
+Zolman BK, Bartel B. 2004. An Arabidopsis indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function. Proceedings of the National Academy of Sciences, USA 101, 1786–1791.
+
+Zolman BK, Silva ID, Bartel B. 2001. The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation. Plant Physiology 127, 1266–1278.

medical/md/PMC2687925.md

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+
+## Book Review
+
+The first essay in the collection is an autobiographical piece by Bliss himself. In it, he describes the course that his career prospects and world-view have taken since his childhood. The son of a physician, Bliss reports that "from the time I could talk I would automatically say that I wanted to grow up to be a doctor." However, he changed his mind at the age of fourteen:
+
+## Book Review By Christine
+
+Zadorozny, McGill University, by Haeman, Li and McKeller, eds University of Toronto Press, March 2008,464 pages ISBN-10: 0802090974 ISBN-13: 978-0802090973 Price: $75.00
+… there was a Sunday afternoon when Dad's and my Scrabble game was interrupted by the appearance at the office door of a policeman with a drunk in tow, the drunk having been in a fight and suffering a badly slashed face. Dad had to sew him up, suturing both inside and outside the cheek, and invited me to watch what would be a demonstration of his surgical skill […] with blood and alcohol fumes everywhere, reflecting on my own complete disinterest in and lack of manual skills, I decided that this was not what I wanted to do in life. And that was the end of my ambition to be a doctor.
+
+Michael Bliss taught and wrote upon a few basic historical themes - medicine, business, and politics - in analyses that were alternatively framed as large-scale events or as individual life stories. In his own academic career, he moved from one special subject to another, showing a certain defiance of narrow academic boundaries.
+
+Thus reads the introduction to Figuring the Social, a collection of essays published by the University of Toronto Press in honour of Canadian historian Michael Bliss. This anthology, edited by E.A. Heaman, Alison Li and Shelley McKellar, consists of dissertations written by students and colleagues of Bliss, who has produced notable works such as The Discovery of Insulin, Plague: A Story of Smallpox in Montreal and a biography of Sir William Osler, entitled William Osler: A Life in Medicine. In addition to his work in the area of medical history in Canada, Bliss has also written extensively about the business and political history of this country. It is, perhaps, due to the wide scope of Bliss's work that the essays presented in this anthology resist categorization under any particular theme. Rather, they vary quite widely in both style and subject matter, from one author's reflection on his professional relationship with Bliss to another's examination of the relationship between Charles Gordon (also known as novelist "Ralph Conner") and Gordon's son, King. While Bliss' dexterity with a wide variety of subjects is to be lauded, however, the attempt of the editors to mirror this diversity results in a compilation of essays that can seem out of place next to one another - a little more thematic cohesion would have given the collection more strength. That being said, there are some thought-provoking essays in this book that are well worth a read by anyone who is interested in Bliss' work, in his philosophies, or in the history of medicine in Canada.
+
+Bliss parallels this moment of revelation with a similar decision made by famous physician, teacher and essayist Sir William Osler. Bliss reports that Osler, the son of a Minister, also forsook his father's trade for another - with the belief that "man's redemption of man" could mean more on earth than the type of redemption that a Minister offered to his parishioners. Bliss situates himself firmly within this tradition, saying that "in looking at the history of medicine we are studying, revealing, and, when it is appropriate, celebrating human achievement aimed at the redemption and salvation of humankind. This is the great satisfaction I find in doing medical history." An excellent essay by Allison Li on the transformation of endocrinology brought on by insulin further highlights Bliss' fascination with the revivifying possibilities afforded by medicine, quoting him as saying that the effects of insulin were the "closest approach to the resurrection of the body that our secular society can achieve." 
+Figuring the Social contains essays on the subject of Michael Bliss' career path and influence as well as dissertations on subjects in politics, business, religion, family, health policy and the practice of medicine in Canada. This latter section contains historical pieces on subjects such as the response of the Canadian 94
+
+![0_image_0.png](0_image_0.png)
+
+government to the thalidomide tragedy, the origins of the Connaught Medical Research Laboratories in Toronto and their influence on Canadian Public Health Policy, and an essay entitled "Comfort, Security, Dignity: Home Care for Canada's Aging Veterans, 1977-2004," which provides an overview on Canada's policies on veteran health care during this time period. Other articles on the subject of health include a history of lobotomy in Ontario, a discussion of medical biography and autobiography as a genre, and an account of the pursuit to develop a mechanical heart undertaken by William Kolff, inventor of the artificial kidney. 
+
+This anthology might have been stronger if it had focused on one particular aspect of Bliss' work. For instance, Bliss' discussion of medical history in his essay is well paired with the historical accounts found in the later chapters on health in Canada. If, as Bliss describes, medicine is an attempt at "man's redemption of man" on earth, which is more successful: the practice of medicine itself or its figuration and chronicling in history? Medicine can prolong the life of individuals - history prolongs our memory of them and can figure them in new ways, beyond what is possible in a lifetime.
+
+According to Bliss, There is the immensely frustrating fact that health care offers only temporary salvation. It buys time, but the time always runs out - even for those virtually resurrected children who outlived every one of the discoverers of insulin. They're all dead now. We can assault morbidity, we can postpone mortality, but we can't change the absolute mortality rate, which is permanently stuck at 100 per cent. Which means, of course, that the deal Osler offers us, of salvation through physical health and health care - he called it a "ministry of health" - is hugely unsatisfactory compared to what Osler's father peddled, which was life everlasting.
+
+The strength of this anthology lies in its exploration of medicine's pursuit to preserve life. This is a goal that is both absolutely worth striving for and, as is illustrated by the dramatic outcomes of the discovery of insulin, is one that medicine can definitely achieve. And yet, as Bliss points out, it is eventually bound to fail as human life cannot be prolonged indefinitely. This is where the historian steps in to record and present these achievements to the generation to come, endowing them with a "life everlasting" of their own. 
+
+Christine Zadorozny (M.D.C.M., 2011) received her B.A. (Hons) in English Literature from the University of Ottawa and her M.A. at Queen's University. She is currently a medical student at McGill University.

medical/md/PMC2948382.md

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+# Diagnosing Malabsorption In The Intensive Care Unit
+
+![0_Image_0.Png](0_Image_0.Png) Martijn B Keur, Albertus Beishuizen And Adriaan A Van Bodegraven*
+
+Address: Departments of Intensive Care and Gastroenterology, VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
+* Corresponding author: Adriaan A van Bodegraven (v.bodegraven@vumc.nl)
+2010, 2:7 (doi:10.3410/M2-7)
+for non-commercial purposes provided the original work is properly cited. You may not use this work for commercial purposes. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/3.0/legalcode), which permits unrestricted use, distribution, and reproduction in any medium, F1000 Medicine Reports The electronic version of this article is the complete one and can be found at: http://f1000.com/reports/medicine/content/2/7
+
+## Abstract
+
+Malabsorption as a result of decreased intestinal function is a frequently occurring problem in intensive care units. Small bowel dysfunction may lead to malnutrition and may predispose patients to infectious complications (sepsis) and may be linked to increased hospitalization duration, morbidity and mortality. There are several small bowel function tests, such as faecal fat excretion and sugar absorption tests, but data specifically applicable to the intensive care setting are limited. Moreover, there are multiple confounders such as altered intestinal permeability, changed transit time and renal dysfunction. A more specific test, fasting plasma citrulline concentration, has poor test characteristics for detection of decreased intestinal function in patients with dysfunctional enterocytes. Therefore, a citrulline generation test was recently developed in order to measure the conversion of glutamine (administered either enterally or intravenously) into citrulline. This conversion takes place exclusively in a properly functioning enterocyte; therefore, citrulline generation upon glutamine stimulation may be a promising biomarker for assessment of the small bowel function.
+
+## Introduction And Context
+
+Malabsorption in intensive care unit (ICU) patients is arbitrarily defined as an intestinal absorption capacity of 85% or less. It is believed to be a frequently occurring problem in ICUs, particularly in patients suffering from sepsis or multiple organ dysfunction [1,2]. Malabsorption is the most important clinical sign of intestinal dysfunction or failure, reflecting various altered aspects of intestinal physiology due to a diseased state. Some of these altered aspects are coordinated intestinal transport
+(motility) and coordinated exocrine digestive functions of various organs like the pancreas, liver and jejunum (enterocytes). Other factors involve the intestinal wall function, like mucosal immunological response and intestinal barrier function, as well as visceral blood flow.
+
+Critically ill patients may be prone to a reduced enterocyte function due mainly to a reduced splanchnic blood flow. This dysfunction may subsequently contribute to (the alleged) bacterial translocation and ultimately sepsis and multi-organ dysfunction syndrome
+(MODS) [3,4]. Simple, clear-cut and validated tests to assess these various aspects of coordinated physiological functions of digestion are not available at the bedside.
+
+Nevertheless, adequate absorption of food is paramount to the improvement of the patient's condition and to the outcome of the critically ill patient [5]. This concise review aims to emphasize the importance of diagnosing malabsorption and to give new insights into how to assess intestinal function.
+
+Current tests In critically ill patients, it may be cumbersome to assess the various aspects of digestion as there are no validated tests to quantify the small bowel function. To meet this challenge, we tend to approach the problem as a failure of the intestinal tract comparable with other organ failures as a part of MODS (such as renal failure).
+
+However, a simple blood test or marker such as creatinine or BUN (blood urea nitrogen) levels is lacking in the evaluation of intestinal function. Thus, secondary variables, such as measurement of intestinal barrier function and intestinal absorption, are usually applied. Relatively common small bowel function tests are the sugar absorption test (SAT), quantification of fat excretion in stools (FES) and bomb calorimetry (BC).
+
+Sugar absorption test Gastrointestinal permeability can be measured by the SAT. During this test, a solution of multiple saccharides is orally administered [6-8]. The applied saccharides have different molecule sizes and are specifically chosen as these cannot be metabolized in the human body. The tight junctions in the tip of the villi allow translocation of small saccharides, whereas translocation of larger saccharides is restricted to the crypts [6]. In a normally functioning small bowel, the permeability for small saccharides is higher than for larger saccharides. However, under pathological conditions, the permeability for small saccharides decreases whereas the permeability for larger saccharides increases [7]. The amount of renallyexcreted saccharides (particularly the large saccharide/
+small saccharide ratio) is an indicator of small bowel absorptive function [7,8]. There are some flaws in this test. By means of SAT, intestinal (paracellular) permeability rather than (metabolic) enterocyte function is assessed [7]. In addition, Oudemans-van Straaten et al.
+
+[9] reported that the SAT is unreliable when used on ICU patients with multiple organ failure because there are various non-permeability-related confounders, such as impaired renal function and diminished intestinal motility. Also, oliguria/anuria limits its use in daily ICU practice.
+
+Quantification of fat excretion in stools This test aims to quantify the FES. The patient is given a diet containing a known high amount of fat to saturate the intestinal mechanisms and to challenge the intestinal maximal absorptive capacity. During this diet, the patient's faeces are sampled for 24 hours or, preferably, 72 hours.
+
+The amount of faecal fat can be measured by the Van de Kamer method, which allows the calculation of fat absorption capacity [10]. The underlying concept of FES
+is that malabsorption of fat correlates with malabsorption in general. However, Wierdsma and van Bodegraven [11]
+found that not all malabsorption states are detected by measuring fat in faeces. Faecal carbohydrate loss significantly contributed to the malnutrition state, and this contribution is overlooked by looking merely at FES [11].
+
+Bomb calorimetry Faecal energy is measured by combustion of a faecal sample and subsequent calculation of 72 hours of faecal energy loss, which is considered the (alleged) gold standard for intestinal digestive function [12,13]. Combining the energy (caloric) intake and the faecal energy loss into a formula allows the calculation of intestinal energy absorption (percentage). Again, data are scarce, specifically in the critically ill, in whom BC is hampered by problems with reliable collection of faeces during a full 3-day period [1].
+
+## Recent Advances
+
+Citrulline is an amino acid that is produced almost entirely from glutamine in the enterocytes. Because this conversion can take place only in a functioning enterocyte, citrulline has been proposed as an attractive and promising biomarker to assess the function of the small intestine [14,15]. Previous studies reported the value of fasting plasma citrulline concentration as a biomarker for enterocyte (intestinal) mass in a wide array of intestinal diseases such as coeliac disease, Crohn's disease, short bowel syndrome and radiation enteritis, and in recipients of intestinal transplants [16-21].
+
+Recently, however, fasting plasma citrulline concentration was found to be a poor predictor of the clinical relevance of decreased intestinal function in patients with short bowel syndrome and, in other words, failed to predict long-term dependence on total parenteral nutrition [22,23].
+
+Citrulline generation test The citrulline generation test (CGT) is a recently developed test to dynamically measure the conversion of glutamine into citrulline over time, following administration of glutamine as a dipeptide with alanine
+(Dipeptiven). Through the administration of a large quantity of glutamine-alanine, the capacity of this enterocytic metabolic pathway is challenged, and this challenge is manifested by a decreased conversion in patients with a decreased enterocyte mass. The CGT is validated in various intestinal diseases [14]. In practice, following a slow intravenous injection of 100 mL of glutamine-alanine, multiple blood samples are drawn at different times to determine the increment (in other words, generation) in serum level of citrulline by using reverse-phase high-performance liquid chromatography
+[24]. The CGT is easily applied in patients with suspected small bowel dysfunction. The citrulline generation [as defined by either the citrulline increase or the AUC (area under the curve)] correlates closely with enterocyte mass and enterocyte function and reflects the amount of functioning enterocytes to generate citrulline. In stable ICU patients, preliminary results from our own group indicate a reduced enterocyte mass that was associated with disease severity.
+
+## Implications For Clinical Practice
+
+The small intestine has many functions, two of which are of major importance in the setting of critical illness: nutrient and energy absorption and function of the intestinal barrier against antigens. The absorption of nutrients, water and drugs is an essential part of improving or regaining health [5]. Malnutrition and insufficient bioavailability of drugs can increase hospitalization stay, morbidity, mortality and costs [5]. The intestinal barrier function is a very important intestinal feature that keeps bacteria from translocating into the blood and thus prevents sepsis from originating from the gut [3,4]. It is obvious that dysfunction of the small intestine may have serious consequences and that identifying those patients who are at risk for developing malabsorption or those who are already malnourished is therefore pivotal.
+
+The most common applied intestinal function tests - faecal, urine and blood tests alike - all have several major flaws (in particular in very ill patients), which prevent these tests from being used and render it very difficult to accurately quantify intestinal function in the ICU setting.
+
+The CGT, a more functional test, measures the enterocyte function and is likely to play an important role in the assessment of intestinal function in the future. The CGT
+is relatively easy to perform but warrants further validation in several ICU patient cohorts by comparing it with one or more of the other small bowel function tests and by taking nutritional status into account.
+
+## Abbreviations
+
+BC, bomb calorimetry; BUN, blood urea nitrogen; CGT, citrulline generation test; FES, fat excretion in stools; ICU, intensive care unit; MODS, multi-organ dysfunction syndrome; SAT, sugar absorption test.
+
+## Competing Interests
+
+The authors declare that they have no competing interests.
+
+## References
+
+1. Strack van Schijndel RJ, Wierdsma NJ, van Heijningen EM, Weijs PJ, de Groot SD, Girbes AR: Fecal energy losses in enterally fed intensive care patients: an explorative study using bomb calorimetry. Am J Gastroenterol 2007, 102:1-6.
+
+2. Carlson GL: Surgical management of intestinal failure. Proc Nutr Soc 2003, 62:711-8.
+
+3. Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M,
+Meddings JB: Increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill ICU patients. Am J Respir Crit Care Med 1998, 158:444-51.
+
+4. Deitch EA: Multiple organ failure: pathophysiology and potential future therapy. Ann Surg 1992, 216:117-34.
+
+5. Martindale RG, McClave SA, Vanek VW, McCarthy M, Roberts P,
+Taylor B, Ochoa JB, Napolitano L, Cresci G; American College of Critical Care Medicine; A.S.P.E.N. Board of Directors: Guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: Society of Critical Care Medicine and American Society for Parenteral and Enteral Nutrition: Executive Summary. Crit Care Med 2009, 37:1757-61.
+
+6. Bjarnason I, MacPherson A, Hollander D: Intestinal permeability:
+an overview. Gastroenterology 1995, 108:1566-81.
+
+7. van Elburg RM, Uil JJ, Kokke FT, Mulder AM, van de Broek WG,
+Mulder CJ, Heymans HS: Repeatability of the sugar-absorption test, using lactulose and mannitol, for measuring intestinal permeability for sugars. J Pediatr Gastroenterol Nutr 1995, 20:184-8.
+
+8. Hessels J, Eidhof HH, Steggink J, Roeloffzen WW, Wu K, Tan G, van de Stadt J, van Bergeijk L: Assessment of hypolactasia and sitespecific intestinal permeability by differential sugar absorption of raffinose, lactose, sucrose and mannitol. Clin Chem Lab Med 2003, 41:1056-63.
+
+9. Oudemans-van Straaten HM, van der Voort PJ, Hoek FJ, Bosman RJ,
+van der Spoel JI, Zandstra DF: Pitfalls in gastrointestinal permeability measurement in ICU patients with multiple organ failure using differential sugar absorption. Intensive Care Med 2002, 28:130-8.
+
+10. Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA: Rapid method for determination of fat in feces. J Biol Chem 1949, 177:347-55.
+
+11. Wierdsma NJ, van Bodegraven AA: Energy losses from a short bowel: not only fat. Ned Tijdschr Geneeskd 2005, 149:381-4.
+
+12. Miller DS, Payne PR: A ballistic bomb calorimeter. Br J Nutr 1959, 13:501-8.
+
+13. Lovelady HG, Stork EJ: An improved method for preparation of feces for bomb calorimetry. Clin Chem 1970, 16:253-4.
+
+14. Peters JHC, Wierdsma NJ, Teerlink T, van Leeuwen PAM, Mulder CJJ,
+van Bodegraven AA: The citrulline generation test: proposal for a new enterocyte function test. Aliment Pharmacol Ther 2008, 27:1300-10.
+
+15. Crenn P, Messing B, Cynober L: Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction. Clin Nutr 2008, 27:328-39.
+
+16. Crenn P, Vahedi K, Lavergne-Slove A, Cynober L, Matuchansky C,
+Messing B: Plasma citrulline: a marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterology 2003, 124:1210-9.
+
+17. Miceli E, Poggi N, Missanelli A, Bianchi P, Moratti R, Corazza GR: Is serum citrulline measurement clinically useful in coeliac disease? Intern Emerg Med 2008, 3:233-6.
+
+18. Rhoads JM, Plunkett E, Galanko J, Lichtman S, Taylor L, Maynor A,
+Weiner T, Freeman K, Guarisco JL, Wu GY: Serum citrulline levels correlate with enteral tolerance and bowel length in infants with short bowel syndrome. J Pediatr 2005, 146:542-7.
+
+19. Lutgens LC, Blijlevens NM, Deutz NE, Donnelly JP, Lambin P, de Pauw BE: Monitoring myeloablative therapy-induced small bowel toxicity by serum citrulline concentration: a comparison with sugar permeability tests. Cancer 2005, 103:191-9.
+
+20. Papadia C, Sherwood RA, Kalantzis C, Wallis K, Volta U, Fiorini E,
+Forbes A: Plasma citrulline concentration: a reliable marker of small bowel absorptive capacity independent of intestinal inflammation. Am J Gastroenterol 2007, 102:1474-82.
+
+21. Pappas PA, Saudubray JM, Tzakis AG, Rabier D, Carreno MR, GomezMarin O, Huijing F, Gelman B, Levi DM, Nery JR, Kato T, Mittal N,
+Nishida S, Thompson JF, Ruiz P: Serum citrulline as a marker of acute cellular rejection for intestinal transplantation. Transplant Proc 2002, 34:915-7.
+
+22. Peters JHC, Wierdsma NJ, Teerlink T, van Leeuwen PAM, Mulder CJJ,
+van Bodegraven AA: Poor diagnostic accuracy of a single fasting plasma citrulline concentration to assess intestinal energy absorption capacity. Am J Gastroenterol 2007, 102:2814-9.
+
+23. Luo M, Fernández-Estívariz C, Manatunga AK, Bazargan N, Gu LH,
+Jones DP, Klapproth JM, Sitaraman SV, Leader LM, Galloway JR,
+Ziegler TR: Are plasma citrulline and glutamine biomarkers of intestinal absorptive function in patients with short bowel syndrome? J Parenter Enteral Nutr 2007, 31:1-7.
+
+24. Teerlink T, van Leeuwen PA, Houdijk A: Plasma amino acids determined by liquid chromatography within 17 minutes. Clin Chem 1994, 40/2:245-9.
+
+Page 3 of 3

medical/md/PMC3165116.md

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+# Comment On Cho Et Al.: Usefulness Of Fdg Pet/Ct In Determining Benign From Malignant Endobronchial Obstruction
+
+Astrid A. M. van der Veldt & Mark Lubberink & Adriaan A. Lammertsma & Egbert F. Smit Received: 26 March 2011 /Accepted: 23 April 2011 / Published online: 18 June 2011
+\# The Author(s) 2011. This article is published with open access at Springerlink.com Dear Sir, With interest we read the study by Cho et al [1], evaluating the usefulness of FDG PET/CT for differentiating malignant endobronchial lesions with distal atelectasis from benign bronchial stenosis. In this retrospective study, they adequately demonstrated the additional value of FDG PET/
+CT in this challenging clinical problem. Besides differentiation of endobronchial lesions, accurate discrimination between malignant tumours and atelectatic lung tissue can facilitate the definition of radiotherapy target volumes. As a result, the size of radiotherapy fields can be decreased [2],
+potentially reducing radiotherapy-induced side-effects.
+
+A reply to this letter can be found at doi:10.1007/s00330-011-2170-y.
+
+A. A. M. van der Veldt (*) : M. Lubberink : A. A. Lammertsma Department of Nuclear Medicine & PET Research, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands e-mail: aam.vanderveldt@vumc.nl E. F. Smit Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands For these purposes, the PET technique may be of additional value to discriminate the obstructive lesion from the atelectatic lung tissue. Although Cho et al [1]
+mentioned different patterns of FDG uptake in atelectatic lung tissue, we wonder whether the FDG uptake in the obstructive lesions could be adequately distinguished from the FDG uptake in the atelectatic lung tissue. As FDG
+uptake is dependent on perfusion for its delivery to tissue, variation in tissue vascularity may contribute to variable FDG uptake in obstructive atelectasis. In a previous PET/
+CT study, we quantified tumour perfusion in lung cancer patients using radiolabelled water ([15O]H2O) [3]. The measured perfusion values in atelectatic lung tissue differed significantly as compared with perfusion values in malignant tumours. Figure 1 shows an example of a patient with relatively high perfusion in atelectatic lung tissue as compared with the malignant tumour. These observations suggest that perfusion measurements may have additional value to discriminate atelectatic lung tissue from malignant lesions. Therefore, we believe that additional perfusion measurements of atelectatic lung tissue may improve diagnosis and radiotherapy planning of malignant endobronchial lesions.
+
+![1_image_0.png](1_image_0.png)
+
+Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
+
+## References
+
+1. Cho A, Hur J, Kang WJ, Cho HJ, Lee JH, Yun M, Lee JD (2011)
+Usefulness of FDG PET/CT in determining benign from malignant endobronchial obstruction. Eur Radiol 21:1077–1087. doi:10.1007/
+s00330-010-2006-1 2. Nestle U, Walter K, Schmidt S, Licht N, Nieder C, Motaref B,
+Hellwig D, Niewald M, Ukena D, Kirsch CM, Sybrecht GW,
+Schnabel K (1999) 18F-deoxyglucose positron emission tomography (FDG-PET) for the planning of radiotherapy in lung cancer:
+high impact in patients with atelectasis. Int J Radiat Oncol Biol Phys 44:593–597 3. Van der Veldt AA, Hendrikse NH, Harms HJ, Comans EF, Postmus PE, Smit EF, Lammertsma AA, Lubberink M (2010) Quantitative parametric perfusion images using 15O-labeled water and a clinical PET/CT scanner: test-retest variability in lung cancer. J Nucl Med 51:1684–1690

medical/md/PMC3168093.md

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+# Case Report
+
+www.ijpm.info
+
+# An Atypical Presentation Of Obsessive Compulsive Disorder With Difficulty In Hearing
+
+Indrapal Singh, Anshul K. Rana, Malay K. Singh, Rakesh K. Tripathi
+
+## Abstract
+
+Obsessive compulsive disorder (OCD) is a common psychiatric disorder which is easily recognized. However, sometimes patients of OCD present in such an atypical or bizarre way that their problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties. We are reporting a case of a man who had first sought opinion in the Department of Ear, Nose and Throat (ENT) for hearing impairment. He was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptom. As no pathology related to ENT or neurology could be detected, he was referred to the Department of Psychiatry. The patient's chief complaints were difficulty in hearing and inability to understand at once. He could be diagnosed as a case of OCD after meticulous evaluation and studying his response to treatment. There was significant improvement in all the presenting symptoms over a period of 6 weeks on 60 mg of fluoxetine.
+
+Key words: *Atypical presentation, hearing impairment, obsessive compulsive disorder*
+
+## Introduction
+
+Obsessive compulsive disorder (OCD) is a common psychiatric disorder (2% - 3% of the general population) which is diagnosed easily most of the time by the psychiatrist or even the physician.[1] It is sometimes very difficult to properly diagnose a person for OCD based only on an interview.[2] The presentation of OCD may be so atypical or unusual that the patients may be referred to different specialties before the actual diagnosis can be made. Neuropsychological studies have consistently found cognitive impairment in the domains of memory and attention in patients of OCD. Anxiety, lack of confidence, indecisiveness, associated clinical symptoms of OCD, along with impaired memory and attention, may further complicate or interfere in the delivery of information.[2,3,4]
+
+## Case Report
+
+A 36-year-old man used to request multiple repetitions of spoken words in order to understand what was said. Relatives and family members advised him to consult an ENT surgeon for hearing impairment. No organic cause of hearing impairment was found. He was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptoms. Even after multiple consultations with the ENT, neurology and medicine specialists, no organic pathology to explain his symptoms could be detected. The patient lost considerable time and money because of delay in diagnosis caused by the atypical presentation to specialties other than Psychiatry. At last, the patient was referred to the Department of Psychiatry for further evaluation. The patient's chief complaints were difficulty in hearing, grasping or comprehending the components of conversations, interactions or clinical interviews at once. He had this problem for the last 6 months. During the interview, almost every question or the instruction had to be repeated slowly 2 to 3 times or more to make him understand. He was anxious and expressed regret repeatedly for the inconvenience caused to the doctor because of his disability. Initially the patient was put on mirtazapine 15 mg/day and clonazepam 0.75 mg/day, keeping in mind the possibility of anxiety disorder NOS (not otherwise specified). There was no significant improvement even after 4 weeks of Department of Geriatric Mental Health, CSM Medical University, Consultant Psychiatrist, RML Hospital, Lucknow, India Address for correspondence: Dr. Anshul K. Rana, A-144, Indira Nagar, Lucknow - 226016, UP, India. E-mail: drakrana@yahoo.com DOI: 10.4103/0253-7176.63581 therapy. The case was reviewed from the diagnosis point of view. Mental state examination of the patient revealed impaired attention and concentration with depressive and anxiety symptoms. Detailed evaluation of the patient revealed that his mind remained full of unnecessary thoughts. However, the patient could not express further details to enable definite diagnosis. The possibility of OCD, predominantly obsessive type, was kept on the basis of answers of leading questions, and he was put on fluoxetine 60 mg/day (titrated from 40 to 60 mg) and clonazepam 0.5 mg/day. There was drastic improvement in all the presenting symptoms, including his ability to grasp and comprehend conversation, along with improvement in anxiety symptoms and clarity in thinking over a period of 6 weeks. The patient was then able to report his pre-treatment problem and the level of improvement comprehensively. He accepted that he was unable to understand or express his own problem before treatment. According to him, there were thoughts keeping his mind busy all the time, to the extent that he was unable to think or communicate his problem effectively. According to him, initially he used to make frequent unsuccessful attempts to get rid of these excessive and unnecessary thoughts. According to the wife of the patient, after treatment the patient was able to hear clearly. The diagnosis could be confirmed as OCD on the basis of patient's spontaneous elaboration of symptoms and the level of improvement.
+
+## Discussion
+
+In this case, the presentation of OCD is atypical, as the chief complaint was problem in hearing. He was able to hear on normal volume of sound, but the sentences needed to be repeated multiple times. He was not hard of hearing. Moreover, no ENT-related or neurological cause of hearing impairment was found in the patient. 
+
+Though hearing difficulty was not his chief subjective problem, he consulted the ENT doctor only after repeated and forced advice by his wife and relatives. The hearing problem as perceived by the patient's friends and relatives was basically a misinterpretation of the patient's problem secondary to his psychiatric illness. Also the patient was unable to express his actual problem at the time of presentation. The patient used to ask for repetition of the spoken sentences multiple times in order to grasp the components of interaction because of his significantly impaired attention. However, there could be the other possibility that this behavior could be a compulsive act of the patient to confirm his doubts. The patient could not confirm the latter. The patient could not express his problem elaborately, which was one of the reasons for not being able to make an early diagnosis. 
+
+There were multiple factors that had complicated and hindered the narration of history by the patient. Lack of clarity of thoughts and associated clinical symptoms of OCD (especially predominantly obsessive type), indecisiveness, anxiety, lack of confidence, along with impaired memory and attention, were the possible factors in this case, as also reported in different studies.[2,3,4] The patient could express details of his illness comprehensively once his above-mentioned signs and symptoms improved significantly. His wife and relatives also reported improvement in his hearing, which was actually the improvement in his attention and OCD symptoms. Such an atypical presentation may lead to misdiagnosis, delay in diagnosis, loss of time and money as the patient may present to other specialties instead of Psychiatry. High suspicion for diagnosis of OCD and response to treatment may help in managing such cases.
+
+## Conclusion
+
+Obsessive compulsive disorder is usually easily recognized, but sometimes its presentation is so atypical or bizarre that the problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties. But if proper evaluation is done, such cases can easily be recognized and treated effectively.
+
+## References
+
+1. Crino R, Slade T, Andrews G. The changing prevalence and severity of OCD criteria from DSM-III to DSM-IV. Am J Psychiatry 2005;162:876-82.
+
+2. Penzel F. Obsessive compulsive disorders. New York: Oxford University; 2000. p. 308.
+
+3. Muller J, Roberts JE. Memory and attention in obsessivecompulsive disorder: A review. J Anxiety Disord 2005; 19:1-28.
+
+4. Deckersbach T, Otto MW, Savage CR, Baer L, Jenike MA. The relationship between semantic organization and memory in obsessive-compulsive disorder. Psychother Psychosom 2000;69:101-7.
+
+Source of Support: Nil, **Conflict of Interest:** None.

medical/md/PMC4120424.md

@@ -0,0 +1,370 @@
+# Is There A Causal Link Between Knee
+
+loading and knee osteoarthritis progression? A systematic review and meta-analysis of cohort studies and randomised trials Marius Henriksen,1 Mark W Creaby,2 Hans Lund,3 Carsten Juhl,3,4 Robin Christensen1,3 To cite: Henriksen M,
+Creaby MW, Lund H, et al. Is there a causal link between knee loading and knee osteoarthritis progression? A
+systematic review and metaanalysis of cohort studies and randomised trials. BMJ Open 2014;4:e005368.
+
+doi:10.1136/bmjopen-2014005368
+▸ Prepublication history and additional material is available. To view please visit the journal (http://dx.doi.org/ 10.1136/bmjopen-2014005368). Received 31 March 2014 Revised 16 June 2014 Accepted 23 June 2014 For numbered affiliations see
+
+![0_image_0.png](0_image_0.png) end of article. Correspondence to Dr Robin Christensen; robin.christensen@frh. regionh.dk
+
+## Abstract
+
+Objective: We performed a systematic review, metaanalysis and assessed the evidence supporting a causal link between knee joint loading during walking and structural knee osteoarthritis (OA) progression.
+
+Design: Systematic review, meta-analysis and application of Bradford Hill's considerations on causation.
+
+Data sources: We searched MEDLINE, Scopus, AMED, CINAHL and SportsDiscus for prospective cohort studies and randomised controlled trials (RCTs) from 1950 through October 2013.
+
+Study eligibility criteria: We selected cohort studies and RCTs in which estimates of knee joint loading during walking were used to predict structural knee OA progression assessed by X-ray or MRI.
+
+Data analyses: Meta-analysis was performed to estimate the combined OR for structural disease progression with higher baseline loading. The likelihood of a causal link between knee joint loading and OA progression was assessed from cohort studies using the Bradford Hill guidelines to derive a 0–4 causation score based on four criteria and examined for confirmation in RCTs.
+
+Results: Of the 1078 potentially eligible articles, 5 prospective cohort studies were included. The studies included a total of 452 patients relating joint loading to disease progression over 12–72 months. There were very serious limitations associated with the methodological quality of the included studies. The combined OR for disease progression was 1.90 (95%
+CI 0.85 to 4.25; I2=77%) for each one-unit increment in baseline knee loading. The combined causation score was 0, indicating no causal association between knee loading and knee OA progression. No RCTs were found to confirm or refute the findings from the cohort studies.
+
+Conclusions: There is very limited and low-quality evidence to support for a causal link between knee joint loading during walking and structural progression of knee OA.
+
+Trial registration number: CRD42012003253
+
+## Strengths And Limitations Of This Study
+
+- We performed the first systematic evaluation of the evidence for a causal link between mechanical loading of the knee during walking and structural progression of knee osteoarthritis.
+
+- This systematic review, meta-analysis, and causation analysis find very limited evidence of a causal link between knee joint loading during walking and structural progression of knee osteoarthritis.
+
+- Few studies were included in the meta-analysis since only a small number of studies have been conducted. Further research is needed.
+
+## Background
+
+Osteoarthritis (OA) is the most common form of arthritis affecting a large section of the population, and is a major cause of illness and disability.1 2 The knee joint is most frequently affected,3 and due to the knee's crucial role in independent ambulation, knee OA leads to considerable disability affecting an individual's participation in society and independent living. Because no cure is available and many people are affected, healthcare costs associated with knee OA are enormous—even higher than for the more high-profile diseases such as diabetes, cancer and cardiovascular diseases.4 With its prevalence on the rise, knee OA poses a substantial socioeconomic and public health burden.
+
+Walking is the most common form of human locomotion, and most people walk every day, resulting in millions of steps per year. Biomechanical loading on the knee joint during walking is estimated to exceed 2–3 times body weight5; thus, knee joint loading is unavoidable component of an independent lifestyle. One widely accepted theory is that higher knee joint loading during walking is causally linked with accelerated structural knee OA progression.6–8 In accordance with this theory, unloading is advocated in an attempt to slow or halt disease progression.9 Unspecific measures of high joint loading, such as excess body mass and body mass index (BMI), have been associated with OA development,10 11 and obesity is believed to act mainly through high mechanical loads,12 although systemic effects have also been indicated.13 However, higher body mass does not necessarily lead to higher joint loading during walking,14 and more specific measures of joint loading are necessary to apply.
+
+Objective estimates of joint loading during walking can be obtained by three-dimensional gait analysis.
+
+Typically, the knee adduction moment (KAM) or biomechanical modelling of compression forces is used.
+
+The KAM has been of particular interest because it reflects the medial to lateral joint load distribution,15 with good face validity with respect to the relative prevalence of medial, as opposed to lateral compartment, tibiofemoral knee OA.16 Despite its common usage, a recent systematic review did not find consistent evidence to the fact that the KAM differs between those with and without knee OA.17 The relationship between knee joint loading during walking and knee OA has been a focus of knee OA-related biomechanical research for 10–20 years.
+
+Studies reveal that symptomatic treatment of knee OA results in increases in knee joint loads during walking,5 18–20 which are unwanted according to the hypothesised structural consequences of increased joint loading. This may generate confusion among researchers, clinicians and patients, who are interested in this information to aid them in prevention, palliation and treatment strategies. Establishing causal relationship among modifiable factors, such as knee joint loading, and disease-specific measures, such as structural disease progression, is imperative in the generation of effective strategies.
+
+Seminal epidemiological work by Hill proposed a set of considerations (the Bradford Hill criteria) to systematically evaluate the existence of a causal link between an exposure and a health outcome.21 These criteria have been previously employed to demonstrate the causal link between smoking and lung cancer,22 and between dietary factors and coronary heart disease,23 and have the capacity to evaluate the link between knee joint loading and structural knee OA progression.
+
+Therefore, the objectives of this study were (1) to systematically evaluate the evidence supporting a causal link between exposure to knee joint loading during walking and structural progression of knee OA based on Hill's considerations on causality21 and (2) to determine which knee joint load variables had been studied sufficiently in randomised controlled trials (RCTs) and found to support the findings of prospective cohort studies.
+
+## Methods
+
+The methods of the study search strategy, inclusion criteria and data analysis were prespecified in a protocol
+(see online supplementary file 1) and pre-registered
+(PROSPERO 2012:CRD42012003253).
+
+We searched MEDLINE, Scopus, AMED, CINAHL and SportsDiscus for prospective cohort studies and RCTs from 1950 through October 2013. Search strategies are available as an online supplementary file 2. The reference lists of the retrieved articles were also searched for additional cohort studies and RCTs. Two reviewers (MH and MWC) independently assessed study eligibility. Excluded studies and reasons for exclusion were recorded. Disagreement was resolved by discussion and consensus. We included original full-length articles pertaining to the association between knee joint loading during walking, measured by three-dimensional gait analysis, and structural disease progression assessed quantitatively or semiquantitatively by X-ray or MRI. We considered only those studies that included participants diagnosed with knee OA that were followed for at least 1 year (necessarily because of the sensitivity of imaging and slow disease progression24). Cohort studies had to include estimates of knee joint loading during walking at baseline and imaging-based assessment of structural disease progression. Clinical trials had to be randomised and compare image-based structural disease progression among different knee joint loading interventions (eg, increase vs decrease), placebo or control.
+
+## Data Extraction And Analysis
+
+The following data were extracted from the studies: (1)
+Study design; (2) Country of origin; (3) Number of subjects; (4) Characteristics of the subjects; (5) Type of knee joint loading estimate; (6) Magnitudes of the estimate; (7) Description of the interventions (if any) and
+(8) Structural disease progression outcome (ie, semiquantitative or quantitative measures on X-ray and/or MRI).
+
+For prospective cohort studies, we extracted estimates of the association between baseline knee joint loading and structural disease progression from baseline to follow-up. A progression group is typically compared to a non-progression group at baseline and the results are typically reported as ORs for disease progression for a one-unit higher baseline knee joint loading. If no OR was reported, we either calculated it from the reported data (if possible), or contacted the study authors and requested for the OR. For the RCTs we aimed to compare relative risks of structural disease progression between knee joint loading modification and control groups.
+
+The individual study results (ie, log OR values and their corresponding SEs) were combined in a random effects meta-analysis model using the generic inverse variance outcome type in Review Manager.25 Heterogeneity between trials was assessed using the standard Q-test statistic (testing the hypothesis of homogeneity), and we present the I2 value, which can be interpreted as the percentage of total variation across the studies due to heterogeneity.26 Methodological quality and risk of bias assessment The methodological quality of the cohort studies was assessed using a published check list.27 Two of the authors (MH and MWC) assessed this individually and judged each criterion to be 'Adequately described',
+'Unclear', or 'Inadequately described'–corresponding to
+'low risk of bias', 'unclear risk of bias', and 'high risk of bias', respectively. Disagreement was resolved by discussion. If an included study was authored by one or more of the current authors, a third reviewer (HL) was asked to perform a quality assessment. One quality assessment item was omitted ("Was a dose–response relationship between exposure and outcome demonstrated?")
+because this item relates to the findings of the study and not the methodological quality, and assessment of dose–
+response forms part of the Bradford Hill criteria for causation (see below). The overall extent of risk of bias and methodological quality within each study was assessed using the GRADE approach to evaluate study limitations.28 Evaluation of evidence for causality Based on the Bradford Hill considerations on causality,21 a causation score was developed and used to systematically evaluate the evidence of a causal link between knee joint loading during walking and structural progression of knee OA. A similar score has previously been derived from the Bradford Hill considerations to assess the causal relationship between dietary factors and coronary heart disease.23 The following criteria were used in the review of the cohort studies and given a score of 1 (criterion satisfied) or 0 (criterion not satisfied):
+1. Strength of association: Associations quantified as a pooled OR ≥5.0, with lower 95% CI above 2.0; the expected direction was defined as 'strong association.' 'Moderate association' is defined as any statistically significant pooled OR (p<0.05). A statistically non-significant pooled OR was defined as 'no association.'
+2. Consistency across studies: An association requires replication in other studies. Consistency is defined as
+≥75% of associations being strong or moderate.
+
+3. Temporality: Refers to temporal relationship of association between exposure and outcome; exposure has to precede outcome. It is difficult to ensure temporal correctness because study participants are assumed to walk daily and are, therefore, exposed to knee joint loading throughout observation periods. We retained this criterion because temporality is necessary to infer causation; absence of temporal relationship between exposure and outcome precludes a causal link. Our analysis accepted studies with a temporally correct design defined as baseline knee joint loading related to disease progression over time from that baseline. The temporality criterion was satisfied when temporal correctness was accepted in ≥75% of the included studies.
+
+4. Biological gradient: When rate of progression increases
+(or decreases) incrementally as dose of exposure increases; provides strong evidence of causal relationship. This criterion is satisfied when ≥75% of tests for a trend pertaining to structural progression outcomes are statistically significant in the expected direction for knee joint loading.
+
+The aforementioned four criteria scores were applied to derive one causation score for each knee joint loading variable. The scores were computed as the unweighted sum of the scores from each of the above criteria, for a possible range of 0–4. A score of 4 is considered strong evidence of a cause-and-effect relationship. A score of 3 is deemed to indicate moderate evidence. A score of 2 or less is considered weak evidence of causation.
+
+A fifth criterion, experimental evidence, was used to examine whether the evidence from the prospective cohort studies was consistent with that from RCTs.
+
+Experimental evidence enhances the probability of causation, and may be used to upgrade or downgrade the calculated causation score.
+
+The following four criteria were omitted:
+Coherence: Causation is more likely if what is observed is supported by and in agreement with the natural history of the disease. This criterion is usually applied when the outcome is assessed by surrogate outcomes.
+
+This criterion is omitted because it is satisfied by default since imaging modalities are surrogate outcomes.
+
+Plausibility relates to the assessment of whether the association is plausible or not. This criterion is omitted because of the highly subjective nature of this criterion.
+
+Specificity relates to the specific response to the exposure. This criterion is omitted because OA is a multistructure disease; an association between knee joint loading and disease progression in multiple structures does not preclude a possible causal relationship.
+
+Analogy relates to the possibility that existing similar association can support causation (eg, Does the same association exist for hip or hand OA? If so, causation may be supported). This criterion is omitted because this review focuses specifically on knee OA.
+
+## Results Study Selection And Characteristics
+
+The search yielded 1078 potentially eligible studies
+(figure 1; references available in online supplementary file 3). Of those, we included five prospective cohort studies published between 2002 and 2013 and involving a total of 452 patients with knee OA29–33 and 0 RCTs.
+
+The diagnostic criteria for knee OA used to determine participant inclusion in individual studies varied. The American College of Rheumatology (ACR) criteria34 were used in one study,32 whereas a combination of
+
+![3_image_0.png](3_image_0.png)
+
+clinical and radiographic signs of knee OA was used as criteria in three studies,29 31 33 and one study did not report the diagnostic criteria used.30 Study characteristics are summarised in table 1.
+
+Joint loading and structural progression measures Four cohort studies29–32 focused on the same knee joint loading estimate: the peak KAM. In these studies, peak KAM was assessed at baseline and related to structural medial disease progression assessed after a median of 15 months from that baseline (12–72 months). In one study,29 the baseline KAM impulse (area under the curve) was also related to medial disease progression. In the fifth study,33 overall knee compression loading was used as load exposure.
+
+Two studies30 31 defined structural disease progression as any worsening in semiquantitative radiographic medial joint space grade.36 One study32 defined structural disease progression as any loss of medial femoral cartilage volume (quantitative MRI) above a measurement error previously established. In another study,33 structural disease progression was assessed from semiquantitative grading of tibiofemoral cartilage loss and bone marrow lesions. In one study,29 structural progression was assessed semi-quantitatively as medial tibiofemoral cartilage loss and medial tibiofemoral bone marrow lesions, and quantitatively as medial tibial cartilage volume loss. For the meta-analysis, we extracted the association (OR) between peak KAM and semiquantitative progression in medial tibiofemoral cartilage loss. No ORs were available for the association between KAM and cartilage volume loss because both were continuous variables. However, the linear regression analysis was extracted for the assessment of the biological gradient criteria.
+
+Association between joint loading and progression ORs were not reported in three studies,31–33 but in32 individual patient data were reported and an OR was calculated. We contacted the authors of ref. 31 asking them to provide an OR if possible. The authors returned a conference abstract including an OR published 4 years prior to the article.35 The abstract recorded 64 patients in contrast to the 57 patients included in ref. 31 The authors could not account for the difference in number of the subjects between the full paper and conference abstract. One study33 grouped participants based on changes in joint loading following weight loss (increased vs reduced loads) and found no between group differences in structural disease progression. Owing to the use of an intervention (weight loss), it was not appropriate to extract OR for baseline loading.
+
+The ORs for structural disease progression ranged from 0.40 to 6.46 for each unit higher baseline peak KAM magnitudes. From the random effects model, the combined OR was estimated to 1.90 (95% CI 0.85 to 4.25). The confidence in this estimate was downgraded due to heterogeneity (I2=77%). Figure 2 summarises the individual and pooled estimates.
+
+In ref 29, the OR for progression of bone marrow lesions was 1.31 (95% CI 0.86 to 1.98) with every unit increment in baseline peak KAM; ORs for progression of cartilage defects and bone marrow lesions were 0.42 (95% CI 0.12 to 1.48) and 1.8 (95% CI 0.63 to 5.17), respectively, with every unit increment in baseline KAM impulse. Methodological quality of the studies One or more criteria were limited in the assessments of risk of selection, detection and attrition biases. Individual risk assessments are presented in online
+
+| Table 1                                                                                                                                                                                                                                                                                                                                                                                                                | Summary of the characteristics of the included studies   |                           |                                        |           |           |    |    |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------|---------------------------|----------------------------------------|-----------|-----------|----|----|
+| Author (year)                                                                                                                                                                                                                                                                                                                                                                                                          | Knee loading                                             | Structural                |                                        |           |           |    |    |
+| country                                                                                                                                                                                                                                                                                                                                                                                                                | variable                                                 | progression measure       | Number of patients (total/progressors/ | Females   |           |    |    |
+| non-progressors)                                                                                                                                                                                                                                                                                                                                                                                                       | (%)                                                      | Reference                 |                                        |           |           |    |    |
+| Miyazaki et al (2002) Japan                                                                                                                                                                                                                                                                                                                                                                                            | Follow-up time (months)                                  |                           |                                        |           |           |    |    |
+| Peak KAM (unit:                                                                                                                                                                                                                                                                                                                                                                                                        | ≥1 grade                                                 |                           |                                        |           |           |    |    |
+| %BW×HT)                                                                                                                                                                                                                                                                                                                                                                                                                | according to Altman atlas                                | sq                        | 72                                     | 74/42/32  | 78        | 30 |    |
+| X-ray                                                                                                                                                                                                                                                                                                                                                                                                                  | 18                                                       | 56 (64*)/41/15            | 59                                     | 31        |           |    |    |
+| sq                                                                                                                                                                                                                                                                                                                                                                                                                     | 35*                                                      |                           |                                        |           |           |    |    |
+| X-ray                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                          |                           |                                        |           |           |    |    |
+| Chang et al                                                                                                                                                                                                                                                                                                                                                                                                            | Peak KAM (unit:                                          | ≥1 grade                  |                                        |           |           |    |    |
+| (2007) USA                                                                                                                                                                                                                                                                                                                                                                                                             | %BW×HT)                                                  | according to Altman atlas |                                        |           |           |    |    |
+| Bennell et al (2011) Australia                                                                                                                                                                                                                                                                                                                                                                                         | Peak KAM (unit:                                          | ≥1 grade                  |                                        |           |           |    |    |
+| %BW×HT)                                                                                                                                                                                                                                                                                                                                                                                                                | medial tibiofemoral cartilage defects                    | sq                        | 12                                     | 138/45/93 | 56        | 29 |    |
+| MRI                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                          |                           |                                        |           |           |    |    |
+| KAM impulse                                                                                                                                                                                                                                                                                                                                                                                                            | Cartilage                                                |                           |                                        |           |           |    |    |
+| (unit: %BW×HT)                                                                                                                                                                                                                                                                                                                                                                                                         | volume loss (mm3 )                                       | q                         | 144/NA/NA                              |           |           |    |    |
+| MRI                                                                                                                                                                                                                                                                                                                                                                                                                    | 12                                                       | 13/6/7                    | 23                                     | 32        |           |    |    |
+| q MRI                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                          |                           |                                        |           |           |    |    |
+| Woollard et al (2011) USA                                                                                                                                                                                                                                                                                                                                                                                              | Peak KAM (unit:                                          | Cartilage                 |                                        |           |           |    |    |
+| Nm/kg)†                                                                                                                                                                                                                                                                                                                                                                                                                | volume loss (mm3 )                                       |                           |                                        |           |           |    |    |
+| Henriksen et al (2013) Denmark                                                                                                                                                                                                                                                                                                                                                                                         | Peak overall knee compression force (unit: N)            | Cartilage loss            | sq                                     | 12        | 157/NA/NA | 89 | 33 |
+| MRI                                                                                                                                                                                                                                                                                                                                                                                                                    | Median: 12                                               | Totals: 452/134/147       | Mean: 67                               |           |           |    |    |
+| *On request, the authors forwarded a conference abstract with additional data based on 64 subjects. The number of progressors/ non-progressors was not available from that abstract. †We converted the data into %BW×HT after requesting for body weight and height data from the authors. KAM, knee adduction moment; BW, body weight; HT, height; q, quantitative; sq, semiquantitative grading; NA, non-applicable. |                                                          |                           |                                        |           |           |    |    |
+
+supplementary appendix A. Across the studies, the comparability of the groups at baseline was generally inadequately or unclearly described (selection bias). Also, descriptions of how reliably the joint load exposures were ascertained were not included in four of the five studies (detection bias), and group differences in loss of subjects were not accounted for (attrition bias). The discrepancy in subject numbers in reports from one cohort,31 35 induced a high risk of selection and attrition bias. Table 2 presents summaries of the quality assessment. Agreement between quality raters was good
+(κ=0.73 (95% CI 0.48 to 0.97)).
+
+## Causality Assessment
+
+The non-significant combined OR indicates no association between knee joint load during walking and structural disease progression. Thus, a score of 0 in the Strength of Association criterion was assigned. The associations were not consistently replicated (50%),
+resulting in a score of 0 in the Consistency criterion. This finding was further supported by the heterogeneity between the studies (I2=77%; figure 2), and the discrepancies between baseline peak KAM magnitudes among progressors and non-progressors across the cohorts
+(figure 3). Temporal correctness was accepted in one cohort.30 After requesting for additional information from the authors of one cohort study,31 we discovered that the article reported only part of the study. The additional information retrieved35 showed that the study consisted of three measurements taken every 18 months. Joint space and Kellgren-Lawrence grades (X-rays) were recorded at all three time points, whereas the KAM was obtained at time points 2 and 3 (18 and 36 months). KAM at time point 2 (18 months) was associated with structural disease progression from 18 to 36 months and structural disease progression from time points 1 to 2
+
+ et al.   5
+
+| Figure 2                                                                                                                                                                                                          | Forest plot of the   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|
+| individual ORs of structural disease progression with every increment in baseline peak knee adduction moment. Weights are from a random effects analysis. Individual and pooled estimates are shown with 95% CIs. |                      |
+
+| Table 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | Summary of the quality assessment (risk of bias)                                           |                                             |         |          |           |    |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------|---------------------------------------------|---------|----------|-----------|----|
+| Bias                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Miyazaki                                                                                   | Chang                                       | Bennell | Woollard | Henriksen |    |
+| et al30                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | et al31 35                                                                                 | et al29                                     | et al32 | et al33  |           |    |
+| type                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Quality criteria                                                                           |                                             |         |          |           |    |
+| Selection                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Were the descriptions of the groups and the distribution of prognostic factors sufficient? | A                                           | I       | I        | A         | A  |
+| Were the groups assembled at a similar point in their disease progression?                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | A                                                                                          | I                                           | I       | A        | A         |    |
+| Were the groups comparable on all important confounding factors?                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | I                                                                                          | I                                           | I       | U        | A         |    |
+| Detection                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Was the joint load estimate                                                                | U                                           | U       | U        | U         | I  |
+| reliably ascertained? Was adequate adjustment made for the effects of these confounding variables?                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | A                                                                                          | I                                           | A       | I        | I         |    |
+| Was outcome assessment                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | A                                                                                          | A                                           | I       | I        | A         |    |
+| blind to exposure status? Was follow-up long enough                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | A                                                                                          | A                                           | A       | A        | A         |    |
+| for the outcomes to occur? What proportion of the                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | U                                                                                          | A                                           | U       | A        | A         |    |
+| cohort was followed-up?*                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                                                            |                                             |         |          |           |    |
+| Attrition                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Were dropout rates and reasons for drop-out similar across groups?                         | I                                           | I       | I        | A         | A  |
+| Overall risk of bias†                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Very serious limitations; high risk of bias                                                | Very serious limitations; high risk of bias |         |          |           |    |
+| Methodological quality                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Low                                                                                        | Low                                         | Low     | Low      | Low       |    |
+| *Adequate=follow-up proportion >80%; unclear 50–80%; inadequate=<50%. †Risk of bias within studies is assessed using GRADE's approach to study limitations: No serious limitation defined as all criteria being adequately described (high methodological quality); serious limitations defined as one criterion being inadequately described or >1 criterion being unclearly described (moderate methodological quality); very serious limitation defined as >1 criterion being inadequately described (low methodological quality). A, adequately described; I, inadequately described; U, unclear. Very serious limitations; high risk of bias Very serious limitations; high risk of bias Very serious limitations; high risk of bias |                                                                                            |                                             |         |          |           |    |
+
+(baseline to 18 months) was associated with increased KAM from 18 to 36 months.35 Thus, temporal correctness cannot be assigned to the observed associations in this study, and the overall Temporality criterion was not satisfied (a 0 score). Biological Gradient was investigated in one cohort29 that showed no positive association between baseline peak KAM and loss of medial tibial cartilage volume over 12 months, resulting in a score of 0. Consequently, the causation score based on the included cohort studies was 0 for the association between peak KAM during walking and structural disease progression in patients with knee OA (table 3).
+
+For the KAM impulse and overall knee compression loading, only one study was available for each exposure,29 33 and therefore no causation scores were calculated for these loading exposures.
+
+As the systematic literature search did not identify any RCTs, the findings from the cohort studies can neither be confirmed nor refuted by experimental evidence.
+
+Hence, the fifth criterion could not be used to upgrade or downgrade the causation scores.
+
+## Discussion
+
+This review is the first to systematically assess—using systematic review, meta-analysis, and the Bradford Hill considerations on causality—whether a causal link exists between knee joint loading during walking and structural disease progression of knee OA. The systematic literature search identified five prospective cohort studies focusing on three estimates of knee joint loading: the peak KAM, KAM impulse and overall tibiofemoral compression force. Using a predefined algorithm, a causation score of 0 was reached, showing that there is no evidence of a causal link between knee joint loading during walking and structural progression of knee OA.
+
+![6_image_0.png](6_image_0.png)
+
+Furthermore, no experimental data from RCTs exist to support or oppose the findings from the cohort studies.
+
+One of the prospective cohort studies33 compared increased versus decreased loads, without demonstrating differences in structural disease progression opposing a causal link.
+
+Indirectly supporting the current findings, a longitudinal randomised trial comparing laterally wedged insoles (previously demonstrated to reduce loading37 38)
+with placebo insoles, showed no group differences in structural disease progression.39 While that study seemingly provided experimental evidence, no joint load exposure data were included to document the assertion that joint loadings were in fact reduced in the 'wedged insole' group. Thus, the study did not fulfil the criteria for inclusion in this review. A recent meta-analysis on symptomatic effects of laterally wedged insoles showed no beneficial effects over neutral insoles, indicating limited effects of laterally wedged insoles.40 However, the null outcome is supported by the results in ref. 33, included in this review, showing neither detrimental effects of increased loading nor beneficial effects of decreased loading over 12 months in a group of knee OA patients.
+
+The quality assessment of the included studies showed high risks of selection, detection and attrition biases (table 3). An important precondition for valid results in cohort studies is that groups should be comparable at baseline. Unless the groups are balanced for relevant baseline characteristics, differences in structural disease progression cannot confidently be attributed to the effects of interest ( joint loads during walking). The included studies revealed significant inadequacies when assessing the comparability of the groups on relevant baseline characteristics, leading to prognostic imbalance.
+
+Four of the five cohort studies did not report how reliably the joint loading estimates were ascertained (detection bias), which could influence the individual results.
+
+Moreover, the loss of subjects at follow-up in two of the five cohort studies was beyond 20%, with the dropout rates and reasons for dropout across groups not adequately accounted for (attrition bias). Finally, the discrepant numbers of subjects presented in different reports from the same cohort31 35 introduced a serious risk of bias and low confidence in the results. The overall low methodological quality of the included studies gives little confidence in the reported estimates of causation.
+
+Despite the shortage of studies reporting a causal link between joint loading and structural disease progression, the conjectured relationship underlies the rationale for many clinical efforts to reduce knee loading during walking in attempts to retard disease progression.
+
+Weight loss, insoles, modified footwear, strengthening exercises, knee braces and gait modifications have been
+
+| Table 3                                                                                                                                                                                                                                                                         | Causation criteria and scores for the identified knee joint loading exposures   | Causation score (number of criteria met)   |            |            |     |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------|--------------------------------------------|------------|------------|-----|
+| Peak KAM                                                                                                                                                                                                                                                                        | 1.90 (0.85 to 4.25)                                                             | 2/4 (50%)                                  | 1/4 (25%)  | 0/4 (0%)   | 0   |
+| KAM impulse                                                                                                                                                                                                                                                                     | 0.42 (0.12 to 1.48)¶                                                            | −**                                        | 0/1 (0%)   | 1/1 (100%) | −†† |
+| Peak overall compression                                                                                                                                                                                                                                                        | No group difference                                                             | −**                                        | 1/1 (100%) | 0/1 (0%)   | −†† |
+| *Strong association is defined as a pooled OR ≥5 with lower 95% CI excluding 2.0. Moderate association is any statistically significant association. Strength* Summary Consistency† Temporality‡ or OR (95% CI) N (%) N (%) Biological gradient§ N (%) Knee joint load exposure |                                                                                 |                                            |            |            |     |
+
+Causation score (number of criteria met)
+Peak KAM 1.90 (0.85 to 4.25) 2/4 (50%) 1/4 (25%) 0/4 (0%) 0 KAM impulse 0.42 (0.12 to 1.48)¶ −** 0/1 (0%) 1/1 (100%) −†† Peak overall compression No group difference −** 1/1 (100%) 0/1 (0%) −††
+*Strong association is defined as a pooled OR ≥5 with lower 95% CI excluding 2.0. Moderate association is any statistically significant association.
+
+†Consistency is defined as ≥75% of associations showing strong or moderate associations.
+
+‡In cohort studies it is difficult if not impossible to ensure temporal correctness because participants in the studies are expected to walk daily and are, therefore, exposed to knee joint loading throughout the observation period. In the current analysis, the temporality criterion is satisfied in studies that relate baseline knee joint loading exposures to disease progression over time from that baseline and demonstrate a statistically significant association with structural disease progression. §Biological gradient is defined as demonstrated when the rate of progression increases (or decreases) incrementally as dose of exposure increases.
+
+¶Estimate based on one cohort study29 assessing baseline KAM impulse to progression of cartilage defects assessed by semiquantitative grading of MRI. **Consistency not possible to assess with only one cohort.
+
+††Causality score not calculated because only one study available.
+
+KAM, knee adduction moment.
+
+suggested as means to lower knee joint loads and thus slow structural disease progression through mechanical pathways,41–46 with effects yet to be proven. The rationale for such efforts is diluted by the current results, not only by the low causality score, but also by the inconsistency across studies in the loading magnitudes between individuals who progress and those that do not. Figure 3 illustrates that patients with a certain baseline peak KAM magnitude in one study progress, whereas those with similar baseline peak KAM magnitudes in other studies do not. These differences in knee joint loading magnitudes between studies may be due to differences in gait analysis protocols across the involved gait labs, for example, in the selection of reference frame convention for the calculation of the KAM47 and footwear condition.44 However, including diversity of measures of the same variable can strengthen the confidence in the results of a systematic review.
+
+The results from the included studies exhibited a high degree of heterogeneity (I2 from the meta-analysis)
+(figure 2). The two cohorts with significant OR30 31 35 both used radiography as the structural outcome assessment, whereas the studies reporting non-significant ORs used MRI.29 32 Furthermore, the two studies reporting significant associations had longer follow-up periods (72 and 18 months), whereas the studies without significant associations assessed disease progression after 12 months. This concurrence of follow-up time and imaging modality precludes conclusions about whether the differences in results are based on follow-up time or imaging modality. Radiography is the current clinical gold standard for assessing structural disease progression, but it has low temporal sensitivity, making long follow-up times necessary. MRI—with its shorter temporal sensitivity—is possibly the more promising tool for assessing single structures and should be used in studies with longer follow-up times. The total number of patients was 452 in five cohorts; that is a very small number of patients when considering the large population and when compared to similar reviews in other chronic diseases. For instance, a recent review of the causal relationship between dietary factors and coronary heart disease included several millions of individuals in 361 cohorts and a multitude of dietary exposures.23 Our study has strengths because we undertook several measures to minimise bias, restricting our review to studies with the strongest causal inference (cohort studies and RCTs), conducted independent assessments of study eligibility, and using predefined criteria to evaluate causation and methodological quality. We may be criticised for using arbitrary OR cut-offs to define strong and moderate association, but identifying the true cut-off for defining clinically meaningful OR values is impossible in a field that has few longitudinal studies to consult. An important limitation is the causality score that applied. The score is a modification of a previous similar score,23 thus it has limited empirical evidence to support its validity. Also, the causality score omitted several of the original causation considerations as proposed by Hill,21 yet the applied method was prespeci-fied. We may also be criticised for analysing any image-based structural deterioration, rather than separating the analyses in X-ray and MRI-based assessments.
+
+However, the analysis plans were also prespecified, without prior knowledge of the number of studies and specifics of the outcomes.
+
+Current guidelines on interpretation of data from observational studies and data combined from repeated observational studies (with a reasonable internal validity)
+would correspond to confidence in the estimate's being
+'Low-quality evidence', but could be upgraded if there is great confidence in the estimates and the effects are large.48 However, the current analysis revealed a large inconsistency among study results, poor precision of the combined analysis, and low methodological quality.
+
+Thus, we downgraded further to 'very low quality evidence'. The inconsistency of the available studies may limit the appropriateness of meta-analyses, yet support the overall conclusion of this systematic literature review.
+
+In conclusion, our systematic review, meta-analyses, and application of Bradford Hill's considerations on causation shows that there is no evidence of a causal link between estimates of knee joint loading during walking and structural progression of knee OA. Future large welldesigned prospective cohorts with subsequent confirmation from randomised trials are strongly recommended.
+
+The current study findings question the rationale behind clinical efforts to reduce knee joint loading during walking as such efforts are currently supported by very low-quality evidence showing no causal link between knee joint loading during walking and progression of knee OA.
+
+## Author Affiliations
+
+1The Parker Institute, Copenhagen University Hospitals Bispebjerg and Frederiksberg, Copenhagen, Denmark 2School of Exercise Science, Australian Catholic University, Brisbane, Queensland, Australia 3Faculty of Health Sciences, Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark 4Department of Rheumatology and Gerontology, Copenhagen University Hospital Gentofte, Copenhagen, Denmark Contributors MH, MWC, HL, CJ and RC were responsible for study concept and design. RC and MH supervised the study. HL and CJ performed the literature search. MH and MWC acquired the data, which were analysed and interpreted by MH, MWC, CJ, HL and RC. MH drafted the manuscript, which was critically revised for important intellectual content by MWC, HL, CJ and RC. RC did the statistical analysis. MH is the guarantor. Funding The Parker Institute holds a grant from The Oak Foundation. Competing interests MH was supported by a grant from The Danish Council for Independent Research|Medical Sciences (Grant number: 10-093704). Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement Extra data can be accessed via the Dryad data repository at http://datadryad.org/ with the doi:10.5061/dryad.b40hm. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
+
+## References
+
+1. Nguyen US, Zhang Y, Zhu Y, et al. Increasing prevalence of knee pain and symptomatic knee osteoarthritis: survey and cohort data.
+
+Ann Intern Med 2011;155:725–32.
+
+2. Felson DT, Naimark A, Anderson J, et al. The prevalence of knee osteoarthritis in the elderly. The Framingham Osteoarthritis Study.
+
+Arthritis Rheum 1987;30:914–18.
+
+3. Lawrence RC, Felson DT, Helmick CG, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum 2008;58:26–35.
+
+4. Aspden RM. Osteoarthritis: a problem of growth not decay?
+
+Rheumatology (Oxford) 2008;47:1452–60.
+
+5. Henriksen M, Simonsen EB, Alkjaer T, et al. Increased joint loads during walking—a consequence of pain relief in knee osteoarthritis. Knee 2006;13:445–50.
+
+6. Andriacchi TP, Mündermann A. The role of ambulatory mechanics in the initiation and progression of knee osteoarthritis. Curr Opin Rheumatol 2006;18:514–18.
+
+7. Felson DT. Osteoarthritis as a disease of mechanics. Osteoarthritis Cartilage 2013;21:10–15.
+
+8. Andriacchi TP. Valgus alignment and lateral compartment knee osteoarthritis: a biomechanical paradox or new insight into knee osteoarthritis? Arthritis Rheum 2013;65:310–13.
+
+9. Lafeber FP, Intema F, Van Roermund PM, et al. Unloading joints to treat osteoarthritis, including joint distraction. Curr Opin Rheumatol 2006;18:519–25.
+
+10. Lohmander LS, Gerhardsson dV, Rollof J, et al. Incidence of severe knee and hip osteoarthritis in relation to different measures of body mass: a population-based prospective cohort study. Ann Rheum Dis 2009;68:490–6.
+
+11. Coggon D, Reading I, Croft P, et al. Knee osteoarthritis and obesity.
+
+Int J Obes Relat Metab Disord 2001;25:622–7.
+
+12. Pottie P, Presle N, Terlain B, et al. Obesity and osteoarthritis: more complex than predicted! Ann Rheum Dis 2006;65:1403–5.
+
+13. Richette P, Poitou C, Garnero P, et al. Benefits of massive weight loss on symptoms, systemic inflammation and cartilage turnover in obese patients with knee osteoarthritis. Ann Rheum Dis 2011;70:139–44.
+
+14. DeVita P, Hortobagyi T. Obesity is not associated with increased knee joint torque and power during level walking. J Biomech 2003;36:1355–62.
+
+15. Zhao D, Banks SA, Mitchell KH, et al. Correlation between the knee adduction torque and medial contact force for a variety of gait patterns. J Orthop Res 2007;25:789–97.
+
+16. Ledingham J, Regan M, Jones A, et al. Radiographic patterns and associations of osteoarthritis of the knee in patients referred to hospital. Ann Rheum Dis 1993;52:520–6.
+
+17. Mills K, Hunt MA, Ferber R. Biomechanical deviations during level walking associated with knee osteoarthritis: a systematic review and meta-analysis. Arthritis Care Res (Hoboken) 2013;65:1643–65.
+
+18. Shrader MW, Draganich LF, Pottenger LA, et al. Effects of knee pain relief in osteoarthritis on gait and stair-stepping. Clin Orthop 2004;421:188–93.
+
+19. Schnitzer TJ, Popovich JM, Andersson GB, et al. Effect of piroxicam on gait in patients with osteoarthritis of the knee. Arthritis Rheum 1993;36:1207–13.
+
+20. Hurwitz DE, Sharma L, Andriacchi TP. Effect of knee pain on joint loading in patients with osteoarthritis. Curr Opin Rheumatol 1999;11:422–6.
+
+21. Hill AB. The environment and disease: association or causation?
+
+Proc R Soc Med 1965;58:295–300.
+
+22. DOLL R, Hill AB. Smoking and carcinoma of the lung; preliminary report. BMJ 1950;2:739–48.
+
+23. Mente A, de KL, Shannon HS, et al. A systematic review of the evidence supporting a causal link between dietary factors and coronary heart disease. Arch Intern Med 2009;169:659–69.
+
+24. Bellamy N, Kirwan J, Boers M, et al. Recommendations for a core set of outcome measures for future phase III clinical trials in knee, hip, and hand osteoarthritis. Consensus development at OMERACT
+III. J Rheumatol 1997;24:799–802.
+
+25. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177–88.
+
+26. Higgins JP, Thompson SG, Deeks JJ, et al. Measuring inconsistency in meta-analyses. BMJ 2003;327:557–60.
+
+27. Khan K, ter Riet G, Popay J, et al. Undertaking systematic reviews of research on effectiveness. STAGE II—conducting the review. PHASE 5—study quality assessment. Undertaking systematic reviews of research on effectiveness: CRD's guidance for carrying out or commissioning reviews. 2nd edn. York: Centre for Reviews and Dissemination, 2001.
+
+28. Guyatt GH, Oxman AD, Vist G, et al. GRADE guidelines: 4. Rating the quality of evidence—study limitations (risk of bias). J Clin Epidemiol 2011;64:407–15.
+
+29. Bennell KL, Bowles KA, Wang Y, et al. Higher dynamic medial knee load predicts greater cartilage loss over 12 months in medial knee osteoarthritis. Ann Rheum Dis 2011;70:1770–4.
+
+30. Miyazaki T, Wada M, Kawahara H, et al. Dynamic load at baseline can predict radiographic disease progression in medial compartment knee osteoarthritis. Ann Rheum Dis 2002;61:617–22.
+
+31. Chang A, Hurwitz D, Dunlop D, et al. The relationship between toe-out angle during gait and progression of medial tibiofemoral osteoarthritis. Ann Rheum Dis 2007;66:1271–5.
+
+32. Woollard JD, Gil AB, Sparto P, et al. Change in knee cartilage volume in individuals completing a therapeutic exercise program for knee osteoarthritis. J Orthop Sports Phys Ther 2011;41:708–22.
+
+33. Henriksen M, Hunter DJ, Dam EB, et al. Is increased joint loading detrimental to obese patients with knee osteoarthritis? A secondary data analysis from a randomized trial. Osteoarthritis Cartilage 2013;21:1865–75.
+
+34. Altman R, Asch E, Bloch D, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039–49.
+
+35. Sharma L, Dunlop D, Andriacchi T, et al. The adduction moment and knee osteoarthritis (OA), a longitudinal study. Arthritis Rheum 2003;48(9 Suppl):S452.
+
+36. Altman RD, Hochberg M, Murphy WA Jr, et al. Atlas of individual radiographic features in osteoarthritis. Osteoarthritis Cartilage 1995;3
+(Suppl A):3–70.
+
+37. Hinman RS, Bowles KA, Bennell KL. Laterally wedged insoles in knee osteoarthritis: do biomechanical effects decline after one month of wear? BMC Musculoskelet Disord 2009;10:146.
+
+38. Hinman RS, Bowles KA, Metcalf BB, et al. Lateral wedge insoles for medial knee osteoarthritis: effects on lower limb frontal plane biomechanics. Clin Biomech (Bristol, Avon) 2012;27:27–33.
+
+39. Bennell KL, Bowles KA, Payne C, et al. Lateral wedge insoles for medial knee osteoarthritis: 12 month randomised controlled trial. BMJ 2011;342:d2912.
+
+40. Parkes MJ, Maricar N, Lunt M, et al. Lateral wedge insoles as a conservative treatment for pain in patients with medial knee osteoarthritis: a meta-analysis. JAMA 2013;310:722–30.
+
+41. Gross KD, Hillstrom HJ. Noninvasive devices targeting the mechanics of osteoarthritis. Rheum Dis Clin North Am 2008;34:755–76.
+
+42. Bennell KL, Kean CO, Wrigley TV, et al. Effects of a modified shoe on knee load in people with and those without knee osteoarthritis.
+
+Arthritis Rheum 2013;65:701–9.
+
+43. Bennell KL, Egerton T, Wrigley TV, et al. Comparison of neuromuscular and quadriceps strengthening exercise in the treatment of varus malaligned knees with medial knee osteoarthritis: a randomised controlled trial protocol. BMC Musculoskelet Disord 2011;12:276.
+
+44. Kemp G, Crossley KM, Wrigley TV, et al. Reducing joint loading in medial knee osteoarthritis: shoes and canes. Arthritis Rheum 2008;59:609–14.
+
+45. Hinman RS, Bennell KL. Advances in insoles and shoes for knee osteoarthritis. Curr Opin Rheumatol 2009;21:164–70.
+
+46. Simic M, Hinman RS, Wrigley TV, et al. Gait modification strategies for altering medial knee joint load: a systematic review. Arthritis Care Res (Hoboken) 2011;63:405–26.
+
+47. Schache AG, Baker R, Vaughan CL. Differences in lower limb transverse plane joint moments during gait when expressed in two alternative reference frames. J Biomech 2007;40:9–19.
+
+48. Guyatt G, Oxman AD, Akl EA, et al. GRADE guidelines: 1.
+
+Introduction-GRADE evidence profiles and summary of findings tables. J Clin Epidemiol 2011;64:383–94.

medical/md/PMC4368931.md

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+# 25 Mg Versus 50 Mg Dose Of Rectal Diclofenac For Prevention Of Post-Ercp Pancreatitis In Japanese Patients: A Retrospective Study
+
+Takeo Yoshihara,1 Masayoshi Horimoto,1 Tetsuhisa Kitamura,2 Naoto Osugi,1 Tatsuro Ikezoe,1 Kaori Kotani,1 Toru Sanada,1 Churi Higashi,1 Daisuke Yamaguchi,1 Makiyo Ota,1 Tatsunori Mizuno,1 Yasukazu Gotoh,1 Yorihide Okuda,1 Kunio Suzuki1 To cite: Yoshihara T, Horimoto M, Kitamura T,
+et al. 25 mg versus 50 mg dose of rectal diclofenac for prevention of post-ERCP pancreatitis in Japanese patients: a retrospective study. BMJ Open 2015;5:
+e006950. doi:10.1136/ bmjopen-2014-006950
+▸ Prepublication history for this paper is available online. To view these files please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2014-006950). Received 18 October 2014 Revised 26 January 2015 Accepted 27 January 2015 1Department of
+
+![0_image_0.png](0_image_0.png)
+
+Gastroenterology, Osaka Saiseikai Senri Hospital, Suita, Osaka, Japan 2Division of Environmental Medicine and Population Sciences, Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Correspondence to Dr Masayoshi Horimoto; horimotomasa@hotmail.com
+
+## Abstract
+
+Objectives: The aim of the present study was to assess the appropriate administration dose of nonsteroidal anti-inflammation drugs to prevent pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP). Importantly, the 100 mg dose of diclofenac recommended in Western countries has not been permitted in Japan.
+
+Design: A retrospective study. Settings: A single centre in Japan. Participants: This study enrolled patients who underwent ERCP at the Department of Gastroenterology, Osaka Saiseikai Senri Hospital, from April 2011 through June 2013, and who received either a 25 or a 50 mg dose of rectal diclofenac after ERCP.
+
+Primary outcome measure: The occurrence of postERCP pancreatitis (PEP). A multivariate regression model was used to assess the effect of the 50 mg dose (the 50 mg group) of rectal diclofenac and to compare it to the occurrence of PEP referring to the 25 mg group.
+
+Results: A total of 155 eligible patients received either 25 mg (84 patients) or 50 mg (71 patients) doses of rectal diclofenac after ERCP to prevent PEP. The proportion of PEP was significantly lower in the 50 mg group than in the 25 mg group (15.5% (11/71) vs 33.3% (28/84), p=0.018). In a multivariate analysis, the occurrence of PEP was significantly lower in the 50 mg group than in the 25 mg group even after adjusting potential confounding factors (adjusted OR=0.27, 95% CI 0.11 to 0.70).
+
+Conclusions: From this observation, the occurrence of PEP was significantly lower among ERCP patients with the 50 mg dose of rectal diclofenac than among those with the 25 mg dose.
+
+## Introduction
+
+Endoscopic retrograde cholangiopancreatography (ERCP) is widely conducted as a therapeutic and diagnostic procedure for hepatobiliary-pancreatic diseases.1–6 However,
+
+## Strengths And Limitations Of This Study
+
+- This study showed that the occurrence of postendoscopic retrograde cholangiopancreatography
+(ERCP) pancreatitis was significantly lower among ERCP patients with the 50 mg dose of rectal diclofenac than among those with the 25 mg dose.
+
+- This study was a retrospective study, so there was the potential of selection bias.
+
+- This study did not assess the effect of non-steroidal anti-inflammation drugs other than diclofenac for prevention of post-ERCP pancreatitis.
+
+the prevalence of ERCP adverse events has become high, and post-ERCP pancreatitis
+(PEP) has occurred in 1–30% of patients who have undergone ERCP. PEP can become severe and lead to death among these patients.1–6 Therefore, many studies on risk factors, preventive manoeuvres and drug administrations for PEP have been conducted worldwide.1–11 Recently, numerous reports have been published on the effectiveness of rectal administration of non-steroidal anti-inflammation drugs (NSAIDs) to prevent PEP.11–15 Several randomised controlled trials revealed that the 100 mg dose of rectal diclofenac/indomethacin after ERCP significantly reduced PEP occurrence compared with nonadministration,11–15 and the rectal administration of NSAIDs after ERCP has been widely accepted worldwide, including in Japan.16 A
+meta-analysis demonstrated that NSAID
+administration for PEP prevention was effective compared with non-administration, and its effectiveness is currently being established.11 However, little is known about the appropriate NSAID dose to prevent PEP.17 In addition, the 100 mg dose of diclofenac recommended in Western countries has not been legally permitted in Japan.16–18 The aim of this study was to compare a 25 mg dose of diclofenac to the 50 mg dose for the prevention of PEP
+among ERCP patients. Our hypothesis is that the occurrence of PEP would be lower in the 50 mg group than in the 25 mg group.
+
+## Methods Study Design
+
+This observation was a single-centre, retrospective study. We enrolled patients who underwent ERCP at the Department of Gastroenterological Medicine, Osaka Saiseikai Senri Hospital, from April 2011 through June 2013, who then received either a 25 or 50 mg dose of rectal diclofenac.
+
+## Settings And Data Collection
+
+Osaka Saiseikai Senri Hospital, located in the northern area of Suita City, Osaka, Japan, has 343 beds, seven attending doctors and four residents in its Department of Gastroenterology (50 beds). Approximately 250 patients with gastroenterological diseases annually undergo ERCP in this hospital. The side-viewing endoscopes used for ERCP were either TJF-260V or JF-260V
+(Olympus Corporation), and the appliance for cannulation was Tandem XL TAPERED TIP and Jagwire (0.035 inch; Boston Scientific Corporation).
+
+Among patients undergoing ERCP, either a 25 or 50 mg dose of rectal diclofenac was administered within 10 min immediately after ERCP for those more likely to develop PEP, based on the judgment of a main ERCP
+operator, and the dose (25 or 50 mg) of rectal diclofenac was decided comprehensively based on patient age, body mass index (BMI) and serum creatinine. In our hospital, during the study period, both sulbactam sodium/cefoperazone sodium 3.0 g/day and gabexate mesilate 0.3 g/day were administered to all patients for at least 2 days after ERCP, and blood tests such as serum pancreatic-type amylase and creatinine were also conducted the day after ERCP. In addition, when a doctor in charge diagnosed or suspected PEP in a patient who underwent ERCP, contrast-enhanced CT scanning and blood tests were conducted.17 Information on characteristics and outcomes of ERCP
+patients was retrospectively extracted from medical records, and the following data were included: gender, age, height, body weight, BMI, serum creatinine concentration; history of hepatobiliary, pancreatic, or gastrointestinal cancers; history of pancreatitis, first conducted ERCP, type of operator (resident or not), duration of ERCP examination, difficult cannulation and ERCP individual procedures (wire insertion into pancreatic duct, cytology of pancreas duct, pancreatography, cytology of bile duct), endoscopic sphincterotomy, endoscopic papillary balloon dilation, choledocholithotomy and stenting to pancreatic duct. Duration of ERCP examination was defined as the time interval from the side-viewing endoscope insertion to its removal,19 and the difficulty of cannulation was determined by a main operator or an attending doctor. The procedure for pancreatic duct was defined as any one of wire insertions into the pancreatic duct, its cytology or pancreatography.
+
+## Study End Point
+
+The primary outcome measure was the occurrence of PEP, defined as the development of both new or worsened abdominal pain and hyperamylasaemia based on the Cotton criteria.1 6 The secondary outcome measure was the occurrence of post-ERCP hyperamylasaemia, defined as a rise in the serum pancreatic-type amylase concentration to more than three times the upper limit of the normal laboratory value (>123 IU/mL) within 24 h after ERCP.1 6 In addition, death, severe acute pancreatitis, acute renal failure and gastrointestinal bleeding were defined as severe adverse events after rectal diclofenac administration.1 6 Severe acute pancreatitis was defined based on the Japanese guideline.17 Acute renal failure was defined as an increase of the serum creatinine ≥3 times over that before ERCP.20
+
+## Statistical Analyses
+
+Patient characteristics and outcomes between the two groups with 25 and 50 mg doses of rectal diclofenac were compared after ERCP. Continuous data were presented as the median (interquartile), differences were analysed by Mann-Whitney test, categorical data were expressed in percentages (n/N) and differences were analysed by χ2 test or Fischer's exact test.
+
+The multivariate logistic regression was used to assess the effect of 50 mg of rectal diclofenac (the 50 mg group) and to compare it to the occurrence of PEP and hyperamylasaemia referring to the 25 mg group.
+
+The ORs and their CIs were calculated. Potential confounding factors based on biological plausibility and previous studies were included in the multivariate analysis.5 6 21 These variables were gender (male, female),
+age (<65, ≥65 years old), BMI (<22, ≥22 kg/m2), type of operator (resident or not), difficult cannulation
+(yes, no), and procedure in pancreatic duct (yes, no). All statistical analyses were performed using SPSS V.22.0J (IBM Corp, Armonk, New York). All tests were two tailed, and p values of <0.05 were considered statistically significant.
+
+## Results
+
+During the study period, a total of 166 patients received either a 25 or 50 mg dose of rectal diclofenac after ERCP to prevent PEP and hyperamylasaemia. Excluding three patients who had pancreatitis and eight who were given NSAIDs before ERCP, 155 patients (84 in the 25 mg group and 71 in the 50 mg group) were eligible for our analysis.
+
+| Table 1                                                                                                                                                  | Characteristics of patients administered a 25 versus 50 mg dose of rectal diclofenac after ERCP Rectal diclofenac Total 25 mg 50 mg   | p Value          |                  |         |      |         |       |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|------------------|------------------|---------|------|---------|-------|
+| Men, % (n/N)                                                                                                                                             | 54.8                                                                                                                                  | (85/155)         | 46.4             | (39/84) | 64.8 | (46/71) | 0.022 |
+| Age (years), median (IQR)                                                                                                                                | 71.0 (63.0–76.0)                                                                                                                      | 73.5 (68.0–80.0) | 67.0 (57.0–74.0) | <0.001  |      |         |       |
+| Elderly aged ≥65, % (n/N)                                                                                                                                | 71.0                                                                                                                                  | (110/155)        | 82.1             | (69/84) | 57.7 | (41/71) | 0.001 |
+| BMI (kg/m2 ), median (IQR)                                                                                                                               | 22.5 (19.5–25.3)                                                                                                                      | 23.2 (21.2–25.6) | 21.9 (19.2–24.4) | 0.019   |      |         |       |
+| BMI <22                                                                                                                                                  | 59.6                                                                                                                                  | (90/151)         | 50.0             | (40/80) | 70.4 | (50/71) | 0.011 |
+| Cancer, % (n/N)                                                                                                                                          | 32.9                                                                                                                                  | (50/155)         | 34.1             | (28/82) | 31.4 | (22/70) | 0.722 |
+| History of pancreatitis, % (n/N)                                                                                                                         | 16.2                                                                                                                                  | (25/154)         | 13.3             | (11/83) | 19.7 | (14/71) | 0.278 |
+| First conducted ERCP, % (n/N)                                                                                                                            | 34.8                                                                                                                                  | (54/155)         | 33.3             | (28/84) | 36.6 | (26/71) | 0.669 |
+| Resident as a main operator, % (n/N)                                                                                                                     | 63.9                                                                                                                                  | (99/155)         | 71.4             | (60/84) | 54.9 | (39/71) | 0.033 |
+| Serum creatinine before ERCP (mg/dL)                                                                                                                     | 0.80 (0.70–0.90)                                                                                                                      | 0.80 (0.70–0.90) | 0.80 (0.70–1.00) | 0.642   |      |         |       |
+| Duration of examination, median (IQR)                                                                                                                    | 36.5 (26.0–51.3)                                                                                                                      | 37.0 (28.0–50.0) | 35.0 (23.0–53.0) | 0.568   |      |         |       |
+| Duration of examination >30 min, % (n/N)                                                                                                                 | 63.6                                                                                                                                  | (98/154)         | 66.3             | (55/83) | 60.6 | (43/71) | 0.463 |
+| Difficult cannulation, % (n/N)                                                                                                                           | 28.4                                                                                                                                  | (44/155)         | 27.4             | (23/84) | 29.6 | (21/71) | 0.763 |
+| Procedures for pancreatic duct, % (n/N)                                                                                                                  | 68.4                                                                                                                                  | (106/155)        | 69.0             | (58/84) | 67.6 | (48/71) | 0.847 |
+| Pancreatic duct wire, % (n/N)                                                                                                                            | 65.2                                                                                                                                  | (101/155)        | 65.5             | (55/84) | 64.8 | (46/71) | 0.929 |
+| Cytology of pancreas duct, % (n/N)                                                                                                                       | 19.4                                                                                                                                  | (30/155)         | 17.9             | (15/84) | 21.1 | (15/71) | 0.608 |
+| Pancreatography, % (n/N)                                                                                                                                 | 31.6                                                                                                                                  | (49/155)         | 34.5             | (29/84) | 28.2 | (20/71) | 0.397 |
+| Cytology of bile duct, % (n/N)                                                                                                                           | 18.7                                                                                                                                  | (29/155)         | 20.2             | (17/84) | 16.9 | (12/71) | 0.596 |
+| EST, % (n/N)                                                                                                                                             | 26.5                                                                                                                                  | (41/155)         | 23.8             | (20/84) | 29.6 | (21/71) | 0.417 |
+| EPBD, % (n/N)                                                                                                                                            | 5.8                                                                                                                                   | (9/155)          | 6.0              | (5/84)  | 5.6  | (4/71)  | 0.605 |
+| Choledocholithotomy, % (n/N)                                                                                                                             | 38.1                                                                                                                                  | (59/155)         | 41.7             | (35/84) | 33.8 | (24/71) | 0.315 |
+| Stenting to pancreatic duct, % (n/N)                                                                                                                     | 2.6                                                                                                                                   | (4/155)          | 2.4              | (2/84)  | 2.8  | (2/71)  | 0.624 |
+| BMI, body mass index; EPBD, endoscopic papillary balloon dilation; ERCP, endoscopic retrograde cholangiopancreatography; EST, endoscopic sphincterotomy. |                                                                                                                                       |                  |                  |         |      |         |       |
+
+Table 1 shows the baseline characteristics and ERCPrelated procedures of eligible patients with the 25 vs 50 mg of rectal diclofenac after ERCP. The proportion of males was significantly higher in the 50 mg group than in the 25 mg group (64.8% vs 46.4%, p=0.022),
+whereas median age was significantly lower in the 50 mg group than in the 25 mg group (67.0% vs 73.5%,
+p<0.001). The 50 mg group had significantly lower BMI
+than the 25 mg group (21.9 vs 23.2, p=0.019). The proportion of ERCP conducted by a resident as main operator was significantly lower in the 50 mg group than in the 25 mg group (54.9% vs 71.4%, p=0.022). Other factors did not differ between the groups.
+
+Among eligible patients receiving rectal diclofenac after ERCP, 25.2% (39/155) had PEP and 43.2%
+(67/155) had post-ERCP hyperamylasaemia. The proportion of PEP was significantly lower in the 50 mg group than in the 25 mg group (15.5% (11/71) vs 33.3% (28/84), p=0.018, figure 1), and the proportion of post-ERCP hyperamylasaemia was also significantly lower in the 50 mg group than in the 25 mg group
+(31.0% (22/71) vs 53.6% (45/84), p=0.005, figure 2).
+
+The concentration of serum pancreatic-type amylase after ERCP was significantly lower in the 50 mg group than in the 25 mg group (54.0 (30.8–189.3) vs 148.0 (40.0–502.0) IU/mL, p=0.006). As for severe events after ERCP, there were no deaths, acute renal failures or gastrointestinal bleeding in either group, but one patient in the 25 mg group had severe acute pancreatitis.
+
+In a multivariate analysis, the occurrence of PEP was
+
+![2_image_0.png](2_image_0.png)
+
+significantly lower in the 50 mg group than in the 25 mg group even after adjusting for potential confounding factors such as sex, age, BMI, the type of operator, difficult cannulation and procedures for pancreatic duct
+(adjusted OR=0.27, 95% CI 0.11 to 0.70, table 2). The occurrence of post-ERCP hyperamylasaemia was also significantly lower in the 50 mg group than in the 25 mg group (adjusted OR=0.35, 95% CI 0.15 to 0.84, table 3).
+
+![2_image_1.png](2_image_1.png)
+
+![3_image_0.png](3_image_0.png)
+
+## Discussion
+
+From a single-centre observational study, we demonstrated that the occurrence of PEP and post-ERCP hyperamylasaemia was significantly lower among ERCP
+patients on the 50 mg dose of rectal diclofenac than among those on the 25 mg dose, which suggested that the effectiveness of diclofenac for PEP was dose dependent. Importantly, the dose of diclofenac administration for patients was limited to a single maximum of 50 mg in Japan,16 17 but the preceding studies could not sufficiently compare the effects of 25 and 50 mg doses of rectal diclofenac for the prevention of PEP among ERCP patients. Therefore, our study provides helpful new clues for actual prevention of PEP at gastroenterological clinical settings in Japan.
+
+The present study showed that the prevention of PEP
+among ERCP patients by diclofenac was dosedependently effective. Previous randomised trials and meta-analyses indicated that 100 mg (not 50 mg) rectal NSAID administration was more effective for PEP prevention than non-administration.11–13 In a randomised
+
+| Table 2                                                                                                                                                                                                                                                                                                    | Association between 25 and 50 mg dose of   |                |       |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------|----------------|-------|
+| rectal diclofenac and post-ERCP pancreatitis OR (95% CI)                                                                                                                                                                                                                                                   | p Value                                    |                |       |
+| Post-ERCP pancreatitis Univariate                                                                                                                                                                                                                                                                          | 0.37                                       | (0.17 to 0.80) | 0.018 |
+| Multivariate Model 1                                                                                                                                                                                                                                                                                       | 0.31                                       | (0.13 to 0.75) | 0.009 |
+| Model 2                                                                                                                                                                                                                                                                                                    | 0.27                                       | (0.11 to 0.70) | 0.007 |
+| ORs were calculated for the 50 mg group referring to the 25 mg group. Model 1: Adjusted for sex, age and BMI. Model 2: Adjusted for sex, age, BMI, type of operator, difficult cannulation and procedures for pancreatic duct. BMI, body mass index; ERCP, endoscopic retrograde cholangiopancreatography. |                                            |                |       |
+
+control trial in Japan, the authors showed that low doses
+(either 25 or 50 mg) of rectal diclofenac were more effective in preventing PEP compared with the placebo group.
+
+16 From a subanalysis of the trial, the occurrence of PEP among patients administered 50 mg of rectal diclofenac tended to be lower than among those given 25 mg, although it was statistically insignificant (0% (0/29) vs 9%
+(2/22), p=0.101). This result was almost consistent with ours. No randomised trials directly compared a 25 mg of rectal diclofenac to a 50 mg dose for the prevention of PEP, but conducting randomised trials to determine the appropriate dose of NSAID administration for the prevention of PEP is important in Japan.
+
+Why does the administration of NSAIDs for ERCP
+patients prevent PEP? Although much of its preventive mechanism is unclear, it is suggested that the activation of trypsin/trypsinogen in acinar cells of the pancreas causes acute pancreatic inflammation by factors such as mechanical stimulus of the pancreatic duct, intestinal fluid flow into the pancreatic duct, increase in pancreatic duct internal pressure or pancreatic tissue pressure by pancreatography and pancreatic fluid congestion by Vater papilla oedema.11 22 23 In particular, phospholipase A2 synthesised in the pancreas regulates inflammation mediators such as prostaglandins, leukotrienes and thromboxanes by arachidonic acid cascade24 and NSAIDs, the cyclo-oxigenase inhibitors, prevent PEP by inhibiting its cascade.12 22–25 The efficacy of NSAIDs including diclofenac is reportedly dose dependent.26–28 This study, showing the dose-dependent effect of diclofenac for PEP prevention, suggested that sufficient doses of NSAIDs are needed to inhibit arachidonic acid cascades for PEP prevention.
+
+Gastrointestinal bleeding and acute renal failure were among the severe adverse events after NSAID administration.15 Preceding studies underscored the very low occurrence of severe adverse events such as gastrointestinal bleeding and acute renal failure after NSAID
+administration for PEP prevention.13 15 16 Even in the present study, there was no gastrointestinal bleeding or acute renal failure regardless of the dose of diclofenac,
+
+| Table 3                                                                                                                                                                                                                                                                                                                             | Association between 25 and 50 mg dose of   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------|
+| rectal diclofenac and post-ERCP hyperamylasaemia OR (95% CI) p Value Post-ERCP hyperamylasaemia Univariate 0.39 (0.20 to 0.75) 0.005 Multivariate Model 1 0.44 (0.21 to 0.92) 0.028 Model 2 0.35 (0.15 to 0.84) 0.018 ORs were calculated for the 50 mg group referring to the 25 mg group. Model 1: Adjusted for sex, age and BMI. |                                            |
+
+and diclofenac administration would thus be acceptable for PEP prevention. However, gastroenterologists in Japan tend to hesitate to use the 50 mg dose of rectal diclofenac for PEP prevention16 17 29 because of concerns such as gastrointestinal adverse events, acute renal failure, and decrease in blood pressure after NSAID
+administration.27–29 However, administering the 50 mg dose of rectal diclofenac for patients without problems such as renal dysfunction before ERCP should be recommended.
+
+NSAID administration for PEP prevention is given a grade A recommendation by the European Society of Gastrointestinal Endoscopy guideline, grade C1 by the Japanese guideline, and a moderate rating by the American College of Gastroenterology (ACG) guideline, respectively.17 30 31 Recently, the number of patients with acute pancreatitis has been excessively increasing,17 32 and its medical cost has also been climbing. Considering the limited medical resources in Japan, the 50 mg dose of rectal diclofenac for PEP prevention will surely become an established method to be administered without hesitation, because NSAIDs including diclofenac are inexpensive and have few adverse effects, and their administration method is also simple and easy. However, large-scale prospective multicentre observational or randomised trials are warranted to confirm our results.
+
+There are several limitations to this study. First, it was conducted retrospectively, and there was the potential of selection bias. For example, the proportion of PEP occurrence in this study was higher than that in previous studies,13 16 because either a 25 or 50 mg dose of diclofenac was selectively administered to ERCP patients who were more likely to develop PEP based on the judgment of a main ERCP operator. Second, our study did not assess the effect of NSAIDs other than diclofenac for PEP prevention. Third, unmeasured confounding factors may have influenced the association between the occurrence of PEP and rectal diclofenac administration among ERCP patients.
+
+## Conclusion
+
+From a single-centre observation in Osaka, this study demonstrated that the occurrence of PEP and post-ERCP hyperamylasaemia was significantly lower among ERCP patients with the 50 mg dose of rectal diclofenac than among those with the 25 mg dose. Further large-scale prospective multicentre observational or randomised controlled trials are warranted to confirm our results.
+
+Acknowledgements The authors are deeply indebted to the staff and physicians of Osaka Saiseikai Senri Hospital. Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None. Ethics approval The study was approved by the institutional review board of Osaka Saiseikai Senri Hospital.
+
+Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http:// creativecommons.org/licenses/by-nc/4.0/
+
+## References
+
+1. Cotton PB, Eisen GM, Aabakken L, et al. A lexicon for endoscopic adverse events: report of an ASGE workshop. Gastrointest Endosc 2010;71:446–54.
+
+2. Freeman ML, Nelson DB, Sherman S, et al. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996;335:
+909–18.
+
+3. Rabenstein T, Hahn EG. Post-ERCP pancreatitis: new momentum.
+
+Endoscopy 2002;34:325–9.
+
+4. Vandervoort J, Soetikno RM, Tham TC, et al. Risk factors for complications after performance of ERCP. Gastrointest Endosc 2002;56:652–6.
+
+5. Freeman ML, DiSario JA, Nelson DB, et al. Risk factors for post-ERCP pancreatitis: a prospective, multicenter study.
+
+Gastrointest Endosc 2001;54:425–34.
+
+6. Kubiliun NM, Adams MA, Akshintala VS, et al. Evaluation of Pharmacologic Prevention of Pancreatitis Following Endoscopic Retrograde Cholangiopancreatography: a Systematic Review. Clin Gastroenterol Hepatol 2015. Published Online 8 Jan 2015. doi:10.1016/j.cgh.2014.11.038 7. Fazel A, Quadri A, Catalano MF, et al. Does a pancreatic duct stent prevent post-ERCP pancreatitis? A prospective randomized study.
+
+Gastrointest Endosc 2003;57:291–4.
+
+8. Andriulli A, Leandro G, Federici T, et al. Prophylactic administration of somatostatin or gabexate does not prevent pancreatitis after ERCP: an updated meta-analysis. Gastrointest Endosc 2007;65:624–32.
+
+9. Tarnasky PR, Palesch YY, Cunningham JT, et al. Pancreatic stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastroenterology 1998;115:1518–24.
+
+10. Singh P, Das A, Isenberg G, et al. Does prophylactic pancreatic stent placement reduce the risk of post-ERCP acute pancreatitis? A meta-analysis of controlled trials. Gastrointest Endosc 2004;60:544–50.
+
+11. Akbar A, Abu Dayyeh BK, Baron TH, et al. Rectal nonsteroidal anti-inflammatory drugs are superior to pancreatic duct stents in preventing pancreatitis after endoscopic retrograde cholangiopancreatography: a network meta-analysis.
+
+Clin Gastroenterol Hepatol 2013;11:778–83.
+
+12. Murray B, Carter R, Imrie C, et al. Diclofenac reduces the incidence of acute pancreatitis after endoscopic retrograde cholangiopancreatography. Gastroenterology 2003;124:1786–91.
+
+13. Elmunzer BJ, Waljee AK, Elta GH, et al. A meta-analysis of rectal NSAIDs in the prevention of post-ERCP pancreatitis. Gut 2008;57:1262–7.
+
+14. Zheng MH, Xia HH, Chen YP. Rectal administration of NSAIDs in the prevention of post-ERCP pancreatitis: a complementary meta-analysis. Gut 2008;57:1632–3.
+
+15. Elmunzer BJ, Scheiman JM, Lehman GA, et al. A randomized trial of rectal indomethacin to prevent post-ERCP pancreatitis. N Engl J
+Med 2012;366:1414–22.
+
+16. Otsuka T, Kawazoe S, Nakashita S, et al. Low-dose cholangiopancreatography pancreatitis: a randomized controlled trial.
+
+J Gastroenterol 2012;47:912–17.
+
+17. Arata S, Takada T, Hirata K, et al. Post-ERCP pancreatitis.
+
+J Hepatobiliary Pancreat Sci 2010;17:70–8.
+
+18. Summary of product characteristics of Voltaren® at Novartis company homepage. http://product.novartis.co.jp/vol/pi/VS1402.pdf (accessed 13 Jun 2014) (In Japanese).
+
+19. Mehta PP, Sanaka MR, Parsi MA, et al. Association of procedure length on outcomes and adverse events of endoscopic retrograde cholangiopancreatography. Gastroenterol Rep 2014;2:
+140–4.
+
+20. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for acute kidney injury. Kidney Int Suppl 2012;2:19–36.
+
+21. Day LW, Lin L, Somsouk M. Adverse events in older patients undergoing ERCP: a systematic review and meta-analysis.
+
+Endoscopy Int Open 2014;02:E28–36.
+
+22. Pezzilli R, Romboli E, Campana D, et al. Mechanisms involved in the onset of post-ERCP pancreatitis. JOP 2002;3:162–8.
+
+23. Tsujino T, Kawabe T, Omata M. Antiproteases in preventing post-ERCP acute pancreatitis. JOP 2007;8:509–17.
+
+24. Mäkelä A, Kuusi T, Schröder T. Inhibition of serum phospholipase-A2 in acute pancreatitis by pharmacological agents in vitro. Scand J Clin Lab Invest 1997;57:401–7.
+
+25. Gross V, Leser HG, Heinisch A, et al. Inflammatory mediators and cytokines—new aspects of the pathophysiology and assessment of severity of acute pancreatitis? Hepatogastroenterology 1993;40:522–30.
+
+26. Giagoudakis G, Markantonis SL. Relationships between the concentrations of prostaglandins and the nonsteroidal antiinflammatory drugs indomethacin, diclofenac, and ibuprofen.
+
+Pharmacotherapy 2005;25:18–25.
+
+27. Richy F, Bruyere O, Ethgen O, et al. Time dependent risk of gastrointestinal complications induced by non-steroidal anti-inflammatory drug use: a consensus statement using a meta-analytic approach. Ann Rheum Dis 2004;63:
+759–66.
+
+28. Rostom A, Goldkind L, Laine L. Nonsteroidal anti-inflammatory drugs and hepatic toxicity: a systematic review of randomized controlled trials in arthritis patients. Clin Gastroenterol Hepatol 2005;3:489–98.
+
+29. Warning and precautions of Voltaren® at Novartis company homepage. http://www.voltaren.jp/m_kinki/kinki_a11.html (accessed 13 Jun 2014) (In Japanese).
+
+30. Dumonceau JM, Andriulli A, Deviere J, et al. European Society of Gastrointestinal Endoscopy (ESGE) Guideline: prophylaxis of post-ERCP pancreatitis. Endoscopy 2010;42:503–15.
+
+31. Tenner S, Baillie J, DeWitt J, et al. American College of Gastroenterology guideline: management of acute pancreatitis. Am J
+Gastroenterol 2013;108:1400–15.
+
+32. American Gastroenterological Association (AGA) Institute on
+"Management of Acute Pancreatits" Clinical Practice and Economics Committee; AGA Institute Governing Board. AGA Institute Medical Position Statement on Acute Pancreatitis. Gastroenterology 2007;132:2019–21.

medical/md/PMC4652769.md

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+# Nucleotide Binding Interactions Modulate Dntp Selectivity And Facilitate 8-Oxo-Dgtp Incorporation By Dna Polymerase Lambda
+
+Matthew J. Burak, Kip E. Guja and Miguel Garcia-Diaz* Received March 17, 2015; Revised July 13, 2015; Accepted July 15, 2015
+
+## Abstract
+
+8-Oxo-7,8,-dihydro-2--deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP
+at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol -) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol -**. We have solved high resolution**
+crystal structures showing how Pol - accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn**-conformation, and is**
+stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 **facilitate incorporation of 8-oxo-dGMP opposite dA and**
+dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol - and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.
+
+## Introduction
+
+DNA is vulnerable to damage by reactive oxygen species
+(ROS). Of the four nucleobases, guanine is the most susceptible to oxidative damage owing to its low redox potential (1). Moreover, guanine oxidation products have a high mutagenic potential (2–6). 8-oxo-7,8-dihydro-2--
+deoxyguanosine (8-oxo-dG) is a major oxidative product present in cells and can arise in either DNA or the free nucleotide pool (7). Accordingly, oxidation of dGTP results in the formation of 8-oxo-7,8,-dihydro-2--deoxyguanosine triphosphate (8-oxo-dGTP). Experimental measurements indicate that 8-oxo-dGTP may account for up to 10% of the total dGTP pool (8). In order to prevent the accumulation of 8-oxo-dGTP, cells encode a pyrophosphohydrolase, MutT/MTH1, which degrades the oxidized nucleotide (9– 11). Despite expression of MutT/MTH1, nucleotide pools can accumulate enough 8-oxo-dGTP to promote mutagenesis and drive cancer progression (12,13). 8-oxo-dGTP is capable of adopting either an anti or syn conformation and thus can base-pair with either cytosine (dC) or adenine
+(dA), respectively (14–17). As a consequence of this, 8-oxodGTP is frequently misincorporated opposite dA *in vitro* by several DNA polymerases (18–25). Thus, *in vivo*, incorporation of 8-oxo-dGMP can lead to an A to C transversion mutation if left unrepaired (9). Conversely, 8-oxo-dGTP incorporation opposite dC can also eventually result in G to T tranversion mutations after subsequent replication (26).
+
+All cells have specialized repair systems to deal with DNA modifications, and the base excision repair (BER)
+pathway is the main mechanism for purging the genome of oxidative lesions such as 8-oxo-dG (27). A specialized form of the BER pathway, initiated by the glycosylase MutY/MTYH, specifically recognizes and repairs 8-oxodG:dA mispairs by removing dA and replacing it with dC,
+enabling subsequent repair of the 8-oxo-dG lesion (28–
+31). However, no repair mechanism exists to cope with misincorporated 8-oxo-dGMP. Significantly, 8-oxo-dGMP
+misincorporation would lead to mutagenic repair by the MutY/MTYH-dependent BER pathway, directing removal of dA and not 8-oxo-dGMP. Thus, 8-oxo-dGMP misincorporation would unavoidably lead to mutagenesis, resulting an A to C transversion (26).
+
+Therefore, preventing misincorporation of 8-oxo-dGMP
+outright is crucial to avoid mutagenesis. In addition to the hydrolytic activity of MutT/MUTH1, discrimination against 8-oxo-dGTP by DNA polymerases is an important factor in the prevention of mutagenesis. Therefore, understanding the mechanistic basis of this discrimination is of significant interest. With the exception of the recent characterization of the interaction between DNA polymerase beta
+(Pol -) and 8-oxo-dGTP (32,33), there is a lack of structural information that might help explain how DNA polymerases avoid 8-oxo-dGTP-induced mutagenesis.
+
+*To whom correspondence should be addressed. Tel: +1 631 444 3054; Fax: +1 631 4449749; Email: miguel.garcia-diaz@stonybrook.edu The family X DNA polymerase lambda (Pol ) plays an important role in mediating cellular tolerance of guanine oxidation (34–36). A key element of the MutY/MTYH
+BER pathway is the ability to readily incorporate dCMP opposite a templating 8-oxodG. In the context of this repair reaction, Pol  is uniquely capable of mediating faithful incorporation of dCMP to complete the repair (23). Pol  also takes advantage of its unique catalytic properties to mediate additional roles in other DNA repair pathways related to oxidative damage, such as non-homologous end joining (NHEJ) (37–41).
+
+Considering that Pol  operates under conditions where oxidative damage is prevalent, it is expected to frequently encounter 8-oxo-dGTP. Moreover, due to its function in NHEJ, Pol  may be responsible for mediating 8-oxodGTP-driven mutagenesis. Curiously, despite its relatively error-free behavior when faced with a templating 8-oxodG,
+Pol  readily misinserts 8-oxo-dGMP opposite dA (23). The contrast between its behavior when incorporating 8-oxodGTP and opposite 8-oxo-dG indicates that the active site of Pol  is asymmetrical, but the structural factors influencing 8-oxo-dGMP misincorporation by Pol  are not well understood.
+
+Here, we report high resolution pre- and post-catalytic crystal structures of Pol  in complex with 8-oxo-dGTP
+and 8-oxo-dGMP, respectively. The structures demonstrate that the oxidized nucleotide can be accommodated in the Pol  active site, where it is stabilized in the pro-mutagenic syn-conformation. Kinetic analysis demonstrates that two residues that partially conform the dNTP binding pocket influence 8-oxo-dGMP incorporation. Ala510 makes a Van der Waals interaction with the base of the incoming nucleotide, whereas Asn513 hydrogen-bonds with the C8carbonyl. Our results indicate that Ala510 and Asn513 differentially impact 8-oxo-dGMP incorporation as well as the balance of purine/pyrimidine incorporation, confirming the importance of these residues in mediating nucleotide selectivity in family X DNA polymerases.
+
+## Materials And Methods Protein Purification
+
+The sequence corresponding to residues 242–575 of human Pol  was previously cloned into the bacterial expression vector pET-22b (42). Site-directed mutagenesis was performed on the Pol  expression vector to generate the A510D, N513A and A510D/N513A mutants. WT, A510D,
+N513A and A510D/N513A Pol  were subsequently expressed in the *E. coli* strain BL21-CodonPlus(DE3)-RIL
+and purified as described (42).
+
+## Oligonucleotides For Crystallography
+
+Oligonucleotides used for the pre-catalytic ternary and post-catalytic nick complexes are shown in Supplementary Figure S1A and B respectively. Oligos were synthesized by solid-state synthesis methods using an automated DNA
+synthesizer. Oligos were subsequently purified by HPLC, ethanol precipitated and quantified by UV absorbance
+(A260). P/T were annealed as previously described in 25 mM Tris–HCl pH 7.5 and 40 mM MgCl2.
+
+## Crystallization
+
+Pre-catalytic ternary complexes were formed using an upstream primer (UP5), downstream primer containing a 5--
+phosphate group (DP4), and template (T8-A) (Supplementary Figure S1A). UP5, DP4 and T8-A (1:1:1) were heated to 80◦C for 10 min before slowly cooling to room temperature in 25 mM Tris–HCl pH 7.5 and 50 mM MgCl2. The resulting primer/templates (P/T) were mixed with WT Pol
+. However, due to the low melting temperature of DP-4
+(16◦C), a second annealing step in the presence of protein was used to ensure the proper formation of a 1-nt gapped substrate. Accordingly, the mixture was heated to 15◦C for 5 min before slowly cooling to 4◦C to ensure proper annealing of DP4. ddCTP and 8-oxo-dGTP/dTTP were sequentially added to the mixture in order to generate a pre-catalytic ternary complex. The resulting mixture (100 l) contained 15 mM Tris–HCl pH 7.5, 75 mM NaCl, 10 mM MgCl2, 1 mM DTT, P/T (0.5 mM), WT Pol  (0.4 mM), 1 mM ddCTP and 3 mM 8-oxo-dGTP/dTTP. Crystals containing 8oxo-dGTP were formed using the hanging drop method by mixing 2 l of the protein/DNA solution containing DNA with 1 l of the reservoir solution containing 0.1 M sodium cacodylate trihydrate pH 7.4 and 1.9 M sodium acetate trihydrate. The crystal was transferred to a solution containing 0.1 M sodium cacodylate trihydrate pH 7.4 and 1.9 M
+sodium acetate trihydrate and 25% (w/v) glycerol and cryocooled in liquid nitrogen prior to data collection. dTTPcontaining crystals were formed as previously stated in solution containing 0.1 M sodium acetate trihydrate pH 4.6 and 2.0 M sodium formate. The crystal was transferred to a solution containing 0.1 M sodium acetate trihydrate pH 4.6 and 2.0 M sodium fomate and 25% w/v glycerol, harvested with a Mitegen MicroMount and cryo-cooled in liquid nitrogen prior to data collection.
+
+Post-catalytic nick complexes were formed using an upstream primer (UP6), DP4 and template T8-A (Supplementary Figure S1B). Pre-hybridized UP6, DP4 and T8-A were mixed with WT Pol  as previously described. 8-Oxo-dGTP
+(4 mM) was subsequently added and the resulting mixture
+(100 l) was equilibrated for 60 min on ice in order to generate a post-catalytic nick complex. Crystals containing 8oxo-dGMP were grown using the hanging drop method by mixing 1 l of the protein/DNA solution containing DNA
+with 1 l of the reservoir solution containing 0.1 M sodium citrate pH 5.6 and 1 M ammonium phosphate. The crystal was transferred to a solution containing 0.1 M sodium citrate pH 5.6, 1.2 M ammonium phosphate and 30% (w/v)
+glycerol, harvested with a Mitegen MicroMount and cryocooled in liquid nitrogen prior to data collection.
+
+## X-Ray Data Collection And Structure Determination
+
+Diffraction data were collected on beamline X25 of the National Synchrotron Light Source at Brookhaven National Laboratory for the dTTP pre-catalytic complex and the 8oxo-dGTP post-catalytic complex, and on beamline 5.0.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory for the 8-oxo-dGTP post-catalytic complex. All datasets were collected at 100 K using a wavelength of 1.0 A. Diffraction data were processed using XDS ( ˚ 43)
+and Aimless (44) as implemented in the autoPROC pipeline
+(45). Phases were obtained by molecular replacement using Phaser (46); search models for the pre- and post-catalytic complexes were created from 1XSN (47) and 1XSP (47),
+respectively. Model building was carried out in Coot (48),
+followed by refinement in Phenix (49), Refmac (50) and BUSTER (51). Diffraction data for the dTTP pre-catalytic complex were strongly anisotropic, with resolution limits
+(defined by CC1/2 and I/σI (44,52) of 2.4 A in ˚ a* and c* reciprocal space directions, but only 2.9 A along the ˚ b* direction. For this reason, an ellipsoidal truncation was carried out using the Anisotropy Diffraction Server (53). Briefly, data were truncated that fell outside an ellipse centered at the reciprocal lattice origin and having vertices at 1/2.4, 1/2.9, and 1/2.4 A along ˚ a*, b*, and c* axes, respectively.
+
+Isotropy was approximated by applying a negative scale factor along b* (−16.8 A˚ 2) with no correction along a* or c*.
+
+These anisotropically scaled data then were used for refinement. In all three structures a small region of the protein surface, far removed from the active site (Loop 3) was disordered. The geometric quality of the refined models was assessed with MolProbity (54) and the structure validation tools in the Phenix suite. Data collection and refinement statistics are shown in Supplementary Table S1.
+
+In order to facilitate more precise structural comparisons, a statistical analysis of atomic coordinate accuracy was carried out using SFCHECK (55). Following the method of Cruickshank (56), the standard deviation of the atomic coordinates can be derived from the properties of the electrondensity map:
+
+## Σ(X) = Σ(Slope)/Curvature
+
+where σ(slope) and curvature are the standard deviation of the slope and curvature of the electron density map at the atomic center. The curvature is approximated by:
+
+$$\mathrm{curvature}=2\pi^{2}\Bigl(\sum h^{2}F_{o b s}\Bigr)\Bigl/a^{2}\,V_{\mathrm{unit,cell}}$$
+
+where a is the unit-cell length and h is the Miller index
+(57,58). The maximal expected coordinate error is calculated by expressing σ(slope) as a function of the difference between Fobs and Fcalc
+
+$$\sigma(\mathrm{slope})=2\pi\left\{\sum\left[h^{2}\left(F_{\mathrm{obs}}-F_{\mathrm{calc}}\right)^{2}\right]\right\}^{1/2}\int a\,V_{\mathrm{unit\_cell}}$$
+
+For any missing reflections, the program uses the average value of σ(F) for the corresponding resolution shell instead of (Fobs - Fcalc). The expected minimal coordinate error is estimated using the experimental σ(F) instead of the difference between the observed and calculated structure factors.
+
+In this case σ(slope) is computed as
+
+$$\sigma(\mathrm{slope})=2\pi\left\{\sum\left[h^{2}\sigma(F)^{2}\right]\right\}^{1/2}\int\!a\,V_{\mathrm{unit\_cell}}$$
+
+If there is no  value for an observed structure factor, the program uses  = 0.04Fobs as the default value, which is roughly the error magnitude usually encountered. The both minimal and maximal estimated errors were calculated to account for the fact that the local quality of the electron density will amost always vary across different regions of a given crystal structure. These values are listed in Supplementary Table S2.
+
+## Oligonucleotides For Steady-State Primer Extension Assays
+
+Oligonucleotides used for steady-state primer extension assays were obtained from Invitrogen and are shown in Supplementary Figure S1C and 1D. Oligos were purified by HPLC and polyacrylamide gel-electrophoresis (PAGE),
+quantified by UV absorbance (A260), and heated to 80◦C for 10 min before slowly cooling to room temperature overnight in 20 mM Tris–HCl pH 7.5 and 150 mM MgCl2.
+
+## Steady-State Primer Extension Assays
+
+Steady-state experiments were performed with an upstream primer 5- labeled with a Cy3 fluorophore (UP20), downstream primer containing a 5--phosphate group (DP14),
+and a template (T35-A/T35-C) (Supplementary Figure S1C
+and 1D). The core sequence of P/T used was the same as the crystallography experiments. Pre-hybridized UP20, DP14 and T35-A/T35-C (1:1.2:1.2) were mixed with either WT
+A510D, N513A or A510D/N513A Pol . The resulting mixture (18 l) contained 50 mM Tris–HCl pH 8.5, 10 mM
+MgCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, P/T (200 nM) and either WT (0.3–5.0 nM), A510D (0.3–5.0 nM),
+N513A (0.3–5.0 nM) or A510D/N513A (0.5–10.0 nM) Pol
+. The protein/DNA mixture was directly added to varying amounts of either dTTP (0–500 M), dGTP (0–500 M) or 8-oxo-dGTP (0–1000 M) to start the polymerization reaction.
+
+Reaction mixtures (20 l) were quenched by the addition of 95% (v/v) formamide, 10 mM EDTA, 0.001% xylene cyanol, 0.001% bromophenol blue (10 l). Extended primers were separated by denaturing (8 M urea) 18% (v/v)
+PAGE. The fluorescence intensity of the bands was quantified using a Typhoon FLA 9000 imager and ImageQuant software.
+
+## Kinetic Analysis Of Primer Extension Assays
+
+The observed rate of nucleotide incorporation (extended primer) was plotted as a function of nucleotide concentration. Steady-state kinetic parameters, Vmax and KM, were determined by fitting the data to the Michaelis–Menten equation: V = Vmax[S]/(KM + [S]). *kcat* was determined with the equation: kcat = Vmax/[E].
+
+## Results And Discussion Pre-Catalytic Structure Of Pol - **With An Incoming 8-Oxodgtp**
+
+In order to understand how 8-oxo-dGTP is misincorporated into DNA, we decided to solve a structure of Pol in complex with an incoming 8-oxo-dGTP in a precatalytic state. Thus, we crystallized Pol  with a 1-nt gapped, dideoxy-terminated DNA substrate and an incoming 8-oxo-dGTP (Figure S1A). We obtained crystals that diffracted to a resolution of 1.95 A. We were able to solve ˚
+the structure using molecular replacement, and subsequent refinement converged to an Rwork and Rfree of 0.1999 and
+
+![3_image_0.png](3_image_0.png)
+
+0.2273 respectively (Supplementary Table S1). The electron density was of sufficient quality to build protein residues 252–575 and all DNA bases, including the incoming 8-oxodGTP. Inspection of the structure immediately reveals that, as expected, 8-oxo-dGTP is bound in the active site (Figure 1A). A Mg2+ atom is present in the dNTP-binding metal (B)
+site. However, a water molecule is occupying the site of the catalytic metal (A), consistent with the absence of the 3-OH
+group from the nucleotide at the primer terminus (47) (Figure 1A).
+
+The density for the 8-oxo-dGTP residue is of high quality, and the C8 carbonyl is clearly observed (Figure 1B). The structure indicates that the incoming 8-oxo-dGTP adopts a syn-conformation and forms a Hoogsteen base-pair with the templating adenine base. Hydrogen-bonds are established between N6 of the adenine and O6 of the guanine
+(3.0 A) and N1 of the adenine and N7 of the guanine (2.9 ˚ A). An intramolecular hydrogen-bond between the N2 and ˚
+a non-bridging oxygen on the -phosphate of 8-oxo-dGTP
+may stabilize the syn-conformation. These interactions and the geometry of 8-oxo-dGTP in the active site of Pol  are consistent with the conformation observed in existing structures of Pol - in complex with 8-oxo-dGTP (32,33).
+
+Interestingly, the geometry of the 8-oxo-dGTP:dA mispair differs from a canonical Watson-Crick base-pair. The R angle for 8-oxo-dGTP is 44.3◦ which is consistent with the syn-conformation of the base in other reported structures (Pol - - 44.2◦) (32,33). However, this differs from that of a dGMPCPP:dC base-pair (54.2◦) (59). The  angle for dA is 55.7◦ and is consistent with an ddTTP:dA base-pair
+(1XSN) (47). This is in agreement with the idea that 8-oxodG adopts an alternate conformation in order to mispair with an adenosine (15,16). Also, the C1-–C1- width of the mispair is 10.7 ± 0.015–0.091 A (where 0.015 and 0.091 ˚ A˚
+correspond to the minimum and maximum estimated errors respectively, Supplementary Table S2) which is larger than for a dTTP:dA base-pair (10.5 ± 0.021–0.094 A, Supple- ˚
+mentary Table S2). Interestingly, this larger base pair width appears to be correlated with the presence of a mispair in the active site in Pol  (dGMPCPP:dT, 10.8 A) ( ˚ 59). However, despite these distortions, Pol  can accommodate the 8oxo-dGTP:dA mispair in the active site without any major conformational rearrangements, as evidenced by the small RMSD between our structure and a reference pre-catalytic complex containing a ddTTP:dA base-pair in the active site
+(1XSN, RMSD of 0.320 A over 261 C ˚  atoms) (47).
+
+## Post-Catalytic Structure Of Pol - **With Incorporated 8-Oxodgmp**
+
+The absence of large-scale subdomain conformational motions during catalysis in Pol  implies that completion of the nucleotidyl transfer reaction and advancement from precatalytic to the post-catalytic complex involves no significant conformational changes, other than stereochemical inversion of the -phosphate (47). We therefore took advantage of this fact in order to determine whether our structure indeed represents a catalytically relevant conformation of 8oxo-dGTP. We decided to crystallize a post-catalytic complex of Pol  with a nicked DNA substrate containing an 8-oxo-dG moiety at the primer terminus (Figure 2A). The crystals of the post-catalytic complex diffracted to a resolution of 2.15 A. The structure was solved by molecular re- ˚
+placement, and refinement converged to an Rwork and Rfree of 0.2144 and 0.2450, respectively (Supplementary Table S1). The electron density was of sufficient quality to build protein residues 251–575 and all DNA bases, including the incorporated 8-oxo-dGMP.
+
+The post-catalytic structure contains both products of the phosphoryl transfer reaction (47). In the structure, 8oxo-dGTP has been turned over, and the incorporated 8oxo-dGMP maintains the syn-conformation. However, the pyrophosphate released by the reaction has diffused away from the active site and has been replaced by a citrate ion.
+
+Citrate is present in high concentration in the crystallization buffer and mimics the conformation of pyrophosphate in other post-catalytic structures (47). Additionally, the citrate is coordinating a Na+ ion that is present in the metal B site (Figure 2A). The metal A is absent in the structure,
+
+![4_image_0.png](4_image_0.png)
+
+consistent with the notion that it dissociates after nucleotide incorporation (47). Superimposition of the pre- and postcatalytic structures reveals that both complexes adopt similar conformations (RMSD of 0.238 A over 236 C ˚  atoms).
+
+Furthermore, the geometry of all active site atoms is conserved (Figure 2B). This confirms that the conformations seen in our structures are catalytically relevant.
+
+## 8-Oxo-Dgtp Is Stabilized By Numerous Interactions
+
+Close inspection of the dNTP binding pocket reveals five residues that interact with 8-oxo-dGTP (Supplementary Figure S2). Three residues form hydrogen-bonding interactions with non-bridging phosphate oxygen atoms in the incoming nucleotide. Arg386 forms a bidentate hydrogenbond (3.0 and 3.2 A) with the ˚ -phosphate while Ser417 and Arg420 each form a hydrogen-bond with the -phosphate
+(2.6 A) and the ˚ --phosphate (3.0 A), respectively. Together, ˚
+these three residues form the phosphate-binding pocket for the incoming nucleotide. However, these interactions are unlikely to confer specificity, since they are established irrespective of the identity of the incoming nucleotide.
+
+Ala510 provides a Van der Waals stacking interaction with the oxidized nucleobase (Figure 3A). This interaction is analogous to what is observed in a complex with undamaged substrates, and is thought to be responsible for the high dNTP binding affinity of Pol  (60). It is however conceivable that the strength of the Ala510 interaction depends on the identity of the incoming dNTP. Consistent with this idea, Ala510 was shown to help discriminate against incorrect nucleotide binding (61). Thus, Pol  may rely on Ala510 to probe for proper nascent base-pair geometry of 8-oxodGTP.
+
+In addition to Ala510, another residue recognizes a unique structural characteristic of 8-oxo-dGTP. Asn513 forms a hydrogen-bonding interaction (3.1 A) with the C8-carbonyl ˚
+of 8-oxo-dGTP (Figure 3B). In the Pol  active site, Asn513 establishes a sequence independent hydrogen-bond with the O2/N3 hydrogen-bond acceptors in the minor groove of the incoming nucleotide (42). These types of minor groove interactions are thought to be crucial to the ability of DNA polymerases to discriminate against non-Watson– Crick base pairs and thus promote fidelity of synthesis (62,63). Similar to what is observed with 8-oxo-dG in the active site of different polymerases (64–67), 8-oxo-dGTP
+may hijack this fidelity checkpoint mechanism in order to stabilize syn-dependent misincorporation. The O8 of 8-oxodGTP (syn) in the DNA minor groove is in a position similar to that of O2 of dTTP for a canonical Watson-Crick base-pair. This suggests that Asn513 stabilizes the lesion in the syn-conformation and thus mediates misincorporation opposite dA. Asn513 is conserved in another family X DNA
+polymerase, human Pol -, where it plays a critical role in facilitating incorporation of 8-oxo-dGMP opposite template dA (19,32,33). Our structural data suggest that this residue could play a similar role in Pol .
+
+## Steady-State Kinetic Analysis Of The Relevance Of Ala510 **For 8-** Oxo-Dgtp Incorporation
+
+As mentioned above, our structural results reveal that 8oxo-dGTP is stabilized by two specific interactions. One of these, Ala510, provides a Van der Waals stacking interaction with the nucleobase of 8-oxo-dGTP. Interestingly, Ala510 in Pol  is replaced by an aspartic acid in Pol - (Asp276), which is believed to restrict dNTP binding (68,69). Thus, to further characterize the role of Ala510 on 8-oxo-dGTP misincorporation in Pol , we constructed an A510D active-site substitution and performed a steady-state kinetic analysis.
+
+Previous data had determined the catalytic efficiency of incorporation of 8-oxo-dGMP opposite dA and dC (23).
+
+However, these studies lacked a comparison to dTMP incorporation. This is relevant, since 8-oxo-dGMP competes with dTTP for binding opposite a templating dA, and thus the relative incorporation efficiency of the two nucleotides ultimately determines the mutagenic potential of 8-oxodGTP *in vivo*. In order to avoid possible sequence-related effects, the core sequence of the P/T used was identical to that of the oligos used for crystallography (Supplementary Figure S1C and 1D). Using these substrates we determined the kinetic parameters [kcat and Km(app)] and catalytic efficiencies [kcat/Km(app)] for both WT and A510D Pol  (Table 1).
+
+A comparison of the catalytic efficiencies for WT Pol revealed a 150-fold difference between dGMP and 8oxodGMP incorporation opposite dC. Conversely, incorporation of 8-oxo-dGMP was only 13-fold less efficient than dTMP opposite dA. The relatively small difference in catalytic efficiencies for 8-oxo-dGMP and dTMP incorporation opposite dA highlights the mutagenic potential of this lesion. Furthermore, comparison of the dA/dC selectivity for 8-oxo-dGTP also revealed that the pro-mutagenic synconformation is preferred in WT Pol  (5:1).
+
+Interestingly, the A510D substitution had a differential effect on all nucleotides tested. With respect to WT protein, dTTP and 8-oxodGTP incorporation opposite dA was reduced by 4.8-fold (Figure 3C) and 5.1-fold (Figure 3D), respectively. This suggests that the A510D substitution may affect 8-oxo-dGTP(syn) in a similar manner to dTTP. However, the same does not hold true for dGTP/8-oxo-dGTP
+opposite dC. Notably, the A510D substitution had the largest effect on the incorporation of 8-oxo-dGTP opposite dC (7.6-fold, Figure 3E) as opposed to dGTP (2.8-fold, Figure 3F). This suggests that Ala510 may preferentially stabilize the *anti*-conformation of 8-oxo-dGTP, promoting its incorporation opposite dC. Accordingly, Ala510 may enhance the ability of Pol  to incorporation 8-oxo-dGMP opposite dC, which could ultimately increase the frequency of G to T tranversion mutations (26).
+
+## Steady State Kinetic Analysis Of The Relevance Of Asn513 **On 8-** Oxo-Dgtp Incorporation
+
+In addition to Ala510, Asn513 also makes a specific interaction with the oxidized nucleobase. To characterize the role of Asn513 on 8-oxo-dGTP misincorporation in Pol , we generated a N513A active-site substitution. Steady-state kinetic parameters [kcat and Km(app)] and catalytic efficiencies
+[kcat/Km(app)] for dTTP/8-oxo-dGTP incorporation opposite dA and dGTP/8-oxo-dGTP opposite dC were determined using N513A Pol  (Table 1). Given the importance of Asn513 during normal polymerization, the N513A substitution expectedly affected the catalytic efficiency for all nucleotides tested. Interestingly, similar to what was observed with Ala510, the effects were differential for all nucleotides tested. With respect to the WT protein, the catalytic efficiency for dTTP decreased by 5.8-fold (Figure 3C)
+in N513A Pol . However, the N513A substitution resulted in a much more drastic effect in the context of 8-oxo-dGMP
+incorporation opposite dA (25-fold, Figure 3D). Also, the catalytic efficiencies of dGMP and 8-oxo-dGMP incorporation opposite dC were reduced by 1.3 (Figure 3E) and 6.4fold (Figure 3F) respectively. Taken together, these results suggest that Asn513 plays an important role in facilitating 8oxo-dGMP incorporation opposite dA, implying that this residue selectively stabilizes the *syn-*conformation of 8-oxodGTP.
+
+Our results suggest that dNTP binding by Pol  is influenced by multiple amino acid residues. Moreover, both Ala510 and Asn513 appear to play critical roles for anti/syndiscrimination. To examine if these residues behave individually or cooperatively, we created a A510D/N513A double substitution (Table 1, Figure 3C–F). As expected, the effects of both substitutions synergistically impacted all nucleotides tested. Interestingly, the drop in catalytic efficiency was nearly identical for 8-oxo-dGMP incorporation opposite dA and dC. This is consistent with the opposite effects of Ala510 and Asn513, facilitating incorporation of the *anti-*and syn-conformations, respectively.
+
+![6_image_0.png](6_image_0.png)
+
+| Table 1. Steady-state kinetic parameters of incorporation of 8-oxo-dGTP opposite dA and dC by DNA polymerase lambda Protein Template dNTP KM (M) kcat (min−1)             | kcat/KM (min−1 M−1)            |             |               |               |
+|-------------|------------|-------------|---------------|---------------|
+| WT          | dTTP       | 4.2 ± 0.7   | 131 ± 7       | 32 ± 4        |
+| 8-oxo-dGTP  | 20 ± 3     | 45.2 ± 3.9  | 2.4 ± 0.3     |               |
+| A510D       | dTTP       | 35 ± 4      | 234 ± 10      | 6.7 ± 0.6     |
+| A           | 8-oxo-dGTP | 120 ± 8     | 56.8 ± 2.8    | 0.47 ± 0.01   |
+| N513A       | dTTP       | 21 ± 4      | 113 ± 1       | 5.5 ± 1.0     |
+| 8-oxo-dGTP  | 41 ± 1.6   | 3.99 ± 0.06 | 0.097 ± 0.003 |               |
+| A510D N513A | dTTP       | 120 ± 16    | 71.8 ± 5.6    | 0.62 ± 0.07   |
+| 8-oxo-dGTP  | 71 ± 2     | 3.00 ± 0.41 | 0.043 ± 0.005 |               |
+| WT          | dGTP       | 1.7 ± 0.1   | 140 ± 22      | 80 ± 10       |
+| 8-oxo-dGTP  | 8.4 ± 0.8  | 4.39 ± 0.04 | 0.53 ± 0.05   |               |
+| A510D       | dGTP       | 2.8 ± 0.5   | 80.0 ± 6.8    | 29 ± 5        |
+| C           | 8-oxo-dGTP | 24 ± 6      | 1.53 ± 0.05   | 0.070 ± 0.014 |
+| N513A       | dGTP       | 2.0 ± 0.2   | 121 ± 2       | 60 ± 4        |
+| 8-oxo-dGTP  | 16 ± 0.47  | 1.31 ± 0.05 | 0.083 ± 0.005 |               |
+| A510D N513A | dGTP       | 6.3 ± 0.8   | 67.2 ± 1.3    | 11 ± 2        |
+| 8-oxo-dGTP  | 120 ± 24   | 1.06 ± 0.04 | 0.009 ± 0.002 |               |
+
+## Structural Features In Pol - **Discourage 8-Oxo-Dgtp Misincorporation**
+
+Although the 8-oxo-dGTP:dA mispair is tolerated in the active site of Pol , kinetic analysis indicates that the insertion efficiency is reduced relative to that of a Watson–Crick basepair (Table 1). In order to determine the structural features discouraging incorporation of 8-oxo-dGTP we crystallized Pol  in complex with undamaged DNA in the exact same sequence context (Supplementary Table S1). We rationalized that this would provide a more accurate basis for comparison to the 8-oxo-dGTP containing structure, since existing structural models either lacked a 3--OH on the incoming nucleotide (1XSN) (47), or used a non-hydrolyzable dNTP
+analog to trap the ternary complex (2PFO) (70). The crystals diffracted with strong anisotropy, to a resolution limit of 2.4 A in ˚ a* and c* reciprocal space directions, but only 2.9 A along the ˚ b* direction. For this reason, an anisotropic correction was carried out using the Anisotropy Diffraction Server [PMID: 24203335]. The structure was solved with molecular replacement and the model was refined to convergence (Rwork/Rfree: 0.1874/0.2358). The electron density was of sufficient quality to build protein residues 252–575 and all DNA bases, including the incoming dTTP.
+
+Comparing our precatalytic structure for 8-oxo-dGMP
+incorporation with this structure indicates that the overall fold of the polymerase is conserved (RMSD of 0.418 over 289 C atoms). However, closer inspection of the active site reveals some important differences. First, the 8-oxo-dGTP
+does not appear to stack as well as dTTP with the base at the primer terminus (Figure 4A). In an attempt to maximize this stacking interaction and accommodate 8-oxo-dGTP in the active site, the templating adenine shifts 0.3 A away ˚
+from the incoming nucleotide (Figure 4B). The direct consequence of this distortion places the templating base 0.4 A away from the minor groove interacting residue, Arg ˚ 517
+(Figure 4B).
+
+Arg517 plays an important role interrogating the nascent base-pair conformation. This residue forms a hydrogenbond with the templating base and adopts a base-pair checking conformation in response to dNTP binding (47).
+
+Figure 4. Structural features that discourage incorporation. (A) Comparison of dTTP and 8-oxo-dGTP stacking interactions in the pre-catalytic structures. The stacking interaction between 8-oxo-dGTP(syn) (magenta) and ddC (cyan) at the primer terminus of a complex is shown in the left panel. Comparison with dTTP (magenta) and the ddC (cyan) in the right panel demonstrates that 8-ox-dGTP stacks more poorly (yellow). (B) Overlay of the nascent base-pair in the pre-catalytic 8-oxo-dGTP and dTTP
+complexes. 8-oxo-dGTP (magenta) appears to displace the templating dA
+away from its canonical position. The direct consequence of this is that dA
+shifts further away from Arg517. The hydrogen-bonding distances for the pre-catalytic 8-oxo-dGTP and dTTP complexes are shown in black and red respectively.
+
+This mechanism appears to be critical for fidelity of synthesis in family X polymerases. Consistently, substitution of Arg517 in Pol  sharply decreases the fidelity of the polymerase as well as the catalytic efficiency of dNTP incorporation (61). Similarly, even subtle alterations of this residue in Pol - result in substantial reduction in the catalytic efficiency (71). Thus, repositioning of the templating adenosine away from Arg517 as observed in our structure would likely cause a loss of insertion efficiency. This provides a structural explanation for the decreased incorporation efficiency observed in our kinetic analysis (Table 1).
+
+## Ala510 And Asn513 Influence The Purine/**Pyrimidine Balance In** Pol -
+
+While Asn513 enhances 8-oxo-dGTP incorporation in Pol
+, this residue also had a differential effect on dTTP and dGTP incorporaton. Steady-state kinetic analysis demonstrated that the catalytic efficiency for dTTP incorporation by N513A Pol  was 5.8-fold (Figure 3C) lower than WT,
+whereas the decrease observed in the case of dGTP incorporation was just 1.3-fold (Figure 3E). Interestingly, the equivalent residue in the family X DNA polymerase from the heat-stable organism *T. thermophilus* (TthPolX) appears to also influence the balance of purine/pyrimidine incorporation (72). Asn513 appears to play a similar role in balancing the purine/pyrimidine bias in Pol .
+
+Strikingly, Ala510 also appears to have a greater effect on pyrimidines than purines. A510D substitution results in a 4.8-fold (Figure 3C) decrease in catalytic efficiency for dTTP incorporation but only a 2.8-fold (Figure 3E)
+decrease for dGTP. In agreement with these results, the A510D/N513A double mutant displays a clear synergistic effect. The double substitution affects dTTP incorporation to a far greater extent (52-fold, Figure 3C) than dGTP
+(7.3-fold, Figure 3E), demonstrating that both residues contribute to balancing purine/pyrimidine incorporation in Pol
+. This indicates that residues in addition to Asn513 are important to achieve the correct purine/pyrimidine balance.
+
+## Conclusion
+
+The free nucleotide pool is susceptible to oxidative damage
+(73). To cope with this oxidative burden, cells encode an enzyme (MutT/MTH1) to sanitize the nucleotide pools by degrading oxidized nucleotides such as 8-oxo-dGTP (10,11).
+
+Despite the presence of this protein, residual 8-oxo-dGTP
+is still capable of promoting mutagenesis (8,12,13). As a result, DNA polymerases such as Pol  may encounter and incorporate this oxidized nucleotide, ultimately leading to mutagenesis.
+
+The structural and kinetic studies presented here reveal how 8-oxo-dGTP evades fidelity checkpoint mechanisms in Pol . Active site residues Ala510 and Asn513 establish critical interactions that help stabilize the incoming nucleotide during polymerization. Asn513 forms a minor groove interaction with the incoming nucleotide. While this interaction is used to ensure that a base-pair with the proper geometry is formed in the active site of the polymerase (42,47),
+this mechanism is co-opted by 8-oxo-dGTP and appears to drive its mutagenic incorporation opposite dA. Asn513 is capable of forming a hydrogen-bond with the C8-carbonyl of 8-oxo-dGTP, thereby stabilizing the oxidized nucleotide in a syn-conformation. Consistently, substitution of asparagine to an alanine eliminates the stabilizing hydrogen-bond and results in a 25-fold reduction in the catalytic efficiency for 8oxo-dGTP misincorporation opposite dA. Conversely, the N513A substitution has only a minor effect on dTTP incorporation opposite dA (5.8-fold). Thus, Asn513 appears to selectively facilitate 8-oxo-dGMP misincorporation.
+
+Asn513 is also conserved in another family X DNA polymerase, Pol - (Asn279). Accordingly, Asn279 also appears to play a role mediating 8-oxo-dGTP-induced mutagenesis.
+
+Strikingly, in comparison to Pol - (1000-fold), the effects of Asn513 on 8-oxo-dGMP misincorporation in Pol  are far less pronounced (25-fold) (19). This implies that minor groove interactions are less critical to stabilize 8-oxo-dGTP
+in a syn-conformation in the Pol  active site. The lesser relative importance of Asn513 with respect to Asn279 is perhaps owed to the differential interactions established by Pol with the incoming nucleotide. Pol  has a higher binding affinity for nucleotides than Pol - (60), which is thought to be largely the result of a Van der Waals contact established between Ala510 and the incoming nucleobase. Conversely, the equivalent residue in Pol -, Asp276, is believed to restrict dNTP binding (68,69).
+
+Our results demonstrate that Ala510 substitution differentially affects incorporation of the nucleotides tested. This implies that Ala510 establishes distinct interactions with each nucleotide and therefore is unequally responsible for their stability in the active site. While an A510D substitution had an nearly equivalent effect on both dTTP (4.8fold) and 8-oxo-dGTP(syn) (5.1-fold), one consequence of this behavior is that substitution of Ala510 selectively impacts 8-oxo-dGTP misincorporation opposite dC. Substitution of Ala510 to aspartic acid results in a greater effect on 8-oxo-dGMP incorporation (7.6-fold) opposite dC than dGMP (2.8-fold). This may be due to an unfavorable electrostatic interaction between the side chain and the C8carbonyl of the nucleotide. As a consequence, Ala510 selectively enhances the incorporation of 8-oxo-dGMP opposite dC. These results are consistent with the increased propensity of 8-oxo-dGTP incorporation opposite dA relative to dC by Pol  (5:1) in comparison to Pol - (24:1) (19,23). Furthermore, this implies that Pol  may be more susceptible to G to T transversion mutations induced by 8-oxo-dGMP incorporation opposite dC than Pol -.
+
+Curiously, the effects of Ala510 and Asn513 on normal nucleotide incorporation appear to have a more pronounced effect on dTMP than dGMP incorporation. The A510D
+substitution results in a greater effect for dTTP (4.8-fold)
+incorporation than dGTP (2.8-fold). Likewise, a N513A
+substitution has a more prominent effect on dTTP (5.8fold) incorporation than dGTP (1.3-fold). Interestingly, the family X DNA polymerase from the heat-stable organism T. thermophilus, TthPolX, contains a serine in the equivalent position of Asn513. The lack of the asparagine in TthPolX restricts the error-prone insertion of 8-oxo-dGTP opposite dA by 10-fold (72). This is thought to be an adaptation to the higher levels of 8-oxo-dGTP in the dNTP
+pool expected at high temperatures. However, the absence of an asparagine residue at this position results in an imbalance in the efficiency of incorporation of purine and pyrimidine nucleotides (72), suggesting that acquiring an asparagine at this position is important for fidelity of incorporation when dealing with undamaged substrates. Accordingly, Asn513 appears to play a role in improving the purine/pyrimidine balance in Pol . Furthermore, our results demonstrate that Ala510 is also important for reducing the purine/pyrimindine bias. Owing to their larger surface area, purines have more stable base-stacking interactions than pyrimidines. Thus, direct interactions with the nucleobase appear to be relatively more important to stabilize the smaller pyrimidines. In that context, the energetics of the Ala510 interaction are likely to depend on the identity of the incoming nucleotide. At the same time, the hydrogen bond established by Asn513 appears to be critical to properly orient the incoming base. Thus, altering the Asn513 interaction in turn affects the Van der Waals contacts established by Ala510. Accordingly, the effect of the double substitution on 8-oxo-dGMP/dTMP incorporation opposite dA is markedly different than the product of both individual single substitutions.
+
+In all DNA polymerases, interactions with the incoming dNTP are essential for catalysis and selectivity. In the Pol  active site, Ala510 and Asn513 cooperate to stabilize the incoming nucleotide. Our results highlight that dNTP
+binding involves multiple interactions and the relative importance of each interaction depends on the specific base being incorporated. Both Ala510 and Asn513 influence 8oxo-dGMP incorporation as well as the balance between purine/pyrimidine incorporation. Thus, the increased catalytic efficiency of incorporation of undamaged nucleotides and purine/pyrimidine balance conferred by these residues comes thus at the price of an increased mutagenic risk in the presence of a significant pool of 8-oxo-dGTP. The described structures and kinetic results provide a framework for understanding 8-oxo-dGTP incorporation by DNA polymerases, and specifically in the context of the Pol  active site. These new structural insights into the catalytic cycle for 8-oxo-dGTP incorporation by Pol  will contribute to our understanding of the mutagenic potential of oxidized nucleotide pools, and the mechanisms by which DNA polymerases cope with and discriminate against these mutagenic events.
+
+## Accession Codes
+
+Atomic coordinates and structure factors for the precatalytic ternary complex and the post have been deposited in the Protein Data Bank with accession codes 4XA5, 4X5V
+and 4XUS.
+
+## Supplementary Data
+
+Supplementary Data are available at NAR Online.
+
+## Acknowledgements
+
+The authors wish to thank all members of the Garcia-Diaz laboratory for insightful discussions and support, as well as Drs. Mark Lukin and Elena Yakubovskaya for oligonucleotide synthesis.
+
+## Funding
+
+National Institutes of Health (NIH) [T32 GM092714 to M.J.B.]; NIH [ES022930 to K.E.G.]; NIH [GM100021 to M.G.D.]; Use of beamline X25 at the National Synchrotron Light Source; Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-98CH10886];
+Use of beamline 5.0.2 at the Advanced Light Source was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy
+[DE-AC02–05CH11231]. Funding for open access charge:
+NIH [GM100021]; Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393).
+
+Conflict of interest statement. None declared.
+
+## References
+
+1. Neeley,W.L. and Essigmann,J.M. (2006) Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem.
+
+Res. Toxicol., 19, 491–505.
+
+2. Kamiya,H., Miura,K., Ishikawa,H., Inoue,H., Nishimura,S. and Ohtsuka,E. (1992) c-Ha-ras containing 8-hydroxyguanine at codon 12 induces point mutations at the modified and adjacent positions.
+
+Cancer Res., 52, 3483–3485.
+
+3. Moriya,M. (1993) Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C–>T.A transversions in simian kidney cells. *Proc. Natl. Acad. Sci.* U.S.A., 90, 1122–1126.
+
+4. Kamiya,H., Murata-Kamiya,N., Koizume,S., Inoue,H., Nishimura,S.
+
+and Ohtsuka,E. (1995) 8-Hydroxyguanine
+(7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene: effects of sequence contexts on mutation spectra. *Carcinogenesis*, 16, 883–889.
+
+5. Le Page,F., Margot,A., Grollman,A.P., Sarasin,A. and Gentil,A.
+
+(1995) Mutagenicity of a unique 8-oxoguanine in a human Ha-ras sequence in mammalian cells. *Carcinogenesis*, 16, 2779–2784.
+
+6. Kamiya,H., Murata-Kamiya,N., Fujimuro,M., Kido,K., Inoue,H.,
+Nishimura,S., Masutani,C., Hanaoka,F. and Ohtsuka,E. (1995)
+Comparison of incorporation and extension of nucleotides in vitro opposite 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene. *Jpn. J. Cancer Res.: Gann*, 86, 270–276.
+
+7. De Bont,R. and van Larebeke,N. (2004) Endogenous DNA damage in humans: a review of quantitative data. *Mutagenesis*, 19, 169–185.
+
+8. Pursell,Z.F., McDonald,J.T., Mathews,C.K. and Kunkel,T.A. (2008)
+Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity. *Nucleic Acids Res.*, 36, 2174–2181.
+
+9. Maki,H. and Sekiguchi,M. (1992) MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. *Nature*, 355, 273–275.
+
+10. Gad,H., Koolmeister,T., Jemth,A.S., Eshtad,S., Jacques,S.A.,
+Strom,C.E., Svensson,L.M., Schultz,N., Lundback,T.,
+Einarsdottir,B.O. *et al.* (2014) MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. *Nature*, 508, 215–221.
+
+11. Huber,K.V., Salah,E., Radic,B., Gridling,M., Elkins,J.M.,
+Stukalov,A., Jemth,A.S., Gokturk,C., Sanjiv,K., Stromberg,K. *et al.* (2014) Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. *Nature*, 508, 222–227.
+
+12. Colussi,C., Parlanti,E., Degan,P., Aquilina,G., Barnes,D.,
+Macpherson,P., Karran,P., Crescenzi,M., Dogliotti,E. and Bignami,M. (2002) The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP
+pool. *Curr. Biol.: CB*, 12, 912–918.
+
+13. Satou,K., Kawai,K., Kasai,H., Harashima,H. and Kamiya,H. (2007)
+Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells. Free Radic. Biol. Med., 42, 1552–1560.
+
+14. Oda,Y., Uesugi,S., Ikehara,M., Nishimura,S., Kawase,Y.,
+Ishikawa,H., Inoue,H. and Ohtsuka,E. (1991) Nmr-Studies of a DNA Containing 8-Hydroxydeoxyguanosine. *Nucleic Acids Res.*, 19, 1407–1412.
+
+15. Kouchakdjian,M., Bodepudi,V., Shibutani,S., Eisenberg,M.,
+Johnson,F., Grollman,A.P. and Patel,D.J. (1991) NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-Oxo-7H-dG(syn).dA(anti)
+alignment at lesion site. *Biochemistry*, 30, 1403–1412.
+
+16. McAuley-Hecht,K.E., Leonard,G.A., Gibson,N.J., Thomson,J.B.,
+Watson,W.P., Hunter,W.N. and Brown,T. (1994) Crystal structure of a DNA duplex containing 8-hydroxydeoxyguanine-adenine base pairs.
+
+Biochemistry, 33, 10266–10270.
+
+17. Lipscomb,L.A., Peek,M.E., Morningstar,M.L., Verghis,S.M.,
+Miller,E.M., Rich,A., Essigmann,J.M. and Williams,L.D. (1995)
+X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine. *Proc. Natl. Acad. Sci. U.S.A.*, 92, 719–723.
+
+18. Einolf,H.J., Schnetz-Boutaud,N. and Guengerich,F.P. (1998)
+Steady-state and pre-steady-state kinetic analysis of 8-oxo-7,8-dihydroguanosine triphosphate incorporation and extension by replicative and repair DNA polymerases. *Biochemistry*,
+37, 13300–13312.
+
+19. Miller,H., Prasad,R., Wilson,S.H., Johnson,F. and Grollman,A.P.
+
+(2000) 8-oxodGTP incorporation by DNA polymerase beta is modified by active-site residue Asn279. *Biochemistry*, 39, 1029–1033.
+
+20. Shimizu,M., Gruz,P., Kamiya,H., Kim,S.R., Pisani,F.M.,
+Masutani,C., Kanke,Y., Harashima,H., Hanaoka,F. and Nohmi,T.
+
+(2003) Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases. *EMBO Rep.*, 4, 269–273.
+
+21. Hanes,J.W., Thal,D.M. and Johnson,K.A. (2006) Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase. *J. Biol. Chem.*, 281, 36241–36248.
+
+22. Yamada,M., Nunoshiba,T., Shimizu,M., Gruz,P., Kamiya,H.,
+Harashima,H. and Nohmi,T. (2006) Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli. *J. Bacteriol.*, 188, 4992–4995.
+
+23. Brown,J.A., Duym,W.W., Fowler,J.D. and Suo,Z. (2007)
+Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2--deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta. J. Mol.
+
+Biol., 367, 1258–1269.
+
+24. Shimizu,M., Gruz,P., Kamiya,H., Masutani,C., Xu,Y., Usui,Y.,
+Sugiyama,H., Harashima,H., Hanaoka,F. and Nohmi,T. (2007) Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta. *Biochemistry*, 46, 5515–5522.
+
+25. Katafuchi,A., Sassa,A., Niimi,N., Gruz,P., Fujimoto,H.,
+Masutani,C., Hanaoka,F., Ohta,T. and Nohmi,T. (2010) Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides. *Nucleic Acids Res.*, 38, 859–867.
+
+26. Zaccolo,M., Williams,D.M., Brown,D.M. and Gherardi,E. (1996) An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. *J. Mol. Biol.*, 255, 589–603.
+
+27. Svilar,D., Goellner,E.M., Almeida,K.H. and Sobol,R.W. (2011) Base excision repair and lesion-dependent subpathways for repair of oxidative DNA damage. *Antioxid. Redox Signal.*, 14, 2491–2507.
+
+28. Aburatani,H., Hippo,Y., Ishida,T., Takashima,R., Matsuba,C.,
+Kodama,T., Takao,M., Yasui,A., Yamamoto,K. and Asano,M. (1997) Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue. *Cancer Res.*, 57, 2151–2156.
+
+29. Williams,S.D. and David,S.S. (1998) Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA. *Nucleic Acids Res.*, 26, 5123–5133.
+
+30. Zharkov,D.O. and Grollman,A.P. (1998) MutY DNA glycosylase:
+base release and intermediate complex formation. *Biochemistry*, 37, 12384–12394.
+
+31. Williams,S.D. and David,S.S. (1999) Formation of a Schiff base intermediate is not required for the adenine glycosylase activity of Escherichia coli MutY. *Biochemistry*, 38, 15417–15424.
+
+32. Batra,V.K., Beard,W.A., Hou,E.W., Pedersen,L.C., Prasad,R. and Wilson,S.H. (2010) Mutagenic conformation of 8-oxo-7,8-dihydro-2--dGTP in the confines of a DNA polymerase active site. *Nat. Struct. Mol. Biol.*, 17, 889–890.
+
+33. Freudenthal,B.D., Beard,W.A., Perera,L., Shock,D.D., Kim,T.,
+Schlick,T. and Wilson,S.H. (2014) Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. *Nature*, 517, 635–639.
+
+34. Maga,G., Villani,G., Crespan,E., Wimmer,U., Ferrari,E., Bertocci,B.
+
+and Hubscher,U. (2007) 8-oxo-guanine bypass by human DNA
+polymerases in the presence of auxiliary proteins. *Nature*, 447, 606–608.
+
+35. Markkanen,E., Castrec,B., Villani,G. and Hubscher,U. (2012) A
+switch between DNA polymerases delta and lambda promotes error-free bypass of 8-oxo-G lesions. *Proc. Natl. Acad. Sci. U.S.A.*,
+109, 20401–20406.
+
+36. Markkanen,E., Dorn,J. and Hubscher,U. (2013) MUTYH DNA
+glycosylase: the rationale for removing undamaged bases from the DNA. *Front. Genet.*, 4, 18.
+
+37. Fan,W. and Wu,X. (2004) DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex. *Biochem. Biophys. Res. Commun.*,
+323, 1328–1333.
+
+38. Lee,J.W., Blanco,L., Zhou,T., Garcia-Diaz,M., Bebenek,K.,
+Kunkel,T.A., Wang,Z. and Povirk,L.F. (2004) Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts. J. Biol.
+
+Chem., 279, 805–811.
+
+39. Nick McElhinny,S.A., Havener,J.M., Garcia-Diaz,M., Juarez,R.,
+Bebenek,K., Kee,B.L., Blanco,L., Kunkel,T.A. and Ramsden,D.A.
+
+(2005) A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. *Mol.* Cell, 19, 357–366.
+
+40. Capp,J.P., Boudsocq,F., Bertrand,P., Laroche-Clary,A., Pourquier,P.,
+Lopez,B.S., Cazaux,C., Hoffmann,J.S. and Canitrot,Y. (2006) The DNA polymerase lambda is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells. *Nucleic* Acids Res., 34, 2998–3007.
+
+41. Akopiants,K., Zhou,R.Z., Mohapatra,S., Valerie,K.,
+Lees-Miller,S.P., Lee,K.J., Chen,D.J., Revy,P., de Villartay,J.P. and Povirk,L.F. (2009) Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts. *Nucleic* Acids Res., 37, 4055–4062.
+
+42. Garcia-Diaz,M., Bebenek,K., Krahn,J.M., Blanco,L., Kunkel,T.A.
+
+and Pedersen,L.C. (2004) A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology. *Mol. Cell*, 13, 561–572.
+
+43. Kabsch,W. (2010) Xds. *Acta Crystallogr. Sect. D, Biol. Crystallogr.*,
+66, 125–132.
+
+44. Evans,P.R. and Murshudov,G.N. (2013) How good are my data and what is the resolution? *Acta Crystallogr. Sect. D, Biol. Crystallogr.*, 69, 1204–1214.
+
+45. Vonrhein,C., Flensburg,C., Keller,P., Sharff,A., Smart,O.,
+Paciorek,W., Womack,T. and Bricogne,G. (2011) Data processing and analysis with the autoPROC toolbox. *Acta Crystallogr. Sect. D, Biol.*
+Crystallogr., 67, 293–302.
+
+46. McCoy,A.J., Grosse-Kunstleve,R.W., Adams,P.D., Winn,M.D.,
+Storoni,L.C. and Read,R.J. (2007) Phaser crystallographic software.
+
+J. Appl. Crystallogr., 40, 658–674.
+
+47. Garcia-Diaz,M., Bebenek,K., Krahn,J.M., Kunkel,T.A. and Pedersen,L.C. (2005) A closed conformation for the Pol lambda catalytic cycle. *Nat. Structu. Mol. Biol.*, 12, 97–98.
+
+48. Emsley,P., Lohkamp,B., Scott,W.G. and Cowtan,K. (2010) Features and development of Coot. *Acta Crystallogr. Sect. D, Biol.*
+Crystallogr., 66, 486–501.
+
+49. Zwart,P.H., Afonine,P.V., Grosse-Kunstleve,R.W., Hung,L.W.,
+Ioerger,T.R., McCoy,A.J., McKee,E., Moriarty,N.W., Read,R.J.,
+Sacchettini,J.C. *et al.* (2008) Automated structure solution with the PHENIX suite. *Methods Mol. Biol.*, 426, 419–435.
+
+50. Murshudov,G.N., Skubak,P., Lebedev,A.A., Pannu,N.S.,
+Steiner,R.A., Nicholls,R.A., Winn,M.D., Long,F. and Vagin,A.A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. *Acta Crystallogr. Sect. D, Biol. Crystallogr.*, 67, 355–367.
+
+51. Smart,O.S., Womack,T.O., Flensburg,C., Keller,P., Paciorek,W.,
+Sharff,A., Vonrhein,C. and Bricogne,G. (2012) Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. *Acta Crystallogr. Sect. D, Biol. Crystallogra.*, 68, 368–380.
+
+52. Karplus,P.A. and Diederichs,K. (2012) Linking crystallographic model and data quality. *Science*, 336, 1030–1033.
+
+53. Sawaya,M.R. (2014) Methods to refine macromolecular structures in cases of severe diffraction anisotropy. *Methods Mol. Biolo.*, **1091**,
+205–214.
+
+54. Chen,V.B., Arendall,W.B. 3rd, Headd,J.J., Keedy,D.A.,
+Immormino,R.M., Kapral,G.J., Murray,L.W., Richardson,J.S. and Richardson,D.C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D, Biol. Crystallogr., 66, 12–21.
+
+55. Vaguine,A.A., Richelle,J. and Wodak,S.J. (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model.
+
+Acta Crystallogra. Sect. D, Biol. Crystallogr., 55, 191–205.
+
+56. Cruickshank,D. (1949) The accuracy of electron-density maps in X-ray analysis with special reference to dibenzyl. *Acta Crystallogr.*, 2, 65–82.
+
+57. Agarwal,R. (1978) A new least-squares refinement technique based on the fast Fourier transform algorithm. *Acta Crystallogr. Sect. A*,
+34, 791–809.
+
+58. Murshudov,G.N., Vagin,A.A. and Dodson,E.J. (1997) Refinement of Macromolecular Structures by the Maximum-Likelihood Method.
+
+Acta Crystallogr. Sect. D, 53, 240–255.
+
+59. Bebenek,K., Pedersen,L.C. and Kunkel,T.A. (2011) Replication infidelity via a mismatch with Watson-Crick geometry. *Proc. Natl.* Acad. Sci. U.S.A., 108, 1862–1867.
+
+60. Garcia-Diaz,M., Bebenek,K., Sabariegos,R., Dominguez,O.,
+Rodriguez,J., Kirchhoff,T., Garcia-Palomero,E., Picher,A.J.,
+Juarez,R., Ruiz,J.F. *et al.* (2002) DNA polymerase lambda, a novel DNA repair enzyme in human cells. *J. Biol. Chem.*, 277, 13184–13191.
+
+61. Brown,J.A., Pack,L.R., Sherrer,S.M., Kshetry,A.K., Newmister,S.A.,
+Fowler,J.D., Taylor,J.S. and Suo,Z. (2010) Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase lambda. *J. Mol. Biol.*, 403, 505–515.
+
+62. Eom,S.H., Wang,J. and Steitz,T.A. (1996) Structure of Taq polymerase with DNA at the polymerase active site. *Nature*, 382, 278–281.
+
+63. Kiefer,J.R., Mao,C., Braman,J.C. and Beese,L.S. (1998) Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. *Nature*, 391, 304–307.
+
+64. Krahn,J.M., Beard,W.A., Miller,H., Grollman,A.P. and Wilson,S.H.
+
+(2003) Structure of DNA polymerase beta with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential. *Structure*, 11, 121–127.
+
+65. Hsu,G.W., Ober,M., Carell,T. and Beese,L.S. (2004) Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA
+polymerase. *Nature*, 431, 217–221.
+
+66. Brieba,L.G., Eichman,B.F., Kokoska,R.J., Doublie,S., Kunkel,T.A.
+
+and Ellenberger,T. (2004) Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase.
+
+EMBO J., 23, 3452–3461.
+
+67. Vasquez-Del Carpio,R., Silverstein,T.D., Lone,S., Swan,M.K.,
+Choudhury,J.R., Johnson,R.E., Prakash,S., Prakash,L. and Aggarwal,A.K. (2009) Structure of human DNA polymerase kappa inserting dATP opposite an 8-OxoG DNA lesion. *PLoS One*, 4, e5766.
+
+68. Beard,W.A. and Wilson,S.H. (2000) Structural design of a eukaryotic DNA repair polymerase: DNA polymerase beta. *Mutat. Res.*, 460, 231–244.
+
+69. Vande Berg,B.J., Beard,W.A. and Wilson,S.H. (2001) DNA structure and aspartate 276 influence nucleotide binding to human DNA
+polymerase beta. Implication for the identity of the rate-limiting conformational change. *J. Biol. Chem.*, 276, 3408–3416.
+
+70. Garcia-Diaz,M., Bebenek,K., Krahn,J.M., Pedersen,L.C. and Kunkel,T.A. (2007) Role of the catalytic metal during polymerization by DNA polymerase lambda. *DNA Repair*, 6, 1333–1340.
+
+71. Werneburg,B.G., Ahn,J., Zhong,X., Hondal,R.J., Kraynov,V.S. and Tsai,M.D. (1996) DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity.
+
+Biochemistry, 35, 7041–7050.
+
+72. Garrido,P., Mejia,E., Garcia-Diaz,M., Blanco,L. and Picher,A.J.
+
+(2014) The active site of TthPolX is adapted to prevent 8-oxo-dGTP misincorporation. *Nucleic Acids Res.*, 42, 534–543.
+
+73. Fraga,C.G., Shigenaga,M.K., Park,J.W., Degan,P. and Ames,B.N.
+
+(1990) Oxidative damage to DNA during aging: 8-hydroxy-2--deoxyguanosine in rat organ DNA and urine. Proc.
+
+Natl. Acad. Sci. U.S.A., 87, 4533–4537.

medical/md/PMC5083326.md

@@ -0,0 +1,52 @@
+# The Earl Of Feversham
+
+Chairman of the Council vffjlfj, National Association for Mental Health, 1946-1"' 
+
+# A Personal Tribute
+
+Lord Feversham has % 
+after a short illness at the ea j age of 56. Tributes have & 
+paid to his career in P? V.j3l agriculture, the Arts and s?\y reform. I want to Pay,hair' tribute to him as the first Co . 
+
+man of The National Ass? 
+
+tion for Mental HealthHe was no figure-head c" ^ 
+man; he saw clearly the sC j of the Association's work { 
+as he judged appropriate 8 t an impetus to develops , 
+Often he backed his judgO1 ^ 
+with gifts from his own P.?c 0f He was behind the opening the Northern Office in 1952'^ 
+creation of the Mental He National Appeal in 1954, appointment of a public formation officer in 1956 a0<* ^ 
+a local associations organise^ 
+1962. In 1961 he began a s* , 
+of discussions on positive me ^ 
+health and as a result oi ^ 
+personal invitation there is J 
+a Seminar in Bristol ai end of September on Counselling Services in Schools. 
+
+As he gained confidence, he began to assume a national role as for the Mental Health Services. In 1958 he initiated the first debate Jn to House of Lords on the Royal Commission's Report on the Law relating Mental Illness and Mental Deficiency. He attended all the sittings w^^nr of Mental Health Act of 1959 was before the Lords, speaking on a nurn amendments. In February 1960 it was at his instigation that the YounghusDtj,e Report was first debated. Last year, in July he initiated a debate on working of the Mental Health Act. .. 
+
+? 1 1 (1 These solemn milestones in his career as our Chairman give very little { 
+of the kind of man he was. He had fastidious tastes and everything a y$ 
+him was of the best; he was never satisfied with second best. In some ^ ^ 
+he was delighted to play his part as a peer and he did it beautifully, was always approachable; he always minded about those who worked foT i a His relationship with all his staff was one of warm friendship. He gift of fun which acted as a leaven to any meeting. Nobody could be 
+
+![0_image_0.png](0_image_0.png)
+
+he are the competing of the competing of the competing of the wide vistes as a sense of the came to the vistas of his Yorkshire moors, plus a feeling that he had just come from a from of his Yorkshire mors, plus a feeling room. But he had probably come from a local associations meeting or from conversations with his friends the Probation Officers. Nothing was too large or too small for his attention.
+
+His interest in mental health—not mental ill-health—inspired all his work for social reform. He drove himself to make speeches because he felt this as something he alone could do. It did not come easily to him. He was e on trable chairman, courteous, patient, humorous, kind : always with an cye on larger issues.
+
+All the world loves a Lord, but very few Lords are as many-sided in their interests or as worthy of love as Lord Feversham. I have written this tribute to my chief and my friend.
+
+## Mary Applebey Editorial The Space Between
+
+It is a common error to assume that the "teenage problem" is that of the present generation and age only. Yet Keats described adolesence as "the space in between in rhich the soul is in a ferment, the if. acter undecided and the way of life uncertain". And, in Henry IV,
+d shakespeare gives a vivid clinical description of adolescent rebellion as parental authority and expectation in his picture of Prince denry of Monmouth. The present dy problem differs in detail and degree rather than in basic principle.
+
+Critical adults There are many reasons, too, why the adult world is basically so critical of the up-and-coming generation; and why we are often reluctant to ne the problem as one for rather than solely of, the adolescent. Not the least of these reasons is that this up-and-coming generation reminding us of our own steadily advancing years; and of the fact that we will be challenged by, and in rivalry with, this new generation when they shortly reach adulthood themselves.
+
+## Try To Understand
+
+The adult need not, and indeed often cannot and should not, agree with the teenager's views on life; but at least he must try to understand the meaning of these views, and the problems which often lie behind them for the adolescent.
+
+The aim of the contributors to this issue has been to highlight and explain some of the specific and special problems of the present-day teenage generation.

medical/md/PMC5136409.md

@@ -0,0 +1,273 @@
+# Original Article The Effect Of Lucite Glass Reinforcement On The Properties Of Conventional Glass-Ionomer Filling Materials
+
+Haleh Kazemi Yazdi 1, Richard Van Noort 2, Mona **Mansouri** 3 1 Dept. of Restorative Dentistry, Dental Branch, Islamic Azad University, Tehran, Iran. 
+
+2 Academic Unit of Restorative Dentistry, University of Sheffield, Sheffield, UK. 
+
+3 Residence of Dept. Operative and Cosmetic Restorative, Dental Branch, Islamic Azad University, Tehran Branch, Tehran, Iran. 
+
+![0_image_0.png](0_image_0.png)
+
+## Abstract
+
+Statement of the **Problem:** The usage of glass ionomer cements (GICs) restorative materials are very limited due to lack of flexural strength and toughness. Purpose: The aim of this study was to investigate the effect of using a leucite glass on a range of mechanical and optical properties of commercially available conventional glass ionomer cement. 
+
+Materials and **Method:** Ball milled 45μm leucite glass particles were incorporated into commercial conventional GIC, Ketac-Molar Easymix (KMEm). The characteristics of the powder particles were observed under scanning electron microscopy. The samples were made for each experimental group; KMEm and lucite- modified KetacMolar easy Mix (LMKMEm) according to manufacturer's instruction then were collected in damp tissue and stored in incubator for 1 hour. The samples were divided into two groups, one stored in distilled water for 24 hours and the others for 1 week.10 samples were made for testing biaxial flexural strength after 1 day and 1 week, with a crosshead speed of 1mm/min, calculated in MPa. The hardness (Vickers hardness tester) of each experimental group was also tested. To evaluate optical properties, 3 samples were made for each experimental group and evaluated with a spectrophotometer. The setting time of modified GIC was measured with Gillmore machine. 
+
+Result: The setting time in LMKMEm was 8 minutes. The mean biaxial flexural strength was LMKMEm/ 1day: 24.13±4.14 MPa, LMKMEm/ 1 week: 24.22±4.87 MPa KMEm/1day:28.87±6.31 MPa and KMEm/1 week: 26.65±5.82 MPa which were not statistically different from each other. The mean Vickers hardness was LMKMEm: 403±66 Mpa and KMEm: 358±22 MPa; though not statistically different from each other. The mean total transmittance (Tt) was LMKMEm: 15.9±0.7, KMEm: 22.3±1.2, the mean diffuse transmittance (Td) was LMKMEm: 12.2±0.5, KMEm: 18.0±0.5 which were statistically different from each other. Conclusion: Leucite glass can be incorporated with a conventional GIC without interfering with setting time. Yet, it did not improve the mechanical and optical properties of the GIC.
+
+Corresponding **Author:** Mansouri M., Dept. Operative and Cosmetic Restorative, Dental Branch, Islamic Azad University, Tehran Branch, Tehran, Iran. Tel: +98-2532942742 Email: dr_monamansouri@yahoo.com Cite this article as: Yazdi H., Van Noort R., Mansouri M. The Effect of Lucite Glass Reinforcement on the Properties of Conventional Glass-Ionomer Filling Materials. J *Dent* Introduction One major advantage of glass ionomer cements (GICs) 
+as a restorative material is bonding to dental tissue and due to this, the prepared cavity for this material can be very conservative. [1] In addition to this, glassionomer has many other advantages such as fluoride release, coefficient of thermal expansion close to tooth structure, durable bond to tooth tissue and biocompatibility. [2-3] In spite of all these advantages, the clinical performance of the material are limited due to other drawbacks: low flexural and fracture toughness, early water sensitivity, low fatigue strength, high surface roughness and poor wear resistance. [2-4] 
+Many investigators have attempted to modify the formulation of GICs to overcome these problems. The modifications have been done on the powder and liquid composition and configuration. [2-6] Modification in powder was different in many aspects, from refinement of glass particle size to incorporation of different particles such as metal, ceramics and fibres. [1-2, 714] Along with modifications in the glass powder of GICs, the matrix has been changed. Some of these modifications are: use of copolymer (carboxylic aciditaconic acid), incorporation of tartaric acid in the liquid part, use of the copolymer with high molecular weight, concentration of poly acid, incorporation of freeze- dried acid copolymer in the cement powder and the use of N-vinylpyrrolidone containing polyacids 
+(NVP). [15-16] Culberston et al. [7] showed that acrylic acid-itaconic acid- NVP (AA/IA/NVP) polymers with diverse molar ratio can produce GICs with increased mechanical properties. Hydroxy apatite (HA) can react with GICs matrix by polyacid hydroxyl group, so incorporation of HA into GICs may improve physical properties and biocompatibility of set cement. Besides, it contributes to the bond strength of GICs due to a similar composition to tooth structure. [10] Lucas et al. [10] illustrated that incorporation of HA in GICs powder increased the fracture toughness significantly so it made more durable bond to dental tissues. Moreover, according to Moshavernia et al. [5] addition of nano HA enhanced the mechanical properties of commercial GIC Fuji II (Fuji II LC, GC Corp.; Tokyo, Japan) and it seems that the bonding property was also enhanced. 
+
+Micro-sized Yttria stabilized zirconia (YSZ) 
+powder was added to Miracle Mix (GC America Inc.; Alsip, IL, USA) powder to improve the mechanical properties. The study showed that the uniform distribution of the glass and YSZ particle in the matrix, which ensured a high packing density of GICs, gave relatively high mechanical properties to YSZ GIC. [17] However, these two last modifications have been done as a patent and there are no commercial brands with these modifications. 
+
+High viscosity conventional GICs like KetacMolar-easy mix (3M ESPE I.D. No.70201119107, Germany), and Fuji IX (GC America Inc.; Alsip, IL, 
+USA) have been introduced recently, designed as an alternative to amalgam for posterior preventive restorations. These show significant improvements in compressive strength and fracture toughness. [15, 18] They can be used as a core build up material under a crown, permanent filling for primary teeth and class I restorations in non-occluded regions. [15, 19] Nevertheless, they are not suitable for high stress areas and still their main problems are low fracture toughness and flexural strength compared to other restorative materials. [18] 
+The aim of this study was to evaluate physical and mechanical properties of a conventional glass ionomer material after substitution a leucite glass for the acid soluble glass in the powder of this material. 
+
+## Materials And Method
+
+The material used in this study was a conventional glassionomer material, Ketac-Molar Easymix(KMEm) (3M ESPE I.D. No.70201119107, Germany), shade 
+(A3). The principle composition of the powder is aluminum-calcium-lanthanum-fluorosilicate glass and the liquid is an aqueous solution of polycarbonic acid (a copolymer from acrylic and maleic acid) and tartaric acid. 
+
+In order to modify the GIC powder, a glass powder with a composition of a leucite glass ceramic (PS4) which was made by a PhD student in Sheffield University Dental School Adult Dental care, was added to KMEm glass ionomer powder. The modified glass powder was ball milled for 10 min and 400 rpm in ball milling machine (Retsch GmbH; PM 100, Germany) to reduce the particles size. To achieve a uniform size of glass filler the leucite glass ceramic was sieved manually with a 45μ sieve (Retsch GmbH; Germany), thus most of the glass had a particle size less than 45 μm. 
+
+The leucite glass ceramic was added to the glassionomer powder in 20 vol%. This addition has been done manually by dry mixing on a paper plate using a metal spatula until an even distribution was achieved. 
+
+The original and modified glass powder was observed under scanning electron microscope (SEM) 
+(X1-20 SEM, Holland). 
+
+A silicone rubber mould was used for fabrication of the samples. The samples were made in a disc with a thickness of 2.5mm and diameter of 12mm.Mixing of the modified powder and the cement liquid was carried out using a metal spatula according to manufacturer's instructions. The mixing time did not exceed 1 min and working time was 3-3.5 min. The cement paste was carried into the mould by metal spatula and pressed using a glass slab. After the material was set, the specimens were collected in a damp tissue and were stored in an incubator (LEEC Compact Incubator LEEC Limited; Nottingham, UK) at a temperature of 37ºC for 1 hour. This was followed by storing the specimens in distilled water for 24 hours. For the biaxial flexural strength test, the specimens were ground and polished down to 600 grit using SiC (Buehler-Met; Metallographic Grinding paper, UK) paper. The final dimensions of specimens were measured with digital micrometer with a precision of 0.01 mm on four central axes and thickness of all samples was 2±0.2mm. 
+
+For the hardness test, the specimens were ground and polished down to 1μm using SiC papers and diamond paste. 
+
+Three samples were made for each experimental group for spectrophotometer test in the silicon rubber mold. The samples were made with a thickness of 2mm and diameter of 16mm. Mixing the modified powder and the cement liquid was carried out by using a metal spatula according to manufacturer's instructions. The mixing time did not exceed 1 min and working time was 3-3.5 min. The cement paste was carried into the mould by metal spatula and was pressed using a glass slab. After the materials were set, the specimens were collected in damp tissue and were stored in an incubator at a temperature of 37ĉ for 1 hour. After that the specimens were stored in distilled water for 24 hours. The specimens were ground and polished down to 600 grit using SiC papers. The thickness of specimens was measured with digital micrometer with a precision of 0.01 mm on three central axes and it was 1±0.1 mm. The specimens were stored in distilled water until the spectrophotometer test was carried out. 
+
+The setting time of modified glass ionomer was measured with Gillmore needle apparatus (Impact Test Equipment Ltd; Scotland, UK) and digital timer. The Gilmore needle works on the principle of two different sized needles with different weight being placed onto the surface of setting cement. Initially a large needle (10 mm) with a weight of (28g) is applied and the working time is determined when the needle no longer indents the surface. A second needle with a smaller diameter (1mm) and larger weight (400g) is then applied to the surface and time when this needle no longer indents the surface is designated as the setting time. Both tests were carried out at ambient temperature 21ºC.
+
+To evaluate flexural strength, ten discs (12mm diameter and 2±0.2 thickness) were made for each experimental group. The biaxial flexural strength was measured after 1day and 1 week. 
+
+The thickness of each specimen was measured in the centre and three peripheral points using a digital micrometer before testing. Flexural property was quantified by biaxial flexural strength test (LLOYD Instrument, LRX 103648, Farham, UK) at a crosshead speed of 1 mm/min (Figure 1). The flexural strength was obtained by measuring the load at fracture in Newton and then calculated in MPa. 
+
+![2_image_0.png](2_image_0.png)
+
+Ten discs were made for each experimental group (N=10). The discs were mounted on the Vickers hardness machine (Vickers Hardness VX Series Tester) and indented with Vickers diamond pyramid at 1 Kg load and the resulting diagonal length was measured. The Vickers hardness in MPa is then obtained using the following equation: HV=1.854 P/d2 Where p is the applied load in Newtons and d is the mean diagonal length in mm. [21] 
+
+![3_image_0.png](3_image_0.png)
+
+Figure 2: Schematic picture of the mechanism of light transmittance detection through the integrating sphere. 
+
+Three sample of each group (N=3) were analyzed by Perkin Elmer Lambda 2 spectrophotometer 
+
+![3_image_2.png](3_image_2.png) with an integrating sphere sensor (Figure 2). The spectral range of the colorimeter was between 380 and 700 nm at 1 nm interval. The data were analyzed by UV 
+WinLab (version 2) software. One-way ANOVA followed by Tukey's pairwise comparison was used for analyzing the data. 
+
+![3_image_3.png](3_image_3.png)
+
+## Result
+
+Characterizations of **powders** 
+The SEM of glass powder in KMEm (3M ESPE, I.D. 
+
+No.70201119107, Germany) (control) and LMKMEm 
+
+![3_image_1.png](3_image_1.png) (experimental) groups are illustrated in Figure 3 and 4. 
+
+The particle size in LMKME was larger as a result of adding 45μm Leucite glass ceramic. However the particle size of the powder was significantly decreased after ball milling and it can be clearly seen in Figure 5. 
+
+Setting **time analysis** 
+The setting time in KMEm GICs (3M, ESPE, I.D. No.70201119107, Germany) is 7 min. Adding 20 vol% leucite glass ceramic to the powder of KMEm GICs increased the setting time. After ball milling, the setting time was decreased to 8 min which was very close to original cements. 
+
+Biaxial **Flexural Strength** 
+The mean biaxial flexural strength of the control (KMEm; 3M, ESPE, I.D. No.70201119107, Germany) and experimental (LMKMEm) groups is summarized in Table 1. 
+
+Table 1: Summary of result of biaxial flexural strength of two glass ionomer cement and storage time (MPa). 
+
+KMEm after 1 day storage recorded a mean biaxial flexural strength of 28.87 MPa, which was the highest. After 1 week storage, the biaxial flexural strength decreased in this group and a mean of 26.65 MPa was recorded for this group. However the differ-
+
+| Materials and                                                                                                | (MPa)   | Min-Max   | SD   |    |
+|--------------------------------------------------------------------------------------------------------------|---------|-----------|------|----|
+| Mean                                                                                                         | (MPa)   | N         |      |    |
+| storage time                                                                                                 |         |           |      |    |
+| KMEm,1 day                                                                                                   | 28.87   | 19.2-37.1 | 6.31 | 10 |
+| LMKMEm,1 day                                                                                                 | 24.13   | 17.4-31.5 | 4.14 | 10 |
+| KMEm,1 week                                                                                                  | 26.65   | 17.6-36.1 | 5.82 | 10 |
+| LMKMEm,1 week                                                                                                | 24.22   | 18.6-32.7 | 4.87 | 10 |
+| KMEm= Ketac-Molar Easymix (Control group), LMKMEm=  Leucite Modified Ketac-Molar Easymix(Experimental group) |         |           |      |    |
+
+ence was not significant. (p> 0.05) For LMKMEm, the biaxial flexural strength has not changed after 1 week storage and a mean of 24.13 MPa and 24.22 MPa was recorded for 1day and 1 week storage respectively. 
+
+One-way ANOVA followed by Tukey's pairwise comparison showed no significant statistical difference between the control and experimental groups(p> 0.05). 
+
+Vickers **Hardness** 
+The calculated Vickers hardness values are summarized in Table 2. 
+
+| Materials                    | Mean  (MPa)   | Min-Max   | SD   |    |
+|------------------------------|---------------|-----------|------|----|
+| (MPa)                        | N             |           |      |    |
+| KMEm  (Control group)        | 358           | 325-399   | 22   | 10 |
+| LMKMEm  (Experimental group) | 403           | 275-533   | 66   | 10 |
+
+Table 2: Summary of the calculated Vickers hardness in two glass ionomer cements groups in MPa. 
+
+LMKMEm GIC recorded a mean Vickers hardness of 402 MPa which is higher than KMEm GIC 
+
+![4_image_0.png](4_image_0.png)
+
+with 358 MPa. One-way ANOVA followed by Tukey's pairwise comparison showed no significant statistical difference between the control and experimental groups (p> 0.05). 
+
+![4_image_1.png](4_image_1.png)
+
+## Optical Properties
+
+Three optical properties of two glass ionomers were analyzed in this study. The result is graphically presented in Figures 6, 7.
+
+The means of 22.3 for total transmittance (Tt) 
+and 18 for diffuse transmittance (Td) and 4.3 for direct transmittance (Tdir) were recorded for KMEm GIC (control group) and the means of Tt, Td and Tdir were recorded 15.9, 12.2 and 3.7 respectively for LMKMEm GIC (experimental group). A decrease in the mean of all optical values was observed in the experimental group according to these data. 
+
+One-way ANOVA followed by Tukey's pairwise comparison showed significant statistical difference of Tt, Td between the control and experimental groups (p< 0.05). Regarding Tdir, one-way ANOVA followed by Tukey's pairwise showed no statistical difference between the control and experimental groups(p> 0.05). 
+
+## Discussion
+
+In this experimental study, 20% volume fraction of a conventional GIC (like KMEm) was replaced with non 
+-reactive leucite glass to try and produce a conventional GIC with improved mechanical properties. Leucite glass was chosen to add to the powder because it is a strong glass with an affinity to poly acrylic acid. [22] 
+Simultaneous with preparing and working with glass ionomer formulation, it is really critical to consider working and setting time. [7] Substitution of reactive powder with non-reactive produced a modified conventional glass ionomer (LMKMEm) with significantly increased setting time (14 minute). The proper setting time for restorative GIC is 6-8 minutes. [23] So to overcome this problem, the powder was ball milled to produce finer grain size to reduce the setting time. 
+
+Finer grain size can speed up the setting reaction because of greater surface. [16] 
+Modification of conventional glass ionomer with leucite glass was a novel approach. Hence the proper milling time and rotating speed for ball milling of the powder had to be done by trial and error. The powder was ball milled with increasing time till the mixing cement had set in proper setting time and was ended up by 10 minute rotating time and 400 rotating speed. The SEM micrographs clearly showed finer grain size of the LMKMEm GICs powder after ball milling (Figure 3, 4). Regarding manipulation properties, LMKMEm- GICs has a setting time of 8 minute which is a 
+
+![5_image_0.png](5_image_0.png)
+
+proper setting time for restorative GIC. Additionally, the LMKMEm GICs powder is mixed to the liquid as the same amount as the KMEm-GICs powder and the viscosity of the paste is the same as well. (Figure 8) 
+In this study, flexural strength test was used to evaluate the mechanical strength of the new cement. Mechanical strength of many brittle dental materials such as cements is evaluated by tensile rather than compressive strength. Crack propagation is the main reason for the failure of these materials which is favored by tensile rather than compressive loading. 
+
+Prosser et al. [24] have recommended that the flexural strength is the most appropriate measurement method for strength evaluation of GICs. They elucidated that, as the fracture would occur in atomic level in GICs matrix by tensile or shear failure; compressive strength basically had no meaning. [24] 
+Although tensile strength is the best method to estimate the mechanical properties of GICs, direct measurement of tensile strength for brittle material has technical problems. Tensile strength measurement by loading in compression (diametrical tensile strength) is only valid if a material does not have significant plastic deformation. So, diametrical tensile strength test usually gives higher values to polyacrylic acid base cements which have some degrees of plasticity. According to these reasons, it has been suggested that the flexural strength tests are the most practical and reliable way for evaluation of tensile strength of brittle materials. However, measurement of compressive and diametrical tensile strength has been reported for GICs, recently. [25] 
+The results in Table 1 show that the mechanical properties of KMEm have not improved with this modification. A mean biaxial flexural strength of 24.13±4.14 was recorded for LMKMEm after 1 day storage in distilled water. In comparison it was 28.87± 6.31 MPa for KMEm. However this difference was not statistically significant. 
+
+It is suggested that different microstructure and variation in composition may have affected the mechanical properties of the GICs. [25] The smaller particle size, smaller and lesser voids and proper bonding of glass fillers and matrix in KMEm GIC appear to have resulted in higher flexural strength. The somewhat larger particle size, lack of proper bond between the filler and matrix and larger and more voids in LMKMEm GIC have caused opposite trends value for flexural strength. Mitsuhashi et al. [3] suggested that the direct tensile strength of resin modified GIC increased significantly by reduction of filler particle size to 5μm. They claimed that the particle size in KMEm - GIC powder is mainly less than 9.6μm, which means that 90% of the particle having a diameter of less than 9.6μm. Additionally 50% of the glass particles are 2.8μm or smaller. [3] Moreover, it has been reported that proper bonding between filler particles and glass matrix is contributing to mechanical properties of set cement by modifying the character of local stress and crack propagation in the matrix. [2] As a result of improper bond between the leucite glass particles and matrix of GIC, the large leucite glass particles might act as a void and contribute to crack propagation in LMKMEm GIC. It can be another reason for decreasing the flexural strength in LMKMEm GIC. Treatment of leucite glass particles with a coupling agent might improve the bonding of this glass particle to the matrix and consequently can improve the mechanical properties of GIC. However further research is necessary to establish this. 
+
+The biaxial flexural strength of KMEm had decreased after 1 week storage in distilled water. Although this reduction was not significant, it was an interesting finding. In this GIC, 5% dried polycarbonate (a copolymer from acrylic and maleic acid) is incorporated into the powder. This leads to greater overall acid concentration in the cement which increases cross-linking and improves mechanical values without a considerable increasing in the initial viscosity. It was reported that GICs based on copolymer of acrylic and maleic acid demonstrate deterioration in flexural strength after water storage. [26] It seems in the glass ionomers with polyacrylic matrix compressive and flexural strength increases after storage in the water, whereas that with copolymers acid does not. 
+
+[27] Generally, the mechanical strength of GIC increases with time. This has been attributed to the maturation of the cement matrix due to further cross-linking because of the formation of new aluminum and calcium polyacrylates and the slow build up of a silica matrix from acid solution of glass particles. [26] 
+The fracture mode of flexural strength samples was also different in KMEm and LMKMEm. All the samples in LMKMEm (experimental groups) regardless to the storage time have been fractured into two pieces. While in KMEm (control groups) a few samples have been fracture into three pieces. It can be attributed to higher flexural strength of control groups comparing to experimental groups. 
+
+In this study, traditional Vickers hardness was used to test the hardness of KMEm-GIC and LMKMEm-GIC. As it was mentioned in the literature review, this was not the best method for testing hardness of GICs because this material is a heterogeneous, biphasic and weak. The applying load was 1 kg which might be very heavy for these materials. But it was the only available method for testing the hardness of the new material. 
+
+According to the result in Table 2, the Vickers hardness of LMKMEm was 403±66 MPa which was higher than KMEm (358 ± 22). This greater value could be attributed to use of extremely hard leucite glass fillers; however, this difference was not statistically significant. Guggenberger et al. [28] reported that the hardness of KMEm reached the level of hybrid composite with the value of 420 MPa. In their study they did not mention the test method they used. Their result was represented in a diagram without showing the standard deviation. According to product pamphlet from ESPE, the surface hardness of KMEm GIC was recorded 420±82 MPa with DIN 53456 standard methods. 
+
+The result of the above mentioned study is different from the outcome of the present study. The difference in the hardness value can be explained by using different testing methods and numerous porosities in our samples. 
+
+The reading of indention was really difficult in this study due to indistinct border of pyramid in the micrograph of Vickers hardness tester (Figures 9 and 10). This phenomenon was explained by Yap AU. et al. [29]They suggested that the pile- up of the material around the indenter due to plasticity of the material is the main cause of this problem. Therefore, to record exact mechanical properties from instrumented indention, a good understanding of the relationship between indentational load and the true area is necessary. [29] 
+The optical properties of new GIC along with the mechanical properties were evaluated. Table 3 shows the mean Tt of 22.3±1.2 and Td of 18.0±0.5 for KME 
+m-GIC compared to 15.9±0.7 and 12.2±0.5 respectively for LMKMEm GIC. A reduction in Tt value and Td value was observed in the LMKMEm GIC which was statistically significant p< 0.05. According to this result LMKMEM GIC is more opaque comparing to KMEm GIC. One of the reasons for opacity of GIC is the mismatch of refractive index of the fillers and matrix. [30] Hence, this reduction can be attributed to more porosity and imperfection in LMKMEM GIC which can increase the refractive index of the matrix. 
+
+Table 3: Summary of calculated three optical properties in two glass-ionomer cements. 
+
+| Optical properties           | Tt                        | Td   | Tdir   |
+|------------------------------|---------------------------|------|--------|
+| Materials                    |                           |      |        |
+| KMEm  (Control group)        | 22.3±1.2 18.0±0.5 4.3±1.3 |      |        |
+| LMKMEm  (Experimental group) | 15.9±0.7 12.2±0.5 3.7±0.2 |      |        |
+
+Filler composition and content also can change the optical properties of direct restorative materials. [31] By very careful evaluation of Figures 6 and 7, a shifting down of Tt and Td can be observed in LMKMEm GIC. The curvature of Tt and Td is the same in both materials but it seems that the curve shifted down in LMKMEm GIC group. It showed that color does not change in the new material because light absorption did not change in all reflected wavelengths. The leucite glass has low refractive index so this opacity could be attributed to different refractive indices of leucite glass and glass ionomer matrix. 
+
+Using leucite glass composition with refractive index closer to the matrix might preclude this problem and more lucent GICs could be produced. However, KMEm is a posterior restorative material and improvement in mechanical properties might have priority compared to optical properties. 
+
+The samples with numerous porosities were the prominent problem in this study. The depth and size of the voids is related to microstructure of the GIC. Some factors such as size and shape of fillers, wet ability of fillers and mixing procedure can affect the number of porosity. [25] Mechanical mixing and using angled nozzle for accurate placement can produce samples with less porosity. 
+
+In this study, new GIC with proper manipulation properties was produced successfully. The mechanical properties of the new material have not improved as a result of problems which were mentioned in the discussion. However, this study could be a good basic study for future investigations. 
+
+## Conclusion
+
+Within the limitations of the present study, the addition of leucite glass into the KMEm GIC produced a modified conventional GIC with reasonable setting time and manipulation properties. The addition of leucite glass to the KMEm GIC did not improve the biaxial flexural strength and hardness of the material. 
+
+The addition of leucite glass to the KMEm GIC cement produced a new material with a higher opacity comparing to the original material. 
+
+## Acknowledgments
+
+This article is based on thesis submitted to the Academic Unit of Restorative dentistry, University of Sheffield, Sheffield, UK in fulfillment of the requirements for master degree. Special gratitude is forwarded to Prof. Richard Van Noort, for his expert advice and academic devotion. 
+
+## Conflict Of Interest
+
+The authors of this manuscript certify that they have no conflict of interest. 
+
+## References
+
+[1] Nagaraja Upadhya P, Kishore G. Glass ionomer cement: The different generations. Trends Biomater Artif Organs. 2005; 18: 158–165. 
+
+[2] Hammouda IM. Reinforcement of conventional glassionomer restorative material with short glassfibers. J Mech Behav Biomed Mater. 2009; 2: 73-81. 
+
+[3] Mitsuhashi A, Hanaoka K, Teranaka T. Fracture toughness of resin-modified glass ionomer restorative materials: effect of powder/liquid ratio and powder particle size reduction on fracture toughness. Dent Mater. 2003; 19: 747-757. 
+
+[4] Ana ID, Matsuya S, Ohta M, Ishikawa K. Effects of added bioactive glass on the setting and mechanical properties of resin-modified glass ionomer cement. Biomaterials. 2003; 24: 3061-3067. 
+
+[5] Moshaverinia A, Ansari S, Moshaverinia M, Roohpour N, Darr JA, Rehman I. Effects of incorporation of hydroxyapatite and fluoroapatite nanobioceramics intoconventional glass ionomer cements (GIC). Acta Biomater. 2008; 4: 432-440. 
+
+[6] Smith DC. Development of glass-ionomer cement systems. Biomaterials. 1998; 19: 467-478. 
+
+[7] Culberston BM. Glass-ionomer dental restoratives. Prog 
+
+Polym Sci. 2001; 26: 577–604. 
+
+[8] McLean JW. Glass-ionomer cements. Br Dent J. 1988; 164: 293-300. 
+[9] Lohbauer U, Walker J, Nikolaenko S, Werner J, Clare A, Petschelt A, Greil P. Reactive fibre reinforced glass ionomer cements. Biomaterials. 2003; 24: 2901-2907. 
+
+[10] Lucas ME, Arita K, Nishino M. Tough-ness, bonding and fluoride-release properties of hydroxyapatite-added glassionomer cement. Biomaterials. 2003; 24: 37873794. 
+[11] Kim DA, Abo-Mosallam HA, Lee HY, Kim GR, Kim HW, Lee HH. Development of a novel aluminum-free glass ionomer cement based onmagnesium/strontiumsilicate glasses. Mater Sci Eng C Mater Biol Appl. 2014; 42: 665-671. 
+
+[12] Shinonaga Y, Arita K, Nishimura T, Chiu SY, Chiu HH, Abe Y, et al. Effects of porous-hydroxyapatite incorporated into glass-ionomer sealants. Dent Mater J. 2015; 34: 196-202. 
+
+[13] Lohbauer U. Dental Glass Ionomer Cements as Permanent Filling Materials?- Properties, Limitations and Future Trends. Materials. 2010; 3: 76-96. 
+
+[14] Moshaverinia A, Roohpour N, Chee WL, Schricker SR. 
+
+A review of powder modifications in conventional 
+glass- ionomer dental cements. J Mater Chem. 2011; 21: 1319-1328. 
+
+[15] Major C. Advance in Glass-Ionomer Cements. 1th ed. 
+
+Berlin, Chicago:Quintessence Publishing Co;1999.p.97. 
+
+[16] Alan D, Wilson J. Glass-Ionomer Cement. 1th ed., Chicago: Quintessence books; 1988. p.247. 
+[17] Gu YW, Yap AUJ, Cheang P, Koh YL, Khor KA. Development of zirconia-glass ionomer cement composites. J Non-Crystalline Solids. 2005; 351: 508-514. 
+
+[18] Hickel R, Dasch W, Janda R, Tyas M, Anusavice K. 
+
+New direct restorative materials. FDI Commission Project. Int Dent J. 1998; 48: 3-16. 
+
+[19] Peez R, Frank S. The physical-mechanical performance of the new Ketac Molar Easymix compared tocommercially available glass ionomer restoratives. J Dent. 2006; 34: 582-587. 
+
+[20] Moshaverinia A, Ansari S, Movasaghi Z, Billington RW, Darr JA, Rehman IU. Modification of conventional glass-ionomer cements with N-vinylpyrrolidone containing polyacids, nano-hydroxy and fluoroapatite to improve mechanical properties. Dent Mater. 2008; 24: 1381-1390. 
+
+[21] Pittayachawan P, McDonald A, Petrie A, Knowles JC. 
+
+The biaxial flexural strength and fatigue property of Lava Y-TZP dental ceramic. Dent Mater. 2007; 23: 1018-1029. 
+
+[22] Jivraj SA, Kim TH, Donovan TE. Selection of luting agents, part 1. J Calif Dent Assoc. 2006; 34: 149-160. 
+
+[23] Robert G. Craig JM. Powers. Restorative Dental Mate rials. 11th ed. St. Louis, Missouri: Mosby; 2002. p.92. 
+
+[24] Prosser HJ, Powis DR, Wilson AD. Glass-ionomer 
+
+cements of improved flexural strength. J Dent Res. 1986; 65: 146-148. 
+[25] Xie D, Brantley WA, Culbertson BM, Wang G. Mechanical properties and microstructures of glassionomer cements. Dent Mater. 2000; 16: 129-138. 
+
+[26] Khouw-Liu VH, Anstice HM, Pearson GJ. An in vitro investigation of a poly(vinyl phosphonic acid) based cement with four conventional glass-ionomer cements. Part 1: Flexural strength and fluoride release. J Dent. 1999; 27: 351-357. 
+
+[27] Williams JA, Billington RW. Changes in compressive strength of glass ionomer restorative materials withrespect to time periods of 24 h to 4 months. J Oral Rehabil. 1991; 18: 163-168. 
+
+[28] Guggenberger R, May R, Stefan KP. New trends in glass-ionomer chemistry. Biomaterials. 1998; 19: 479483. 
+
+[29] Yap AU, Wang X, Wu X, Chung SM. Comparative hardness and modulus of tooth-colored restoratives: a depth-sensing micro in dentation study. Biomaterials. 2004; 25: 2179-285. 
+
+[30] Noort RV. Introduction to Dental Materials. 3th ed. 
+
+Sheffield UK: Mosby Elsevier; 2007. p. 90. 
+
+[31] Engqvist H, Lööf J, Uppström S, Phaneuf MW, Jonsson JC, Hermansson L, et al. Transmittance of a bioceramic dental restorative material based on calciumaluminate. J 
+
+Biomed Mater Res B Appl Biomater. 2004; 69: 94-98. 

medical/md/PMC5183298.md

@@ -0,0 +1,107 @@
+does not regard the tetrachloride as being a perfectly good anesthetic ; but he thinks that, in several instances, it may bo advantageously mixed with chloroform, as it has some properties which are antagonistic to those of the latter substance, and may, therefore, modify its action. The use of tetrachloride of carbon may be made to precede that of chloroform ; and, although he invites further experiments upon the point, Dr. Sansom thinks that a mixture of one part of the tetrachloride of carbon and six of chloroform will constitute a safe, as well as agreeable, anesthetic.— British Medical Journal, Sept. 7, 1867.
+
+ On the Resin of the Veratrum viride and its Therapeutical action.  By Dr. Srom , of Wurzburg.—The resin of the Veratrum viride employed by Dr. Stohr is an extract from the root of the plant, which in the dry state ought to yield a bright brown, faintly-smelling powder, with a slight taste. The cases in which Dr. Stohr employed this resin included several of pneumonia, one of acute rheumatism, one of measles, and one of emphysema, in the last of which there were very distressing palpitations of the heart. In this last case the patient took four pills, each containing one-sixth of a grain of the resin with one-eighth of a grain of morphia, at intervals of half-an-hour.
+
+the third dose the temperature of the extremities was reduced, the frequency of the pulse was lowered, the breathing became quieter, and the palpitations ceased in two hours, and after the fourth pill nausea supervened. The general effects produced by the resin of the Veratrum viride were similar to those just mentioned—namely, reduction of the temperature, of the pulse, and of the frequency of respiration. Dr. Stohr considers that the use of the resin is indicated only in those diseases in which the pulse and the temperature are unusually high, because in such cases a very rapid remedial action is desirable, and the unpleasant effects are outweighed by a quickly successful result. When the medicine is employed, its rapid and energetic action must be recollected, and the patient should be carefully watched. Schmidt's Jahrbiicher der Gesammten Medicin, December 10, 1866.
+
+REPORT ON PATHOLOGY AND PRINCIPLES AND
+PRACTICE OF MEDICINE.
+
+BY FRANCIS C. WEEB, M.D., F.L.S.,
+Member of the Royal College of Physicians, Physician to the Great Northern Hospital.
+
+On the Pathogeny of Fatty Transformations, with Experimental Researches on the Action of Phosphorus on the Living Tissues. By Dr. L. Ranvier.—The author combats the idea accepted by Virchow, that in the case of fatty transformation following inflammation, the former may be considered a part of the inflammatory process.
+
+Numberless facts show that inflammation and fatty degeneration, Dstead of being connected, are in reality opposed. In inflammation 0 the subcutaneous cellular tissue fatty cells lose the fat which they c9ntain; their nuclei and the little mass of enclosed protoplasm 8ne place by division to an abundant production of cells, which fill old adipose vesicle. In acute osteitis we And the fatty medulla transformed in like manner into proliferous tissue. In acute or 
+]oriC ^u^ammations of joints, the cartilage cells, which, physioogically, contain fat, are deprived of it as long as cellular prolifera-continues. ^1IS ^appearance of fat in elements which physiog'cally contain it is met not only iu inflammatory processes, u a also in all active new formations. Thus, when new growths te their point of departure from cellulo-adipose tissue, or from niedulla of bone, they determine the disappearance of fat in t^e ce^3 which they invade. But a more important fact still is at when, under the influence of a pathological cause, fatty traus- 
+?rmation has invaded certain cells, it is observed that these 
+_ e is may free themselves from the fat which they contain under fi !nfluence inflammation, provided it supervene before the e lular elements are completely destroyed by the degeneration, evertheless, at the end of the inflammatory process, and in the last age of every new formation, a fatty transformation is observed of per abundant elements. This transformation must not be attricd to the original irritative action, but rather to an alteration of u rxtion, for it never supervenes when the cells are in full prolifera-It occurs only when the former movement is arrested, and 
+"en nutritive changes go on with difficulty in those elements of 
+!ch the number is excessive in proportion to the vascular supP J' The first day of a catarrhal inflammation the exudation is lansparent, and the numberless cellules which it contains afford t Jundant evidence of active multiplication, but do not enclose a ngfe fatty granule. Later, when the exudation becomes yellow a|1(| ?paque, the contents of the cells are nearly all granules and g ooules of fat. The author sums up the results of his experiments on animals and observations in the following conclusions: The pro-oplasm of cells appears to be the seat of changes in, and of the e aboration of the materials brought by the blood; it is also in the protoplasm that fat is at first deposited. The presence of fat in a ce which does not apparently contain it in the normal state proceeds from a diminution of the nutritive movement of that cell. If e movement is rendered active by irritation, fat disappenrs. Cer-ain cells have considerable activity in the elaboration of fat, but it 
+?es not follow that they form it from other materials with which ney are brought in contact, for blood contains fat in a metamor-phosed state, that is to say, in a state of combination or of saponifi-cation. Fat-cells, the cells of the liver, &c., seem then to bring back such metamorphosed fat to the neutral or insoluble state. In dead 
+?tuses retained some weeks in the uterine cavity, metamorphosed at becomes apparent to microscopic observation in the liver, the Sidneys, the heart, and cartilage cells. Phosphorus determines fatty ransformations, because it enfeebles the nutrition of histological e ements.? Gazette Medicate de Paris, July 6 and 13, 1867. 
+
+On a Form of Fever ivhich Prevailed throughout certain Districts of Western Malwa in 1865. By H. C. Brodrick, M.D., 1st Regiment Central India Horse.?The author was employed by the Indian Government to report on a very fatal form of fever known in the districts of Western Malwa by the name of Oujrati Beemari, and amongst the Mussulmans as Yerkan or Feelia, both terms indicating jaundice. The disease is described as an ardent fever, without remissions, attended with congestion of the liver, pain in the side, and jaundice, and with congestion of the lungs or pneumonia, indi-cated by difficulty of breathing, cough, and bloody expectoration. 
+
+The skin and conjunctivae were yellow, and the perspiration stained the clothes of a yellow colour. The disease prevailed pari passu with cholera, and the symptoms of both seem to have been con-founded by the native population. The author believes it to have been a form of jungle fever, without remissions. Relapses, however, were observed. The disease was generally fatal on the third day. In one village visited, which originally contained fifty houses, twenty-five had been desolated by the fever. There was no evidence that it was contagious. The author has treated several cases in the Cavalry Hospital at Augger of a fever which he believes to be identical with the Gujrati Beemari. They were very unpromising cases, but yielded to quinine in doses of from ten to twenty grains, with animal broths and alcohol. The author suggests that this disease may be identical with Palee plague.?Madras Quarterly Journal, October, 1866. 
+
+On Leucocythoemia and the Pathogeny of Hemorrhages and Thromboses which supervene in that affection. By MM. Auguste Olltvier and Louis Ranyier.?The authors observe that Yirchow, and the principal writers of Germany who have discussed the pathological changes in leucocythcemia, have considered the presence of white globules between the hepatic cells or the renal tubuli as the result of hyperplasie of the interstitial connective tissue of the organs. This interpretation seems at first sight to account for the enormous pro-duction of white globules, although it does not explain the manner in which they penetrate the circulatory system. Since the researches of Reeling hausen there has been a disposition to consider the plas-matic network of the connective tissue as the origin of the lymphatics, and to explain the presence of globules in their final ramifications, by supposing that they are penetrated by the cells of that tissue. It follows that every multiplication of these cells would throw a great number of globules into the lymph, and thence into the blood. On the other hand, we know that lymphatic tumours may develope in a large number of organs without determining leucocythoemia. The observations of the authors have led them to give a different inter-pretation to the facts. In a case of leucocythoemia they believe they have observed, both in the liver and in the kidney, a connection of continuity between the interior of the bloodvessels and the islets formed in the stroma of these organs by an accumulation of white globules. Hence the presence of white globules in these islets results from capillary haemorrhage, and moreover, in the kidney they cceeded in discovering some red globules mixed, in small propor-ar?n''Wl^ wkite. As to organized lymphatic new formations, they 
+^ 
+e inclined to consider them as having two origins?the one a yperpJasie 0f the connective tissue, the other the formation of a ymphatic tissue developed at the expense of extravasated white ca? !!i They ^??k 011 ^e white globules as embryonic cells, pable of forming a new tissue. In the majority of cases the esS?u.ce red globules in the meshes of the connective tissue is v P j by the rapid destruction of these globules when extra-ceh i Case examined by the authors exhibited multiple o-ra h?morrhages, and also coagulations of blood in the crural and er arteries. The latter are frequent in leucocythoemia. According to tl? } l61"' Wou^ be due to a chemical alteration of the plasma of 6 b*??d, although it is difficult to conceive how the same modifica-on could produce at once haemorrhage and thrombosis. The authors er the following interpretation : The red globules of the blood re i ;re easily in the capillaries because they are smooth and the11 White globules are larger in man, rugous, and possess property of adhering. If the white globules become very merous capiHary circulation will be difficult; and if, as in the of tf ^ ?kservedj the number of white globules is larger than that h< 
+le re<?> and their size attains ^ of a millimetre, as frequently 
+^ouw\S ^ ^eucocJ^oemia> they think that the capillary circulation jj u be extremely slackened, and even suppressed in some organs. 
+
+a _,nce there will be a detention of globules in the capillary network, St t>a s.u?c^ent tension to determine the rupture of the vessels. 
+
+, ??ati?n of blood will equally account for coagulation in the erial or venous trunks. The most powerful of the causes pro-thrombosis is arrest or slackening of the circulation.?Gaz. 
+
+e lca^G Paris, June 15, 1867. 
+
+^le influence of Disease upon the Weight of the Body. By Thos. 
+
+a^T10^' Sterne, Paris.?The author's observations were undertaken 
+?o a Charite, under the direction of Prof. Monneret. In regard acute diseases, two principal periods are noticed?those of loss as reP,arati?n- Whilst the period of loss lasts the weight diminishes, an 
+^ 
+] 
+8' raPidly> and from the proportion of its diurnal variations ' ea may be formed of the relative gravity of the case. The dail 
+?r ^aS S6en a decrease in typhoid fever of more than 500 grammes x'1 ^ reparation the increase of weight is at times sudden and ti exPected, and is ushered in before other symptoms would warrant littl au.uoVnc?meiit of convalescence. This fact is so striking that but wl 6 ati?n need be felt in declaring convalescent the patient u os? weight begins to ascend, even though, from the indications s ^a y accepted, this announcement may seem premature. The ries of weights taken during convalescence furnish the means of 
+^ 
+no wing whether this state of reparation will be short or tedious, as hpS if k rapid augmentation accompanies a speedy return to a h, while the contrary obtains when the intermediary condition aut^601? health and disease is destined to drag on slowly. The or has made these observations on weight coincide with thermometric investigation, and has found that an elevation of temperature is attended by a diminution of weight; or in other terms, the higher the fever the lower is the figure given by the scales. The author's paper contains a number of observations of the variations of weight in different acute and chronic diseases.?Southern Journal of Medical Sciences, August, 1867. 
+
+Case of Cholesteatoma. By Dr. F. Bate man.?T. H?, zet. 2 6, a tall, well-made farm labourer; admitted into Norwich and Norfolk Hospital on July 7,1866. History.?Been married four years ; had one child twenty-one months old; never had syphilis; had always been a steady man, and had enjoyed good health until nine months after marriage. First morbid symptom, a shaking first of right hand, then of left; this was followed in a few months by frontal headache. About a year ago weakness of legs set in and inability to guide himself, but intelligence and memory remained unimpaired; about this time slight embarrassment of speech was observed, this was followed by dimness of vision, which resulted in complete blind-ness, the right eye being first affected. Symptoms observed on admis-sion.?Staggering gait; no actual paralysis of limbs, but perhaps a little less muscular power on right than on left side; sensation un-impaired ; difficult micturition ; no want of power over rectum. He complains of severe frontal headache, is deaf on the right side ; there is double amaurosis, the pupils act, though sluggishly; no apparent disease of the eye itself. Intelligence and memory unimpaired; he converses coherently, with well-selected words, but he has an un-natural jerking, spasmodic manner of speaking, as if the same want of power of co-ordination in the act of volition which is apparent in his gait extends to the expression of his ideas. After a few days the frontal headache increased in intensity, he became drowsy, and died suddenly on the eleventh day after admission. Necropsy, thirteen hours after death.?Body well nourished ; lungs sligthly adherent at both apices ; heart healthy; kidneys, liver, and spleen intensely con-gested. Brain congested, but extremely firm. Arachnoid contained five and a half ounces of cerebro-spinal fluid ; ventricles dilated with fluid. On removing the brain, " a glistening tumour was found at the base of the cranium, adherent to and apparently springing from the posterior surface of the petrous portion of the right temporal, and from the basilar process of the occipital bones." " Tumour was flattened from above downwards; two and a quarter inches in its longest, one and a quarter in its shortest diameter; its surface uneven, of a white silvery appearance, resembling mother-o'-pearl." 
+It was invested with a membrane of the thickness of ordinary paper, which enclosed, as in a cyst, a substance of the thickness and consistence of wax, the central portion being of a dirty straw-colour, not unlike tallow, but becoming paler, of an almost pure white, and firmer as it approached the circumference of the tumour. It had no farther adhesion, and there was no trace of vessels in its structure. 
+
+It lay against the under-surface of the right lobe of the cerebellum, where an excavation existed for its reception; it pressed on this lobe, also on the right side of the medulla oblongata, the right crus cerebelli, and the right side of the poos varolii in its whole extent. 
+
+A portion examined microscopically exhibited a number of cells, crystals of cholesterine, and fat-globules. A portion was examined by Dr. Beale and pronounced cholesteatoma. The author refers the unsteadiness of gait to impairment of the functions of the cerebellun by pressure; the amaurosis to pressure by the anterior extremity ol the growth upon the posterior and inferior surface ol the right tha-lamus opticus, and, consequently, upon the origin of the optic nerve 
+(the double amaurosis being explicable by the decussation of the fibres of the optic nerve at the commissure); and the deafness to pressure on the portio mollis, as its winds round the corpus resti-forme, and also at its entrance into the meatus auditorius internus. 
+
+The author believes this tumour to be the largest of this description and in this situation recorded. The ataxia ot articulation he thinks Was probably due to pressure on the origin of the glossopharyngeal and hypoglossal nerves.?JBeetle's Archives, No. xvi, vol. iv. 
+
+Anatomico-pathological Considerations on Abscess of the Brain. 
+
+?By M. E. GiNTitAC.?Suppuration and ramollissement are the modes of alteration by which encephalitis terminates, or under which it most frequently presents itself, since out ot 410 cases there were only 29 in which these conditions were not established; suppuration numbers rather more examples than inflammatory ramollissement. Kamol-lissement is notably more frequent in the cortical substance, in the anterior lobes of the brain, in the corpora striata and optic thalami, and in the pons varolii. Suppuration has been more frequently observed in the middle and posterior lobes of the brain, and in the cerebellum. It is also the most frequent termination of cerebral or cerebellar inflammation accompanying alteration ot the bones of the skull, and principally of the petrous portion of the temporal^ Pus which forms in the brain may be exuded on its surface, or disseminated io its tissue, or collected at one or more points ol its extent. 1 he first mode is rare; the second is sometimes observed with ramollissement, to which it gives a yellow-greenish tinge, great fluidity, and a peculiar odour. The third (abscess) is the most common. Lebert divides abscesses of the brain into multiple and solitary. But in circumscribed localities abscesses are not always solitary?sometimes two, but rarely a larger number are met with in the middle or posterior lobe, or in the cerebellum. Xiebert has noted that the left hemisphere was more often affected than the right, twenty-thiee to eighteen. M. Gintrac has collected ninety-three cases of abscess ot the right half of the encephalon, and eighty-five of the left. It is principally in the medullary substance of the different lobes ot the cerebrum that purulent collections are found, whilst the cortical substance is almost exempt. But the cerebellum, which is abun antly provided with grey substance, is pre-eminently subject to abscess, and the pons very little so, although principally formed ot white matter. Cerebral abscesses,however,arise principally in the me-dullary substance, which, although less vascular than the g, ey> is not, therefore,less apt to inflame and suppurate. 1 he volume of the abscess is sometimes considerable, varying from the size of a nut to that ot a 80?xl. 
+
+pigeon's egg, a hen's egg, and an orange. The author has seen the hemi-sphere converted almost entirely into a sac full of pus. The form of the abscess is usually rounded or ovoid. When two or more abscesses are contiguous, they generally remain separated by partitions of medullary substance. But communications may be established between them. 
+
+In some instances several foci communicate and run together, pro-ducing a cavity of irregular form, and of anfractuous or multilocular appearance. The pus contained in cerebral abscesses is usually greenish-yellow or olive-green, thick, creamy, or liquid and ropy. 
+
+It may contain a pultaceous reddish matter; it may be inodorous, but in a large number of instances it is very fetid, although it has had no communication with the air. It is generally alkaline; under the microscope it presents ordinary characters, but granules are more abundant than the proper pus cells. The matter is sometimes in immediate contact with the cerebral substance, the surface of which may be softened or hardened, injected, red, or greyish, or yellowish, or granular; or sometimes it is enclosed in a cyst. 
+
+This cyst is formed of a vascular membrane of variable thickness, white, reddish, or greyish in colour. The more recent the cyst is, the thinner and softer it is. From the thirtieth to the sixtieth day and later it becomes thick and dense, and appears formed of two or three membranes. Of these, the external has a cellular appearance, the middle may be dense, resistant, fibrous, or cellulo-fibrous and vascular ; the internal is soft, and like a mucous membrane. This is sometimes covered by a sort of thick concretion of pus. The cyst is sometimes easily detached from the cerebral substance, at other times it is closely united. The cysts differ from those which are formed round hasmorrhagic clots. The latter are agents of secretion, absorption, and resolution. Those which enclose abscesses do not fulfil like functions. Their membrane is rather pyogenic than absorbent. Abscesses are rarely found diminishing and on the road.to cure; but although such a termination is exceedingly rare, it is not impossible. Cerebral abscesses have a tendency to open towards the points where there is the least resistance. Pus generally makes its way towards the ventricles, and these cavities are generally found more or less filled with it. Very rarely the matter appears at the surface of the brain. Cerebral abscesses evacuated by the nostrils have been reported, but the minute details fail, or pua is said to have been evacuated by the ear. But it is worthy of note that in certain cases where the cavities of the internal ear enclosed pus, the cerebral abscess itself did not communicate with the interior of the petrous bone, of which the surface was entire, and the dura mater intact. Nevertheless, it is more common to trace the con-nection between the contemporaneous affections of the ear, the membranes, and the brain.?Journal de MSdecine de Bordeaux. 
+
+Gazette MSdicale de Paris, July 27. 
+
+On Convulsive Tremor. By Dr. William A. Hammond.?Under this term Dr. Hammond describes a paroxysmal affection, of which the following case may be taken as an example :?J. S?, a gentle-man, set. 35. Ordinarily had nothing to complain of on the score of health. Appetit? was good, and all functions performed with regularity; but two or three times during the day he would be seized with severe and unrestrainable muscular tremor, involving the head and all the muscles of the trunk and arms. At the same time there would be slight headache and vertigo, and an intense feeling of anxiety. There was no I03S of consciousness, not even for an instant, nor inability to walk or to direct any muscle, and no confusion of thought. After the paroxysm had lasted fifteen or twenty minutes it gradually passed off, leaving him in a profuse perspiration. During his first interview with Dr. Hammond he was seized with a paroxysm which is thus described :?" He was seized with as much suddenness as though he were struck with an epileptic fit. His head shook violently, the muscles of his face were convulsed, his arms and hands trembled, and his gluteal muscles contracted so powerfully as to cause him to move convulsively up and down in the chair. His lower extremities were altogether free from spasm or convulsion. Upon putting my hand on his wrist, 1 found that every tendon was in action, and in the arm, hand, neck, and face, the vibration of the muscular fibres could be distinctly seen and felt. The thermometer applied to the axillae marked 101? 
+
+Fahr., and the sesthesiometer showed an increased sensibility of the skin of the face, neck, hands, and ail the upper parts of the body examined. The respiration was quickened and the pulse was increased from 80 to 95 per minute. During the paroxysm he con-versed rationally, but with some difficulty, owing to the action in the muscles of the neck, mouth, and chest." In this case the attacks seem to have been brought on by venereal excesses, the first paroxysm having supervened during coitus. He rapidly improved under treat-ment, which consisted of a seton in the nape of the neck (to counteract supposed cerebellar irritation), thirty grains of bromide of potas-sium three times a day, and the application of the constant galvanic current. Two other cases of somewhat similar character are related, in which a similar treatment proved also of use.?New York Medical Journal, June, 1867. 
+
+On the Influence of Narrowing of the Pulmonary Orifice on the Formation of Pulmonary Tubercles. By M. Lebeet. Presented to the Academy of Sciences.?The author, in the course of his re-searches on mechanical influences in the etiology of tuberculous affections, has been struck by the frequency of the occurrence of pulmonary tubercle in cases of congenital narrowing, either of the cone or the orifice of the pulmonary artery. This coincidence has been observed, in isolated cases, by Favre, Travers, Gregory, Louis, and Creveld, and during the last twenty years it has been noticed in one third of the observed cases, often under circumstances in which no other etiological element of tuberculisation could be sup-posed to exist. The author has been able to collect twenty-four facts of this kind, a large number if the relative rarity of this affection be taken into account. The frequent development of tubercles in connection with this condition is the more striking, as nothing is more rare than to meet with pulmonary tubercles in alterations of the orifices of the left heart. Three forms of congenital narrow-ing of the pulmonary artery are to be distinguished?primitive narrowness of the vessel, which is furnished only with two valves, narrowing of the pulmonary arterial cone, and narrowing of the orifice. The last two are the results of endocarditis or myocarditis, and as ordinarily the interventricular septum is deficient, this inflammation must take place before the end of the third month of intra-uterine life. The foramen ovale remains often also open, the arterial duct more rarely. Hence, the circulation through the lungs is irregular and incomplete. Dilatation of the bronchial, cesophagean and coronary arteries of the heart, also of the sub-clavian, furnishes only an incomplete collateral circulation, whence arises imperfect nutrition of the lungs, so that they are observed to be small and incompletely developed. This irregular, unequal, incomplete circulation in places, gives rise to pulmonary tubercle, whilst the most intense and extended pulmonary hyperemia from alteration of the bicuspid and tricuspid orifices does not conduce to its development. The author knows neither age nor disease which presents this proportion, i. e. of one third affected with pulmonary tubercle, and hence he can only refer it to pulmonary stenosis. 
+
+The character of the tubercular attack is protracted, progressive, and fatal. Haemoptysis is frequent, and it is ordinarily the left lung that is first attacked, and not the right, which the author thinks is the lung which ordinarily suffers first. The anatomical characters are the same as in other forms of tuberculisation.? 
+
+Gazette Medicale de Paris, July 27, 1867. 
+
+Rupture of the Tricuspid Valves of the Heart from Fright. By Alfred Hitchcock, M.D.?M. H?, set. 2 years and 9 months, of a sanguine and very nervous temperament, was frightened in the middle of the night of July 16th, 1865, by an alarm suddenly sounded by the steam fire-whistle in the immediate vicinity of the house where she was sleeping. She awoke in terror, screaming and panting for breath, with suffocating paroxysms. She died seventy-eight hours after the fright. The symptoms present were vomiting, dyspnoea, lividity of skin, absence or great feebleness and irregu-larity of the radial pulse. The heart sounds were too feeble to be heard, or too tumultuous and irregular to be identified. There was loud moist bronchial and tracheal rattle. 
+
+Autopsy.?"The right auriculo-ventricular valves were lacerated and broken in their substance ; also several of the carnece columnce and chordae tendinece; and the endocardiac serous membrane in the vicinity was ecchymosed and elevated by the infiltration of blood underneath and around the points of ruptured tissue. The right auricle and ventricle, by the destruction of the tricuspid valves, had become one continuous pouch, wThich contained a small quantity of coagula."?Boston Med. and Surg. Journ. New York Med. Journal, May, 1867. 
+
+The Condition of the Blood after Death from SnaJce Bite. By Professor G-. B. Hautord, M.D.?In a case of poisoning by the bite of the cobra-di-capella, and in animals bitten by poisonous snakes, Dr. Halford has found that the blood contains a number of nucleated cells. In a dog killed by the injection of cobra poison under the skin, these cells were of a perfectly circular form, with a diameter on the 
+:
+average of 1707 of an inch. The nucleus was nearly round, and 7707 of an inch broad. On the second day, on applying magenta, a minute spot became visible at some part of the circumference of the cell.
+
+This was visible in all. Of these cells there were many millions ;
+they were present in the blood of every part of the body, but in much greater quantity in the dark congested parts of the lungs. In the poison of the snake itself, Dr. Halford found, besides epithelium and much molecular matter, nucleated cells of the same size as those seen in the blood of the dog, but no spot or macula was seen in the circumference. Dr. Halford believes that when a person is mortally bitten by the cobra-di-capella, molecules of living germinal matter are thrown into the blood, and speedily grow into cells, and as rapidly multiply, so that in a few hours millions upon millions are produced at the expense, probably, of the oxygen absorbed into the blood during respiration ; hence the gradual decrease and ultimate extinction of combustion, and chemical change in every other part of the body, followed by coldness, sleepiness, insensibility, slow breathing, and death. Dr. Halford suggests that cholera may be due to the presence of foreign cells in the blood, and refers to an observation by Dr. James Cowan, published in the
+' Edinburgh Monthly Review,' of some peculiar bodies in the blood of a cholera patient, and also to an observation by Virchow that the white corpuscles are increased in cholera, in support of the hypothesis.— Pamphlet, Melbourne. - Int On the Formation of the so-called Cells the By J. GA. G B. Horton, M.D. Londen, Essentials of the Principles and Practice of Medicine. By H. Hartshorne, M.D.
+
+An Practical Treatise on Shock after Surgical Operations and Injuries, with
+special reference to Shock caused by
+Hardwicke, London. 1867. pp. 88.
+
+Royal Society,' Nos. 89 and 94.) 1867.
+
+Lectures on Clinical Medicine. By A.
+
+Trousseau. Of Translated fand o Edited by P. V. Bazire, M.D. Hardwicke, London.
+
+Iluncan, M.D. (From ' Transactions of The Treatment of Aneurysm by Electrolysis. 18 By Drs. J. Duncan and T. R.

medical/md/PMC5280726.md

@@ -0,0 +1,36 @@
+lP" Applied Anatomy of the Nervous System: Being a Study of this Portion of the Human Body from a Standpoint of its general Interest and Practical Utility in Diagnosis, designed for as a lext-book and a Work of Reference. By Ambrose L. 
+
+-^anney, A.M., M.D., Professor of the Anatomy and Physiology o the Nervous System in the New York Post-Graduate Medical c iool and Hospital, etc., etc. Second Edition. London: H. 
+
+1^. Lewis: 1888. 
+
+an^T ^ diagnosis disease of the nervous system, more than of an t group of morbid conditions, that a precise knowledge of the rp,0lny and physiology of the structures involved is essential, lab lau recently devised methods of research, and to the an (|UIS ?^a ^ar?e body of able investigators, our knowledge of the cor- ^ie nervous system is being daily extended and pi ? f ' as a necessary consequence, our knowledge of its Cai!?Sy is likewise widening and becoming more precise. There Sei> V10 ^ou^t that it is largely due to this advance of our purely infl knowledge?although we must also fully recognise the tion-^T better clinical and pathological methods of investigar is ^le diagnosis of many diseased conditions of the brain aPt)r VV- S? Wonderfully accurate as to enable us to operate with 
+^?Xln?a*;e certainty of success. 
+
+l?gical 
+? ^ie most recent results of anatomical and physiotliese researc^) and at the same time showing the bearing of beCOrng^0n explanation of various pathological conditions, thus is Well08.a de.s^eratum with every practitioner; and such a want second edition of Dr Ranney's admirable chana-e -^naiomy and Physiology of the Nervous System. The must al m t^le second edition are so radical and extensive that it 
+^hi/11^ regai'ded as a new work. 
+
+*endenr - \n mos^ t'ie recent works upon the nervous system the finical been somewhat to magnify the pathological and P'^iolo^?8 ^ie subject a* the expense of the anatomical and 
+*'le Srea?1^ ^ I^nney's treatise the reverse holds good; and ll0i'mal' 61 J-a.rt ^ie W01'k is devoted to the elucidation of the divero-e,,00110!1^0118' structural and functional, while the possible After tC6S m ^ese are merely sketclied in outline. 
+
+brain are ^nera introduction, the anatomy and physiology of the nerves ar ilsc|lssed w^b great fulness, in Part II. the cranial fUncti0nse f? Wlt^' while in Parts III. and IV. the structure and 
+^ full and? 116 C01"d and spinal nerves are carefully described, corisid a j?Urate index accompanies the book?a fact which must The field a Stron? rec?mmendation to such a work of reference. 
+
+and the ai tl C?^eied *3 necessarily very wide, and ever extending; in brinp-ino-T-18-t0 be COngr<atulated on having so well succeeded no means ln!ormati?n abreast of recent research. We are EDiNRTIn' S SUrPr'sed to find many important investigations unH MED- JOURK., VOL. XXXIV*.?NO. IV. 2 X 
+noticed. We observe that the author does not mention Professor Hamilton's interesting observations on the corpus callosum, which at least clearly show that all the callosal fibres are not commissural to corresponding parts of the two hemispheres. An intentional or accidental omission, however, of more practical importance is Hare's very admirable method of localizing the various cerebral sulci. The more complicated and less satisfactory method of Fere is described.
+
+Similar omissions might be noticed, but in a work of such size these are almost inevitable.
+
+To the medical man we can recommend the book as a most useful work of reference.
+
+A Handbook of the Diseases of the Eye. By HENRY R. SWANZY,
+A.M., M.B., F.R.C.S.I. Second Edition. London: H. K.
+
+Lewis.
+
+A GOOD many important additions have been made by Mr Swanzy in the second edition of his excellent handbook. The additions appear to be most numerous in the chapters on the Diseases of the Retina and Optic Nerve, and on Amblyopia. Many points in connexion with these affections have received more general attention of late years, and we are glad to find such full reference to them in a text-book which has gained such a welldeserved popularity. No better book could be put in the hands of students than this one by Mr Swanzy. It is concise and practical, and is written from the point of view of a man not only of large experience of ophthalmic surgery, but of excellent judgment as well. The first edition has been so rapidly exhausted, that a more complete review is hardly called for. Suffice it to say, that while retaining all the characteristics on account of which we felt confident in recommending it to the readers of the Edinburgh Medical Journal, enough new matter has been added to the book to bring it thoroughly up to date.
+
+Ueber Gemüthsbewegungen. Eine Psycho-Physiologische Studie. Von Dr C. Lange, Kopenhagen. Autorisirte uebersetzung von Dr H. Kurella. Leipzig, 1887. As Danish is not much cultivated in this country, those who wish to study Dr Lange's treatise on the expression of the emotions will be glad to avail themselves of Dr Kurella's translation. Having compared it with the original, we are able to say that it is both clear and faithful. Dr Lange gives a striking description of the sensible effects of the passions upon the bodily frame.
+
+Most of these he traces to the action of the sympathetic system in constricting and dilating the vessels of different parts of the body¬ While giving due tribute to the extraordinary powers of observation shown by Darwin in his work On the Expression of the

medical/md/PMC5412800.md

@@ -0,0 +1,176 @@
+CASE REPORT
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_1.png](0_image_1.png)
+
+# Case Report: A New Method For Treatment Of Permethrin - Resistant Head Lice Farhad Jahangiri Department Of Pathobiology, School Of Veterinary Medicine, Shiraz University, 1731, Shiraz 71345, Iran
+
+Correspondence Farhad Jahangiri, Department of Veterinary Medicine (Veterinary Parasitology), University of Shiraz, Shiraz University, P.O. Box 1731, Shiraz 71345, Iran. Tel: +98 711 2288660; Fax: +98 711 2286950; E-mail: Jahangirifarhad@gmail.com Funding Information No sources of funding were declared for this study.
+
+Received: 7 June 2016; Revised: 5 November 2016; Accepted: 13 February 2017 Clinical Case Reports 2017; 5(5): 601–604 doi: 10.1002/ccr3.899
+
+## Key Clinical Message
+
+Head lice (Pediculus humanus capitis) infestation is an important disease worldwide, especially in children under 11 years old. Permethrin is the most common insecticide for treatment of human pediculosis, but nowadays resistance to permethrin and other pyrethroids has caused many treatment failures. The second treatment of choice is malathion.
+
+## Keywords
+
+Head lice, malathion, pyrethroids.
+
+## Introduction
+
+Lice are blood-sucking insects of the order Anoplura.
+
+Each species of lice dwell in a particular host and a particular part of body [1]. Each louse survives for about 30 days [2]. The female louse produces about 130 eggs
+(nits) in its life. After about 9 days, the eggs hatch to nymph. The nymph molts several times over the course of 15 days and becomes an adult louse [3]. Away from host, mature lice survive just for 3 days, but this survival time is about 10 days for nits [2, 4]. Lice spread from person to person when people's heads touch or after sharing things such as hats, combs, clothing, headbands, brushes, or barrettes [4–6]. Control of this insect worldwide depends on the repeated or continued applications of pyrethroids (permethrin), organophosphates
+(malathion), carbamate (carbaryl), organochlorine pesticides (lindane), and in some countries ivermectin, benzyl alcohol (Ulesfia), and spinosad (Natroba) [7, 8]. Resistance to the common pediculicides may have contributed to the recent surge in head lice infestations [4, 9]. Although Pediculus humanus capitis (head lice) is not an important vector of microorganisms, it is a major social and economic concern in North America and worldwide [10]. Infestation with head lice has been a chronic problem throughout the world for thousands of years, with an estimated 6–12 million cases of pediculosis occurring each year in the United States alone, most of which affect girls [1, 11]. The annual cost of head lice in the United States may be as high as 1 billion $
+because of costs for treatment, appointments with a provider, prescription medications, time spent by school nurses to inspect students, and time of work for caregivers who must stay home with their children during the infestation [12].
+
+## Case Report
+
+Recently, we were confronted with some permethrin –
+resistant pediculosis in a large family. There were 13 females and seven males in this family. All of the females were infested but the males were not. Lice were present just on the head, and no lice were found in body inspection. Patients ranged in age from 3 to 60 years old. All of the patients of the family had already been treated by permethrin with no satisfactory result. Patients had just primary clinical signs of itching, and there were no other clinical or biological signs.
+
+The second treatment of choice for human pediculosis is malathion, but because of poisoning probability in children, attempts were made to find a replacement for malathion. Ultimately, ethanol was chosen and applied to the hair of the family members. 96% ethanol was sprayed on to the patient's hair. After ethanol spraying, a nylon bag was used to cover the patient's hair. The plastic cover was removed after 10 min and the hair washed. Three days after treatment, all patients were considered completely treated, and there were no lice and clinical signs of infestation. Based on life cycle of the lice, treatment was repeated 2 weeks later.
+
+## Results And Discussion
+
+Contrary to the first treatment with permethrin, 3 days after alcohol application there was no live nit or louse on the heads of patients, all of the patients were satisfied, and they had no itching or other clinical signs on the head.
+
+In treatment of infective diseases, it is important to consider safety, efficacy, availability, expense, and ease of application [7]. Permethrin, malathion, and lindane are usual insecticides for treatment of head lice in the world [7]. In treatment failure with pyrethrin or permethrin, malathion is the second treatment of choice. Orally administered ivermectin has been reported to be efficacious, but it is not currently a US Food and Drug Administration - approved pediculicide [7, 13]. Ivermectin 0.5% lotion (Sklice) was approved by the FDA in 2012. It is indicated for the treatment of head lice in patients aged 6 months and older [14]. Benzyl alcohol 5% lotion (Ulesfia) was approved in 2009 by the FDA
+for the treatment of head lice in patients aged 6 months and older [15]. It is the first non-neurotoxic agent and has a unique mechanism of action. Benzyl alcohol works by "suffocating" lice because it prevents the closing of the respiratory spiracles via obstruction, leading to asphyxiation. It does not have ovicidal activity [15]. Spinosad 0.9% topical suspension (Natroba) is a new pediculicide that was approved in January 2011 for treatment in patients aged 4 years and older. Although the suspension has been shown to be effective in patients as young as 6 months of age, systemic safety has not been evaluated for subjects younger than 4 years [16]. Many researches have been carried out for finding an effective and safe treatment for head lice. For example, Plant oils have been suggested as an alternative source of materials for insect control because they constitute a potential source of bioactive chemicals and are commonly used as fragrances and as flavoring agents for foods and beverages [17–19], but till now, an effective commercial result has not been found.
+
+Insecticides are usually applied in very low dosage, about 1%, because they have a narrow safety border. This application at low dosage creates a drug resistance field.
+
+Also, some of the treatments, for example, permethrin in treatment recommended dosage has a slight ovicidal effect that increases the probability of resistance occurrence. On the other hand, increasing the dosage of drug can cause poisoning in patients. Lindane is effective on eggs of lice, but lindane resistance among head lice has been reported in the United Kingdom, the Netherlands, Panama, and the United States [4, 20, 21]. Easy absorption of lindane to skin can cause neurotoxicity and anemia in patients
+[22–24]. For this reason, this treatment should be applied with extreme caution in patients with a history of seizure and in HIV infected patients [24–26]. Other side effects of Lindan are mild edema, pruritus, stinging, burning, rash, erythema, and tingling [6]. Malathion has been distributed in a 0.5% formulation as Ovide (Medicis).
+
+Malathion resistance has also been reported [24, 27]. Malathion can cause neurotoxicity in humans [28]. Use of malathion in patients younger than 6 years is not recommended, and its use is contraindicated for neonates and infants because of increased permeability of the scalp
+[29]. Ivermectin, benzyl alcohol (Ulesfia), and spinosad
+(Natroba) are new approved drugs, but they are not available for human use in all countries. Benzyl alcohol can be used in patients aged 6 months, but it does not have ovicidal activity [15]. Also, the cost of new treatments (ivermectin, benzyl alcohol, and spinosad) is much greater than that of old treatments [30]. Generally, the majority of new and old mentioned treatments have resistant probability problem in low dosage and poisoning probability problem in high dosage. Another property of insecticides is poison durability. For treatment of many of the external arthropod infestations, especially in animals, a chemical poison with high residual protection is a good and necessary property of pesticide; but in head lice infestation, this is not necessary or good property because durability of the poison can aggravate the risk of poisoning especially in children.
+
+Ethanol, which is commonly called drinking alcohol, has many good properties regarding the efficacy, safety, cheapness, availability, patient preference, and ease of application features that are important factors for treatment selection. High percentage usage of spraying ethanol
+(even to 100%) bring many benefits. The first is its fast effect. The second is its ovicidal effect [31, 32]. The third benefit is the very low probability of resistance occurrence. Ethanol has very low risk of poisoning occurrence because it evaporates rapidly and its pharmaceutical residues are very low. So this treatment can be applied to children up to 3 years old with exact monitoring till alcohol evaporation. Although some side effects, especially carcinogenicity, have been expressed for ethanol [33],
+topical usage of ethanol once or twice could not provoke such problems. While many kinds of alcoholic drinks are available in markets, alcohol is cheaper than other treatments and can be the treatment of choice for head lice in epidemic conditions or outbreaks of head lice infestation.
+
+## Authorship
+
+FJ: performed all the work.
+
+## Conflict Of Interest None Declared. References
+
+1. Centers for Disease Control and Prevention. 2010, November 2. Parasites: Head lice. Available at http:// www.cdc.gov/parasites/lice/head/index.html (accessed 6 March 2017).
+
+2. Burkhart, C., B. Stantiewicz, I. Pchalek, M. Kruge, and C.
+
+Burkhart. 1999. Molecular composition of the louse sheath. J. Parasitol. 85:559–561.
+
+3. Aronson, S. S., and T. R. Shope. 2009. Managing infectious diseases in child care and schools: a quick reference guide, 2nd ed. American Academy of Pediatrics, Elk Grove Village, IL.
+
+4. Meinking, T. 1999. Infestations. Curr. Probl. Dermatol.
+
+11:73–120.
+
+5. Witkowski, J., and L. Parish. 1997. What's new with the management of head lice. Infect. Med. 14:287–289.
+
+6. Dodd, C. 2001. Interventions for treating head lice
+(Cochrane Review). Cochrane Database Syst. Rev. 4:
+CD 001165.
+
+7. Jones, K., and J. C. English. 2003. Review of common therapeutic options in the United States for the treatment of pediculosis capitis. Clin. Infect. Dis. 36:1355–1361.
+
+8. Dolianitis, C., and R. Sinclair. 2002. Optimal treatment of head lice: is a no-nit policy justified? Clin. Dermatol.
+
+20:94–96.
+
+9. Sceck, A. 1997. "Super lice" force a second look at treatment options. Dermatol. Times 18:3–4.
+
+10. Williams, L. K., A. Reichart, W. R. MacKenzie, A. W.
+
+Hightower, and P. A. Blake. 2001. Lice, nits, and school policy. Pediatrics 107:1011–1015.
+
+11. Chosidow, O. 2000. Scabies and pediculosis. Lancet 355:819–826.
+
+12. McCormack, P. L. 2011. Spinosad: In Pediculosis capitis.
+
+Am. J. Clin. Dermatol. 12:349–353.
+
+13. Chosidow, O., B. Giraudeau, J. Cottrell, A. Izri, R.
+
+Hofmann, S. Mann, et al. 2010. Oral ivermectin versus malathion lotion for difficult-to-treat head lice. N. Engl. J.
+
+Med. 362:896–905.
+
+14. Sanofi Pasteur. 2012. Highlights of prescribing information
+[Sklice lotion prescribing guide]. Available at http://
+www.accessdata.fda.gov/drugsatfda_docs/Label/2012/
+202736s000lbl.pdf (accessed 8 March 2017).
+
+15. Lexi-Comp Online. 2012. Lexi-Drugs. Lexi-Comp, Hudson, OH.
+
+16. Therapeutic Research Center. 2011. Commentary: Natroba
+(Spinosad) for the treatment of head lice. (DetailDocument \#270904.) Pharmacist's Letter/Prescriber's Letter.
+
+17. Isman, M. B. 2000. Plant essential oils for pest and disease management. Crop Prot. 19:603–608.
+
+18. Isman, M. B. 2001. Pesticides based on plant essential oils for management of plant pests and diseases international symposium on development of natural pesticides from forest resources. pp. 1–9. Korea Forest Research Institute, Seoul, Korea.
+
+19. Yang, Y. C., H. Y. Choi, W. S. Choi, J. M. Clark, and Y. J.
+
+Ahn. 2004. Ovicidal and adulticidal activity of Eucalyptus globules leaf oil terpenoids against Pediculushumanus capitis (Anoplura: Pediculidae). J. Agric. Food Chem.
+
+52:2507–2511.
+
+20. Maunder, J. 1971. Resistance to organochloride insecticides in head lice and trials using alternative compounds. Med.
+
+Officer. 125:27.
+
+21. Abramowicz, M. 1995. Drugs for parasitic infections. Med.
+
+Lett. Drugs Ther. 37:99–108.
+
+22. Friedman, S. 1987. Lindane neurotoxic reaction in nonbullous congenital ichthyosiform erythroderma. Arch.
+
+Dermatol. 123:1056–1058.
+
+23. Pollack, R., A. Kiszewski, P. Armstrong, C. Hahn, N. Wolfe, HA. Rahman, et al. 1999. Differential permethrin susceptibility of head lice sample in the United States and Borneo. Arch. Pediatr. Adolesc. Med. 153:969–973.
+
+24. Downs, A. M., K. A. Stafford, I. Harvey, and G. C. Coles.
+
+1999. Evidence for double resistance to permethrin and malathion in head lice. Br. J. Dermatol. 141:508–511.
+
+25. Paller, A. 1993. Scabies in infants and small children.
+
+Semin. Dermatol. 12:3–8.
+
+26. Hoke, A., and H. Maibach. 1999. Scabies management: a current perspective. Cutis 64(Suppl.):2–14.
+
+27. Burkhart, C. G., and C. N. Burkhart. 2000. Clinical evidence of lice esistance to over-the-counter products. J.
+
+Cutan. Med. Surg. 4:199–201.
+
+28. Beauvais, S.L., S.B. Jones, S.K. Brewer, and E.E. Little 2000.
+
+Physiological measures of neurotoxicity of diazinon and malathion to larval rainbow trout (Oncorhynchus mykiss)
+and their correlation with behavioral measures.
+
+Environmental Toxicology and Chemistry. 19:
+1875–1880.
+
+29. Taro Pharmaceuticals. 2005. Ovide lotion: Highlights of prescribing information. Available at http://www.accessda ta.fda.gov/drugsatfda_docs/nda/2005/076790_ORIGINAL%
+20APPROVAL_PACKAGE.pdf (accessed 8 March 2017).
+
+30. Eisenhower, C., and E. A. Farrington. 2012. Advancements in the treatment of head lice in pediatrics. J. Pediatr.
+
+Health Care 26:451–461.
+
+31. Lover, M. J., A. J. Singer, D. M. Lynch, and W. E. Rhodes.
+
+1983. U.S. Patent No. 4,368,207. Patent and Trademark Office, Washington, DC: U.S.
+
+32. Perez-Esandi, M. V., C. W. Colli, and P. M. Schantz. 1974.
+
+The ovicidal effect of selected chemicals against eggs of Echinococcusgranulosus. Bull. World Health Organ. 51:550.
+
+33. Lachenmeier, D. W. 2008. Safety evaluation of topical applications of ethanol on the skin and inside the oral cavity. J. Occup. Med. Toxicol. 3:1.

medical/md/PMC5704672.md

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+# Effects Of Brief Communication Skills Training For Workers Based On The Principles Of Cognitive Behavioral Therapy A Randomized Controlled Trial Norio Sasaki, Md, Hironori Somemura, Phd, Saki Nakamura, Phd, Megumi Yamamoto, Phd, Manabu Isojima, Md, Issei Shinmei, Phd, Masaru Horikoshi, Phd, And Katsutoshi Tanaka, Phd
+
+Objective: Stimulating communication is an important workplace issue. We investigated the effects of a brief communication skills training (CST)
+program based on the principles of cognitive behavioral therapy (CBT). Methods: A randomized controlled trial was conducted on 206 white-collar workers. The intervention group underwent a 2-hour CST group training conducted by an occupational physician. Result: The results of the intention-to-treat analysis using a mixed-effects model showed that there was a significant interaction between group and time observed for the item
+''thinking together to solve problems and issues'' (P ¼ 0.02). The effect size (Cohen d) was 0.35 (95% confidence interval, 0.07 to 0.62). Conclusions: The present study suggests that a brief CST based on the principles of CBT could improve the communication behavior of workers.
+
+S timulating communication is becoming an important workplace issue. Previous observational studies have shown that effective communication among workers is significantly correlated with their mental health level.1–5 Moreover, studies indicate that effective communication was associated with an innovative workplace environment.6 In addition, a correlation between communication and work safety has been suggested.7 Several communication skills trainings (CSTs) have been given to health care providers such as doctors and nurses working in medical settings.8–13 These CSTs for health care providers were shown to be able to change the attitudes of these health care providers, improving their relationships with patients and increasing the level of satisfaction of patients and families involved.14–18 To date, however, no intervention study has examined the effectiveness of CST on nonmedical workers. Effective CSTs in the workplace are to promote communication behaviors to investigate solutions together with others to deal with many problems or issues.
+
+CSTs conducted in medical settings to date have typically been performed quite thoroughly, at 3 days to 2 weeks in total and with single sessions of intervention lasting 2 to 8 hours. A correlation between length of training and effectiveness has been reported.19 However, the amount of time provided for workers for training at the workplace is usually limited to a single training session of 2 to 3 hours. Therefore, an effective CST program that could feasibly be followed by workers in a shorter time period is essential.
+
+Here, we conducted a brief CST program based on the principles of cognitive behavioral therapy (CBT) and investigated whether it would be effective in improving the communicative behaviors to solve problems that arise in the workplace.
+
+METHODS
+Participants and Procedure This study was conducted as an in-house training program for section chiefs and staff members at a single company in the service industry in the Tokyo Metropolitan Area. Because of their small number, managers were not included in the study population. As section chiefs and their subordinates usually work together as colleagues, they were included. A request to participate in this research was sent in a company email, which explained the purpose and the methods of the study. After obtaining informed consent, the participants were assigned to an intervention or control group at random. In February 2015, the intervention group underwent group training on communication skills, with follow-up 1 month after the intervention. After the follow-up was completed, the control group was also provided with an identical intervention due to ethical considerations. No exclusion criteria were set. This study was approved upon by the Kitasato University School of Allied Health Sciences Ethics Committee and the Health and Safety Commission of the workplace concerned.
+
+Intervention The training content structure is summarized in Table 1.
+
+Training was identical for section chiefs and staff members. The intervention was a 120-minute group training on communication skills. The group training was conducted by an occupational physician who had received 10 hours of training from a specialist in CBT.
+
+The educational materials used in the present training were developed by reference to the CST guide20 created by the National Center for Cognitive Behavior Therapy and Research, National Center of Neurology and Psychiatry. The content of the group training is broken down into the following three sections: the communication model, empathy and support to start relationships with others, and guiding people in using questions to solve problems. In the communication model section, people learned that there is a process for creating relationships that enables communication. Initially, people learn the importance of the progression of communication that consists of gathering information from others and assessing it, building rapport, and proceeding to solving problems.
+
+Next, in the empathy and support to start relationships section, the listener comes to understand the importance of identifying the other person's emotions in order to create a relationship of mutual trust with the speaker. An ''emotion identification sheet'' is used to train participants in identifying emotions. They then learn that it is possible to ''create an empathetic and cooperative relationship'' through the use of empathetic and supportive expressions.
+
+JOEM -  61
+
+Sasaki et al JOEM - TABLE 1. Contents of the Program Topics Contents
+In guiding others by asking questions to solve problems section, the questioning technique known as the Socratic method is employed, and the speaker guides the listener to discover answers for problems. The Socratic method, also known as Socratic debate, is often used in CBT.21 This is a form of cooperative argumentative dialogue between individuals, which is characterized by the asking and answering of open-ended questions to promote critical thinking and new ideas to solve problems. The questions used focused on the ''problems'' the other person was having and on four areas: ''emotions,'' ''cognition,'' ''behaviors,'' and ''a person's body.''
+To familiarize subjects with the Socratic method, training was conducted to allow people to deduce specific answers, externalize their problem, and connect it to specific support.
+
+All of these exercises were performed in groups and were conducted primarily through role playing. The exercises used examples that could occur in the workplace, such as problems with human relationships at work, concerns about the way that a job is done, and worries about not being able to accomplish an objective. This content was set up so that the participants would be able to feel it as being real. We attempted to allow for more effective learning during the role playing by asking participants to write their conversation down on paper.
+
+Outcome Assessment The outcome was assessed by evaluating the differences between the groups 1 month after the training using original selfadministered questionnaires. The questionnaire had five items with 10 possible responses to each. The items were as follows: ''I am able to have an attitude of empathy and support,'' ''I am able to talk with others while accepting their opinions,'' ''I can skillfully ask others about problems and issues,'' ''I am able to think together with others to solve problems and issues,'' and ''I am able to communicate smoothly.'' The responses ranged from ''1 point: It does not apply at all'' to ''10 points: It is very applicable.''
+1. Communication Model in Psychotherapy 1.1 Necessity of Communication Skills
+(10 min)
+Giving an explanation of the necessity of learning basic techniques for communication that will provide a foundation for providing supportive assistance.
+
+Giving an explanation of the fact that communication is the foundation of human relationships and gaining an overall understanding of communication.
+
+1.2 The Significance and Progression of Communication items (10 min)
+Learning the basic communication model in which broad information is gathered from others and assessed, rapport is built, and then problem solving is undertaken.
+
+Learning that by understanding the model, they will actually be able to put it into practice.
+
+2. Empathy and support that starts relationships 2.1 Establishing Relationships where No Conflict Occurs (20 min)
+Understanding the principles of relationships and learning the factors for human relationships where there are no conflicts. Understanding that it is important to identify the emotions of the other person. Emotion identification training using the ''emotion identification sheet.''
+2.2 Making the Relationship Work with Empathy (20 min)
+Creating human relationships by communicating in a way where people listen to each other's emotions and by giving them instructions using empathetic expressions. Concretely practicing examples of being forceful and examples of giving someone else instructions using the dialog sheet.
+
+3. Guiding problem-solving through questions 3.1 Defining Guided Discovery (20 min) Gaining an understanding that it is necessary to intervene in problems that someone else has in order to step forward beyond the stage of relationship building. Learning the progression of guidance that allows a person to discover the answer to something that they want to solve on their own.
+
+This is done by presenting a plan for a solution to the problem.
+
+3.2 How to Use the Socratic Method
+(40 min)
+Gaining an understanding of the purpose of the Socratic method as uncovering the unrealistic portions of the speaker's thoughts or cognitions and then allowing them to notice the patterns and habits in their thinking.
+
+Questions are asked to deduce a specific answer, based on the five points used for devising questions, such as quantification, comparison, other points of view, evidence, and materialization. Thereafter, practice guiding another so that that person can discover the answer for themselves.
+
+Randomization An independent researcher who had no direct contact with the participants used random computer-generated numbers to divide the participants into the two groups. The assessors were masked during analysis regardless of which group the participant was assigned to. The participants were aware of their assignment due to the nature of the intervention conducted.
+
+Statistical Analysis In a systematic review of the CST given to medical personnel, the effect size was taken to be a Cohen d of 0.37 [95%
+confidence interval (95% CI): 0.23 to 0.60].19 From calculations assuming an effect size of 0.37, an a error of 0.05, and a b error of 0.20, we deduced that a sample size of 92 people per group would be necessary. The intervention effect was evaluated by investigating the significance of the interaction between group and time, using the mixed-effects models (group, time, and group  time interaction as fixed effects, and randomized participants as random effect). The effect size was investigated by comparing the difference in outcome score values (adjusted for baseline outcome value) of the two groups 1 month after intervention. To satisfy the intention-to-treat (ITT) principle, which states that all participants should be analyzed just as they were assigned, a multiple imputation method was employed to replace the missing outcome data on the assumption that the data could be considered to be missing at random.22 The following items were investigated as baseline attributes:
+age, gender, consecutive years of service, job title (section chief or staff member), type of job, hours of overtime a month, average sleeping hours on weekdays, and the six-item Kessler psychological distress scale (K6) score. The K6 scale is widely used to assess psychological stress, with the score obtained from a simple selfrating questionnaire on symptoms of depression and anxiety experienced over the previous month.23 The reliability and validity of the 62 - 
+Japanese version of the K6 questionnaire utilized in this study have been verified.24 Work-related stress and job satisfaction were also investigated using a visual analog scale. When the data items were continuous variables, the t test was used for assessment, and Fisher exact test was used to assess the category variables. All tests used P value less than 0.05 as indicative of a significant difference. Statistical analyses were conducted using commercial software (SPSS Statistics 22 and SPSS Missing Values 22; IBM Corp, Armonk, NY).
+
+RESULTS
+Study Flow Figure 1 shows the flow of the study. One of the 207 workers did not provide consent to participate in the research, as it did not work with their schedule. Excluding this person, all 206 people were randomly assigned into two groups, with 103 people each assigned to the control and intervention groups. Of the 103 people in the intervention group, 68 (66.0%) attended the group training and 81 (78.6%) responded to the questionnaire 1 month after the intervention. Of the 103 people in the control group, 80 people
+(77.7%) responded to the questionnaire 1 month after the intervention.
+
+Baseline Characteristics Baseline attributes of participants were based on questionnaire responses obtained from all participants (Table 2). There was no significant demographic difference observed between the two groups. A total of 44.6% of participants were male (44.6% in the intervention group and 44.6% in the control group) with an average age of 38.4 years (38.4 in the intervention group and 38.4 in the control group). No significant difference between the two groups was observed for any item assessing consecutive years of service, job title, type of job, number of overtime hours worked, and average sleep time. There was also no difference observed in K6 scores, work-related stress, job satisfaction, and the items ''attitude of empathy and support,'' ''acceptance of others' opinions,''
+Intervention Effects The results of the intervention are summarized in Table 3. A
+significant interaction was observed between group and time for the item ''thinking together to solve problems'' (F1, 171.2¼ 5.82, P ¼ 0.02). The difference between the scores of both groups 1 month after training was 0.30 points, and the effect size (Cohen d) was 0.35 (95% CI, 0.07 to 0.62).
+
+In contrast, a tendency toward significant improvement was observed for the intervention group with regard to ''attitude of empathy and support,'' ''acceptance of others' opinions,'' ''skillfully asking others about problems,'' or ''ability to communicate smoothly, with interactions between group and time having the respective values F1, 166.7 ¼ 2.92, P ¼ 0.09; F1, 164.0 ¼ 3.58, P ¼ 0.06; F1, 172.5 ¼ 3.77, P ¼ 0.05; and F1, 164.5 ¼ 3.72, P ¼ 0.06.
+
+DISCUSSION
+The results of this study indicate that a brief CST effectively improved communication behavior by helping workers to think together with others to solve problems and issues. The CST in the present study was not simply meant to foster communication skills that improve relationships with other people; rather, the purpose of this program is to promote communication behaviors to solve problems that arise in the workplace. Participants mainly work in jobs that require investigating solutions together with others to deal with many problems or issues. Thus, learning communication skills to solve problems meets the needs of the people who took the training course. It is possible that this made it easy for the effects of the training to be reflected as results.
+
+In addition, this training focused on three basic components of communication skills, making use of a concise program that could be understood step by step. For participatory training focused on role play, exercises contained content that is likely to be of interest to participants, such as those that occur in real workplaces.
+
+The training instructor in charge was occupational physician who Enrolled in the study(n=207)
+
+![2_image_0.png](2_image_0.png)
+
+FIGURE 1. Consort flowchart of participants.
+
+| TABLE 2. Baseline Characteristics in the Intervention and Control Groups Total (n ¼ 206) Intervention (n ¼ 103)                                                                                     | Control (n ¼ 103)                                                                                                                       | P            |            |      |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------|------------|------------|------|
+| Gender male, n (%)                                                                                                                                                                                  | 92 (44.6)                                                                                                                               | 46 (44.6)  | 46 (44.6)  | 1.00 |
+| Age average, years (SD)                                                                                                                                                                             | 32.0 (5.5)                                                                                                                              | 32.2 (5.4) | 31.7 (5.6) | 0.58 |
+| Years employed in current work, n (%) <1                                                                                                                                                            | 24 (11.7)                                                                                                                               | 12 (11.7)  | 12 (11.7)  | 0.37 |
+| 1   < 3                                                                                                                                                                                                     | 100 (48.5)                                                                                                                              | 48 (46.6)  | 52 (50.5)  |      |
+| 3   < 5                                                                                                                                                                                                     | 55 (26.7)                                                                                                                               | 33 (32.0)  | 22 (21.4)  |      |
+| 5                                                                                                                                                                                                     | 20 (9.7)                                                                                                                                | 8 (7.8)    | 12 (11.7)  |      |
+| Missing                                                                                                                                                                                             | 7 (3.4)                                                                                                                                 | 2 (1.9)    | 5 (4.9)    |      |
+| Job title, n (%) Staff member                                                                                                                                                                       | 150 (72.8)                                                                                                                              | 76 (73.8)  | 74 (71.8)  | 0.75 |
+| Section chief                                                                                                                                                                                       | 56 (27.2)                                                                                                                               | 27 (26.2)  | 29 (28.2)  |      |
+| Number of subordinates, n (%) 1   < 3                                                                                                                                                                                                     | 6 (2.9)                                                                                                                                 | 1 (1.0)    | 5 (4.9)    | 0.33 |
+| 3   < 5                                                                                                                                                                                                     | 10 (4.9)                                                                                                                                | 7 (6.8)    | 3 (2.9)    |      |
+| 5   < 10                                                                                                                                                                                                     | 30 (14.6)                                                                                                                               | 17 (16.5)  | 13 (12.6)  |      |
+| 10                                                                                                                                                                                                     | 10 (2.9)                                                                                                                                | 2 (1.0)    | 8 (4.9)    |      |
+| Missing                                                                                                                                                                                             | 0                                                                                                                                       | 0          | 0          |      |
+| Type of job, n (%) Office job                                                                                                                                                                       | 56 (27.2)                                                                                                                               | 25 (24.3)  | 31 (30.1)  | 0.44 |
+| Technical job                                                                                                                                                                                       | 28 (13.6)                                                                                                                               | 14 (13.6)  | 14 (13.6)  |      |
+| Development job                                                                                                                                                                                     | 22 (10.7)                                                                                                                               | 12 (11.7)  | 10 (9.7)   |      |
+| Professional job                                                                                                                                                                                    | 96 (46.6)                                                                                                                               | 48 (46.6)  | 48 (46.6)  |      |
+| Others                                                                                                                                                                                              | 3 (1.5)                                                                                                                                 | 3 (2.9)    | 0 (0.0)    |      |
+| Missing                                                                                                                                                                                             | 1 (0.5)                                                                                                                                 | 1 (1.0)    | 0 (0.0)    |      |
+| Hours of overtime a month, n (%) <20                                                                                                                                                                | 118 (57.3)                                                                                                                              | 57 (55.3)  | 61 (59.2)  | 0.21 |
+| 20  < 45                                                                                                                                                                                                     | 70 (34.0)                                                                                                                               | 39 (37.9)  | 31 (30.1)  |      |
+| 45  < 80                                                                                                                                                                                                     | 8 (3.9)                                                                                                                                 | 5 (4.9)    | 3 (2.9)    |      |
+| 80                                                                                                                                                                                                     | 3 (1.5)                                                                                                                                 | 0 (0.0)    | 3 (2.9)    |      |
+| Missing                                                                                                                                                                                             | 7 (3.4)                                                                                                                                 | 2 (1.9)    | 5 (4.9)    |      |
+| Nighty hours of sleep on weekdays, n (%) <4                                                                                                                                                         | 0 (0.0)                                                                                                                                 | 0 (0.0)    | 0 (0.0)    | 0.13 |
+| 4   < 5                                                                                                                                                                                                     | 20 (9.7)                                                                                                                                | 13 (12.6)  | 7 (6.8)    |      |
+| 5   < 6                                                                                                                                                                                                     | 83 (40.3)                                                                                                                               | 48 (46.6)  | 35 (34.0)  |      |
+| 6   < 7                                                                                                                                                                                                     | 76 (36.9)                                                                                                                               | 30 (29.1)  | 46 (44.7)  |      |
+| 7   < 8                                                                                                                                                                                                     | 16 (7.8)                                                                                                                                | 8 (7.8)    | 8 (7.8)    |      |
+| 8                                                                                                                                                                                                     | 4 (1.9)                                                                                                                                 | 2 (1.9)    | 2 (1.9)    |      |
+| Missing                                                                                                                                                                                             | 7 (3.4)                                                                                                                                 | 2 (1.9)    | 5 (4.9)    |      |
+| K6 scorey , mean (SD)                                                                                                                                                                               | 5.6 (4.7)                                                                                                                               | 5.4 (4.5)  | 5.8 (4.8)  | 0.57 |
+| Work-related stress, mean (SD)z                                                                                                                                                                     | 4.4 (2.7)                                                                                                                               | 4.4 (2.6)  | 4.5 (2.7)  | 0.80 |
+| Job satisfaction, mean (SD)z                                                                                                                                                                        | 6.1 (2.6)                                                                                                                               | 5.9 (2.6)  | 6.2 (2.5)  | 0.44 |
+| Attitude of empathy and support, mean (SD)§                                                                                                                                                         | 7.1 (1.8)                                                                                                                               | 7.1 (1.8)  | 7.2 (1.8)  | 0.55 |
+| Acceptance of the others' opinions, mean (SD)§                                                                                                                                                      | 7.1 (1.8)                                                                                                                               | 7.0 (1.7)  | 7.2 (1.8)  | 0.38 |
+| Skillfully asking others about problems, mean (SD)§                                                                                                                                                 | 5.8 (1.8)                                                                                                                               | 5.7 (1.7)  | 5.9 (1.9)  | 0.34 |
+| Thinking together to solve problems, mean (SD)§                                                                                                                                                     | 7.0 (1.8)                                                                                                                               | 6.9 (1.9)  | 7.1 (1.8)  | 0.42 |
+| Ability to communicate smoothly, mean (SD)§                                                                                                                                                         | 6.6 (1.7)                                                                                                                               | 6.6 (1.7)  | 6.7 (1.7)  | 0.67 |
+| SD, standard deviation.  Independent t test for difference between groups for continuous measures and Fisher exact test for differences between groups in categorical characteristics. y The six-item Kessler psychological distress scale. z Evaluated using visual analogue scale from 0 to 10, with 0 indicating not at all, and 10 indicating very much. § Scored on a scale 1 to 10, with 1 indicating not at all, and 10 indicating very well.                                                                                                                                                                                                     |                                                                                                                                         |            |            |      |
+| knew workplace conditions well. This physician conducted their training using an easy-to-understand vocabulary. The participants were familiar with group discussions and were able to deepen their | Further, the percentage of people who underwent the training session was low, partially because it overlapped with a busy time at work. |            |            |      |
+
+knew workplace conditions well. This physician conducted their training using an easy-to-understand vocabulary. The participants were familiar with group discussions and were able to deepen their understanding of communication skills by engaging in a lively exchange of opinions. Despite the short-term nature of the training, results showing significant differences were produced.
+
+Although there was a trend toward significant improvement observed for the four items ''I am able to have an attitude of empathy and support,'' ''I am able to talk with others while accepting their opinions,'' ''I can skillfully ask others about problems and issues,'' and ''I am able to communicate smoothly,'' no statistically significant difference between groups was shown. One possible reason for this is that the training time was too short to allow the participants to gain an adequate understanding of all content.
+
+Further, the percentage of people who underwent the training session was low, partially because it overlapped with a busy time at work.
+
+A review of 13 articles on CSTs by Barth and Lannen19 provided to medical professionals working in oncology showed that training health professionals by CST is a promising approach to change communication. In a systematic review by McGilton et al,25 the CST intervention demonstrated a positive change in medical professionals' communication behaviors, communication skills, and knowledge about communication. However, the CSTs used in medical settings required an intervention time period of several days to several weeks. Given this, the previous reviews have stated that furtherinvestigation based on economic considerations and feasibility is required.19,25 64 - 
+
+| Mean Scores (SE)                                                             |                 |            |             |                     |
+|-------------------------------------------------------------|-----------------|------------|-------------|---------------------|
+| Intervention                                                | Control         | Group  Time            | Effect Size |                     |
+| Interactiony                                                | (95% CI)                 |            |             |                     |
+| Group (n ¼ 103)                                             | Group (n ¼ 103) |            |             |                     |
+| Attitude of empathy and support                             | 7.2 (0.08)      | 7.0 (0.08) | 0.09        | 0.25 (0.03 to 0.52)                     |
+| Acceptance of the others' opinions                          | 7.1 (0.07)      | 6.9 (0.08) | 0.06        | 0.24 (0.03 to 0.52                     |
+| Skillfully asking others about problems and issues          | 6.0 (0.14)      | 5.8 (0.14) | 0.05        | 0.14 (0.14 to 0.40)                     |
+| Thinking together to solve problems and issues              | 7.1 (0.08)      | 6.8 (0.09) | 0.02        | 0.35 (0.07 to 0.62) |
+| Can communicate smoothly                                    | 6.7 (0.11)      | 6.5 (0.11) | 0.06        | 0.18 (0.09 to 0.46)                     |
+| CI, confidence interval; SE, standard error.  Adjusted for baseline score of each outcome. y P value assessed using linear mixed models, including group, time, and group x time as fixed factors; participants as a random factor. 1. Outcomes were evaluated using a questionnaire of our design whose reliability and validity have not been assessed. This may A characteristic of our study was that the CST program                                                             |                 |            |             |                     |
+| offered was brief and could be conducted by an occupational |                 |            |             |                     |
+
+A characteristic of our study was that the CST program offered was brief and could be conducted by an occupational physician. Education in the workplace should be brief to minimize interruption and ensure provision to as many employees as possible.
+
+To enable implementation in a workplace setting and to reduce the economic and time constraints of employees, the program was simplified even more than previous studies in medical settings. On the contrary, concern for benefits being compromised due to oversimplification is therefore justified. To counteract any detractions due to the simplicity of our program, we should have utilized homework or E-learning as supplemental materials.
+
+The contents of the CST in the present study were based on the principles of CBT. CBT in medical settings to date has mainly involved high-intensity CBT centered on individual psychotherapy. However, in recent years, low-intensity CBT that employs the Internet, books, distributed materials, or the provision of information or education through group training has been shown to have an effect on reducing psychological distress26,27 and absenteeism due to mental illness.28,29 In addition, significant reductions in the level of depression have been indicated in randomized controlled trials (RCTs) that employed problem-solving methods. CBT emphasizes that the listener should create an empathetic and cooperative relationship to increase the subject's ability to cope with problems by working on dysfunctional cognition.30 Repairing dysfunctional cognition might improve their flexibility regarding new ways of thinking and action, help them accept their role in a problem, find more solutions for the problem, and take action appropriate to a given situation.31,32 In this study, the components of CBT were proactively incorporated into the CST.
+
+In observational studies, it has been suggested that the improvement of communication skills between workers and superiors could be related to an increase in work satisfaction and reduced work-related stress.33–35 In future studies, it will be necessary to examine the effects of a CST on increasing job satisfaction and work-related stress. Furthermore, the improvement of workers' performance of duties is an extremely important topic for businesses. Cross-sectional studies examining the factors that determine an innovative workplace atmosphere identified a correlation between a better environment for communication and a more innovative workplace.6 Thus, improving workers' communication skills could potentially contribute to improving worker performance and productivity.
+
+As we have seen, effective communication has been shown to correlate with many key factors in the workplace. Therefore, our finding that a brief CST produced a recognizable improvement in workers' communication behaviors is considered to be significant.
+
+Limitations The following are limitations of this study:
+1. Outcomes were evaluated using a questionnaire of our design whose reliability and validity have not been assessed. This may have caused measurement bias. Although a communications skills questionnaire specifically for physician–patient communication has been verified for reliability and validity, a similar evaluation scale for general workers is not available. To evaluate the impact of CST training on health care professionals' communication behaviors, video or audio recordings are mostly used in clinical settings.19 Information obtained by recording devices for both self-evaluation and evaluation by others has been used to assess the effect of CST training on communication behaviors.
+
+2. The relevance of the results is not completely clear. We believe that our results, although their effect size was small, are important, because improvement of communication behaviors is suggested to be associated with reducing workers' stress and increasing job satisfaction. However, we investigated changes in communication behaviors and not outcomes such as workers' well-being, and were therefore unable to show whether the improvement in communication behavior by CST was associated with changes in workers' well-being.
+
+3. The time period in which the training was conducted overlapped with a busy period at work, such that the rate of participation in the training program was 66.0%. It is necessary to avoid busy periods and to create a plan to increase the number of training sessions available. This will make it easier for participants to attend.
+
+4. There were instances where people in the intervention group and the control group both worked at the same workplace, so it is possible that there was some type of information exchange that took place.
+
+5. The observational period was quite short, such that the long-term effects were not sufficiently verified. Due to time constraints imposed by workplace convenience, this study's follow-up period was short-term. A study with a longer follow-up period is needed to confirm the effectiveness of CST in the workplace.
+
+6. We conducted this CST without distinction between section chiefs and staff members. Although they work together and the management role of section chief is limited, some difference in the level of effects may have been detected due to differences between section chiefs and staff members in their need and responsibility to effectively communicate with others.
+
+CONCLUSION
+This study suggests that a simple CST program as part of work has the effect of improving communication behavior by helping workers to think together with others to solve problems and issues. It has been suggested that communication within the scope of one's work relates to stress in the workplace and that the improvement of workplace communication through CST training could potentially lead to an increase in the level of mental health of
+-  65 REFERENCES
+1. Mizoue T, Takahashi H, Shazuki A, Kubota S, Mishima N, Nagata S.
+
+Relationships among self-management skills, communication with superiors, and mental health of employees in a Japanese worksite. Ind Health.
+
+2003;41:335–337.
+
+2. Shigemi J, Mino Y, Tsuda T, Babazono A, Aoyama H. The relationship between job stress and mental health at work. Ind Health. 1997;35:29–35.
+
+3. Ikegami K, Tagawa Y, Mafune K, Hiro H, Nagata S. Effectiveness of mental health training including active listening for managers. Sangyo Eiseigaku Zasshi. 2008;50:120–127.
+
+4. Mineyama S, Tsutsumi A, Takao S, Nishiuchi K, Kawakami N. Supervisors' attitudes and skills for active listening with regard to working conditions and psychological stress reactions among subordinate workers. J Occup Health.
+
+2007;49:81–87.
+
+5. Kawakami N, Kobayashi Y, Takao S, Tsutsumi A. Effects of web-based supervisor training on supervisor support and psychological distress among workers: a randomized controlled trial. Prev Med. 2005;41:471–478.
+
+6. Ko¨hler T, Janssen C, Plath SC, et al. Communication, social capital and workplace health management as determinants of the innovative climate in German banks. Int J Public Health. 2010;55:561–570.
+
+7. Parker SK, Axtell CM, Turner N. Designing a safer workplace: importance of job autonomy, communication quality, and supportive supervisors. J Occup Health Psychol. 2001;6:211–228.
+
+8. Yamagishi M1, Kobayashi T, Nagami M, Shimazu A, Kageyama T. Effect of web-based assertion training for stress management of Japanese nurses. YJ Nurs Manag. 2007;15:603–607.
+
+9. Szmuilowicz E, el-Jawahri A, Chiappetta L, Kamdar M, Block S. Improving residents' end-of-life communication skills with a short retreat: a randomized controlled trial. Palliat Med J. 2010;13:439–452.
+
+10. Amiel GE, Ungar L, Alperin M, Baharier Z, Cohen R, Reis S. Ability of primary care physician's to break bad news: a performance based assessment of an educational intervention. Patient Educ Couns. 2006;60:10–15.
+
+11. Lienard A, Merckaert I, Libert Y, et al. Factors that influence cancer patient' anxiety following a medical consultation: impact of a communication skills training programme for physicians. Psycho-oncology. 2008; 17:488–496.
+
+12. Alelwani SM, Ahmed YA. Medical training for communication of bad news:
+a literature review. J Educ Health Promot. 2014;23:3–51.
+
+13. Børøsund E, Cvancarova M, Moore SM, Ekstedt M, Ruland CM. Comparing effects in regular practice of e-communication and web-based self-management support among breast cancer patients: preliminary results from a randomized controlled trial. JMIR. 2014;16:e295.
+
+14. Delvaux N, Razavi D, Marchal S, Bre´dart A, Farvacques C, Slachmuylder JL.
+
+Effects of a 105 hours psychological training program on attitudes, communication skills and occupational stress in oncology: a randomised study. Br J Cancer. 2004;90:106–114.
+
+15. Smith RC, Lyles JS, Mettler J, et al. The effectiveness of intensive training for residents in interviewing. A randomized, controlled study. Ann Intern Med.
+
+1998;128:118–126.
+
+16. Abdollah D, Kobra A, Tayyebeh M. Assessing the effectiveness of interpersonal communication skills training on job satisfaction among nurses in Al-Zahra Hospital of Isfahan, Iran. Iran J Nurs Midwifery Res. 2012; 17:290–295.
+
+17. Langewitz WA, Eich P, Kiss A, Wo¨ssmer B. Improving communication skills a randomized controlled behaviorally oriented intervention study for residents in internal medicine. Psychosom Med. 1998;60:268–276.
+
+18. Fawole OA, Dy SM, Renee F, et al. A systematic review of communication quality improvement interventions for patients with advanced and serious illness. J Gen Intern Med. 2013;28:570–577.
+
+19. Barth J, Lannen P. Efficacy of communication skills training courses in oncology: a systematic review and meta-analysis. Ann Oncol. 2011;22:
+1030–1040.
+
+20. Horikoshi M. The ABCs of Communication Skills. Tokyo: National Center of Neurology and Psychiatry; 2013, 10–46.
+
+21. Froja´n-Parga M, Calero-Elvira A, Montan˜o-Fidalgo M. Study of the Socratic method during cognitive restructuring. Clin Psychol Psychother. 2011;18:
+110–123.
+
+22. Donders A, van der Heijden G, Stijnen T, Moons K. Review: a gentle introduction to imputation of missing values. J Clin Epidemiol. 2006; 59:1087–1091.
+
+23. Kessler RC, Barker PR, Colpe LJ, et al. Screening for serious mental illness in the general population. Arch Gen Psychiatry. 2003;60:184–189.
+
+24. Furukawa TA, Kawakami N, Saitoh M, et al. The performance of the Japanese version of the K6 and K10 in the World Mental Health Survey Japan. Int J Methods Psychiatr Res. 2008;17:152–158.
+
+25. McGilton KS, Boscart V, Fox M, Sidani S, Rochon E, Sorin-Peters R.
+
+A systematic review of the effectiveness of communication interventions for health care providers caring for patients in residential care settings. Worldviews Evid Based Nurs. 2009;6:149–159.
+
+26. Kimura R, Mori M, Tanaka K, et al. Effect of a brief training program based on cognitive behavioral therapy in improving work performance: a randomized controlled trial. J Occup Health. 2015;57:169–178.
+
+27. Mori M, Tajima M, Kimura R, et al. A web-based training program using cognitive behavioral therapy to alleviate psychological distress among employees: randomized controlled pilot trial. JMIR Res Protoc. 2014;3:e70.
+
+28. Grime PR. Computerized cognitive behavioural therapy at work: a randomized controlled trial in employees with recent stress-related absenteeism.
+
+Occup Med. 2004;54:353–359.
+
+29. Kraaijeveld RA, Schaafsma FG, Boot CR, Shaw WS, Bu¨ltmann U, Anema JR. Implementation of the Participatory Approach to increase supervisors' self-efficacy in supporting employees at risk for sick leave; design of a randomised controlled trial. BMC Public Health. 2013;13:750.
+
+30. David Daniel E, Dirk L, Leif B, et al. Efficacy of an internet-based problemsolving training for teachers: results of a randomized controlled trial. Scand J
+Work Environ Health. 2014;40:582–596.
+
+31. Fresco DM, Rytwinski NK, Craighead LW. Explanatory flexibility and negative life events interact to predict depression symptoms. J Soc Clin Psychol. 2007;26:595–608.
+
+32. Crowe M, Whitehead L, Carlyle D, et al. The process of change in psychotherapy for depression: helping clients to reformulate the problem.
+
+J Psychiatr Ment Health Nurs. 2012;19:681–689.
+
+33. Eguchi H, Tsuda Y, Tsukahara T, Washizuka S, Kawakami N, Nomiyama T.
+
+The effects of workplace occupational mental health and related activities on psychological distress among workers: a multilevel cross-sectional analysis. J Occup Environ Med. 2012;54:939–947.
+
+34. Ikegami K, Tahara H, Yamada T, Mafune K, Hiro H, Nagata S. Effects of a mental health training program for manufacturing company managers.
+
+J UOEH. 2010;32:141–153.
+
+35. Tsutsumi A, Takao S, Mineyama S, Nishiuchi K, Komatsu H, Kawakami N.
+
+Effects of a supervisory education for positive mental health in the workplace: a quasi-experimental study. J Occup Health. 2005;47:226–235.

medical/md/PMC5822332.md

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+
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+![0_image_0.png](0_image_0.png)
+
+Swowtr* 
+
+![0_image_1.png](0_image_1.png)
+
+REVIEWS. 
+
+Works on Physiological and Pathological Chemistry By Simon, Day, Griffiths, Rees, &c. 
+
+{Continued from our last.) 
+Bile.?The following observations by Dr Day, on the uses of the bile, may tend to correct a popular error. 
+
+" That the bile is not merely an excrementitions fluid, intended to remove effete matter from the blood, but that it is a secretion essential to the animal economy, was rendered almost certain by the experiments of Berzelius, and Theyer and Schlosser, which showed that the human feces contained much too small a quantity of a substance resembling bile, to justify the idea that it is evacuated in this manner. A further proof that the bile is absorbed and not excreted, is afforded by an examination made by Enderlin, of the ash yielded by the contents of the different portions of the intestinal canal of a hare. He found that the ash from the contents of the duodenum alone effervesced on the addition of an acid, thus showing that the choleate of soda (which yields the carbonate on incineration) is absorbed before reaching the jejunum. 
+
+Schwann has recently established this opinion, beyond a doubt, by a series of well-devised experiments on dogs. He tied the ductus communis choledochus and at the same time formed a fistulous opening in the gall-bladder, by which the bile escaped externally. His most important conclusions are : ]st, That when the bile does not get into the bowel, its absence is generally perceptible in dogs about the third day, by a marked diminution in weight; and 2dly, That unless the channel for the conveyance of bile to the duodenum is re-established, symptoms of deficient nutrition, wasting, debility, &c., ensue, and death is the ultimate consequence."?Vol. ii. p. 26. 
+
+Although Blondlot has subsequently repeated Schwann's experi-ments with very different results, we are still inclined to believe that the view maintained by Dr Day is the correct one. In fact, that gentleman gives a further proof of the correctness of his view in his Report on the Progress of Animal Chemistry in the 1st Volume of Banking's Half-yearly Abstract, p. 345. It is there stated that the fasces of a healthy man, when extracted with spirit and treated by Pettenkofer's test, did not yield the slightest reac-tion, whilst on previously adding a little bile to the fieces, it was perfectly developed. In cases of diarrhoea, and in the evacuation produced by calomel, this test would, however, always detect the presence of bile. 
+
+The chemistry of healthy and diseased bile has been carefully studied by Frerichs. (Day's Reports, in Vols. ii. and iii. of Ranking.) The bile in the two following cases was obtained from healthy men, killed by severe accidents. 
+
+| 1.                                                                                                  | 2.    |
+|-----------------------------------------------------------------------------------------------------|-------|
+| 8600                                                                                                | 85-92 |
+| 14-00                                                                                               | 14-08 |
+| 10-22                                                                                               | 9-14  |
+| 0-16                                                                                                | 0-26  |
+| 0-32                                                                                                | 0-92  |
+| 0-65                                                                                                | 0-79  |
+| Water,  Solid residue,  Bilate (or choleate) of soda,  Cholesterine,  Margarine and oleine,  Salts, |       |
+
+Milk.?The fourth chapter?on the milk?contains all that is known on that subject. We must content ourselves with simply extracting the following easy method of analysing that secretion. 
+
+" When milk is stirred with about one-fourth of its weight of finely powdered gypsum, and heated to 212?, it is entirely coagulated ; and if the whole is then evaporated to dryness, a brittle mass is obtained that is easily reducible to powder. From the powder the butter may be extracted with ether ; the sugar of milk and soluble salts may be removed by hot alcohol of 0 85 ; while the caseate and sulphate of lime, and insoluble salts remain undissolved. The alcoholic solution scarcely exhibits any perceptible opacity on the addition of chloride of barium, showing that no error in the result is occasioned by any of the gypsum being taken up by the alcohol. 
+
+" About 100 grains of gypsum, and four times its weight of milk, answer very well. The soluble salts extracted from the milk by the alcohol may easily be determined by maceration ; and since their amount is to that of the insoluble salts in the average proportion of 5 to 7, the amount of the latter may at least be found approximately, and the ascertained weight of the sugar and casein corrected accordingly. But if it be required to determine the salts with perfect accuracy, it is best to incinerate a weighed quantity of milk, and to analyse the residue."?Vol. ii. p. 46. 
+
+Urine.?The chapter on the urine, which extends over 240 pages, is undoubtedly one of the most perfect treatises on the subject ever written. Its value is greatly enhanced by the additions of Dr Day. 
+
+In p. 526, we find a description of a new method for the quanti-tative determination of urea. It is founded on the circumstance, that if a solution of urea be heated with concentrated sulphuric acid, its amount may be determined from the sulphate of ammonia that is formed. The reaction is shown in the equation,? 
+
+C2N2H402 -f 2HO+2 (SOg,HO), = 2 (NH40,S03)-f2 C02. 
+
+The following is the best method of proceeding. The urine must be mixed with about half its weight of concentrated sulphuric acid. 
+
+The mixture must be kept in a state of moderate ebullition; and when the temperature reaches 392 degrees, there ensues an evolution of carbonic acid in small bubbles. The cessation of the dis-engagement of gas indicates that the urea is completely decomposed. 
+
+The residue, which presents a black, charred appearance, is then thoroughly extracted with water, and the solution filtered. The clear filtrate is evaporated in the water-bath, and the sulphate of ammonia treated with alcohol and bichloride of platinum. 
+
+Since urine contains salts of potash and ammonia, which will, of course, likewise precipitate upon the addition of bichloride ofplatinum, it is necessary to determine the exact proportion in which these salts are present in the urine under examination. For this purpose, a separate weighed portion of urine is precipitated with bichloride of platinum, and the amount of precipitate subtracted from the former. The extractive matters normally present in the urine exercise no adverse influence in determining the urea in this manner. 
+
+The following method of obtaining hippuric acid from human urine is given by Dr Day. 
+
+" Evaporate the urine till there is a copious deposition of salts ; add strong alcohol, and place the mixture in a stoppered bottle. With the aid of a gentle heat (for instance, by placing the bottle in warm water), we ensure the solution of the urea, the lactates (if any are present), and the hippurates, in the alcohol, whilst the urates remain with the insoluble constituents. When the super-natant fluid is perfectly clear, it must be decanted, evaporated very nearly to dryness, and re-dissolved in hot water. If a stream of chlorine be passed through the aqueous solution, the urea is destroyed ; and, by gradual concen-tration, and the addition of a little free mineral acid, we obtain crystals of hippuric acid."?Vol. ii. p. 117. 
+
+The effect of diet on the urinary secretion has been studied by many physiologists. The most trustworthy experiments are those of Lehmann, instituted on himself. The following table gives the mean daily amount of the various solid constituents during different systems of diet:? 
+
+```
+                            Mixed Animal Vegetable Non-nitrogen-diet. diet. diet. ous diet. 
+Solid constituents G7*82 87*44 59-24 41*G8 grammes. 
+   Urea 32*50 53*20 22*48 15*41 
+   Uric acid 1*18 1*48 1*02 0*73 ... 
+   Lactic acid and lactates 2*72 2*17 2*68 5*82 
+   Extractive matters 10*49 5*20 16*50 11*85 
+
+```
+
+The urine in typhus should be submitted to daily examination. 
+
+Schonlein's opinion, that the urine in the regular course of typhus is at first dark and very acid, subsequently neutral, and even alka-line, and finally again becomes acid at the commencement of con-valescence, is confirmed by several cases given by Simon:? 
+
+" In one case the urine became faintly alkaline on the seventh day after admission ; it remained either alkaline or neutral for seven or eight days; and then became faintly acid and gradually clearer as soon as the patient exhibited symptoms of convalescence. 
+
+" In a second (very severe) case, the urine remained acid till the twenty-first day; it then became neutral, and afterwards alkaline for the space of ten or eleven days, when it returned to its normal reaction. 
+
+" In two other cases the urine became alkaline previously to the fourteenth day of the disease ; in one of them the secretion was so thoroughly saturated with carbonate of ammonia, and evolved so disgusting an odour, as to be per-ceptible over the whole ward. This urine deposited a considerable sediment of pus or mucus, mixed with the phosphates of lime and magnesia, and effervesced briskly on the- addition of an acid. In one of these cases the urine re-mained alkaline for fourteen, and in the other for twenty-one days, before it resumed its acid reaction. Both cases recovered. 
+
+" It is worthy of notice that a deposition of urate of ammonia not unfre-quently precedes the occurrence of alkalinity and the appearance of the earthy phosphates, which, as Schonlein remarks, may be regarded as the precursors of a favourable change."?P. 24G. 
+
+During the mild form of typhus prevalent at Berlin, in the year 1843, he noticed these changes in several cases, and in fact, when from being alkaline, the urine again became acid, and at the same time clear and abundant, there was scarcely any risk in giving a favourable prognosis. 
+
+The Urine in Gout.?The following observations on the urine in two cases of gout will be read with interest, as they not only indi-cate the chemical character of the renal secretion in that disease, but likewise show the degree to which it is modified by the administration of benzoic acid. In the first case, before the use of benzoic acid, the urea amounted to 30*16$ and the uric acid to 2*14$, and afterwards they amounted to 28*21$ and 2*22$ respec-tively. In the second case the urea and uric acid amounted to 26*56$ and 1*66$ before the use of the acid, and 26*61$ and 1*59$ 
+afterwards. 
+
+These analyses are insufficient to show that benzoic acid exerts any influence on the amount of urea or uric acid. The clinical experiments of Froreip and others indicate, however, that it is a valuable remedy in various forms of arthritis. 
+
+The urine in diabetes meets with all due consideration from Simon and Dr Day. Simon quotes Henry's tables for determining the solid constituents of diabetic urine by the application of the urinometer, and states that his own experience coincides with that of Dr G. O. 
+
+Rees, in showing that the table gives tolerably accurate results. 
+
+We have ourselves devoted a good deal of attention to the urinary secretion in diabetes, and having always adopted the formula of our accurate and trust-worthy friend and preceptor, Dr Christison, in preference to that of Henry, we were not a little pleased to find how thoroughly its correctness has been established by Dr Day, who has shown, by more than 200 observations, that Christison's formula gives more correct results in health and disease generally than either those of Becquerel or Henry. As Dr Day's essay appeared more than two years ago in the Lancet, and his conclusions have been adopted in Dr Golding Bird's excellent treatise on 
+" Urinary De-posits," we are surprised to find that Dr G. O. Rees has not modified his opinions in the recent edition of his work. 
+
+The Urine in Syphilis.?In his observations on the urine in syphilis, Dr Day quotes a case in which Heller analysed the renal secretion of a patient, who was taking two scruples of iodide of potassium and half a grain of iodine daily. As iodine is always in a state of combination when it occurs in the secretions, this quantity may be regarded as equivalent (chemically) to 40*625 grains of the iodide. From the urine of twenty-four hours, Heller succeeded in obtaining 38*G89 grains of the iodide; hence, the whole was re-moved by the kidneys, with the exception of nearly two grains, which were partly distributed to the saliva, sweat, nasal mucus, &c. 
+
+and partly retained in the blood. 
+
+Tlie Urine in Pregnancy.?In the early volumes of this Journal there were several papers on the peculiar character of the urine during pregnancy. We do not find any notice of them in Simon's 
+" Animal Chemistry;" but notwithstanding this apparent over-sight, we believe that, on the whole, the work we have before us contains the best account yet published, of the urine in this state. 
+
+And, in connection with this subject, we may notice a slight over-sight on the part of Dr G. O. Rees. At page 200, he observes that 
+" the butyric acid has been observed but in one specimen of urine, and it is a matter of doubt, whether it be not rather an accidental than a necessary constituent of the fluid." He probably refers to the case noticed by Berzelius, but if he will turn to page 33.1 of the second volume of Simon, he will find that that chemist states, that in the course of twenty-four hours, a white viscid sediment was generally deposited from the urine during pregnancy, and that, on washing this sediment with water, and then treating it with alcohol and a little sulphuric acid, a disagreeable fruit-like odour, reminding him of butyric ether, was developed. And in the following page, we find that Lehmann, in experimenting on the urine of a pregnant woman, not only detected butyric acid by its odour, but actually obtained brilliant crystals of butyrate of baryta. 
+
+1847.] PHYSIOLOGICAL AND PATHOLOGICAL CHEMISTRY. G73 On the passage of medicinal and other substances into the urine The remarks on this subject, from the pen of the editor, are de-serving of an attentive perusal. We regret that Ave have only space for the following brief extract on the means of detecting quinine in the urine. 
+
+" Quinine, when administered in large doses, has been noticed in the urine by Piorry, Landerer, and others. The best test for its presence is the iodated iodide of potassium, consisting of four parts of iodide of potassium, one of iodine, and ten of water. The precipitate afforded by this reagent with disul-phate of quinine, is very insoluble in water, is not affected by an excess of the test, and is readily soluble in alcohol. It is of a yellowish brown colour, and forms a turbidity or sediment according to the amount of the alkaloid in the urine. When the quantity is very small, there is merely an olive tint produced on the addition of the test.''?Vol. ii. pp. 338, 339. 
+
+The eighth and ninth chapters treat of the secretion of the lachrymal, Meibomian, and ceruminous glands, and the secretions of the generative organs. 
+
+The tenth chapter enters fully into the consideration of the various modifications of feces in health and disease, and terminates with the analyses of certain vomited matters. One of the most singular points connected with ftecal chemistry, is the large amount of fat discharged from the bowels in diabetes. Simon found the fat in a case of this nature, to amount to 34$ of the dried faeces; the ordinary amount being, according to Percy, about 12?. 
+
+The eleventh chapter treats of the component parts of the animal body,?the bones, teeth, cartilage, cellular tissue, tendons, ligaments, skin, hair, crystalline lens, arteries, veins, muscles, nervous matter, fat, and glands. We can only say of it, as, indeed, we may say of almost every part of the work, that it contains nearly every thing that is known of the subjects on which it treats. 
+
+The two last chapters are devoted to the consideration of solid and fluid morbid products, to the chemical composition of which we intend devoting another article on some future occasion. 
+
+The English Edition of Simon's Animal Chemistry is, undoubt-edly, the most perfect work on the subject in this, or perhaps any language. When we compare the therein contained chapter on the Blood with the article on the same subject in Dr Copland's Dic-tionary, we cannot fail to be struck with the energy and perseverance displayed in this department of medicine by our German brethren during the last few years. The learned encyclopaedist is distin-guished for the zeal (to say nothing of the judgment) with which he collects materials for his elaborate articles ; and yet, on referring to his Essay on the Blood, we do not find a single analysis of that fluid in a morbid condition ; and what he tells us regarding its nor-mal state is, in the highest degree, vague and unsatisfactory. 
+
+There were several passages in the work of Dr Rees, which we had marked for extraction or comment. The extracts must, how-ever, be omitted, and our comments must be very brief. In the NEW SERIES.?NO. IX. MARCH 1847. 3 I 
+description of the analysis of the blood (see page 27), he obtains a residue, which " consists of albumen, combined with soda." If he believes that the blood does contain such a compound, he should, at all events, have stated the views of the Giessen school on this subject. Enderlin's experiments (see page 596) seem to afford strong evidence against the existence of such a compound in the blood. 
+
+In a note to page 32, we read that " iron exists as a mere trace in serum, and, when found, is probably owing to accidental admix-ture with a small number of blood corpuscles. What says Mulder on this point? In page 337 of Fromberg's translation of " The Chemistry of Vegetable and Animal Physiology," it is written that 
+" other constituents of the blood besides hjematin contain iron." 
+We cannot allow the " examination of blood supposed to contain urea" to pass without one or two remarks. In the first place, we have already shown, that the occurrence of crystals in the alcoholic extract, after the addition of nitric acid, does not afford a decisive proof of the existence of urea; and, secondly, we cannot ascertain the proportion of urea by the method given for that purpose by Dr Rees, or, indeed, by any other method we are acquainted with. 
+
+Did Dr Rees never read of an experiment performed by Marchand, showing that there is a peculiar difficulty in the precise determination of this constituent ? 
+
+^ In his observations on the probability " that bile exists in serum in its perfect state," we should have been glad to have met with something about Pettenkofer's Test; and we cannot agree with him in his statement, that " we know of no method at present of accurately ascertaining the proportion of sugar in diabetic blood." 
+If the sugar exist in more than a mere trace, its amount may be ascertained by the carbonic acid which it yields on fermentation. 
+
+Finally, Dr Rees tells us that " the only compound, calculus containing cystine, as a constituent, is contained in the Collection of Guy's Hospital." We like to see men take a pride in their own hospitals and their own museums, but this is really carrying the feeling a little too far. If Dr Rees had simply told us that such a calculus was to be found in that museum, he would have been con-veying to his readers an interesting piece of information, but when we happen to know that such calculi have also been described and analysed by Bley, and Henry, and Yellowly, we cannot help feeling that there is an occasional laxity and carelessness in his statements, in which a writer of his acknowledged ability and learning ought not to indulge. We have already noticed one or two similar over-sights. Notwithstanding these slight defects, the book is a good one; and to those who simply desire to acquire a knowledge of the manipulations requisite for the separation of the most important constituents of the blood and urine, we do not know that we can recommend a safer guide. 
+
+The work of Dr Griffith is of altogether a different character. 
+
+His information is brought down to a much later date than that of Dr Rees. We fear, however, that the extreme conciseness of his manual will render it a difficult book to those not already acquainted with the subject We cannot conclude without expressing a wish that animal che-mistry were cultivated more generally than it is. We want physiological and pathological chemists to assist in building up a solid and exact basis for the healing art. We want in this country enthusiastic young men who, like Simon and Schercr, will be content for a time to work in hospitals, analysing the solid and fluid constitu-ents of the body, in health and disease. Such labours must inevit-ably acquire for them a fame and reputation, that they will find it impossible to reach by other paths. The chemical lectures at pre-sent delivered in our colleges and schools should constitute a portion of that preliminary education necessary for a proper study of all the arts and sciences ; there should be reserved for the medical student a course of physiological and pathological chemistry, more especially brought to bear upon the explanation of the causes, signs, and treatment of disease. Such is one of the elements necessary for the cultivation of a rational system of medicine, and we look forward to the period when this great fact will be universally ac-knowledged. In the meantime, let us do all in our power to stimulate perseverance and talent to occupy themselves on subjects best calculated for the public good; and we are satisfied that in the present day, there is not one which is so likely to yield beneficial and important results as that of animal chemistry. 
+
+On Diseases of the Skin. By Erasmus Wilson, F.R.S., &c. 
+
+Second Edition. London, 1847. 
+
+Notwithstanding all that has been written and published on the subject of skin diseases, it must be confessed that the great bulk of the profession is still veiy imperfectly informed regarding their diagnosis and treatment. We do not now allude so much to the febrile eruptions, which, from their frequency and danger, neces-sarily force themselves upon the attention of every medical man, as to the less alarming and more chronic affections to which the skin is exposed. And yet we have known ignorance in this de-partment of medicine exasperate the progress of other maladies, render life miserable, destroy those social relations and ties so dear to all men, and finally ruin the reputation of more than one otherwise well informed practitioner. The cause of this must be sought for in the absence of all instruction regarding it in our large schools, and the non-existence in this country of hospitals set aside and properly furnished for the treat ment of this class of disorders. There are no diseases which more require that preliminary instruction in them should be practical, than those which affect the skin. Books and plates, after all, teach very little. For the most part they are compilations; and from the unnecessary multiplication of forms and varieties, so far from extricating the reader from the difficulties which surround the subject, only tend to increase his bewilderment and perplexity. 
+
+Skin diseases vary so much in their nature and external characters, that a classification of them is essential. No kind of description, either verbal or written, could be made without one. But here is the difficulty. Shall we choose an artificial, a natural, or a pathological arrangement? We do not hesitate a moment in our choice. 
+
+For the purposes of the practitioner, that is with reference to diagnosis and treatment, we are of opinion that the classification of Willan and Bateman, as modified by Biett, is the only one worth a moment's consideration. A natural arrangement presupposes great knowledge of the subject, and is therefore useless to the 
+.? ; student. A pathological arrangement presupposes a knowledge of 
+... ' the nature of skin diseases, and this, with regard to many of them, 
+>?' . we do not possess, and it must necessarily be faulty. On the other 
+? hand, the artificial arrangement of Willan, notwithstanding its imperfections and inconveniences, can be at once understood by, and is tolerably easy for, the practical student; and this classifica-tion, certainly rendered more perfect by the modifications of Biett, is the one which best answers the purposes both of teacher and student, and will enable the latter to acquire a knowledge of forms and varieties more readily than any other. 
+
+Mr Wilson denominates his classification a natural one. It is founded on a mixture of anatomy, physiology, and pathology. For instance, the four great divisions refer to the anatomy of the skin. 
+
+They are, 1st, Diseases of the Derma; 2d, of the Sudoriparous Glands ; 3d, of the Sebiparous Glands; 4th, of the Hairs and Hair Follicles. We do not object to this primary anatomical division; on the contrary, it may be considered good, if it were possible to separate precisely the diseases belonging to each. Most of the subdivisions, however, are very unphysiological and unpathological. 
+
+YVc arc at once struck with the following extraordinary arrangement of inflammatory diseases of the derma:? 
+
+" inflammationCongestive? 
+
+Specific?Rubeola?Scarlatina?V ariola?V aricella?V accinia. 
+
+Non- Specific?Erysipelas?U rticaria?Roseola?Erythema. 
+
+Effusive? 
+
+A sthcnic?Pem phigus?Rupia. 
+
+Sthcnic?Herpes?Eczema?Sudamina. 
+
+Suppurative?Impetigo?Ecthyma. 
+
+Depositive?Strophulus?Lichen?Prurigo. 
+
+Squamous?Lepra?Psoriasis?Pityriasis. 
+
+From Parasitic Animalcules?Scabies."?Pref., p. xxii. 
+
+mations, arc novel. Let us endeavour to understand their mean-ing?and, first, what are we to comprehend by a congestive inflammation ? All pathologists, whilst they allow that congestion precedes inflammation, have been most careful to separate the two lesions. Does Mr Wilson affirm that a congestion, that is, an un-usual distension of the vessels with blood, is an inflammation ? We cannot think so. On the other hand, does he mean to assert, that there can be any acute inflammation without congestion? We hope not. Surely, then, the term congestive inflammation is some-what contradictory. Under this head he has arranged Variola, Varicella, and Vaccinia. What, then, we should like to know, is suppurative inflammation ? On turning to page 220, we are told it is characterized by the formation of pus occupying the surface of the derma, and producing an elevation of it to a limited extent. 
+
+Why, then, are variola and vaccinia not placed in this group ? Be- ..." 
+cause, says Mr Wilson, they are torn from their natural affinities, 
+" and, for this reason, I have thought it proper to transfer the genus ; ^ 
+to the group of eruptive fevers."?P. 221. Now, we do not object * .. * 
+to Mr Wilson's forming a group of eruptive fevers, but we strongly . y,i. 
+
+protest against his placing pustular diseases under such an unmean- 
+'? 
+
+. * '?' 
+ing head as Congestive Inflammation. 
+
+Is there any method of drawing a pathological distinction between an effusive and a suppurative inflammation? We think not. Examine the contents of a herpetic vesicle, and of an ecthymatous pustule, and both will be found to contain pus. True, the vesicular and the pustula? have been long separated, and we understand by the former a disease in which the vesicles present a stage in which the liquor sanguinis has not yet fully passed into pus. It must be evident, however, that such a distinction can have no real patholo-gical value, for, as regards their inflammatory phenomena, that is, the formation of small collections of pus, they are the same. Why the pus of Herpes should be longer in forming than that of Ecthyma, or why the latter should leave a cicatrix, whilst the former does not, we are ignorant. Again, the suppurative action of Herpes is identical with that of Variola or Vaccinia; but why a pus globule of these last should have the power of regenerating a similar disease, whilst one of the latter has not, we know nothing. But it is exactly on account of our ignorance of such points, which consti-tute the true nature of these affections, that it is impossible to found a classification on a pathological basis. 
+
+The term depositive inflammation, we are told, p. 236, has been selected only in the absence of a more suitable word. Assuredly that chosen is not a good one, inasmuch as every inflammation is depositive. It is employed by Mr Wilson, however, " to express that condition of the inflamed membrane in which plastic lymph is exuded by the capillary rete into the tissue of the derma, so as to give rise to the production of small hard elevations of the skin, or pimples." We beg leave to deny that, in strophulus, lichen, or prurigo, a deposit is exuded in any way analogous to plastic lymph, as Mr Wilson may easily satisfy himself by a microscopic examination. 
+
+What a squamous inflammation is the author has not thought proper to inform us; for, on turning to p. 261, we find no attempt at definition. The scales are composed of epidermic cells, which, not being vascular, cannot become inflamed. A squamous inflam-mation, therefore, cannot mean inflammation of the scales. Are we to understand by it an inflammation of the skin, in which the scales peel off in increased quantity ? If so, Scarlatina and Erysipelas ought to be classified under this head. The nature and meaning of this newly discovered inflammation is a perfect mystery to us, and we therefore strongly object to it. We are told " that hypertrophy of the derma in the squamous diseases is very evident." Why, then, not place them with callosities and corns, which are also hypertrophies of the derma ? 
+
+Another subdivision of inflammation is that from parasitic animalcules. But is the inflammation produced in this way different from that originating in any other ? Or is it consistent with the principles of classification to divide inflammation in some cases according to its results, as effusive or suppurative, and in others according to its causes ? Again, if animal parasites are to consti-tute a ground of distinction, why may not vegetable parasites claim a similar notice? Do these, in the course of their development, never cause inflammation of the derma ? Most assuredly they do, as we have proved innumerable times. 
+
+With regard to vegetable parasites, however, we have to charge Mr Wilson with a total unacquaintance with the subject. Whilst he has personally examined into diseases caused by animal para-sites, he seems to have entirely neglected those of vegetable origin. It is true he quotes passages from the writings of Gruby and Hughes Bennett, but his own opinion seems to be, that the structures described have no analogy with those of the vegetable wrorld. Speaking of the favus crust, in the preface, p. xx., he says, 
+" Its vegetable nature is hypotheticaland, when treating of the pathology of favus, he remarks,? 
+
+" However closely the fungous growth here described may resemble a plant, its vegetable nature is very far from being established. The simplest forms of animals are composed, like the mycodermis, of cells, variously connected to-gether ; and subsequent research may prove the growth under consideration to be of a similar nature. To my mind there is nothing improbable in the sup-position of the origin of the growth from morbidly developed epidermal cells of the hair-follicle, or from those of the sebaceous substance. In a preceding section of this work, I have shown that the latter are susceptible of considerable alteration, and that in this state they assume an appearance widely different from that of their normal condition. Mr Busk also entertains doubts with regard to the vegetable nature of the mycodermis, and deduces an opinion favourable to his opinion, from the chemical analysis of the crusts of favus given by Tlienard, who found them composed of ? 
+
+```
+Albumen 
+Gelatine 
+Phosphate of Lime 
+Water and loss 
+
+```
+
+70 
+.17 
+. 5 
+. 8 100 
+" Dr Carpenter, in his ' Principles of Physiology' (p. 453), speaking on the same subject, remarks,?' It has been assumed that the organization is vegetable, because it (mycodermis) consists of a mass of cells capable of extending themselves by the ordinary process of multiplication. But it must be remembered that the vesicular organization is common to animals as well as to plants, being the only form that manifests itself at an early period of development in either kingdom, and remaining throughout life in those parts which have not undergone a metamorphosis for special purposes. Hence, to speak of porrigo favosa, or any similar disease, as produced by the growth of a vegetable within the animal body, appears to the author a very arbitrary assumption; the simple fact being, in regard to this and many other structures of a low type, that they present the simplest or most general kind of organization.'"?Pp. 430, 431. 
+
+Mr Wilson has taught anatomy, and is the author of an ana-tomical manual of no small repute. How any one possessing a knowledge of the elementary structures of the animal tissues could have published such a passage surpasses our comprehension. It immediately follows an accurate description of the vegetations by Gruby, in which we are told that they consist of roots and branches dividing dichotornously, composed of jointed tubes with transverse septa, containing granules, and giving off at their extremities seeds or sporules. 
+
+We have placed this passage in italics purposely. Where, we ask Mr Wilson, is there an animal tissue which presents a similar struc-ture, and where is the vegetable conferva that does not ? Knowing, as Mr Wilson seems to do, that such accurate observers and laborious inquirers as Remak, Schonlein, Gruby, Fuchs, Henle, Vogel, and many others, have pronounced in favour of the vegetable nature of these growths, we can only regard his citing as an authority, in favour of his own opinion, a writer like Dr Carpenter, to be either a piece of innocent badinage, or a specimen of tine irony. The fact is, that there is no animal tissue either healthy or diseased, which in any way resembles the structure of the favus crust. 
+
+In the present edition, Mr Wilson has separated ichthyosis from warts, corns, and other hypertrophies of the derma. He assigns, as a reason for this, his conviction that the disease is a concretion of altered sebaceous substance? 
+
+" In an instance of this affection which fell under my observation about ten years since, I had the opportunity of examining the skin after the death of the patient from visceral disease. In this case, the scales were remarkable for their thickness ; after being well washed, they were greyish in colour upon the surface, but white beneath, and evidently consisted of concreted sebaceous substance. On removing a portion of the epiderma by maceration, the ducts of the sebiparous glands and hair-follicles were found distended with inspissated white secretion, and had a very beautiful and brilliant appearance, projecting like cones of pearl from the under surface of the membrane. The derma presented a number of small deep pits, corresponding with these dilated ducts. 
+
+The mouths of the distended excretory ducts opened upon the surface of the epiderma, some immediately beneath, and in the middle of the scales, and others by their borders. In the former situation, they could be seen as small white points through the scale, and still more evidently when the epiderma was separated by maceration. 
+
+From the careful examination of this case, of which a preparation is now before me, and of others which I have subsequently observed, I have been led to the conclusion, that the scales, in this disorder, increase in thickness, in two ways, firstly, by additions to the free surface, by means of the secretion poured out in the linear furrows of the skin, and, consequently, between the scales ; and, secondly, by additions successively made to the attached surface by the effusion of inspissated secretion beneath them. In the preparation before me, the growth of the scales by both of these processes is distinctly evident." 
+Supposing Mr Wilson's view to be correct, which, as regards all the forms of ichthyosis, we are disposed to doubt, it must be ac-knowledged that the disorder is in every way allied to the squama?. 
+
+Thus, while under the head of congestive inflammation, he mixes up the exanthemata and pustulae on account of their natural affinities, he dissociates ichthyosis from psoriasis, on account of a theory he has formed with regard to the formation of the scales. 
+
+Under the head of disorders of the vascular tissue, Mr Wilson has placed vascular n?evi and purpura. Yet what evidence have we that purpura is a disorder of the vascular tissue ? On turning to page 316, we find the following passage? 
+
+" Purpura is a morbid state of the capillary system, characterized by the effusion of blood into the different tissues of the body, this effusion giving rise to the formation of sanguineous patches in considerable numbers, and of various sizes. The capillary vessels of the skin participate in this morbid disposition ; 
+hence purpura has obtained a place, by courtesy, among cutaneous disorders." 
+Surely there is nothing here to prove that the capillary system is affected in purpura. We have been in the habit of considering it as dependent on some lesion of the blood, and the observations of Liston, Lane, and others would seem to countenance this opinion. 
+
+Of this we. are quite satisfied, that the capillary vessels have never been shown to be diseased, and hence another discrepancy in the pathological arrangement of our author. 
+
+But it is unnecessary to criticise the classification of Mr Wilson further. We think enough has been said to show that his arrange-ment is not very happy, or consistent with our present knowledge of pathology. Most assuredly, in departing from the principles of arrangement introduced by Willan, he has not given us one that is more simple or correct; but, on the contrary, by the formation of unpathological terms, has thrown increased mystification and confusion on the whole subject. 
+
+The matter of classification is at all times important, and we are well aware of its difficulties. Hence, perhaps, our dislike to the derangement of that which is well established and easily understood* It is curious that writers on dermatology should have acquired their knowledge by following the system of Willan and Bateman, and afterwards endeavoured to supersede it by a natural or pathological arrangement. We observe this in the writings of Alibert, Cazenave, Hebra, and now of Mr Wilson. No doubt that system has its faults, and as our knowledge becomes perfect we naturally endea-vour to arrange it upon a scientific rather than upon an artificial basis. We hope that the efforts which are making in this direction will ultimately be crowned with success. But we must bide our time. Science is not yet sufficiently advanced to solve those difficulties which lie on the threshold of the path, and all the attempts which have been made to form classifications of skin diseases, not by mastering these difficulties, but by putting them aside, have invariably failed, and produced no permanent influence on the minds of the profession. 
+
+Turning from the classification and general pathology of Mr Wilson to the special treatises of which his book is composed, we feel much pleasure in stating that many of its individual parts are carefully compiled, and that some give evidence of considerable research and original investigation. Among the latter, we would especially point out the introductory chapter on the Anatomy and Physiology of the Skin, the section on Diseases of the Sebiparous Glands, and the two concluding chapters on the Itch Insect, and the " Steatozoon" Folliculorum. 
+
+We cannot discover in the work any comprehensive notions respecting the treatment of skin diseases. One of the evidences of an improved pathology will be the introduction of general principles to guide us in our efforts at cure. As it is, we find only in the publication before us the same empirical formula, and reliance upon contradictory individual experience, as in most books on the same subject. 
+
+The following are some of the author's observations on the treatment of chronic eczema:? 
+
+" In the, chronic form of eczema, when the disease is obstinate, and resists our common methods of treatment, it becomes necessary to modify the state of the constitution by various means ; as, for instance, by a course of hydriodate of potash, of mercury, of Donovan's solution, of liquor arsenicalis, or of tincture of cantharides. 
+
+" In employing arsenic and cantharides as therapeutic agents, it will be necessary to watch their effects with care, and bear in mind the serious symp-toms which may result from their abuse. Should any of these symptoms be apparent, namely, nervous disorder, and disorder of the alimentary canal, in the case of arsenic,?or of the urinary system, in the case of cantharides, the medicines must be immediately laid aside, either permanently, or to be resumed after a few days, according to the judgment of the practitioner. Whene^r we put a stop to the exhibition of arsenic, and return to it again, it is necessary to begin with a smaller dose than that at which we left off. Arsenic, when it acts upon the nervous system, performs the part of an alterative, but when its effects are directed upon the digestive system, it appears to me to act, like can-tharides, upon the mucous membrane of the kidney, viz. by counter-irritation, by exciting inflammatory action in the interior, and thus determining from the surface. 
+
+" The best formula for the exhibition of cantharides is one in which it is NEW- SERIES.?NO. IX. MARCH 1847. 3 K 
+combined wiflh equal parts of compound tincture of camphor, and taken in tincture of cinchona."?Pp. 212, 213. 
+
+We have found that arsenic and cantharides act very well to-gether. Equal parts of Fowler's solution, and the tincture of the latter drug, may be mixed together, of which ten drops form a dose to begin with for an adult. The local remedies available in chronic eczema are, according to Mr Wilson, very numerous. We give them exactly as they are set down by him:? 
+
+" Sulphuret of potash in lotion or ointment, lime water, bichloride of mercury in weak solution, calamine ointment, zinc ointment, sulphate of copper oint-ment, tannin ointment, white precipitate ointment, red precipitate ointment, calomel ointment with watery extract of opium, carbonate of lead ointment, tar ointment, sulphur ointment, and mercurial ointment. 
+
+"To relieve the pain and pruritus which accompany the eruption, the fol-lowing remedies as local applications may be tried, viz. acidulated lotions, alkaline lotions, lotion of super-acetate of lead, emulsion of bitter almonds, or of hydrocyanic acid, lotions of opium or hyoscyamus, camphor mixture, infusion of dulcamara, &c."?P. 213. 
+
+We do not think that much benefit in practice is likely to be obtained by following directions, if such they can be called, like those just given. Certainly chronic eczema is a most rebellious disorder; but, according to our experience, is in no way benefited by irritating applications like red precipitate ointment. We have found alkaline lotions the best local method of treatment, but then the parts should be kept continually moist, by means of saturated cloths, well covered with oil-silk to prevent evaporation. 
+
+The following are the author's observations on the treatment of lepra:? 
+
+" The internal remedies recommended for lepra are, a course of purgatives ; 
+bichloride of mercury in decoction of sarsaparilla, or dulcamara; hydriodate of potash ; tincture of cantharides ; liquor arsenicalis ; liquor potassae; sulphuric acid ; decoctions of guaiacum, mezereum, and elm bark ; infusion of nettles; milk of sulphur, &c. 
+
+" Of these remedies, that on which I place the greatest reliance is the liquor arsenicalis, or the liquor hydriodatis hydrargyri et arsenici (? 446). The former of these solutions in doses of five drops, and the latter ten to thirty, three times a day, with meals. The latter is the milder remedy of the two, and rarely produces any disagreeable constitutional effects. Of course it is necessary, in using arsenical preparations of every kind, to counsel the avoid-ance of acids, fruits, and vegetables, and to explain to the patient the symptoms which call for the suspension or omission of the remedy. The effect of arsenic on the leprous patches is, in the first instance, to increase their redness, activity, and heat, and subsequently to diminish these symptoms, and render them brownish and dull. When the latter change takes place, the eruptions quickly fade and disappear. The medicine requires to be taken regularly, and to be persisted in for several months."?P. 268. 
+
+" The local remedies are, lotions of sulphuret of potash, alkaline baths, vapour baths and douches, sea-bathing, spirituous solution of bichloride of mercury, zinc ointment, white precipitate ointment, calomel ointment, nitrate of mercury ointment, ointments of acetate and phosphate of mercury, of sul-phate and deutoxide of antimony, ioduret of sulphur ointment, from ten to twenty grains to the ounce, creosote, blisters, nitrate of silver, &c. M. Gibert speaks favourably of an ointment of the ioduret of ammonia, in the proportion of a drachm to an ounce, and also of the ointments of Anthrakokali and Fuli-gokali. In the employment of these applications, care must be taken, in acute cases, to use them only after the reduction of the local excitement, by means of fomentations, emollient baths, &c., and then only of moderate strength. In chronic cases, however, they may be employed from the commencement, and in a more concentrated form, with the vifew of modifying the diseased structures. 
+
+"' My own practice,' says a distinguished author (Rayer) on cutaneous diseases, ' is to begin with the white precipitate ointment, or with that of the protochloride of mercury, unless in those cases where the disease is of very long standing, when I try the ioduret of sulphur in preference.' 
+" M. Emery of Saint Louis has lately recalled the attention of practitioners to an old but valuable application in leprous affections, namely, tar. Finding, however, that this remedy was objectionable on account of its colour and odour, he had recourse to one of the products of tar, concrete napthaline, which afforded him the most successful results. The preparation which he employs is an ointment composed of Napthaline two to four parts. 
+
+Lard thirty parts. 
+
+This he applies to the diseased skin, on folds of linen, night and morning. The ointment is highly stimulating, and has a powerful smell, which quickly passes away. By means of the napthaline ointment M. Emery succeeded in curing eight patients out of fourteen, in from five weeks to three months."?Pp. 270,271. 
+
+Our own experience is decidedly more in favour of the preparations of pitch as local applications than of any other. Napthaline is a cleanly means of employing it, and deserves a trial. We have seen, in the wards of Dr Hebra at Vienna, scaly diseases success-fully treated by infusions of tar, made with boiling water in the usual manner, of which a pint was drank daily. In this manner, the disorders yielded as rapidly as if the remedy had been applied externally, and, of course, without the unpleasantness resulting from the latter mode of treatment. 
+
+In the treatment of lupus, Mr Wilson has found the best results from a prolonged course of liquor hydriodatis hydrargyri et arsenici. 
+
+He seems to be unacquainted with the extensive experiments lately made at the Hopital St Louis, by M. Emery and others, which have shown the great benefits to be derived from the internal administration of cod-liver oil in these cases. 
+
+The following are the author's observations on the treatment of ring-worm:? 
+
+" The treatment of trichosis offers two indications: firstly, to correct the deranged nutrition by constitutional means: secondly, to excite a new action in the skin by means of moderately stimulating local remedies. 
+
+" In effecting the first of these objects, diet, exercise, clothing, and ventila-tion, are important and powerful agents. And their beneficial influence may be assisted by tonic medicinal remedies, such as the mineral acids, quinine, steel, vegetable bitters, &c., given separately or combined. I have found the syrup of quinine and steel a valuable medicine in this complaint, and one which suits the taste of children better than other remedies. The kind of tonic must, however, be left to the judgment of the practitioner, and be determined by its effect on the economy. 
+
+" In directing the local treatment, I have found it useful to commence with some moderately powerful stimulant, such as acetum cantharidis, or the stronger acetic acid, which I apply once to the diseased spots ; and, after the tenderness has abated, anoint the patches daily with some moderately stimu-lating ointment, such as the unguentum hydrargyri nitratis diluted with ceratum cetacei. Another ointment which I have found of great service, is one composed of a drachm of sulphate of zinc to an ounce of simple cerate. 
+
+When an objection exists to ointments, a lotion of bichloride of mercury or sulphate of zinc will be found useful, correcting the dryness which follows the employment of lotions by means of pomatum or cold cream. It is beneficial to wash the head with soap once a day, and, when dried, to anoint it with pomatum. I regard the keeping the scalp constantly moistened with some oleaginous matter as an important adjuvant to cure. 
+
+" The principle of local treatment being, as I have above stated, moderately stimulant, the remedies applicable to this disease, with advantage, are very numerous, and an extensive choice is left to the practitioner ; so that no excuse exists for using such as are disagreeable in their appearance, odour, or effects. 
+
+The preparations of iodine, for example, are open to objection, on account of the stain which they leave behind, and they are in no wise more useful in their effects than more elegant compounds. A remedy which I frequently use in this disease is liquor ammoniae and olive oil, accommodating the quantity of the alkali to the amount of stimulation which I desire to effect. This preparation has the advantage of being cleanly and easily removed by washing, while it clears the patches more completely than any other that I am acquainted with. Another elegant, as well as useful application, is a pomatum of cantha-ridine, prepared at my request by Mr Savory of Bond Street."?Pp. 419, 420. 
+
+It may be worth while knowing the treatment which has proved very successful in Heriot's Hospital School in Edinburgh,? so' successful, indeed, that the disease is now very rarely seen there. 
+
+A piece of very coarse brown paper is rolled into a cone, and the larger end set on fire. The smoke is directed on the diseased scalp. 
+
+In a little while a drop of empyreumatic oil collects at the extremity of the cone, which should be allowed to fall on the affected part. 
+
+When it separates, a cure is often produced. Should other means fail, it may be thought worth while to try this. 
+
+We could continue making extracts and commentaries, but are now obliged to conclude. This we do, with the remark, that our present mode of treating chronic skin diseases is most unsatisfactory, and often irrational; and that we cannot hope to see greater cer-tainty introduced into our practice, until the pathology of these disorders is further advanced. The microscope has here a fertile field to cultivate. Doubtless, some of the fruits have already been plucked by Kemak, Gruby, Hebra, and our author; but there is still a rich harvest to be gathered in by him who will diligently cultivate the soil. 
+
+1. School for Deaconesses for the Sick and Poor, and other Charitable Institutions at Kaiserswerth on the Rhine. Extract from the Journey of a Tourist. (Rev. C. Major.) Berlin : 1843. 
+
+2. Institution of Protestant Deaconesses or Nursing Sisters at Kaiserswerth, on the Rhine, in Prussia. London : 1846. 
+
+3. Passing Visits to Distant Charitable Institutions. Die Barmberzigen Schwestern. Extracted by permission from Lowe's Edin-burgh Magazine. Edinburgh: 1846. 
+
+In the admirable report given by Dr Balfour, on homoeopathic treatment in Dr Fleischmann's Hospital, Vienna, which appeared in the British and Foreign Medical Review, for October last, a passage occurs well worthy the attention of every medical man, and more especially deserving to be seriously pondered by those who have learnt that the giving of drugs is neither the only nor the most important duty which he has to discharge to his patient. The passage is as follows:? 
+
+" In taking into consideration the adjuvants to treatment, the religious character of the establishment must not be forgotten. The greater part of the patients are Roman Catholics. These find themselves surrounded by all the consolations of religion?by every thing which, in their opinion, tends to insure in the event of death a speedy passage of the soul to the realms of bliss. Their minds, thus set at ease with respect to futurity, are less gloomy and desponding, and, consequently, react less unfavourably upon the body than in opposite circumstances. Nay, looking upon their nurses as self-devoted in the service of Heaven and suffering humanity, they cannot but believe that the blessing of the Almighty will rest upon their labours; and being the object of those labours they naturally enough appropriate a portion of this blessing to them-selves, and imagine that their recovery can hardly fail to be promoted by being the inmates of such an institution. In support of this opinion I may state, that though most of the patients were young, and many dangerously ill, I 
+never heard one expression indicative of a fear of death, nor one murmur, how-ever slight, unless extorted by the extremest pain, and even then, it was more an aspiration after relief than a grieving at suffering. The severer the disease the more closely do they grasp their rosaries and crucifixes. So long as they are able to read, prayer books are constantly in their hands, and even in the intervals of delirium, nay, often, in the cases of women especially, during delirium itself, the exercises of repeating prayers, or snatches of them, is their occupation. The superiority of attendance is also one great advantage in favour of this hospital, independently of the important fact just stated, that the nurses are spiritual as well as temporal comforters." 
+This truthful statement of what any one may witness in those hospitals where the patients are nursed by the Sisters of Charity, may well induce us to ask the question, why it is that the profes-sors of a religion which we have been taught to regard as erroneous, should set Protestants such an example in self-denial, and works and labours of love:? 
+
+" Observations of this nature made many Protestants of these countries feel the duty of setting before the eyes of their Roman Catholic neighbours, some examples of charitable works carried on by Protestants without the aid of false doctrine, or monastic vows. The Rev. Mr Fliedner embraced the idea with much fervour?and has been liberally supported in his undertaking by many Christians all over Germany." 
+This quotation will explain the origin of the interesting institu-tion, the benevolent and successful operations of which it is the intention of the pamphlets at the head of this article to record ; 
+our object at present, however, is more immediately to show the immense advantage in a medical point of view, which would be conferred on our hospitals, and the sick generally, by the introduction of such an institution into this country. 
+
+The immediate object of the institution will be best explained by another extract from the same pamphlet:? 
+
+" But long after this office had ceased, the Roman Catholics endeavoured to fill the breach by their ' Sisters of Charity,' who have given evidence of their usefulness in our provinces likewise, whilst they are employed in almost every public hospital in France. Owing, however, to their very small number, and particularly to their education in monasteries, the recluse principles, which they often endeavour to spread, these Sisters of Charity of the Romish congre-gation are insufficient to fill this important office under all circumstances, and according to apostolical principles never can be admitted to serve the Protestant Church nor its members. The object of the school for deaconesses in Kaiserswerth is to correct this deficiency, by educating converted female Christians for deaconesses, to be sent out among the poor and sick of all denominations, for usefulness either in public hospitals or private houses ; they may likewise be appointed by Protestant churches, who feel the duty'of renewing this aposto-lical institution in their congregations. 
+
+" It appears that several females, after having been very useful in the hospital of Kaiserswerth, have been sent out to different public hospitals or private families, where they continue to labour with much success in a truly evangelical spirit. Others have been appointed as deaconesses by Presbyterian ministers in their own congregations, and it is to be hoped that these churches will gradually feel the good effect of this very useful institution, which owes its existence to the charity of Mr Fliedner, and the liberal contribution of many Christians all over Germany, among whom I remark the name of the whole Royal family of Prussia, the Duchess of Orleans, and the King of Wir-temberg, whom Mr Fliedner has interested in his benevolent enterprises." 
+The rules of the institution we shall extract from the second pamphlet on our list:? 
+
+" The rules of the establishment at Kaiserswerth are the following :?The candidates must not be under eighteen years of age, and serve from six months to two years for probation. After this probationary time, those among them who have been found fit individuals for the work of Christ, receive, during divine service, a solemn christian blessing, and then enter upon their duties as deaconesses at the infirmary, which contains from 100 to 110 beds. They engage themselves to serve at least five years, after which time they are allowed to leave, or may renew their engagement. It is understood, that if nearer, personal, or family duties, should make them wish for a change of situation during that period, every reasonable facility is granted to them for that purpose by the direction, vested in a committee. They receive no salary: a very moderate annual sum is paid by the institution or family they serve to the institution at Kaiserswerth, which defrays their personal wants, enables them to keep themselves decent and respectable, and entirely provides for those whose health has suffered in consequence of their hard service.'' 
+In order that the deaconesses might be properly trained to the duties of their responsible office, Mr Fliedner established near his own house a small infirmary, where the wants of the sick poor could be attended to, while, at the same time, the nurses were acquiring that information, which was to fit them for more exten-sive spheres of duty. During the nine years which have elapsed since the hospital was opened, it has received about 1900 patients, of both sexes, and of all religious denominations. 
+
+We cannot forbear extracting the following truly eloquent and touching passage from the last work on our list, where the scenes enacted in this hospital are painfully contrasted with those which are too frequently witnessed in similar institutions in this country, which takes so large an amount of credit for christian and en-lightened benevolence :? 
+
+" The office of these ' sisters of charity' (in the best sense of the word they are so), which elevates them above the common sick nurse, and engages them in concerns which touch on eternity, is that of reading the Scriptures to the sick and aged, and dropping a word of consolation into the languid ear, while they minister to the bodily wants. This they are authorised and expected to do, so that, instead of doing it by stealth, as a pious sick nurse may do in our hospitals; or, instead of railing on the poor sufferer who cries out in concern for his soul's health, as an impious one has sometimes been known to do, they breathe balm while they turn the pillow, and speak of the way of reconciliation while they endeavour to lull pain. Barmhcrzigen Schwestem, in the true sense of that emphatic title, they soothe the distressed?they speak of sin and the fountain opened, without speaking of penances or confessional, and teach to adore Him who laid down his life for the perishing, without interposing crucifixes, or Mary, or saint, or angel. They are by the bed in the midnight hour, and can seize the moment of coolness and clearness to speak to the afflicted?a moment which neither chaplain, nor medical man, nor friendly visiter, may be so happy as to hit upon ; and, while they are forbidden to be preachers, their living actions, their christian bearing, and their faithful advices, are calculated to drop like balm on the wounded spirit, and have, in many cases, accomplished good which we may justly call incalculable, for its consequences are eternal." 
+Hard as the labour is, and irksome as the duty must often be found, we rejoice to be enabled to state that numbers have come forward willing to undertake it, and many of these from a rank of life, which shows that they could not have been actuated by any selfish considerations :? 
+
+" The deaconesses are not only of the lower and middle classes, but several also of the higher and highest ranks of life. One young Baroness of the Grand Duchy of Mecklenberg has just been educated at Kaiserswerth, and is now destined to be the matron of the large new model hospital at Berlin, lately established by the King of Prussia, in which, at least, thirty deaconesses will find work, and which is to become a great nursery for training deaconesses to serve in the different parts of the monarchy. The institution at Kaiserswerth has been called upon to send, at least, twelve trained deaconesses without delay to that model hospital. Two other ladies of high rank are at present at Kaiserswerth, devoting themselves to the same offices. Some nurses have also been educated at Kaiserswerth for Switzerland, for France, and for Holland, and the claims from many parts of the continent for deaconesses from Kaiserswerth are so numerous, that this establishment cannot satisfy them all. It results from the testimonies of the administration and the medical officers of those public institutions, and is a fact of general notoriety, that wherever those deaconesses have been intrusted with the care of an hospital or of a branch of the same, a visible change for the better takes place in all departments, and the satisfaction, the gratitude, and the blessings of the patients, follow those self-devoted nurses everywhere." 
+We think the passages we have quoted are amply sufficient to exhibit the nature and design of the institution, and putting its religious character, for the present, out of consideration, we would ask our professional brethren whether such an institution, properly organized and conducted, would not prove to them an inestimable boon. Could we see the patients in our hospitals treated with kindness and consideration, no longer left to the mercies of hire-lings often of the worst characters, and generally selfish to a degree ; 
+could we be sure that our orders were strictly attended to, and, if neglected, that such neglect were reported to us; had we the comfortable conviction that cheerfulness and universal kindness reigned in our wards, with how much greater satisfaction could we discharge our duties, and how much more frequently should we expect a beneficial result. Nor are the evils we would remedy, confined to hospitals alone. How often is wealth itself unable to procure the luxury of kind and considerate attention; how almost impossible to obtain conscientious and disinterested service. If Mrs Harris be an imaginary character, Mrs Gamp is by no means so, and were such moral nuisances swept away, and their places supplied by active, intelligent, and well educated nurses, the phy-sician would obtain a coadjutor of inestimable value, and the patient an attendant above all price. 
+
+Let medical men proclaim the boon which such an institution would confer, let them press it on the attention of all who have time or talents, means or opportunities to prove it, and we do not despair before long of seeing among us, a school where those who have the necessary gifts may be trained for the best offices of christian charity. Those who are familiar with the working of the Kaiserswerth Hospital, have not been slow to appreciate its advan-tages, and to endeavour to extend its sphere of usefulness. At the first public meeting in London, in 1845, for the purpose of found-ing the German Hospital there, a wish expressed by Chevalier Bunsen, Prussian Ambassador, 
+" That some of the Protestant deaconesses of Pastor Fliedner's Institution at Kaiserswerth (near Dusseldorf, for the sick and poor), might come from the Rhine to England for the service of that institution, has been carried into effect in these last days. Four of these deaconesses, educated at the mother institution of Kaiserswerth, arrived last week in London, accompanied by the Rev. Mr Fliedner, the founder of that institution, and have entered upon their duties as nursing sisters, one of them in the capacity of matron of the establish-ment. 
+
+" This new hospital, which is going on exceedingly well, and has, since its opening on the 18th October last, already benefited some hundreds of poor sick Germans, can now be expected to become a still greater blessing to the inmates. 
+
+For these christian sisters, in the strength of faith working by love, have undertaken, under the direction of the committee, the management of the hospital, and are ready to show no less humility than self-devotion in its service. Yea, it is to be hoped (to use the expressions of the Rev. Dr M'Caul and the Venerable Archdeacon Robinson at the above-mentioned public meet-ing), that this plan of Protestant nursing sisters will soon be imitated in the English hospitals, where, no less than in other countries, such self-denying sisters are extremely wanted, according to the general testimony of persons of experience." 
+We cannot conclude without one other extract from the last of our authorities. No one has had better opportunities of observing the general habits of her sex than the accomplished writer?would that her advice were seriously pondered. How many are the complaints, formidable alike to the sufferer and her friends, and exhausting both to body and mind, which might be altogether prevented, or when they did occur, alleviated, if not removed, by suitable and engrossing occupation. Luxury, refinement, and indolence are slaying their thousands, what but self-denial or Christian principles can preserve them:? 
+
+" In this country we lack a little of the German simplicity, and are so nice about distinctions of rank, and what belongs to our supposed station in society, that it may excite strong displeasure if we siy that there are many single women in Scotland, of the excellent of the earth, who are not so useful in the Church as they might be; that the reason of this is their want of proper guidance in selecting their work, and of support in its prosecution ; and that the deaconess' status in society, and the style of character and bearing ex-pected from her, is exactly what is wanted to confer the necessary energy and steadiness." 
+We rejoice to see, from the article in Lowe's Magazine, that the subject is attracting attention in Edinburgh, and we earnestly trust that the members of our profession will not be behind-hand in pro-moting the formation of such an institution, which might surely be engrafted without great expense on some of our existing medical charities. 
+
+Ueber die Amputation am Fussgelenke. Von Dr Franz Chelius. 
+
+Mit vier Tafeln. Heidelberg, 1846. 4to. 
+
+On Amputation of the Ankle Joint. By Dr Francis Chelius; with Four Plates. Heidelberg, 1846. 4to. 
+
+Dr Chelius, during a short visit he made to Edinburgh in the summer of 1846, saw Professor Syme perform amputation at the ankle joint. He had also an opportunity of examining three other cases in various stages, after the operation had been completed. He tells us that the results of this operation excited his astonishment, and convinced him that it was one of the most brilliant and valuable with which modern surgery had been enriched. On his return to Heidelberg he found a case of diseased tarsus, in the Surgical Clinic, in which amputation of the leg had already been determined on. His father, the well known and distinguished professor of surgery, permitted him to perform Mr Syme's operation, which was done without accident, and with the most perfect success. 
+
+NEW SERIES.?NO. IX. MARCH 1847. 3 L 
+The present essay constitutes Dr Chelius' inaugural dissertation. 
+
+It is appropriately dedicated to the surgeon, whom he shows, from a learned critical analysis of all that has been written on the subject, has the merit of originating it, viz. Professor Syme. It gives an excellent description of the mode in which it should be performed, divided into five stages, and concludes with the case which was so successful in his hands. It is illustrated with four very well executed lithographic engravings, exhibiting how the incisions should be made, with a copy of the stump in the instance referred to. 
+
+So much has been said in this Journal concerning the operation, that it is unnecessary for us to analyse minutely the excellent essay of Dr Chelius. It is in the highest degree creditable to him, and is exactly that kind of inaugural dissertation we should expect to see in a young surgeon of talent, and one who has been educated under such a distinguished preceptor as its author can boast of. 
+
+With respect to the operation itself, we sincerely join in his con-cluding remark; and trust that, owing to his admirable monograph, it will soon be adopted and firmly rooted in the surgical practice of Germany. 
+
+Hand Book of Human Anatomy, General, Special, and Typographical. 
+
+Translated from the original German of Dr Alfred Yon Behr, and adapted to the use of the English Student, by John Birkett, F.R.C.S. of England, &c. London, 1846. 
+
+The plan of this work is admirably conceived, but is rather too vast for the purposes of a manual. On the whole, however, we can confidently recommend it as an excellent guide to the medical student during the first year of his studies. 
+
+Elements of Chemistry, including the Actual State and Prevalent Doctrines of the Science. By the late Edward Turner, M.D., 
+F.R.S.L. and E. Eighth Edition. Edited by Baron Liebig, and William Gregory, M.D., F.R.S.E., &c. Part I.?In-organic Chemistry. London, 1847. 
+
+This work is now universally considered as one of the standard books on Chemistry, a reputation which it has maintained through seven editions, and which the eighth, now published, is in every way calculated to confirm. 

medical/md/PMC5889981.md

@@ -0,0 +1,67 @@
+# Current Practices Of Percutaneous 
+
+![0_Image_0.Png](0_Image_0.Png) Coronary Intervention In Korea Between 2011 And 2015
+
+## Han Saem Jeong , Md, And Soon Jun Hong **, Md, Phd**
+
+Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, Seoul, Korea
+► See the article "Trends, Characteristics, and Clinical Outcomes of Patients Undergoing Percutaneous Coronary Intervention in Korea between 2011 and 2015" in volume 48 on page 310.
+
+The number of percutaneous coronary intervention (PCI) procedures for coronary artery disease has dramatically increased as a result of technical and pharmacological advances. As well as PCI procedures in patients with acute coronary syndromes (ACSs), stable angina is one of the most common indications for PCI. In ACS, previous trials have shown that an invasive strategy of early and appropriate revascularization improved clinical outcomes.1)
+However, the value of PCI in preventing major adverse cardiac events compared with optimal medical therapy alone in patients with stable angina is controversial.2) The Clinical Outcomes Utilizing Revascularization and Aggressive Drug Evaluation (COURAGE) trial and the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial failed to show a benefit of PCI over optimal medical therapy in patients with stable angina.3) PCI in patients with stable angina has decreased significantly after COURAGE and BARI 2D trials.4)
+Moreover, the baseline characteristics of the patients who received PCI have changed over time. Consequently, identifying national trends is important to develop national clinical guidelines and implementation of these guidelines into clinical practice. In Korea, diversity in ethnicity is quite different from either Europeans or Americans. In addition, recent westernization has influenced lifestyle, diet, and disease patterns in Korea. In this aspect, Han et al.5) provided valuable evidence of the national trend for PCI in Korea. The authors5) reported a large nationwide data showing the current practice of PCI in patients with angina pectoris and acute myocardial infarction (AMI) in Korea, and they have well summarized current trends, characteristics, and clinical outcomes of PCI in Korea between 2011 and 2015. This study showed that comorbidities such as hyperlipidemia increased in patients with angina pectoris and AMI from 2011–2012 to 2014–2015. On the other hand, the incidence of hypertension decreased from 2011 to 2015, but other diseases including diabetes and congestive heart failure did not show significant changes in both groups. Proportion of male patients undergoing PCI slightly increased from 2011 to 2015. In comparison to recent Japanese and U.S. nationwide registry,6) prevalance of hypertension, dyslipidemia, and diabetes mellitus in patients undergoing PCI were relatively low in Korea (56.0% in Korea vs. 74.6% in Japan vs. 81.8% in the USA, 37.8% vs. 63.0% vs. 80.0%, and 33.1% vs. 43.3% vs. 35.8%, respectively). However, in-hospital mortality was relatively higher in Korea when compared to Japan and the USA (2.6% vs. 0.9% vs. 1.1%, respectively). Mean age and Editorial
+
+| Received: Feb 14, 2018 Accepted: Mar 5, 2018 Correspondence to Soon Jun Hong, MD, PhD   |
+|-----------------------------------------------------------------------------------------|
+
+Correspondence to Soon Jun Hong, MD, PhD Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, 73, Inchon-ro, Seongbuk-gu, Seoul 02841, Korea. E-mail: psyche94@gmail.com Copyright © 2018. The Korean Society of Cardiology This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ORCID iDs Han Saem Jeong https://orcid.org/0000-0002-6035-4332 Soon Jun Hong https://orcid.org/0000-0003-4832-6678 Conflict of Interest The authors have no financial conflicts of interest. Author Contributions Conceptualization: Hong SJ; Formal analysis: Hong SJ; Investigation: Hong SJ; Methodology: Hong SJ; Supervision: Jeong HS; Validation: Jeong HS; Writing - original draft: Jeong HS, Hong SJ; Writing - review & editing: Jeong HS. The contents of the report are the author's own views and do not necessarily reflect the views of the *Korean Circulation Journal*.
+
+proportion of male were comparable with U.S. cohort (64.4 vs. 64.8 years, 69.7% vs. 67.2%, 
+
+![1_image_0.png](1_image_0.png)
+
+respectively). The proportion of patients over 75 years was silmilar with the USA data.4) In UK 
+registry between 2007 and 2013, comorbities in patients undergoding PCI in UK were different from Korea,7) higher hypercholesterolemia (52.5%) and less diabetes mellitus (17.6%).
+
+There was a significant 10% increase in the numbers of PCI for angina from 2011–2012 to 2014–2015 in Korea. However, the number of PCI for AMI decreased marginally between 2011–2012 and 2014–2015. After peak volume in 2006 in the USA, PCI volume in patient with stable angina pectoris dramatically dropped in 2011 after COURGAGE trial.4) The PCI 
+volume for MI was either steady or showed an increase with time in the USA. In Korean data, the prevalence of risk profiles such as hypertension and diabetes in patients with AMI were steady or decreased slightly between 2011 and 2015. The number of PCI procedures in patients with angina pectoris increased between 2011 and 2015. Recent efforts for educating cardiovascular diseases in general population might have been correlated with the early need for PCI procedures in Korea. Moreover, technical medical advances led to perform PCI in those patients with multiple comorbidities with complex coronary morphology.8)
+Nonetheless, as the authors pointed out, the use of optimal medical therapy remained suboptimal in Korea. Greater acceptance in both physicians and patients to adopt optimal medical therapy and implementation of appropriateness criteria for PCI in physicians should be emphasized. Recommended pharmacological therapies such as antiplatelet agent, statin, angiotensin converting enzyme (ACE) inhibitors/angiotensin receptor blockers (ARBs), and beta blocker in patients with angina and AMI will improve future prognosis.9) Market share of drug-eluting stents (DESs) in Korea was about 93% throughout 2011 and 2015, in comparison to relatively higher use (about 25% in 2010) of bare-metal stents (BMSs) in the USA and UK data.4)7) Current practice pattern of mostly using DES for PCI in Korea reflected better angiographic and clinical outcomes of newer-generation DESs over BMS.10) The limitation of this study is that unstable angina was not well defined. However, this study provided a comprehensive national trend for PCI in Korea. It is clear from the worldwide available evidence that PCI in patients with either angina or AMI has improved quality of life and clinical outcomes. This study by Han et al.5) will provide evidence for achieving consensus on PCI in the Korean population with angina and AMI, and will provide fundamental references for future PCI trials in Korea.
+
+## References
+
+1. Anderson JL, Adams CD, Antman EM, et al. 2012 ACCF/AHA focused update incorporated into the ACCF/
+AHA 2007 guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. *J Am Coll Cardiol* 2013;61:e179-347. 
+
+PUBMED | CROSSREF
+2. Pursnani S, Korley F, Gopaul R, et al. Percutaneous coronary intervention versus optimal medical therapy in stable coronary artery disease: a systematic review and meta-analysis of randomized clinical trials. Circ Cardiovasc Interv 2012;5:476-90. 
+
+PUBMED | CROSSREF
+3. Fihn SD, Blankenship JC, Alexander KP, et al. 2014 ACC/AHA/AATS/PCNA/SCAI/STS focused update of the guideline for the diagnosis and management of patients with stable ischemic heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, and the American Association for Thoracic Surgery, Preventive Cardiovascular Nurses Association, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. *J Am Coll* Cardiol 2014;64:1929-49. 
+
+PUBMED | CROSSREF
+4. Bangalore S, Gupta N, Genereux P, Guo Y, Pancholy S, Feit F. Trend in percutaneous coronary 
+
+![2_image_0.png](2_image_0.png)
+
+intervention volume following the COURAGE and BARI-2D trials: insight from over 8.1 million percutaneous coronary interventions. *Int J Cardiol* 2015;183:6-10. 
+
+PUBMED | CROSSREF
+5. Han S, Park GM, Kim YG, et al. Trends, characteristics, and clinical outcomes of patients undergoing percutaneous coronary intervention in Korea between 2011 and 2015. *Korean Circ J* 2018;48:310-21. 
+
+CROSSREF
+6. Tsai TT, Patel UD, Chang TI, et al. Validated contemporary risk model of acute kidney injury in patients undergoing percutaneous coronary interventions: insights from the National Cardiovascular Data Registry Cath-PCI Registry. *J Am Heart Assoc* 2014;3:e001380. 
+
+PUBMED | CROSSREF
+7. O'Neill D, Nicholas O, Gale CP, et al. Total center percutaneous coronary intervention volume and 30-day mortality: a contemporary national cohort study of 427 467 elective, urgent, and emergency cases. *Circ* Cardiovasc Qual Outcomes 2017;10:e003186. 
+
+PUBMED | CROSSREF
+8. Singh M, Rihal CS, Gersh BJ, et al. Twenty-five-year trends in in-hospital and long-term outcome after percutaneous coronary intervention: a single-institution experience. *Circulation* 2007;115:2835-41. 
+
+PUBMED | CROSSREF
+9. Messerli FH, Mancia G, Conti CR, Pepine CJ. Guidelines on the management of stable angina pectoris: 
+executive summary: the task force on the management of stable angina pectoris of the European society of cardiology. *Eur Heart J* 2006;27:2902-3. 
+
+PUBMED | CROSSREF
+10. Bangalore S, Amoroso N, Fusaro M, Kumar S, Feit F. Outcomes with various drug-eluting or bare metal stents in patients with ST-segment-elevation myocardial infarction: a mixed treatment comparison analysis of trial level data from 34 068 patient-years of follow-up from randomized trials. Circ Cardiovasc Interv 2013;6:378-90. 
+
+PUBMED | CROSSREF

medical/md/PMC5895985.md

@@ -0,0 +1,189 @@
+
+## Evaluation Of The Prognostic Value Of Multimodal Intraoperative Monitoring In Posterior Fossa Surgery Patients With Cerebellopontine Angle Tumors
+
+Mohsen Dalvandi (1), Ali Nazemi Rafie (1), Alireza Kamali (2), Alireza Jamshidifard (3)
+(1) Department of Neurosurgery, Arak University of Medical Sciences, Arak; (2) Department of Anesthesiology, Arak University of Medical Sciences, Arak; (3) Department of Neurology, Arak University of Medical Sciences, Arak Iran.
+
+This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
+
+## Abstract
+
+The aim of this study was to compare the outcomes of four intraoperative monitoring approaches in order to compare their prognostic value in surgical complications of the facial nerve. This quasi-experimental study was conducted on 25 patients with Cerebellopontine Angle (CPA) tumors, who were hospitalized in the Valiasr Hospital, affiliated to the Arak University of Medical Sciences, Arak, Iran. The degree of the facial nerve paralysis was assessed based on the House-Brackmann (HB) facial nerve grading system. The Orbicularis oris and Oculi muscles were evaluated by electromyography (EMG) before and after the surgery. Monitoring the facial nerve was performed by the EMG, Facial Nerve Antidromic Potentials (FNAPs), Transcranial Electrical Stimulation (TES), and Brainstem Auditory Evoked Potentials (BAEPs) methods. The mean HB score was 0.36.6±5.83, indicating that the function of the facial muscle was abnormal in all the patients pre-surgery. The comparison of the four monitoring approaches six months after surgery showed that the predictive value of facial nerve TES was higher than the other approaches (P<0.005). Furthemroe, 4% of the patients showed normal function in the facial muscle after the surgery. In addition, 8%, 56%, and 32% of the patients had mild abnormalities, moderate to severe paralysis in the facial muscles, and severe facial nerve dysfunction, respectively. In summary, 36% of the patients showed a mild abnormalities in the follow-up period, and full recovery was observed in 28% of the cases. In conclusion, the transcranial motor evoked potential (TCMEP) is a useful method for monitoring the facial nerve during the surgery along with the continuous EMG recordings.
+
+Key Words: Multimodal IOM, CPA Tumor Resection, Facial Nerve, Acoustic nerve, Predictive Value Eur J Transl Myol 28 (1): 124-131, 2018 One of the main problems of the neurological surgery is the damage to the peripheral or central nerves. Cerebellopontine angle (CPA) tumors are the most common neoplasm in the posterior fossa, which can lead to unilateral hearing loss, speech impediments, disequilibrium, and other loss of motor control.1In some cases such as those with meningioma, the patient's life is seriously at risk; therefore, the therapeutic measures should be performed as soon as possible for these patients.2,3 There are some factors playing an important role in the treatment process of this tumor. These factors include age, tumor size, time of tumor diagnosis, cranial nerve condition before surgery, surgical route, the adhesion of the facial nerve sheath, and previous underlying diseases 4. Damage to the cranial nerve during tumor removal is inevitable in some cases, especially in the tumors bigger than 4 cm. Therefore, the facial nerve damage may be widespread, leading to practical problems and changing the emotional and social factors in the patient's future life.
+
+The use of the intraoperative neurophysiological monitoring (IOM) is a common approach to monitor the functional integrity of certain neural structures during the neurological surgery. This approach aims to pervent the damage caused by the manipulation of the nervous tissue,5,6 however, it is not very common in Iran compared to other countires.
+
+Monitoring the facial nerve during the removal of such tumors as vestibular schwannoma and other posterior fossa tumors can help not only reduce the post-surgery facial nerve damage, but also improve the patient's quality of life and decrease the further medical costs. Therefore, it is necessary to perform some studies assessing the exact criteria for monitoring and determining the respective applicable approaches. In this regard, no accurate method has been proposed for estimating the prognostic value of these approaches in facial nerve function after the CPA surgery. EMG is a well-known technique for monitoring the facial nerve during the CPA operation. This method is used for the registration of facial muscle responses to the electrical and mechanical stimulations.7 However, due to the excessive sensitivity of EMG technique in the administration of neuromuscular blockers, the application of additional methods is required. Antidromic facial nerve potentials (FNAPs), transcranial motor evoked potentials (TCMEPs), and brainstem auditory evoked potentials (BAEPs) are other methods for continuous monitoring of the facial nerve.8. All these methods are effective in the prevention of the damage during the surgery and the prediction of the patient's condition post-surgery. Despite the growing trend of IOM in the cervical spine surgery, there is limited data on the sensitivity, specificity, and predictive values of these techniques in detecting new neurological deficits. Although IOM has a long history, its application has only recently become widespread.9 Accordingly, the commercial IOM machine and its educational programs were recently developed in Iran. Although IOM has no impact on the pathology of the patients, it can affect the process of maintaining the anatomical integrity during the surgery. With this background in mind, the present study aimed to compare the functions of the four intraoperative monitoring approaches in order to compare their prognostic values in surgical complications of the facial nerve.
+
+## Materials And Methods Ethical Approval
+
+The present study was performed based the Codes of Medical Ethics released by the Ministry of Health and Medical Education, Iran and Declaration of Helsinki. In this study, all approaches were performed based on the international standard protocol. The stages and techniques of this study were clearly explained to the patients, and their informed consent was obtained. Moreover, they were assured about the confidentiality of their information and the possibility to leave the study at any stage of the project. In addition, no money was obtained from the patients until the sixth month of operation. Participants This quasi-experimental study was conducted on 32 patients with CPA tumor, who were hospitalized in the Valiasr hospital affiliated to the Arak University of Medical Sciences, Arak, Iran. Out of the 32 patients selected by the convenience sampling method, 25 cases were candidates for surgery. The exclusion criteria included suffering from congenital deafness, facial paralysis, diabetes mellitus, or addiction, and those undergoing radio or re-surgery six months after surgery.
+
+## Study Design
+
+The patients were selected based on the inclusion criteria to minimize the sampling error or bias risk. In the first step, the degree of the facial nerve paralysis was assessed based on the House-Brackmann (HB) facial nerve grading system. Additionally, the Orbicularis oris and Oculi muscles were evaluated by the EMG prior to the surgery. To determine the type and size of tumor, certain para-clinic tests, such as computed tomography scan and magnetic resonance imaging with or without contrast, were performed by neurologist colleagues. Subsequently, the patients were referred for the surgery. In accordance with the monitoring methods, the anesthetic technique was performed using the Los Angeles Anesthesia Friendliness Scale protocol (modified in 5/17/2004). The resection surgery was performed through the retrosigmoid and middle fossa approaches. The facial VII and acoustic VIII nerves were assessed before, during, and six months after the surgery. Due to the lack of data normality, the obtained data of each patient was paired with his/her data in the four stages. To minimize the bias risk of other confounding variables, the temperature of operating room and that of the rooms the patients were kept in before and after operation was fixed at 25°C. Furthermore, all the patients were operated by the same surgeon. During surgery, the antidromic sensory nerve action potentials of the facial nerve in the CPA were repeatedly recorded by the FNAPs method. The electrical stimulation was performed by corkscrew on the Cz, C3, and C4 regions through the transcranial electrical stimulation (TES) approach. The motor evoked potentials (MEP) of the Orbicularis Oris and Oculi muscles were recorded by the EMG. About four to six transcranial stimulations were performed (typically at the intervals of 2-4 ms) with an intensity of 300-500 mA in the pre-specified regions of the skull by the TES approach. The location of the electrodes during the intraoperative electromyographic monitoring of cranial nerves is shown in Table 1. The extended and freerunning EMG of the orbicularis oris and oculi muscles were also recorded during the surgery. The EMG activity in response to direct electrical stimulation of nerve and spontaneous activities were recorded using a monitoring device (Viasys/Nicolet, USA) containing 52 canals. The other invwstigations were performed pre- and postsurgery by the EMG and BAEP evaluation through electrodiagnostic 4-channel system. These devices were placed in the Laboratory of Clinical Neurophysiology at Medical School of Mashhad University of Medical 
+
+| Table 1. Location of the electrodes in intraoperative  electromyographic monitoring of cranial Cranial nerve Muscle V Masseter VII Buccalis, Mentalis  Frontalis, Zygomaticus, IX Soft palate X Vocal cord XI Trapezius   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Sciences, Mashhad, Iran, and neurosurgical operating rooms of Valiasr Hospital. The stimulation parameters and recording process were controlled by the computers connected to the device. Time and amplitude parameters were listed based on mSe and mV, respectively, recorded to two decimal places on the computer screen. Our devices were equipped with amplifier system and digital converter; therefore, they were automatically calibrated in each registration stage. Prior to surgery, pure tone and absolute threshold of hearing (ATH) were measured. 
+
+Subsequently, the speech discrimination score (SDS) was obtained, which was repeated three weeks and six months after the surgery. The SDS was standardized by Moslehi et al. in 2001.
+
+10. The BAEPs of both ears were recorded by stimulating the electrodes fixed in the patients' ears before, during, and after the surgery. The auditory status was monitored by the IOM device and the use of installed speakers of ear canal under surgery providing 31 stimulations per second, which was simultaneously recorded by the electrodes placed on the scalp from both ear canals. The electrodes were placed in both ears and vertex (Cz), and the filtration was adjusted within 1503,000 HZ. The mean of each recorded signal of BAEP 
+was 1,000-4,000 stimulations, which was repeated 
+
+| Table 2. Change of the facial nerve before and after surgery based on House-Brackmann facial nerve grading scale Cases Pre-surgery Post-surgery After 3 weeks After 6 weeks 1 0.875 1 1 2 0.625 0.75 0.875 3 0.75 0.5 1 4 0.625 0.125 1 5 0.375 0.75 0.625 6 0.75 0.125 1 7 0.125 0.25 0.875 8 0.125 0 0.875 9 0.25 0 0.75 10 0.125 0.25 0.875 11 0.25 0.25 0.875 12 0.5 0.875 1 13 0.125 0 0.875 14 0.25 0 0.75 15 0.25 0.125 0.875 16 0.125 0.25 0.625 17 0.5 0.25 0.875 18 0.375 0.875 1 19 0.875 0.5 1 20 0.75 0.5 1 21 0.25 0 0.75 22 0.25 0 0.625 23 0.375 0.25 0.875 24 0.25 0 0.75 25 0.0 0 0.75   |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+almost every 2 min. The presence of signal and wave V 
+latency were reported. The changes of V waveform and the lack or presence of wave V latency (more than 1 m) 
+were reported to the surgeon. As a result, the surgeon was recommended to change the position of the retractor on the cerebellum and reduce its stretch as long as the change in signal be compensated. This lasted for a few seconds, and the signal appeared again. In the BAEP technique, more than a millisecond delay for the wave V 
+latency was considered significant. Moreover, the audiometry, pure-tone average, and SDS were evaluated and recorded for each patient before and after the surgery by an audiologist. Based on the HB scale, the degree of the facial nerve paralysis of the Orbicularis and Oculi muscles was evaluated through the EMG in the third week and sixth month of the surgery. All data were registered in the medical records of the patients, and then analyzed in the EXCEL and SPSS software using the data regression tests.
+
+## Results
+
+About 76% of the patients were female. The mean age of the participants was 50.05±10.81 years. Out of the 25 subjects, 23 patients were diagnosed with vestibular schwannoma, 1 with neurofibromatosis, and 1 with recurrent tumor. Furthermore, meningioma was observed in two males. The mean tumor size was 36.64±5.83 mm, ranging within 24-45 mm. About 76% and 24% of the tumors were removed through the retro-sigmoid and middle cranial fossa approaches, respectively. The change of the facial nerve paralysis degree before and after the surgery based on the HB is shown in Table 2. The mean HB score was 0.36.6±5.83, therefore, the function of the facial muscle was abnormal in all the patients prior to the surgery. Before the surgery, the mean values of ATH and SDS were 43.58±15.97 dB and 38.08± 10.43 in the affected side, respectively. Six months after the surgery, the mean ATH was 10.49±31.76 dB, which was increased in the affected side of eight patients and decreased in seventeen patients. Furthermore, the mean of SDS was 59.92±13.96 after six months, showing an increase in the affected side of the patients. The comparison of ATH scores showed that there was a significant difference in the hearing quality before and after the operation (P<0.005). Likewise, the SDS was significantly different before and after the surgery (P<0.005). However, the ATH and SDS scores showed no correlation with the HB score before and after the operation (P>0.005). The comparison of the facial nerve paralysis degree of the patients six months after the surgery demonstrated no significant relationship between the EMG findings and spikes recorded in the free-running EMG. Furthermore, there was no significant relationship between the facial status and latency changes of BAEP wave and the presence or lack of wave V. The changes of audiology test results in both ears before and after the surgery are shown in Table 3. There was a weak correlation between the HB score and amplitude response of FNAP during the surgery before removing the tumor 
+(r=0.67, P=0.005). The evaluation of the reflexes and evoked responses of Orbicularis Oris and Oculi after the operation and six months after tumor removal revealed a direct weak correlation between the HB score and amplitude response of FNAP (r=0.59, P=0.005). 
+
+Additionally, a positive correlation was observed before removing the tumor and six months after the operation between the HB score and TES amplitude response of facial muscles (r=0.78, P=0.005). Similarly, a high direct correlation was observed between the HB score and TES amplitude response of facial muscles in the affected side and normal side of each patient after tumor removal 
+(r=0.807, P=0.005). 
+
+The positive prognostic values of the four monitoring approaches were measured using the following formula: The predictive values of anti-dromic facial nerve 
+
+![3_image_0.png](3_image_0.png) stimulation, facial nerve TES, free-running EMG, and BAER were 36.3, 0.87, 0.43, and 0.6, respectively. The comparison of these four approaches six months after the surgery showed that the predictive value of the facial nerve TES was higher than those of the other approaches. In the patients with the HB score of one, a low incidence of facial nerve involvement was reported postsurgery.However, in some cases, the recovery of facial nerve function was observed immediately after the surgery. In addition, all the patients were found to have such recovery six months after the operation, rendering the mean HB of 0.86±0.12 post-surgery. Following the surgery and getting discharged from the hospital, 4% of the patients were observed to have normal function in the facial muscle. In addition, 8%, 56%, and 32% of the patients had mild abnormalities, moderate to severe paralysis in the facial muscles, and severe facial nerve dysfunction, respectively. In summary, 36% of the patients showed mild abnormalities in the follow-up, and full recovery was observed in 28% of the cases. 
+
+Furthermore, two patients one of whom suffering from Ependymoma and the other one having a history of Cerebral Vascular Accident passed away during the follow-up due to bleeding in the brain stem and spontaneous bleeding unrelated to the surgery on the opposite side (in the supratentorial), respectively.
+
+## Discussion
+
+As the findings of our study indicate, the transcranial stimulation of the facial nerve is a useful technique to monitor the facial nerve performance during CPA operation. Other similar studies have also confirmed this finding.4,11-13 Our data showed that the hearing quality
+
+| Table 3. Changes of audiology test results in both ears before and after the surgery  Cases Pre-surgery Six months post-surgery PTA audiometry (dB) SDS (%) PTA audiometry (dB)   | SDS (%)   |        |          |        |          |        |      |    |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|--------|----------|--------|----------|--------|------|----|
+| Normal                                                                                                                                                                            | Affected  | Normal | Affected | Normal | Affected | Normal |      |    |
+| side                                                                                                                                                                              | side      | side   | side     | side   | side     | side   | side |    |
+| 1                                                                                                                                                                                 | 8.16      | 26.28  | 64       | 52     | 8        | 43     | 64   | 56 |
+| 2                                                                                                                                                                                 | 12.64     | 22.45  | 72       | 64     | 12.21    | 54     | 72   | 72 |
+| 3                                                                                                                                                                                 | 10.58     | 27.34  | 66       | 42     | 10.32    | 40     | 66   | 54 |
+| 4                                                                                                                                                                                 | 5.9       | 24.64  | 60       | 46     | 6        | 37     | 60   | 54 |
+| 5                                                                                                                                                                                 | 8.6       | 38.46  | 52       | 32     | 8        | 62     | 52   | 44 |
+| 6                                                                                                                                                                                 | 11.5      | 38.82  | 68       | 48     | 10       | 35     | 68   | 56 |
+| 7                                                                                                                                                                                 | 5.7       | 36.80  | 76       | 44     | 5.3      | 30     | 76   | 56 |
+| 8                                                                                                                                                                                 | 9.7       | 41.34  | 68       | 32     | 9.5      | 22     | 68   | 48 |
+| 9                                                                                                                                                                                 | 9.4       | 46.62  | 42       | 30     | 9.7      | 26     | 42   | 46 |
+| 10                                                                                                                                                                                | 12.3      | 40.86  | 54       | 38     | 12.5     | 30     | 54   | 50 |
+| 11                                                                                                                                                                                | 7.4       | 48.22  | 44       | 28     | 7        | 20     | 44   | 40 |
+| 12                                                                                                                                                                                | 6.6       | 58.36  | 54       | 36     | 6.2      | 32     | 54   | 52 |
+| 13                                                                                                                                                                                | 7.9       | 47.93  | 42       | 22     | 8        | 18     | 42   | 44 |
+| 14                                                                                                                                                                                | 14.3      | 52.65  | 56       | 30     | 14.5     | 30     | 56   | 40 |
+| 15                                                                                                                                                                                | 5.9       | 70.62  | 60       | 34     | 6        | 34     | 60   | 54 |
+| 16                                                                                                                                                                                | 10.4      | 48.38  | 64       | 36     | 10       | 32     | 64   | 48 |
+| 17                                                                                                                                                                                | 11.7      | 39.18  | 70       | 40     | 12       | 36     | 70   | 56 |
+| 18                                                                                                                                                                                | 9.7       | 30.41  | 78       | 48     | 9.5      | 36     | 78   | 64 |
+| 19                                                                                                                                                                                | 8.5       | 20.36  | 86       | 56     | 8.5      | 35     | 86   | 64 |
+| 20                                                                                                                                                                                | 6.8       | 18.38  | 88       | 48     | 7        | 26     | 88   | 80 |
+| 21                                                                                                                                                                                | 7.8       | 68.92  | 46       | 32     | 8        | 24     | 46   | 50 |
+| 22                                                                                                                                                                                | 11.6      | 66.38  | 36       | 28     | 11       | 30     | 36   | 36 |
+| 23                                                                                                                                                                                | 13.4      | 70.36  | 54       | 32     | 13       | 20     | 54   | 40 |
+| 24                                                                                                                                                                                | 11.9      | 44.86  | 56       | 28     | 12       | 24     | 56   | 52 |
+| 25                                                                                                                                                                                | 8.5       | 60.88  | 42       | 26     | 8.2      | 18     | 42   | 40 |
+| Affected                                                                                                                                                                          |           |        |          |        |          |        |      |    |
+| PTA: pure-tone average, SDS: speech discrimination score                                                                                                                          |           |        |          |        |          |        |      |    |
+
+and speech discrimination were improved six months after the operation. Furthemore, a high direct correlation was observed between the HB score and TES amplitude response of facial muscles in the affected side and normal side of the patients after tumor removal. In our study, the TCMEP was performed to measure the optimal stimulation parameters of the facial nerve by using multipulse stimulation of 3-5-pulse trains, duration of 0.2-0.5 ms, and interstimulus interval of 4 ms as performed in the pervious studies.4, 11-13. The comparison of the affected and normal sides of facial nerve gives an optimal estimation from the nerve clinical practice. In our study, the highest electromyographic responses between the two sides of facial nerve were observed when comparing the TES responses of facial muscles. Therefore, the post-surgery stimulation of motor cortex of facial nerve results in improved state of descending motor pathways. The TCMEP findings had the highest correlation with the six-month follow-up results than the other approaches. Regarding this, the TCMEP can be introduced as a successful intraoperative neural monitoring in case it is applied by an experienced neuroanesthesiology team. Moreover, the TCMEP data are useful in distinguishing the subjects with initial paresis/paralysis as well as the recovery of their facial function in the long-term. Cosetti et al. evaluated the efficiency of the TCMEP in monitoring the facial nerve. To this aim, they performed TES technique on 52 patients under the CPA surgery. Similar to our study, the facial status was predicted immediately after the surgery using the HP scale and the changes of electromyographic responses of stimulated muscle before and after the tumor removal. The obtained results of the mentioned study showed that the changes of the electromyographic responses of the facial nerve immediately after the surgery should not be reduced to more than 57%, 
+compared to that of the pre-surgery. Therefore, they concluded that more than 57% changes in the facial nerve may cause problems in the long-term.7 The weakness of the TCMEP approach is its intermittent feature since this approach is performed every 10 min; therefore, the use of the EMG is constantly suggested. The EMG can present a feedback from the environmental damages, which have shown to have a significant correlation with surgery outcomes in terms of the facial nerve.14 The FNMEP is a standard approach confirmed by the National Institutes of Health, USA, as a proper technique to avoid damaging the facial nerve and facilitate long-term prediction of its performance. In our study, this method was used to predict the facial nerve status after the operation. In a study conducted by Fukuda et al., the FNMEP was used, and the corkscrew was installed on the C3, C4, and Cz regions. The motor responses of the orbicularis oris and oculi were recorded, which were caused by180-550 V supramaximal stimulations. In the mentioned study, there was a correlation between the post-surgery facial nerve function and FNMEP ratios. Accordingly, in case the FNMEP ratio remained > 50%, the patient had a satisfactory facial nerve function.13 Our findings demonstrated that the intraoperative FNMEP monitoring could be useful for predicting the facial nerve function post-surgery. This new method is a valuable adjunct to the conventional facial nerve monitoring techniques. Totally, intraoperative monitoring is an indispensible part of the acoustic neuroma surgery that leads to significant improvements in the functional outcomes of CPA surgery and prevents the facial paralysis.15 According to our results, severe facial nerve dysfunction was observed immediately after the surgery in about one third of the patients. However, the facial function was fully recovered in about one third of cases in the followup period; accordingly, these patients obtained HB grade I or II at the follow-up evaluation. Similar to our study, in the study carrieod out by Cosetti et al, complete paralysis and severe facial nerve dysfunction were observed in 32% of the patients immediately after the operation. Furthemore, the facial function was recovered in more than one fifth of the patients.7 Temporary motor nerve conduction block, neuropraxia or axonotmesis, edema, vasoconstriction, and herpes virus reactivation are introduced as the main causes of immediate paresis or paralysis in the patients with CPA tumor; however, the etiology of this problem have not fully discovered yet. Some of these causes (e.g., conduction block) may rapidly improve; nevertheless, in some cases, the paresis or paralysis may persist for months and even, as can be seen in case of neuropraxia or axonometsis, the patient may not achieve the pre-operative function of facial nerve.16 Immediately after surgery, the occurance of facial nerve paralysis or enhancement of HB score is possible; however, the causes are not clear. These conditions may be due to neurapraxia and temporary conduction block of the facial nerve or perioperative trauma, which are usually recovered a day or two after the surgery. Nonetheless, neurapraxia and axonotmesis injuries may continue for a few months.9 Since the CPA 
+surgery may be associated with main communication problems and ocular complications, the recognition of the patients at risk is very important due to the necessity of the prevention of such complications in these patients. 
+
+The relationship between TCMEP, HB grading, and other variables, such as age, tumor size, diagnosis time, and preoperative symptoms, are not clear yet. In our study, the investigation of the association between the TCMEP and other approaches showed a positive correlation between the HB score and amplitude responses of FNAP and TES. The predictive value of these variables in terms of the facial nerve are confirmed by similar studies16,17 The outcome of the patients with CPA is largely affected by the rate of tumor adhesion to the facial nerve sheath and facial nerve, which was not possible to check in this study. However, the relationship between these variables was assessed in our study. The data on facial nerve function cannot be achieved through continuous EMG or direct electrical stimulation. Nonetheless, the presence of a stimulating probe is necessary to gain information during the surgery, which can be transferred by the TCMEP monitoring. In the present study, the BAEP approach resulted in the improvement of the hearing quality and speech discrimination six months after the operation. This finding was also confirmed in a study conducted by Yamakami et al.18 However, in a study carried out by Rampp et al., the auditory steady-state response was suggested as a more effective tool in the assessment of the hearing quality and speech discrimination.19. One of the advantages of BAEP approach is the low probability of the anesthetics effectiveness on the BAEP responses. 
+
+As the findings of this study revealed, the TCMEP was a useful method for monitoring the facial nerve during the surgery, which could predict the facial nerve complications along with continuous EMG. Therefore, this method cannot be considered as a complete technique for monitoring the facial nerve during the CPA surgery since it should be simultaneously used along with other monitoring approaches. However, the TCMEP 
+approach is more favorable for monitoring the facial nerve in the posterior fossa surgery.
+
+## List Of Acronyms
+
+CPA: Cerebellopontine Angle HB: House-Brackmann EMG: Electromyography FNAPs: Facial Nerve Antidromic Potentials TES: Transcranial Electrical Stimulation BAEPs: Brainstem Auditory Evoked Potentials TCMEP: Transcranial motor evoked potential ATH: Absolute threshold of hearing SDS: Speech discrimination score IOM: Intraoperative neurophysiological monitoring MEP: The motor evoked potentials
+
+## Author'S Contributions
+
+Each author contributed in equal part to the manuscript.
+
+## Acknowledgments And Funding
+
+Hereby, we extend our gratitude to the Deputy of Research and Technology as well as all the members and experts of the Research Council of Mashhad University of Medical Sciences for approving the project proposal. We also wish to appreciate all the patients participating in this study. Other thanks go to the personnel of operating room and anesthesiology department of the hospital. Additionally, we would like to thank Dr. A.R. Ashtiani, from department of Neurology for his cooperation. Funding: None.
+
+## Conflict Of Interest
+
+The authors declare no conflicts of interests derived from the outcomes of this stud.
+
+## Ethical Publication Statement
+
+We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
+
+## Corresponding Author
+
+Ali Nazemi Rafie, Department of Neurosurgery, Arak University of Medical Sciences, Arak, Iran. Email: nazemirafie.ali@gmail.com E-mails of co-authors Mohsen Dalvandi: Mdalvandi@yahoo.com Alireza Kamali: Alikamaliir@yahoo.com Alireza Jamshidifard: arjfard@yahoo.com
+
+## References
+
+1. Son DW, Choi CH, Cha SH. Epidermoid tumors in the cerebellopontine angle presenting with trigeminal neuralgia. J Korean Neurosurg Soc 2010;47:271-7. doi: 10.3340/jkns.2010.47.4.271. Epub 2010 Apr 30.
+
+2. Nakamura M. Facial and cochlear nerve function after surgery of cerebellopontine angle meningiomas. Samii's Essentials in Neurosurgery, Springer 2014:321-32.
+
+3. House JW, Brackmann DE. Facial nerve grading system. Otolaryngol Head Neck Surg.1985;93(2):146-7.
+
+4. Acioly MA, Liebsch M, Carvalho CH, et al. 
+
+Transcranial electrocortical stimulation to monitor the facial nerve motor function during cerebellopontine angle surgery. Neurosurgery 2010;66:354-62.
+
+5. Fehlings MG, Brodke DS, Norvell DC, Dettori JR. 
+
+The evidence for intraoperative neurophysiological monitoring in spine surgery: does it make a difference? Spine 2010;35:S37-S46.
+
+6. Lall RR, Lall RR, Hauptman JS, et al. 
+
+Intraoperative neurophysiological monitoring in spine surgery: indications, efficacy, and role of the preoperative checklist. Neurosurgical focus 2012;33:E10.
+
+7. Cosetti MK, Xu M, Rivera A, et al. Intraoperative transcranial motor-evoked potential monitoring of the facial nerve during cerebellopontine angle tumor resection. J Neurol Surg B Skull Base. 2012;73:308-15. doi: 10.1055/s-0032-1321507.
+
+8. Galloway GM, Nuwer MR, Lopez JR, Zamel KM. 
+
+Intraoperative neurophysiologic monitoring: Cambridge University Press, 2010.
+
+9. Kim S-M, Kim SH, Seo DW, Lee KW. 
+
+Intraoperative neurophysiologic monitoring: basic principles and recent update. J Korean Med Sci 2013;28:1261-9. doi: 10.3346/jkms.2013. 28.9.1261. Epub 2013 Aug 28.
+
+10 Mosleh M. Development and evaluation of a speech recognition test for Persian speaking adults. Bimonthly Audiology - Tehran University of Medical Sciences. 2001;9:72-6.
+
+11. Akagami R, Dong CC, Westerberg BD. Localized transcranial electrical motor evoked potentials for monitoring cranial nerves in cranial base surgery. 
+
+Neurosurgery. 2005;57:78-85. discussion 78-85.
+
+12. Dong CC, MacDonald DB, Akagami R, et al. 
+
+Intraoperative facial motor evoked potential monitoring with transcranial electrical stimulation during skull base surgery. Clin neurophysiol 2005;116:588-96.
+
+13. Fukuda M, Oishi M, Takao T, et al. Facial nerve motor-evoked potential monitoring during skull base surgery predicts facial nerve outcome. J Neurol Neurosurg Psychiatry 2008;79:1066-70. doi: 10.1136/jnnp.2007. 130500. Epub 2008 Feb 1.
+
+14. Prell J, Rachinger J, Scheller C, et al. A Real‐Time Monitoring System for the Facial Nerve. Neurosurgery 2010;66:1064-73.
+
+15. Youssef AS, Downes AE. Intraoperative neurophysiological monitoring in vestibular schwannoma surgery: advances and clinical implications. Neurosurgical focus 2009;27(4):E9.
+
+16 Bernat I, Grayeli AB, Esquia G, et al. Intraoperative electromyography and surgical observations as predictive factors of facial nerve outcome in vestibular schwannoma surgery. Otol Neurotol 2010;31:306-12. doi: 10.1097/MAO. 0b013e3181be6228.
+
+17. Esquia-Medina GN, Grayeli AB, Ferrary E, et al. 
+
+Do facial nerve displacement pattern and tumor adhesion influence the facial nerve outcome in vestibular schwannoma surgery? Audiol Neurootol 2013;18:406-13. doi: 10.1159/000 354115. Epub 2013 Oct 23.
+
+18. Yamakami I, Ushikubo O, Uchino Y, et al. 
+
+Intraoperative monitoring of hearing function in the removal of cerebellopontine angle tumor: auditory brainstem response and cochlear nerve compound action potential. No shinkei geka Neurological surgery 2002;30:275-82.
+
+19. Rampp S, Rensch L, Simmermacher S, et al. 
+
+Intraoperative auditory steady-state monitoring during surgery in the cerebellopontine angle for estimation of postoperative hearing classes. J Neurosurg 2017;127:559-568. doi: 
+10.3171/2016.7.JNS16460. Epub 2016 Oct 14.
+
+Received for publication: January 25, 2018 Revision received: February 5, 2018 Accepted for publication: February 7, 2018 

medical/md/PMC5922562.md

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+# Risk Prediction In Stable Cardiovascular Disease Using A High-Sensitivity Cardiac Troponin T Single Biomarker Strategy Compared To The **Esc-Score**
+
+Moritz Biener,1 Evangelos Giannitsis,1 Manuel Kuhner,1 Thomas Zelniker,1 Matthias Mueller-Hennessen,1 Mehrshad Vafaie,1 Kiril M Stoyanov,2 Franz-Josef Neumann,2 Hugo A Katus,1 Willibald Hochholzer,2 Christian Marc Valina2
+► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ openhrt-2017-000710).
+
+To cite: Biener M, Giannitsis E, 
+Kuhner M*, et al*. Risk prediction in stable cardiovascular disease using a high-sensitivity cardiac troponin T single biomarker strategy compared to the ESCSCORE*. Open Heart* 2018;5:e000710. doi:10.1136/ openhrt-2017-000710 Received 2 September 2017 Revised 25 November 2017 Accepted 27 March 2018 1Zentrum für Innere Medizin, Klinik für Kardiologie, Angiologie und Pneumologie, Universitätsklinikum Heidelberg, Heidelberg, Germany 2Klinik für Kardiologie und Angiologie II, UniversitätsHerzzentrum Freiburg Bad Krozingen, Bad Krozingen, Germany Correspondence to Dr Evangelos Giannitsis; evangelos_giannitsis@med.uniheidelberg.de Abstract Objective To evaluate the prognostic performance of high-sensitivity cardiac troponin T (hs-cTnT) compared with the ESC-SCORE. Methods We included low-risk outpatients with stable cardiovascular (CV) disease categorised into need for nonsecondary and secondary prevention. The prognostication of hs-cTnT at index visit was compared with the European Society of Cardiology-Systematic COronary Risk Evaluation (ESC-SCORE) with respect to all-cause mortality (ACM) and two composite endpoints (ACM, acute myocardial infarction (AMI) and stroke and ACM, AMI, stroke and rehospitalisation for acute coronary syndrome (ACS) and decompensated heart failure (DHF)).
+
+Results Within a median follow-up of 796 days, a total of 16 deaths, 32 composite endpoints of ACM, AMI and stroke and 83 composite endpoints of ACM, AMI, stroke, rehospitalisation for ACS and DHF were observed among 693 stable low-risk outpatients. Using C-statistics, measurement of hs-cTnT alone outperformed the ESCSCORE for the prediction of ACM in the entire study population (Δarea under the curve (AUC) 0.221, p=0.0039) 
+and both prevention groups (non-secondary: ΔAUC 0.164, p=0.0208; secondary: ΔAUC 0.264, p=0.0134). For the prediction of all other secondary endpoints, hs-cTnT was at least as effective as the ESC-SCORE, both in secondary and non-secondary prevention. Using continuous and categorical net reclassification improvement and integrated discrimination improvement, hs-cTnT significantly improved reclassification regarding all endpoints in the entire population and in the secondary prevention cohort. In non-secondary prevention, hs-cTnT improved reclassification only for ACM. The results were confirmed in an independent external cohort on 2046 patients. Conclusions Hs-cTnT is superior to the multivariable ESC-SCORE for the prediction of ACM and a composite endpoint in stable outpatients with and without relevant CV disease. Trial registration number NCT01954303; Pre-results.
+
+Introduction Cardiac troponins are recommended as the preferred biomarkers for the diagnosis of 
+
+## Key Questions
+
+What is already known about this subject?
+
+► Elevated values of high sensitivity troponins are indicative of adverse outcome irrespective of the underlying reason.
+
+What does this study add?
+
+► This study compares for the first time the prognostic value of high sensitivity cardiac troponin T in relation to the European Society of CardiologySystematic COronary Risk Evaluation.
+
+How might this impact on clinical practice?
+
+► Multivariable risk scores are rarely used in daily clinical practice due to inconvenience.
+
+► In contrast, high sensitivity troponins are widely available and provide an easy-to-use risk stratification tool.
+
+► They might therefore facilitate risk stratification with a better acceptance in daily clinical practice.
+
+acute myocardial infarction (AMI1). After more sensitive and highly sensitive troponin assays replaced conventional cardiac troponin assays almost 10 years ago, AMI 
+could be diagnosed earlier with a more accurate discrimination between unstable angina 
+(UA) and non-ST elevation myocardial infarction.2 3 Furthermore, a higher prevalence of elevated troponins due to myocardial injury for non-coronary and non-cardiac reasons is observed.4 5 Irrespective of the underlying release mechanism, elevated troponin levels were found to be highly predictive for cardiovascular (CV) endpoints including mortality.6–8 A high predictive value of cardiac troponin has also been confirmed in stable coronary artery disease in large trials like the Prevention of Events with ACE 
+Inhibition (PEACE)9 or the Heart Outcomes Prevention Evaluation (HOPE) study.10 While several multivariable clinical risk scores have been prospectively validated for the risk stratification of patients with an acute coronary syndrome (ACS),11 12 there is no clinical risk score for individualising secondary prevention in patients with coronary artery disease (CAD), CV disease, peripheral artery disease (PAD) or with a history of remote AMI. In contrast, several risk scores have been developed and validated for risk assessment in primary prevention such as the Framingham Score13 and the European Society of Cardiology-Systematic COronary Risk Evaluation (ESC-SCORE)14 which are recommended by European guidelines.15 The ESC-SCORE has been validated in a large cohort of patients, ethnicities and for the endpoint CV mortality.14 Unfortunately, these multivariable scores are rarely used in clinical routine.16 17 Cardiac troponins are widely available in primary care and in hospitalised patients, provide plausible biological information on underlying pathophysiology, are established in the diagnosis of ACS,1 improve risk assessment in ACS,18 atrial fibrillation19 and pulmonary embolism20 and may guide therapeutic management in several acute and chronic diseases. It is tempting to speculate that a single biomarker-based risk prediction tool may be better adopted for prognostication than a complex multivariate score. Therefore, the aim of this study was to compare the performance of high-sensitivity cardiac troponin T 
+(hs-cTnT) alone against the European guideline recommended ESC-SCORE in a stable outpatient population presenting with or without relevant CV disease, that is, patients who require secondary prevention measures or not.
+
+## Methods Study Population
+
+We screened 965 individuals presenting to the outpatient unit of Hugo A Katus at the Department of Cardiology of the University Hospital of Heidelberg between June 2009 and June 2010. These patients were either healthy (almost healthy with minor manifestation of CV 
+disease) or asymptomatic with or without stable symptoms or signs of CV disease. Patients without obvious CV 
+disease, those with chronic arrhythmias like atrial fibrillation, pacemaker follow-up visits or arterial hypertension were summarised into the non-secondary prevention group, whereas patients with a history of documented AMI, previous percutaneous coronary intervention (PCI), previous coronary artery bypass graft 
+(CABG), angiographically confirmed CAD (defined as >50% luminal obstruction), asymptomatic or symptomatic peripheral arterial disease (PAD), previous transient ischemic attack (TIA) or stroke were categorised into secondary prevention. Individuals who were potentially belonging to both groups were classified as secondary prevention. Patients received routinely ECG, carotid scan, blood testing and stress tests. The latter included exercise ECG, stress echocardiography and stress MRI. Imaging tests were performed at the discretion of the attending cardiologist including coronary CT, cardiac MRI, pulmonary function tests and Ankle-Brachial-Index measurements.
+
+All patients with acute manifestations of CV disease such as recent ACS or acute heart failure were excluded to avoid a confounding effect of acute hs-cTnT elevation.
+
+The diagnostic workup comprised a 12-lead-ECG, 
+stress testing (ECG, echocardiography or MRI), carotid ultrasonography, CT coronary angiography, cardiac MRI, pulmonary function testing as well as Holter ECG 
+and 24hours blood pressure recording at the discretion of the treating physician. Furthermore, laboratory testing including hs-cTnT, blood count, clinical chemistry and coagulation tests were performed. A detailed description of the study population is available elsewhere.21 Left ventricular (LV) function was determined via echocardiography and categorised according to the ejection fraction (EF) into normal (≥55%), mildly reduced (45%– 54%), moderately reduced (30%–44%) and severely reduced (<30%). If available, MRI was used to determine LV function. Pulmonary artery pressure was measured by echocardiography or right heart catheterisation. Aortic valve stenosis was quantified by two-dimensional echocardiography or heart catheterisation.
+
+All patients of the original cohort were seen by the same physicians (HAK and EG) and treated according to the current guidelines. Inclusion criteria were a stable clinical course, a documented hs-cTnT value on index visit and at least one follow-up visit. Stable disease was defined by the absence of recent ACS, coronary intervention or acute cardiac decompensation since the previous visit. Patients with a history of heart transplantation were excluded.
+
+Follow-up was accomplished by hospital records, questionnaires, phone calls and death certificates. All scheduled and unscheduled visits or hospital admissions were recorded during follow-up. Due to the retrospective character of this analysis, informed consent was not possible and had not to be obtained according to a statement of the ethics committee of the University of Heidelberg.
+
+## Excelsior Cohort
+
+The main findings of the original study cohort were tested in an independent cohort of 2046 stable patients of the Effect of Clopidogrel Loading and Risk of PCI (EXCELSIOR) trial. A detailed description of the methodology and patient characteristics have been published by Hochholzer *et al*.
+
+22 In this study, subjects were recruited from consecutive patients referred for an elective invasive coronary angiography due to suspected stable CAD. Individuals were then categorised into a secondary and non-secondary prevention category according to the presence or absence of significant coronary artery stenosis >50% (see online supplementary file 1).
+
+## Outcome Measures
+
+Three prognostic endpoints were defined: (1) all-cause mortality (ACM); (2) a composite of ACM, AMI and stroke and (3) a composite of ACM, AMI, stroke and rehospitalisation for ACS and decompensated heart failure (DHF). 
+
+ACM was defined as death from any cause including CV 
+and non-CV conditions. AMI was defined according to the European Society of Cardiology third universal definition of myocardial infaction1 including ST-segment elevation and non-ST-segment elevation myocardial infarction. Stroke was defined according to the Updated definition of stroke for the 21st century of the American Heart Association/American Stroke Association.23 ACS 
+was defined according to the European Society of Cardiology Guidelines on the management of ACSs in patients presenting without persistent ST-segment elevation24 and the European Society of Cardiology Guidelines for the management of AMI in patients presenting with ST-segment elevation.25 DHF was defined by presentation to the emergency department with symptoms of heart failure including angina or dyspnoea requiring diuretic therapy.
+
+## Laboratory Measurements
+
+Only troponin measurements during routine presentations were considered. Hs-cTnT concentrations were tested immediately from fresh blood samples. Cardiac troponin was measured in serum on a COBAS E411 using the hs-cTnT assay by Roche Diagnostics. The limit of blank and limit of detection have been determined to be 3ng/L and 5ng/L.26 The 10% CV was determined at 13ng/L in 100 measurements in the authors' laboratory. The interassay CV was 8% at 10ng/L and 2.5% at 100ng/L. The intra-assay CV was 5% at 10ng/L and 1% at 100ng/L. The hs-cTnT assays were not affected by a lot to lot variation that occurred in 2009 and 2010.27
+
+## Esc-Score
+
+The CV risk was calculated for every patient according to the original publication of the ESC-SCORE14 considering the risk variables at index visit. Individual risk was calculated according to the ESC-SCORE charts expressed by the 10-year risk for a fatal CV event. The recalibrated low risk chart was used in this population as recommended by the guidelines.15
+
+## Statistical Analysis
+
+Due to the pilot character of this observational study with a prespecified recruitment period of 12 months that was defined by the implementation of hs-cTnT as the routine laboratory troponin assay at the University of Heidelberg, a sample size calculation was not performed.
+
+Variables were tested for normal distribution using the D'Agostino-Pearson test and were presented either as means±SDdeviation, or as medians with 25th and 75th percentiles. Discrete variables were compared using Χ2 or Fisher's exact test. Continuous variables were compared using either Student's t-test for parametric or MannWhitney U test for non-parametric variables. Alternatively, we used analysis of variance (ANOVA) after logarithmic transformation of the data. If the ANOVA test was found positive (p<0.05), then the Student-Newman-Keuls test for pairwise comparison of subgroups was applied.
+
+We defined three endpoints to assess the prognostic performance: (1) ACM, (2) a composite endpoint of ACM, recurrent AMI and stroke and (3) a composite endpoint of ACM, recurrent AMI, stroke and hospitalisation for ACS or DHF.
+
+The prognostic performance of the ESC-SCORE and hs-cTnT for the defined endpoints was evaluated by C-statistics plotting and receiver operating characteristic (ROC) curves comparing the differences of the areas under the curve (AUC). The risk was determined by the 10-year risk for a fatal CV event as indicated by the ESC-SCORE charts and the hs-cTnT value on index visit. AUCs were compared using the test of DeLong *et al*.
+
+28 To determine the additional value of hs-cTnT in the prediction of the defined endpoints, we performed a logistic regression analysis and compared the prognostic performance of the combined variable (hs-cTnT+ESC-SCORE) with the performance of the ESC-SCORE in an ROC analysis.
+
+In order to evaluate benefits in risk prediction, reclassification analyses were performed to quantify improvements in model performance (in terms of correct reclassification to respective risk categories) after addition of hs-cTnT.29 Therefore, the categorical and the continuous net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) were calculated. Risk categories for the respective endpoint were set as 0%–10% for low risk, 10%–20% for intermediate risk and ≥20% for high risk according to the recommendations of the National Cholesterol Education Programme.30 Statistical analyses were performed with SPSS V.20.0, MedCalc V.11.1 (MedCalc software, Mariakerke, Belgium), R V.3.1.2 (R Core Team, Vienna, Austria), as well as the R package 'PredictABEL'.31 32 All tests were two-tailed and a p value of <0.05 was considered statistically significant.
+
+## Results Baseline Characteristics
+
+We screened 965 patients presenting to the outpatient clinic of HAK. A total of 63 subjects were excluded due to missing data (n=47) or an unstable clinical course 
+(n=16). Of the remaining 902 stable outpatients, 11 individuals were excluded due to a missing hs-cTnT value and 179 patients could not be included because of less than two visits to the outpatient clinic. Finally, 19 patients were not recruited since they had a history of heart transplantation (figure 1). The final study cohort comprised 693 patients, of which 333 were classified as non-secondary prevention and 360 as secondary prevention.
+
+Follow-up was available in 686 patients (98.9%) with a median time of 796 days (25th; 75th percentile: 631–935 days). A median of three (25th; 75th percentile: 2–4) 
+
+![3_image_0.png](3_image_0.png)
+
+scheduled ambulatory visits with a median of three (25th; 75th percentile: 2–4) hs-cTnT measurements were documented. hs-cTnT levels exceeding the 99th percentile 
+(ie, 14ng/L) on index visit were found in 146 patients (21.1%). A history of an ACS was documented in 27% of all patients. Seven out of 693 patients (1%) had an AMI within 6 months before index visit with a median time since last AMI of 62.6 months (95%CI 48.5 to 79.2) before index visit.
+
+The baseline characteristics split by cardiac troponin below or exceeding the 99th percentile are displayed in table 1. Most baseline characteristics differed significantly. Patients with values >14ng/L were older, were more likely to be male, had more often CV risk factors such as diabetes mellitus or arterial hypertension and were more often smokers. In addition, they had more often a history of CAD, AMI, CABG, cardiomyopathy, heart failure, arrhythmias, valvular heart disease, vascular and pulmonary diseases, renal failure and stroke/TIA. 
+
+Patients with hs-cTnT values exceeding the 99th percentile had a higher CV risk as indicated by the ESC-SCORE. 
+
+Most patients were seen because of a former ACS (25.4%, median time since last ACS: 62.5 months, 95%CI 28.9 to 160.9 months) or stable angiographically confirmed significant CAD with or without a history of elective coronary revascularisation (26.4%). Five patients (0.7%) were seen following a cerebrovascular event (TIA/stroke). Echocardiography (92.6%) and ECG (94.1%) were performed in almost every patient, and an ECG stress test (64.4%) was performed in the majority of patients.
+
+## General Outcome Characteristics
+
+In the overall cohort, we observed 16 deaths (ACM), 32 patients with ACM, AMI or stroke and 83 patients with ACM, AMI, stroke and rehospitalisation for ACS or DHF. Total mortality of the entire cohort was 2.03% during follow-up (796 days) corresponding to a total annual mortality rate of 0.93%.
+
+The prognostic performance of the ESC-SCORE and hs-cTnT in secondary and non-secondary prevention is displayed in table 2.
+
+The ESC-SCORE performed superior regarding the prediction of ACM in the non-secondary prevention group compared with the secondary prevention group (figure 2A). Regarding the composite endpoint of ACM, AMI and stroke, we also observed a better performance of the ESC-SCORE in non-secondary prevention compared with secondary prevention patients (figure 2B), whereas the prognostic performance for the prediction of the composite endpoint ACM, AMI, stroke, rehospitalisation for ACS or DHF was comparable between both groups (figure 2C).
+
+Hs-cTnT performed better in the prediction of ACM in the non-secondary prevention group compared with the secondary prevention group, whereas the prognostication of the composite endpoints of ACM, AMI and stroke and ACM, AMI, stroke, rehospitalisation for ACS or DHF was comparable.
+
+In the prediction of ACM, the hs-cTnT value on index visit (AUC 0.878, 95% CI 0.805 to 0.950) outperformed the ESC-SCORE (AUC 0.657, 95% CI 0.526 to 0.789) in the overall study population (ΔAUC 0.221, p=0.0039, figure 3A). Hs-cTnT was also superior to the ESC-SCORE in the prediction of ACM in non-secondary prevention (hs-cTnT: AUC 0.962, 95% CI 0.924 to 1.000; ESC-SCORE: AUC 0.798, 95% CI 0.664 to 0.931; ΔAUC 0.164, p=0.0208, figure 3B). In secondary prevention, with 0.264 (p=0.0134, figure 3C) the delta between hs-cTnT (AUC 0.836, 95% CI 0.734 to 0.937) and the ESC-SCORE (AUC 0.572, 95% CI 0.389 to 0.755) was even higher.
+
+Hs-cTnT and the ESC-SCORE had a comparable performance regarding the prediction of the composite endpoint ACM, AMI and stroke in secondary (ΔAUC 
+hs-cTnT vs ESC-SCORE: 0.110, p=NS, online supplementary figure 1) and non-secondary prevention (ΔAUC hs-cTnT vs ESC-SCORE: 0.119, p=NS, online supplementary figure 2).
+
+The prognostic performance of hs-cTnT to predict the composite endpoint ACM, AMI, stroke, rehospitalisation for ACS or DHF was higher in the secondary prevention group (ΔAUC hs-cTnT vs ESC-SCORE: 0.136, p=0.02), 
+
+| Coronary artery disease                                                                                                                                                                                                                   |                                                                   |                          |                          |              |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|--------------------------|--------------------------|--------------|
+| Table 1                                                                                                                                                                                                                                   | Baseline characteristics according to troponin status All (n=693) | Troponin ≤14ng/L (n=547) | Troponin >14ng/L (n=146) |              |
+| Presentations (quantity)                                                                                                                                                                                                                  | 3 (3–5)                                                           | 3 (3–5)                  | 4 (3–6)***               |              |
+|                                                                                                                                                                                                                                           | Outpatient clinic                                                 | 3 (2–4)                  | 3 (2–4)                  | 3 (2–4)      |
+| Admission to hospital                                                                                                                                                                                                                     | 0 (0–1)                                                           | 0 (0–1)                  | 0 (0–1)***               |              |
+| Admission to emergency department                                                                                                                                                                                                         | 0 (0–0)                                                           | 0 (0–0)                  | 0 (0–1)***               |              |
+| Age (years)                                                                                                                                                                                                                               | 66.3±12.3                                                         | 64.2±11.9                | 74.3±10.3***             |              |
+| Male gender, n (%)                                                                                                                                                                                                                        | 519 (74.9)                                                        | 391 (71.5)               | 128 (87.7)***            |              |
+| Risk factors, n (%) Diabetes mellitus                                                                                                                                                                                                     | 136 (19.7)                                                        | 92 (16.9)                | 44 (30.1)***             |              |
+| Dyslipidaemia                                                                                                                                                                                                                             | 580 (84.9)                                                        | 455 (84.4)               | 125 (86.8)               |              |
+| Hypertension                                                                                                                                                                                                                              | 574 (84.3)                                                        | 442 (82.3)               | 132 (91.7)**             |              |
+| Smoking                                                                                                                                                                                                                                   | 32 (13.1)                                                         | 4 (6.2)                  | 28 (15.6)***             |              |
+|                                                                                                                                                                                                                                           | Ex-smoker                                                         | 100 (41.0)               | 71 (39.7)                | 29 (44.6)    |
+| Family history of coronary artery disease                                                                                                                                                                                                 | 258 (52.9)                                                        | 224 (55.6)               | 34 (40.0)**              |              |
+| Medical history, n (%) Coronary artery disease                                                                                                                                                                                            | 420 (60.6)                                                        | 311 (56.9)               | 109 (74.7)***            |              |
+| Acute myocardial infarction                                                                                                                                                                                                               | 185 (26.7)                                                        | 136 (24.9)               | 49 (33.6)*               |              |
+|                                                                                                                                                                                                                                           | Percutaneous coronary intervention                                | 293 (42.3)               | 229 (41.9)               | 64 (44.1)    |
+|                                                                                                                                                                                                                                           | Coronary artery bypass graft                                      | 99 (14.3)                | 54 (9.9)                 | 45 (30.8)*** |
+| Cardiomyopathy                                                                                                                                                                                                                            | 83 (12.0)                                                         | 50 (9.1)                 | 33 (22.6)***             |              |
+| Chronic heart failure                                                                                                                                                                                                                     | 208 (30.0)                                                        | 156 (28.5)               | 52 (35.6)                |              |
+| Dysarhythmia                                                                                                                                                                                                                              | 200 (28.9)                                                        | 140 (25.6)               | 60 (41.1)***             |              |
+| Valvular disease                                                                                                                                                                                                                          | 472 (68.1)                                                        | 358 (65.4)               | 114 (78.1)**             |              |
+| Arterial occlusive disease                                                                                                                                                                                                                | 128 (18.5)                                                        | 71 (13.0)                | 57 (39.0)***             |              |
+| Pulmonary disease                                                                                                                                                                                                                         | 80 (11.5)                                                         | 56 (10.2)                | 24 (16.4)*               |              |
+| Kidney disease                                                                                                                                                                                                                            | 60 (8.7)                                                          | 28 (5.1)                 | 32 (21.9)***             |              |
+| History of stroke                                                                                                                                                                                                                         | 45 (6.5)                                                          | 29 (5.3)                 | 16 (11.0)*               |              |
+| Rheumatic disease                                                                                                                                                                                                                         | 13 (1.9)                                                          | 8 (1.5)                  | 5 (3.4)                  |              |
+| Cancer                                                                                                                                                                                                                                    | 78 (11.3)                                                         | 57 (10.4)                | 21 (14.4)                |              |
+| Other                                                                                                                                                                                                                                     | 229 (33.0)                                                        | 211 (38.6)               | 18 (12.3)***             |              |
+| Clinical chemistry High-sensitivity cardiac troponin T (ng/L)                                                                                                                                                                             | 6 (3–13)                                                          | 4 (3–8)                  | 21 (17–29)***            |              |
+| N-terminal pro b-type natriuretic peptide (ng/L)                                                                                                                                                                                          | 193 (84–484)                                                      | 153 (78–374)             | 443 (197–1472)***        |              |
+| Creatinine (mg/dL)                                                                                                                                                                                                                        | 1.0 (0.9–1.1)                                                     | 0.9 (0.8–1.1)            | 1.0 (1.0–1.0)***         |              |
+| Estimated glomerular filtration rate (mL/min/1.73m2 )                                                                                                                                                                                     | 82.9±30.0                                                         | 87.3±28.8                | 65.9±28.2***             |              |
+| Medication, n (%) Antihypertensive therapy                                                                                                                                                                                                | 576 (83.1)                                                        | 438 (80.1)               | 138 (94.5)***            |              |
+| Diuretics                                                                                                                                                                                                                                 | 256 (36.9)                                                        | 177 (32.4)               | 79 (54.1)***             |              |
+| Acetylsalicylic acid                                                                                                                                                                                                                      | 395 (57.0)                                                        | 324 (59.2)               | 71 (48.6)*               |              |
+| Clopidogrel                                                                                                                                                                                                                               | 103 (14.9)                                                        | 76 (13.9)                | 27 (18.5)                |              |
+| Statins                                                                                                                                                                                                                                   | 500 (72.2)                                                        | 385 (70.4)               | 115 (78.8)               |              |
+| Antiarrhythmics                                                                                                                                                                                                                           | 414 (59.7)                                                        | 299 (54.7)               | 115 (78.8)***            |              |
+| Insulin                                                                                                                                                                                                                                   | 19 (2.7)                                                          | 7 (1.3)                  | 12 (8.2)***              |              |
+| Oral antidiabetics                                                                                                                                                                                                                        | 78 (11.3)                                                         | 52 (9.5)                 | 26 (17.8)**              |              |
+| Other                                                                                                                                                                                                                                     | 316 (45.6)                                                        | 253 (46.3)               | 63 (43.2)                |              |
+| European Society of Cardiology-Systematic COronary Risk                                                                                                                                                                                   | 2.7±1.9                                                           | 2.4±2.0                  | 3.1±1.7***               |              |
+| Evaluation (points) *P<0.05 compared with high-sensitivity cardiac troponin T ≤14ng/L, **P<0.01, ***P<0.001. Presentations: quantity of presentations to outpatient clinic, emergency department or hospital admissions during follow-up. |                                                                   |                          |                          |              |
+
+Open Heart
+
+| Table 2                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Prognostic performance of the ESC-SCORE and high-sensitivity cardiac troponin T in non-secondary and secondary   |                      |                |       |        |                |          |      |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------|----------------------|----------------|-------|--------|----------------|----------|------|
+| prevention                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Non-secondary prevention                                                                                         | Secondary prevention |                |       |        |                |          |      |
+| AUC                                                                                                                                                                                                                                                                                                                                                                                                                                                        | SE                                                                                                               | 95%CI                | AUC            | SE    | 95%CI  | ∆AUC           | P values |      |
+| All-cause mortality ESC-SCORE                                                                                                                                                                                                                                                                                                                                                                                                                              | 0.798                                                                                                            | 0.0681               | 0.664 to 0.931 | 0.572 | 0.0933 | 0.389 to 0.755 | 0.226    | 0.05 |
+| High-sensitivity troponin T                                                                                                                                                                                                                                                                                                                                                                                                                                | 0.967                                                                                                            | 0.0161               | 0.935 to 0.999 | 0.830 | 0.0516 | 0.729 to 0.931 | 0.137    | 0.01 |
+| All-cause mortality, acute myocardial infarction, stroke (endpoint 2) ESC-SCORE 0.794 0.0732 0.650 to 0.937                                                                                                                                                                                                                                                                                                                                                | 0.590                                                                                                            | 0.0689               | 0.455 to 0.725 | 0.204 | 0.04   |                |          |      |
+| High-sensitivity troponin T                                                                                                                                                                                                                                                                                                                                                                                                                                | 0.675                                                                                                            | 0.1350               | 0.412 to 0.939 | 0.696 | 0.0521 | 0.594 to 0.798 | 0.021    | NS   |
+| All-cause mortality, acute myocardial infarction, stroke, rehospitalisation for acute coronary syndrome or decompensated heart failure (endpoint 3) ESC-SCORE 0.591 0.0699 0.454 to 0.728 0.525 0.0428 0.442 to 0.609 0.066 NS High-sensitivity troponin T 0.668 0.0757 0.519 to 0.816 0.661 0.0372 0.588 to 0.734 0.001 NS AUC, area under the curve; ESC-SCORE, European Society of Cardiology-Systematic COronary Risk Evaluation; NS, not significant. |                                                                                                                  |                      |                |       |        |                |          |      |
+
+whereas no difference was observed in the non-secondary prevention group (ΔAUC hs-cTnT vs ESC-SCORE: 0.077, p=NS).
+
+Reclassification analysis according to hs-cTnT status 
+(>14ng/L at index visit) is displayed for the entire population as well as the secondary and non-secondary prevention group in the online supplementary table 1. Briefly, hs-cTnT significantly improved reclassification regarding all endpoints in the overall population and in the secondary prevention cohort. In non-secondary prevention, hs-cTnT improved reclassification only for ACM.
+
+We found no statistically significant difference of hs-cTnT concentrations in men compared with women regarding all endpoints (ACM: men: 49.2±95.1ng/L, women: 26.0±20.3ng/L, p=NS; ACM, AMI, stroke: men: 30.0±65.6, women: 16.8±19.1, p=NS; ACM, AMI, stroke, rehospitalisation for ACS or DHF: men: 23.4±44.7, women: 8.7±7.4, p=NS).
+
+## Discussion
+
+Our study provides several important findings and supports the significant predictive role of hs-cTnT for risk stratification of patients with stable CAD. First, hs-cTnT outperformed the multivariable ESC-SCORE 
+for the prediction of ACM in non-secondary as well as in secondary prevention. Second, hs-cTnT provides reliable prognostic information on composite endpoints that also include soft outcome measures like readmission with ACS or DHF. Third, hs-cTnT provided consistent prognostic information in an external cohort with a different baseline risk.
+
+We decided to compare the prognostication of hs-cTnT 
+to the ESC-SCORE because it is validated in a large cohort across different countries for the occurrence of CV mortality as an objective and robust endpoint14 and is therefore recommended by ESC guidelines.15 The high prognostic performance of hs-cTnT in stable CV disease has been reported in many studies including large multicenter trials like the PEACE9 or the HOPE study.10 Our group had recently reported about the strong independent predictive value of hs-cTnT in the same low risk cohort of asymptomatic or stable patients with a broad spectrum of CV diseases visiting an outpatient clinic for routine regular check-ups.21 In another recently published analysis, we reported on the performance of hs-cTnT compared with established multivariable scores that are used in some European regions (Prospective Cardiovascular Münster Study (PROCAM) Score) or in 
+
+![5_image_0.png](5_image_0.png)
+
+![6_image_0.png](6_image_0.png)
+
+the US (Framingham Scores) in primary prevention.33 In consistency with the both previous studies, hs-cTnT predicted ACM with an AUC of 0.878 and thus—as expected—significantly better than the ESC-SCORE in secondary prevention. To our surprise, hs-cTnT also performed significantly better in non-secondary prevention, which is the domain of CV risk scores including the ESC-SCORE. Although our findings need to be confirmed in larger prospective cohorts, our results provide a strong hypothesis generating finding that could improve adoption of risk stratification tools both in primary and secondary prevention. For the latter, there is no universally established risk stratification tool that allows risk estimation after the subacute phase of ACS or after diagnosis of a relevant CV or cerebrovascular disease manifestation.
+
+Although the ESC-SCORE has not been validated for soft endpoints such as rehospitalisation for DHF, AMI 
+or recurrent ACS, we compared the prognostic information incorporated in a single hs-cTnT value as compared with a multivariable score and consistently confirmed our findings on the role of hs-cTnT to predict ACM. In agreement with the previous findings6 34 35 and our own published work on this low risk cohort,21 the prognostic performance of hs-cTnT to predict a variety of softer endpoints than ACM was only moderate with an AUC of only 0.707 for the composite endpoint of ACM, AMI and stroke, and 0.671 for the combined endpoint of ACM, AMI, stroke and rehospitalisation for DHF or ACS. Our results using traditional C-statistics were substantiated by reclassification analyses including continuous and categorical NRI as well as IDI.
+
+A general shortcoming of CV risk scores is that their predictive performance strongly depends on the similarity of the population in which they are applied to the original derivation cohort. An impaired performance has been reported particularly when the baseline risk differs from the original population.36–38 European guidelines therefore recommend the use of national recalibrations of the ESC-SCORE that consider the general population's risk.15 Nevertheless, this score has not been evaluated in non-caucasian populations and predicts only the endpoint 10-year CV mortality.14 In contrast, the predictive ability of hs-cTnT is more consistent showing an excellent performance across a broad spectrum of stable or asymptomatic patients including low-risk cohorts,39–41 chronic heart failure,42 asymptomatic high risk patients10 and in the general population.7 8 Since risk scores are only rarely used in daily clinical practice,16 17 the use of hs-cTnT within a single-biomarker strategy may facilitate the prognostication of low-risk patients and increase the acceptance of risk stratification in daily practice. Nevertheless, although higher hs-cTnT values have been associated with an increased risk for CV endpoints, it may not be directly modified by lifestyle changes in contrast to some of the variables of the ESC-SCORE like hypertension or smoking status.
+
+Our results were validated in a large independent study cohort of 2046 stable patients with documented coronary anatomy from the EXCELSIOR trial.22 In the EXCELSIOR cohort, a lower prognostic performance of hs-cTnT 
+for the prediction of ACM as compared with our study cohort was found (AUC 0.759, 95% CI 0.740 to 0.777 vs AUC 0.878 95% CI0.805 to 0.950). A reason for this observation might be the higher baseline risk since all individuals of the EXCELSIOR cohort underwent coronary angiography. Furthermore, the annual mortality was more than twice as high compared with our cohort (2.1 vs 0.9%). Irrespective of the higher baseline risk, hs-cTnT also outperformed the ESC-SCORE in the prediction of ACM and non-fatal AMI in the EXCELSIOR trial. The use of hs-cTnT in addition to the ESC-SCORE could significantly improve risk classification of patients. Multiple subgroup analyses of this cohort demonstrated that this finding was consistent throughout the whole population of this study. We therefore assume that our results may also be applied to other cohorts including populations at higher baseline risk.
+
+In conclusion, our study suggests that hs-cTnT is a reliable and convenient risk predictor with a superior performance compared with the ESC-SCORE in non-secondary and secondary prevention that may be applied in a broad spectrum of patients in primary care. It is thereby easier and more convenient to use in daily clinical practice.
+
+## Limitations
+
+The findings of this registry are limited by a relatively small sample size and small event rates, particularly occurrence of few all-cause deaths. However, our findings are consistent with the previous findings across the spectrum of stable CV disease and plausible due to the comprehensive integration of different pathophysiological aspects reflected by elevated cardiac troponin in blood. Our findings were validated in an independent large registry consistently confirming our results. Nevertheless, we believe that our results are hypothesis generating and would benefit from additional external validation in prospective trials or registries. Furthermore, there is large controversy regarding the role of sex-specific cut-offs that are being recommended by the Universal Myocardial Infarction Definition Task Force.1 Currently, sex-specific cut-offs have neither been recommended by ESC guidelines nor have they been validated prospectively. In the absence of convincing data supporting an improvement of diagnostic and prognostic reclassification,43 we preferred to use a single, sex-independent cut-off at the 99th percentile of a healthy reference population.26 Acknowledgements We thank Mrs Heidi Deigentasch, Mrs Melanie Hütter, Mrs Elisabeth Mertz and Mrs Monika Arnold for their valuable support regarding data management and follow-up. Contributors All authors contributed to the manuscript.
+
+Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None declared. Patient consent Not required. Ethics approval Ethics Committee, University Hospital Heidelberg. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/ licenses/by-nc/4.0/ © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
+
+## References
+
+1. Thygesen K, Alpert JS, Jaffe AS, *et al*. Third universal definition of myocardial infarction. *Eur Heart J* 2012;33:2551–67.
+
+2. Keller T, Zeller T, Peetz D, *et al*. Sensitive troponin I assay in early diagnosis of acute myocardial infarction. *N Engl J Med* 2009;361:868–77.
+
+3. Reichlin T, Hochholzer W, Bassetti S, *et al*. Early diagnosis of myocardial infarction with sensitive cardiac troponin assays. N Engl J Med 2009;361:858–67.
+
+4. Agewall S, Giannitsis E, Jernberg T, *et al*. Troponin elevation in coronary vs. non-coronary disease. *Eur Heart J* 2011;32:404–11.
+
+5. Giannitsis E, Katus HA. Cardiac troponin level elevations not related to acute coronary syndromes. *Nat Rev Cardiol* 2013;10:623–34.
+
+6. Saunders JT, Nambi V, de Lemos JA, *et al*. Cardiac troponin T 
+measured by a highly sensitive assay predicts coronary heart disease, heart failure, and mortality in the Atherosclerosis Risk in Communities Study. *Circulation* 2011;123:1367–76.
+
+7. de Lemos JA, Drazner MH, Omland T, *et al*. Association of troponin T detected with a highly sensitive assay and cardiac structure and mortality risk in the general population. *JAMA* 2010;304:2503–12.
+
+8. deFilippi CR, de Lemos JA, Christenson RH, *et al*. Association of serial measures of cardiac troponin T using a sensitive assay with incident heart failure and cardiovascular mortality in older adults. JAMA 2010;304:2494–502.
+
+9. Omland T, de Lemos JA, Sabatine MS, *et al*. A sensitive cardiac troponin T assay in stable coronary artery disease. *N Engl J Med* 2009;361:2538–47.
+
+10. Kavsak PA, McQueen MJ. Sensitive and high sensitivity cardiac troponin I concentrations in the Heart Outcomes Prevention Evaluation (HOPE) study--a high risk population. *Clin Chim Acta* 2010;411:1832.
+
+11. Fox KA, Dabbous OH, Goldberg RJ, *et al*. Prediction of risk of death and myocardial infarction in the six months after presentation with acute coronary syndrome: prospective multinational observational study (GRACE). BMJ 2006;333:1091.
+
+12. Antman EM, Cohen M, Bernink PJ, *et al*. The TIMI risk score for unstable angina/non-ST elevation MI: A method for prognostication and therapeutic decision making. *JAMA* 2000;284:835–42.
+
+13. D'Agostino RB, Vasan RS, Pencina MJ, *et al*. General cardiovascular risk profile for use in primary care: the Framingham Heart Study. Circulation 2008;117:743–53.
+
+14. Conroy RM, Pyörälä K, Fitzgerald AP, *et al*. Estimation of ten-year risk of fatal cardiovascular disease in Europe: the SCORE project. Eur Heart J 2003;24:987–1003.
+
+15. Piepoli MF, Hoes AW, Agewall S, *et al*. 2016 European Guidelines on cardiovascular disease prevention in clinical practice: The Sixth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of 10 societies and by invited experts) Developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR). Atherosclerosis 2016;252:207–74.
+
+16. Sposito AC, Ramires JA, Jukema JW, *et al*. Physicians' attitudes and adherence to use of risk scores for primary prevention of cardiovascular disease: cross-sectional survey in three world regions. *Curr Med Res Opin* 2009;25:1171–8.
+
+17. Müller-Riemenschneider F, Holmberg C, Rieckmann N, *et al*. Barriers to routine risk-score use for healthy primary care patients: survey and qualitative study. *Arch Intern Med* 2010;170:719–24.
+
+18. Mueller M, Celik S, Biener M, *et al*. Diagnostic and prognostic performance of a novel high-sensitivity cardiac troponin T assay compared to a contemporary sensitive cardiac troponin I assay in patients with acute coronary syndrome. *Clin Res Cardiol* 2012;101:837–45.
+
+19. Stoyanov KM, Giannitsis E, Biener M, *et al*. Prognostic value of elevated high-sensitivity cardiac troponin T in patients admitted to an emergency department with atrial fibrillation. *Europace* 2017.
+
+20. Giannitsis E, Katus HA. Biomarkers for clinical decisionmaking in the management of pulmonary embolism. *Clin Chem* 2017;63:91–100.
+
+21. Biener M, Giannitsis E, Lamerz J, *et al*. Prognostic value of elevated high-sensitivity cardiac troponin T levels in a low risk outpatient population with cardiovascular disease. *Eur Heart J Acute* Cardiovasc Care 2016;5.
+
+22. Hochholzer W, Valina CM, Stratz C, *et al*. High-sensitivity cardiac troponin for risk prediction in patients with and without coronary heart disease. *Int J Cardiol* 2014;176:444–9.
+
+23. Sacco RL, Kasner SE, Broderick JP, *et al*. An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 2013;44:2064–89.
+
+24. Roffi M, Patrono C, Collet JP, *et al*. 2015 ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent st-segment elevation of the European Society of Cardiology (ESC). *Eur Heart J* 2016;37:267–315.
+
+25. Ibanez B, James S, Agewall S, *et al*. 2017 ESC guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: the Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC). *Eur Heart J* 2018;39:119–77.
+
+26. Giannitsis E, Kurz K, Hallermayer K, *et al*. Analytical validation of a high-sensitivity cardiac troponin T assay. *Clin Chem* 2010;56:254–61.
+
+27. Apple FS, Jaffe AS. Clinical implications of a recent adjustment to the high-sensitivity cardiac troponin T assay: user beware. Clin Chem 2012;58:1599–600.
+
+28. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. *Biometrics* 1988;44:837–45.
+
+29. Pencina MJ, D'Agostino RB, D'Agostino RB, *et al*. Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond. *Stat Med* 2008;27:157–72. discussion 207-12.
+
+30. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). *JAMA* 2001;285:2486–97.
+
+31. Team RC. R: A language and environment for statistical computing. 
+
+2014 http://www.R-project.org/.
+
+32. Kundu S, Aulchenko YS, van Duijn CM, *et al*. PredictABEL: an R package for the assessment of risk prediction models. Eur J Epidemiol 2011;26:261–4.
+
+33. Biener M, Giannitsis E, Kuhner M, *et al*. Prognostic value of highsensitivity cardiac troponin T compared with risk scores in stable cardiovascular disease. *Am J Med* 2017;130.
+
+34. Omland T, Pfeffer MA, Solomon SD, *et al*. Prognostic value of cardiac troponin I measured with a highly sensitive assay in patients with stable coronary artery disease. *J Am Coll Cardiol* 2013;61:1240–9.
+
+35. Eggers KM, Lagerqvist B, Oldgren J, *et al*. Pathophysiologic mechanisms of persistent cardiac troponin I elevation in stabilized patients after an episode of acute coronary syndrome. *Am Heart J* 2008;156:588–94.
+
+36. Scheltens T, Verschuren WM, Boshuizen HC, *et al*. Estimation of cardiovascular risk: a comparison between the Framingham and the SCORE model in people under 60 years of age. Eur J Cardiovasc Prev Rehabil 2008;15:562–6.
+
+37. Selvarajah S, Kaur G, Haniff J, *et al*. Comparison of the Framingham Risk Score, SCORE and WHO/ISH cardiovascular risk prediction models in an Asian population. *Int J Cardiol* 2014;176:211–8.
+
+38. Versteylen MO, Joosen IA, Shaw LJ, *et al*. Comparison of Framingham, PROCAM, SCORE, and Diamond Forrester to predict coronary atherosclerosis and cardiovascular events. *J Nucl Cardiol* 2011;18:904–11.
+
+39. Beatty AL, Ku IA, Christenson RH, *et al*. High-sensitivity cardiac troponin T levels and secondary events in outpatients with coronary heart disease from the Heart and Soul Study. *JAMA Intern Med* 2013;173:763–9.
+
+40. Koenig W, Breitling LP, Hahmann H, *et al*. Cardiac troponin T measured by a high-sensitivity assay predicts recurrent cardiovascular events in stable coronary heart disease patients with 8-year follow-up. *Clin Chem* 2012;58:1215–24.
+
+41. Giannitsis E, Spanuth E, Horsch A, *et al*. High-sensitivity cardiac troponin T and N-terminal pro-B-type natriuretic peptide predict mortality in stable coronary artery disease: results from the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. *Clin* Chem Lab Med 2013;51:2019–28.
+
+42. Nagarajan V, Hernandez AV, Tang WH. Prognostic value of cardiac troponin in chronic stable heart failure: a systematic review. *Heart* 2012;98:1778–86.
+
+43. Mueller-Hennessen M, Lindahl B, Giannitsis E, *et al*. Diagnostic and prognostic implications using age- and gender-specific cut-offs for high-sensitivity cardiac troponin T - sub-analysis from the TRAPIDAMI study. *Int J Cardiol* 2016;209:26–33.

medical/md/PMC5954647.md

@@ -0,0 +1,154 @@
+
+## The Relationship Between Perceived Social Support In The First Pregnancy And Fear Of Childbirth Abstract
+
+Background: Numerous empirical evidences have shown that social and environmental circumstances and social relations have an important impact on pregnancy outcomes, women's ability to cope with stressful situations, and childbirth pain management. The present study was conducted to determine the relationship between perceived social support and fear of childbirth. **Materials and Methods:** The present descriptive correlational study was conducted on 270 nulliparous pregnant women who referred for pregnancy care in 2016. The subjects were selected through convenience sampling method. The data collection tools were a demographic characteristics form, the Multidimensional Scale of Perceived Social Support (MSPSS), and the Wijma Delivery Expectancy/Experience Questionnaire (W‑DEQ). The collected data were analyzed using descriptive and inferential statistics tests. **Results:** The mean perceived social support score of the participants was 77.90 and a significant correlation was observed between the perceived social support score and fear of childbirth score (β = −0.18, p = 0.004). The participants' mean score of fear of childbirth was 36.8. The results of study after the evaluation of the effect of predictive variables on the fear of childbirth are as follows: pregnancy preparation classes: *(β* = 3.50*, p* = 0.220); observation of natural childbirth videos: (β = 5.26, p = 0.040); and use of educational software: *(β* =5.82, p = 0.080). **Conclusions:** In order to reduce the fear of childbirth, women's social support structure during pregnancy should be assessed. Moreover, demographic characteristics form the structure and determine the extent of individuals' social network and, through the evaluation of these characteristics during pregnancy, the rate of effective support can be predicted in individuals. Keywords: *Childbirth, fear, Iran, pregnancy, social support*
+
+## Introduction
+
+Childbirth is a social occurrence all around the world and can be considered as the happiest occurrence in any parent's life. Becoming a parent is accompanied by obtaining a high social status, and the majority of parents describe it as a positive experience. However, pregnancy in itself is a traumatic occurrence and the mother who encounters can potentially be stressful.[1]
+The birth of the first child, in particular, is categorized as an extremely stressful event in psychosocial stress tables.[2] Concern about the adverse physical–psychological effects of pregnancy and fear of childbirth can be noted as two of the stressors of this period. It has been estimated that 1 out of 5 pregnant women have fear of childbirth. Moreover, approximately 6–13% of pregnant women experience severe and debilitating fear, which, in addition to physical outcomes, can result This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercialShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. 
+
+For reprints contact: reprints@medknow.com in psychological complications such as depression and anxiety, negative mood after childbirth, eating disorders,[3] and even uninformed selection of delivery method. It has been proven that fear of childbirth has an important role in the selection of cesarean section by Iranian women.[4] Based on the statistics reported by the World Health Organization (WHO), the prevalence of cesarean section was 46% in Iran in 2014; however, the recommendation of the WHO for an acceptable prevalence of cesarean section by 2014 was a maximum of 5–15%.[5]
+Fear of childbirth is an important issue during pregnancy and during and after childbirth, and psychological and social factors have an effective role in its creation.[6] The results of the study by Fisher et al.[7] illustrated that fear of childbirth has two dimensions: individual and social dimension. The social dimension has received less attention in most studies,[6]
+How to cite this article: Azimi M, Fahami F, 
+Mohamadirizi S. The relationship between perceived social support in the first pregnancy and fear of childbirth. Iranian J Nursing Midwifery Res 2018;23:235-9.
+
+Received: September, 2016. **Accepted:** September, 2017.
+
+## Masoomeh Azimi1, Fariba Fahami2, Soheila Mohamadirizi2
+
+1Student *Research Center,* 
+School of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran, 2Nursing and Midwifery Care Research Center, Faculty of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran Address for correspondence: Ms. Fariba Fahami, Nursing and Midwifery Care Research Center, Faculty of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran. E‑mail: *Fahami@nm.mui.ac.ir*
+
+![0_image_0.png](0_image_0.png)
+
+although it has been found that the social dimension not only affects women's identity but also their perception of childbirth as an abstract event. Social relations, the unofficial support networks of pregnant women, and strong support by midwives can strengthen women's perception of childbirth as a physiological and controllable process.[7] On the contrary, researches have shown that Iranian women's perception of childbirth is derived from their culture and values and is rooted in the environment in which they live.[8] It must be noted that social support has two main dimensions: received and perceived social support. Received social support is the rate of support received by the individual. Perceived social support is the individual's evaluation of the availability of support when necessary and required and is a qualitative‑mental and measurable concept.[9] It is affected by culture, evolution, personality, and support systems.[7] Theoreticians believe relationships are not a source of social support unless the individual perceives them as a suitable and available source for fulfilling their needs.[10]
+Social support is an interpersonal relationship which provides individuals with psychological help when required. Those who provide this help include family members, neighbors, colleagues, relatives, and the treatment team, who provide mothers with psychological support and information.[1] Relatives, especially the spouse, illustrate their high regard for the pregnant woman through their support for her and this gives her hope. Previous studies have shown that social support has a significant relationship with reduction of stress during pregnancy,[11] and the lack of support has a relationship with unwillingness to breastfeed,[12] low IQ in infants,[13] postpartum depression (PPD),[14] depression during pregnancy,[15] and cleft palate incidence.[16] Some researchers have reported contradictory results regarding this issue. Some studies have shown that sometimes the support provided is inappropriate, untimely, and contrary to the mother's wishes.[17] Other studies have illustrated that this support was ineffective. Jirka et al. found no difference in the rate of social support of pregnant and nonpregnant couples; they received the same rate of support.[18]
+Due to the importance of the role of perceived social support in individuals' psychological wellbeing, the contradictory results of previous studies, the role of cultural difference in social support patterns,[19] and the important role of fear of childbirth in the selection of cesarean section by Iranian women,[4] the present study was undertaken. The present study was conducted to determine the possible challenges regarding mothers' social support and the role of predictive variables on fear of childbirth. It is hoped that with the presentation of the results to prenatal caregivers plans can be made for the improvement of mothers' social support, and thus, a small step can be taken toward the improvement of mothers' health and their empowerment.
+
+## Materials And Methods
+
+The present descriptive correlational study was conducted on 270 nulliparous pregnant women who referred to health and treatment centers in Isfahan, Iran, for pregnancy care in 2016. The division of perceived social support in the population into two groups of adequate social support and lack of adequate social support required at least 135 subjects from each population and a total of 270 individuals to observe, with a 90% probability, a significant difference of 0.4 in mean score of fear of childbirth between the two populations at 5% significance level. The study environment consisted of the health and treatment centers of Isfahan. The centers were selected through cluster‑nonrandom and quota sampling methods after obtaining the approval of the Ethics Committee of Isfahan University of Medical Sciences, Isfahan. These centers were selected due to the possibility of easy access to study subjects. The inclusion criteria consisted of nulliparous pregnancy, Persian nationality, gestational age of 35 weeks and higher, lack of recognized psychological disorders, such as depression and mood disorder (these data were retrieved from the mothers' pregnancy care file), and low‑risk pregnancy such as lack of chronic diseases like heart disease, hypertension, and diabetes. In addition, the subjects were questioned regarding participation in childbirth preparation classes, use of computer training programs, and observation of natural childbirth videos; these variables are effective on the mean score of fear of childbirth and their impact was studied in regression analysis. Data were collected through self‑report method and if necessary through interviews using three questionnaires. The data collection tools were a demographic characteristics form, the Multidimensional Scale of Perceived Social Support (MSPSS),[20] and the Wijma Delivery Expectancy/ Experience Questionnaire (W‑DEQ).[21] The demographic characteristics form assessed the age and occupation of the mother and her spouse, education level, and economic status. The MSPSS is a 12‑item tool that evaluates perceived social support in the three dimensions of family (questions 3, 4, 8, 11), friends (questions 6, 7, 9, 12), and specific individual (questions 1, 2, 5, 10). This tool is scored based on seven‑point Likert scale (completely agree = 7; agree = 6; somewhat agree = 5; I have no opinion = 4; somewhat disagree = 3; disagree = 2; completely disagree = 1). The minimum and maximum obtained scores of the MSPSS are, respectively, 12 and 84, and higher scores represent higher social support.[20] The W‑DEQ consists of 16 items scored based on a four‑point Likert scale (lack of fear = 1; low = 2; average = 3; high = 4). Its total score ranges from 16 to 64 and higher scores represent greater fear.[21]
+The validity and reliability of the MSPSS were reported for the first time by Zimet et al.
+
+[20] (Cronbach's alpha = 0.88). In the study by Gao[22] performed on 236  ¦  ¦  ¦ 
+68 nulliparous pregnant women in China, the validity of the MSPSS was approved through content validity and its reliability was obtained through the calculation of Cronbach's alpha ( = 0.85). The validity and reliability of the MSPSS have also been approved in Iran.[8] The validity and reliability of the W‑DEQ were obtained for the first time by Wijma et al.[21] (Cronbach's alpha = 0.89). In the study by Carlson on 406 nulliparous pregnant women in Sweden, the validity and reliability of the W‑DEQ were calculated through content validity and Cronbach's alpha (α = 0.92), respectively.[23] The validity and reliability of this questionnaire have also been approved in Iran.[24]
+After obtaining permission and an introduction letter, the researcher referred to the intended treatment centers, presented the introduction letter to the authorities of the centers, and explained the goals of the study to the personnel of the centers. The researcher referred to the health and treatment centers every day during working hours[8‑13] and selected women who referred for regular pregnancy care and had the inclusion criteria through simple sampling method. The questionnaires were distributed among the subjects and their completion method was explained to them. Before the completion of the questionnaire, a suitable environment was prepared for the participants. The questionnaires were completed in one stage and through self‑report method and, if necessary, through interviews. After collecting the completed questionnaires, the subjects were thanked and catered for. The collected data were analyzed using descriptive (mean, median, prevalence distribution, and percentage) and analytical statistics (Pearson coefficient, and regression) at 95% confidence interval in Statistical Package for the Social Sciences (SPSS) software (version 16, SPSS Inc., Chicago, IL, USA).
+
+## Ethical Considerations
+
+Ethical principles were respected in all stages of the study. The objectives of the study were fully explained to the participants. Mothers who were willing to participate in the study were entered into the study after obtaining informed written consents from them. The subjects were completely free to leave the study at any moment of its execution. Moreover, they were assured of the confidentiality of their information.
+
+## Results
+
+In the present study, the mean age of the women was 26 years. The mean age of their spouses was 30 years. The education level of most of the participants was higher than diploma (41.30%) and university degree (41.35%). The majority of women were homemakers (85.92%) and most of their spouses were employed (87.71%). In addition, most of the subjects had an average economic status (93%). Their mean MSPSS score was 77.90%; the scores of support by family, friends, and spouse were 28.11, 22.42, and 27.73, respectively. The mean W‑DEQ score of the subjects was 36.81%. The results of single‑variable linear regression analysis showed that perceived social support, age, and spouse's age were predictors of fear of childbirth; 1 point increase in the perceived social support score, 1 year increase in the mother's age, and 1 year increase in the spouse's age resulted in the significant reduction of the mean score of fear of childbirth by, respectively, 0.23, 0.73, and 0.84 points. Education, mother's occupation, spouse's employment, lack of participation in pregnancy preparation classes, and lack of use of pregnancy education software did not cause a significant difference in the fear of childbirth. Nevertheless, household income significantly reduced the fear of childbirth, but observation of natural childbirth videos significantly increased the fear of childbirth [Table 1].
+
+| Table 1: Predictors of fear of giving birth: Results of  univariate linear regression   |        |              |         |
+|-----------------------------------------------------------------------------------------|--------|--------------|---------|
+| Predictors of fear of giving                                                            | β      | 95% CI       | p       |
+| birth Perceived social support                                                          | −0.18  | −0.39-−0.07  | 0.004   |
+| Age                                                                                     | −0.73  | −1.31-0.16   | 0.013   |
+| Age of husband                                                                          | −0.84  | −1.47-0.21   | 0.009   |
+| Education Illiterate                                                                    | -      | -            | -       |
+| High school                                                                             | 1.43   | −37.64-40.49 | 0.943   |
+| College (Diploma)                                                                       | 3.40   | −35.34-42.15 | 0.863   |
+| University                                                                              | −5.38  | −44.11-33.36 | 0.785   |
+| Occupation Teaching related                                                             | -      | -            | -       |
+| Government staff                                                                        | −9.58  | −28.92-9.75  | 0.330   |
+| Workers                                                                                 | −9.17  | −51.52-33.19 | 0.670   |
+| Housewife                                                                               | 3.92   | −13.58-21.41 | 0.659   |
+| Husband occupation Employed                                                             | -      | -            | -       |
+| Unemployed                                                                              | 7.81   | −0.23-15.85  | 0.057   |
+| Family income <US$ 250                                                                  | -      | -            | -       |
+| US$ 250-US$ 500                                                                         | −4.89  | −10.15-0.36  | 0.068   |
+| US$ 500-US$ 750                                                                         | −13.10 | −25.80-−0.39 | 0.043   |
+| > US$ 750                                                                               | 5.65   | −14.04-25.34 | 0.572   |
+| Participating in giving birth education Yes                                             | -      | -            | -       |
+| No                                                                                      | 3.49   | −2.10-9.09   | 0.220   |
+| Using giving birth program Yes                                                          | -      | -            | -       |
+| No                                                                                      | 5.82   | −0.77-12.42  | 0.083   |
+| Watching natural giving birth video Yes                                                 | -      | -            | -       |
+| No                                                                                      | 5.26   | 0.22-10.30   | 0.0.041 |
+
+ ¦  ¦  ¦  237
+
+## Discussion
+
+The rate of social support in the present study was 77.90%, which was in agreement with the results of the studies by Sadeghe,[8] Jamshidimanesh,[25] and Gao.[22] In the study by Sadeghe, the mean perceived social support score was 62.71 and the scores of support by family, friends, and spouse were 22.53, 16.56, and 23.61, respectively.[8] In the study by Gao,[22] the mean perceived social support score was 68%. The similarity of the findings in the study by Gao to the present study may be due to the fact that China is also an eastern country with a traditional culture in which the family is the main unit of society. In eastern societies, women have an important and high status in the family and the majority of families with different cultures and statuses provide pregnant women with suitable support. In the present study, family support was the most important source of social support and being in the family environment was an unconditional membership in a support group.[25] Moreover, the support of the spouse is an important source which creates a sense of respect, self‑esteem, and safety during pregnancy.[8]
+The mean score of fear of childbirth in the present study was 36.8%, which was in agreement with the results of the studies by Khorsande[3] and Nasiri.[26] In the study by Khorsande,[3] the mean score of fear of childbirth of nulliparous women was 26%. In the study by Nasiri,[26]
+the mean score of fear of childbirth was 33.45. From these results it can be inferred that the W‑DEQ is a valid tool that is effective in determining women with fear of childbirth and planning to control their fear. If the pregnant mother imagines she cannot control the situation and believes herself to be unable to tolerate the pain, the resulting fear and anxiety will cause her to accept cesarean section without medical indication. In addition, she will not be immune to the negative effects of stress on health. There was a negative significant relationship between the perceived social support score of and fear of childbirth. In this respect, the study by Fisher et al. showed that social relations, unofficial support networks of pregnant women, and the strong support by midwives can strengthen women's belief that childbirth is a physiological and controllable process, and thus, result in psychological wellbeing and reduced fear of childbirth.[7] Furthermore, Pasha and Faramarze reported that one‑third of women experienced pregnancy as a stressful event and the most unpleasant experience of pregnancy was related to the third trimester. They also found that social support had a negative significant relationship with pregnancy stress; with increase in social support score, pregnancy stress decreased (p < 0.05).[11] Najafe et al.[27] observed a negative significant relationship between pregnancy preparation classes and fear of childbirth (p < 0.001). However, this relationship was not significant in the present study. The difference between Najafi et al. study and the present study may be due to the fact that the study by Najafi et al. was an interventional study or due to the difference in the quality of pregnancy preparation classes. Today, one of the causes of the increase in the prevalence of cesarean section in the world is the fear of childbirth. Furthermore, the statistics of cesarean section in Iran are higher than global standards.[5] Thus, pregnancy preparation classes centered on pregnant women with fear of childbirth can be an effective measure in the reduction of fear of childbirth and the promotion of natural childbirth. No similar study was found on the relationship between observation of natural childbirth videos and fear of childbirth. However, the results of the present study (conducted for the first time in Iran) can reinforce the belief that common natural childbirth videos have not been prepared with the aim of education and empowerment of women. It can also be stated that these videos can increase tension, and thus, inefficiency and fear. Through the conveyance of this important issue to pregnant mothers by health personnel and informing them, a small step can be taken toward decreasing the fear of childbirth. The results of the study by Hamzekhane[28]
+suggest that educational software can be more effective in increasing the awareness and knowledge of individuals compared to traditional education methods. Nevertheless, they did not decrease the fear of childbirth in the present study. The findings of the present research were studied along with its limitations such as cultural factors, beliefs, and traditions which could impact the responses of the subjects and could not be controlled by the researcher.
+
+## Conclusion
+
+The results of the present study showed that demographic factors determine the receiving of social support, because these factors form the structure and determine the extent of individuals' social network. It has been proven that lower income, education, economic status, and occupational position are accompanied by a smaller and less diverse social network. Thus, the rate of effective support in pregnant mothers can be predicted through the evaluation of the demographic factors of the support process from the beginning of pregnancy until after childbirth. Furthermore, through informing the treatment team of these results they can be asked to assess clients with characteristics such as low age of mother and spouse, low education, and low income in terms of perceived social support dimensions throughout their pregnancy. The continuous assessment of this support trend from the beginning of pregnancy until after childbirth by the treatment team resulted in the empowerment of women and reduced fear of childbirth which will, in turn, result in increased psychological wellbeing among mothers and reduced prevalence of cesarean section.
+
+238  ¦  ¦  ¦ 
+Acknowledgement The article was derived from the Master's thesis with project number 395096, Isfahan University of Medical Sciences, Isfahan, Iran.
+
+Financial support and sponsorship Isfahan University of Medical Sciences. Conflicts of interest Nothing to declare.
+
+## References
+
+1.	 Nazare S, Salare P, Mazlom R, Mehram F. The comparative study of social support postpartum mothers multiparity in the first and second month after delivery. Public Health Research Khorasa 2014;8:1‑9.
+
+2.	 Pournamdarian S, Birashk B, Asgharnejad Farid A. The clarification of contribution of meta‑cognitive beliefs in explaining the symptoms of depression, anxiety and stress in nurses. Knowledge & Research in Applied Psychology. 2012;13:86‑94.
+
+3.	 Khorsande M, Vakilian K, Masole M. The reasons natural childbirth. J Fasa Univ of Medi Sci 2014;4:161‑6.
+
+4.	 Zahede M, Asemane O, Mahmodyan H. Given the role of medical in assistants in reducing caesarean sections; designed and evaluated the psychometric questionnaire to measure attitudes of residents towards elective caesarean sections and vaginal births. Iranian Journal of Medical Ethics and History 2015;8:59‑71.
+
+5.	 Word Report on Knowledge for Better Health, Strengthening Health Systems, World Health Organization 2004; Available form: URL: www.who.int/rpc/meeting/publ/en/. [Last accessed on 2009 Aug 29].
+
+6.	 Taheri Z, Khorsande M, Amire M, Hasanzade A. Investigate the causes of childbirth in pregnant woman in Shahrekord. Univ of Medi Scirafsanjan 2015;14:345‑50.
+
+7.	 Fisher C, Hauckb Y, Fenwickb J. How social context impacts on women's fears of childbirth. Soc Sci Med 2006;63:64‑75.
+
+8.	 Sadeghe H, Mosave S, Jahde F, et al. The relationship between perceived social support with the consent of the pregnant women referred to health center martyr Akbarabadi Tehran. Journal of Preventive Care in Nursing and Midwifery 2013;4:54‑64.
+
+9.	 Gulacti F. The effect of perceived social support on subjective well‑being. Procedia‑Social and Behavioral Sciences 2010;2:3844‑9.
+
+10.	 Chadwick KA, Collins PA. Examining the relationship between social support availability urban center size, and self‑perceived mental health of recent immigrants to Canada: A mixed‑methods analysis. Soc Sci Med 2015;128:220‑30.
+
+11.	 Pasha H, Faramarze M. The role of social support in predication of stress in pregnancy. Journal Babol Univ of Medi Sci 2015;17:52‑60.
+
+12.	 Hildingsson I, Tingvall M, Rubertsson C. Partner support in the childbearing period - A follow up study. Women Birth 2008;21:141‑8.
+
+13.	 Theodore RF, Thompson JM, Waldie KE, Bercroft DM,
+Robinson E, Wild CJ, et al. Determinants of cognitive ability at 7 years: A longitudinal case control study of children born small‑for‑gestational age at term. Eur J Pediatr 2009;168:1217‑24.
+
+14.	 Spoozak L, Gotman N, Smith MV, Belanger K, Yonkers KA.
+
+Evaluation of a social support measure that may indicate risk of depression during pregnancy. J Affect Disord 2009;114:216‑23.
+
+15.	 Nore R, Kareme N, Mohamade M. The relationship Between Prenatal depression with Social Support and satisfaction. Journal Univ Tehran Iran 2013;1:10‑15.
+
+16.	 Ma J, Huang YQ, Yao C, Ma SQ, Meng T, Ma M, et al. Parental Health and Social Support in the First Trimester of Pregnancy and the Risk of oral Cleft. Plast Reconstr Surg 2015;135:212‑8.
+
+17.	 Garcia MF, Ramirez M, Jariego MI. Social support and locus of control as predictors of psychological well‑being in Moroccan and Peruvian immigrant women in Spain. International Journal of Intercultural Relations 2002;26:287‑310.
+
+18.	 Jirka J, Schuett SH, Foxall MJ. Loneliness and Social Support in infertile couples. J Obstet Gynecol Neonatal Nurs 1996;25:55‑60.
+
+19.	 Logsdon M. Social and professional support for pregnancy and parenting women. MCN Am J Matern Child Nurs 2003;28:371‑6.
+
+20.	 Zimet GD, Dahlem NW, Zimet SG, Farley GK. The multidimensional scale of perceived social support. Journal of Personality Assessment 1998;52:30‑41.
+
+21.	 Wijma K, Wijma B, Zar M. A new questionnaire for the measurement of fear of childbirth (Psychometric aspects of the W‑DEQ). J Psychosom Obstet Gynecol 1998;19:84‑97.
+
+22.	 Gao L, Sun K, Chan SW. Social support and parenting self‑efficacy among Chinese women in the perinatal period. Midwifery 2014;30;532‑8.
+
+23.	 Carlsson M, Ziegert K, Nissen E. The relationship between childbirth self‑efficacy and aspects of well‑being, birth interventions and birth outcomes. Midwifery 2015;31:1000‑7.
+
+24.	 Khorsandi M, Ghofranipour F, Heydarnia A, Faghih Zadeh S,
+Vafaei M, Rousta F, et al. The effect of childbirth preparation classes on childbirth fear and normal delivery among primiparous women. Arak Medical University Journal 2008;11:9.
+
+25.	 Jamshidimanesh M, Astaraki L, Moghadam ZM, Taghizade Z,
+Haghane H. Maternal‑fetal attachment and its associated factor. Hayat 2012;18:33‑45.
+
+26.	 Nasiri F, Sharifi SH. The relationship between personality type and childbirth in pregnant women. IJOGI 2013;16:18‑25.
+
+27.	 Najafe F, Abozar K, Jafarzade F, Rahnama P, Gholame B.
+
+Corporate communications at antenatal classes with fear and type of delivery. Tehran Nursing and Midwifery School (Hayat) 2015;21:30‑40.
+
+28.	 Hamzekhane M, Hamidzade A, Vasegh F. The effect of computerized educational program on self‑efficacy of pregnant women with postpartum adjustment. Journal of Knowledge and Health 2013;9:13‑20.

medical/md/PMC6030411.md

@@ -0,0 +1,39 @@
+# Radiation Nephritis: 99Mtc Hydroxydiphosphonate Bone Scan, 99Mtc Dimercaptosuccinic Acid Renal Scan, And 18F-Fdg Pet/Ct Findings
+
+Hye Lim Park1, Ie Ryung Yoo1, Yeon Sil Kim2, Jin-Hyoung Kang3, and Ji Eun Lee3 A 20-year-old male had Ewing's sarcoma of the right paraspinal region 
+(T12–L2 vertebra level) and was treated with chemotherapy and radiation therapy (RT). 99mTc hydroxydiphosphonate bone scan was performed 4 months after the end of treatment. 
+
+Intense uptake was noted in both kidneys (Fig. 1). On the fluorodeoxyglucose 
+(FDG) positron emission tomography/ computed tomography (PET/CT) for response evaluation, mild uptake was also seen in both kidneys (Fig. 2A and 2B). On 99mTc dimercaptosuccinic acid 
+(DMSA) scan, photon defects were noted in both kidneys (Fig. 2C). When we reviewed the RT plan, the medial aspect of both kidneys was included in the radiation field (Fig. 2D and 2E). On contrast-enhanced CT, low-densities were noted in both kidneys (Fig. 2F). 
+
+We thus diagnosed radiation nephritis (RN). On 6-month follow-up bone scan, the previously noted intensity on both kidneys was decreased.
+
+RN is commonly seen following RT 
+of intra-abdominal tumors or bony metastasis of spine. The histologic findings are vascular sclerosis with tubular ischemic change. Tubular dysfunction and ischemic change explain the photon defect on DMSA scan and the delayed enhancement and retention of contrast on CT. Delayed excretion of radiopharmaceuticals could 
+
+![0_image_0.png](0_image_0.png) cause increased uptake on bone scan. 
+
+CT and bone scan findings were previously reported, but PET/CT findings were reported in only one case. 
+
+Although the exact mechanism of increased activity on PET/CT remains uncertain, delayed tracer excretion via parenchymal injury could be a major cause. Radiation-induced inflammation can also cause. However, the lack 
+
+IMAGE OF INTEREST
+Korean J Intern Med 2018;33:837-838 https://doi.org/10.3904/kjim.2017.145 1Division of Nuclear Medicine, Department of Radiology, 2Department of Radiation Oncology, 3Division of Medical Oncology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Received: March **21, 2017** Revised : May **14, 2017** Accepted: May **14, 2017**
+Figure 1. On 99mTc hydroxydiphosphonate bone scan 4 months after radiotherapy for evaluation of metastasis, intense linear band-like uptake was noted in the medial aspect of both kidneys (arrows). 
+
+Correspondence to Ie Ryung Yoo, M.D. Tel: +82-2-2258-1552 Fax: +82-2-2258-1575 E-mail: iryoo@catholic.ac.kr Copyright © 2018 The Korean Association of Internal Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
+
+pISSN 1226-3303 eISSN 2005-6648 http://www.kjim.org
+
+![1_image_0.png](1_image_0.png)
+
+of evidence of inflammation in other tissues included in the radiation field and the decreased blood flow to the kidneys on enhanced CT indicate that the inflammation was a minor contributor.
+
+Focal renal activity on PET/CT could be misinterpreted as malignancy. Differential diagnostic criteria for distinguishing RN from malignancy include a RT history, a linear uptake pattern, and a sharp margin of uptake. 
+
+Lesion locations were the medial or upper portion of the kidney. Clinicians need to exclude RN when linear increases in renal activity are incidentally detected on FDG PET/CT.
+
+## Conflict Of Interest
+
+No potential conflict of interest relevant to this article was reported.

medical/md/PMC6113417.md

@@ -0,0 +1,92 @@
+# Metabolic Reprogramming After Left Ventricular Assist Device
+
+Remodeling Without Recovery of Cardiac Energetics* J. Eduardo Rame, MD, MPHIL,
+a Emma J. Birks, MD, PHDb Despite impressive changes at the organ, tissue and cellular level in myocardial reverse remodeling with left ventricular assist device support, reprogramming the presumed "endstage" human heart toward the nonfailing phenotype has not been convincingly demonstrated (1–5). Many have questioned the possibility of achieving sustained myocardial "recovery" using our current technology, leading to a call for further investigation by a recently gathered National Heart, Lung, and Blood Institute task force (6). Metabolic adaptations have been reported in the calorie-restricted, exercising, ischemic, and failing myocardium—and recently, the lipidomic signature of the end-stage failing, nondiabetic human heart has revealed rarefied lipid stores and evidence for a newly discovered shift to ketones as an alternative energy fuel (7–10).
+
+## See Page 432
+
+Enter the highly anticipated work by Diakos et al.
+
+(11) to fill in the translational knowledge gaps between myocardial recovery and metabolic adaptation after left ventricular assist device (LVAD) support. The authors performed targeted and untargeted quantitation of metabolites with mass spectroscopy to characterize the metabolic phenotype of myocardial reverse remodeling with mechanical unloading in nonfailing donor hearts, as well as paired myocardial samples obtained before LVAD and before transplantation after LVAD support. Their analyses revealed the following signatures: 1) increased glycolytic flux without a commensurate increase in glucose oxidation resulting in increased myocardial pyruvate and shuttled lactate levels; 2) a persistent disruption in the tricarboxylic acid (TCA) cycle characterized by splitting between first-span and secondspan levels of intermediates; 3) a deficit in mitochondrial oxidative capacity with decreased State III
+and State IV respiration in the chronically failing heart not improved with mechanical unloading; and 4) decreased density and altered structure of mitochondria in failing hearts pre- and post-mechanical unloading. The authors (11) reason that the decoupling of the utilization of glucose in the failing myocardium—glycolysis from oxidation—is linked with deficits in mitochondrial function, both in the cycling of intermediary metabolites and oxidative phosphorylation. In order to interpret the significance of these findings, we first need to step back and understand the current work in the context of recent advances in our understanding of the metabolic adaptation of the failing heart. Second, we need to ask exactly what are the mechanisms behind this remodeling in the adaptive metabolism of the failing heart that fall well short of recovering the cardiac energetics with mechanical assist device support?
+
+Muscle, both cardiac and skeletal, has a high degree of plasticity to the changing conditions challenging its environment. In response to a change in loading conditions and loss of contracting mass and function, metabolic adaptations have been demonstrated in the failing mammalian heart including decreased utilization of fatty acids with a shift to the more oxygen-sparing energetic substrate of glucose
+(12–14). The work by Diakos et al. (11) convincingly demonstrates decreased levels of glucose-1phosphate and glucose-6-phosphate in the endstage failing myocardium, which is indicative of a decreased utilization of glucose, and is consistent with decreased glycolysis in advanced heart failure.
+
+The importance of this finding, coupled with the demonstration that with mechanical unloading there is an increase in the levels of glycolytic metabolites is extremely important, insofar as it shows that there is not a uniform shift to increased glucose metabolism across the broad spectrum of heart failure. Instead, these data support the dynamic model of metabolic adaptation across the different stages of heart failure, with specific metabolic signatures apparent in responders versus non-responders. Indeed, although not large enough for analysis, the subgroup of patients with significant myocardial functional improvement after mechanical unloading did show greater mitochondrial functional and structural improvement than the "nonresponders." A study investigating the metabolic phenotype of nonselective adrenergic receptor blockade in chronic human heart failure with 18-fluoro-deoxyglucose positron emission tomography demonstrated an increased uptake of glucose in the patients with the most significant degree of myocardial reverse remodeling and improved left ventricular ejection fraction (15). Thus, an improved myocardial phenotype may shift more towards increased reliance on glucose metabolism.
+
+The work by Diakos et al. (11) extends the observation of myocardial plasticity in metabolic adaptation to the end-stage failing heart transitioning to mechanical support and provides evidence of increased glucose utilization through glycolysis but without the capacity to couple this with the oxidation of pyruvate and increased oxidative metabolism.
+
+When considering the impact of these results on future investigation, an important question concerns the mechanism of how glycolytic flux becomes uncoupled from glucose oxidation. This uncoupling results in a bottleneck increasing the myocardial levels of pyruvate and lactate above the nonfailing levels after a period of mechanical unloading. Could the increase in glycolysis be the result of improving myocardial insulin resistance that has been reported after LVAD therapy (16,17), with the bottleneck at pyruvate representing a failure to release the brake on pyruvate dehydrogenase (PDH) caused by the excess circulating fatty acids? In essence, the effects of the Randle Cycle (18)—inhibition of glucose oxidation by fatty acid uptake and utilization—may persist until the complete reversal of the heart failure syndrome with subsequent normalization of the peripheral metabolic milieu. Alternatively, is the increased concentration of glycolytic metabolites the result of a "shift back to glucose" and away from ketone metabolism (7,8), as peripheral insulin signaling improves, effectively decreasing or halting hepatic ketogenesis? If so, there may be a novel link switching cardiac substrate utilization on/off under failing conditions which accompanies the metabolic reprogramming to an earlier stage of heart failure marked by a preferential metabolism of glucose.
+
+Diakos et al. (11) have correctly identified abnormalities in mitochondrial structure and function in the failing heart that persist after a period of mechanical unloading, invoking this broader mechanism as the reason for the impaired oxidation of glucose and the decoupling from glycolysis. They present very convincing data that the TCA cycle, which was recently demonstrated to be disrupted in advanced heart failure with a decreased ratio of succinyl-CoA
+(an early second-span intermediate) to acetyl-CoA (the proximal metabolic point of entry), remains abnormal despite effective mechanical unloading with LVAD therapy (8). A proposal is made by the authors that mechanisms involving anaplerosis and cataplerosis interacting with amino acid metabolism can explain the changes in the intermediate metabolites of the Krebs cycle. However, what remains unexplained is the difference in post-LVAD levels between first-span (remain decreased) and secondspan (restored to non-failing levels) intermediates.
+
+Precisely why are the metabolites after succinyl-CoA
+restored as the end-stage failing heart remodels with mechanical unloading? This may again represent a "switch away from ketones" because succinyl-CoA
+is no longer shuttled from the TCA cycle to serve as the CoA donor in the rate-limiting step of ketone oxidation through the enzyme SCOT (succinyl-CoA:3oxoacid CoA transferase) that was demonstrated to be significantly up-regulated in the end-stage failing human heart (8) (Figure 1). Thus, the active oxidation of ketones results in a depletion of succinyl-CoA
+while reversing this state through a decrease or halting of ketogenesis restores the myocardial pool of succinyl-CoA and second-span TCA cycle intermediates. Given the importance of succinylation and acetylation in the transcriptional and enzymatic control of important metabolic pathways, the myocardial concentration of succinyl-CoA may play a key role in the regulation of metabolic adaptations across the various stages of heart failure.
+
+What are the therapeutic implications of this work in the field of myocardial recovery with LVAD therapy? It should be noted that the present study by Diakos et al. (11) focused on the effects of mechanical unloading rather than the metabolic signature of
+
+![2_image_0.png](2_image_0.png)
+
+recovery because these were paired samples from patients bridged to transplantation. We are approaching the final follow-up phase of the prospective, multicenter study of myocardial reverse remodeling with LVAD and standard optimized heart failure therapies (ReSTAGE HF [Remission from Stage D Heart Failure]). The ReSTAGE-HF study was designed to evaluate the rate of recovery when centers optimize pump speed for unloading and use optimal heart failure therapy. By including intermittent testing of underlying function at set time points in a common protocol the rate of recovery under "best standard" conditions will be determined. Once this has been established the next phase will be to consider adjunctive therapies, that is, drugs and strategies that can be added, to firstly further promote reverse remodeling and enhance the rate and degree of myocardial reverse remodeling, and secondly and importantly, to enhance its durability. Previously, the Harefield, United Kingdom, group successfully added clenbuterol as an adjunctive second stage of the therapy, which was done to enhance the durability of recovery (1). Adding a period of cardiac reloading before explantation is also likely to be beneficial, and the work by these authors suggests this might have beneficial effects on mitochondrial structure and function. Like exercise, which has been shown to improve mitochondrial function, cardiac reloading may prove to be an important addition to recovery protocols. The current study also suggests that novel drugs targeting mitochondrial function and enhancement of metabolic adaptation might serve as important additions to recovery protocols to improve the myocardial function and durability of the therapeutic approach. Starting with this important contribution by Diakos et al. (11), further understanding of the metabolic phenotypes resulting from the reversal of heart failure is likely to enhance, not only our rate of myocardial recovery, but the durability after explantation that may be the real target in our efforts to reprogram the failing human heart.
+
+REPRINT REQUESTS AND CORRESPONDENCE: Dr. J. Eduardo Rame, Cardiovascular Institute, University of Pennsylvania, 11th Floor South Tower, 3400 Civic Center Boulevard, Philadelphia, Pennsylvania 19104.
+
+E-mail: Eduardo.rame@uphs.upenn.edu.
+
+## References
+
+1. Birks EJ, Tansley PD, Hardy J, et al. Left ventricular assist device and drug therapy for the reversal of heart failure. N Engl J Med 2006;355:
+1873–84.
+
+2. Dipla K, Mattiello JA, Jeevanandam V, Houser SR,
+Margulies KB. Myocyte recovery after mechanical circulatory support in humans with end-stage heart failure. Circulation 1998;97:2316–22.
+
+3. Drakos SG, Wever-Pinzon O, Selzman CH, et al. Magnitude and time course of changes induced by continuous-flow left ventricular assist device unloading in chronic heart failure: insights into cardiac recovery. J Am Coll Cardiol 2013;61:
+1985–94.
+
+4. Klotz S, Barbone A, Reiken S, et al. Left ventricular assist device support normalizes left and right ventricular beta-adrenergic pathway properties. J Am Coll Cardiol 2005;45:668–76. 5. Klotz S, Foronjy RF, Dickstein ML, et al. Mechanical unloading during left ventricular assist device support increases left ventricular collagen cross-linking and myocardial stiffness. Circulation 2005;112:364–74.
+
+6. NHLBI Working Group on Advancing the Science of Myocardial Recovery with Mechanical Circulatory Support. 2016. Available at: http:// www.nhlbi.nih.gov/research/reports/nhlbi-working -group-advancing-science-myocardial-recovery-mech anical-circulatory-support. Accessed September 18, 2016.
+
+7. Aubert G, Martin OJ, Horton JL, et al. The failing heart relies on ketone bodies as a fuel. Circulation 2016;133:698–705.
+
+8. Bedi KC Jr., Snyder NW, Brandimarto J, et al. Evidence for intramyocardial disruption of lipid metabolism and increased myocardial ketone utilization in advanced human heart failure. Circulation 2016;133:706–16.
+
+9. Kolwicz SC Jr., Airhart S, Tian R. Ketones step to the plate: a game changer for metabolic remodeling in heart failure? Circulation 2016;133:
+689–91.
+
+10. Taegtmeyer H. Failing heart and starving brain: ketone bodies to the rescue. Circulation 2016;134:265–6.
+
+11. Diakos NA, Navankasattusas S, Abel ED, et al. Evidence of glycolysis up-regulation and pyruvate mitochondrial oxidation mismatch during mechanical unloading of the failing human heart:
+implications for cardiac reloading and conditioning. J Am Coll Cardiol Basic Trans Science 2016;1:
+432–44.
+
+12. Davila-Roman VG, Vedala G, Herrero P, et al.
+
+Altered myocardial fatty acid and glucose metabolism in idiopathic dilated cardiomyopathy. J Am Coll Cardiol 2002;40:271–7.
+
+13. Taylor M, Wallhaus TR, Degrado TR, et al. An evaluation ofmyocardial fatty acid and glucose uptake using PET with [18F]fluoro-6-thia-heptadecanoic acid and [18F]FDG in patients with congestive heart failure. J Nucl Med 2001;42:55–62.
+
+14. Tenenbaum A, Fisman EZ. Impaired glucose metabolism in patients with heart failure:
+pathophysiology and possible treatment strategies. Am J Cardiovasc Drugs 2004;4:
+269–80.
+
+15. Wallhaus TR, Taylor M, DeGrado TR, et al. Myocardial free fatty acid and glucose use after carvedilol treatment in patients with congestive heart failure. Circulation 2001;103:
+2441–6.
+
+16. Chokshi A, Drosatos K, Cheema FH, et al. Ventricular assist device implantation corrects myocardial lipotoxicity, reverses insulin resistance, and normalizes cardiac metabolism in patients with advanced heart failure. Circulation 2012;125:
+2844–53.
+
+17. Rame JE. Chronic heart failure: a reversible metabolic syndrome? Circulation 2012;125:
+2809–11.
+
+18. Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1:
+785–9.
+
+## Key Words Cardiac Conditioning,
+
+cardiac metabolism, left ventricular assist device

medical/md/PMC6256180.md

@@ -0,0 +1,160 @@
+# Inhibition Of Osteoblast Differentiation By Ritonavir
+
+YOSHITAKA WAKABAYASHI1, YUSUKE YOSHINO2, KAZUNORI SEO2, ICHIRO KOGA2, TAKATOSHI KITAZAWA2 and YASUO OTA2 1Department of Infectious Diseases, The University of Tokyo Hospital, Tokyo 113‑8655; 2Department of Internal Medicine, Teikyo University, Tokyo 173‑8606, Japan Received March 30, 2018; Accepted September 26, 2018
+
+## Doi: 10.3892/Br.2018.1154
+
+Abstract. Osteoporosis is one of the chronic complications seen in human immunodeficiency virus (HIV)‑infected patients, and affects patients at high prevalence. The causes of osteoporosis in HIV‑infected patients are multiple, and include chronic HIV 
+infection, living habits such as smoking and alcohol consumption, and antiretroviral drug use. Among antiretroviral drugs, protease inhibitors have been reported to be associated with osteoporosis. However, it remains to be determined how anti‑HIV drugs affect osteoblast differentiation. In the current study, MC3T3‑E1 cells, a mouse osteoblastic cell line, were cultured in osteoblast differentiation medium with or without different protease inhibitors (ritonavir, lopinavir, darunavir or atazanavir), and alkaline phosphatase (ALP) activity and the expression of Runt‑related transcription factor 2 (Runx2) were analyzed. The ALP activity in MC3T3‑E1 cells cultured with ritonavir was significantly reduced compared with that in cells in only osteoblast inducer reagent, indicating that ritonavir inhibited osteoblast differentiation. Meanwhile, ALP activity was not reduced in cells cultured with any of the other inhibitors. In addition, ritonavir inhibited the expression of Runx2, a key regulator of osteoblast differentiation, in the early period of osteoblast differentiation. To the best of our knowledge, this is the first study to demonstrate that ritonavir inhibits osteoblast differentiation in *vitro*. The present findings may explain the mechanism of osteopenia induced by combination antiretroviral therapy involving protease inhibitors.
+
+## Introduction
+
+The progress of combination antiretroviral therapy (cART) has led to increased life expectancy of patients infected with human immunodeficiency virus (HIV)(1). However, as life expectancy has increased, chronic complications have become key challenges (2). Osteoporosis is among the chronic complications seen in HIV‑infected patients; the prevalence of osteoporosis is reportedly three times higher among HIV‑infected patients than non‑HIV‑infected patients (3). The reported prevalence rate of osteopenia in HIV‑infected cohorts ranges from 22 to 71%, and that of osteoporosis from 3 to 33% (4). The causes of osteoporosis in HIV‑infected patients are multiple, and include chronic HIV infection, living habits such as smoking and alcohol consumption, and antiretroviral drug use (5). Among antiretroviral drugs, protease inhibitors have been reported to be associated with osteoporosis, and antiretroviral regimens containing protease inhibitors can accelerate osteopenia and osteoporosis (6).
+
+Osteoporosis is caused by an imbalance of bone resorption and formation (7). Osteoclasts have a role in bone forma-tion, and osteoblasts that are derived from mesenchymal stem cells are responsible for bone formation (8). Osteoblast differentiation is regulated by transcription factors including Runt‑related transcription factor 2 (Runx2) (9). Runx2 is a positive regulator of genes associated with bone matrix proteins including collagen type I α 1/2 chain (9), and triggers the expression of major bone matrix genes during the early stages of osteoblast differentiation (9). As such, Runx2 is considered to serve a central role in skeletal development and be associated with osteoporosis (9). However, it remains to be determined specifically how anti‑HIV drugs affect osteoblast differentiation. Thus, in the present study, the influence of anti‑HIV drugs on osteoblast differentiation was examined in *vitro*.
+
+## Materials And Methods
+
+Cell line and culture. The clonal mouse osteoblastic cell line, MC3T3‑E1 subclone 14, was purchased from American Type Culture Collection (Manassas, VA, USA). The MC3T3‑E1 cells were cultured at 37˚C in 5% CO2 in Dulbecco's modi-fied Eagle's medium (DMEM; Sigma‑Aldrich; Merck KGaA, 
+Darmstadt, Germany) with 10% fetal bovine serum (Hyclone; GE Healthcare Life Science, Logan, UT, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin. To induce osteoblast differentiation, MC3T3‑E1 cells were cultured at 37˚C in 5% CO2 in DMEM with osteoblast inducer reagent containing 1% L‑ascorbic acid, 2% β‑glycerophosphate and 0.2% hydrocortisone (Takara Bio, Inc., Otsu, Japan). The medium was changed every other day.
+
+Anti‑HIV drugs. The HIV protease inhibitors ritonavir, darunavir, atazanavir and lopinavir were purchased from Toronto Research Chemicals, Inc. (Toronto, ON, Canada). These drugs were prepared as stock solutions in methanol. All prepared anti‑HIV drugs were mixed with osteoblast differentiation medium to adjust to the maximum serum concentration 
+(Cmax) that these drugs achieve when they are administered at treatment doses in adult HIV patients. Cmax values of protease inhibitors were based on those on the University of Liverpool website, www.hiv‑druginteraction.org. The Cmax values were: Ritonavir, 11.20 µg/ml; lopinavir, 9.60 µg/ml; darunavir, 6.50 µg/ml; and atazanavir 3.15 µg/ml. Alkaline phosphatase (ALP) activity. MC3T3‑E1 cells were seeded at a density of 1x104 cells/well in a 96‑well microplate in normal culture medium. To induce differentiation, the medium was replaced by DMEM with osteoblast inducer reagent after 1 day in the presence or absence of each protease inhibitor, respectively. The cells were incubated for 7 and 9 days in four types of medium: i) DMEM; ii) DMEM with osteoblast inducer reagent; iii) DMEM with osteoblast inducer reagent and an anti‑HIV drug; and iv) DMEM with vehicle (methanol). For subsequent treatments with ritonavir, MC3T3‑E1 cells were treated with various concentrations (0.1, 0.5, 1.0, 5.0 and 10.0 µg/ml ritonavir) and for various durations (10.0 µg/ml ritonavir for 9, 11 and 14 days). All cells were cultured at 37˚C in a 5% CO2 atmosphere. ALP activity was evaluated using a TRACP & ALP double‑staining kit (Takara Bio, Inc.) according to the manufacturer's protocol. Briefly, the plates were incubated with kit reagents at 37˚C for 15 min, absorbance was measured at 405 nm, and ALP activity was calculated from a standard value (bone ALP; Takara Bio, Inc.). The total protein concentration of each cell was determined by a DC™ Protein Assay (Bio‑Rad Laboratories, Inc., Hercules, CA, USA), and the activity data were normalized to the total protein concentration. Alizarin Red staining. MC3T3‑E1 cells were seeded at a density of 4x104 cells/well in a 24‑well plate. The cells were incubated with 10.0 µg/ml ritonavir or control mediums as above. Mineralization of MC3T3‑E1 cells was confirmed by using an Alizarin Red S staining kit (Cosmo Bio, Co., Ltd., Tokyo, Japan) after 7, 14, 21 and 28 days of culture. Then, cells were washed three times with phosphate‑buffered saline, with 500 µl of 100% methanol added to each well, and incubated at 4˚C for 20 min. Alizarin Red solution was added to each well and incubated at room temperature for 5 min. Each well was then washed again three times and directly observed. Total RNA extraction and cDNA synthesis. MC3T3‑E1 cells seeded at 1x105 cells/well in a 12‑well plate were incubated with 10.0 µg/ml ritonavir or control mediums as above for 3, 5 and 7 days. Total RNA was extracted from the MC3T3‑E1 cells by adding 350 µl Tripure (Roche, Basel, Switzerland) to each well. RNA was isolated according to the manufacturer's instructions. Total RNA was reverse‑transcribed into cDNA 
+by using ReverTra Ace® qPCR RT Master Mix (Toyobo Life Science, Osaka, Japan) according to the manufacturer's instructions.
+
+Quantitative polymerase chain reaction (qPCR). Runx2 gene expression in MC3T3‑E1 cells was examined by qPCR. β‑actin was used as an internal control gene. The qPCR was performed with a Roche LightCycler 480 system using a LightCycler 480 Probe Master kit (Roche Diagnostics, Basel, Switzerland). Reactions were performed in a final total volume of 20 µl containing 1x LightCycler 480 Probe Master mix, 0.1 µM of each forward and reverse primer, 3.8 µl distilled H2O and 5 µl of the cDNA template. Specific primers for mouse Runx2 and β‑actin were designed using the Universal ProbeLibrary (Roche Diagnostics). The primers used in the current study were as follows: For Runx2, forward, 5'‑cgtgtcagcaaagcttctttt‑3' and reverse, 5'‑ggctcacgtcgctcatct‑3'; and for β‑actin, forward, 5'‑tgacaggatgcagaaggaga‑3' and reverse, 5'‑cgctcaggaggagcaatg‑3'. The amplification conditions were as follows: Denaturation at 95˚C for 5 min, 45 cycles of amplification (95˚C for 10 sec, 60˚C for 30 sec and 72˚C for 1 sec), and cooling at 50˚C for 10 sec. Relative quantification of target gene expression was determined using the E‑Method (Efficiency Method) from the LightCycler 480 software (10). Lactate dehydrogenase (LDH) activity assay. LDH activity was measured to determine MC3T3‑E1 cell viability following treatment with ritonavir. MC3T3‑E1 cells were plated in 12‑well plates (1x105cells/well) and incubated in DMEM 
+with osteoblast inducer reagent in the presence or absence of ritonavir (10 µg/ml). On day 8, culture medium was replenished and cells were cultured again for 24 h. Cell medium and lysate extracted with radioimmunoprecipitation assay buffer (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were collected on day 9, and cell viability was evaluated using Cytotoxicity detection KitPLUS (Roche Diagnostics) according to the manufacturer's protocol. Cells in DMEM only were defined as a control. The absorbance was measured at 490 nm, and cell viability was calculated using the following formula: Cell viability (%) = {1‑[(absorbance of medium ‑ absorbance of control medium)/(absorbance of lysate ‑ absorbance of control lysate)]} x100. Statistical analysis. Significant differences among samples were determined by one‑way analysis of variance with post hoc Tukey's honest significant difference test, with P<0.05 considered to indicate statistical significance. At least three samples were tested in three independent experiments. All results are presented as means ± standard error of the mean. The data analysis was performed using JMP software version 12.2 (SAS Institute, Inc., Cary, NC, USA).
+
+## Results
+
+Osteoblast differentiation is inhibited by ritonavir. As documented, osteoblasts are differentiated by a medium that contains L‑ascorbic acid, β‑glycerophosphate and steroid (11). Since ALP activity is enhanced during osteoblast differentiation in *vitro*, ALP may be used as an osteogenic differentiation marker (12). First, the present study observed that ALP activity was enhanced in MC3T3‑E1 cells at 7 days after the addition of DMEM with osteoblast inducer reagent compared with in cells cultured in DMEM only (P<0.05; data not shown). Subsequently, the effect of protease inhibitors on 
+
+![2_image_0.png](2_image_0.png)
+
+osteoblast differentiation was examined. MC3T3‑E1 cells were cultured in osteogenic differentiation medium in the presence or absence of the protease inhibitors ritonavir, lopinavir, darunavir and atazanavir 3.15 µg/ml at their Cmax for 9 days, and then ALP activity was examined. The ALP activity of MC3T3‑E1 cells cultured with ritonavir was significantly reduced compared with that of the control cells in osteoblast induction medium alone (P<0.05). However, ALP activity was not reduced in cells cultured with the other anti‑HIV drugs (Fig. 1A). These results indicated that the protease inhibitor ritonavir inhibited osteoblast differentiation.
+
+Next, the relationship between ritonavir concentration and osteoblast differentiation was examined. MC3T3‑E1 cells were treated with various concentrations of ritonavir (0.1, 0.5, 1.0, 5.0 and 10.0 µg/ml). ALP activity was significantly reduced in MC3T3‑E1 cells cultured with 5.0 or 10.0 µg/ml ritonavir compared with the controls in osteoblast induction medium alone (P<0.05); however, lower concentrations of ritonavir did not inhibit ALP activity (Fig. 1B).
+
+The time course of ALP activity in MC3T3‑E1 cells cultured with ritonavir was also examined. MC3T3‑E1 cells were cultured in osteogenic differentiation medium with or without 10.0 µg/ml ritonavir for 9, 11 and 14 days. ALP activity was significantly reduced following ritonavir treatment at each time point (P<0.05; Fig. 1C). Effect of ritonavir on the expression of Runx2 in MC3T3‑E1 cells. RUNX2 is the key gene responsible for the differentiation of human mesenchymal cells into osteoblasts; it upregulates bone matrix proteins including ALP in the early phase of osteoblast differentiation (13). MC3T3‑1 cells were cultured in osteogenic differentiation medium with or without 10.0 µg/ml ritonavir for 3, 5 and 7 days, and the mRNA expression of Runx2 was measured by reverse transcription‑qPCR. On days 3 and 5, the mRNA levels of Runx2 were significantly reduced in differentiating cells cultured with ritonavir compared with in the differentiating controls (P<0.05); however, no significant differences were observed on day 7 (Fig. 2). These results indicated that ritonavir inhibited expression of Runx2 in the early phase of osteoblast differentiation. Osteoblast mineralization is inhibited by ritonavir. In the process of bone formation, osteoblasts need to differentiate and mineralize. Thus, whether ritonavir inhibited osteoblast mineralization was examined. MC3T3‑E1 cells were cultured in DMEM with osteoblast inducer reagent with or without 10 µg/ml ritonavir for 7, 14, 21 and 28 days. Then, cells were stained with Alizarin Red S to assess mineralization. At 7 and 14 days, cells treated with DMEM or with osteoblast induction medium, with or without ritonavir, exhibited no positive staining indicative of mineralization. At 21 and 28 days, cells treated with osteoblast induction medium with or without vehicle exhibited increasing mineralization, while those treated with DMEM only or osteoblast induction medium with ritonavir were not (Fig. 3). These results indicated that ritonavir suppressed or delayed osteoblast mineralization. Cell viability of MC3T3‑E1 cells treated with ritonavir. The cytotoxicity of ritonavir was assessed via measuring LDH activity. MC3T3‑E1 cells were cultured in osteogenic differentiation medium with or without 10.0 µg/ml ritonavir for 9 days, and the levels of LDH activity were measured. 
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+There were no statistically significant differences among the groups (Fig. 4). These results showed that ritonavir did not exert cytotoxic effects in MC3T3‑E1 cells.
+
+## Discussion
+
+Osteoporosis is one of the complications seen in patients with HIV. HIV itself suppresses osteogenesis (14). Several in *vivo* studies have reported that osteopenia and osteoporosis are common among patients treated with antiretroviral drugs (15‑18). Bedimo et al (15) reported that cumulative exposure to tenofovir and protease inhibitors, particularly lopinavir/ritonavir, was an independent predictor of increased risk of osteoporotic fracture in HIV patients on cART. Further, it was shown in the Data collection on Adverse events of anti‑HIV Drugs study that protease inhibitor use was one of the risk factors for osteoporosis (16,17). Duvivier et al (18) reported that decreased lumbar spine bone mineral density was more pronounced in patients receiving protease inhibitors (indinavir/ritonavir or lopinavir/ritonavir) compared with other antiretroviral drugs. By contrast, a cross‑sectional study identified decreased bone density in HIV‑positive patients irrespective of treatment with or without protease inhibitors (19). Thus, the association between osteoporosis and protease inhibitors in *vivo* is yet to be confirmed.
+
+The MC3T3‑E1 cell line is a pre‑osteoblast line derived from mouse calvaria (20). It is established to exhibit a time‑dependent and sequential expression of osteoblast characteristics analogous to in *vivo* bone formation, and is used as a bone differentiation and mineralization model in *vitro* (21). In the present study, ritonavir was indicated to affect the pathway of osteoblast differentiation and the time course of differentiation. There are a number of in *vitro* reports describing the relationship between certain antiretroviral drugs and osteoblast differentiation (21,22). Jain and Lenhard reported that two protease inhibitors, lopinavir and nelfinavir, decreased osteoblast ALP activity and gene expression in human mesenchymal stem cells (21). Another report demonstrated that ALP activity decreased significantly in human osteoblast cultures following exposure to nelfinavir and indinavir (22). Santiago et al has previously reported ritonavir may facilitate osteoclast differentiation (23); however, other findings have suggested that ritonavir could inhibit osteoclast formation and function (24). Thus, the exact effect of RTV on osteoblast cells remained unknown.
+
+Ritonavir was originally used for its antiviral action, but is now used as a booster of other protease inhibitors. The therapeutic adult dose of ritonavir is 600 mg twice a day. When 600 mg ritonavir is taken twice a day, the mean maximum and minimum serum concentrations (Cmax and Cmin) of ritonavir have been determined as 11.2±3.6 and 3.7±2.6 µg/ml, respectively (25). In the present study, osteoblast differentiation was inhibited by 5.0 and 10.0 µg/ml ritonavir. These results indicated the possibility that administration of therapeutic doses of ritonavir may inhibit osteoblast differentiation in *vivo*. However, as a booster, ritonavir is used at a dose of 100 to 200 mg a day, and the expected ritonavir Cmax is ~1.5 µg/ml, and therefore osteoblast differentiation appears unlikely to be inhibited in *vivo*. However, to our knowledge there are no reports of a pharmacokinetic analysis of ritonavir in bone. Thus, osteoblast differentiation may be suppressed in bone when the booster dose of ritonavir is administered.
+
+In the present study, ritonavir suppressed the expression of Runx2 mRNA. To the best of our knowledge, this is the first study to report a suppressive effect of ritonavir on the expression of Runx2, and that ritonavir may affect osteoblast differentiation and mineralization in MC3T3‑E1 cells. There are numerous pathways associated with osteoblast differentiation including the bone morphogenetic protein BMP and parathyroid hormone pathways (26). Runx2 is an essential transcription factor required for osteogenesis; Runx2‑knockout mice exhibit a complete absence of mature osteoblasts and ossification (26). In particular, Runx2 is a key regulator of osteoblast differentiation and regulates the expression of several osteoblastic genes, including collagen 1, osteopontin, osteocalcin and bone sialoprotein, to induce differentiation (13). In primary osteoblast cells isolated from the calvariae of rats, study has found ALP activity to be significantly decreased by Runx2 small interfering RNA treatment when cells were treated with osteoblast differentiation medium that included ascorbic acid and β‑glycerophosphate (27), indicating that Runx2 expression is upstream of ALP. In the present study, the expression of Runx2 mRNA was suppressed on days 3 and 5, but not on day 7 of treatment with ritonavir in osteoblast differentiation medium. Prior to day 7, the ALP activities of cells with or without differentiation medium did not change (not shown). But ALP activities were suppressed on day 9 or later of treatment with ritonavir in osteoblast differentiation medium. These results collectively indicate that ritonavir suppressed Runx2 directly or other regulators of osteoblast differentiation upstream of Runx2, and, as a result, ALP activity was suppressed.
+
+The present results also suggested inhibition of osteoblast mineralization by ritonavir. However, bone formation takes 3 to 4 months (28), and cells in the current study were cultured for only 28 days (4 weeks). Thus, the possibility that ritonavir simply delayed osteoblast mineralization cannot be excluded.
+
+In conclusion, the present study is seemingly the first to demonstrate that ritonavir may inhibit osteoblast differentiation in *vitro*. Expression of Runx2 was suppressed by ritonavir in MC3T3‑E1 cells. This may explain the mechanism of osteopenia induced by cART involving protease inhibitors.
+
+## Acknowledgements
+
+Not applicable.
+
+## Funding
+
+No funding was received.
+
+## Availability Of Data And Materials
+
+The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
+
+## Authors' Contributions
+
+YW, YY, KS, IK, TK and YO designed the study. YW processed the experimental data, performed the analysis, drafted the manuscript and produced the figures. All authors discussed the results and contributed to the final manuscript.
+
+## Ethics Approval And Consent To Participate
+
+Not applicable. Patient consent for publication Not applicable.
+
+## Competing Interests
+
+The authors declare that they have no competing interests.
+
+## References
+
+1. Lohse N, Hansen AB, Gerstoft J and Obel N: Improved survival in HIV‑infected persons: Consequences and perspectives. J Antimicrob Chemother 60: 461‑463, 2007.
+
+2. Maartens G, Celum C and Lewin SR: HIV infection: 
+Epidemiology, pathogenesis, treatment, and prevention. Lancet 384: 258‑271, 2014.
+
+3. Brown TT and Qaqish RB: Antiretroviral therapy and the prevalence of osteopenia and osteoporosis: A meta‑analytic review. AIDS 20: 2165‑2174, 2006.
+
+4. Del Carpio‑Cano FE, Dela Cadena RA and Sawaya BE: HIV and bone disease: A perspective of the role of microRNAs in bone biology upon HIV infection. J Osteoporos 2013: 571418, 2013.
+
+5. McComsey GA, Tebas P, Shane E, Yin MT, Overton ET, Huang JS, 
+Aldrovandi GM, Cardoso SW, Santana JL and Brown TT: Bone disease in HIV infection: A practical review and recommen- dations for HIV care providers. Clin Infect Dis 51: 937‑946, 2010.
+
+6. Tebas P, Powderly WG, Claxton S, Marin D, Tantisiriwat W, 
+Teitelbaum SL and Yarasheski KE: Accelerated bone mineral loss in HIV‑infected patients receiving potent antiretroviral therapy. AIDS 14: F63‑F67, 2000.
+
+7. Kanis JA: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis: Synopsis of a WHO report. WHO Study Group. Osteoporos Int 4: 368‑381, 1994.
+
+8. Endo I and Mastumoto T: Bone and stem cells: Regulatory mechanism of mesenchymal stem cell differentiation to osteo- blasts. Clin Calcium 24: 555‑564, 2014 (In Japanese).
+
+9. Komori T: Regulation of bone development and maintenance by Runx2. Front Biosci 13: 898‑903, 2008.
+
+10. Tellmann G: The E‑Method: A highly accurate technique for gene‑expression analysis. Nat Methods 3: i‑ii, 2006.
+
+11. Jaiswal N, Haynesworth SE, Caplan AI and Bruder SP: Osteogenic differentiation of purified, culture‑expanded human mesenchymal stem cells in vitro. J Cell Biochem 64: 295‑312, 1997.
+
+12. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, 
+Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 284: 143‑147, 1999.
+
+13. Xiao ZS, Liu SG, Hinson TK and Quarles LD: Characterization of the upstream mouse Cbfa1/Runx2 promoter. J Cell Biochem 82: 647‑659, 2001.
+
+14. Cotter EJ, Malizia AP, Chew N, Powderly WG and Doran PP: 
+HIV proteins regulate bone marker secretion and transcription factor activity in cultured human osteoblasts with consequent potential implications for osteoblast function and development. AIDS Res Hum Retroviruses 23: 1521‑1530, 2007.
+
+15. Bedimo R, Maalouf NM, Zhang S, Drechsler H and Tebas P: 
+Osteoporotic fracture risk associated with cumulative exposure to tenofovir and other antiretroviral agents. AIDS 26: 825‑831, 2012.
+
+16. Friis‑Møller N, Reiss P, Sabin CA, Weber R, Monforte A, 
+El‑Sadr W, Thiébaut R, De Wit S, Kirk O, Fontas E, et al; DAD Study Group: Class of antiretroviral drugs and the risk of myocardial infarction. N Engl J Med 356: 1723‑1735, 2007.
+
+17. Flint OP, Noor MA, Hruz PW, Hylemon PB, Yarasheski K, 
+Kotler DP, Parker RA and Bellamine A: The role of protease inhibitors in the pathogenesis of HIV‑associated lipodystrophy: Cellular mechanisms and clinical implications. Toxicol Pathol 37: 65‑77, 2009.
+
+18. Duvivier C, Kolta S, Assoumou L, Ghosn J, Rozenberg S, 
+Murphy RL, Katlama C and Costagliola D; ANRS 121 Hippocampe study group: Greater decrease in bone mineral density with protease inhibitor regimens compared with nonnucleoside reverse transcriptase inhibitor regimens in HIV‑1 infected naive patients. AIDS 23: 817‑824, 2009.
+
+19. Amiel C, Ostertag A, Slama L, Baudoin C, N'Guyen T, 
+Lajeunie E, Neit‑Ngeilh L, Rozenbaum W and De Vernejoul MC: 
+BMD is reduced in HIV‑infected men irrespective of treatment. J Bone Miner Res 19: 402‑409, 2004.
+
+20. Kodama H, Amagai Y, Sudo H, Kasai S and Yamamoto S: 
+Establishment of a clonal osteogenic cell linefrom newborn mouse calvaria. Jap J Oral Biol 23: 899‑901, 1981.
+
+21. Jain RG and Lenhard JM: Select HIV protease inhibitors alter bone and fat metabolism ex vivo. J Biol Chem 277: 19247‑19250, 2002.
+
+22. Malizia AP, Cotter E, Chew N, Powderly WG and Doran PP: HIV 
+protease inhibitors selectively induce gene expression alterations associated with reduced calcium deposition in primary human osteoblasts. AIDS Res Hum Retroviruses 23: 243‑250, 2007.
+
+23. Santiago F, Oguma J, Brown AM and Laurence J: Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir. Biochem Biophys Res Commun 417: 223‑230, 2012.
+
+24. Wang MW, Wei S, Faccio R, Takeshita S, Tebas P, Powderly WG, 
+Teitelbaum SL and Ross FP: The HIV protease inhibitor ritonavir blocks osteoclastogenesis and function by impairing RANKL‑induced signaling. J Clin Invest 114: 206‑213, 2004.
+
+25. Ltd AL: Norvir® Tablets Summary of Product Characteristics. 
+
+2010.
+
+26. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, 
+Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, et al: 
+Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89: 755‑764, 1997.
+
+27. Lin L, Shen Q, Leng H, Duan X, Fu X and Yu C: Synergistic inhibition of endochondral bone formation by silencing Hif1α and Runx2 in trauma‑induced heterotopic ossification. Mol Ther 19: 1426‑1432, 2011.
+
+28. Sims NA and Martin TJ: Coupling the activities of bone formation and resorption: A multitude of signals within the basic multicellular unit. Bonekey Rep 3: 481, 2014.
+
+This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License.

medical/md/PMC6827196.md

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+# Improvement Of Solubility And Yield Of Recombinant Protein Expression In E. Coli **Using A Two-Step System**
+
+Tahereh Sadeghian-Rizi1,2, Azade Ebrahimi1,2, Fatemeh Moazzen1**, Hesam Yousefian2**, 
+and Ali Jahanian-Najafabadi1,* 
+1Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran. 2Student Research Committee, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran. 
+
+## Abstract
+
+Overexpression of recombinant proteins in *Escherichia coli* results in inclusion body formation, and consequently decreased production yield and increased production cost. Co-expression of chaperon systems accompanied by recombinant protein is a general method to increase the production yield. However, it has not been successful enough due to imposed intense stress to the host cells. The aim of this study was to balance the rate of protein production and the imposed cellular stresses using a two-step expression system. 
+
+For this purpose, in the first step, green fluorescent protein (GFP) was expressed as a recombinant protein model under control of the T7-TetO artificial promoter-operator, accompanied by Dnak/J/GrpE chaperon system. Then, in the next step, TetR repressor was activated automatically under the control of the stress promoter ibpAB and suppressed the GFP production after accumulation of inclusion bodies. Thus in this step incorrect folded proteins and inclusion bodies are refolded causing increased yield and solubility of the recombinant protein and restarting GFP expression again. Total GFP, soluble and insoluble GFP fractions, were measured by Synergy H1 multiple reader. Results showed that expression yield and soluble/insoluble ratio of GFP have been increased 5 and 2.5 times using this system in comparison with the single step process, respectively. The efficiency of this system in increasing solubility and production yield of recombinant proteins was confirmed. The two-step system must be evaluated for expression of various proteins to further confirm its applicability in the field of recombinant protein production. 
+
+Keywords: Chaperons; *E. coli*; GFP; Inclusion bodies; Protein solubility; Recombinant proteins. 
+
+## Introduction
+
+Recombinant proteins have many applications in the course of molecular biology, food industries, and pharmaceutical companies (1). The universality of the genetic codons permits protein expression in heterologous organisms (2). *Escherichia coli* (*E. coli)* is one of the most frequently used production host due to the extensive information of its physiological and genetics processes, ease of cultivation, low cost of its growth, and achievement of high cell density fermentations (3,4). Overexpression of the recombinant protein in this organism often leads to incorrect folding, aggregation, and inclusion body formation. Limitation 
+*Corresponding author: A. Jahanian-Najafabadi Tel: +98-3137927056, Fax: +98-3136680011 Email: jahanian@pharm.mui.ac.ir of the host chaperon capacity may be one of 
+
+![0_image_2.png](0_image_2.png)
+
+the causes of this problem (5). Chaperons help proper folding of the newly synthesized proteins and refolding aggregated and misfolded proteins to the native conformation as protein quality control system in cells (6). 
+
+DnaK and GroEL/ES systems are two main chaperon systems in the *E. coli* cells (7-9). 
+
+General approaches for increasing protein production yields are based on the expression of recombinant proteins accompanied by chaperons whose unbalanced expression cause severe stress in host cells lowering the success of the system (10,11). 
+
+Other researchers designed expression processes to equilibrate protein production and cellular stresses. Previously, a two-step procedure reported by Marco *et al.* showed efficient enhancement of solubility. 
+
+In the first step of this procedure the four chaperon systems were coordinately co-overproduced with recombinant proteins to optimized folding. Then in the next step protein biosynthesis was inhibited by removal of isopropyl β- d-1-thiogalactopyranoside (IPTG) to permit chaperone-mediated refolding of the misfolded and aggregated proteins *in vivo*. This strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold (12). Disadvantage of this method is the need of external intervention for protein expression inhibition which is impractical and time consuming with expensive process in the industrial scale. Dragosits *et al.* presented a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. In their study the soluble/insoluble ratio of recombinant protein increased but total protein yield decreased (13). In the present research we investigated the latter system by some modifications in the expression vectors, expression conditions, and in the presence of DnaK/J/GrpE chaperon system for increasing protein solubility without decreasing total protein yield. In this manner, recombinant protein production was suppressed by the precise feedback system in the appropriate time by the cell itself and after refolding of the misfolded and aggregated proteins, expression of recombinant protein was restarted.
+
+ 
+
+## Materials And Methods Bacterial Strains
+
+E. coli strains used in this study were XL1-blue and BL21 [DE3]. All strains were grown on LB agar plates or in LB broth supplemented with 100 μg/mL ampicillin, 50 μg/mL kanamycin and/or 34 μg/mL 
+chloramphenicol as necessary. 
+
+## Plasmid Construction
+
+GFP and TetR plasmids The schematic presentation of the produced recombinant vectors is represented in Fig. 1. 
+
+Briefly, to produce the presented constructs, Tet operator (TetO) sequence was inserted downstream of the T7 promoter sequence and a ribosome binding site sequence of the pET28a vector (13). Then, the GFP coding sequence was codon optimized for expression in *E. coli BL21*(DE3) cells and cloned downstream of the ribosome binding site sequence. Finally, the corresponding DNA 
+sequence was ordered to be synthesized by Biomatik Company (Canada) and cloned into the BglII and XhoI restriction sites of the pET28a plasmid. 
+
+The TetR repressor sequence was also codon optimized for optimal expression in E. coli BL21 and was cloned under the control of stress-sensitive IbpAB promoter (PIbpAB) and a mutant weak ribosome binding site sequence. Finally, the corresponding DNA 
+sequence was ordered to be synthesized by Biomatik Company (Canada) and cloned to the *BglI*I and XhoI restriction sites of the pET15-b plasmid. All of the above cloning procedures were authenticated by restriction endonuclease digestion and finally by DNA sequencing (14). The confirmed pET28a derived plasmid was designated as pET28-T7-
+TetO-GFP and pET15b as pET15b-IPAB-TetR plasmids. 
+
+Under this circumstance, GFP is expected to be expressed under the control of T7 promoter until formation of inclusion bodies enough for activation of PIbpAB
+promoter, which results in the production of TetR protein, and consequently inhibition of GFP production via activation of Tet-O 
+operator on the pET28-GFP. 
+
+## Chaperon Plasmid
+
+In order to verify the effect of simultaneous application of the chaperones on the production yield of recombinant GFP protein produced by the mentioned controllable system, commercial vector pKJE7 from Takara Company (Takara, Japan) was used for expression of DnaK/J/GrpE chaperon system under control of L-arabinose promoter. 
+
+![2_image_0.png](2_image_0.png)
+
+Fig. 1. The schematic presentation of the produced recombinant vectors. A, pET15b-TetR vector and B, pET28a-GFP vector
+Protein expression Co-transformation of the plasmids was performed by electroporation using Electro-competent *E. coli BL21 (DE3)* cells. Then, the cells were cultivated in the presence of suitable antibiotics in accordance with the plasmids each cell harbored. For the one-step procedure, a single colony from the cells transformed with the pET28a-GFP was used to inoculate 5 mL of LB medium supplemented with kanamycin (50 µg/mL) and incubated overnight at 37 °C and 180 rpm. The overnight culture (500 µL) was used to inoculate the 50 mL LB medium supplemented with kanamycin (50 µg/mL) and incubated in conditions mentioned above. At an optical density of 0.6 at 600 nm, GFP expression was induced by addition of 1 mM IPTG and cells were further incubated overnight at 20 °C. 
+
+Expression of GFP was also optimized based on tuning of three variables, including temperature (25, 30, and 37 °C), 
+IPTG concentration (0, 0.5, 1, and 1.5 mM), and post-induction cultivation time (2, 4, 6, 8, and 24 h) (15). For the two-step procedure, a single colony of the cells transformed with the pET28a-GFP, pET15b-TetR, and pKJE7 was cultured in 5 mL of LB medium supplemented with kanamycin (50 µg/mL), ampicillin (100 µg/mL), and chloramphenicol 
+(25 µg/mL), and incubated overnight as mentioned above. L-arabinose (0.4 g/mL) was added first to induce chaperon system expression, and IPTG (1 mM) was added at an optical density of 0.6 at 600 nm. Then, the cells were further incubated overnight at 20 °C. GFP expression was investigated in various combinations of pET28a-GFP with the two other plasmids 
+(pET15b-TetR and pKJE7) (12,13). In fact, host cell was transformed with the plasmids in 3 combinations including combination 1, pET28a-GFP and pET15bTetR, for evaluation of the effect of TetR on the GFP expression yield and soluble/insoluble ratio; combination 2, pET28a-GFP and pKJE7, for evaluation of the effect of DnaK/J/GrpE 
+chaperon system on GFP expression yield and soluble/insoluble ratio; and combination 3, pET28a-GFP, pET15b-TetR, and pKJE7, for investigation of the two-step procedure effect on total GFP expression and soluble/insoluble ratio. 
+
+## Protein Purification And Analysis Of Soluble And Insoluble Protein Ratio
+
+Preparation of soluble and insoluble GFP fractions were performed according to the method based on single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form (16). Briefly, the cells were harvested at 5000 g for 20 min at 4 °C, 
+resuspended in 15 mL phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 12H2O, 2 mM KH2PO4, pH 8.0), and then lysed by sonication on ice. 
+
+The lysate was centrifuged at 12,000 g for 20 min at 4 °C. The clarified supernatant was collected as soluble fraction of GFP 
+and retained for analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis 
+(SDS-PAGE) and fluorescence measurement. 
+
+The insoluble pellet was resuspended in 20 mL 
+washing buffer (50 mM tris-Hcl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% Triton™ 
+X-100, 1M urea) and then analyzed by SDS-PAGE. The pellet was washed with 20 mL washing buffer for three times, then washed with PBS buffer to remove TritonTM X-100 detergent and centrifuged at 12000 g for 20 min at 4 °C. For subsequent solubilization and refolding, the purified inclusion body pellet was resuspended in PBS buffer containing 2 M urea, frozen at -20 °C for overnight, thawed at room temperature, and centrifuged at 12,000 g for 15 min at 4 °C. Supernatant was collected as insoluble fraction of GFP and retained for analysis by SDS-PAGE and fluorescence measurement. 
+
+## Fluorescence Measurements 
+
+![3_Image_0.Png](3_Image_0.Png)
+
+The total GFP, soluble, and insoluble GFP amounts were measured using Synergy H1 Multiple Reader (BioTek, USA) using the following settings: 37 °C, excitation wavelength 485 nm, emission wavelength 535 nm and gain 75. The GFP fluorescence values were reported as GFP fluorescence per optical density at 600 nm (13). 
+
+Statistical analyses Data are presented as mean ± standard deviation (SD) of at least three experiments. Statistical significance was tested by two-tailed t-test and two-way ANOVA test. Analyses were performed using GraphPad Prism 8.0.2 software. P < 0.05 was considered significant. 
+
+## Results Optimization Of Gfp Expression In One-Step Procedure
+
+Expression of the GFP protein by the transformed *E. coli* BL21 cells with pET28a-GFP plasmid was verified by fluorescent microscopy (Fig. 2A). Then, the optimized conditions for GFP expression was investigated by evaluating the effects of temperature, IPTG concentration, and post-induction incubation time. As shown in Fig. 2B, the maximal GFP expression was achieved at 25 °C, 1 mM IPTG 
+and a post-induction incubation time of 16 h (results related to the effect of time are not presented). 
+
+![4_image_0.png](4_image_0.png)
+
+## Effect Of Two-Step Procedure On Gfp Expression
+
+Figure 3 shows the effect of TetR 
+expression on GFP production. We observed GFP expression was increased 2.4 times where the feedback loop, encoded by pET15b-TetR, was induced (Fig. 3A). 
+
+Expression of TetR under control of stress-sensitive IbpAB promoter translated to increased soluble/insoluble ratio of the produced GFP (2.6 times higher than the expression of GFP in the absence of TetR) 
+(Fig. 3B,3C). As shown in Fig. 3A-3C, 
+co-expression of DnaK/J/grpE with GFP did not exhibit notable effect on the yield of total GFP expression compared with the two other groups but a significant increase of soluble/insoluble ratio was observed 
+(3 times). Figure 3A-3C shows that total GFP expression was increased 5 times using the two-step expression procedure in comparison with GFP expression in the absence of TetR and DnaK/J/GrpE. On the other hand, the main fraction of total GFP 
+was in the soluble form (soluble/insoluble ratio 8.5). 
+
+ 
+
+## Discussion
+
+Bacterial inclusion bodies are insoluble protein aggregates formed in the bacterial cytoplasm and periplasm during the overproduction of recombinant proteins, especially those from mammalian or viral origin (17,18). Factors including high copy number of target gene, strong promoter, as well as high temperature during protein expression, and high inducer concentrations favor high level expression of recombinant protein and increase the possibility of the inclusion body formation (19-22). 
+
+Reducing environment of bacterial cytosol, amino acid sequence of protein with highly hydrophobic proteins, lack of post-translational modification, and eukaryotic chaperones also favor this event (23). 
+
+Inclusion bodies formation imposes a great obstacle in the production and downstream processing of recombinant proteins (24). Solubilization and refolding of inclusion bodies into bioactive form is a challenging task. If an efficient and convenient way of decreasing formation of inclusion bodies without decreasing protein production yield could be developed, it would reduce the need of excess chromatographic purification steps and downstream processing costs (25). 
+
+At the midstream level, co-expression of folding modulators such as chaperones, lowering the transcription rate, and reducing the growth temperature have been thoroughly explored strategies which have been successful for an important number of specific proteins but they do not offer generic solutions to this matter (26-28). Co-expression of molecular chaperones and foldases from accessory plasmids with no further control results in imbalanced expression of these proteins and has detrimental effects on the production process (29,30). Dragosits *et al.* developed a synthetic expression platform that enables the cell to shut down the recombinant protein production mechanism automatically by itself through stress-induced feedback to address challenges such as balancing protein production and cellular stress. This system limited stressful protein expression and increased the total soluble fraction but significantly decreased the protein yield. 
+
+The authors suggested further investigation on promoter and repressor engineering, other recombinant proteins, and different inducer concentrations for avoiding such loss (13). 
+
+In another study, Marco *et al.* used a two-step procedure that shows the strongest enhancement of the solubility. In the first step of this procedure the four chaperon systems were coordinately co-overproduced with recombinant proteins to optimized folding and in the next step protein biosynthesis was inhibited by removal of IPTG to permit chaperone mediated refolding of the misfolded and aggregated proteins *in vivo*. This strategy increased the solubility of 64 different heterologous proteins up to 42 folds without decreasing total protein yield (12). 
+
+Disadvantage of this method is requirement of external intervention to inhibit protein expression and an impractical and expensive process in industrial scale. We combined the two methods with some modifications. We substituted pNf-TetR with pET15b-TetR and pET23b-GFP with pET28a. In this study, both soluble/insoluble ratio and total GFP expression was increased (2.5 and 5 times, respectively), while in the study of Dragosits et al. soluble/insoluble ratio was increased up to 1.5 times but total GFP expression was decreased 1.4 times as explained earlier. This may be due to the use of pET15b vector for cloning of TetR, which retains similar copy number to pET28a as carrier of GFP encoding sequence in comparison with pNF. 
+
+Second reason may be related to the use of DnaK/J/GrpE chaperon system and increasing of chaperon capacity of bacterial cells which resulted in folding of misfolded and aggregated GFP, suppression of stress-sensitive IbpAB promoter and excessive expression of TetR as suppressor of GFP expression and resuming of its expression. It would be interested to test the efficacy of this system with other recombinant proteins. 
+
+## Conclusion
+
+ 
+Altogether, in the present study, the use of an automatic negative feed-back system, combined with co-expression of major cytosolic chaperon system DnaK/J/GrpE, resulted in an increased solubility and total recombinant protein expression. 
+
+Therefore, this system is very applicable, beneficial, and cost-effective in recombinant protein expression field of biotechnology. 
+
+Further studies on using this system for expression of various recombinant therapeutic proteins are undergoing in our laboratory. 
+
+## Acknowledgments
+
+This study was conducted by Tahereh Sadeghian, and aslo Azadeh Ebrahimi which was financially supported by Student Research Committee of School of Pharmacy and Pharmaceutical Sciences, and Research Deputy of Isfahan University of Medical Sciences, Isfahan, Iran through the Grants No. 
+
+194105 and 194296, respectively. 
+
+## References
+
+1. Ben-Nissan G, Vimer S, Warszawski S, Katz A, 
+Yona M, Unger T, *et al*. Rapid characterization of secreted recombinant proteins by native mass spectrometry. Commun Biol. 2018;1(1):213-224. 
+
+2. Kyratsous CA, Silverstein SJ, DeLong CR, 
+Panagiotidis CA. Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in *Escherichia coli*. 
+
+Gene. 2009;440(1-2):9-15. 
+
+3. Chen R. Bacterial expression systems for recombinant protein production: *E. coli* and beyond. Biotechnol Adv. 2012;30(5):1102-1107. 
+
+4. Panda AK. Bioprocessing of therapeutic proteins from the inclusion bodies of *Escherichia coli*. 
+
+Adv Biochem Eng Biotechnol. 2003;85:43-93. 
+
+5. Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, 
+et al. Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-stepdenaturing and refolding method. PLoS One. 
+
+2011;6(7):e22981. 
+
+6. Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14(10):630-642. 
+
+7. Calloni G, Chen T, Schermann SM, Chang HC, 
+Genevaux P, Agostini F, *et al.* DnaK functions as a central hub in the *E. coli* chaperone network. 
+
+Cell Rep. 2012;1(3):251-264. 
+
+8. Aguilar-Rodríguez J, Sabater-Muñoz B, MontagudMartínez R, Berlanga V, Alvarez-Ponce D, Wagner A, *et al*. The molecular chaperone DnaK is a source of mutational robustness. Genome Biol Evol. 2016;8(9):2979-2991. 
+
+9. Kedzierska S, Staniszewska M, Wegrzyn A, 
+Taylor A. The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from *Escherichia coli* cells. FEBS Lett. 1999;446 (2-3):331-337.
+
+10. Gopal GJ, Kumar A. Strategies for the production of recombinant protein in *Escherichia coli*. Protein J. 
+
+2013;32(6):419-425. 
+
+11. Xiao S, Shiloach J, Betenbaugh MJ. Engineering cells to improve protein expression. Curr Opin Struct Biol. 2014;26:32-38. 
+
+12. de Marco A, Deuerling E, Mogk A, Tomoyasu T, 
+Bukau B. Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 2007;7:32-40. 
+
+13. Dragosits M, Nicklas D, Tagkopoulos I. A synthetic biology approach to self-regulatory recombinant protein production in *Escherichia coli*. J Biol Eng. 2012;6(1):2-11. 
+
+14. Jahanian-Najafabadi A, Soleimani M, Azadmanesh K, Mostafavi E, Majidzadeh AK. Molecualr cloning of the capsular antigen F1 of Yersinia pestis in pBAD/gIII plasmid. Res Pharm Sci. 2015;10(1): 
+84-89. 
+
+15. Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, 
+et al. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in *E. coli*. 
+
+BMC Biotechnol. 2015;1(15):13-21. 
+
+16. Qi X, Sun Y, Xiong S. A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microb Cell Fact. 2015;14:24-35. 
+
+17. Garcia-Fruitos E. Inclusion bodies: a new concept. 
+
+Microb Cell Fact. 2010;9:80-82. 
+
+18. Rodriguez-Carmona E, Cano-Garrido O, 
+Seras-Franzoso J, Villaverde A, Garcia-Fruitos E. Isolation of cell-free bacterial inclusion bodies. 
+
+Microb Cell Fact. 2010;9:71-79. 
+
+19. Singh A, Upadhyay V, Upadhyay AK, Singh SM, 
+Panda AK. Protein recovery from inclusion bodies of *Escherichia coli* using mild solubilization process. Microb Cell Fact. 2015;14:41-50. 
+
+20. Carrio MM, Villaverde A. Localization of chaperones DnaK and GroEL in bacterial inclusion bodies. J Bacteriol. 2005;187:3599-3601. 
+
+21. Chang CC, Song J, Tey BT, Ramanan RN. 
+
+Bioinformatics approaches for improved recombinant protein production in *Escherichia coli*: protein solubility prediction. Brief Bioinform. 
+
+2014;15(6):953-962. 
+
+22. Rosano GL, Morales ES, Ceccarelli EA. 
+
+New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 2019;28(8):1412-1422 23. Carrio MM, Cubarsi R, Villaverde A. Fine architecture of bacterial inclusion bodies. 
+
+FEBS Lett. 2000;471(1):7-11. 
+
+24. Rosano GL, Ceccarelli EA. Recombinant protein expression in *Escherichia coli*: advances and challenges. Front Microbiol. 2014;5:172-188. 
+
+25. Salehinia J, Sadeghi HMM, Abedi D, Akbari V. 
+
+Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 
+
+single-chain antibody fragment inclusion bodies. Res Pharm Sci. 2018;13(6):566-574. 
+
+26. Garcia-Fruitos E, Sabate R, de Groot NS, Villaverde A, Ventura S. Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J. 
+
+2011;278(14):2419-2427. 
+
+27. Liu L, Yang H, Shin HD, Chen RR, Li J, Du G, et al. How to achieve high-level expression of microbial enzymes: strategies and perspectives. Bioengineered. 2013;4(4):212-223. 
+
+28. Martínez-Alonso M, González-Montalbán N, 
+García-Fruitós E, Villaverde A. Learning about protein solubility from bacterial inclusion bodies. 
+
+Microb Cell Fact. 2009;8(1):4-8. 
+
+29. Martínez-Alonso M, García-Fruitós E, 
+Ferrer-Miralles N, Rinas U, Villaverde A. Side effects of chaperone gene co-expression in recombinant protein production. Microb Cell Fact. 2010;9(1): 64-69.
+
+30. Gasser B, Saloheimo M, Rinas U, Dragosits M, 
+Rodríguez-Carmona E, Baumann K, *et al.* Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact. 2008;7(1):11-28. 

medical/md/PMC7252995.md

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+# Prompt Recognition Of Infectious Pulmonary Tuberculosis Is Critical To Achieving Elimination Goals: A Retrospective Cohort **Study**
+
+Courtney Heffernan ,1 James Barrie,2 Alexander Doroshenko,3 Mary Lou Egedahl,1 Catherine Paulsen,1 Ambikaipakan Senthilselvan,4 Richard Long1 To cite: Heffernan C, Barrie J, 
+Doroshenko A, *et al*. Prompt recognition of infectious pulmonary tuberculosis is critical to achieving elimination goals: a retrospective cohort study. *BMJ Open Resp Res* 2020;7:e000521. doi:10.1136/
+bmjresp-2019-000521 ► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ bmjresp-2019-000521). Received 29 October 2019 Revised 16 April 2020 Accepted 29 April 2020 © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
+
+1Department of Medicine; Division of Pulmonary Medicine, University of Alberta, Edmonton, Alberta, Canada 2Department of Radiology and Diagnostic Imaging, University of Alberta, Edmonton, Alberta, Canada 3Department of Medicine; Division of Preventive Medicine, University of Alberta, Edmonton, Alberta, Canada 4School of Public Health, University of Alberta, Edmonton, Alberta, Canada Correspondence to Dr Richard Long; rlong@ualberta.ca
+
+## Abstract
+
+Introduction All pulmonary tuberculosis (PTB) cases are presumed to be infectious to some degree. This spectrum of infectiousness is independently described by both the acidfast bacilli smear and radiographic findings. Smear-positive patients with chest radiographic findings that are typical for adult-type PTB are believed to be most infectious.
+
+Hypothesis Characterisation of the presumed most infectious PTB case is possible by reference to readily available clinical features and laboratory results.
+
+Methods Retrospective cohort study of adult, culturepositive PTB cases (151 smear-positive; 162 smear-negative) 
+diagnosed between 1 January 2013 and 30 April 2017 in Canada. We describe cases according to demographic, clinical and laboratory features. We use multivariable multinomial logistic regression to estimate the relative risk ratio (RRR) with 95% CI of features associated with an outcome of smear-positive PTB, characterised by 'typical' chest radiograph findings.
+
+Results Being Canadian-born, symptomatic, having a subacute duration of symptoms and broad-spectrum antibiotic prescriptions were all more commonly associated with smear-positive than smear-negative disease (36% vs 20%; 95% vs 63%; 88% vs 54%; and 59% vs 28%, 
+respectively). After combining smear status and radiographic features, we show that smear-positive patients with typical chest radiographs were younger, had a longer duration of symptoms (RRR 2.41; 95% CI 1.01 to 5.74 and 2.93; 95% CI 
+1.20 to 7.11, respectively) and were less likely to be foreignborn, or have a moderate to high-risk factor for reactivation 
+(RRR 0.40; 95% CI 0.17 to 0.92 and 0.18; 95% CI 0.04 to 0.71, respectively) compared with smear-negative patients with atypical chest radiograph findings.
+
+Conclusion A clear picture of the presumed most infectious PTB case emerges from available historical and laboratory information; vigilance for this presentation by front-line providers will support elimination strategies aimed at reducing transmission.
+
+Introduction Elimination of tuberculosis (TB) requires a dual-pronged approach. Both interrupting transmission by promptly diagnosing and 
+
+## Key Messages
+
+What is the key question?
+
+► Can we identify a highly infectious pulmonary tuberculosis (TB) case in high-income, low-TB incidence settings?
+
+What is the bottom line?
+
+► A combination of readily available clinical (n=6) and laboratory (n=2) features describes the prototypical infectious case of pulmonary TB.
+
+Why read on?
+
+► The timely diagnosis of infection-spreading cases, especially by front-line providers, is critical to interrupting transmission, and thus, to meeting TB elimination targets.
+
+treating infection-spreading cases, and preventing TB among those already infected but who have not yet developed a disease are necessary. As TB becomes less common in high-income, low-incidence settings, the prompt diagnosis of infection-spreading cases by front-line physicians and nurses becomes increasingly difficult. These cases are mainly bacillary positive (smear-positive, culturepositive and smear-negative, culture-positive) 
+pulmonary TB (hereafter 'PTB'). Among culture-positive PTB cases, those with smearpositive disease are 5–10 times more likely to generate a secondary case than those who are smear-negative.1–3 In addition, among persons with smear-positive PTB, those with 'typical' findings on chest radiograph (defined below) are 10 times more likely to generate a secondary case than smearpositive cases without such features.4 Thus, both the smear status and the presentation of disease on chest radiograph contribute independently to transmission.4–7 Six features from the medical history and two readily available laboratory tests (the chest radiograph and complete blood count (CBC)) have emerged, both within the literature and clinical practice, as being critical to suspecting PTB rather than disease attributable to other respiratory pathogens, and subsequently ordering the collection of sputum or other specimens for mycobacteriological examination.8 9 The relative predictive value of each variable on an outcome of PTB in a specialised TB clinic has previously been shown.10 What has not been shown is how well the combination of these features differentiates the highly infectious case from the minimally infectious case, among whom the collection of a sputum is imperative for achieving public health goals.
+
+The objective of this current study is to distinguish smear-positive and smear-negative PTB by describing the relative frequency of the aforementioned features in a well-defined cohort of adult patients. While all patients with PTB are presumed infectious to some degree, it is the smear-positive case with typical chest radiographic findings whose timely diagnosis offers the greatest potential for interrupting transmission, and impacting public health. Meeting this objective could establish a clinical threshold for front-line providers to consider the diagnosis and seek subsequent laboratory confirmation, which is important to TB elimination efforts.11
+
+## Methods
+
+This study was performed in Alberta—population over 4.25million in 2016 (Statistics Canada)—one of four major immigrant-receiving provinces in Canada, the other three being British Columbia, Ontario and Quebec. In Alberta, in 2016, the crude incidence rate of TB was 5.6 per 100000 population. University-based pulmonary and infectious disease physicians staff three dedicated public health clinics that provide TB services to the province.9 Language line (translation) services are available as necessary. Introduction of an assessment form into the two clinics supported by University of Alberta staff occurred in 2013. It records relevant information for each referred case. We identified a cohort of consecutive, adult (age >14 years), culture-positive pulmonary cases diagnosed between 1 January 2013 and 30 April 2017 from these two clinics. We supplemented data collected through the assessment form with findings from the Provincial Laboratory for Public Health, which performs all mycobacteriology in the province. Per standard of care, patients whose initial sputum specimens were smear-negative had three additional specimens collected at the time of reporting of the first positive culture to confirm that they were still smear-negative on the start date of treatment, which, in Alberta, is the date of diagnosis. The initial isolate of each case was genotyped using mycobacterial interspersed repetitive units, and laboratory cross-contamination was excluded in all patients with a single, smear-negative, culture-positive specimen.12 Basic demographic and clinical information is used to describe the cohort of patients in this study. These data included: age at diagnosis (15–64 years, >64 years), sex, population group (Canadian born and foreign born), disease type (new active vs relapse/retreatment) and HIV status (positive, negative). We then examined the following eight explanatory variables, which we hypothesise describe the most infectious case of PTB.
+
+Clinical characteristics included, as above, six pieces of information related to patient histories: (1) symptoms, (2) duration of symptoms, (3) absence of dyspnoea, (4) broad-spectrum antibiotic prescriptions in the 6months antedating diagnosis, (5) epidemiological risk of exposure to, or infection with, *Mycobacterium tuberculosis*, and 
+(6) risk factors for the reactivation of latent TB infection 
+(LTBI). Symptoms, if present, were reported as respiratory (dry cough, productive cough, haemoptysis or chest pain), constitutional (fever, night sweats, weight loss or fatigue) or both.13 The absence of dyspnoea is known to favour a diagnosis of PTB over community-acquired pneumonia; parallel reductions in ventilation and perfusion at sites of PTB disease are known to preserve gas exchange.14–16 We documented antibiotic prescriptions in individual records, and confirmed their prescription for respiratory complaints by cross-referencing the cohort against the provincial (Alberta Health) stakeholder registry, which, in turn, reports physician claims for the reimbursement of services provided.
+
+Epidemiological risks include: a history of TB, and whether it was adequately treated, a history of overt contact with PTB, and whether it was adequately assessed (eg, tuberculin skin test or interferon gamma release assay) and treated, migration from a high-incidence country of birth, or travel and work to/within a high-incidence country, an occupational history (eg, healthcare worker), 
+and/or social risk factors such as homelessness, incarceration and alcohol/substance misuse where non-overt exposures might occur. Online supplementary table 1 provides a description of social factors including homelessness, incarceration and alcohol/substance misuse. 
+
+We reported the presence or absence of one or more 'moderate' or 'high risk' factors for the reactivation of LTBI, as described in the Canadian Tuberculosis Standards.17 Finally, we report the results of two routine laboratory tests performed at the time of diagnosis: the CBC and the chest radiograph. The results of the CBC are grouped into four patterns based on the presence or absence of anaemia, and/or leucocytosis.18 19 An experienced chest radiologist, blind to the smear status of the patients, re-read the posterior-anterior and lateral chest images performed at the time of diagnosis. In the context of this study, d frontal images were divided into four quadrants by halving a perpendicular drawn from the apex of the lung to the dome of the hemidiaphragm. Documented were the presence or absence of (1) parenchymal infiltrates and their dominant location—no distinction was made between infiltrates that were airspace, interstitial, nodular or some combination of these; (2) cavities, defined as a gas-filled space within pulmonary consolidation, a mass, or a nodule; (3) 
+adenopathy—hilar, mediastinal or both; and (4) pleural effusion.
+
+As previously reported, we categorised patients as having 'typical' versus 'atypical' radiographs based on the characteristics listed above.4 20 For patients with infiltration localised to, or predominantly in the upper long zones, with or without cavitation, but with no discernable intrathoracic adenopathy, the radiograph is 'typical' for adult-type PTB. In patients with (1) no abnormality; (2) intrathoracic adenopathy with or without parenchymal disease; (3) a localised or predominant lower lung zone infiltrate, with or without cavitation; (4) an isolated pleural effusion; and (5) a miliary (diffuse micronodular) pattern, the radiograph is 'atypical' for adult-type PTB. We report extent of disease on chest radiograph as normal, minimal, moderately, far advanced or miliary according to criteria established by the US National Tuberculosis and Respiratory Disease Association.21
+
+## Statistical Analysis
+
+We describe cases by smear status according to each of the aforementioned features. We tested the difference in proportions, by smear status, of each feature between two independent samples using the direct test, Fisher's exact test or χ2 test as appropriate. Thereafter, we generated a combined outcome of smear status and radiographic findings, creating an outcome for multinomial logistic regression that was a nominal variable with four responses: atypical smear-negative, typical smear-negative, atypical smear-positive and typical smear-positive, with atypical smear-negative considered to be the baseline group. The model includes all features and confounders considered clinically relevant to making a timely diagnosis of TB from a review of the literature. The total sample size of 313 in this study achieves 80% power or greater to detect an effect size (W) of 0.19 or greater for comparison of proportions between four groups using 3 df, and χ2 test with a significance level (alpha) of 0.05.22 We performed all statistical analyses using STATA V.14, StataCorp 2015. There were no missing data within this cohort. Patient and public involvement We did not involve patients and the public in this study given its retrospective design, but we anticipate that the findings presented herein will lead to benefits for future cohorts of patients.
+
+## Results
+
+There were 313 patients in the cohort, 151 (48%) smearpositive and 162 (52%) smear-negative. Being Canadianborn was associated with a greater likelihood of having smear-positive than smear-negative disease (36% vs 20%); otherwise there were no statistically significant differences in the age, sex, disease type,or HIV status between these two groups (see table 1). The leading countries of birth of foreign-born patients were the Philippines (38%), India (16%), Vietnam (5%) and Somalia 
+(5%); Bhutan, Ethiopia and China each contributed 3% of foreign-born patients.
+
+As shown in table 2, being symptomatic with either respiratory, constitutional or both types of symptoms at the time of diagnosis was more likely in smear-positive than smear-negative patients (95% vs 63%). Cough (dry or productive) and haemoptysis were more common in smear-positive than smear-negative patients (83% vs 45%, and 25% vs 6%, respectively). Chest pain was equally uncommon in both groups (11% vs 9%). Dyspnoea was also relatively uncommon in both groups (28% vs 14%). Each of the major constitutional symptoms was more common in smear-positive than smear-negative patients (weight loss, 50% vs 28%; fever, 45% vs 20%; night sweats, 24% vs 10%; and fatigue, 21% vs 12%). Finally, having a combination of respiratory and constitutional symptoms (70% vs 28%) and a duration of symptoms ≥2 weeks (88% vs 54%) were statistically significantly more common in smear-positive than smear-negative patients. Among smear-positive and smear-negative patients with any symptoms, 93% and 86%, respectively, had symptoms for ≥2 weeks (data not shown).
+
+We dichotomised possible combinations of antibiotic prescription, epidemiological risk and reactivation risk, and describe their associations with an outcome of either smear-positive or smear-negative disease (table 3). A history of broad-spectrum antibiotic prescription for a respiratory complaint in the 6 months preceding the date of diagnosis was more likely among smear-positive patients (57% vs 23%); of those, multiple prescriptions were also more likely among smear-positive patients (49% vs 36%—data not shown). Macrolides, penicillins and fluoroquinolones were, in order of frequency, the three major classes of antibiotics prescribed. If one assumes that antibiotic prescriptions for respiratory complaints, in the months immediately preceding their diagnosis, were due to PTB then there were missed opportunities to diagnose these patients. In absolute numbers, there were 291 antibiotic prescriptions in the 6 months antedating a TB diagnosis; 208 among persons ultimately diagnosed with smear-positive PTB and 83 among persons ultimately diagnosed with smear-negative PTB (data not shown).
+
+Almost all patients in both groups (95%) had some epidemiological risk. The distribution of social risk factors was similar in smear-positive and smear-negative patients. Notably, however, they were much more common among Canadian-born than among foreign-born patients (see online supplementary table 1). A moderate to high risk of reactivation was similarly uncommon in both smearpositive and smear-negative patients (6% vs 9%).
+
+Anaemia was more common in smear-positive than smear-negative cases (65% vs 42%). Anaemia was usually of the chronic disease, iron deficiency varieties or both (data not shown). A leucocytosis was uncommon in both groups, but more uncommon in smear-negative cases 
+
+| Table 1                                                                                                                       | Demographic and clinical characteristics of patients with pulmonary tuberculosis by smear status Smear status* Positive Negative n (%) n (%)   | P value‡   |            |           |
+|-------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|------------|------------|-----------|
+| Assessed, n                                                                                                                   | 313                                                                                                                                            | 151        | 162        |           |
+| Age  15–64 years                                                                                                              | 248                                                                                                                                            | 120 (79.4) | 128 (79.0) | 0.38      |
+|                                                                                                                               | >64 years                                                                                                                                      | 65         | 31 (20.6)  | 34 (21.0) |
+| Sex  Male                                                                                                                     | 173                                                                                                                                            | 86 (57.0)  | 87 (53.8)  | 0.56      |
+|                                                                                                                               | Female                                                                                                                                         | 140        | 65 (43.0)  | 75 (46.2) |
+| Population group†  Foreign-born                                                                                               | 226                                                                                                                                            | 97 (64.3)  | 129 (79.7) | 0.002     |
+|                                                                                                                               | Canadian-born                                                                                                                                  | 87         | 54 (35.7)  | 33 (20.3) |
+| Disease type  New active                                                                                                      | 280                                                                                                                                            | 135 (89.4) | 145 (89.5) | 0.97      |
+|                                                                                                                               | Relapse/retreatment                                                                                                                            | 33         | 16 (10.6)  | 17 (10.5) |
+| HIV status  Negative                                                                                                          | 300                                                                                                                                            | 147 (97.4) | 153 (94.5) | 0.19      |
+|                                                                                                                               | Positive                                                                                                                                       | 13         | 4 (2.6)    | 9 (5.5)   |
+| Total                                                                                                                         |                                                                                                                                                |            |            |           |
+| Characteristics                                                                                                               | n                                                                                                                                              |            |            |           |
+| *Smear-positive patients had to have one or more airway secretion specimens, on or before the start date of treatment, with a |                                                                                                                                                |            |            |           |
+
+*Smear-positive patients had to have one or more airway secretion specimens, on or before the start date of treatment, with a semiquantitative smear size of 1+ or greater. †Canadian-born includes 61 indigenous patients and 26 Canadian-born non-indigenous patients. Among the foreign-born patients, one smear-positive and 28 smear-negative were referred to public health through the immigration medical screening surveillance mechanism. ‡Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences.
+
+(21% and 7%). The mean and median haemoglobin in smear-positive males and females was 127 and 126g/L, and 112 and 112g/L, respectively. In smear-negative males and females, it was 135 and 142g/L, and 123 and 124g/L, respectively. The mean and median leucocyte count was 8.6×109/L and 7.8×109/L in smear-positive cases, and 7×109/L and 7×109/L in smear-negative cases. The reference range for a normal haemoglobin is 135–175g/L and 120–160g/L for males and females, respectively. The reference range for a normal leucocyte count is 4.0–11.0×109/L.
+
+Chest radiographic features that are 'typical' for adulttype PTB, bilateral disease, cavitation and moderate or far-advanced disease were all more common in smearpositive than smear-negative patients (78% vs 36%; 
+56% vs 21%; 62% vs 6%; and 81% vs 22%, respectively). Among smear-positive patients, those with cavitation had a statistically significantly higher semiquantitative smear grade (3+ to 4+) vs (1+ to 2+), 82% vs 46% than those without cavitation (data not shown). Only three patients, one smear positive and two smear negative, had a miliary pattern.
+
+Table 4 shows the findings of our multinomial logistic regression. An absence of dyspnoea was more likely among smear-negative patients with typical chest radiograph findings than smear-negative patients with atypical chest radiograph findings; lower lung zone disease and dependent effusions—atypical findings on chest radiograph—are presumed to more negatively affect gas exchange (relative risk ratio (RRR) 4.71, 95% CI 1.69 to 13.18). Young age and a subacute or chronic duration of symptoms were more greatly associated with smearpositive patients who have typical chest radiographs than smear-negative patients with atypical chest radiographs (RRR 2.41; 95% CI 1.01 to 5.74, and RRR 2.93; 95% CI 1.20 to 7.11, respectively), while being foreign born and having a moderate to high-risk factor for reactivation were less commonly associated with smear-positive PTB with typical chest radiographs than smear-negative patients with PTB with atypical chest radiographs (RRR 0.40; 95% CI 0.17 to 0.92, and RRR 0.18; 95% CI 0.04 to 0.71, respectively) (see table 4 and online supplementary table 2). All major symptoms, respiratory and constitutional, were more commonly associated with typical presentation, cavitation, bilateral and moderate to advanced disease on chest radiograph (see online supplementary table 3).
+
+## Discussion
+
+It has previously been established that of the information readily available to front-line providers, three pieces are key to triggering suspicion of PTB. These include symptoms, especially if both respiratory and constitutional and subacute or chronic, and *typical* chest radiographic features; a public health trigger is an epidemiological 
+
+| Table 2                                                                                                                                                                                                     | Symptoms and their duration in patients with pulmonary tuberculosis by smear status Smear status Total   |            |            |            |        |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------|------------|------------|------------|--------|
+| Characteristics                                                                                                                                                                                             | n                                                                                                        | Positive   | Negative   |            |        |
+| n (%)                                                                                                                                                                                                       | n (%)                                                                                                    | P value*   |            |            |        |
+| Assessed, n                                                                                                                                                                                                 | 313                                                                                                      | 151        | 162        | -          |        |
+| Symptomatic  Yes                                                                                                                                                                                            | 245                                                                                                      | 143 (94.7) | 102 (63.0) | <0.001     |        |
+|                                                                                                                                                                                                             | No                                                                                                       | 68         | 8 (5.3)    | 60 (37.0)  |        |
+| Symptoms  Respiratory                                                                                                                                                                                       | 217                                                                                                      | 133 (88.1) | 84 (51.8)  | <0.001     |        |
+|                                                                                                                                                                                                             | Cough (dry)                                                                                              | 149        | 90 (59.6)  | 59 (36.4)  |        |
+|                                                                                                                                                                                                             | Cough (productive)                                                                                       | 50         | 36 (23.8)  | 14 (8.6)   |        |
+|                                                                                                                                                                                                             | Haemoptysis                                                                                              | 46         | 37 (24.5)  | 9 (5.5)    |        |
+|                                                                                                                                                                                                             | Chest pain                                                                                               | 31         | 17 (11.2)  | 14 (8.6)   |        |
+|                                                                                                                                                                                                             | Absence of dyspnoea                                                                                      |            |            |            |        |
+|                                                                                                                                                                                                             | Yes                                                                                                      | 249        | 109 (72.2) | 140 (86.4) | <0.001 |
+|                                                                                                                                                                                                             | No                                                                                                       | 64         | 42 (27.8)  | 22 (13.6)  |        |
+|                                                                                                                                                                                                             | Constitutional                                                                                           | 176        | 113 (74.8) | 63 (38.8)  | <0.001 |
+|                                                                                                                                                                                                             | Weight loss                                                                                              | 121        | 76 (50.3)  | 45 (27.7)  |        |
+|                                                                                                                                                                                                             | Fever                                                                                                    | 101        | 68 (45.0)  | 33 (20.3)  |        |
+|                                                                                                                                                                                                             | Night sweats                                                                                             | 52         | 36 (23.8)  | 16 (9.8)   |        |
+|                                                                                                                                                                                                             | Fatigue                                                                                                  | 51         | 31 (20.5)  | 20 (12.3)  |        |
+|                                                                                                                                                                                                             | Both†                                                                                                    |            |            |            |        |
+|                                                                                                                                                                                                             | Yes                                                                                                      | 151        | 105 (69.5) | 46 (28.4)  | <0.001 |
+|                                                                                                                                                                                                             | No                                                                                                       | 162        | 46 (30.5)  | 116 (71.6) |        |
+| Duration of symptoms  ≥2 weeks                                                                                                                                                                              | 221                                                                                                      | 133 (88.0) | 88 (54.3)  | <0.001     |        |
+|                                                                                                                                                                                                             | <2weeks                                                                                                  | 92         | 18 (12.0)  | 74 (45.7)  |        |
+| *Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. †Patients with some combination of both respiratory and constitutional symptoms. |                                                                                                          |            |            |            |        |
+
+risk of having or acquiring LTBI (see figure 1). Our study shows that although a public health risk was similarly common in smear-positive and smear-negative cases (95% and 97%, respectively), clinical triggers (symptoms: 95% vs 63%, and typical chest radiographic features: 78% vs 36%) were much more common in smear-positive cases; fortuitous given that smear positivity and typical radiographic features are presently the major determinants of infectiousness, and therefore the public health risk PTB poses.
+
+1–4 7 Other variables, including broad-spectrum antibiotic prescription history, reactivation risk factors and the CBC findings, are supportive in considering the likelihood of PTB.
+
+Individually, each of the features identified in the multinomial logistic regression model as distinguishing 
+(typical) smear-positive cases from (atypical) smearnegative cases has precedent in the literature, including younger age23 24; symptoms and their duration25–27; less likely to be foreign-born14 28; and less likely to have a moderate to high-risk factor for reactivation.29–32 When combined, the features have utility for describing the prototypical smear-positive patient with typical chest radiographic features: a young middle-aged man or woman with an epidemiological risk for LTBI, and a subacute or chronic duration of symptoms, a history of broad-spectrum antibiotic prescriptions, rarely having a risk factor for reactivation, and with an anaemia and a normal or low leucocyte count on CBC.
+
+In the future, semiquantitative results from the real-time PCR method, Xpert MTB/RIF assay, may replace smear microscopy and its current role as a proximate measure of infectiousness.33 34 There is, however, no expectation that this technology will impact the picture of the prototypical infectious case described herein. By contrast, cough aerosol technology might. For reasons that are only speculative at this time, some patients, whether smear positive or smear negative, are better able to generate an infectious aerosol than others.35 Therefore, once available, this technology is expected to further clarify the picture of the most infectious cases. The earlier the detection of these cases, the lesser the transmission and the smaller the reservoir of LTBI, the seedbed of future cases.
+
+Table 3 Broad-spectrum antibiotic use, epidemiological risk, reactivation risk and laboratory features in patients with 
+
+pulmonary tuberculosis by smear status
+
+| pulmonary tuberculosis by smear status                                                                                     | Smear status                                   |            |            |            |        |
+|----------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|------------|------------|------------|--------|
+| Total                                                                                                                      |                                                |            |            |            |        |
+| Characteristics                                                                                                            | n                                              | Positive   | Negative   |            |        |
+| n (%)                                                                                                                      | n (%)                                          | P value*   |            |            |        |
+| Assessed, n                                                                                                                | 313                                            | 151        | 162        | -          |        |
+| Antibiotic use†  Yes                                                                                                       | 124                                            | 86 (56.9)  | 38 (23.5)  | <0.001     |        |
+|                                                                                                                            | No                                             | 189        | 65 (43.1)  | 124 (76.5) |        |
+| Epidemiological risk‡  Yes                                                                                                 | 301                                            | 144 (95.4) | 157 (96.9) | 0.48       |        |
+|                                                                                                                            | No                                             | 12         | 7 (4.3)    | 5 (3.1)    |        |
+| Reactivation risk§  Yes                                                                                                    | 23                                             | 9 (5.9)    | 14 (8.6)   | 0.35       |        |
+|                                                                                                                            | No                                             | 290        | 142 (94.1) | 148 (91.4) |        |
+| Complete blood count¶  Normal haemoglobin; leucocytosis**                                                                  | 8                                              | 7 (4.6)    | 1 (0.6)    | 0.03       |        |
+|                                                                                                                            | Normal haemoglobin; normal/low leucocyte count | 139        | 46 (30.4)  | 93 (57.4)  | <0.001 |
+|                                                                                                                            | Anaemia and leucocytosis**                     | 36         | 25 (16.5)  | 11 (6.7)   | 0.01   |
+|                                                                                                                            | Anaemia and normal/low leucocyte count         | 130        | 73 (48.5)  | 57 (35.3)  | 0 .01  |
+| Chest radiographs  Typical appearance††                                                                                    | 176                                            | 118 (78.1) | 58 (35.8)  | <0.001     |        |
+|                                                                                                                            | Bilateral‡‡                                    | 118        | 84 (55.6)  | 34 (21.1)  | <0.001 |
+|                                                                                                                            | Cavitary                                       | 103        | 93 (61.5)  | 10 (6.1)   | <0.001 |
+|                                                                                                                            | Extent: moderate to far-advanced disease§§     | 159        | 123 (81.4) | 36 (22.2)  | <0.001 |
+| *Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. |                                                |            |            |            |        |
+
+*Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. †Refers to broad-spectrum antibiotic use in the 6months preceding the date of diagnosis (the start date of treatment). ‡Epidemiological risk for exposure to *Mycobacterium tuberculosis*. §Moderate to high-risk factors for the reactivation of tuberculosis in those with latent tuberculosis (TB) infection, according to the Canadian Tuberculosis Standards. ¶The reference range for a normal haemoglobin is 135–175g/L and 120–160g/L for males and females, respectively. The reference range for a normal leucocyte count is 4.0–11.0x109/L. Anaemia and leucocytosis among smear-positive cases as compared with smear-negative cases (65% vs 42% and 21% vs 7%) were significant at p≤0.05, using Pearson's χ2 test. **Fisher's exact test and χ2 test used to test for differences.
+
+††Typical=upper lung zone predominant infiltrate, with or without cavitation, but no discernable adenopathy. ‡‡Of the 176 cases without bilateral disease, 27 had a normal chest X-ray (CXR): 25 smear negative and 2 smear positive. One of the 25 smear-negative cases with a 'normal' CXR had evidence of intrathoracic adenopathy and normal-appearing lung parenchyma. §§Includes three cases with a miliary pattern (see text).
+
+It is a common misconception that smear-negative disease is early disease, and smear-positive disease is late disease and that the extent of disease on chest radiograph is an indication of its duration, that is, that a minimal lesion is early disease and a moderate or advanced lesion is old or chronic disease. Years ago, Kurt Toman used data from longitudinal studies, and periodic X-ray surveys of large populations to dispel this idea.27 He concluded that both types of disease—advanced smear-positive TB and minimal, only culture-positive TB—usually developed within the same time frame; thus, making a diagnosis at first presentation an important piece of the elimination puzzle. A recent proof-of-concept study supports his findings of different phenotypic expressions of disease.20 The proportion of smear-positive and smear-negative patients in our study that were asymptomatic (5% and 37%, respectively) is remarkably similar to what he reported across diverse ethnic groups many years ago.36–38 The strengths of this study include the completeness of the data, made possible by the use of an assessment form and the centralised nature of the programme, and the validation of key variables, for example, the attribution of smear status, radiographic features and broad-spectrum antibiotic prescriptions. Consecutive patients in the jurisdiction and time period of the study were included. This may be considered both a strength (reflecting programmatic realities and an unfiltered comparison of smearpositive and smear-negative disease) and a weakness 
+(the pathway to diagnosis in smear-negative cases may be through active vs passive case finding activities such as Table 4 Multivariable multinomial regression showing the associations between demographic and clinical characteristics to a combined smear, and radiographic outcome
+
+| Age (years) Sex Population group Absence of dyspnoea Duration of symptoms* Antibiotics† Epidemiological risk§ Reactivation risk¶ CBC** Demographic  and clinical  characteristics   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Atypical (base) Typical Atypical **Typical** n (%) n (%) RRR (95%CI) n (%) RRR (95%CI) n (%) **RRR (95%CI)**
+
+Assessed, n 51 111 33 118 Age (years) >64 15 (23.1) 19 (29.2) 1.00 14 (21.5) 1.00 17 (26.2) 1.00 15��64 36 (14.5) 92 (37.1) 1.68 (0.71 to 3.92) 19 (7.6) 0.56 (0.20 to 1.55) 101 (40.7) 2.41 (1.01 to 5.74) Sex Female 22 (15.7) 53 (37.9) 1.00 13 (9.3) 1.00 52 (37.1) 1.00 Male 29 (16.8) 58 (33.5) 0.77 (0.38 to 1.56) 20 (11.6) 1.15 (0.45 to 2.93) 66 (38.2) 0.90 (0.44 to 1.83) Population group Canadian born 12 (13.8) 21 (24.1) 1.00 8 (9.2) 1.00 46 (52.9) 1.00 Foreign born 39 (17.3) 90 (40.0) 1.00 (0.42 to 2.37) 25 (11.0) 1.09 (0.36 to 3.32) 72 (31.7) 0.40 (0.17 to 0.92) Absence of dyspnoea No 15 (23.4) 7 (11.0) 1.00 16 (25.0) 1.00 26 (40.6) 1.00 Yes 36 (14.4) 104 (41.8) 4.71 (1.69 to 13.18) 17 (6.8) 0.59 (0.22 to 1.60) 92 (37.0) 1.85 (0.79 to 4.34) Duration of symptoms* <2 weeks 18 (19.5) 56 (60.9) 1.00 3 (3.3) 1.00 15 (16.3) 1.00 ≥2weeks 33 (14.9) 55 (24.9) 0.68 (0.31 to 1.75) 30 (13.6) 3.86 (0.93 to 15.92) 103 (46.6) 2.93 (1.20 to 7.11) Antibiotics† No 35 (18.5) 89 (47.1) 1.00 13 (6.9) 1.00 52 (27.5) 1.00 Yes‡ 16 (12.9) 22 (17.7) 0.74 (0.31 to 1.75) 20 (16.1) 2.14 (0.79 to 5.82) 66 (53.2) 1.76 (0.79 to 3.89) Epidemiological risk§ None/low 24 (16.3) 45 (30.6) 1.00 17 (11.6) 1.00 61 (41.5) 1.00 Moderate to high 27 (16.3) 66 (39.8) 0.89 (0.42 to 1.87) 16 (9.6) 1.20 (0.46 to 3.11) 57 (34.3) 0.77 (0.36 to 1.61) Reactivation risk¶ None/low 44 (15.2) 104 (35.9) 1.00 28 (9.7) 1.00 114 (39.3) 1.00 Moderate to high 7 (30.4) 7 (30.4) 0.43 (0.13 to 1.43) 5 (21.7) 1.24 (0.32 to 4.80) 4 (17.4) 0.18 (0.04 to 0.79) CBC** Low risk 5 (11.4) 7 (15.9) 1.00 5 (11.4) 1.00 27 (61.4) 1.00 High risk 46 (17.1) 104 (38.7) 1.26 (0.36 to 3.28) 28 (10.4) 0.64 (0.15 to 2.61) 91 (33.8) 0.49 (0.16 to 1.48)
+
+*The presence of 'symptoms' and the 'duration of symptoms' were considered collinear—here we report just on the duration of symptoms. †One or more broad-spectrum antibiotics prescribed in the 6months prior to diagnosis. ‡One or more broad-spectrum antibiotic prescription(s) within six months of the PTB diagnosis with no, or a limited and unsustained 
+
+therapeuticbenefit.
+
+§Epidemiological risks none/low=no known occupational risk, travel history, migration from endemic nation, place of residence in a high-incidence indigenous community, recent contact with a known TB case plus no history of TB; OR, a history of TB/latent TB infection (LTBI) that has been adequately treated. Moderate to high=occupational risk, travel history, migration from endemic nation, place of residence in a high-incidence 
+
+indigenous community, recent contact with a known tuberculosis (TB) case and/or a positive tuberculin skin test (TST)/interferon gamma release 
+
+assay (IGRA) or a history of TB that was inadequately treated or with no record of treatment. ¶Per the Canadian Tuberculosis Standards, 7th Edition. **Low risk=combined normal haemoglobin; leucocytosis and anaemia; leucocytosis. High risk=combined normal haemoglobin; normal/low 
+
+leucocyte count and anaemia; normal/low leucocyte count.
+
+| Smear negative                                                                                                                        | Smear positive   |             |                      |             |                      |             |                     |
+|---------------------------------------------------------------------------------------------------------------------------------------|------------------|-------------|----------------------|-------------|----------------------|-------------|---------------------|
+| Atypical (base)                                                                                                                       | Typical          | Atypical    | Typical              |             |                      |             |                     |
+| n (%)                                                                                                                                 | n (%)            | RRR (95%CI) | n (%)                | RRR (95%CI) | n (%)                | RRR (95%CI) |                     |
+| Assessed, n                                                                                                                           | 51               | 111         | 33                   | 118         |                      |             |                     |
+| >64                                                                                                                                   | 15 (23.1)        | 19 (29.2)   | 1.00                 | 14 (21.5)   | 1.00                 | 17 (26.2)   | 1.00                |
+| 15–64                                                                                                                                 | 36 (14.5)        | 92 (37.1)   | 1.68 (0.71 to 3.92)  | 19 (7.6)    | 0.56 (0.20 to 1.55)  | 101 (40.7)  | 2.41 (1.01 to 5.74) |
+| Female                                                                                                                                | 22 (15.7)        | 53 (37.9)   | 1.00                 | 13 (9.3)    | 1.00                 | 52 (37.1)   | 1.00                |
+| Male                                                                                                                                  | 29 (16.8)        | 58 (33.5)   | 0.77 (0.38 to 1.56)  | 20 (11.6)   | 1.15 (0.45 to 2.93)  | 66 (38.2)   | 0.90 (0.44 to 1.83) |
+| Canadian born                                                                                                                         | 12 (13.8)        | 21 (24.1)   | 1.00                 | 8 (9.2)     | 1.00                 | 46 (52.9)   | 1.00                |
+| Foreign born                                                                                                                          | 39 (17.3)        | 90 (40.0)   | 1.00 (0.42 to 2.37)  | 25 (11.0)   | 1.09 (0.36 to 3.32)  | 72 (31.7)   | 0.40 (0.17 to 0.92) |
+| No                                                                                                                                    | 15 (23.4)        | 7 (11.0)    | 1.00                 | 16 (25.0)   | 1.00                 | 26 (40.6)   | 1.00                |
+| Yes                                                                                                                                   | 36 (14.4)        | 104 (41.8)  | 4.71 (1.69 to 13.18) | 17 (6.8)    | 0.59 (0.22 to 1.60)  | 92 (37.0)   | 1.85 (0.79 to 4.34) |
+| <2 weeks                                                                                                                              | 18 (19.5)        | 56 (60.9)   | 1.00                 | 3 (3.3)     | 1.00                 | 15 (16.3)   | 1.00                |
+| ≥2weeks                                                                                                                               | 33 (14.9)        | 55 (24.9)   | 0.68 (0.31 to 1.75)  | 30 (13.6)   | 3.86 (0.93 to 15.92) | 103 (46.6)  | 2.93 (1.20 to 7.11) |
+| No                                                                                                                                    | 35 (18.5)        | 89 (47.1)   | 1.00                 | 13 (6.9)    | 1.00                 | 52 (27.5)   | 1.00                |
+| Yes‡                                                                                                                                  | 16 (12.9)        | 22 (17.7)   | 0.74 (0.31 to 1.75)  | 20 (16.1)   | 2.14 (0.79 to 5.82)  | 66 (53.2)   | 1.76 (0.79 to 3.89) |
+| None/low                                                                                                                              | 24 (16.3)        | 45 (30.6)   | 1.00                 | 17 (11.6)   | 1.00                 | 61 (41.5)   | 1.00                |
+| Moderate to high                                                                                                                      | 27 (16.3)        | 66 (39.8)   | 0.89 (0.42 to 1.87)  | 16 (9.6)    | 1.20 (0.46 to 3.11)  | 57 (34.3)   | 0.77 (0.36 to 1.61) |
+| None/low                                                                                                                              | 44 (15.2)        | 104 (35.9)  | 1.00                 | 28 (9.7)    | 1.00                 | 114 (39.3)  | 1.00                |
+| Moderate to high                                                                                                                      | 7 (30.4)         | 7 (30.4)    | 0.43 (0.13 to 1.43)  | 5 (21.7)    | 1.24 (0.32 to 4.80)  | 4 (17.4)    | 0.18 (0.04 to 0.79) |
+| Low risk                                                                                                                              | 5 (11.4)         | 7 (15.9)    | 1.00                 | 5 (11.4)    | 1.00                 | 27 (61.4)   | 1.00                |
+| High risk                                                                                                                             | 46 (17.1)        | 104 (38.7)  | 1.26 (0.36 to 3.28)  | 28 (10.4)   | 0.64 (0.15 to 2.61)  | 91 (33.8)   | 0.49 (0.16 to 1.48) |
+| *The presence of 'symptoms' and the 'duration of symptoms' were considered collinear—here we report just on the duration of symptoms. |                  |             |                      |             |                      |             |                     |
+
+immigration medical surveillance).39 A limitation of this study includes the relatively small cohort, which is typical in low-incidence settings. A second limitation includes the retrospective study design, which affects the interpretation of the strength of relationship between clinical features and outcomes, though collection of the data evaluated in this study is by protocol and we are confident in their accuracy. This study describes infectious TB cases in a highincome, low-incidence country where the disease is increasingly uncommon. We anticipate this description will contribute to the prompt submission of specimens for 
+
+![7_image_0.png](7_image_0.png)
+
+Figure 1 Smear-positive pulmonary tuberculosis (TB) cases with typical chest radiographs. This figure describes a diagnostic loop with two entry points: (1) report of one or more classical pulmonary tuberculosis symptoms, and (2) report of a chest radiograph abnormality consistent with adult-type pulmonary tuberculosis. Proceeding clockwise, the percentages describe the proportion of patients with one or more classical symptoms who had each of the other features shown; proceeding counterclockwise the percentages describe the proportion of patients who had a chest radiograph abnormality consistent with adult-type pulmonary tuberculosis with each of the other features. CXR, chest X-ray.
+
+confirmation by mycobacteriological testing. The prompt recognition of these cases is possible but takes a collaborative effort that includes front-line providers. Such input is understood to be a high-income, low-incidence country effort to follow the WHO/Stop TB Partnership roadmap to engage all care providers in TB prevention and care.11 Limiting the potential for transmission will ultimately aid TB elimination efforts. Electronic recording of the readily available clinical features proposed in this work along with computer-aided detection of adult-type PTB on chest radiographs could conceivably automate this process.
+
+Acknowledgements The authors are very grateful to the staff of the Edmonton TB Clinic, the Provincial TB Clinic and the Tuberculosis Program Evaluation and Research Unit for assistance in assembling the data presented in this study. Contributors CH and RL conceived this study. CH analysed and interpreted the data and provided initial drafts. JB interpreted the radiographs and was involved in critical review. AD and AS provided assistance with statistical methods and critical reviews. MLE and CP abstracted and collated relevant clinical data and provided critical reviews. RL interpreted the chest radiographs and provided expert oversight and critical reviews for all the drafts. Funding An Alberta Lung Association/Respiratory Health Strategic Clinical Health Network Studentship, and the University of Alberta Pulmonary Research Group award provided financial support to the lead author for her studies, of which this work represents one part. Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
+
+Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available upon reasonable request. All data are available upon reasonable request made to the corresponding author. Subsequent secondary analyses will be subject to appropriate REB approvals as necessary. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iD Courtney Heffernan http://orcid.org/0000-0003-1241-7201
+
+## References
+
+1 Behr MA, Warren SA, Salamon H, *et al*. Transmission of Mycobacterium tuberculosis from patients smear-negative for acidfast bacilli. *Lancet* 1999;353:444–9.
+
+2 Hernández-Garduño E, Cook V, Kunimoto D, *et al*. Transmission of tuberculosis from smear negative patients: a molecular epidemiology study. *Thorax* 2004;59:286–90.
+
+3 Tostmann A, Kik SV, Kalisvaart NA, *et al*. Tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the Netherlands. *Clin Infect Dis* 2008;47:1135–42.
+
+4 Lau A, Barrie J, Winter C, *et al*. Chest radiographic patterns and the transmission of tuberculosis: implications for automated systems. 
+
+PLoS One 2016;11:e0154032.
+
+5 Catanzaro A. Nosocomial tuberculosis. *Am Rev Respir Dis* 1982;125:559–62.
+
+6 Bailey WC, Gerald LB, Kimerling ME, *et al*. Predictive model to identify positive tuberculosis skin test results during contact investigations. *JAMA* 2002;287:996–1002.
+
+7 Golub JE, Bur S, Cronin WA, *et al*. Delayed tuberculosis diagnosis and tuberculosis transmission. *Int J Tuberc Lung Dis* 2006;10:24–30.
+
+8 Long R. Making a timely diagnosis of pulmonary tuberculosis. *Can* Respir J 2015;22:317–21.
+
+9 Long R, Heffernan C, Gao Z, *et al*. Do "virtual" and "outpatient" public health tuberculosis clinics perform Equally Well? A ProgramWide Evaluation in Alberta, Canada. *PLoS One* 2015;10:e0144784.
+
+10 Heffernan C, Doroshenko A, Egedahl ML, *et al*. Predicting pulmonary tuberculosis in immigrants: a retrospective cohort study. *ERJ Open* Res 2018;4. doi:10.1183/23120541.00170-2017. [Epub ahead of print: 20 Apr 2018].
+
+11 World Health Organization and STOP TB Partnership. PublicPrivate mix for TB prevention and care: a roadmap. WHO/CDS/
+TB/2018.32. Geneva, Switzerland: WHO, 2018. http://www.who.int/ tb/publications/2018/ppmroadmap 12 Small PM, McClenny NB, Singh SP, *et al*. Molecular strain typing of *Mycobacterium tuberculosis* to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. *J Clin Microbiol* 1993;31:1677–82.
+
+13 van't Hoog AH, Meme HK, Laserson KF, *et al*. Screening strategies for tuberculosis prevalence surveys: the value of chest radiography and symptoms. *PLoS One* 2012;7:e38691.
+
+14 Wisnivesky JP, Kaplan J, Henschke C, *et al*. Evaluation of clinical parameters to predict *Mycobacterium tuberculosis* in inpatients. Arch Intern Med 2000;160:2471–6.
+
+15 Samb B, Henzel D, Daley CL, *et al*. Methods for diagnosing tuberculosis among in-patients in eastern Africa whose sputum smears are negative. *Int J Tuberc Lung Dis* 1997;1:25–30.
+
+16 Long R, Maycher B, Dhar A, *et al*. Pulmonary tuberculosis treated with directly observed therapy: serial changes in lung structure and function. *Chest* 1998;113:933–43.
+
+17 The Canadian Lung Association and the Public Health Agency of Canada. *The Canadian tuberculosis standards*. 7th edn, 2013. http://
+www.publichealth.gc.ca/tuberculosis 18 Morris CD. The radiography, haematology and biochemistry of pulmonary tuberculosis in the aged. *Q J Med* 1989;71:529–3.
+
+19 Morris CD, Bird AR, Nell H. The haematological and biochemical changes in severe pulmonary tuberculosis. *Q J Med* 1989;73:1151–9.
+
+20 Long R, Asadi L, Heffernan C, *et al*. Is there a fundamental flaw in Canada's post-arrival immigrant surveillance system for tuberculosis? *PLoS One* 2019;14:e0212706.
+
+21 Falk A, O'Connor JB, Pratt PC, *et al*. Classification of pulmonary tuberculosis. In: Diagnostic standards and classification of tuberculosis. 12th edn. New York: National Tuberculosis and Respiratory Disease Association, 1969: 68–76.
+
+22 Cohen J. *Statistical power analysis for the behavioral sciences*. 2nd edn. Hillside, NJ: Lawrence Erlbaum Associates, 1998.
+
+23 Perez-Guzman C, Torres-Cruz A, Villarreal-Velarde H, *et al*. 
+
+Progressive age-related changes in pulmonary tuberculosis images and the effect of diabetes. *Am J Respir Crit Care Med* 2000;162:1738–40.
+
+24 Chan CH, Woo J, Or KK, *et al*. The effect of age on the presentation of patients with tuberculosis. *Tuber Lung Dis* 1995;76:290–4.
+
+25 Cohen R, Muzaffar S, Capellan J, *et al*. The validity of classic symptoms and chest radiographic configuration in predicting pulmonary tuberculosis. *Chest* 1996;109:420–3.
+
+26 Al-Tawfiq JA, Saadeh BM. Radiographic manifestations of culturepositive pulmonary tuberculosis: cavitary or non-cavitary? Int J 
+Tuberc Lung Dis 2009;13:367–70.
+
+27 Toman K. *Tuberculosis: case finding and chemotherapy: questions* and answers. Geneva: World Health Organization, 1979.
+
+28 Phoa L-L, Teleman MD, Wang Y-T, *et al*. Characteristics of patients with delayed diagnosis of infectious pulmonary tuberculosis. Respirology 2005;10:196–200.
+
+29 Jones BE, Young SM, Antoniskis D, *et al*. Relationship of the manifestations of tuberculosis to CD4 cell counts in patients with human immunodeficiency virus infection. *Am Rev Respir Dis* 1993;148:1292–7.
+
+30 Skogberg K, Ruutu P, Tukiainen P, *et al*. Effect of immunosuppressive therapy on the clinical presentation and outcome of tuberculosis. Clin Infect Dis 1993;17:1012–7.
+
+31 Kiyan E, Kilicaslan Z, Gurgan M, *et al*. Clinical and radiographic features of pulmonary tuberculosis in nonAIDS immunocompromised patients. *Int J Tuberc Lung Dis* 2003;7:764–70.
+
+32 Wilcke JT, Askgaard DS, Nybo Jensen B, *et al*. Radiographic spectrum of adult pulmonary tuberculosis in a developed country. Respir Med 1998;92:493–7.
+
+33 Lee H-S, Kee S-J, Shin J-H, *et al*. Xpert MTB/RIF assay as a substitute for smear microscopy in an intermediate-burden setting. 
+
+Am J Respir Crit Care Med 2019;199:784–94.
+
+34 Van Deun A, Tahseen S, Affolabi D, *et al*. Sputum smear microscopy in the Xpert® MTB/RIF era. *Int J Tuberc Lung Dis* 2019;23:12–18.
+
+35 Jones-López EC, Namugga O, Mumbowa F, *et al*. Cough aerosols of Mycobacterium tuberculosis predict new infection: a household contact study. *Am J Respir Crit Care Med* 2013;187:1007–15.
+
+36 Banerji D, Andersen S. A sociological study of awareness of symptoms among persons with pulmonary tuberculosis. Bull World Health Organ 1963;29:665–83.
+
+37 Styblo K, Dankova D, Drepela J, *et al*. Epidemiological and clinical study of tuberculosis in the district of Kalin, Czechoslovakia. *Bull* WHO 1967;37:819–74.
+
+38 Shimao T. A study of the mode of detection of newly registered pulmonary tuberculosis patients with special reference to their symptoms. In: Reports on medical research problems of the Japan anti-tuberculosis association. , 1974: 21, 17.
+
+39 Ward HA, Marciniuk DD, Pahwa P, *et al*. Extent of pulmonary tuberculosis in patients diagnosed by active compared to passive case finding. *Int J Tuberc Lung Dis* 2004;8:593–7.

medical/md/PMC7592778.md

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+# Traf4 Enhances Osteosarcoma Cell Proliferation And Invasion By Akt Signaling Pathway Weitao Yao, Xin Wang, Qiqing Cai, Songtao Gao, Jiaqiang Wang, And Peng Zhang
+
+Department of Bone and Soft Tumor, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China TRAF4, or tumor necrosis factor receptor-associated factor 4, is overexpressed in several cancers, suggesting a specific role in cancer progression. However, its functions in osteosarcoma are unclear. This study aimed to explore the expression of TRAF4 in osteosarcoma tissues and cells, the correlation of TRAF4 to clinical pathology of osteosarcoma, as well as the role and mechanism of TRAF4 in osteosarcoma metastasis. The protein expression levels of TRAF4 in osteosarcoma tissues and three osteosarcoma cell lines, MG-63, HOS, and U2OS, were assessed. Constructed TRAF4 overexpression vectors and established TRAF4 overexpression of the U2OS cell line. Cell proliferation, cell invasion, protein levels, and TRAF4 phosphorylations were assessed following TRAF4 transfection, as well as the effects of TRAF4 siRNA on cell proliferation and invasion. The results show that TRAF4 protein levels in osteosarcoma tissues were significantly higher than that in normal bone tissues. Importantly, an obvious upregulation of TRAF4 was found in carcinoma tissues from patients with lung metastasis compared with patients without lung metastasis. Consistently, a similar increase in TRAF4 mRNA and protein was also demonstrated in the osteosarcoma cell lines MG-63, HOS, and U2OS compared to normal bone cells, hFOB1.19. When TRAF4 was overexpressed in U2OS cells, cell proliferation was significantly enhanced, accompanied by an increase in Ki67 expression and colony formation. Compared with the control and vector-treated groups, TRAF4 transfection increased the invasion potential of U2OS cells (p < 0.05).
+
+Interestingly, TRAF4 transfection significantly enhanced the phosphorylation of Akt. After blocking Akt with its specific siRNA, TRAF4-induced cell proliferation and invasion were dramatically attenuated. In summary, our findings demonstrated that TRAF4 enhances osteosarcoma cell proliferation and invasion partially by the Akt pathway. This work suggests that TRAF4 might be an important target in osteosarcoma. Key words: Osteosarcoma; TRAF4; Cell proliferation; Cell invasion; Akt signaling pathway Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, with a peak incidence at the age of 15–19 years (1,2). Osteosarcoma is strongly invasive and has a high tendency to metastasize early (3). Unfortunately, treating metastatic osteosarcoma is a challenge in oncology and is the major cause of treatment failure and death (4).
+
+Tumor necrosis factor receptor-associated factors
+(TRAFs) were initially discovered as adaptor proteins that regulate cell life and death (5). So far, seven members (TRAF1-7) have been identified, and each member shares a common structural domain at the C-terminus (5,6). TRAFs form a complex network in which each TRAF is able to interact with and influence the function of many signaling molecules, either directly or through interactions with other TRAF family members (6). TRAF4 is a unique member of the TRAF protein family (7); it was initially identified in breast carcinoma (8). TRAF4 is required during embryogenesis in key biological processes including the formation of the trachea, the development of the axial skeleton, and the closure of the neural tube (9). TRAF4 deficiency leads to severe developmental alterations (10). TRAF4 is likely to be involved in signal transduction as it directly or indirectly mediates the downstream signal molecules. For instance, TRAF4 downregulates innate immune responses through its direct binding to NOD2 and inhibits NOD2-induced NF- B activation (11) and restricts IL-17-mediated pathology and signaling processes (12). TRAF4 stimulates MEKK4 kinase activity to regulate JNK activation (13).
+
+Recently, TRAF4 has been considered as an oncogene as it is overexpressed in a wide range of human malignancies, including breast cancer, lung cancer, colon adenocarcinomas, melanomas, neurogenic tumors, and lymphomas
+(14). Accumulating evidence indicates that TRAF4 plays critical role in breast cancer, such as antiapoptotic (15), and promotes cell migration (16). It has been reported that TRAF4 interacts with PIPs and acts as a negative regulator of tight junctions and increases cell migration
+(17). Moreover, TRAF4 promotes TGF- receptor signaling and drives breast cancer metastasis (18) and TRAF4 overexpression-induced degradation of p53 leading to poor prognosis in breast cancer patients (19). TRAF4 is also involved in maintaining tumorigenic properties in lung cancer; TRAF4 is required for EGF-induced Akt activation, and its deficiency markedly impairs the activity of Akt signaling and Akt-mediated lung cancer glycolysis (20). However, few studies have focused on the function of TRAF4 in osteosarcoma.
+
+In the present study, we aimed to assess the expression of TRAF4 in osteosarcoma tissue and cell lines, that is, MG-63, HOS, and H2OS. The role and the underlying mechanism of TRAF4 in osteosarcoma cell proliferation and invasion were also explored.
+
+MATERIALS AND METHODS
+
+## Specimens
+
+Fresh osteosarcoma tissue specimens were collected from 62 patients who underwent osteosarcoma resection operation in our hospital between July 2012 and March 2014. In addition, 22 normal bone tissue specimens were collected. Patients with osteosarcoma were matched for gender, age, and lung metastasis (Table 1). The patients with osteosarcoma included 42 males and 20 females; 44 cases were younger than 23 years old, while 20 patients were older than 23 years old. Without any preoperative treatment, all 36 cases were pathologically diagnosed with osteosarcoma postoperatively. There were 41 cases with lung metastases, while 21 others had no metastases.
+
+The specimens were preserved in liquid nitrogen immediately for subsequent testing. The specimen collection procedure was performed with informed patient consent and the approval of the Medical Ethics Committee. Cell Lines and Cell Culture
+
+## The Immortalized Human Osteoblast Cell Line Hfob1.19 And The Human Osteosarcoma Cell Lines Mg-63, Hos,
+
+and U2OS were purchased from American Type Culture Collection (ATCC). Three osteosarcoma cell lines (MG63, U2OS, and HOS) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin at 37°C in a humidified 5% CO2 incubator. The immortalized human osteoblast cell line (hFOB1.19) was grown in DMEM/Ham's F-12 containing 10% and geneticin (400 mg/ml) at a temperature of 34°C. These cells were maintained in the appropriate medium and passaged every 3 days. Construction of Expression Vectors and Cell Transfection Total RNA from U2OS cells were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), based on the manufacturer's instructions. The cDNA was synthesized by reverse transcription of total RNA, using the PrimeScript® RT reagent kit (Takara, Dalian, China) with oligo-dT primers, according to the manufacturer's protocol. Then, the open reading frame of TRAF4 cDNA was cloned and inserted into the pcDNA3.1 vector (Invitrogen) to construct the recombinant pcDNA3.1–TRAF4 expression vector. Control cells and pcDNA3.1 vector-transfected cells were also prepared. For cell transfection, cells were cultured to 60% confluence, and transfection was performed using the FuGENE HD transfection reagent (Roche, Indianapolis, IN, USA) method, as suggested by the manufacturer. siRNA Transfection Akt siRNA and control siRNA (con-siRNA) were purchased from Cell Signaling (Beverly, MA, USA). For transfection, 5 × 104 cells were seeded in each cell of 24well microplates, grown for 24 h to reach 30–50% confluence, and then incubated with a mixture of siRNA and Lipofectamine 2000 reagent (Invitrogen) in 100 ml of serum-free OPTI-MEM according to the manufacturer's instructions. The transfection efficiency was examined by real-time PCR and Western blotting. Western Blotting The proteins were extracted from tissue and cells using RIPA lysis buffer (Beyotime, Nantong, China). The protein Table 1. Correlation of TRAF4 Expression With Pathologic Parameter of Osteosarcoma
+
+| Pathologic Parameter   | Case   | Positive Rate (%)   | 2      | p     |
+|------------------------|--------|---------------------|--------|-------|
+| Normal tissues         | 22     | 2 (9.10%)           | 31.466 | 0.000 |
+| Carcinoma tissues      | 62     | 48 (77.42%)         |        |       |
+| Gender Male            | 42     | 32 (76.19%)         | 0.010  | 0.919 |
+| Female                 | 20     | 15 (75.00%)         |        |       |
+| Age <23                | 44     | 34 (77.27%)         | 0.388  | 0.533 |
+| >23                    | 20     | 14 (70.00%)         |        |       |
+| Lung metastasis        | 41     | 38 (92.69%)         | 11.038 | 0.001 |
+| Nonmetastasis          | 21     | 12 (57.14%)         |        |       |
+
+concentration in the lysates was determined using a BCA protein assay kit (Beyotime, Nantong, China). Western blotting was performed according to standard methods.
+
+Proteins were separated by SDS-PAGE and then transferred onto nitrocellulose membranes (GE Healthcare, Germany) in a semidry transblot apparatus using transfer buffer. Membranes were blocked with 10% defatted milk in PBS at 4°C overnight and incubated with the primary antibodies for 60 min. The primary antibodies are as follows: mouse anti-TRAF4 antibody (Santa Cruz, CA, USA), mouse anti- -actin antibody (KangChen Bio-tech, Shanghai, China), rabbit anti-Akt antibody, and rabbit anti-phospho-Akt (p-Ser473) antibody (Cell Signaling Technology, Danvers, MA, USA). After washing with TBST buffer, the membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (KangChen Bio-tech, Shanghai, China). ECL
+reagent (Beyotime) was used for detection. All experiments were performed in triplicate, and the results were normalized according to -actin. Real-Time Quantitative PCR
+Total RNA and cDNA was obtained as described above. Real-time quantitative PCR (RT-PCR) reactions were performed on a Bio-Rad iQ5 real-time thermal cycler using SYBR® Premix Ex Taq™ II kit (Takara, Dalian, China). The PCR primers specific for the TRAF4 was designed as previously reported (21): 5 -AGGAG TTCGTCTTTGACACCATC-3 (forward), 5 -CTTTGA ATGGGCAGAGCACC-3 (reverse); those for -actin were 5 -CAACTTGATGTATGAAGGCTTTGGT-3 (forward) and 5 -ACTTTTATTGGTCTCAAGTCAGTGTA
+CAG-3 (reverse). These primers were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR procedure was as follows: polymerase activation for 30 s at 95°C; 40 cycles of amplification each consisting of 95°C for 5 s and 60°C for 20 s; and one cycle of dissociation consisting of 95°C for 15 s, 60°C for 30 s, and 95°C
+for 15 s. All reactions were performed in triplicate, and the results were represented as the relative mRNA expression calculated according to the 2−DDCT method (22).
+
+MTT Assay Cell proliferation was determined by the MTT assay.
+
+Cells were seeded into 96-well plates, washed twice with PBS, and 10 µl of MTT was added to each well. Then, the cells were incubated at 37°C for 2 h, and 100 µl DMSO was added to dissolve the formazan crystals. Absorbance was measured at 560 nm with a SpectraMax Paradigm Multi-Mode Reader (Molecular Devices, Austria). Soft Agar Assay
+
+## The Soft Agar Assay Was Performed According To A Previous Report (23). The Control Group, The Vector-Transfected
+
+group, and the TRAF4-transfected group cells were evenly seeded into 100-mm petri dishes with soft agar. The cells were allowed to grow for 3 weeks until colonies formed. Colonies were stained with crystal violet and counted under a microscope using OpenCFU 3.8-BETA software (24). Cell Invasion Assay The in vitro invasive properties of cells were evaluated using a Transwell chamber assay. According to the manufacturer's protocol, chambers coated with Matrigel (50 µl per filter) (BD, USA) are incubated at 37°C for 30 min to create a Matrigel membrane, and divide the chamber into upper and lower compartments. Serum-free DMEM was added to the upper chambers, and DMEM containing 10% fetal calf serum was added to the lower chambers. The control, the vector transfected, and the TRAF4-transfected cells were cultured for 48 h and transferred to the upper chambers (1 × 105 cells per Transwell). After 24 h of incubation, cells that had migrated to the lower surface were fixed in 90% alcohol and stained with 0.08% crystal violet. Statistical Analysis The SPSS version 19.0 software was used to analyze the related data with chi-square test or t test. The results were considered statistically significant if p < 0.05.
+
+RESULTS
+The Expression of TRAF4 in Human Osteosarcoma Tissue To investigate whether TRAF4 was highly expressed in osteosarcoma tissue, we analyzed the protein level of TRAF4 in normal and different osteosarcoma patient tissues by Western blotting. The results showed that the TRAF4 expression level in osteosarcoma tissue was more than a 2.5-fold increase compared to normal bone tissue. Moreover, TRAF4 expression in osteosarcoma with lung metastatic tissue was more than twofold higher compared with tissue without lung metastases (Fig. 1). These results indicate that TRAF4 might be critical in osteosarcoma. TRAF4 Is Upregulated in Human Osteosarcoma Cell Lines To further verify the expression of TRAF4 in normal bone and osteosarcoma cells, we used RT-PCR to detect TRAF4 mRNA levels and Western blotting to analyze the protein levels in hFOB1.19, MG-63, HOS, and U2OS cell lines. RT-PCR analysis showed that the mRNA levels of TRAF4 in three osteosarcoma cell lines were higher than that in normal cells (Fig. 2A). Consistent with the results of mRNA levels, the protein levels in different osteosarcoma cells were higher than that in normal cells
+(Fig. 2B). It is noteworthy that both the mRNA and protein levels of TRAF4 in U2OS cells were higher than those in MG-63 and HOS cells.
+
+![3_image_0.png](3_image_0.png)
+
+## Expression Levels Of Traf4 In Traf4-Transfected
+
+U2OS Cells On the basis of our observations that TRAF4 is more highly expressed in U2OS cells than other two osteosarcoma cell lines, MG-63 and HOS (Fig. 2), we generated TRAF4-overexpressing U2OS cells to investigate the function of TRAF4 in osteosarcoma cells. After TRAF4 transfection, the mRNA and protein levels of TRAF4 were significantly increased (Fig. 3). Therefore, these results confirm that TRF4-overexpressing cells were successfully established.
+
+## The Effect Of Traf4 Overexpression On Cell Growth
+
+In order to investigate the role of TRAF4 in osteosarcoma cell growth, cell proliferation was assessed in TRAF4-transfected U2OS cells. The MTT analysis revealed that TRAF4-transfected cells possessed almost twofold higher cell proliferative ability than the other two groups
+(p < 0.05) (Fig. 4A). No differences were detected between the control and vector control cells. Ki67 is a nuclear protein that is expressed in proliferating cells (25)
+and may be required for maintaining cell proliferation. It had been used as a marker for cell proliferation in cancers
+
+![3_image_1.png](3_image_1.png)
+
+24
+
+![4_image_0.png](4_image_0.png)
+
+including osteosarcoma (26–28). We further analyzed the Ki67 protein expression levels and revealed that TRAF4 overexpression promoted the Ki67 protein expression
+(Fig. 4B). Additionally, colony formation was also detected by the soft agar assay. We found that U2OS cells transfected with TRAF4 yielded more colonies than the control and vector-transfected cells ( p < 0.05) (Fig. 4C).
+
+trol and vector-transfected groups.
+
+The Effect of TRAF4 Overexpression on Cell Invasion To further assess the function of TRAF4 on osteosarcoma cells, U2OS cell invasion was detected after TRAF4 transfection. As shown in Figure 5, forced
+
+![5_image_0.png](5_image_0.png)
+
+TRAF4 expression notably upregulated the number of invading cells from 120 to 197 compared to the control group. No difference was found between the control and vector groups (p < 0.05). Hence, these results indicate that TRAF4 is a positive regulator of osteosarcoma cell invasion. TRAF4 Enhances Osteosarcoma Cell Viability and Invasion Partially by the Akt Signaling Pathway The Akt signaling plays a major role in tumorigenesis by regulating cell proliferation, colony formation, cell cycle, cell survival, cell invasion, and metabolism (23,29–31). The fact that TRAF4 enhanced osteosarcoma cell growth and cell invasion was demonstrated in our previous results. To further investigate the underlying mechanism involved in this process, we investigated Akt signaling. Interestingly, the TRAF4-transfected cells exhibited higher Akt phosphorylation levels than the other two groups (p < 0.05) (Fig. 6A). To further explore the function of Akt signaling in TRAF4-induced osteosarcoma cell proliferation and invasion, Akt siRNA was used to knock down Akt level in U2OS cells. After silencing Akt signaling with its specific siRNA, the effect of TRAF4 on enhanced cell viability (p < 0.05)
+(Fig. 6B) and invasion (p < 0.05) (Fig. 6C) was obviously decreased. These results indicate that TRAF4 enhances osteosarcoma cell proliferation and invasion partly through the Akt signaling pathway.
+
+DISCUSSION
+Osteosarcoma is a leading malignant tumor in childhood and adolescence. It is a highly aggressive tumor that metastasizes to the lung (4). TRAF4 was first identified overexpressed in breast cancer in 1995 (8). In the following years, accumulating evidence indicated that TRAF4 is highly expressed in a range of human malignancies and tumors (32). However, its role and mechanism in osteosarcoma remains undefined. In this study, our results
+
+![5_image_1.png](5_image_1.png) showed that TRAF4 was highly expressed not only in osteosarcoma tissue but also in different osteosarcoma cell lines such as MG-63, HOS, and U2OS, indicating a potential critical role of TRAF4 in the development of osteosarcoma.
+
+Cell proliferation in cancer is associated with cancer development and prognosis. In order to explore the role of TRAF4 in osteosarcoma cell proliferation, we generated TRAF4-overexpressing U2OS cells and assessed cell proliferation. The results showed that TRAF4 overexpression dramatically promoted cell proliferation. We also detected the expression of protein Ki67, which is a marker for proliferating cells (33). Consistent with speculation, TRAF4 promoted Ki67 protein expression. Moreover, we also found that TRAF4 overexpression promoted colony formation. Our data demonstrate that TRAF4 plays an important role in promoting osteosarcoma cell growth.
+
+A common feature of osteosarcoma is that it metastasizes primarily to the lung (34). Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis (35). Thus, the invasion ability of tumor cells is closely related to tumor metastasis. Previous studies reported that TRAF4 promotes TGFreceptor signaling and drives breast cancer metastasis
+(18); moreover, TRAF4 modulates tight junctions and promotes cell migration (17). Our results reveal that TRAF4 expression was higher in osteosarcoma tissue from a patient with lung metastases than in those without metastases, indicating that TRAF4 is a critical molecule during osteosarcoma metastasis. Further in vitro analysis confirmed that TRAF4 overexpression enhanced cell invasion, providing evidence of the positive role of TRAF4 in osteosarcoma metastasis.
+
+The serine/threonine kinase Akt is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli (30). As a key molecule of this pathway, Akt is relevant to cancer cell survival, proliferation (increased cell number), and growth (increased cell size) (30,36,37). To further clarify the underlying mechanism involved in TRAF4-induced osteosarcoma cell proliferation and invasion, we detected Akt phosphorylation after TRAF4 transfection. As expected, TRAF4 upregulation promoted the activation of the Akt signaling pathway. When this signaling was blocked with Akt siRNA, cell proliferation induced by TRAF4 was decreased. It has been reported that Akt can promote cell invasion in prostate cancer (38) and by increased motility and metalloproteinase production in the HT1080 cell line (39).
+
+To further explore the mechanism of TRAF4 in promoting osteosarcoma cell invasion, we analyzed Akt function on TAFR4-induced cell invasion. The results show that Akt siRNA impaired the TAFR4-dependent enhancement of osteosarcoma cell invasion. Together, these results suggest that TRAF4 enhances osteosarcoma cell proliferation and invasion partially by the Akt signaling pathway.
+
+In summary, TRAF4 was confirmed in this study to be overexpressed in human osteosarcoma. Furthermore, TRAF4 may facilitate osteosarcoma progression by enhancing osteosarcoma cell proliferation and invasion partially through the Akt signaling pathway. Our research suggests that TRAF4 plays a critical role in osteosarcoma and may serve as a potential target against osteosarcoma.
+
+ACKNOWLEDGMENT: The authors declare no conflicts of interest.
+
+REFERENCES
+1. Vijayamurugan, N.; Bakhshi, S. Review of management issues in relapsed osteosarcoma. Expert Rev. Anticancer 14: 151–161; 2014.
+
+2. Bielack, S.; Carrle, D.; Hardes, J.; Schuck, A. Paulussen, M. Bone tumors in adolescents and young adults. Curr. Treat. Options Oncol. 9:67–80; 2008.
+
+3. Harting, M. T.; Blakely, M. L. Management of osteosarcoma pulmonary metastases. Semin. Pediatr. Surg. 15:25–29; 2006.
+
+4. Zwaga, T.; Bovee, J. V.; Kroon, H. M. Osteosarcoma of the femur with skip, lymph node, and lung metastases. Radiographics 28:277–283; 2008.
+
+5. Arch, R. H.; Gedrich, R. W.; Thompson, C. B. Tumor necrosis factor receptor-associated factors (TRAFs)—A family of adapter proteins that regulates life and death. Gene Dev. 12:2821–2830; 1998.
+
+6. Bradley, J. R.; Pober, J. S. Tumor necrosis factor receptorassociated factors (TRAFs). Oncogene 20:6482–6491; 2001. 7. Kedinger, V.; Rio, M. C. TRAF4, the unique family member. Adv. Exp. Med. Biol. 597:60–71; 2007.
+
+8. Regnier, C. H.; Tomasetto, C.; Moog-Lutz, C.; Chenard, M.
+
+P.; Wendling, C.; Basset, P.; Rio, M. C. Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma. J. Biol. Chem. 270:25715– 25721; 1995.
+
+9. Régnier, C. H.; Masson, R.; Kedinger, V.; Textoris, J.; Stoll, I.; Chenard, M.-P.; Dierich, A.; Tomasetto, C.; Rio, M.-C. Impaired neural tube closure, axial skeleton malformations, and tracheal ring disruption in TRAF4-deficient mice. Proc. Natl. Acad. Sci. USA 99:5585–5590; 2002.
+
+10. Shiels, H.; Li, X.; Schumacker, P. T.; Maltepe, E.; Padrid, P. A.; Sperling, A.; Thompson, C. B.; Lindsten, T. TRAF4 deficiency leads to tracheal malformation with resulting alterations in air flow to the lungs. Am. J. Pathol. 157: 679–688; 2000.
+
+11. Marinis, J. M.; Homer, C. R.; McDonald, C.; Abbott, D. W.
+
+A novel motif in the Crohn's disease susceptibility protein, NOD2, allows TRAF4 to down-regulate innate immune responses. J. Biol. Chem. 286:1938–1950; 2011.
+
+12. Zepp, J. A.; Liu, C.; Qian, W.; Wu, L.; Gulen, M. F.; Kang, Z.; Li, X. Cutting edge: TNF receptor-associated factor 4 restricts IL-17-mediated pathology and signaling processes. J. Immunol. 189:33–37; 2012.
+
+13. Abell, A. N.; Johnson, G. L. MEKK4 is an effector of the embryonic TRAF4 for JNK activation. J. Biol. Chem. 280: 35793–35796; 2005.
+
+14. Camilleri-Broet, S.; Cremer, I.; Marmey, B.; Comperat, E.;
+Viguie, F.; Audouin, J.; Rio, M. C.; Fridman, W. H.; Sautes-Fridman, C.; Regnier, C. H. TRAF4 overexpression is a common characteristic of human carcinomas. Oncogene 26:142–147; 2006.
+
+15. Zhang, X.; Wen, Z.; Mi, X. Expression and anti-apoptotic function of TRAF4 in human breast cancer MCF-7 cells. Oncol. Lett. 7:411–414; 2014.
+
+16. Wang, X.; Jin, C.; Tang, Y.; Tang, L. Y.; Zhang, Y. E.
+
+Ubiquitination of tumor necrosis factor receptor-associated factor 4 (TRAF4) by smad ubiquitination regulatory factor
+
+TRAF4 ENHANCES OSTEOSARCOMA CELL PROLIFERATION AND INVASION 27
+1 (Smurf1) regulates motility of breast epithelial and cancer cells. J. Biol. Chem. 288:21784–21792; 2013.
+
+17. Rousseau, A.; McEwen, A. G.; Poussin-Courmontagne, P.;
+Rognan, D.; Nomine, Y.; Rio, M. C.; Tomasetto, C.; Alpy, F. TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration. PLoS
+Biol. 11:e1001726; 2013.
+
+18. Zhang, L.; Zhou, F.; Garcia de Vinuesa, A.; de Kruijf, E. M.;
+Mesker, W. E.; Hui, L.; Drabsch, Y.; Li, Y.; Bauer, A.; Rousseau, A.; Sheppard, K. A.; Mickanin, C.; Kuppen, P. J.; Lu, C. X.; Ten Dijke, P. TRAF4 promotes TGF- receptor signaling and drives breast cancer metastasis. Mol. Cell 51: 559–572; 2013.
+
+19. Yi, P.; Xia, W.; Wu, R.-C.; Lonard, D. M.; Hung, M.-C.;
+O'Malley, B. W. SRC-3 coactivator regulates cell resistance to cytotoxic stress via TRAF4-mediated p53 destabilization. Genes Dev. 27:274–287; 2013.
+
+20. Li, W.; Peng, C.; Lee, M. H.; Lim, D.; Zhu, F.; Fu, Y.;
+Yang, G.; Sheng, Y.; Xiao, L.; Dong, X.; Ma, W.; Bode, A.
+
+M.; Cao, Y.; Dong, Z. TRAF4 is a critical molecule for Akt activation in lung cancer. Cancer Res. 73:6938–6950; 2013.
+
+21. Shen, J.; Qiao, Y.; Ran, Z.; Wang, T. Different activation of TRAF4 and TRAF6 in inflammatory bowel disease. Mediat. Inflamm. 647936:28; 2013.
+
+22. Schmittgen, T. D.; Livak, K. J. Analyzing real-time PCR
+data by the comparative CT method. Nat. Protoc. 3: 1101–1108; 2008.
+
+23. Zhang, K.; Tian, F.; Zhang, Y.; Zhu, Q.; Xue, N.; Zhu, H.;
+Wang, H.; Guo, X. MACC1 is involved in the regulation of proliferation, colony formation, invasion ability, cell cycle distribution, apoptosis and tumorigenicity by altering Akt signaling pathway in human osteosarcoma. Tumour Biol. 35(3):2537–2548; 2014.
+
+24. Geissmann, Q. OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS
+One 8:e54072; 2013.
+
+25. Romero, Q.; Bendahl, P. O.; Klintman, M.; Loman, N.;
+Ingvar, C.; Ryden, L.; Rose, C.; Grabau, D.; Borgquist, S. Ki67 proliferation in core biopsies versus surgical samples —A model for neo-adjuvant breast cancer studies. BMC Cancer 11:341; 2011.
+
+26. Hernández-Rodríguez, N. A.; Correa, E.; Sotelo, R.;
+Contreras-Paredes, A.; Gomez-Ruiz, C.; Green, L.; Mohar, A. Ki-67: A proliferative marker that may predict pulmonary metastases and mortality of primary osteosarcoma.
+
+Cancer Detect. Prev. 25:210–215; 2001.
+
+27. Li, B.; Yang, Y.; Jiang, S.; Ni, B.; Chen, K.; Jiang, L.
+
+Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/Akt pathway. Int. J Oncol. 41: 1809–1819; 2012.
+
+28. Urruticoechea, A.; Smith, I. E.; Dowsett, M. Proliferation marker Ki-67 in early breast cancer. J. Clin. Oncol. 23: 7212–7220; 2005.
+
+29. Brazil, D. P.; Park, J.; Hemmings, B. A. PKB binding proteins: Getting in on the Akt. Cell 111:293–303; 2002.
+
+30. Manning, B. D.; Cantley, L. C. Akt/PKB signaling: Navigating downstream. Cell 129:1261–1274; 2007.
+
+31. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Targeting the phosphoinositide 3-kinase pathway in cancer. Nat. Rev. Drug Discov. 8:627–644; 2009.
+
+32. Camilleri-Broet, S.; Cremer, I.; Marmey, B.; Comperat, E.;
+Viguie, F.; Audouin, J.; Rio, M. C.; Fridman, W. H.; Sautes-Fridman, C.; Regnier, C. H. TRAF4 overexpression is a common characteristic of human carcinomas. Oncogene 26:142–147; 2007.
+
+33. Santisteban, M.; Reynolds, C.; Barr Fritcher, E. G.; Frost, M. H.; Vierkant, R. A.; Anderson, S. S.; Degnim, A. C.; Visscher, D. W.; Pankratz, V. S.; Hartmann, L. C. Ki67: A time-varying biomarker of risk of breast cancer in atypical hyperplasia. Breast Cancer Res. Tr. 121:431–437; 2010.
+
+34. Longhi, A.; Errani, C.; De Paolis, M.; Mercuri, M.; Bacci, G. Primary bone osteosarcoma in the pediatric age: State of the art. Cancer Treat. Rev. 32:423–436; 2006.
+
+35. Yamaguchi, H.; Wyckoff, J.; Condeelis, J. Cell migration in tumors. Curr. Opin. Cell Biol. 17:559–564; 2005.
+
+36. Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3-kinase-akt pathway in human cancer. Nat. Rev. Cancer 2: 489–501; 2002.
+
+37. Zhao, G.; Cai, C.; Yang, T.; Qiu, X.; Liao, B.; Li, W.; Ji, Z.; Zhao, J.; Zhao, H.; Guo, M.; Ma, Q.; Xiao, C.; Fan, Q.; Ma, B. MicroRNA-221 induces cell survival and cisplatin resistance through PI3K/Akt pathway in human osteosarcoma. Plos One 8:e53906; 2013.
+
+38. Shukla, S.; MacLennan, G. T.; Hartman, D. J.; Fu, P.;
+Resnick, M. I.; Gupta, S. Activation of PI3K-Akt signaling pathway promotes prostate cancer cell invasion. Int. J. Cancer 121:1424–1432; 2007.
+
+39. Kim, D.; Kim, S.; Koh, H.; Yoon, S. O.; Chung, A. S.;
+Cho, K. S.; Chung, J. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J. 15:1953–1962; 2001.

medical/md/PMC7718532.md

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+# Scientific Article
+
+www.advancesradonc.org
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_1.png](0_image_1.png)
+
+Patterns of Failure After Adjuvant Stereotactic Body Radiation Therapy for Pancreatic Cancer With Close or Positive Margins Ankur K. Patel, MD,a Joshua L. Rodríguez-López, MD,a Nathan Bahary, MD, PhD,b Amer H. Zureikat, MD,c Steven A. Burton, MD,a Dwight E. Heron, MD, MBA, FACRO, FACR,a and Adam C. Olson, MD, MSa,*
+Departments of aRadiation Oncology, bMedicine, Division of Oncology, and cSurgery, Division of GI Surgical Oncology, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Received 4 March 2020; revised 31 July 2020; accepted 19 August 2020
+
+## Abstract
+
+Purpose: There is no consensus on treatment volumes for adjuvant stereotactic body radiation therapy (SBRT) for pancreatic cancer.
+
+Herein, we report patterns of failure after pancreatic SBRT for close/positive margins, which may inform target volume design.
+
+Methods and Materials: An institutional review board-approved retrospective review of patients with pancreatic adenocarcinoma treated with adjuvant SBRT for close/positive margins from 2009 to 2018 was conducted. Patterns of failure were defined as local (LF)
+within the tumor bed, regional (RF) within lymph nodes or anastomoses, or distant (DF). The cumulative incidence of locoregional failure was calculated using the cumulative incidence function accounting for the competing risk of death. LFs were mapped to the planning target volume (PTV) and classified as in-field (completely within the PTV), marginal (partially within the PTV), or out-of-field
+(completely outside the PTV). The location of LFs was compared with the Radiation Therapy Oncology Group 0848 contouring atlas to determine whether standard postoperative radiation therapy volumes would have included the LF.
+
+Results: Seventy-six patients were treated with adjuvant SBRT for close (51.3%) or positive (48.7%) margins. Most (81.6%) received 36 Gy in 3 fractions, with a median PTV volume of 17.8 cc (interquartile range, 12.1-25.6). With a median follow-up of 17.0 months
+(interquartile range, 7.3-28.4), crude rates of first isolated LF, isolated RF, and DF þ/e LF or RF were 9.2%, 6.6%, and 56.6%,
+respectively. Two-year cumulative incidences of LF, RF, locoregional failure, and DF were 34.9%, 30.8%, 49.2%, and 60.4%,
+respectively. Of 28 reviewable LFs, 21.4% were in-field while the remainder were completely outside (60.7%) or partially outside
+(17.9%) the PTV. Most LFs (92.9%) would have been encompassed by the Radiation Therapy Oncology Group consensus target volumes.
+
+Conclusions: After adjuvant pancreatic SBRT for close/positive margins, the majority of LFs were outside the PTV but within contemporary target volumes for conventional radiation therapy.
+
+ 2020 Published by Elsevier Inc. on behalf of American Society for Radiation Oncology. This is an open access article under the CC
+BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+## Introduction
+
+Pancreatic cancer is a highly lethal malignancy, responsible for the fourth-highest cancer death rate in the United States. Despite advances in management, 5-year overall survival (OS) remains dismal at 9%.1 Resection remains the most important curative modality; however, 80% to 85% of patients are unresectable at presentation.2,3 Of resectable patients, 20% to 30% have positive margins
+(R1) and fare worse than patients with negative margins
+(R0), even with adjuvant chemotherapy.4-12 Adjuvant radiation therapy (RT) may thus have a role after R1 resection and for other high-risk patients; however, studies examining adjuvant RT have produced conflicting results.9,12-20 The role of adjuvant RT is currently being examined in Radiation Therapy Oncology Group (RTOG)
+0848.21 One concern with adjuvant conventionally fractionated RT is the delay to initiating systemic chemotherapy, owing to time needed to deliver RT over 5 to 6 weeks as well as potential interruptions in treatment from treatment-induced toxicities. Micrometastatic spread before adjuvant chemotherapy may offset any locoregional control benefits with adjuvant RT. For these reasons, adjuvant stereotactic body radiation therapy (SBRT)
+delivered over a few fractions may be an attractive option for select patients. Definitive pancreatic SBRT provides local control rates of 70% to 80%.22-24 Adjuvant pancreatic SBRT provides similar local control with mild toxicities, though the body of evidence for adjuvant SBRT is limited.25-27 A previous report of a prospective trial from our institution involving 50 patients who received adjuvant pancreatic SBRT for close/positive margins showed 2-year local and regional control of 77%
+and 73%, respectively. Acute grade 3þ toxicity was 4.1%, and no late grade 3þ toxicity or changes in patientreported quality of life were evident.25 Although SBRT may provide some advantages over conventionally fractionated RT, 1 disadvantage to SBRT
+is the size limit on treatment volumes. Dose constraints of adjacent organs, chiefly the duodenum, limit the ability to dose-escalate in this region. Thus, SBRT target volumes are often limited to areas of highest risk of recurrence (ie, the positive margin or critical vascular interfaces); coverage of regional lymph nodes (LNs) and other areas at risk for harboring microscopic disease are usually omitted. Furthermore, although guidelines exist to standardize treatment volumes with adjuvant conventionally fractionated RT, no such consensus has been reached for pancreatic SBRT volumes. Contouring guidelines for postoperative conventionally fractionated RT were developed based on studies examining patterns of failure (POF), and recommend inclusion of surgical anastomoses and regional LNs (Table 1).28 Limited treatment volumes with SBRT that omit areas adjacent to the tumor or tumor bed may not be adequate in preventing adjacent local or regional recurrences.
+
+Few studies have reported POF after pancreatic SBRT
+to inform appropriate treatment volumes.29,30 No studies, to our knowledge, have examined failure locations relative to treatment volumes after adjuvant pancreatic SBRT.
+
+Herein we report POF in patients treated with adjuvant pancreatic SBRT for close/positive margins.
+
+## Methods And Materials Eligibility
+
+An institutional review board-approved retrospective review of consecutive patients treated between October 2009-February 2018 with adjuvant pancreatic SBRT for close/positive margins was conducted. During this time, many patients were treated in a prospective observational trial at our institution , results of which have been previously reported.25 Patients in this trial were included in this retrospective review if they met inclusion criteria.
+
+Demographic, clinical, pathologic, and treatment details were collected. Included patients had resectable or borderline resectable pancreatic adenocarcinoma and were treated with resection and adjuvant pancreatic SBRT
+for close/positive margins. Resectability definitions were based on National Comprehensive Cancer Network guidelines available at the time of treatment. Generally, resectable patients had no arterial tumor contact and/or
+-180 venous tumor contact without vein contour irregularity, while borderline resectable patients had arterial tumor contact -180 and/or venous tumor contact >180 or -180 with contour irregularity that was amenable to vein reconstruction. Patients underwent pancreaticoduodenectomy or distal pancreatectomies for pancreatic tail tumors  robotic assistance. Positive surgical margins included viable tumor cells present at any resection margin. Close margins included viable tumor cells present within 2 mm of any resection margin. Patients received adjuvant or neoadjuvant chemotherapy or both per physician discretion and patient tolerance.
+
+Experimental systemic therapy was allowed in institutional protocols based on eligibility and patient/physician preference.
+
+## Stereotactic Body Radiation Therapy
+
+Patients were simulated supine with arms raised and vacuum lock bag immobilization. Axial computed tomography (CT) images with 1.25 mm slice thickness were obtained. Intravenous and/or enteral contrast was administered per physician discretion. Four-dimensional CT data were acquired to evaluate target motion. The clinical target volume (CTV) was delineated in conjunction with the operating surgeon and included the area of close/positive margin, clips/fiducials, and other areas at high risk of microscopic disease based on the pathology report. Abutting vasculature was only included if that specific vessel margin was close/positive and was otherwise not routinely included. Elective regional LNs were not included in the CTV. The planning target volume (PTV) included margins for set-up error and target motion and was altered accordingly to respect critical structure tolerance. All patients were treated with 3 fractions of SBRT, every other day, to a dose of 27 to 36 Gy using daily cone beam CT for image guidance. Patients were typically seen 10 to 12 weeks post-SBRT, then every 3 months for 1 year, with subsequent follow-up per physician discretion. CT scans were typically obtained at each follow-up or if there was a concern for recurrence or progression.
+
+## Endpoints And Statistical Analysis
+
+Clinical notes and radiologic images/reports were reviewed to capture recurrences. For patterns of failure analysis, failures were classified as local (LF), regional
+(RF), locoregional (LRF), or distant (DF) as deemed by the reading radiologist and/or treating physician in radiology reports and/or clinical notes. LFs were recurrences within the operative bed. RFs were recurrences within the regional LNs (new or enlarging LNs) or anastomosis.
+
+LRFs were defined as local and/or regional failures. DFs were recurrences in nonregional LNs or distant organs.
+
+The date of recurrence was backdated to the initial imaging study demonstrating any radiographic evidence of progression. Both the site of first failure and the cumulative incidence of all failure types were collected. All patients were evaluated for LF and RF from SBRT completion to death or loss of follow-up, regardless of progression at other sites.
+
+SBRT treatment plans were accessed to map LFs relative to the PTV. In-field LFs were LFs completely within the PTV. Marginal failures were LFs partially within the PTV, and out-of-field were LFs completely outside the PTV. The location of LFs and RFs was compared with the RTOG 0848 consensus volumes for postoperative treatment of pancreatic cancer28 to determine whether consensus volumes for conventionally fractionated postoperative RT would have included the LF or RF. Recommended contours of each region of interest and CTV expansions were considered when determining whether an LF/RF would have been encompassed by the RTOG consensus volumes (Table 1). To successfully map LFs and RFs relative to the PTV and the RTOG
+consensus volumes, the location of each failure relative to the preoperative tumor location and measured distances between the edge of failures and prominent abdominal anatomy (eg, celiac artery [CA], superior mesenteric artery [SMA], portal vein, anastomoses) were recorded and compared with the location and measured distances of the PTV volumes and RTOG consensus volumes relative to the same anatomic landmarks. Patients without available SBRT plans or imaging evidence of LF/RF were excluded from analysis of failure location.
+
+Crude failure rates were calculated from the number of each failure type within the cohort. Time-to-event analysis was calculated from the date of SBRT completion to the date of the event. OS was calculated using KaplanMeier methods. The cumulative incidence function was used to calculate the cumulative incidence of LF, in-field LF, marginal LF, out-of-field LF, RF, LRF, and DF þ/e LF or RF from the last day of SBRT treatment to the date of failure, accounting for the competing risk of death.31-33 Differences in time to LF by type of LF were analyzed with the log-rank test. Cox proportional hazards regression was used to test the effect of demographic, clinical, pathologic, and SBRT treatment details on the risk of LF
+and in-field LF. Log-minus-log plots were used to confirm the proportional hazards assumption. As a sensitivity analysis, continuous variables were also tested as binary categorical variables stratified by the median value. Variables with P values < .10 on univariate analysis were entered into the multivariate model, which was run with backward stepwise selection. P values < .05 were considered statistically significant. Statistical analysis was conducted with Statistical Package for the Social Sciences
+(SPSS) Statistics for Windows version 25.0 (SPSS Inc, Chicago, IL) and R version 3.6.2.34
+
+## Results
+
+Seventy-six patients were treated with adjuvant SBRT
+for close/positive margins. Table 2 outlines cohort characteristics. Most patients (56.6%) were male and had pancreatic head tumors (80.3%). Most (59.2%) received neoadjuvant chemotherapy, most commonly gemcitabine/
+nab-paclitaxel (48.9%). The majority (81.6%) received adjuvant chemotherapy, most commonly gemcitabine
+(41.9%).
+
+Three-quarters of patients were resectable and 25.0%
+were borderline resectable. Most patients (86.8%) underwent pancreaticoduodenectomy. Close margins were seen in 51.3%; positive margins were seen in 48.7%. The most common positive/close margin was retroperitoneal
+(52.6%). Twenty patients (26.3%) had multiple positive/ close margins. More than 3/4 (77.6%) had pathologic T2 disease. Pathologic nodal staging was variable: pN0, pN1, and pN2 was seen in 26.3%, 39.5%, and 34.2%, respectively.
+
+Adjuvant SBRT was delivered a median 2.2 months after surgery (interquartile range [IQR], 1.7-3.0). Most patients (81.6%) received 36 Gy in 3 fractions. Median PTV was 17.8 cc (IQR, 12.1-25.6). Median equivalent 1200 A.K. Patel et al Advances in Radiation Oncology: NovembereDecember 2020
+
+| Table 1                                                                                                                                                                                                                                                                             | Radiation Therapy Oncology Group consensus treatment volumes for conventionally fractionated postoperative                                 |                    |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
+| pancreatic radiation therapy.28 Region of interest                                                                                                                                                                                                                                  | RTOG* recommendations for                                                                                                                  | Expansions for CTV |
+| volume delineation                                                                                                                                                                                                                                                                  |                                                                                                                                            |                    |
+| Postoperative bed                                                                                                                                                                                                                                                                   | Based on preoperative tumor location and                                                                                                   | 0.5-1.0 cm                    |
+| pathology reports; include surgical clips.                                                                                                                                                                                                                                          |                                                                                                                                            |                    |
+| Anastomoses                                                                                                                                                                                                                                                                         | Pancreaticojejunostomy (PJ), choledochaljejunostomy, or hepaticojejunostomy. Do not include pancreaticogastrostomy if present.             | 0.5-1.0 cm         |
+| Abdominal nodal regions Celiac artery (CA)                                                                                                                                                                                                                                          | Most proximal 1.0-1.5 cm                                                                                                                   | 1.0-1.5 cm         |
+| Superior mesenteric artery (SMA)                                                                                                                                                                                                                                                    | Most proximal 2.5-3.0 cm                                                                                                                   | 1.0-1.5 cm         |
+| (includes peripancreatic nodes)                                                                                                                                                                                                                                                     |                                                                                                                                            |                    |
+| Porta hepatic                                                                                                                                                                                                                                                                       | Portal vein (PV) segment that runs anterior and                                                                                            | 1.0-1.5 cm         |
+| anteromedial to the inferior vena cava.                                                                                                                                                                                                                                             | 2.5-3.0 cm to the right, 1.0 cm to the left, 2.0-2.5 cm anteriorly, and 0.2 cm posteriorly toward the anterior edge of the vertebral body. |                    |
+| Abbreviations: CTV Z clinical target volume; RTOG Z Radiation Therapy Oncology Group (RTOG). Para-aortic Aorta from most cephalad aspect of CA, PJ, or PV (whichever is most cephalad), to bottom of L2; cover to bottom of L3 if needed to encompass extent of preoperative tumor. |                                                                                                                                            |                    |
+
+dose in 2-Gray-per-fraction using alpha:beta Z 10 and biologically effective dose using alpha:beta Z 10 was 66.0 Gy (IQR, 66.0-66.0) and 79.2 Gy (IQR, 79.2-79.2),
+respectively. No patient had a significant interruption during SBRT; the median time elapsed during the SBRT
+course was 6.0 days (IQR, 5.0-7.0).
+
+Median follow-up after SBRT completion was 17.0 months (IQR, 7.3-28.4). Median OS was 17.3 months (95%
+confidence interval, 11.0-23.5 months). Fifty-seven patients (75.0%) experienced a recurrence at any site during follow-up. Table 3 outlines POF. Upon examination of sites of first failure, isolated DF was predominant (30.2%, n Z
+23). LF was a component of first failure in 23 patients.
+
+Crude rates of isolated LF, isolated RF, and DF þ/e LF or RF at first failure were 9.2%, 6.6%, and 56.6%, respectively. A total of 32 (42.1%), 24 (31.6%), 42 (55.3%), and 50 patients (65.8%) developed a LF, RF, LRF, or DF, respectively, during follow-up. Upon examination of LF or RF, 18 patients (23.7%) had an LF  DF but without RF, 10 patients (13.2%) had an RF  DF but without LF, and 14 patients (18.4%) had both LF and RF  DF. The 2-year cumulative incidence of LF, RF, LRF, or DF, accounting for the competing risk of death, was 34.9%, 30.8%, 49.2%,
+and 60.4%, respectively (Fig 1). Univariate Cox regression did not reveal any statistically significant predictors of LF
+(Table 4), although a trend was seen for positive pancreatic neck margin (P Z .056).
+
+Of 32 patients with an LF during follow-up, 3 patients had unavailable imaging of the LF, and 1 patient had an unavailable SBRT plan. Thus, 28 of 32 patients (87.5%)
+were analyzed for POF relative to treatment volumes.
+
+Only 21.4% (n Z 6) of LFs were in-field; the remaining were out-of-field (60.7%, n Z 17) or marginal (17.9%, n Z 5). The 2-year cumulative incidence of in-field, marginal, and out-of-field LFs was 7.3%, 5.8%, and 17.9%,
+respectively. There were no differences in time to LF
+based on type of LF; median time to failure for in-field, marginal, and out-of-field LFs was 5.0, 8.0, and 11.8 months, respectively (P Z .477). All 6 patients with an in-field LF received 36 Gy in 3 fractions and had larger CTV (median, 15.2 cc; IQR, 12.4-16.7) and PTV (median, 27.5 cc; IQR, 22.0-33.9) volumes relative to the entire cohort. However, no variables correlated with risk of in-field LF (Table 4).
+
+Because RTOG consensus volumes cover areas at risk for LF and RF, every LF (n Z 28) and RF (n Z 24) with available imaging was examined to assess its location relative to recommended RTOG consensus volumes.
+
+Most LFs (92.9%, n Z 26) would have been encompassed by the RTOG consensus volumes for postoperative conventional RT. Two LFs (7.1%) were deemed to be outside RTOG consensus volumes. Both were infiltrative masses that spread along and cuffed the distal SMA >5 cm beyond the SMA origination from the aorta, and thus outside the caudal extent of the recommended SMA target volume.
+
+Upon examination of RFs, 62.5% (n Z 15) would have been encompassed by RTOG consensus volumes.
+
+Of 9 RFs deemed to be outside RTOG consensus volumes, 5 were in high para-aortic or gastrohepatic LNs located 1 cm above the recommended cephalad border of the para-aortic LN target volume, 2 were in para-aortic LNs below the recommended caudal extent of the paraaortic LN volume, and 2 were in mesenteric LNs in the anterior or lower abdomen along the distal SMA, beyond the recommended SMA nodal target volume. The 2-year cumulative incidence of LRF within RTOG consensus volumes was 39.3%.
+
+| Table 2                                                  | Demographic, clinical, pathologic, and treatment   |
+|----------------------------------------------------------|----------------------------------------------------|
+| detail characteristics of cohort Characteristic          | Value (n Z 76)                                     |
+| Age at diagnosis (median, IQR)                           | 66.0 (57.3-73.0)                                   |
+| Sex (n, %) Male                                          | 43 (56.6)                                          |
+| Female                                                   | 33 (43.4)                                          |
+| Tumor location (n, %) Body                               | 8 (10.5)                                           |
+| Head                                                     | 61 (80.3)                                          |
+| Tail                                                     | 2 (2.6)                                            |
+| Uncinate process                                         | 4 (5.3)                                            |
+| Neck                                                     | 1 (1.3)                                            |
+| Resectability (n, %) Resectable                          | 57 (75.0)                                          |
+| Borderline resectable                                    | 19 (25.0)                                          |
+| Surgery type (n, %) Pancreaticoduodenectomy              | 66 (86.8)                                          |
+| Distal pancreatectomy                                    | 10 (13.2)                                          |
+| Margin status (n, %) Positive                            | 37 (48.7)                                          |
+| Close (1 mm)                                             | 37 (48.7)                                          |
+| Close (2 mm)                                             | 2 (2.6)                                            |
+| Location of positive/close margin (n, %) Retroperitoneal | 40 (52.6)                                          |
+| Vascular groove                                          | 35 (46.1)                                          |
+| Pancreatic neck                                          | 11 (14.5)                                          |
+| Chemotherapy (n, %) Neoadjuvant                          | 45 (59.2)                                          |
+| Gemcitabine                                              | 2 (4.4)                                            |
+| FOLFIRINOX                                               | 6 (13.3)                                           |
+| Gemcitabine/nab-paclitaxel                               | 22 (48.9)                                          |
+| Gemcitabine/hydroxychloroquine                           | 9 (20.0)                                           |
+| Other                                                    | 6 (13.3)                                           |
+| Adjuvant                                                 | 62 (81.6)                                          |
+| Gemcitabine                                              | 26 (41.9)                                          |
+| FOLFIRINOX                                               | 4 (6.5)                                            |
+| Gemcitabine/capecitabine                                 | 10 (16.1)                                          |
+| Gemcitabine/nab-paclitaxel                               | 13 (21.0)                                          |
+| FOLFOX                                                   | 4 (6.5)                                            |
+| Other                                                    | 5 (8.1)                                            |
+| Pathologic T stage (AJCC 8th edition) (n, %) pT1a        | 1 (1.3)                                            |
+| pT1c                                                     | 6 (7.9)                                            |
+| pT2                                                      | 59 (77.6)                                          |
+| pT3                                                      | 10 (13.2)                                          |
+| Pathologic N stage (AJCC 8th edition) (n, %) pN0         | 20 (26.3)                                          |
+| pN1                                                      | 30 (39.5)                                          |
+| pN2                                                      | 26 (34.2)                                          |
+| SBRT regimen (n, %) 36 Gy in 3 fractions                 | 62 (81.6)                                          |
+| 30 Gy in 3 fractions                                     | 13 (17.1)                                          |
+| 27 Gy in 3 fractions                                     | 1 (1.3)                                            |
+| EQD210 (median, IQR)                                     | 66.0 (66.0-66.0)                                   |
+| BED10 (median, IQR)                                      | 79.2 (79.2-79.2)                                   |
+| (continued on next page)                                 |                                                    |
+
+| Table 2 (continued) Characteristic   | Value (n Z 76)      |
+|--------------------------------------|---------------------|
+| CTV volume (median, IQR)             | 9.7 cc (6.8-14.4)   |
+| PTV volume (median, IQR)             | 17.8 cc (12.1-25.6) |
+| PTV V100% (median, IQR)              | 94.8% (90.3-98.1)   |
+| Months from surgery to SBRT start    | 2.2 (1.6-3.0)       |
+| (median, IQR)                        |                     |
+| Days elapsed from SBRT start to end  | 6.0 (5.0-7.0)       |
+| (median, IQR)                        |                     |
+| Abbreviations: AJCC Z American Joint Committee on Cancer; BED10 Z biologically effective dose using an alpha/beta ratio of 10; CTV Z clinical target volume; EQD210 Z equivalent dose in 2 Gray per fraction using an alpha/beta ratio of 10; IQR Z interquartile range; PTV Z planning target volume; PTV V100% Z percent of PTV that received 100% of the prescription dose; SBRT Z stereotactic body radiation therapy.                                      |                     |
+
+## Discussion
+
+In this single-institution retrospective analysis of adjuvant pancreatic SBRT for close/positive margins, we identified a high rate of LRF outside the SBRT PTV but within contemporary RT target volumes. Although the predominant POF at first failure was isolated DF (30.2%),
+42.1% of patients developed an LF during follow-up. The 2-year cumulative incidence of LF was 34.9%. Thus, despite macroscopic resection, targeted RT, and systemic therapy, patients with close/positive margins were at substantial risk for developing an LF. LFs within the PTV were infrequent (6 of 28 patients); the 2-year cumulative incidence of in-field LFs was 7.3%. Most LFs were completely outside the PTV (60.7%). Over 90% of LFs would have been encompassed by RTOG consensus volumes for postoperative conventionally fractionated RT
+for pancreatic cancer.
+
+This study is the first, to our knowledge, that describes POF after adjuvant SBRT. Published studies examining POF with pancreatic SBRT are limited to patients treated in the definitive or neoadjuvant setting, and few studies have detailed examination of failures relative to treatment volumes or abdominal vasculature.29,30,35 A Chinese study of >500 medically inoperable patients treated with pancreatic SBRT revealed that most LFs occurred near the CA, SMA, or splenic artery for pancreatic tail tumors29; 80% and 95% of failures near the CA or SMA
+occurred within 11 and 13 mm of the vessel, respectively.
+
+These failures likely would have been included within the consensus postoperative target volumes for conventionally fractionated RT. In-field failures (>80% of volume
+
+| Table 3                                                                                  | Patterns of failure with adjuvant SBRT Crude rates   | Cumulative incidence (95% CI)   |                   |                   |
+|------------------------------------------------------------------------------------------|------------------------------------------------------|---------------------------------|-------------------|-------------------|
+| n (%)                                                                                    | 6-month                                              | 12-month                        | 24-month          |                   |
+| Location of failure based on clinical documentation (n Z 76) No failure                  | 19 (25.0)                                            |                                 |                   |                   |
+| Local failure                                                                            | 32 (42.1)                                            | 17.2% (9.7-26.5)                | 29.3% (19.5-39.8) | 34.9% (24.3-45.8) |
+| Regional failure                                                                         | 24 (31.6)                                            | 13.2% (6.7-21.2)                | 23.9% (15.0-34.0) | 30.8% (20.7-41.5) |
+| Locoregional                                                                             | 42 (55.3)                                            | 26.3% (17.0-36.6)               | 40.8% (29.7-51.5) | 49.2% (37.4-59.9) |
+| Distant failure                                                                          | 50 (65.8)                                            | 33.1% (22.7-43.4)               | 50.5% (38.7-61.2) | 60.4% (48.2-70.5) |
+| Location of local failure in patients with available plans and imaging (n Z 28) In-field | 6 (21.4)                                             | 4.2% (1.1-10.8)                 | 5.7% (1.8-12.9)   | 7.3% (2.7-15.2)   |
+| Marginal                                                                                 | 5 (17.9)                                             | 2.9% (0.1-8.9)                  | 5.8% (1.9-13.2)   | 5.8% (1.9-13.2)   |
+| Out-of-field                                                                             | 17 (60.7)                                            | 8.0% (3.3-15.6)                 | 14.8% (7.8-23.9)  | 17.9% (10.1-27.6) |
+| Abbreviations: CI Z confidence interval; SBRT Z stereotactic body radiation therapy.     |                                                      |                                 |                   |                   |
+
+![5_image_0.png](5_image_0.png)
+
+within the prescription isodose) occurred in 22.9%; this number mirrors the in-field failure rate in this study of 21.4%. The distance from primary tumors to retroperitoneal recurrences was larger than the distance from primary tumors to the CA (11.7 vs 9.0 mm, respectively),
+suggesting that asymmetrical expansion of SBRT target volumes to include the local retroperitoneal space may be warranted. Kharofa et al30 reported POF in 18 resectable or borderline resectable patients treated with neoadjuvant pancreatic SBRT (33 Gy in 5 fractions) to the tumor and entire circumference of the abutting vessel. After interim analysis showed 2 of 8 patients failed locally at the vessel at the PTV margin, the remaining patients received 25 Gy in 5 fractions simultaneously to an additional PTV
+covering the entire pancreatic head/body, with extension of vessel coverage to the CA origin and SMA. With the limitations of a small sample size, the combined PTV
+approach led to longer 1-year PFS (60% vs 25%; P Z
+.03), 2-year OS (67% vs 25%; P Z .03), and lower 1-year cumulative incidence of LF (38% vs 70%; P Z .45),
+raising the question of whether elective coverage of vessel origins with SBRT reduces the risk of failures at the margin of the primary PTV.
+
+There are no consensus pancreatic SBRT volumes.
+
+Results from our study support consideration for expanding treatment volumes if feasible. Indeed, initial results from our institutional prospective trial of adjuvant pancreatic SBRT for close/positive margins found CTV
+volume was the only predictor for freedom from LF on multivariate analysis (hazard ratio, 1.095; 95% confidence interval, 1.005-1.193; P Z .038).25 However, the tradeoff between expanding target volumes and irradiating more normal tissue must be carefully evaluated. A study by Dholakia et al36 generated hypothetical adjuvant intensity modulated radiation therapy and SBRT volumes encompassing the majority of LFs in >200 patients after pancreaticoduodenectomy. CTV volumes encompassed either 90% of LFs (CTV90) or 80% of LFs (CTV80) based on specific asymmetrical expansions around CA and SMA
+contours. With no further margins for setup error, PTV80 and PTV90 volumes ranged from 123.0 to 139.6 cc and 183.2 to 215.7 cc, respectively. These volumes are significantly larger than those in our study but may represent a practical strategy to expand treatment volumes while minimizing irradiation of normal tissue. Although the authors report critical structure dose constraints were met with 25 Gy in 5 fractions to the PTV90 with a simultaneous integrated boost to 33 Gy to PTV80, no patients were actually treated using the hypothetical plans.
+
+The aforementioned studies highlight the commonality of LFs along vessels after SBRT and underline the importance of detailed POF examinations. Studies reporting LFs after pancreatic SBRT use different LF
+definitions, which may inadvertently include nodal failures e the Chinese and Dholakia studies defined LFs as Advances in Radiation Oncology: NovembereDecember 2020 Patterns of failure after pancreatic SBRT 1203
+
+| Table 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | Univariate Cox regression for predictors of LF in all patients (n Z 76) as deemed by the reading radiologist and/or   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|
+| treating physicians and for predictors of in-field LF in patients with documented LF who had available plans and imaging (n Z 28) Univariate analysis Variable HR for LF (95% CI) P value HR for in-field LF (95% CI) P value Age at diagnosis 1.00 (0.96-1.03) .769 1.06 (0.96-1.17) .259 Sex .565 .441 Male Reference Reference Female 0.81 (0.40-1.66) 1.88 (0.38-9.42) Tumor location .999 .650 Body Reference Reference Head 1.05 (0.36-3.04) 0.33 (0.06-1.81) Tail 1.44 (0.16-13.04) NR Uncinate process 1.05 (0.19-5.80) NR Neck NR NR Resectability .127 .856 Resectable Reference Reference Borderline resectable 1.83 (0.84-3.96) 1.17 (0.21-6.60) Surgery type .600 .501 Pancreaticoduodenectomy Reference Reference Distal pancreatectomy 1.27 (0.52-3.10) 1.79 (0.33-9.82) Margin status .862 .395 Positive Reference Reference Close (1 mm) 0.87 (0.42-1.78) 0.87 (0.14-5.29) Close (2 mm) 0.62 (0.08-4.79) 4.26 (0.37-49.65) Retroperitoneal margin .279 .465 Negative Reference Reference Positive/close 1.48 (0.73-3.03) 1.89 (0.34-10.51) Vascular groove margin .256 .717 Negative Reference Reference Positive/close 0.66 (0.32-1.35) 0.73 (0.13-4.02) Pancreatic neck margin .056 .847 Negative Reference Reference Positive/close 2.30 (0.98-5.42) 0.81 (0.09-7.08) Pathologic tumor size (cm) 1.10 (0.82-1.46) .538 0.39 (0.11-1.43) .155 Neoadjuvant chemotherapy .624 .390 No Reference Reference Yes 0.84 (0.42-1.70) 2.57 (0.30-22.07) Adjuvant chemotherapy .183 No Reference Reference Yes 2.25 (0.68-7.42) NR CTV volume (cc) 1.02 (0.98-1.05) .414 1.01 (0.94-1.08) .760 PTV volume (cc) 1.01 (0.98-1.04) .414 1.03 (0.97-1.09) .334 PTV V100% (%) 1.01 (0.97-1.05) .565 1.04 (0.92-1.16) .589 BED10 1.00 (0.96-1.06) .760 1.17 (0.72-1.92) .562 EQD210 1.01 (0.95-1.07) .760 NR .818 Abbreviations: BED10 Z biologically effective dose using an alpha/beta ratio of 10; CI Z confidence interval; CTV Z clinical target volume; EQD210 Z equivalent dose in 2 Gray per fraction using an alpha/beta ratio of 10; HR Z hazard ratio; LF Z local failure; NR Z not reportable; PTV Z planning target volume; PTV V100% Z percent of PTV that received 100% of the prescription dose. |                                                                                                                       |
+
+failures in between the diaphragm and bottom of L3, while Kharofa et al considered LFs as failures within the SBRT volume or within institutional fractionated volumes that electively covered the regional vasculature. In our analysis, because no elective nodal volumes were treated, we constrained the definition of LF to failures within the operative bed based on preoperative tumor location to minimize inclusion of nodal failures. Despite this constrained definition, LFs were identified in 42.1% of patients after SBRT targeted to close/positive margins.
+
+Thus, true LFs occur in a significant proportion of patients, despite SBRT targeting the areas of highest risk of recurrence. Of 72 patients with available imaging and SBRT plans, only 6 (8.3%) experienced an LF within the treatment volume; the majority of LFs were outside or marginal to the treatment volume. This suggests issues with volume and not dose, and thus expanding treatment volumes to include all portions of the pancreas/pancreatic bed and abutting vasculature may be warranted. Because RTOG consensus volumes encompassed most LFs as well as RFs in this study, adopting a similar philosophy with SBRT treatment volumes may be warranted if feasible.
+
+We note several limitations of this study, including its retrospective nature, relatively small sample size, and the subjective nature of mapping LFs and RFs to PTV volumes and RTOG consensus volumes. The latter issue was mitigated by systematically noting the location and distance of failures relative to abdominal vasculature, which allowed for more precise mapping.
+
+## Conclusions
+
+In patients with resectable or borderline resectable adenocarcinoma of the pancreas who received adjuvant SBRT for close or positive margins, the majority of LFs occurred outside the PTV. Future trials involving adjuvant SBRT or hypofractionated RT should consider expansion of treatment volumes if feasible.
+
+## References
+
+1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7-30.
+
+2. Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet.
+
+2004;363:1049-1057.
+
+3. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N
+Engl J Med. 2014;371:1039-1049.
+
+4. Konstantinidis IT, Warshaw AL, Allen JN, et al. Pancreatic ductal adenocarcinoma. Ann Surg. 2013;257:731-736.
+
+5. Gnerlich JL, Luka SR, Deshpande AD, et al. Microscopic margins and patterns of treatment failure in resected pancreatic adenocarcinoma. Arch Surg. 2012;147:753-760.
+
+6. Delpero JR, Bachellier P, Regenet N, et al. Pancreaticoduodenectomy for pancreatic ductal adenocarcinoma: A
+French multicentre prospective evaluation of resection margins in 150 evaluable specimens. HPB. 2014;16:20-33.
+
+7. Neoptolemos JP, Palmer DH, Ghaneh P, et al. Comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer (ESPAC-4): A
+multicentre, open-label, randomised, phase 3 trial. Lancet. 2017; 389:1011-1024.
+
+8. Stocken DD, Bü Chler MW, Dervenis C, et al. Meta-analysis of randomised adjuvant therapy trials for pancreatic cancer. Br J
+Cancer. 2005;92:1372-1381.
+
+9. Herman JM, Swartz MJ, Hsu CC, et al. Analysis of fluorouracilbased adjuvant chemotherapy and radiation after pancreaticoduodenectomy for ductal adenocarcinoma of the pancreas:
+Results of a large, prospectively collected database at the Johns Hopkins Hospital. J Clin Oncol. 2008;26:3503-3510.
+
+10. Parikh AA, Maiga A, Bentrem D, et al. Adjuvant therapy in pancreas cancer: Does it influence patterns of recurrence? J Am Coll Surg. 2016;222:448-456.
+
+11. Ghaneh P, Kleeff J, Halloran CM, et al. The impact of positive resection margins on survival and recurrence following resection and adjuvant chemotherapy for pancreatic ductal adenocarcinoma.
+
+Ann Surg. 2019;269:520-529.
+
+12. Morganti AG, Falconi M, Van Stiphout RGPM, et al. Multi-institutional pooled analysis on adjuvant chemoradiation in pancreatic cancer. Int J Radiat Oncol Biol Phys. 2014;90:911-917.
+
+13. Smeenk HG, Van Eijck CHJ, Hop WC, et al. Long-term survival and metastatic pattern of pancreatic and periampullary cancer after adjuvant chemoradiation or observation: Long-term results of EORTC trial 40891. Ann Surg. 2007;246:734-740.
+
+14. Kaiser MH, Ellenberg SS. Pancreatic cancer: Adjuvant combined radiation and chemotherapy following curative resection. Arch Surg.
+
+1985;120:899-903.
+
+15. Neoptolemos JP, Stocken DD, Friess H, et al. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. N Engl J Med. 2004;350:1200-1210.
+
+16. Tesfaye AA, Philip PA. Further evidence of effective adjuvant combined radiation and chemotherapy following curative resection of pancreatic cancer. Gastrointestinal Tumor Study Group. Cancer. 1987;59:2006-2010.
+
+17. Van Laethem JL, Hammel P, Mornex F, et al. Adjuvant gemcitabine alone versus gemcitabine-based chemoradiotherapy after curative resection for pancreatic cancer: A randomized EORTC-4001322012/FFCD- 9203/GERCOR phase II study. J Clin Oncol. 2010; 28:4450-4456.
+
+18. Schmidt J, Abel U, Debus J, et al. Open-label, multicenter, randomized phase III trial of adjuvant chemoradiation plus interferon alfa-2b versus fluorouracil and folinic acid for patients with resected pancreatic adenocarcinoma. J Clin Oncol. 2012;30:4077-4083.
+
+19. Kooby DA, Gillespie TW, Liu Y, et al. Impact of adjuvant radiotherapy on survival after pancreatic cancer resection: An appraisal of data from the national cancer data base. Ann Surg Oncol. 2013;20:
+3634-3642.
+
+20. Shinde A, Verma V, Li R, et al. The role of sequential radiation following adjuvant chemotherapy in resected pancreatic cancer. J Gastrointest Oncol. 2019;10:462-473.
+
+21. Gemcitabine hydrochloride with or without erlotinib hydrochloride followed by the same chemotherapy regimen with or without radiation therapy and capecitabine or fluorouracil in treating patients with pancreatic cancer that has been removed by surgery. Available at: https://clinicaltrials.gov/ct2/show/NCT01013649. Accessed January 9, 2020.
+
+22. Chuong MD, Springett GM, Freilich JM, et al. Stereotactic body radiation therapy for locally advanced and borderline resectable pancreatic cancer is effective and well tolerated. Int J Radiat Oncol Biol Phys. 2013;86:516-522.
+
+23. Mahadevan A, Jain S, Goldstein M, et al. Stereotactic body radiotherapy and gemcitabine for locally advanced pancreatic cancer. Int J Radiat Oncol Biol Phys. 2010;78:735-742.
+
+24. Herman JM, Chang DT, Goodman KA, et al. Phase 2 multiinstitutional trial evaluating gemcitabine and stereotactic body radiotherapy for patients with locally advanced unresectable pancreatic adenocarcinoma. Cancer. 2015;121:1128-1137.
+
+25. Bernard ME, Sutera PA, Iarrobino NA, et al. Initial results of a prospective study of adjuvant pancreatic stereotactic body radiation therapy for close or positive margins. Adv Radiat Oncol. 2019;4: 294-301.
+
+26. Rwigema J-CM, Parikh SD, Heron DE, et al. Stereotactic body radiotherapy in the treatment of advanced adenocarcinoma of the pancreas. Am J Clin Oncol. 2011;34:63-69.
+
+27. Rwigema JCM, Heron DE, Parikh SD, et al. Adjuvant stereotactic body radiotherapy for resected pancreatic adenocarcinoma with close or positive margins. J Gastro Cancer. 2012;43:70-76.
+
+28. Goodman KA, Regine WF, Dawson LA, et al. Radiation Therapy Oncology Group consensus panel guidelines for the delineation of the clinical target volume in the postoperative treatment of pancreatic head cancer. Int J Radiat Oncol. 2012; 83:901-908.
+
+29. Zhu X, Ju X, Cao Y, et al. Patterns of local failure after stereotactic body radiation therapy and sequential chemotherapy as initial
+
+## Advances In Radiation Oncology: Novemberedecember 2020 Patterns Of Failure After Pancreatic Sbrt 1205
+
+treatment for pancreatic cancer: Implications of target volume design. Int J Radiat Oncol Biol Phys. 2019;104:101-110.
+
+30. Kharofa J, Mierzwa ML, Olowokure OO, et al. Pattern of marginal local failure in a phase II trial of neoadjuvant chemotherapy and stereotactic body radiation therapy for resectable and borderline resectable pancreas cancer. Am J Clin Oncol. 2019;42:247-252.
+
+482-482.
+
+31. Austin PC, Lee DS, Fine JP. Introduction to the analysis of survival data in the presence of competing risks. Circulation. 2016;133:601-609.
+
+32. Scrucca L, Santucci A, Aversa F. Competing risk analysis using R: An easy guide for clinicians. Bone Marrow Transplant. 2007;40:381-387.
+
+33. Kim HT. Cumulative incidence in competing risks data and competing risks regression analysis. Clin Cancer Res. 2007;13:559-565.
+
+34. R Core Team. R: A Language and Environment for Statistical Computing.
+
+Vienna, Austria: R Foundation for Statistical Computing; 2019.
+
+35. Baine MJ, Sleightholm R, Lin C. Incidence and patterns of locoregional failure after stereotactic body radiation therapy for pancreatic adenocarcinoma. Pract Radiat Oncol. 2019;9:e29-e37.
+
+36. Dholakia AS, Kumar R, Raman SP, et al. Mapping patterns of local recurrence after pancreaticoduodenectomy for pancreatic adenocarcinoma: A new approach to adjuvant radiation field design. Int J
+Radiat Oncol Biol Phys. 2013;87:1007-1015.

medical/md/PMC7776371.md

@@ -0,0 +1,27 @@
+Conclusion: TNFα and IL-18 in combination with CMV viral load may be useful in predicting likelihood of TI-CMV. This is important in situations where tissue biopsies are not feasible, and adds to our diagnostic capability for TI-CMV in SOT recipients.
+
+Disclosures: Stuart C.  Ray, MD, **miDiagnostics, Inc.** (Board Member, Research Grant or Support) Robin K. Avery, MD, **Aicuris** (Scientific Research Study Investigator)**Astellas** (Scientific Research Study Investigator)**Chimerix** (Scientific Research Study Investigator)**Merck** (Grant/Research Support, Scientific Research Study Investigator)**Oxford Immunotec** (Scientific Research Study Investigator)**Qiagen** (Scientific Research Study Investigator)**Takeda/Shire** (Scientific Research Study Investigator) 687. Iatrogenic Kaposi's Sarcoma Following Treatment with Vedolizumab Susanne O. Ajao, n/a1; HAMID SHAABAN, n/a2; Rajasingam Jayasingam, n/a2; 1St. Michael's Medical Center, Bloomfield, New Jersey 2St. Michael Medical Center, Newark, New Jersey Session: P-27. Diagnostics: Virology Background: Kaposi's sarcoma (KS) is a vascular tumor caused by human herpes virus-8 infection (HHV-8) commonly involving the skin. We report a case of a patient with controlled HIV who developed Kaposi's sarcoma on vedolizumab.
+
+![0_image_1.png](0_image_1.png)
+
+(UC), Hodgkin's lymphoma in remission and HIV presented with complaints of abdominal pain and bloody diarrhea. He had new tender lesions on his left foot which was absent at his previous admission a month ago. The lesions started as macules and later progressed to tender lesions. Mesalamine and oral corticosteroids were previously prescribed without symptomatic relief and he was started on vedolizumab 2 months ago. On physical exam he had abdominal tenderness, tender cervical and inguinal lymph nodes, and dark macules on his feet. Digital rectal exam revealed bloody mucoid stool. Laboratory showed white blood cell count of 12,600/mm3, ESR of 132 mm/
+hr and CRP of 4.6 mg/dL. His CD4 T-cell count was 873 cells/mm3 and viral load was 50 copies per milliliter. Cervical lymph node biopsy showed polymorphous population of lymphocytes but was negative for malignant cells. Biopsy of the foot plaques showed atypical intradermal vascular and spindle cell proliferation positive for HHV-8 and for vascular marker CD34. The results were consistent with the diagnosis of KS. Serology was also positive for HHV-8 with high viral titers of 74 copies/mL. Colonoscopy showed severe proctitis with deep ulcerations in a continuous pattern in the rectum with a normal sigmoid colon. Follow up colonoscopy showed improved proctitis and he was started on doxorubicin to treat KS with improvement of the foot lesion a month later following treatment.
+
+Dark macules on the sole of both feet
+
+Figure 3a: Spindle cells with irregular small vessel proliferation and red blood cell 
+
+![0_image_0.png](0_image_0.png)
+
+extravasation between tumor cells Figure 3b: Immunohistochemical stain showing ![0_image_2.png](0_image_2.png)
+
+of lymphocytes to the inflamed tissue. It is approved for the treatment of IBD and has shown efficacy and safety. The iatrogenic form of Kaposi's sarcoma occurs in patients on immunosuppressive therapy, as this patient.
+
+Conclusion: Patients with IBD on immunosuppressive drugs should be followed up closely and screened for latent viral infections prior to initiating therapy. As in the patient, HHV-8 should be recognized as a likely underlying opportunistic infection in immunocompromised patients with IBD.
+
+Disclosures: **All Authors**: No reported disclosures
+
+## 688. Marginal Structure Models To Estimate The Effect Of Cytomegalovirus Infection On Hospitalization Among Children Undergoing Allogeneic Hematopoietic Cell Transplantation
+
+Yun Li, PhD1; Arman Oganisian, MS2; Craig L. K. Boge, MPH3; Molly Hayes, PharmD, BCPS3; Alexander Newman, MD4; Brian T. Fisher, DO, MPH, MSCE5; Brian T. Fisher, DO, MPH, MSCE5; 1University of Pennsylvania, Philadelphia, Pennsylvania; 2Department of Biostatistics, Epidemiology and Informatics, philadelphia, Pennsylvania; 3Children's Hospital of Philadelphia, Philadelphia, PA; 4Ann and Robert H Lurie Children's Hospital of Chicago, Chicago, IL; 5Children's Hospital of Philadelphia; Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Session: P-27. Diagnostics: Virology Background: Children receiving an allogeneic hematopoietic cell transplant 
+(HCT) are at risk for cytomegalovirus (CMV) infection in the post-transplant period, necessitating routine surveillance for CMV. Some patients will not have CMV detected 

medical/md/PMC8028335.md

@@ -0,0 +1,509 @@
+pubs.acs.org/JPCA Article
+
+![0_image_0.png](0_image_0.png)
+
+# Assessment Of The Ab Initio Bethe−Salpeter Equation Approach For The Low-Lying Excitation Energies Of Bacteriochlorophylls And Chlorophylls Zohreh Hashemi And Linn Leppert*
+
+ABSTRACT: Bacteriochlorophyll and chlorophyll molecules are crucial building blocks of the photosynthetic apparatus in bacteria, algae, and plants. Embedded in transmembrane protein complexes, they are responsible for the primary processes of photosynthesis:
+excitation energy and charge transfer. Here, we use ab initio manybody perturbation theory within the GW approximation and Bethe−Salpeter equation (BSE) approach to calculate the electronic structure and optical excitations of bacteriochlorophylls a, b, c, d, and e and chlorophylls a and b. We systematically study the effects of the structure, basis set size, partial self-consistency in GW, and the underlying exchange−correlation approximation and compare our calculations with results from time-dependent density functional theory, multireference RASPT2, and experimental literature results. We find that optical excitations calculated with GW+BSE are in excellent agreement with experimental data, with an average deviation of less than 100 meV for the first three bright excitations of the entire family of (bacterio)chlorophylls.
+
+Contrary to state-of-the-art time-dependent density functional theory (TDDFT) with an optimally tuned range-separated hybrid functional, this accuracy is achieved in a parameter-free approach. Moreover, GW+BSE predicts the energy differences between the low-energy excitations correctly and eliminates spurious charge transfer states that TDDFT with (semi)local approximations is known to produce. Our study provides accurate reference results and highlights the potential of the GW+BSE approach for the simulation of larger pigment complexes.
+
+- INTRODUCTION
+Electronic excitations form the foundation of some of the most fundamental natural processes. In photosynthesis, plants, algae, and bacteria convert solar energy into chemical energy, utilizing a cascade of coupled energy and charge transfer excitations that are performed by pigment−protein complexes with high quantum efficiency. Bacteriochlorophyll (BCL) and chlorophyll (CL) molecules are among the most important building blocks of these pigment−protein complexes.1 They are responsible for the absorption and transfer of excitation energy and for the charge separation necessary for establishing a proton gradient that eventually drives the synthesis of chemical energy in plants and bacteria.2 Accurately calculating the electronic structure and excitations of these molecules from first principles is the prerequisite for understanding their interactions with each other and with the surrounding proteins and, consequently, energy and charge transfer in natural photosynthesis.
+
+BCL and CL molecules constitute a family of substituted tetrapyrroles with varying absorption properties depending on conjugation and the number and nature of substitutions. CL a and b are present in plants and green algae, whereas green
+
+ACCESS Metrics & More Article Recommendations *sı Supporting Information
+bacteria mostly rely on BCL c, d, and e for excitation energy transfer and BCL a for concentrating excitations close to the reaction center of the photosynthetic unit.3 BCL a is also the main pigment in purple bacteria, whose light harvesting apparatus and reaction center are among the most thoroughly studied natural light-harvesting systems.4 The optical excitation spectrum of these pigments possesses two characteristic absorption bands: (1) the Q band in the visible part of the spectrum, composed of excitations Qy and Qx with high- and low-oscillator strength, respectively and (2) the B (or Soret) band in the near ultraviolet.
+
+In the field of finite organic and biological molecular systems, neutral excitations and optical spectra are predominantly calculated using time-dependent density functional theory (TDDFT). In conjunction with model Hamiltonian
+
+Received: February 10, 2021
+
+![0_image_2.png](0_image_2.png) Revised: February 16, 2021 Published: March 3, 2021
+
+![0_image_1.png](0_image_1.png)
+
+approaches, TDDFT has been employed for the simulation of large photosynthetic pigment−protein complexes.5,6 The accuracy of its approximations and implementations has been tested for a variety of biochromophores.7,8,47 However, TDDFT's standard approximations are inadequate for describing long-range charge transfer excitations9 and highenergy Rydberg states10 due to self-interaction errors and an incorrect asymptotic behavior. Exchange−correlation (xc)
+functionals that contain long-range exact exchange, such as optimally tuned range-separated hybrid functionals (OTRSH), can be employed as a remedy in such cases,11,12 but the use of such functionals requires a tedious per-system tuning procedure.
+
+Multireference wavefunction-based methods have scarcely been used for molecules as large as BCL and CL. Vertical excitation energies of CL a, based on ADC(2) and different coupled cluster approaches show a spread of ∼0.4 eV, strongly depending on the method, basis set, and structural model used in these calculations.13 −15 In 2016 and 2019, Anda et al.
+
+reported multistate RASPT2/RASSCF excitation energies of several BCL units within the light-harvesting system 2 of a purple bacterium.16,17 A RASPT2 approach was also combined with electrostatic embedding of fixed-point charges to simulate the effect of the protein environment on excitation energies of the same system by Segatta et al.18 While these reports constitute important advances in the use of wavefunctionbased methods for complex biological molecules, they were performed with relatively small basis sets and show a dependence on the choice of the restricted active space (RAS).
+
+The ab initio Bethe−Salpeter equation (BSE) approach, when rigorously based on many-body Green's function theory, is an alternative method for describing neutral excitations of correlated many-electron systems.19 It is based on a framework of charged excitation energies that correspond to electron addition and removal energies and that are most frequently calculated within the GW approximation. The GW+BSE
+approach has been shown to be successful in predicting the optical spectra of bulk solids20,21 and low-dimensional materials.22 In recent years, it has also begun to be applied to finite systems, such as small molecules,23−25 and larger molecular complexes,26−28 for which its accuracy has been shown to be comparable to single-reference wavefunction methods for both localized and charge transfer excitations,29 at a substantially reduced computational cost.
+
+In this article, we assess the accuracy of the ab initio GW
++BSE approach for the Qy, Qx, and the first bright B excitation of several members of the BCL and CL family and the chemically closely related bacteriochlorin (BC) molecule. We compare two different approaches for approximating the electronic self-energy Σ = iGW: (1) G0W0, a one-shot method, in which the zeroth-order Green's function G0 and screened Coulomb interaction W0 are constructed from a DFT
+eigensystem and directly used to correct DFT eigenvalues perturbatively and (2) partially self-consistent GW (evGnWn),
+in which the corrected eigenvalues are used to iteratively recalculate G and/or W until self-consistency is reached. We compare our results to TDDFT calculations with the local density approximation (LDA), two global hybrids and an OTRSH functional, with RASPT2 literature results,16,17 and with experimental data.30,31 We find that the GW+BSE approach used in a partially selfconsistent manner results in excitation energies in the visible and near-ultraviolet within less than 100 meV from experiment for the entire family of pigments studied here. Our results are almost completely independent of the DFT eigensystem used as an input for the GW+BSE calculations. In fact, even a simple and computationally inexpensive LDA starting point leads to excellent agreement with experiment and eliminates spurious charge transfer excitations between Qy and Qx that TDDFT
+with (semi)local functionals produces. Contrary to TDDFT,
+GW+BSE also correctly predicts the energy difference between the two Q-band excitations, a crucial prerequisite for understanding the coupling of excitations in systems consisting of more than one pigment. Finally, we show that differences between evGnWn+BSE and state-of-the-art TDDFT calculations using an OT-RSH functional can be explained almost entirely based on differences in how electron−hole interactions are described by the xc kernel of TDDFT and the BSE kernel, respectively. Eigenvalue differences as computed with evGnWn and DFT with an OT-RSH functional are almost identical.
+
+The remainder of this article is structured as follows: we start by briefly reviewing the GW+BSE approach and report computational details and numerical convergence. We then show the effect of different DFT starting points and partial selfconsistency on the excitation energies of the BC molecule.
+
+After this, we discuss our results for BCL a, b, c, d, and e and CL a and b, followed by a comparison with literature results and an in-depth discussion of differences between our GW
++BSE and TDDFT results for BCL a.
+
+- METHODS
+GW+BSE Approach. In Green's function-based manybody perturbation theory, the calculation of charged excitations, corresponding to electron removal and addition energies, is based on knowledge of the exact interacting singleparticle Green's function G, which can, in principle, be computed from a set of self-consistent integro-differential equationsHedin's equationslinking G to the electronic self-energy Σ, the screened Coulomb interaction W, the irreducible polarizability χ, and the vertex function Γ.
+
+32 The lowest-order expansion of Σ with respect to W leads to the GW approximation, in which the electronic self-energy Σ = iGW.
+
+33 Quasiparticle (QP) eigenvalues can be obtained by solving Ä
+
+Ç
+ÅÅÅÅÅÅÅÅÅ
+$$\begin{array}{l}{{-\frac{\hbar^{2}}{2m}\nabla^{2}\,+\,V_{\mathrm{ion}}({\bf r})\,+\,V_{\mathrm{H}}({\bf r})\,\biggr]\!\varphi_{n}^{\mathrm{QP}}({\bf r})}}\\ {{+\,\int\!\mathrm{d}{\bf r}^{\prime}\Sigma({\bf r},\,{\bf r}^{\prime};\,\varepsilon_{n}^{\mathrm{QP}})\varphi_{n}^{\mathrm{QP}}({\bf r}^{\prime})\,=\,\varepsilon_{n}^{\mathrm{QP}}\varphi_{n}^{\mathrm{QP}}({\bf r})}}\end{array}$$
+$\left(1\right)$. 
+here, Vion is the ionic potential, VH is the Hartree potential, and εn QP and φn QP are QP energies and wavefunctions, respectively.
+
+To avoid the high computational cost of a self-consistent solution of eq 1, the GW approach is commonly used within a one-shot scheme, in which G0 and W0 are constructed from a
+(generalized) Kohn−Sham (gKS) eigensystem obtained from a preceding DFT calculation. We use the notation G0W0@gKS
+to refer to G0W0 based on the gKS eigensystem (φn gKS;εn gKS)
+computed with the xc functional Exc gKS. In this approach, QP
+corrections are calculated to first order in Σ as
+
+$$(\varepsilon_{n}^{\mathrm{qp}})\,-\,V_{\mathrm{xc}}|\varphi_{n}^{\mathrm{gKS}}\rangle$$
+$$=\varepsilon_{n}^{\mathrm{gKS}}+\,\langle\varphi_{n}^{\mathrm{gKS}}|\Sigma$$
+
+n n ( ) V n n n QP gKS gKS QP
+xcgKS ε = + ⟨ |Σ − | *ε φε φ* ⟩ (2)
+where Vxc is the xc potential, and it is assumed that φn QP ≈ φn gKS.
+
+While the G0W0 approach has been used with much success, in particular, for the calculation of band gaps and band structures of solids, a well-known and well-documented dependence on the gKS eigensystem used to construct G0 Karlsruhe basis sets def2-SVP, def2-TZVP, and def2-TZVPP
+
+![2_image_0.png](2_image_0.png)
+
+and their corresponding auxiliary basis sets.42 Figures 1 and S2 show the convergence of the HOMO−
+LUMO gap and Qy and Qx excitation energies as a function of and W0 limits its predictive power.34−36 Partial self-consistency in the QP eigenvalues can often mitigate this problem. In eigenvalue self-consistent GW, the gKS eigenvalues used to construct G and/or W are replaced with those from the output of a prior GW step; the self-energy corrections are then iterated until the QP eigenvalues converge. This approach, which we call evGnWn in the following (n refers to the number of iterations), has been shown to remove much of the starting point dependence for a range of different systems.37,38 The BSE is an equation for the two-particle electron−hole Green's function and allows for the calculation of the polarizability including electron−hole interactions through the screened Coulomb interaction W. In practice, the BSE is usually solved by neglecting the frequency dependence of W. Within this static approximation, it can be written in a form equivalent to Casida's equations of TDDFT
+
+k
+$${\binom{A}{-\ B\ -\ A}}{\binom{X^{s}}{Y^{s}}}=\Omega_{s}{\binom{X^{s}}{Y^{s}}}$$
+− − s = Ω(3)
+k
+where Ωs represents neutral excitation energies and (Xs, Ys) the corresponding eigenvectors.19 A and −A represent resonant and antiresonant transitions that can be expressed as
+
+$$A_{i a}^{j b}=(\varepsilon_{a}^{\mathrm{QP}}-\varepsilon_{i}^{\mathrm{QP}})\delta_{i j}\delta_{a b}-2(i a|j b)+W_{i j}^{a b}(\omega=0)$$
+and that are coupled through $B$ and $-B$, defined as . 
+$$B_{i a}^{j b}=-2(i a l b j)\,+\,W_{i i}^{a b}(\omega=0)$$
+ij
+ab =− | + = ω (5)
+for singlet excitations. In these expressions, i and j are occupied and a and b are unoccupied states and (ia|bj) stands for
+
+$$(i a l b j)=\iint\mathrm{d}\mathbf{r}\mathrm{d}\mathbf{r}^{\prime}\varphi_{i}^{\mathrm{QP}}(\mathbf{r})\varphi_{a}^{\mathrm{QP}}(\mathbf{r}){\frac{1}{|\mathbf{r}-\mathbf{r}^{\prime}|}}\varphi_{j}^{\mathrm{QP}}(\mathbf{r}^{\prime})\varphi_{b}^{\mathrm{QP}}(\mathbf{r}^{\prime})$$
+
+(6)
+Note that φi QP = φi gKS, whenever the G0W0 or evGnWn approaches are used to construct A and B.
+
+Computational Details. Our calculations of charged and neutral excitations were performed using the GW+BSE and TDDFT implementation in the open-source MOLGW software package (version 2B), which relies on Gaussian basis functions.39 We used the frozen-core approximation throughout, which changes excitation energies by less than 1 meV. We also employed the resolution-of-the-identity (RI) method, in order to reduce the calculation of four-center integrals to two- and three-center integrals. For BCL a, the RI changes the QP highest occupied molecular orbital
+(HOMO)−lowest unoccupied molecular orbital (LUMO)
+gap by less than 50 meV using a 6-31G basis set and BHLYP as a starting point, but we expect the effect of the RI to be even smaller for the larger basis sets used in the remainder of this article.40 To further reduce the computational cost of the evaluation of the GW polarizability, we use the single-pole approximation (see the Supporting Information for details).
+
+The Tamm−Dancoff approximation, which corresponds to neglecting the B matrix elements in eq 3 is not used as it consistently increases both GW+BSE and TDDFT results by
+∼0.3 eV, in agreement with previous findings.6,27 We tested the influence of the Gaussian basis set size on HOMO−LUMO gaps and Qy and Qx excitations of BCL a
+(using a structure from ref 16) with G0W0@BHLYP+BSE,
+considering seven different basis sets, namely, the Pople basis sets 6-31G, 6-311G, 6-311++G**, and 6-311++G(2d,2p),
+combined with the DeMon auxiliary basis set,41 and the
+
+$$({\mathfrak{I}})$$
+
+$$\left({4}\right)$$
+
+the inverse number of basis functions, 1/Nbasis for GW+BSE
+and TDDFT, respectively (raw data are presented in Tables S1 and S2). We find that the HOMO−LUMO gap depends significantly more on 1/Nbasis than Qy and Qx excitation energies and that TDDFT results are less sensitive to the choice of the basis set than GW+BSE. Based on these tests, we use the 6-311++G(2d,2p) basis set for all calculations reported in the following. We estimate the error in the GW(+BSE)
+HOMO−LUMO gap and the Qy and Qx excitation energies by linearly extrapolating to an infinite basis set. By excluding the very small 6-31G and 6-311G basis sets from these fits, we obtain extrapolated values of 3.57 eV for the HOMO−LUMO
+gap, 1.11 eV for Qy, and 1.81 eV for Qx. We conclude that using the 6-311++G(2d,2p) basis set for all further calculations, we likely overestimate GW(+BSE) HOMO−
+LUMO gaps and Qy and Qx excitation energies by ∼0.1 eV
+with respect to the complete basis set limit. Conversely, the use of the single-pole approximation leads to a similar underestimation of the HOMO−LUMO gap and the Qy and Qx excitations, resulting in a fortuitous cancellation of errors.
+
+We test the effect of different xc functionals on our TDDFT
+and GW+BSE results. We use the LDA, two global hybrid functionals (B3LYP and BHLYP), and the range-separated hybrid (RSH) functional ωPBE. In RSH functionals, the Coulomb repulsion is separated into a short-range part and a long-range part, for numerical convenience expressed as
+
+$${\frac{1}{r}}={\frac{1\,-\,\operatorname{erf}(a r)}{r}}\,+\,{\frac{\operatorname{erf}(a r)}{r}}$$
+
+1 1 erf( ) erf( ) ω ω = − + (7)
+where ω is called the range separation parameter. The ωPBE
+functional uses PBE exchange in the short range and the exact exchange energy in the long range, allowing for a selfinteraction-free description at large electron−electron distances. We obtain the range separation parameter ω through the tuning procedure outlined in ref 43, in which ω is chosen such that the HOMO eigenvalue is as close as possible to the negative ionization potential both for the neutral and anionic system. Consequently, and by construction, the resulting HOMO−LUMO gap is a very good approximation to the fundamental gap of the neutral molecule. We use the Q-Chem code and a 6-31G(d,p) basis set for the tuning.44 The tuned range separation parameters for all systems discussed in the following can be found in Table S3.
+
+- RESULTS AND DISCUSSION
+Bacteriochlorin. To validate our methodological setup and investigate the starting point dependence of the G0W0 approach and the effect of eigenvalue self-consistency on our calculated HOMO−LUMO gaps and excitation energies, we start by examining the BC molecule, for which GW+BSE
+results have been reported in ref 27. We use a BC structure from ref 27 and denote the lowest energy excitations Qx and Qy, according to the direction of their transition dipole moments. Table 1 contains our calculated HOMO−LUMO
+
+Table 1. HOMO−LUMO Gaps, Qx and Qy Excitation
+
+Energies (in eV), and the Corresponding Oscillator
+
+Strengths, Γx and Γy, for BC Calculated with the 6-311+
+
++G(2d,2p) Basis Set
+
+method xc functional H-L gap Qx Γx Qy Γy
+
+TDDFT LDA 1.38 2.04 0.18 2.39 0.03
+
+B3LYP 2.17 2.06 0.23 2.51 0.04 BHLYP 3.27 1.93 0.28 2.55 0.04
+
+ωPBE 4.38 1.87 0.23 2.42 0.05
+
+G0W0+BSE LDA 4.15 1.21 0.09 1.67 0.04
+
+B3LYP 4.36 1.44 0.14 1.97 0.04 BHLYP 4.56 1.67 0.19 2.23 0.05
+
+ωPBE 4.59 1.64 0.19 2.26 0.05
+
+evGnW0+BSE LDA 4.31 1.41 0.13 1.97 0.04
+
+B3LYP 4.43 1.54 0.16 2.12 0.05 BHLYP 4.56 1.66 0.19 2.24 0.05
+
+ωPBE 4.57 1.62 0.18 2.27 0.04
+
+evGnWn+BSE LDA 4.42 1.51 0.17 2.21 0.05
+
+B3LYP 4.55 1.63 0.19 2.24 0.05 BHLYP 4.60 1.69 0.20 2.27 0.05
+
+ωPBE 4.56 1.61 0.18 2.26 0.04
+
+Expa 1.60 2.30
+
+aData for bacteriopheophorbide from refs.31,27
+
+gaps, Qx and Qy excitation energies, and oscillator strengths using TDDFT and several flavors of the GW+BSE approach. We find that, as expected, (generalized) Kohn−Sham HOMO−LUMO gaps show a large dependence on the xc functional, with LDA, B3LYP, and BHLYP leading to a significantly lower HOMO−LUMO gap and ωPBE leading to a HOMO−LUMO gap similar to the HOMO−LUMO gap calculated with G0W0 and eigenvalue-self-consistent evGnWn. In turn, Qy and Qx excitation energies from TDDFT are considerably less dependent on the xc functional than HOMO−LUMO gaps. In agreement with previous studies, we find that TDDFT overestimates the experimental values for The Journal of Physical Chemistry A pubs.acs.org/JPCA Article Qx and Qy by up to ∼0.4 eV, depending on the xc functional.27 TDDFT with the OT-RSH ωPBE is in best agreement with experiment, overestimating it by ∼0.2 eV for both excitations.
+
+We further find that G0W0@LDA+BSE underestimates Qx by 0.4 eV and Qy by 0.6 eV, whereas the use of a BHLYP and ωPBE starting point results in excitations within 0.1 eV of the experimental results. In accordance with prior studies, we observe that most of the starting point dependence of the G0W0+BSE results is inherited from the starting point dependence of the HOMO−LUMO gaps.25 In order to investigate the effect of eigenvalue selfconsistency in the GW+BSE approach, we tested the effect of updating the eigenvalues in the construction of G only
+(evGnW0) and of both G and W (evGnWn). Eigenvalue selfconsistency in G alone only slightly changes the results as compared to G0W0. In contrast, full eigenvalue self-consistency largely eliminates the starting point dependence. In particular, using an LDA starting point results in excitation energies within 0.1 eV from experimentsimilar to the ωPBE starting point, but at considerably reduced computational cost. In Table S4, we report similar results for the more complex pigment BCL a. In the remainder of this article, we therefore focus primarily on eigenvalue self-consistent results based on LDA and ωPBE starting points.
+
+Excitation Energies of Bacteriochlorophylls and Chlorophylls. Next, we turn to reporting the vertical excitation energies of several members of the BCL and CL family of pigments. All structures were obtained from ref 45 and geometry-optimized using DFT as implemented in the Turbomole code with a def2-TZVP basis set and the B3LYP xc functional.46 Atomic coordinates of all relaxed structures can be found in the Supporting Information. We used both LDA
+and ωPBE starting points for our evGnWn+BSE and ωPBE for our TDDFT calculations. Unlike the Qx excitation of BCL a and b, which has significant oscillator strength, the Qx excitation of BCL c−e is dark. Following ref 8, we therefore also compare our calculations with experimental results for the higher-energy B band.31 We report the vertical excitation energies and corresponding oscillator strengths of the first six excitations of all pigments in Tables S6 and S7. In these calculations, we included a total of 20 excitations, in order to ensure that the higher lying excitations are well converged.
+
+Table 2 demonstrates that evGnWn+BSE is in excellent agreement with experiment for the entire family of BCL and CL molecules. The MAE is about 50 meV for the Qy and Qx and between 100 and 200 meV for the B excitation. Our evGnWn+BSE results also accurately reflect the spectral shifts of the Qy excitation when comparing different BCL pigments with each other. For example, the BCL b molecule differs from BCL
+a through an ethyliden side group, which shifts the Qy excitation by 40 meV to the red. This red shift is perfectly reproduced in our GW+BSE calculations. This is the first main result of this study. The second one is that our results are essentially independent of the DFT eigensystem used as an input for the GW+BSE approach: a computationally inexpensive LDA starting point results in the same level of agreement with experiment as the more tedious ωPBE
+calculation that involves a system-dependent tuning procedure for the range separation parameter ω. This is in stark contrast to TDDFT. TD-LDA leads to spurious excitations with charge transfer character in between Qy and Qx, as well as slightly above Qx, depending on the structure, as discussed below and in the literature.47 TDDFT with the optimally tuned ωPBE
+
+| GW@LDA+BSE                                                                                                           | GW@ωPBE+BSE   | TD-ωPBE   | expb   |      |      |      |      |      |      |      |      |      |
+|----------------------------------------------------------------------------------------------------------------------|---------------|-----------|--------|------|------|------|------|------|------|------|------|------|
+| molecule                                                                                                             | Qy            | Qx        | B      | Qy   | Qx   | B    | Qy   | Qx   | B    | Qy   | Qx   | B    |
+| BCL a                                                                                                                | 1.52          | 2.08      | 3.25   | 1.50 | 2.10 | 3.16 | 1.75 | 2.16 | 3.33 | 1.60 | 2.15 | 3.46 |
+| BCL b                                                                                                                | 1.48          | 2.07      | 2.95   | 1.45 | 2.09 | 3.05 | 1.69 | 2.15 | 3.19 | 1.56 | 2.14 | 3.37 |
+| BCL c                                                                                                                | 1.85          | 2.05      | 2.94   | 1.84 | 2.11 | 3.02 | 2.05 | 2.21 | 3.21 | 1.88 | 2.89 |      |
+| BCL d                                                                                                                | 1.90          | 2.15      | 2.94   | 1.89 | 2.21 | 3.05 | 2.08 | 2.29 | 3.19 | 1.90 | 2.93 |      |
+| BCL e                                                                                                                | 2.01          | 2.04      | 2.78   | 1.96 | 2.13 | 2.88 | 2.10 | 2.23 | 3.02 | 1.92 | 2.72 |      |
+| CL a                                                                                                                 | 1.85          | 2.13      | 2.91   | 1.86 | 2.19 | 3.02 | 2.06 | 2.29 | 3.16 | 1.87 | 2.14 | 2.88 |
+| CL b                                                                                                                 | 1.95          | 2.17      | 2.79   | 1.93 | 2.20 | 2.85 | 2.10 | 2.29 | 2.97 | 1.92 | 2.26 | 2.72 |
+| MAE                                                                                                                  | 0.05          | 0.06      | 0.12   | 0.04 | 0.05 | 0.17 | 0.17 | 0.05 | 0.25 |      |      |      |
+| a GW+BSE results are based on eigenvalue self-consistent evGnWn. b Experimental results in diethyl ether from ref 8. |               |           |        |      |      |      |      |      |      |      |      |      |
+
+![4_image_0.png](4_image_0.png)
+
+Table 2. Qy, Qx and First B Band Excitation Energy of BCLs and CLs Calculated Using a 6-311++G(2d,2p) Basis Seta results in good agreement with experiment for all three excitations, albeit with slightly higher MAEs of 170, 50, and 250 meV for Qy, Qx, and B, respectively.
+
+In Figure 2, we plot the difference between our calculated results and experiment, averaged over all three excitations, to further highlight qualitative differences between evGnWn+BSE
+and TDDFT. For BCL a and BCL b, evGnWn+BSE, on average, underestimates experiment by ∼100 meV, whereas the average TDDFT deviation is close to zero, because TDDFT
+slightly overestimates the Qy and Qx excitations but underestimates the B excitation of these pigments. For all other BCL
+and the two CL molecules studied here, we consistently find that the average deviation of evGnWn+BSE is significantly smaller than that of TDDFT. Similar to our results for the BC molecule and to other benchmark studies of complex organic molecules,6 TDDFT tends to overestimate all three excitations by between 200 and 300 meV. evGnWn+BSE is in much closer agreement with experiment for these pigments, on average, overestimating their excitation energies by less than 100 meV. We stress again that these results are independent of the DFT
+starting point, whereas our TDDFT results rely on a persystem tuning procedure.
+
+Our results are in excellent agreement with correlated excited-state methods for those systems for which such studies have been reported, primarily CL a and BCL a. ADC(2)
+excitation energies of the first three excitations of CL a reported by Suomivuori et al. are 1.97, 2.11, and 2.95 eV,
+within ∼0.1 eV of our evGnWn@LDA+BSE results.13 In another study by the same authors, the ADC(2) Qy excitation energy of histidin-ligated BCL a was reported to be 1.46 eV,
+again within ∼0.1 eV of our results, although it should be noted that the structures of ligated and free-standing BCL a slightly differ, leading to excitation energy differences of 10−30 meV at the ADC(2) level.14 Furthermore, Sirohiwal et al. used a pair-natural orbital coupled cluster approach to study CL a and reported Qy and Qx excitation energies of 1.75 and 2.24 eV, respectively, for CL a, also within ∼0.1 eV of our GW+BSE
+results for these excitations.15 Not only the absolute energies of Qy and Qx excitations are important for understanding and predicting excitation energy and charge transfer in photosynthetic systems but also their relative energy difference, ΔQx−Qy
+, plays a role, in particular, in coupled systems of several pigment units. It is therefore reassuring that evGnWn+BSE predicts ΔQx−Qy in very good agreement with experiment, with a deviation of only 10 meV for BCL a, BCL b, and CL a and 120 meV for CL b for the LDA starting point and a slightly larger deviation of, on average, 60 meV for the ωPBE starting point. TDDFT based on ωPBE tends to underestimate ΔQx−Qy
+, by, on average, 110 meV for these four pigments. For BCL a, we also show in Table 3 that ΔQx−Qy strongly depends on the xc functional used in the TDDFT calculations, primarily because of the strong dependence of the Qx excitation on the amount of exact exchange, which can be seen by comparing the results based on the LDA (0% of exact exchange), B3LYP (∼23%), and BHLYP
+Table 3. Difference between Qx and Qy Excitation Energies
+(in eV) Using TDDFT and evGnWn+BSE for BCL a
+
+| method     | xc functional   | ΔQx−Qy   |
+|------------|-----------------|----------|
+| evGnWn+BSE | LDA             | 0.57     |
+| B3LYP      | 0.54            |          |
+| BHLYP      | 0.54            |          |
+| ωPBE       | 0.59            |          |
+| TDDFT      | LDA             | 0.25     |
+| B3LYP      | 0.40            |          |
+| BHLYP      | 0.64            |          |
+| ωPBE       | 0.43            |          |
+| exp8       | 0.55            |          |
+
+(50%). As before, evGnWn+BSE is in excellent agreement with experiment and almost independent of the underlying xc functional.
+
+The experimental results reported in Tables 2 and 3 are based on measurements in diethyl ether, whereas our calculations are for gas-phase molecules. To approximately account for the effect of the solvent, we extracted experimental reference values for Qy and Qx excitations from a study by Limantara et al.,30 in which electronic absorption spectroscopy was used to obtain Qy and Qx for a large number of nonpolar and polar solvents at room temperature. This study reports regression lines for Qy and Qx excitations of BCL a as a function of R(n) = n2 − 1/n2 + 2, where n is the refractive index of the solvent. The extrapolated values for n = 1 (vacuum) are 1.68 eV (nonpolar) and 1.67 eV (polar) for the Qy and 2.25 eV (nonpolar) and 2.21 eV (polar) for the Qx excitation. Based on these regression parameters, we estimate that the experimental reference values in Table 2 lie ∼50−70 meV below the gas-phase excitation energies. We also calculated the Qy and Qx excitation energies of BCL a with TDDFT (using ωPBE), approximating solvent effects with the COSMO approach as implemented in Turbomole. We used a dielectric constant of 4.33ε0 corresponding to the value in diethyl ether. COSMO red-shifts the Qy and Qx excitation energies by 70 and 50 meV, respectively, supporting our estimate. We conclude that solvent effects are smallwithin the numerical accuracy of our GW+BSE calculationsand do not change our main conclusions. Note that we also neglect the effects of temperature and the 0−0 vibrational energy contribution in our comparison with experimental results. Exact agreement of our calculated results with experiment is therefore not expected.
+
+Bacteriochlorophyll a. In the remainder of this paper, we will use the BCL a molecule as a case study to compare to available computational literature results for this pigment, discuss the origin of differences between our evGnWn+BSE and TDDFT results, and comment on the effects of the choice of structure on excitation energies.
+
+Comparison with RASPT2. For the Qy and Qx excitations of BCL a, we compare our GW+BSE and TDDFT calculations to multistate, second-order perturbation theory (RASPT2)
+calculations by Anda et al.16,17 For this comparison, we use the molecular geometry reported in ref 16, which is a BCL a unit from the light-harvesting system LH2 of Rhodoblastus acidophilus. This structure was extracted from an experimental X-ray crystallographic structure of the LH2 complex (unit 302 within the structure 1NKZ in the RCSB Protein Data Bank).48 The phytyl tail was truncated and replaced by a hydrogen atom, and no further geometry optimization was carried out. In the following, we will call this structure "A". Our geometryoptimized version of "A", which we relaxed using DFT as
+
+![5_image_0.png](5_image_0.png)
+
+implemented in the Turbomole code with a def2-TZVP basis set and B3LYP,46 will be called "R". A visual comparison between "A" and "R" is shown in Figure 3. The large differences that we observe between these two structures are unsurprising, given that we perform our geometry optimizations without taking into account the protein environment in which BCL a "A" is embedded in vivo. Table 4 shows our GW
+Table 4. HOMO−LUMO Gaps and Qy and Qx Excitation Energies (in eV) for BCL a Structure "A"
++BSE and TDDFT results for "A" in comparison with the RASPT2 excitation energies from refs.16,17 We find, as before, that when eigenvalue self-consistency is used in GW, HOMO−
+LUMO gaps and Qy and Qx excitation energies differ by a maximum of 0.1 eV. Most notably, however, our GW+BSE
+excitation energies substantially differ from those calculated with RASPT2, with Qy 0.4 eV and Qx 0.5 eV lower than the RASPT2 result.
+
+We find that about half of this difference can be traced back to the use of a smaller basis set (ANO-RCC-vDZP) in ref 16.
+
+Repeating our evGnWn@LDA+BSE calculation with the same basis, we obtain excitation energies of 1.38 eV for Qy and 2.18 eV for Qx. In line with previous studies, we also find that TDDFT with global hybrid functionals (B3LYP and BHLYP)
+results in similar excitation energies to RASPT2 for the Qy excitation.17,49 We hypothesize that this agreement is fortuitous. The optimally tuned RSH functional ωPBE has been shown to better describe singlet excitation energies of a
+
+| method/basis set           | xc   |      |      |      |
+|----------------------------|------|------|------|------|
+| functional                 | H-L  |      |      |      |
+| evGnWn+BSE/6-311++G(2d,2p) | LDA  | 3.62 | 1.17 | 1.90 |
+| evGnWn+BSE/ANO-RCC-vDZP    | LDA  | 3.76 | 1.38 | 2.18 |
+| TDDFT/6-311++G(2d,2p)      | LDA  | 0.92 | 1.59 | 1.99 |
+| RASPT2/ANO-RCC-vDZP        | 1.61 | 2.40 |      |      |
+
+wide variety of organic compounds as compared to global hybrid functionals50−52 and is more than 0.1 eV lower in energy than the RASPT2 Qy excitation energy. Similar trends have also been shown for CL a, where DFT-based multireference CI, just as TDDFT with global hybrid functionals, tends to overestimate experiment by ∼0.2 eV for the Qy and Qx excitation.53 All in all, given that comparisons with experimental data are complicated for an in vivo structure such as "A", we consider it most likely that our GW+BSE
+calculations underestimate the excitation energies of structure
+"A" by ∼0.1 eV, similar to our results for gas-phase BCL a
+(Table 2). The remaining deviations could be attributed to the multireference character of the Qy excitation16 and the choice of the RAS.
+
+We also note that our GW+BSE results reproduce the energetic order and relative energy differences of the Qy excitation of other BCL units within the LH2 ring that RASPT2 predicts, when using the ANO-RCC-vDZP basis set.
+
+However, the use of the significantly larger 6-311++G(2d,2p)
+basis leads to substantially larger excitation energy differences between these units (Table S7). Finally, it is worth mentioning that our GW+BSE calculations reproduce the relatively large energy difference ΔQx−Qy ≈ 0.8 eV that RASPT2 predicts, whereas TDDFT excitation energy differences are much less sensitive to details of the structure, with ΔQx−Qy ≈ 0.5 eV (using ωPBE) similar to the gas-phase structure of BCL a. We speculate that a geometry optimization of structure "A" within its protein environment would result in a smaller ΔQx−Qy for both RASPT2 and GW+BSE.
+
+Role of the Electron−Hole Kernel. We find that the difference between GW+BSE and TDDFT excitation energies can be traced back almost entirely to differences in how electron−hole interactions are described in both schemes. The Qy excitation is primarily (∼90%) a HOMO → LUMO
+transition, and the HOMO−LUMO gaps, as calculated with DFT-ωPBE and evGnWn@ωPBE, differ by only 0.02 meV
+(Table 3). In fact, the density of states (DOS) in the energy range relevant for both the Qy and the Qx excitations based on evGnWn@ωPBE and DFT-ωPBE eigenvalues are almost identical (see Figure 4). To further test our hypothesis, we construct the statically screened Coulomb interaction Wij ab (see eq 4) and solve the BSE based on a DFT-ωPBE eigensystem
+(instead of first computing QP eigenvalues using eq 2). We obtain values for the Qy and Qx excitation that are only 20 meV
+higher and 40 meV lower than the full GW+BSE solution, respectively, for structure "A". Similarly, for structure "R", the results are within less than 10 and 50 meV for the Qy and Qx excitation, respectively. This observation confirms that differences between the GW+BSE and TDDFT excitation energies are primarily due to differences in the xc and the BSE kernel.
+
+Generally, the overestimation of excitation energies that we observe with TDDFT is in line with results for other organic π chromophores such as rhodamine and rosamine54 and of phenothiazine dyes,55 for which it has been linked to an insufficient treatment of differential electron correlation between the ground and excited states by most TDDFT xc kernels.56 Charge Transfer Excitations with TD-LDA and GnWn@LDA
++BSE. Finally, motivated by the excellent performance of evGnWn@LDA+BSE, we compare G0W0@LDA+BSE, evGWLDA+BSE, and TD-LDA results for structures "A" and "R" of BCL a. Figure 5 shows the excitation spectrum calculated at
+
+The Journal of Physical Chemistry A pubs.acs.org/JPCA Article
+
+![6_image_0.png](6_image_0.png)
+
+these levels of theory. TD-LDA's severe underestimation of charge transfer excitations is well known9 and leads to spurious excitations with charge transfer character at energies between Qy and Qx for BCL a.
+
+47 Our comparison of structures "A" and
+"R" shows that while the energy of Qy and Qx is changing only slightly when TD-LDA is used, the relative position of these spurious low-oscillator strength excitations depends strongly on the structure. G0W0@LDA+BSE results in a very different, albeit no more reassuring, picture. For both structures, the first excitation already appears at energies below or around 1 eV
+and its oscillator strength is considerably lower than with TDLDA; for structure "A", the oscillator strength of Qy is even lower than that of Qx. For structure "R", excitations 2, 3, and 4 have similar, very low, oscillator strength. However, already at the G0W0@LDA+BSE level, no charge transfer excitations are found between Qy and Qxa consequence of the inherent nonlocality of the BSE kernel. Finally, for both structures, eigenvalue self-consistency pushes all excitations to significantly higher energies and results in a quantitatively correct description of Qy and Qx.
+
+Inspection of the DOS calculated with DFT-xc, G0W0@xc, and GnWn@xc (xc = LDA, ωPBE) shown in Figure 4 is instructive for understanding the contribution of eigenvalue differences to the TDDFT and GW+BSE excitation energies.
+
+The G0W0@LDA DOS underestimates the HOMO−LUMO
+gap and the energy difference between the HOMO and HOMO − 1. In contrast, there is virtually no difference between the HOMO, HOMO − 1, and LUMO energies as calculated with DFT-ωPBE, G0W0@ωPBE, evGnWn@ωPBE,
+and evGnWn@LDA. As expected, the DFT-LDA DOS is markedly different, not only underestimating the HOMO− LUMO gap but also significantly underestimating the energy differences between the HOMO − 1, HOMO − 2, and HOMO − 3. Notably, the spurious dark states between Qy and Qx that TD-LDA predicts have significant contributions from transitions involving these lower occupied states.
+
+The Journal of Physical Chemistry A pubs.acs.org/JPCA Article
+
+![7_image_0.png](7_image_0.png)
+
+- CONCLUSIONS
+In this article, we performed a systematic first-principles study of the electronic structure and excitations of seven members of the (bacterio)chlorophyll family, which we validated through comparison with calculated and experimental literature results.
+
+The GW+BSE approach, when used in a partially selfconsistent fashion, is in excellent agreement with experiment for excitations in the visible and near-ultraviolet part of the spectrum. GW+BSE also correctly predicts the energy difference between the low-energy Qy and Qx excitations of these pigments, relevant for the description of the coupling between pigment complexes, present in the light harvesting units and reaction centers of plants and bacteria and crucial for excitation energy and charge transfer. Most importantly, our results are almost entirely independent of the DFT eigensystem used as an input for the GW+BSE calculations. A computationally inexpensive LDA starting point leads to similar results as a more involved optimally tuned ωPBE
+starting point.
+
+It should be noted that the GW approach, despite its implementation using Gaussian basis functions and the use of the RI approximation in MOLGW and other codes, remains a major bottleneck of these calculations due to its O(N4) scaling with system size. Furthermore, our results highlight that the GW approach, more so than DFT, requires careful convergence with respect to the basis set size. This limits its applicability to systems with a few (B)CL pigments at most, until algorithms with better scaling become more widely available.57−59 Our study joins a growing number of results, demonstrating that the GW+BSE approach can accurately predict neutral excitations of complex molecules without empirical parameters.29 With new approaches for combining GW+BSE with large-scale molecular mechanics simulations28 and polarizable continuum embedding60 emerging, an accurate
+
+prediction of excitation energy and charge transfer in complex molecular environments is within reach.
+
+- ASSOCIATED CONTENT
+*sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpca.1c01240.
+
+Atomic coordinates (ZIP) Additional convergence data, tuned range-separation parameters, and excitation energies of all molecules discussed in the main manuscript (PDF)
+- AUTHOR INFORMATION
+Corresponding Author Linn Leppert − MESA+ Institute for Nanotechnology, University of Twente, 7500 AE Enschede, The Netherlands; Institute of Physics, University of Bayreuth, Bayreuth 95440, Germany; orcid.org/0000-0002-4361-4382; Email: l.leppert@utwente.nl Author Zohreh Hashemi − Institute of Physics, University of Bayreuth, Bayreuth 95440, Germany Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jpca.1c01240
+
+## Notes
+
+The authors declare no competing financial interest.
+
+- ACKNOWLEDGMENTS
+The authors are grateful for helpful discussions with C. Filippi. This work was supported by the Bavarian State Ministry of Science and the Arts through the Collaborative Research Network Solar Technologies go Hybrid (SolTech), the Elite Network Bavaria (ENB), and through computational resources provided by the Bavarian Polymer Institute (BPI).
+
+- REFERENCES
+(1) Blankenship, R. E. Molecular Mechanisms of Photosynthesis, 2nd ed.; Wiley Blackwell: Oxford, 2014.
+
+(2) Hu, X.; Ritz, T.; Damjanovic, A.; Autenrieth, F.; Schulten, K. ́
+Photosynthetic Apparatus of Purple Bacteria. Q. Rev. Biophys. 2002, 35, 1−62.
+
+(3) Croce, R.; van Amerongen, H. Natural Strategies for Photosynthetic Light Harvesting. Nat. Chem. Biol. 2014, 10, 492−501.
+
+(4) Cogdell, R. J.; Gall, A.; Köhler, J. The Architecture and Function of the Light-Harvesting Apparatus of Purple Bacteria: From Single Molecules to In Vivo Membranes. Q. Rev. Biophys. 2006, 39, 227−
+324.
+
+(5) Jang, S.; Mennucci, B. Delocalized Excitons in Natural LightHarvesting Complexes. Rev. Mod. Phys. 2018, 90, 035003.
+
+(6) Shao, Y.; Mei, Y.; Sundholm, D.; Kaila, V. R. I. Benchmarking the Performance of Time-Dependent Density Functional Theory Methods on Biochromophores. J. Chem. Theory Comput. 2020, 16, 587−600.
+
+(7) Sundholm, D. A Density-Functional-Theory Study of Bacteriochlorophyll b. Phys. Chem. Chem. Phys. 2003, 5, 4265−4271.
+
+(8) Vokácová, Z.; Burda, J. V. Computational Study on Spectral ̌
+Properties of the Selected Pigments from Various Photosystems: Structure-Transition Energy Relationship. J. Phys. Chem. A 2007, 111, 5864−5878.
+
+(9) Dreuw, A.; Head-Gordon, M. Failure of Time-Dependent Density Functional Theory for Long-Range Charge-Transfer Excited States: The Zincbacteriochlorin-Bacteriochlorin and Bacteriochlorophyll-Spheroidene Complexes. J. Am. Chem. Soc. 2004, 126, 4007−
+4016.
+
+(10) Tozer, D. J.; Handy, N. C. Improving Virtual Kohn-Sham Orbitals and Eigenvalues: Application to Excitation Energies and Static Polarizabilities. J. Chem. Phys. 1998, 109, 10180−10189.
+
+(11) Kuritz, N.; Stein, T.; Baer, R.; Kronik, L. Charge-Transfer-Like π -> π * Excitations in Time-Dependent Density Functional Theory:
+A Conundrum and Its Solution. J. Chem. Theory Comput. 2011, 7, 2408−2415.
+
+(12) Kümmel, S. Charge-Transfer Excitations: A Challenge for Time-Dependent Density Functional Theory That Has Been Met. Adv. Energy Mater. 2017, 7, 1700440.
+
+(13) Suomivuori, C.-M.; Fliegl, H.; Starikov, E. B.; Balaban, T. S.;
+Kaila, V. R. I.; Sundholm, D. Absorption Shifts of Diastereotopically Ligated Chlorophyll Dimers of Photosystem I. Phys. Chem. Chem.
+
+Phys. 2019, 21, 6851−6858.
+
+(14) Suomivuori, C.-M.; Winter, N. O. C.; Hättig, C.; Sundholm, D.;
+Kaila, V. R. I. Exploring the Light-Capturing Properties of Photosynthetic Chlorophyll Clusters Using Large-Scale Correlated Calculations. J. Chem. Theory Comput. 2016, 12, 2644−2651.
+
+(15) Sirohiwal, A.; Berraud-Pache, R.; Neese, F.; Izsák, R.; Pantazis, D. A. Accurate Computation of the Absorption Spectrum of Chlorophyll a with Pair Natural Orbital Coupled Cluster Methods.
+
+J. Phys. Chem. B 2020, 124, 8761−8771.
+
+(16) Anda, A.; Hansen, T.; De Vico, L. Multireference Excitation Energies for Bacteriochlorophylls A within Light Harvesting System 2.
+
+J. Chem. Theory Comput. 2016, 12, 1305−1313.
+
+(17) Anda, A.; Hansen, T.; De Vico, L. Qy and Qx Absorption Bands for Bacteriochlorophyll a Molecules from LH2 and LH3. J. Phys.
+
+Chem. A 2019, 123, 5283−5292.
+
+(18) Segatta, F.; Cupellini, L.; Jurinovich, S.; Mukamel, S.; Dapor, M.; Taioli, S.; Garavelli, M.; Mennucci, B. A Quantum Chemical Interpretation of Two-Dimensional Electronic Spectroscopy of LightHarvesting Complexes. J. Am. Chem. Soc. 2017, 139, 7558−7567.
+
+(19) Onida, G.; Reining, L.; Rubio, A. Electronic Excitations:
+Density-Functional versus Many-Body Green's-Function Approaches.
+
+Rev. Mod. Phys. 2002, 74, 601.
+
+The Journal of Physical Chemistry A pubs.acs.org/JPCA Article
+(20) Albrecht, S.; Reining, L.; Del Sole, R.; Onida, G. Ab Initio Calculation of Excitonic Effects in the Optical Spectra of Semiconductors. Phys. Rev. Lett. 1998, 80, 4510.
+
+(21) Rohlfing, M.; Louie, S. G. Electron-Hole Excitations and Optical Spectra from First Principles. Phys. Rev. B: Condens. Matter Mater. Phys. 2000, 62, 4927.
+
+(22) Qiu, D. Y.; Da Jornada, F. H.; Louie, S. G. Optical Spectrum of MoS2: Many-Body Effects and Diversity of Exciton States. Phys. Rev.
+
+Lett. 2013, 111, 216805.
+
+(23) Grossman, J. C.; Rohlfing, M.; Mitas, L.; Louie, S. G.; Cohen, M. L. High Accuracy Many-Body Calculational Approaches for Excitations in Molecules. Phys. Rev. Lett. 2001, 86, 472−475.
+
+(24) Tiago, M. L.; Kent, P. R. C.; Hood, R. Q.; Reboredo, F. A.
+
+Neutral and Charged Excitations in Carbon Fullerenes from FirstFrinciples Many-Body Theories. J. Chem. Phys. 2008, 129, 084311.
+
+(25) Bruneval, F.; Hamed, S. M.; Neaton, J. B. A Systematic Benchmark of the Ab Initio Bethe-Salpeter Equation Approach for Low-Lying Optical Excitations of Small Organic Molecules. J. Chem. Phys. 2015, 142, 244101.
+
+(26) Palummo, M.; Hogan, C.; Sottile, F.; Bagalá, P.; Rubio, A. Ab Initio Electronic and Optical Spectra of Free-Base Porphyrins: The Role of Electronic Correlation. J. Chem. Phys. 2009, 131, 084102.
+
+(27) Duchemin, I.; Deutsch, T.; Blase, X. Short-Range to LongRange Charge-Transfer Excitations in the ZincbacteriochlorinBacteriochlorin Complex: A Bethe-Salpeter Study. Phys. Rev. Lett.
+
+2012, 109, 167801.
+
+(28) Wehner, J.; Brombacher, L.; Brown, J.; Junghans, C.; Çaylak, O.; Khalak, Y.; Madhikar, P.; Tirimbo, G.; Baumeier, B. Electronic ̀
+Excitations in Complex Molecular Environments: Many-Body Green's Functions Theory in VOTCA-XTP. J. Chem. Theory Comput. 2018, 14, 6253−6268.
+
+(29) Blase, X.; Duchemin, I.; Jacquemin, D. The Bethe-Salpeter Equation in Chemistry: Relations with TD-DFT, Applications and Challenges. Chem. Soc. Rev. 2018, 47, 1022−1043.
+
+(30) Limantara, L.; Sakamoto, S.; Koyama, Y.; Nagae, H. Effects of Nonpolar and Polar Solvents on the Qx and Qy Energies of Bacteriochlorophyll a and Bacteriopheophytin a. Photochem. Photobiol.
+
+1997, 65, 330−337.
+
+(31) Scheer, H.; Inhoffen, H. The Porphyrins; Dolphi, Ed.; Academic Press: New York, 1978; Vol. II.
+
+(32) Fetter, A.; Walecka, J. Quantum Theory of Many-Particle Systems; MacGraw-Hill: New York, 1971.
+
+(33) Hedin, L. On Correlation Effects in Electron Spectroscopies and the GW Approximation. J. Phys.: Condens. Matter 1999, 11, R489.
+
+(34) Jiang, H.; Gomez-Abal, R. I.; Rinke, P.; Scheffler, M. FirstPrinciples Modeling of Localized d States with the GW@LDA+U
+Approach. Phys. Rev. B: Condens. Matter Mater. Phys. 2010, 82, 045108.
+
+(35) Liao, P.; Carter, E. A. Testing Variations of the GW
+Approximation on Strongly Correlated Transition Metal Oxides:
+Hematite (α-Fe2O3) as a Benchmark. Phys. Chem. Chem. Phys. 2011, 13, 15189−15199.
+
+(36) Marom, N.; Caruso, F.; Ren, X.; Hofmann, O. T.; Körzdörfer, T.; Chelikowsky, J. R.; Rubio, A.; Scheffler, M.; Rinke, P. Benchmark of GW Methods for Azabenzenes. Phys. Rev. B: Condens. Matter Mater. Phys. 2012, 86, 245127.
+
+(37) Jacquemin, D.; Duchemin, I.; Blase, X. Benchmarking the Bethe-Salpeter Formalism on a Standard Organic Molecular Set. J.
+
+Chem. Theory Comput. 2015, 11, 3290−3304.
+
+(38) Kaplan, F.; Weigend, F.; Evers, F.; Van Setten, M. J. OffDiagonal Self-Energy Terms and Partially Self-Consistency in GW
+Calculations for Single Molecules: Efficient Implementation and Quantitative Effects on Ionization Potentials. J. Chem. Theory Comput.
+
+2015, 11, 5152−5160.
+
+(39) Bruneval, F.; Rangel, T.; Hamed, S. M.; Shao, M.; Yang, C.;
+Neaton, J. B. MOLGW 1: Many-Body Perturbation Theory Software for Atoms, Molecules, and Clusters. Comput. Phys. Commun. 2016, 208, 149−161.
+
+(58) Vlcek, V.; Rabani, E.; Neuhauser, D.; Baer, R. Stochastic GW ̌
+Calculations for Molecules. J. Chem. Theory Comput. 2017, 13, 4997−
+5003.
+
+(59) Förster, A.; Visscher, L. Low-Order Scaling G0W0 by Pair Atomic Density Fitting. J. Chem. Theory Comput. 2020, 16, 7381−
+7399.
+
+(60) Duchemin, I.; Guido, C. A.; Jacquemin, D.; Blase, X. The Bethe-Salpeter Formalism with Polarisable Continuum Embedding: Reconciling Linear-Response and State-Specific Features. Chem. Sci.
+
+2018, 9, 4430−4443.
+
+(40) van Setten, M. J.; Caruso, F.; Sharifzadeh, S.; Ren, X.; Scheffler, M.; Liu, F.; Lischner, J.; Lin, L.; Deslippe, J. R.; Louie, S. G.; Yang, C.; Weigend, F.; Neaton, J. B.; Evers, F.; Rinke, P. GW100: Benchmarking G0W0 for Molecular Systems. J. Chem. Theory Comput.
+
+2015, 11, 5665−5687.
+
+(41) Godbout, N.; Salahub, D. R.; Andzelm, J.; Wimmer, E.
+
+Optimization of Gaussian-Type Basis Sets for Local Spin Density Functional Calculations. Part I. Boron Through Neon, Optimization Technique and Validation. Can. J. Chem. 1992, 70, 560−571.
+
+(42) Zheng, J.; Xu, X.; Truhlar, D. G. Minimally Augmented Karlsruhe Basis Sets. Theor. Chem. Acc. 2011, 128, 295−305.
+
+(43) Stein, T.; Eisenberg, H.; Kronik, L.; Baer, R. Fundamental Gaps in Finite Systems from Eigenvalues of a Generalized Kohn-Sham Method. Phys. Rev. Lett. 2010, 105, 266802.
+
+(44) Shao, Y.; Gan, Z.; Epifanovsky, E.; Gilbert, A. T.; Wormit, M.;
+Kussmann, J.; Lange, A. W.; Behn, A.; Deng, J.; Feng, X.; et al.
+
+Advances in Molecular Quantum Chemistry Contained in the QChem 4 Program Package. Mol. Phys. 2015, 113, 184−215.
+
+(45) Oviedo, M. B.; Negre, C. F. A.; Sánchez, C. G. Dynamical Simulation of the Optical Response of Photosynthetic Pigments. Phys.
+
+Chem. Chem. Phys. 2010, 12, 6706−6711.
+
+(46) Turbomole, V7.4 2019, a development of University of Karlsruhe and Forschungszentrum Karlsruhe GmbH, 2019. www. turbomole.org.
+
+(47) Schelter, I.; Foerster, J. M.; Gardiner, A. T.; Roszak, A. W.;
+Cogdell, R. J.; Ullmann, G. M.; De Queiroz, T. B.; Kümmel, S.
+
+Assessing Density Functional Theory in Real-Time and Real-Space as a Tool for Studying Bacteriochlorophylls and the Light-Harvesting Complex 2. J. Chem. Phys. 2019, 151, 134114.
+
+(48) Papiz, M. Z.; Prince, S. M.; Howard, T.; Cogdell, R. J.; Isaacs, N. W. The Structure and Thermal Motion of the B800-850 LH2 Complex from Rps. acidophila at 2.0 Å Resolution and 100 K: New Structural Features and Functionally Relevant Motions. J. Mol. Biol.
+
+2003, 326, 1523−1538.
+
+(49) List, N. H.; Curutchet, C.; Knecht, S.; Mennucci, B.; Kongsted, J. Toward Reliable Prediction of the Energy Ladder in Multichromophoric Systems: A Benchmark Study on the FMO LightHarvesting Complex. J. Chem. Theory Comput. 2013, 9, 4928−4938.
+
+(50) Kronik, L.; Stein, T.; Refaely-Abramson, S.; Baer, R. Excitation Gaps of Finite-Sized Systems from Optimally Tuned Range-Separated Hybrid Functionals. J. Chem. Theory Comput. 2012, 8, 1515−1531.
+
+(51) Refaely-Abramson, S.; Baer, R.; Kronik, L. Fundamental and Excitation Gaps in Molecules of Relevance for Organic Photovoltaics from an Optimally Tuned Range-Separated Hybrid Functional. Phys.
+
+Rev. B: Condens. Matter Mater. Phys. 2011, 84, 075144.
+
+(52) Jacquemin, D.; Moore, B.; Planchat, A.; Adamo, C.;
+Autschbach, J. Performance of an Optimally Tuned Range-Separated Hybrid Functional for 0-0 Electronic Excitation Energies. J. Chem.
+
+Theory Comput. 2014, 10, 1677−1685.
+
+(53) Parusel, A. B. J.; Grimme, S. A Theoretical Study of the Excited States of Chlorophyllaand Pheophytina. J. Phys. Chem. B 2000, 104, 5395−5398.
+
+(54) Moore, B.; Schrader, R. L.; Kowalski, K.; Autschbach, J.
+
+Electronic π-to-π* Excitations of Rhodamine Dyes Exhibit a TimeDependent Kohn-Sham Theory "Cyanine Problem". ChemistryOpen 2017, 6, 385−392.
+
+(55) De Queiroz, T. B.; De Figueroa, E. R.; Coutinho-Neto, M. D.;
+Maciel, C. D.; Tapavicza, E.; Hashemi, Z.; Leppert, L. First Principles Theoretical Spectroscopy of Methylene Blue: Between Limitations of Time-Dependent Density Functional Theory Approximations and its Realistic Description in the Solvent. J. Chem. Phys. 2021, 154, 044106.
+
+(56) Moore, B.; Autschbach, J. Longest-Wavelength Electronic Excitations of Linear Cyanines: The Role of Electron Delocalization and of Approximations in Time-Dependent Density Functional Theory. J. Chem. Theory Comput. 2013, 9, 4991−5003.
+
+(57) Neuhauser, D.; Gao, Y.; Arntsen, C.; Karshenas, C.; Rabani, E.;
+Baer, R. Breaking the Theoretical Scaling Limit for Predicting Quasiparticle Energies: The Stochastic GW Approach. Phys. Rev. Lett.
+
+2014, 113, 076402.

medical/md/PMC8061605.md

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+
+## Sliding Inguinal Hernia In A Case Of Fournier'S Gangrene: A **Rare Case**
+
+Abstract Fournier's gangrene and inguinal hernia, both are common diseases. However, inguinal hernia in a case of Fournier's gangrene is rarest of rare cases. Only one such case is reported in literature. We present one such rare case. A 50‑year–old man presented with blackish discoloration of scrotum with inguinoscrotal swelling. It was associated with pus discharge and foul smell. There were noassociated co morbidities. The patient was a chronic smoker from 40 years. Serial debridement was done until the wound became healthy and free of infection. After infection control, the patient was operated and hernioplasty was done. Keywords: *Debridement, Fournier's, gangrene, hernia, inguinoscrotal swelling*
+
+## Introduction
+
+Fournier's gangrene is a necrotizing fasciitis characterized by a polymicrobial infection of the soft tissues of the perineum, external genitalia and perianal region.[1] It can arise following minor injuries or procedures in the perineal area, such as a bruise, scratch, or opening of a periurethral abscess. Many patients have concurrent illnesses that diminish their defenses, most notably diabetes mellitus and alcoholism. There is a mixed infection of aerobic and anaerobic bacteria in a fulminating inflammation of the subcutaneous tissues which results in an obliterative arteritis of the arterioles to the scrotal skin resulting in gangrene. The condition can spread rapidly to involve the fascia and skin of the penis, perineum, and abdominal wall. Infection spreads rapidly with small necrotic areas of skin which, if untreated, coalesce to involve the entire scrotal and penile coverings, which may then slough, leaving the testes exposed. There may be crepitus and a foul‑smelling exudate. The patient typically becomes septic in a short period of time. Fournier's gangrene is a surgical emergency. Initial management involves intravenous fluid resuscitation and early use of broad spectrum intravenous antibiotics. Urgent wide surgical excision of the dead and infected tissue is essential and the extent of the internal necrosis is typically much This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
+
+For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com greater than the external appearances. Supportive care is essential.
+
+## Case Report
+
+A  50‑year‑Gentleman presented with a complaint of blackish discoloration of scrotum from 20 days. The patient initially developed pain and pustule over scrotum which later progressed to involving whole of the scrotum and was associated with foul smell, blackish discoloration, and fever. The patient was diagnosed with Fournier's Gangrene and debridement of scrotum was done [Figure 1a]. The patient had also left inguinal hernia and it was irreducible. The patient was chronic smoker from 40 years. There was no significant past medical or surgical history. On examination, the patient was vitally stable. Irreducible left inguinal hernia with Fournier's gangrene of scrotum was present. Patient's hemoglobin was on the lower side (8.4 g %) and total leukocyte count was raised (31,000/
+mm3). Serial debridement was done and culture and sensitivity of tissue was sent [Figure 1b]. Broad spectrum antibiotic started early and later, the patient was shifted on antibiotic according to sensitivity test. Dressings were done twice a day to manage the Fournier's Gangrene. Patient's clinical picture and blood parameters improved significantly. Patient was operated for irreducible inguinal hernia after scrotal infection was controlled and debridement with reduction of hernial sac with anatomical repair of hernia (hernioplasy) 
+Meghraj Kundan, Vinaysheel Priyadarshi, Chintamani Department of General Surgery, VMMC and Safdarjung Hospital, New *Delhi, India* Submitted: 09‑Dec‑2019 Revised: 02‑Dec‑2020 Accepted: 03-Jan-2021 Published: 08‑Apr‑2021 Address for correspondence: Dr. Vinaysheel Priyadarshi, Post Graduate Resident, Department of Surgery, VMMC and Safdarjung Hospital, New Delhi. E‑mail: *drmeghrajkundan@* gmail.com
+
+![0_image_0.png](0_image_0.png)
+
+How to cite this article: Kundan M, Priyadarshi V, 
+Chintamani. Sliding inguinal hernia in a case of Fournier's gangrene: A rare case. Int J App Basic Med Res 2021;11:106-7.
+
+![1_image_0.png](1_image_0.png)
+
+under general anaesthesia was done. Per-operative findings– Dense adhesions were present between gangrenous testis and caecum. Caecum was the content of the hernial sac. Specimen was sent for histopathological examination [Figure 1c]. The patient improved and discharged. Patient was followed up after 2 weeks and then after 3 months. The patient had no complaints.
+
+## Discussion
+
+Fournier's gangrene was first described by Baurienne in 1764 and is named after a French venereologist, Jean Alfred Fournier following five cases he presented in clinical lectures in 1883.[2] Initially, it was considered as rapidly progressing idiopathic necrotizing soft‑tissue infection of scrotum. However, later, it was concluded that it is the infection of perineal and genital region, typically seen in elderly, diabetic, or otherwise immune‑compromised and is known to be frequently due to a symbiotic polymicrobial infection. Females can also acquire this infection but there is a male predominance. Sliding hernia is the hernia in which one of the walls of the hernial sac is formed by a viscous. Approximately 3%–5% of all indirect inguinal hernias are sliding hernias. Sigmoid colon being the most common slided organ. Other common slided organs are retroperitoneal appendix, caecum, and urinary bladder. In 1995, Laor et al.
+
+[3] developed Fournier's Gangrene Severity Index to predict the prognosis of the patients. The treatment of Fournier's gangrene is radical debridement of the gangrene until no infected tissue is left, supported by broad spectrum antibiotics and followed by skin grafting. The treatment of sliding hernia is surgery. Depending on the setting of treating center, surgeon and the patient, treatment can either be done by open surgeries or by laparoscopic surgeries.
+
+## Conclusion
+
+Inguinoscrotal hernia in a case of Fournier's gangrene is extremely rare. No literature states the proper treatment for such condition. Fournier's gangrene should managed first as it is a life‑threatening condition if inguinal hernia is uncomplicated. After control of infection, the patient can be taken for surgery to manage the hernia. It can either be done as open surgery using high inguinal approach or can be done laparoscopically. However, in case of complicated inguinal hernia with Fournier's gangrene, further study is needed.
+
+## Declaration Of Patient Consent
+
+The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
+
+## References
+
+1. Yecies T, Lee DJ, Sorbellini M, Ramasamy R. Penile Fournier's gangrene. Urology 2013;82:e31.
+
+2. Fournier JA. Fournier 1832‑1914. Gangrene foudroyante de la verge (overwhelming gangrene). Sem Med 1883. Dis Colon Rectum 1988;31:984‑8.
+
+3. Laor E, Palmer LS, Tolia BM, Reid RE, Winter HI. Outcome prediction in patients with Fournier's gangrene. J Urol 1995;154:89‑92.

medical/md/PMC8080457.md

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+
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_1.png](0_image_1.png)
+
+![0_image_3.png](0_image_3.png)
+
+![0_image_5.png](0_image_5.png)
+
+Review 
+
+![0_image_2.png](0_image_2.png)
+
+Epidural analgesia and related ethical issues among pregnant women: 
+Literature review Sawsan Abuhammad *, Esraa Alholi School of Nursing, Jordan University of Science and Technology, Irbid, Jordan 
+
+| ARTICLE INFO  Keywords:  Informed consent  Epidural analgesia  Pregnant women  And antenatal period   |
+|-------------------------------------------------------------------------------------------------------|
+
+| ABSTRACT  Objective: This study aims to review the literature regarding epidural analgesia and related ethical issues among  pregnant women.  Method: The authors were searched many electronic databases (CHINAL, Ovid Midline, Pub MED and Google  Scholar, Science Direct Database, Biomed) under the following keywords (Informed consent, epidural analgesia,  pregnant women, and antenatal period). The several quantitative studies published in English were reviewed and  analyzed. The authors were analyzed the studies using a literature review matrix subheading to author, years  published, type of study, variables, design and methodology, finding, conclusion, limitations for practices and  limitations for studies.  Results: The themes that emerged from the literature review were: difference between patient and provider in  perceptions of informed consent toward EA during childbirth process; factors affecting the women choice of  using EA during childbirth process and awareness regarding using of EA in childbirth among women in antenatal  clinics.  Conclusion: The process of signing of informed consent for EA in women during childbirth is linked to many  ethical responsibilities from women and healthcare providers. The informed consent practice can be enhanced by  putting greater emphasis on antenatal information, providing suitable timing of information and developments  in recent methods of information release and transfer.   |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+## 1. **Introduction**
+
+Pain through childbirth is main concern of most women during childbirth in the world. Childbirth pain is the most extreme pain that most women may experience during their life [1]. Many women have increased awareness of their right to enhance the quality of care through childbirth by using an appropriate pain relief way [2,3]. Currently, epidural anesthesia [EA] is the most effective way to control pain without any risk to the mother or the fetus [4]. However, EA is still not a completely and acceptable practice for many healthcare providers in developing countries despite several benefits of this method [4]. Normal vaginal delivery is an excruciating event resulting from frequent uterine contractions [5]. National Institute for Clinical Excellence [6] in United Kingdom recommends educating pregnant women about the accessibility of useful analgesia methods during childbirth to ensure that they obtain the most favorable experience through childbirth. The information regarding childbirth pain and way to control it are relatively in intuitive stages in developing countries [7]. Insufficient information, antenatal dialogue, and practical experience are still the major concerns towards the practice of EA among obstetricians and healthcare providers. The main key source of receiving information regarding childbirth analgesia methods is from family and friends [8]. Therefore, it is the responsibility of the decision maker to improve the quality of EA 
+practicing by providing consistent information about childbirth analgesia. There are many advantages of increasing awareness regarding EA 
+among women which may prevent women from choosing cesarean section to avoid painful childbirth [8]. Because this intervention of healthcare provider holds risks and benefits, women are put in serious position at a critical time when they are vulnerable, in addition to the time needed to attain a lot of information to maintain and support their choices. Accordingly, increasing emphasis on the significance of the decision-making process to the childbirth process is required to enhance prenatal outcomes [12]. Women need to be informed about the advantages and disadvantages connected with EA through childbirth process 
+[14]. Ethically, pregnant women have the right to obtain informed consent; there is an obligation to have all information regarding EA that 
+* Corresponding author. 
+
+E-mail address: Shabuhammad@just.edu.jo (S. Abuhammad). 
+
+https://doi.org/10.1016/j.amsu.2021.102305 Received 23 March 2021; Received in revised form 6 April 2021; Accepted 7 April 2021 Available online 13 April 2021 2049-0801/© 2021 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+![0_image_4.png](0_image_4.png)
+
+journal homepage: www.elsevier.com/locate/amsu include advantage and disadvantage of using this way to control pain [9]. 
+
+In many countries, it is the responsibility of the anesthesiologist to get the informed consent from the women for any surgical procedure. This practice is inadequate because it is affected by many ethical factors related to the client and anesthesiologist if they fail to gain ethical informed consent, and this obligate the anesthesiologist to reassess the understanding of the patient [10–12]. The EA consent form is habitually oral and just obtained prior to insertion; therefore, this study aims to assess the process of obtaining informed consent regarding EA among pregnant women. This study aims to review the literature regarding epidural analgesia and related ethical issues among pregnant women. 
+
+## 2. **Method** 2 2.1. Searching Strategies
+
+Electronic search using the following databases to collect data about informed consent toward epidurals among pregnant women: science direct, PubMed, Google Scholar, Google advanced search with the following search words: Informed Consent, Epidural Analgesia, Antenatal Period, Pregnant Women, Risk Factors, Benefits, Characteristics, Knowledge, Practice, Nurses, Midwives, and Perception. The authors extended the searching to the reference list of each article they found. The articles were systematic review, randomized controlled trial (RCT), crosssectional study, cohort study and descriptive study. The inclusion criteria were articles written in English language with focusing on the main keywords listed previously. The emerging themes from the literature review were: Differences among Patient and Health Care Providers; Factors Affecting the Women Choice of Using EA During Childbirth Process, and Awareness Regarding Using of EA in Childbirth Process among Pregnant Women in Antenatal Clinics. 
+
+## 3. **Results**
+
+3.1. Difference between patient and provider in perceptions of informed consent toward EA during childbirth process The first theme that emerged from the literature review was difference between patient and provider in perceptions of informed consent. In a comparative study to determine the variation of informed decisionmaking awareness toward EA during childbirth among patient and provider. They found a considerable difference between provider and patient in defining and recognizing pain relief methods. Providers were expected to support the rights of women toward informed decisions compare to the women who are expected to respect the healthcare providers decisions [15]. In another research study to compare the differences in decision-making among health care providers and women regarding utilizing EA throughout childbirth process. It was obvious that choosing of EA depend on the information that women were received from the healthcare providers [15,16]. 
+
+## 3.2. Factors Affecting The Women Choice Of Using Ea During Childbirth Process
+
+There were many studies were described factors that affecting choice of using EA during childbirth [6,8,16,17]. For example, a prospective cross-sectional study was conducted to assess factors linked to the refusing of EA among 933 among women. This study was collected information about their parity, literacy level, reasons for seeking or refusing EA, a resource of information and satisfaction. This study demonstrated that universal reasons were misconceptions regarding the nature of EA (65.9%) and lacking information about the choice of EA 
+(20.5%); familiarity with the side effects of EA (70.5%). For participants used EA during childbirth, their choices for having EA were directed by healthcare providers [8]. In two studies in Nigeria and Norway, to compare the characteristics between women who received EA and who not. They found that the education level was higher with women who had received EA during childbirth compared to none. Moreover, women who received childbirth preparation classes are more likely to use EA during childbirth [22,23]. Another national antenatal survey among 8233 found that refusing to seek EA was greater in poor women such as none employed women or less than bachelor's degree [16]. In comparison, a survey study among 320 participants found that 80% of women receiving an epidural during childbirth. The identified factors for using EA include prior epidural use, partner preference, age, language, type of insurance, duration of childbirth, and education [17]. 
+
+In summary, the main reasons for avoiding EA are lack of knowledge and awareness, misconceptions about EA, and desire to experience vaginal delivery [8]. The recommendation of National Institute for Clinical Excellence to educate pregnant women about availability of effective analgesia in childbirth process and find the factors that impacted their decision to choose EA during childbirth [6]. 
+
+3.3. Awareness regarding using of EA in childbirth among women in antenatal clinics Another theme that emerged from this literature review was awareness regarding EA in childbirth among women in antenatal clinics 
+[7,8,18,20–24]. A descriptive study among multipara pregnant women found that many misconceptions and worries linked with EA use. Braun, Skene, & Merry [20] anticipated three methods to address the impeding factors for obtaining informed consent from healthcare providers that should described in preadmission clinic. These clinics require to inform women regarding pain-relief options during childbirth. A cross-sectional study among 350 women attending antenatal classes aimed to identify the awareness and utilization of EA during childbirth between April 2016 and July 2016 found that only 43.3% of the participants were aware of using EA during childbirth. However, only 7.5% used EA. The results from this study focus on developing a strategy to enhance performance and quality of EA by information providing during pregnancy 
+[8]. The improving of antenatal information was required teamwork with sufficient resources [7]. 
+
+A study among 40 pregnant and newly birthing women regarding how media, friends, and family informing decision-making for women for using EA during childbirth. The results of the study show that women define birth based on previous experiences. These experiences impact the women's choices regarding use of EA during childbirth process [23]. 
+
+Yet, it is implicit that women's choices of delivery would be enhanced if women informed correctly and professionally regarding EA. Stewart et al. experimental study found that most women in both standard care and intervention groups would receive the information regarding EA 
+from anesthesiologist prior to childbirth. They found that women need to obtain both verbal and written information from healthcare providers prior to childbirth to formulate a decision regarding using EA (26). It is the responsibility of healthcare to give an adequate information about pain relief options during childbirth to empower pregnant women to make an informed choice. 
+
+## 3.4. Critique
+
+There were many limitation and gaps in the literature regarding informed consent for EA during childbirth, these limitations are small sample size or focusing on homogeneous sample that could limit the generalizability of the findings [8,17,19,20]. For example, a study by Harkins et al. [17] regarding the impact of women characteristics on healthcare providers choice of having informed consent regarding EA. This study weakens by using homogenous sample from English-literate, married, and highly educated women. 
+
+Similarly, Sitras et al. [25] utilize an investigation to inspect how women gain knowledge regarding informed consent of EA during childbirth. A limitation is the generalizability of the findings that restricted to Norwegian-speaking women who were Caucasian sample. 
+
+Another possible limitation of this study is not using validated instrument. A study in Northern British Columbia where EA is available and physicians directed most of the clinics that improved the chance of using EA, which limit the generalizability the results to other areas [25]. 
+
+## 3.5. Implications And Recommendation
+
+The study's results draw attention for the importance of legal and ethical principles toward the childbirth process. Healthcare providers should assist women regarding decisions toward EA. It is vital for healthcare providers to find the best method to improve woman understanding of option during childbirth process. Lastly, it is the responsibility of healthcare providers to guarantee the understanding of EA during informed consent. It is recommended that providers spend more effort in implementing "shared decision-making practices". 
+
+## 4. **Conclusion**
+
+The process of signing of informed consent for EA in women during childbirth is linked to many ethical responsibilities from women and healthcare providers. The informed consent practice can be enhanced by putting greater emphasis on antenatal information, providing suitable timing of information and developments in recent methods of information release and transfer. Changes in existing practice may well be helpful to women to better adhere to the legal standards of obtaining informed consent. There is a need for future research which may add further information to enhance awareness regarding EA during childbirth. 
+
+## Ethical Approval
+
+No ethical approval is required. 
+
+Sources of funding No funding Author contribution All authors were participated in all stages of this paper. 
+
+Consent No consent is required. 
+
+Registration of research studies 1. Name of the registry: 
+2. Unique Identifying number or registration ID: 
+3. Hyperlink to your specific registration (must be publicly accessible and will be checked): 
+
+## Guarantor
+
+All authors were participated in all steps of this paper. 
+
+## Declaration Of Competing Interest
+
+No conflict of interest. 
+
+## References
+
+[1] L.O. Lawani, J.N. Eze, O.B. Anozie, C.A. Iyoke, N.N. Ekem, Obstetric analgesia for vaginal birth in contemporary obstetrics: a survey of the practice of obstetricians in Nigeria, BMC Pregnancy Childbirth 14 (1) (2014 Dec) 1–6. 
+
+[2] E.O. Ogboli-Nwasor, S.E. Adaji, Between pain and pleasure: pregnant women's knowledge and preferences for pain relief in labor, a pilot study from Zaria, Northern Nigeria, Saudi J. Anaesth. 8 (Suppl 1) (2014 Nov) S20. 
+
+[3] E. Ogboli-Nwasor, S.E. Adaji, S.B. Bature, O.S. Shittu, Pain relief in labor: a survey of awareness, attitude, and practice of health care providers in Zaria, Nigeria, J. Pain Res. 4 (2011) 227. 
+
+[4] R.V. Shidhaye, M.V. Galande, V.B. Bangal, S.S. Joshi, U.R. Shidhaye, Awareness and attitude towards labour analgesia of Indian pregnant women, Anaesth. Pain Intensive Care 16 (2) (2012) 131–136. 
+
+[5] H. Aksoy, B. Yücel, U. Aksoy, G. Acmaz, T. Aydin, M.A. Babayigit, The relationship between expectation, experience and perception of labour pain: an observational study, SpringerPlus 5 (1) (2016 Dec) 1–5. 
+
+[6] NICE Intrapartum care for healthy women and babies, Clin. Guidelines (CG 190) 
+(2014 Dec) 1–103. 
+
+[7] A.R. Braun, L. Skene, A.F. Merry, Informed consent for anaesthesia in Australia and New Zealand, Anaesth. Intensive Care 38 (5) (2010 Sep) 809–822. 
+
+[8] A. Prakash, A. Yadav, H.M. Karim, S.K. Sahoo, P. Jena, K. Aman, Knowledge, awareness and acceptance of labor analgesia among antenatal women in a remote Island: a questionnaire based study, J. Adv. Med. Med. Res. (2017 May 31) 1–7. 
+
+[9] Authority, Informed consent, NHSLA Risk Alert 4 (2) (2014) 53–58. 
+
+[10] G.N. Jackson, T. Sensky, P. Reide, S.M. Yentis, The capacity to consent to epidural analgesia in labour, Int. J. Obstet. Anesth. 20 (3) (2011 Jul 1) 269–270. 
+
+[11] B.M. Broaddus, S. Chandrasekhar, Informed consent in obstetric anesthesia, Anesth. Analg. 112 (4) (2011 Apr 1) 912–915. 
+
+[12] Sakala C, Corry MP. Evidence-based Maternity Care: what it Is and what it Can Achieve. 
+
+[14] V.A. Entwistle, I.S. Watt, Patient involvement in treatment decision-making: the case for a broader conceptual framework, Patient Educ. Counsel. 63 (3) (2006 Nov 1) 268–278. 
+
+[15] H.B. Goldberg, A. Shorten, Differences between patient and provider perceptions of informed decision making about epidural analgesia use during childbirth, J. Perinat. Educ. 23 (2) (2014 Jan 1) 104–112. 
+
+[16] Fisher KT. reportWomen's Recall of Information Received Relating to Labour Epidural Analgesia at an Academic Hospital: a Pilot Study (Doctoral dissertation). 
+
+[17] J. Harkins, B. Carvalho, A. Evers, S. Mehta, E.T. Riley, Survey of the factors associated with a woman's choice to have an epidural for labor analgesia, Anesthesiol. Res. Pract. (2010 Jan 1) 2010. 
+
+[18] P.O. Ezeonu, O.B. Anozie, F.A. Onu, C.U. Esike, J.E. Mamah, L.O. Lawani, R. 
+
+C. Onoh, E.O. Ndukwe, R.L. Ewah, R.O. Anozie, Perceptions and practice of epidural analgesia among women attending antenatal clinic in FETHA, Int. J. Wom. Health 9 (2017) 905. 
+
+[19] A. Barakzai, G. Haider, F. Yousuf, A. Haider, N. Muhammad, Awareness of women regarding analgesia during labour, J. Ayub Med. Coll. Abbottabad 22 (1) (2010 Mar 1) 73–75. 
+
+[20] L. Ray, F. Goffinet, M. Palot, M. Garel, B. Blondel, 681:'Choice'of delivery without epidural analgesia in women at low risk in France Camille, Am. J. Obstet. Gynecol. 197 (6) (2007 Dec 1) S195. 
+
+[21] C.O. Imarengiaye, B.N. Olagbuji, M.C. Ezeanochie, Akhideno II, Clinical correlates of women requesting labour epidural analgesia in a tertiary hospital in Nigeria, Niger. Postgrad. Med. J. 20 (3) (2013 Sep 1) 214–217. 
+
+[22] S.B. Munro, A. Hui, E.A. Gemmell, N. Torabi, A.S. Johnston, P.A. Janssen, Evaluation of an information pamphlet for women considering epidural analgesia in labour, J. Obstet. Gynaecol. Can. 40 (2) (2018 Feb 1) 171–179. 
+
+[23] C. Malacrida, Always, already-medicalized: women's prenatal knowledge and choice in two Canadian contexts, Curr. Sociol. 63 (5) (2015 Sep) 636–651. 
+
+[24] S.J. Brubaker, H.E. Dillaway, Medicalization, natural childbirth and birthing experiences, Sociol. Compass 3 (1) (2009 Jan) 31–48. 
+
+[25] V. Sitras, J. Saltyt ˇ e ˙ Benth, M. Eberhard-Gran, Obstetric and psychological characteristics of women choosing epidural analgesia during labour: a cohort study, PloS One 12 (10) (2017 Oct 18), e0186564. 

medical/md/PMC8129987.md

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+Contents lists available at ScienceDirect 
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_2.png](0_image_2.png)
+
+![0_image_4.png](0_image_4.png)
+
+![0_image_7.png](0_image_7.png)
+
+# Molecular Genetics And Metabolism Reports 
+
+![0_Image_3.Png](0_Image_3.Png)
+
+![0_Image_5.Png](0_Image_5.Png)
+
+![0_Image_6.Png](0_Image_6.Png)
+
+journal homepage: www.elsevier.com/locate/ymgmr 
+
+![0_image_8.png](0_image_8.png)
+
+Short Communication 
+
+![0_image_1.png](0_image_1.png)
+
+Successful pregnancy in a woman with glycogen storage disease type 6 Sarah Catharina Grünert *, Stefanie Rosenbaum-Fabian , Luciana Hannibal , Anke Schumann , 
+Ute Spiekerkoetter Department of General Paediatrics, Adolescent Medicine and Neonatology, Medical Centre- University of Freiburg, Faculty of Medicine, Mathildenstraße 1, 79106 Freiburg, Germany ARTICLE INFO 
+ABSTRACT 
+
+| Keywords:  Glycogen storage disease type 6  GSD VI  Pregnancy  Ketotic glycogen storage disease  Glycogen phosphorylase deficiency  PYGL   |
+|--------------------------------------------------------------------------------------------------------------------------------------------|
+
+Glycogen storage disease type VI is caused by biallelic variants in the *PYGL* gene that result in hepatic glycogen phosphorylase deficiency. The disorder is clinically characterized by hepatomegaly and recurrent ketotic hypoglycemia from infancy. Although most patients reach adulthood without major complications, no pregnancies in women with GSD VI have been reported so far. We report on a successful pregnancy in a GSD VI patient that resulted in a healthy offspring and describe the pre- and perinatal management. 
+
+## 1. **Introduction**
+
+Glycogen storage disease type VI (GSD VI, OMIM \#232700) is a rare inherited disorder of glycogen metabolism caused by mutations in the PYGL gene resulting in deficiency of hepatic glycogen phosphorylase 
+(PYGL) [1–4]. The incidence is approximately 1:65,000–1:85,000 [1]. 
+
+Patients with GSD VI usually present in childhood with hepatomegaly and recurrent hypoglycemia. Other clinical symptoms comprise impaired growth resulting in short stature, osteopenia, delayed puberty and hepatic fibrosis [1,2]. Typical laboratory findings include hypoglycemia with hyperketonemia, elevated transaminase concentrations, hyperlipidemia, and reduced prealbumin concentration [1]. Most adults with GSD VI are asymptomatic [2]. As GSD VI is typically a rather mild disease, women with GSD VI usually reach the child-bearing age without major complications. Nevertheless, to our knowledge, no pregnancies in patients with GSD VI have been reported so far. 
+
+## 2. **Case Presentation**
+
+The patient is the first child of non-consanguineous parents. Clinical signs of hypoglycemia including sweating and shakiness were reported from early infancy throughout childhood. During early childhood the patient required feeding every 2 h, also at night. Hepatomegaly and elevated transaminases were first noted at 3 years. At the age of 6, a liver biopsy was performed and the diagnosis of GSD VI was made based on reduced activity of hepatic PYGL. Until age 13, the patient received regular cornstarch doses during the night, and no episodes of severe hypoglycemia were reported in the further course. From puberty until the age of 36, the patient was lost to follow-up. When she first presented to our metabolic centre at the age of 36, mild hepatomegaly was present, but otherwise she was in good metabolic control. She was on a selfchosen low-carb diet to reduce weight (body weight 113 kg, BMI 39.1 kg/m2). Mutation analysis of the PYGL gene was performed and yielded compound heterozygosity for the variants c.475G > C (p.Gly159Arg) 
+and c.697G > A (p.Gly233Ser). The diet was switched to a protein-rich diet with reduction of simple sugars (protein 30% of total energy). At age 38, the patient became pregnant after fertility treatment. Apart from ovarian hyperstimulation syndrome, the pregnancy was uncomplicated. 
+
+Her maximum fasting time during the night was 11 h with blood glucose levels above 4.2 mmol/L in the morning and ketone levels <0.1 mmol/L 
+with rare exceptions. The patient took a protein-rich bedtime snack or alternatively 30 g of uncooked corn starch. No severe hypoglycemias occurred throughout pregnancy (lowest documented glucose level 3.7 mmol/L), however, no conctinuos glucose monitoring was performed. In compliance with our recommendation, no oral glucose tolerance test was performed in the second trimester. Weight gain during pregnancy was 25 kg (weight before pregnancy 100 kg, BMI 34.6 kg/m2). In the 37th week of gestation, she spontaneously delivered a healthy baby boy 
+* Corresponding author at: Department of Paediatrics, Adolescent Medicine and Neonatology, University Medical Center, Mathildenstraße 1, 79106 Freiburg, Germany. 
+
+E-mail addresses: sarah.gruenert@uniklinik-freiburg.de (S.C. Grünert), stefanie.rosenbaum-fabian@uniklinik-freiburg.de (S. Rosenbaum-Fabian), luciana. 
+
+hannibal@uniklinik-freiburg.de (L. Hannibal), anke.schumann@uniklinik-freiburg.de (A. Schumann), ute.spiekerkoetter@uniklinik-freiburg.de (U. Spiekerkoetter). https://doi.org/10.1016/j.ymgmr.2021.100770 Received 10 March 2021; Received in revised form 30 April 2021; Accepted 1 May 2021 2214-4269/© 2021 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+S.C. Grünert et al. 
+
+(weight 3400 g (76th centile), length 52 cm (71th centile), head circumference 32.5 cm (6th centile)). Management in the peripartal period included an intravenous glucose-electrolyte infusion (starting dose 10 g glucose/h), and regular blood glucose and ketone measurements in the mother during labour. Under this regimen, blood glucose remained stable throughout delivery. The boy could be breastfed without any problems and shows normal development at age 5 months. 
+
+With him being a heterozygous carrier of the disease, no special precautions needed to be taken. 
+
+## 3. **Discussion**
+
+GSD VI is a rare disorder with only about 50 cases reported. Due to the rather benign character and good prognosis of the disease, more and more patients will reach child-bearing age. To our knowledge, this is the first pregnancy of a woman with GSD VI. 
+
+To ensure an adequate supply of nutrients and energy to the unborn foetus, several alterations in maternal physiology occur during pregnancy mainly influenced by placental hormones [5]. While the first half of pregnancy is an anabolic state associated with increased insulin sensitivity and fat deposition in adipose tissue, the second half is characterized by a maternal insulin resistant state in order to preserve maternal glucose for foetal metabolism. Additionally, hepatic glucose production increases to meet the growing demands of both the foetus and the mother [6]. As women with GSD VI may not be able to match glucose production with increasing energy demands, patients with GSD 
+VI are at risk for metabolic derangements with more pronounced hypoglycemia and hyperketonemia during pregnancy [7]. Careful monitoring and optimal dietetic treatment are therefore necessary throughout pregnancy to maintain maternal blood glucose levels in the normal range for the safety and proper development of the fetus [8]. For GSD type I, it has been shown that good metabolic control before conception and throughout pregnancy is directly related to successful outcomes [8]. In patients with GSD III it has been shown that maternal hypoglycemia may be associated with intrauterine growth restriction and low birth weight [7], and similar relations can be expected in GSD VI. The mainstay of therapy in GSD VI is the prevention of prolonged fasting, a protein-rich diet and supplementation with uncooked corn starch, if necessary. Treatment and monitoring have to be adapted on an individual basis. In the non-GSD population, oral glucose tolerance tests are routinely performed in the second trimester to screen for gestational diabetes. It is important to note that this test is contraindicated in women with GSDs, as fasting prior to the ingestion of 75 g of concentrated glucose is required for proper interpretation [8]. Fasting, however, poses a high risk of hypoglycemia on pregnant GSD women, and a large bolus of concentrated glucose can cause metabolic instability and result in lactic acidosis [8,9]. To prevent hypoglycemia during delivery, peripartum management should include administration of a high glucose-electrolyte infusion and regular monitoring of blood glucose and ketones during labour. Optimal planning and interdisciplinary collaboration between metabolic physicians and gynecologists is necessary to guarantee a safe setting for mother and child. 
+
+## 4. **Conclusions**
+
+Our case demonstrates that successful pregnancy is possible in GSD 
+VI. Careful monitoring during pregnancy and delivery is necessary to minimize the risk of recurrent hypoglycemia for both mother and child. 
+
+## Acknowledgement
+
+We are grateful to the patient for her support of this publication. This work was supported by the Metabolic Division in the University Children's Hospital, which is part of the Freiburg Center for Rare Diseases. 
+
+Several authors of this publication are members of the European Reference Network for Rare Hereditary Metabolic Disorders (MetabERN) - Project ID No 739543. We acknowledge David Weinstein, Terry Derks and Ulrike Steuerwald for their clinical advice with respect to glycogen storage diseases and many fruitful discussions. The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding programme Open Access Publishing. 
+
+## References
+
+[1] E. Labrador, D.A. Weinstein, Glycogen storage disease type VI, in: M.P. Adam, H. 
+
+H. Ardinger, R.A. Pagon, S.E. Wallace, L.J. Bean, K. Stephens (Eds.), GeneReviews®, 
+University of Washington, Seattle, Seattle (WA), 1993. http://www.ncbi.nlm.nih. gov/books/NBK5941/. Accessed 4 Sep 2020. 
+
+[2] Margaret A. Chen, David A. Weinstein, Glyogen storage diseases: diagnosis, treatment and outcome, Trans. Sci. Rare Dis. 1 (2016) 45–72. 
+
+[3] S. Chang, M.J. Rosenberg, H. Morton, C.A. Francomano, L.G. Biesecker, Identification of a mutation in liver glycogen phosphorylase in glycogen storage disease type VI, Hum. Mol. Genet. 7 (1998) 865–870. 
+
+[4] B. Burwinkel, H.D. Bakker, E. Herschkovitz, S.W. Moses, Y.S. Shin, M.W. Kilimann, Mutations in the liver glycogen phosphorylase gene (PYGL) underlying glycogenosis type VI, Am. J. Hum. Genet. 62 (1998) 785–791. 
+
+[5] N.F. Butte, Carbohydrate and lipid metabolism in pregnancy: normal compared with gestational diabetes mellitus, Am. J. Clin. Nutr. 71 (5 Suppl) (2000), 1256S–61S. 
+
+[6] S.C. Kalhan, L.J. D'Angelo, S.M. Savin, P.A. Adam, Glucose production in pregnant women at term gestation. Sources of glucose for human fetus, J. Clin. Invest. 63 
+(1979) 388–394. 
+
+[7] R. Ramachandran, Y. Wedatilake, C. Coats, F. Walker, P. Elliott, P.J. Lee, et al., 
+Pregnancy and its management in women with GSD type III - a single Centre experience, J. Inherit. Metab. Dis. 35 (2012) 245–251. 
+
+[8] I.A. Ferrecchia, G. Guenette, E.A. Potocik, D.A. Weinstein, Pregnancy in women with glycogen storage disease Ia and Ib, J. Perinat. Neonatal. Nurs. 28 (2014) 26–31. 
+
+[9] J. Fernandes, J.F. Koster, W.F. Grose, N. Sorgedrager, Hepatic phosphorylase deficiency. Its differentiation from other hepatic glycogenoses, Arch. Dis. Child. 49 
+(1974) 186–191. 
+
+2

medical/md/PMC8311701.md

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+# The Gutenberg Revolution In Cardiovascular Medicine
+
+Personalized 3D Printing in Planning Aortic Valve Surgery* Gianluca Torregrossa, MD, Andrea Amabile, MD
+The invention of the printing press had a significant democratizing effect on society, known as The Gutenberg Revolution. The simple yet extraordinary concept of movable types paved the way for the era of mass communication, which permanently altered the structure of society.
+
+The relatively unrestricted circulation of printed ideas transcended borders and ultimately led to the spread of revolutions such as the Protestant Reformation, the Age of Enlightenment, and the establishment of the industrial society in Europe. By 1620, the English philosopher Francis Bacon wrote in his Instauratio Magna that the 3 inventions that "have altered the face and state of the world" were gunpowder, the nautical compass and the printing press
+(1). Indeed, the printing press was a key factor in establishing a community of scientists who could easily disseminate their discoveries through scholarly journals, and eventually helped standardize language, grammar, and spelling in the scientific world.
+
+However, when Johannes Gutenberg built the first printing press in approximately 1436, he was certainly not aware of the immense revolution his invention would have been responsible for: Gutenberg died penniless and his presses were impounded by his
+
+![0_image_0.png](0_image_0.png)
+
+creditors.
+
+Similarly, 3-dimensional (3D) printing has entered clinical research as a tool with potentials that clinicians have not completely explored yet. The application of 3D-printed patient-specific models in cardiovascular medicine has a potential role in several areas (2,3). This technology represents a powerful teaching tool, which can rapidly convey a complex anatomic arrangement to trainees or which can make patients reach a higher level of awareness about their structural heart disease.
+
+Also, 3D-printed models represent an outstanding tool for surgical or interventional preoperative planning and simulation; they can also be used as functional models to investigate intracardiac flow, and they have a tangible role in testing and innovating new surgical and transcatheter devices.
+
+In this issue of JACC: Case Reports, Shearn et al. (4)
+from the Bristol Heart Institute reported the use of 3D-printed, computed tomography–derived, patientspecific aortic root models for the preoperative planning of neocuspidization surgery (Ozaki procedure) in 2 patients with bicuspid aortic valve (BAV). Neocuspidization surgery has gained popularity over the past 10 years as a novel technique in which, during surgery, the autologous pericardium of a patient is molded into 3 different leaflets to create a competent aortic valve (5). Potential advantages of such technique are several: first, the absence of a stent allows molding of a neo-aortic valve with excellent hemodynamics made by all biological material, so theoretically it is less prone to develop endocarditis; second, the design of the valve creates a large coapting surface between the leaflets allowing to maintain a competent valve even if the aortic root will grow (children) or dilate in the future. Finally, ISSN 2666-0849 https://doi.org/10.1016/j.jaccas.2020.05.033 the use of autologous pericardium offers a potential superior durability respect of the xenogenic biological material used for commercially available prosthetic valves, making this technique attractive for young patients with aortic valve disease. All these advantages are waiting to be reported and verified by groups other than Ozaki himself who has the largest experience and has published excellent midterm results of this technique.
+
+Shearn et al. (4) should be complimented for their excellent short-term outcomes in applying 3D printing technology to the surgical planning of the Ozaki procedure in BAV morphology. BAV anatomy represents indeed an extra technical challenge for the neocuspidization technique in which a trileaflet valve needs to be constructed in a previously bicuspid annulus by creating a new commissure within the aortic root: the success of this type of procedure heavily relies on the technical experience of the surgeon, as demonstrated by the midterm outcomes of Prof. Ozaki (6). The possibility given by 3D printing technology to bench test the surgery on a patientspecific anatomy represents a unique opportunity for aiming at high-quality reconstructions.
+
+Interestingly, the authors claimed that the presized leaflets were larger than those ultimately implanted in vivo in both patients raising some concern about the fidelity of the model created.
+
+Nevertheless, it would certainly be worth further investigating the efficacy of this 2-step approach in a
+
+## References
+
+1. Boruchoff DA. The three greatest inventions of modern times: an idea and its public. In: Hock K,
+Mackenthu G, editors. Entangled Knowledge: Scientific Discourses and Cultural Differences.
+
+Münster and New York: Waxmann, 2012;133–63.
+
+2. Giannopoulos AA, Mitsouras D, Yoo SJ, et al.
+
+Applications of 3D printing in cardiovascular diseases. Nat Rev Cardiol 2016;13:701–18.
+
+3. Vukicevic M, Mosadegh B, Min JK, et al. Cardiac 3D printing and its future directions. J Am Coll Cardiol Img 2017;10:171–84.
+
+4. Shearn AIU, Ordoñez MV, Rapetto F, et al. Rapid prototyping flexible aortic models aids sizing of valve leaflets and planning the Ozaki repair. J Am Coll Cardiol Case Rep 2020;2:1137–40.
+
+5. Ozaki S, Kawase I, Yamashita H, et al. Reconstruction of bicuspid aortic valve with autologous pericardium. Circ J 2014;78:1144–51.
+
+6. Ozaki S, Kawase I, Yamashita H, et al. Midterm outcomes after aortic valve neocuspidization with glutaraldehyde-treated autologous pericardium.
+
+J Thorac Cardiovasc Surg 2018;155:2379–87.
+
+7. Arjmand SA, Pourghorban R, Tehrai M, et al.
+
+Comparison of aortic root dimension changes during cardiac cycle between the patients with and without aortic valve calcification using ECG-gated 64-slice and dual-source 256-slice computed tomography scanners: results of a multicenter study.
+
+Int J Cardiovasc Imaging 2013;29:1391–400.
+
+KEY WORDS 3D printing, aortic valve surgery, neocuspidization, Ozaki technique larger series of patients (comparing the observedversus-expected leaflet sizing and evaluating the long-term follow-up), with a standardized computed tomography scan protocol and cycle time acquisition for the 3D modeling (because the aortic root anatomy changes in shape throughout the cardiac cycle [7]).
+
+Although the tendency in cardiovascular medicine has been focused on investigating different treatments for a specific disease (surgical aortic valve replacement vs. transcatheter aortic valve replacement, repair vs. replacement, mechanical vs. biological, coronary artery bypass grafting vs. percutaneous coronary intervention, surgery vs. percutaneous treatment); the future of medicine is shifting to a clinical practice extremely oriented to a patientspecific, tailored approach, in which bench simulation will guide which device or which technique can better address that patient's specific anatomy and disease.
+
+The paper by Shearn et al. (4) perfectly represents this paradigm: from bedside to bench, and from bench to bedside again.
+
+ADDRESS FOR CORRESPONDENCE: Dr. Gianluca Torregrossa, Division of Minimally Invasive and Robotic Cardiac Surgery, Department of Surgery, University of Chicago Medicine, 5758 S. Maryland Avenue, Chicago, Illinois 60637. E-mail: gianluca. torregrossa@gmail.com.

medical/md/PMC8506526.md

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+Original Article
+
+![0_image_0.png](0_image_0.png)
+
+# Acy1 Regulating Pten/Pi3K/Akt Signaling In The Promotion Of Non-Small Cell Lung Cancer Progression
+
+## Hong Chen1#, Wei Wang2#, Caizhi Xiao1, Dongqin Xia1, Fangfei Li1**, Shaoyong Liu1**
+
+1Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing, China; 2Key Laboratory for Biorheological Science and Technology of Ministry of Education (Chongqing University), Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing, China Contributions: (I) Conception and design: H Chen, W Wang; (II) Administrative support: H Chen, W Wang; (III) Provision of study materials or patients: C Xiao, D Xia; (IV) Collection and assembly of data: H Chen, W Wang, F Li, S Liu; (V) Data analysis and interpretation: H Chen, W Wang, C Xiao, D Xia; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.
+
+\#These authors contributed equally to this work. 
+
+Correspondence to: Wei Wang. Key Laboratory for Biorheological Science and Technology of Ministry of Education (Chongqing University), 
+Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, 181 Hanyu Road, Chongqing 400044, China. Email: wangweixiayanai@163.com.
+
+Background: Non-small cell lung cancer (NSCLC) has a poor prognosis and is the most common cause of cancer-related deaths worldwide. Aminoacylase 1 (ACY1) plays a promoting role in some cancers, but its role in NSCLC is still unclear. 
+
+Methods: Immunohistochemistry, Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting assays were used to determine ACY1 expression patterns in NSCLC tissues and cell lines. The clinical significance of ACY1 in NSCLC was evaluated by χ 2 test and Kaplan-Meier analysis. MTT, flow cytometry, wound healing, and Transwell assays were performed to assess cell growth, apoptosis, migration, invasion, and tumorigenesis under different treatments. Male athymic BALB/C nude mice were used for xenotransplantation experiments.
+
+Results: The results showed that ACY1 expression was elevated in NSCLC tissue samples and cells, and high ACY1 expression predicted an advanced clinical process and shorter overall survival in patients with NSCLC. Overexpression of ACY1 significantly increased cell growth, migration, invasion, and tumorigenesis, and reduced cell apoptosis, indicating that ACY1 functions as an oncogene in NSCLC. Moreover, ACY1 decreased phosphatase and tensin homolog (PTEN) expression, increased its ubiquitination, and activated PI3K/AKT signaling. Overexpression of PTEN diminished the effects of ACY1 upregulation on cell tumorigenesis promotion.
+
+Conclusions: This study reveals that ACY1 may promote the progression of NSCLC via activating PI3K/AKT signaling in a PTEN-dependent manner. Our study may provide a better understanding of the pathogenesis and development of NSCLC. 
+
+Keywords: Aminoacylase 1 (ACY1); phosphatase and tensin homolog (PTEN); PI3K/AKT; non-small cell lung cancer (NSCLC) Submitted May 30, 2021. Accepted for publication Aug 16, 2021. doi: 10.21037/atm-21-3127 View this article at: https://dx.doi.org/10.21037/atm-21-3127
+
+## Page 2 Of 13 Introduction
+
+Non-small cell lung cancer (NSCLC), including adenocarcinoma and squamous cell carcinoma, accounts for 80–85% of all lung cancer cases and is the most common cause of cancer-related deaths globally (1,2). NSCLC has been identified as a highly heterogeneous type of cancer, harboring multiple genetic aberrations within its various subtypes (3). Despite recent advances in clinical and experimental oncology, the prognosis of lung cancer remains poor. Approximately 35% of NSCLC cases are diagnosed at stage IV, resulting in an overall 5-year survival rate of only 15% (1,4). Therefore, further understanding of the mechanisms related to NSCLC 
+progression is needed to develop novel therapeutic strategies.
+
+Aminoacylase 1 (ACY1) is a homodimeric zinc-binding metalloenzyme which is widely expressed in mammalian tissues. It catalyzes the hydrolysis of Na-acylated amino acids to L-amino acids and an acyl group (5). Recent studies have reported the role of ACY1 in tumorigenesis in several types of human cancer. For example, Miller et al. (6) found that ACY1 expression was reduced in a significant number of small-cell lung cancer (SCLC) 
+cell lines. Additionally, Hwa and Balabanov *et al.* (7,8)
+reported low ACY1 expression in renal clear cell carcinoma. 
+
+Wei *et al.* (9) revealed that the expression of ACY1 was decreased in hepatocellular carcinoma (HCC) tissues and that it functioned as a tumor suppressor. However, ACY1 was reported to be overexpressed in colorectal cancer (CRC), and knockdown of ACY1 significantly inhibited proliferation and induced cell apoptosis (10,11). It is showed that in neuroblastoma, ACY1 has the effect of inhibiting proliferation and metastasis (12). Nevertheless, the roles of ACY1 in NSCLC remain unclear.
+
+The PI3K/AKT/mTOR pathway is a well-studied signal pathway which plays a key role in the modulation of many cellular functions, such as cell proliferation, apoptosis, metastasis, and invasion (13,14). The hyperactivation of PI3K/AKT/mTOR can be detected in most cancers including NSCLC and is associated with cancer progression (15). Phosphatase and tensin homolog (PTEN), a tumor suppressor, has been reported to induce the dephosphorylation of the phospholipid products of PI3K, thereby resulting in the repression of PI3K/AKT/ mTOR signaling (15,16). Studies have confirmed that PTEN expression is regulated by number of genes which exert oncogenic or suppressive roles in the development 
+
+## Chen Et Al. Effects Of Acy1 On Nsclc
+
+of NSCLC, such as long noncoding RNA growth arrestspecific 5 (*GAS5*) (17), transforming growth factor beta (TGF-β) (18), and tumor protein 53 target gene 1 (*TP53TG1*) (19).
+
+Although the expression pattern of ACY1 has been found to be reduced in SCLC (6), to our knowledge, its function and clinical significance in NSCLC has not been clarified. In this study, we aimed to determine the expression profile of ACY1 in NSCLC, to evaluate the association between ACY1 expression and the clinical process and prognosis of patients with NSCLC, and to investigate whether PTEN/
+PI3K/AKT/mTOR signaling is involved *in vitro* and in vivo. It is hoped that the findings of this study can aid in the development of new methods for the diagnosis and treatment of NSCLC.
+
+We present the following article in accordance with the ARRIVE reporting checklist (available at https://dx.doi. org/10.21037/atm-21-3127).
+
+## Methods Tissue Samples
+
+Twenty paired NSCLC and the adjacent normal tissue samples were obtained from patients with NSCLC who underwent pneumonectomy and did not receive any form of chemoradiotherapy. All tissue samples were stored at 
+−80 ℃ immediately after resection. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Ethics Committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital (Chongqing, China) and informed consent was taken from all the patients.
+
+## Immunohistochemistry
+
+First, the tissues were cut into 4-μm slides. After deparaffinization and treatment to remove the endogenous peroxidase activity, antigen retrieval, and blocking with goat serum, the sections were incubated with the primary antiACY1 antibody (no. AF2900, R&D Systems, Minneapolis, MA, USA) at a 1:100 dilution. The corresponding secondary antibody (Zhongshanqinqiao, Beijing, China) was then added and counterstained with Harris hematoxylin. Cell nuclei were dyed with 3,3'-diaminobenzidine (DAB; Serva, Heidelberg, Germany) at a 1:5,000 dilution.
+
+| Table 1 Primer sequence Genes Sequence ACY1 sense-5'-CAGCGACAGGAGAGTGAGC-3' ACY1 antisense-5'-CACATCCGTGTGGGAGTTGA-3 PI3K sense-5'-AAATGCTTGGGGTGGAAGGG-3' PI3K antisense-5'-ATGTATTCAGTTCAATTGCAGAAGG-3' AKT sense-5'-GAAGGACGGGAGCAGGC-3' AKT antisense-5'-TGTACTCCCCTCGTTTGTGC-3' mTOR sense-5'-CTTAGAGGACAGCGGGGAAG-3' mTOR antisense-5'-TCCAAGCATCTTGCCCTGAG-3' PTEN sense-5'-CAGCAGCTTCTGCCATCTCT-3' PTEN antisense-5'-TGCTTTGAATCCAAAAACCTTACT-3' GAPDH sense-5'-CCACTAGGCGCTCACTGTTCT-3' GAPDH antisense-5'-GCATCGCCCCACTTGATTTT-3' ACY1, aminoacylase 1; PI3K, phosphatidylinositol 3 kinase; AKT,   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+ACY1, aminoacylase 1; PI3K, phosphatidylinositol 3 kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog deleted on chromosome ten; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
+
+## Cells Culture
+
+Human embryonic lung WI-38 cells, along with NSCLC cell lines HCC2935, A549, and H1975, were obtained from the American Type Culture Collection (ATCC, VA, USA). WI-38 cells were grown in Eagle's Minimum Essential Medium; A549 cells in F-12K medium; and HCC2935 and H1975 cells in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/
+streptomycin. All reagents used for cell culture were obtained from Thermo Fisher Scientific (Danvers, MA, USA). 
+
+## Cell Transfection
+
+The lentivirus vector shRNA-ACY1 used to silence ACY1 expression in NSCLC cells and its negative control (shNC) were purchased from OriGene (Beijing, China; no. 
+
+TL315053). The overexpressing lentivirus vectors used to upregulate PTEN (OE-PTEN) and ACY1 (OE-ACY1) were obtained from GenePharma (Shanghai, China). Firstly, the gene fragments were amplified, and the amplified fragments were connected to the lentivirus and packaged. 
+
+To establish stable sh-ACY1- or OE-ACY1-transfected cell lines, H1975 cells were transfected with sh-ACY1 or OE-ACY1and then cultured with 8 μg/mL puromycin for 2 weeks, with the medium being changed every 2 days. Meanwhile, 100 μg/mL of G418 was used to select cells with OE-PTEN transfection.
+
+## Real Time Quantitative Polymerase Chain Reaction Assay
+
+RNA was extracted from cells and tissues with an Oligotex mRNA Mini Lit (Qiagen, Valencia, CA, USA). The RNA was then reverse-transcribed into complement DNA (cDNA) with a PrimerScript RT reagent kit (TransGen Biotech, Beijing, China) in accordance with the instructions. Polymerase chain reaction (PCR) amplification was conducted using a SYBR Premix Ex TaqTM II kit 
+(TransGen Biotech, Beijing, China) in a ViiATM 7 realtime fluorescence quantitative PCR system. Expression of messenger RNA (mRNA) was normalized to that of GAPDH. The relative expressions of mRNA were analyzed with the 2-∆∆CT method. Primer sequences were showed int *Table 1*.
+
+## Western Blotting Assay
+
+Cells in different groups and tissues were lysed with RIPA 
+buffer (Thermo Scientific) at 4 ℃ for 30 minutes. Proteins were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane by electroblotting. Nonfat milk was used to block the PVDF membrane at 4 ℃ overnight. 
+
+The membrane was then incubated with the primary antibodies at 4 ℃ overnight. The antibodies were showed in *Table 2*. The membranes were then incubated at room temperature for 1.5 hours with the secondary antibody (1:2,000, Abcam). GAPDH was used as the internal reference. Target bands of proteins were visualized with an efficient chemiluminescence (ECL) western blotting kit (Millipore, Boston, MA, USA). Relative expression of proteins was assessed with the 2−∆∆CT method.
+
+## Immunoprecipitation Assay
+
+NSCLC cells were treated with sh-NC, sh-ACY1, OENC, or OE-ACY1 for 48 hours before being rinsed with cold phosphate-buffered saline (PBS) and lysed in immunoprecipitation (IP) lysis buffer (Thermo Fisher Scientific). The cell lysate containing 200 μg proteins 
+
+## Page 4 Of 13 Chen Et Al. Effects Of Acy1 On Nsclc
+
+| Table 2 Antibodies Names                                                                                                     | Dilution   | Item No.   | Company                                     |
+|------------------------------------------------------------------------------------------------------------------------------|------------|------------|---------------------------------------------|
+| ACY1                                                                                                                         | 1:500      | AF2900     | R&D Systems, MN, USA                        |
+| PI3K                                                                                                                         | 1:500      | 4252       | Cell Signaling Technology, Beverly, CA, USA |
+| p-PI3K                                                                                                                       | 1:500      | 4228       | Cell Signaling Technology, Beverly, CA, USA |
+| AKT                                                                                                                          | 1:500      | 9272       | Cell Signaling Technology, Beverly, CA, USA |
+| p-AKT                                                                                                                        | 1:500      | 13038      | Cell Signaling Technology, Beverly, CA, USA |
+| mTOR                                                                                                                         | 1:500      | ab2732     | Abcam, Cambridge, MA, USA                   |
+| PTEN                                                                                                                         | 1:500      | 9552       | Cell Signaling Technology, Beverly, CA, USA |
+| Ub                                                                                                                           | 1:500      | 3933       | Cell Signaling Technology, Beverly, CA, USA |
+| GAPDH                                                                                                                        | 1:500      | TA-08      | Zhongshanjinqiao, Beijing, China            |
+| ACY1, aminoacylase 1; PI3K, phosphatidylinositol 3 kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, |            |            |                                             |
+
+was incubated with Dynabeads protein G for 1 hour, and incubated overnight at 4 ℃ with 2 μg of antibody against PTEN, PI3K, AKT, and mTOR, which was followed by incubation with Dynabeads protein G for 1 hour. The immune complex was then loaded onto gels using Ub antibody. 
+
+## Mtt Assay
+
+For the MTT assay, 3×103 cells (with 200 μL growth medium) were seeded into 96-well plates overnight. Subsequently, MTT (Merck, Germany) was added and incubated at 37 ℃ for 4 hours. After this, 150 μL of dimethyl sulfoxide (DMSO) was added for 10 minutes. The absorbance was measured at 570 nm.
+
+## Flow Cytometry Assay
+
+Cells in different groups were seeded into 6-well plates 
+(5×105). After 24 hours of treatment, cells were digested with 0.25% trypsin and resuspended in binding buffer. Following this, 5 μL of Annexin V-FITC (Annexin V-FITC Apoptosis Detection kit, BD Biosciences, San Jose, CA, USA) and 5 μL of propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) were added to each sample and incubated at 2–8 ℃ for 5 minutes in the dark. Fluorescence-activated apoptotic cell assays were conducted with a flow cytometer (BD FACSVantage SE, BD Biosciences Co., Franklin Lakes, NJ, USA). The sum of the data of Q2 and Q3 was the apoptosis rate (%).
+
+## Wound Healing Assay
+
+NSCLC cells were seeded into 6-well plates at a 2×105 cells/well density and transfected with sh-NC, sh-ACY1, and OE-NC or OE-ACY1. The scratch was made using a sterile 20-μL pipette tip, and the medium was replaced with a serum-free medium until the cells reached a confluent monolayer. Cells were then incubated at 37 ℃ for 24 hours. 
+
+The cells were examined and photographed under phase contrast microscopy at 0 and 24 hours. The rate of cell mobility inhibition (%) was calculated as follows: cell mobility = scratch width (24 hours)/scratch width (0 hour) ×100%.
+
+## Transwell Chamber With Matrigel Assay
+
+Cell invasion was detected by transwell chambers (8 μm pore size; BD Biosciences, San Diego, CA, USA) 
+precoated with Matrigel (BD Biosciences). Next, 1.0×105 cells resuspended in 200 μL of serum-free medium were inoculated into the upper chamber of the transwell. Meanwhile, 600 μL of medium containing 15% FBS was added to the lower chamber. The cells were stained with 1% crystal violet for 20 minutes and then washed with PBS. The invaded cells were counted with a microscope after 48 hours.
+
+## Xenotransplantation Assay
+
+A total of 12 four-week-old male athymic BALB/C nude mice (Weitonglihua. Co., Ltd., Beijing, China) were used 
+
+to assess cell tumorigenesis. The animals were kept in a professional laboratory for nude mice in an atmosphere of 24±1 ℃ with 60%±5% relative humidity. A random number table method was used to divide the mice into 4 groups (N=3): control, sh-ACY1, OE-ACY1, and OEACY1+OE-PTEN. In detail, 5×106 H1975 cells with stable control, sh-ACY1, OE-ACY1, or OE-ACY1+OE-PTEN transfection were resuspended in 200 μL PBS and then injected into the armpit area of the nude mice. Twentyeight days posttransplantation, the mice were sacrificed, and the tumors were collected for weighing. During the experiment, care was taken to minimize the pain of the mice without affecting the results of the experiment. No deaths occurred during the experiment. Euthanasia was administered when the diameter of the tumor was greater than 2 cm (not involved in this experiment). Protocols involving animals were approved by the ethical committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, and animal experiment was conducted in compliance with Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital guidelines for the care and use of animals.
+
+## Statistical Analysis
+
+Each measurement was obtained in 3 parallel tests. All experimental data are presented as the mean ± SD. Statistical analyses were performed on GraphPad Prism 7 software with one-way analysis of variance followed by the Tukey's test for multiple groups and Student's t-test for 2 groups. χ 2 test and Kaplan-Meier analysis were used to evaluate the clinical significance of ACY1 in NSCLC. 
+
+Pearson Correlation Coefficient was used to verify the correlation between ACY1 and PTEN in NSCLC tissues. A P value <0.05 was considered a statistically significant difference.
+
+## Results Acy1 Expression Was Increased In Nsclc Tissues And Cells
+
+We first identified the expression patterns of ACY1 and PTEN in NSCLC tissues and cells. Increased ACY1 expression was observed in NSCLC tissues when compared to the adjacent normal tissues in both protein and mRNA 
+levels, as determined by immunohistochemistry (*Figure 1A*), 
+RT-PCR (*Figure 1B*), and western blotting (*Figure 1C*). 
+
+The expression of PTEN protein in NSCLC tissues was significantly lower than that of adjacent normal tissues 
+(*Figure 1C*). In NSCLC tissues, the trends of PTEN 
+and ACY1 were opposite. In addition, ACY1 and PTEN 
+expression was also detected in NSCLC cell lines by realtime PCR (RT-PCR) and western blotting assays. ACY1 expression levels were increased in human NSCLC cell lines HCC2935, A549, and H1975 compared to those in human embryonic lung WI-38 cells, while PTEN mRNA 
+and protein were decreased (*Figure 1D,1E*). These results suggest that ACY1 was deregulated in NSCLC.
+
+## High Acy1 Expression Predicted An Advanced Clinical Features And Shorter Overall Survival For Patients With Nsclc
+
+To investigate the role of ACY1 in NSCLC progression, we then assessed its clinical significance in NSCLC. A total of 91 patients with NSCLC were divided into 2 groups according to the expression levels of ACY1, among whom 60 had high ACY1 expression (high expression group) and 31 had low expression (low expression group). As shown in *Table 3,* there were no significant differences in gender, age, or histological type, but patients with ACY1 high expression had significantly lower differentiation (P=0.011), lymph node metastasis (P=0.002), and advanced tumor-node-metastasis (TNM) stage (P=0.006). Moreover, patients in the high expression group had shorter overall survival than those in the low expression group (*Figure 1F*). 
+
+In addition, the expression of ACY1 and PTEN in NSCLC tissues were negatively correlated (*Figure 1G*). 
+
+These results suggest that ACY1 may have the potential to predict the clinical process and outcome of NSCLC 
+patients. 
+
+## Overexpression Of Acy1 Facilitated Nsclc Cell Conversion To A Malignant Phenotype
+
+We investigated the effects of ACY1 in the progression of NSCLC *in vitro* via gain/loss-of-function assays. ACY1 expression was decreased to 20–30% when HCC2935 and H1975 cells were transfected with sh-2, while it was increased to 350–380% in mRNA levels and 200–210% 
+in protein levels when cells were transfected with OEACY1 (*Figure 2A,2B*). Compared with controls, sh-ACY1 transfection significantly increased cell proliferation, and sh-ACY1 impaired cell proliferation in HCC2935 (*Figure 2C*) 
+and H1975 cells (*Figure 2D*). Cell apoptosis was decreased 
+
+![5_image_0.png](5_image_0.png)
+
+when HCC2935 and H1975 cells were transfected with OEACY1, and it was increased when ACY1 was downregulated 
+(*Figure 2E*). In addition, ACY1 upregulation promoted cell migration and invasion, and knockdown of ACY1 caused the opposite to occur (*Figure 3A-3C*). We also detected the effects of ACY1 on epithelial-mesenchymal transition (EMT)-marked proteins. The results showed that when ACY1 was inhibited, the expression of E-cadherin protein increased, and the expression of N-cadherin and Vimentin protein decreased (*Figure 3D,3E*). Overexpression of ACY1 produced the opposite result. Together, these findings demonstrated that ACY1 functioned as an oncogene in NSCLC.
+
+## Overexpression Of Acy1 Promoted The Activation Of Pi3K/ Akt Signaling Through Downregulation Of Pten
+
+Subsequently, we investigated the effects of ACY1 expression on the activation of PI3K/AKT signaling. Compared with the control group, ACY1 upregulation significantly increased the protein expression of p-PI3K, p-AKT, and mTOR, whereas it reduced PTEN expression (*Figure 4A*) 
+and showed no obvious influence in the mRNA levels of PI3K, AKT, mTOR, or PTEN (*Figure 4B*). To further explore the mechanism of ACY1 in regulating PTEN/PI3K/ AKT signaling, we used an IP assay to assess the interaction between ACY1 and PTEN/PI3K/AKT signaling-related proteins. The results showed that ACY1 could only combine 
+
+| Table 3 Association of ACY1 expression and the clinicopathologic  features of patients with NSCLC ACY1 expression Variable P value High Low Gender 0.649 Male 37 21 Female 23 10 Age (y) 0.186 <60 38 15 ≥60 22 16 Histological type 0.512 Squamous cell carcinoma 32 14 Adenocarcinoma 28 17 Differentiation 0.011 Well 12 14 Moderate 22 12 Poor 26 5 Lymph node metastasis 0.002 No 28 25 Yes 32 6 TNM stage 0.006 I 10 11 II 17 14 III 33 6 NSCLC, non-small cell lung cancer; ACY1, aminoacylase 1;  TNM, tumor-node-metastasis.   |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+with PTEN, and this interaction was weakened when ACY1 was overexpressed in H1975 cells (*Figure 4C*). This might have been due to ACY1 inducing the degradation of PTEN protein. Moreover, ACY1 upregulation promoted the protein degradation of PTEN protein (*Figure 5A*), and increased the ubiquitination of PTEN protein (*Figure 5B*), 
+and vice versa. Overall, these results demonstrated that ACY1 upregulation could promote PI3K/AKT signaling activation via downregulation of PTEN.
+
+## Acy1 Enhanced Nsclc Cell Tumorigenesis Via Downregulating Pten
+
+We further examined the effects of ACY1/PTEN in the tumorigenesis of NSCLC cells. Tumorigenesis was significantly enhanced in H1975 cells during stable upregulation of ACY1, while it was decreased at the time of ACY1 downregulation. Overexpression of PTEN 
+counteracted the enhancement of H1975 cell tumorigenesis induced by ACY1 upregulation (*Figure 6A,6B*), illustrating that ACY1 promoted cell tumorigenesis via downregulating PTEN in NSCLC.
+
+## Discussion
+
+The American Joint Committee on Cancer (AJCC) (20,21) has reported that up to 70% of NSCLC patients are incurable due to the extent of cancer at the time of diagnosis. A standard treatment for NSCLC, platinumbased chemotherapy, also shows low response rates of 17–32% and overall survival of 7.4–11.3 months for NSCLC patients at advanced stages (22). Therefore, it is crucial to further investigate the underlying mechanisms of NSCLC. This study focused on understanding the role and mechanism of ACY1, revealed the high fold change in the protein and mRNA expression levels of ACY1 in NSCLC tissues and cells, and showed that ACY1 has an oncogenic role in NSCLC. To our knowledge, there is a lack of studies on the clinical significance of ACY1 in NSCLC. However, the present study demonstrated that NSCLC patients with ACY1 high expression were prone to show low differentiation (P=0.011), lymph node metastasis (P=0.002), and advanced TNM stage (P=0.006), as well as poor prognosis, which was consistent with previous studies concerning the role of ACY1 in CRC (23). 
+
+ACY1 is commonly found in the kidneys and the brain. 
+
+Its deregulation has been associated with autism and convulsions (24,25). ACY1 has also been reported to play a role in the deacetylation of aflatoxin epoxy derivatives and thereby influence cell detoxification function (26). Moreover, studies have found ACY1 to be strongly implicated in carcinogenesis, with different roles in different types of cancers. For instance, low expression of ACY1 was found in HCC tissues, and ACY1 expression was significantly associated with tumor invasiveness; furthermore, overexpression of ACY1 in HCC BEL7402 cells was shown to significantly inhibit cell viability and invasiveness (9), revealing that ACY1 may function as a tumor suppressor. Conversely, ACY1 was reported to be overexpressed in CRC, and knockdown of ACY1 significantly inhibited cell proliferation and induced cell apoptosis (10,11). In contrast to the ACY1 expression Page 8 of 13
+
+![7_image_0.png](7_image_0.png) 
+
+profiles in SCLC (6), we found that ACY1 expression was obviously increased in NSCLC tissues and cells in both protein and mRNA levels. To measure the effects of this increase of ACY1 in NSCLC progression, gain/loss-offunction assays were performed in 2 NSCLC cell lines, H1975 and HCC2935. The results showed that cell growth, migration, invasion, and tumorigenesis were enhanced, while cell apoptosis was decreased when ACY1 was upregulated, indicating that ACY1 has a tumor-promoting role in NSCLC. 
+
+In addition, we investigated the mechanism of ACY1 in promoting the progression of NSCLC. We observed that ACY1 could activate PI3K/AKT signaling through downregulating PTEN. Studies have demonstrated that 
+
+Annals of Translational Medicine, Vol 9, No 17 September 2021 **Page 9 of 13**
+
+![8_image_0.png](8_image_0.png)
+
+![9_image_0.png](9_image_0.png)
+
+PTEN protein expression is significantly reduced in poorly differentiated NSCLC tumors compared to moderately differentiated NSCLC tumors. This is one of the main contributors to PI3K/AKT pathway activation and resistance to tyrosine kinase inhibitors (TKIs), including the most common chemotherapy drugs for NSCLC, such as gefitinib, erlotinib, and afatinib (27,28). Furthermore, NSCLC patients with PTEN low expression level always have lower survival rates than those with PTEN high expression (29). This study, along with previous studies, illustrates the crucial role of PTEN in NSCLC. To further investigate the deregulation of PTEN and clarify the mechanism of ACY1 in NSCLC, we carried out western blotting and IP assays and found that ACY1 could interact with PTEN protein but not mTOR, AKT, or PI3K, and could decrease its expression via accelerating its ubiquitination and degradation. Moreover, we observed that PTEN upregulation weakened the effect of ACY1 on tumorigenesis promotion in NSCLC H1975 cells, suggesting that ACY1 facilitated the progression of NSCLC 
+through PTEN deletion. Although previous studies have reported that activation of ERK1/2 and expression of TGF-β are implicated in ACY1-induced tumor cell proliferation (9,30), the present study revealed, for the first time, that the PTEN/PI3K/AKT pathway may also be the target signaling of ACY1 in carcinogenesis. Based on this, we speculated that in NSCLC, the upregulation of ACY1 protein might activate the PI3K/AKT/mTOR pathway by inducing degradation of PTEN protein, thereby promoting cell proliferation and metastasis capabilities, and inhibiting apoptosis. However, the molecular mechanism underlying the manner in which ACY1 induces PTEN ubiquitination and degradation still needs to be explored by further indepth analysis.
+
+In conclusion, this study suggests that ACY1 may function as an oncogene in NSCLC via regulating the PTEN/PI3K/AKT signaling pathway. It also demonstrates the clinical significance of ACY1 in NSCL, specifically, how ACY1 expression may be closely associated with an advanced clinical process and shorter overall survival for 
+
+![10_image_0.png](10_image_0.png)
+
+![10_image_1.png](10_image_1.png)
+
+## Page 12 Of 13
+
+patients with NSCLC. Our study aims to provide a better understanding of the pathogenesis and development of NSCLC. However, further exploration into the mechanism of ACY1 influence in the occurrence and progression of lung cancer is needed.
+
+## Acknowledgments
+
+Funding: The study was funded in part by Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital (No. ZY201701001).
+
+## Footnote
+
+Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://dx.doi. org/10.21037/atm-21-3127 Data Sharing Statement: Available at https://dx.doi.
+
+org/10.21037/atm-21-3127 Conflicts of Interest: All authors have completed the ICMJE 
+uniform disclosure form (available at https://dx.doi. org/10.21037/atm-21-3127). The authors have no conflicts of interest to declare.
+
+Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Ethics Committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital (Chongqing, China) and informed consent was taken from all the patients. Protocols involving animals were approved by the ethical committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, and animal experiment was conducted in compliance with Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital guidelines for the care and use of animals.
+
+Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-
+
+## Chen Et Al. Effects Of Acy1 On Nsclc
+
+commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). 
+
+See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
+
+## References
+
+1. Dempke WC. Targeted Therapy for NSCLC--A Doubleedged Sword? Anticancer Res 2015;35:2503-12.
+
+2. Ettinger DS, Wood DE, Akerley W, et al. Non-Small Cell Lung Cancer, Version 6.2015. J Natl Compr Canc Netw 2015;13:515-24.
+
+3. Jiao Z, Yu A, He X, et al. Bioinformatics analysis to determine the prognostic value and prospective pathway signaling of miR-126 in non-small cell lung cancer. Ann Transl Med 2020;8:1639.
+
+4. Wu KL, Tsai MJ, Yang CJ, et al. Liver metastasis predicts poorer prognosis in stage IV lung adenocarcinoma patients receiving first-line gefitinib. Lung Cancer 2015;88:187-94.
+
+5. Heese D, Löffler HG, Röhm KH. Further characterization of porcine kidney aminoacylase I reveals close similarity to 'renal dipeptidase'. Biol Chem Hoppe Seyler 1988;369:559-66.
+
+6. Miller YE, Minna JD, Gazdar AF. Lack of expression of aminoacylase-1 in small cell lung cancer. Evidence for inactivation of genes encoded by chromosome 3p. J Clin Invest 1989;83:2120-4.
+
+7. Balabanov S, Zimmermann U, Protzel C, et al. Tumourrelated enzyme alterations in the clear cell type of human renal cell carcinoma identified by two-dimensional gel electrophoresis. Eur J Biochem 2001;268:5977-80.
+
+8. Hwa JS, Park HJ, Jung JH, et al. Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis. J Korean Med Sci 2005;20:450-5.
+
+9. Wei X, Li J, Xie H, et al. Proteomics-based identification of the tumor suppressor role of aminoacylase 1 in hepatocellular carcinoma. Cancer Lett 2014;351:117-25.
+
+10. Shi H, Hayes MT, Kirana C, et al. Overexpression of aminoacylase 1 is associated with colorectal cancer progression. Hum Pathol 2013;44:1089-97.
+
+11. Shi H, Hood KA, Hayes MT, et al. Proteomic analysis of advanced colorectal cancer by laser capture microdissection and two-dimensional difference gel electrophoresis. J Proteomics 2011;75:339-51.
+
+12. Chen Z, Gao J, Sun J, et al. Aminoacylase 1 (ACY1) Mediates the Proliferation and Migration of 
+
+Neuroblastoma Cells in Humans Through the ERK/
+Transforming Growth Factor β (TGF-β) Signaling Pathways. Med Sci Monit 2021;27:e928813.
+
+13. Ersahin T, Tuncbag N, Cetin-Atalay R. The PI3K/AKT/
+mTOR interactive pathway. Mol Biosyst 2015;11:1946-54.
+
+14. Fan X, Huang H, Ji Z, et al. Long non-coding RNA 
+MEG3 functions as a competing endogenous RNA of miR-93 to regulate bladder cancer progression via PI3K/ AKT/mTOR pathway. Transl Cancer Res 2020;9:1678-88.
+
+15. Pérez-Ramírez C, Cañadas-Garre M, Molina M��, et al. 
+
+PTEN and PI3K/AKT in non-small-cell lung cancer. Pharmacogenomics 2015;16:1843-62.
+
+16. Xia M, Tong JH, Ji NN, et al. Tramadol regulates proliferation, migration and invasion via PTEN/PI3K/ AKT signaling in lung adenocarcinoma cells. Eur Rev Med Pharmacol Sci 2016;20:2573-80.
+
+17. Cao L, Chen J, Ou B, et al. GAS5 knockdown reduces the chemo-sensitivity of non-small cell lung cancer (NSCLC) cell to cisplatin (DDP) through regulating miR-21/PTEN axis. Biomed Pharmacother 2017;93:570-9.
+
+18. Shen H, Guan D, Shen J, et al. TGF-β1 induces erlotinib resistance in non-small cell lung cancer by downregulating PTEN. Biomed Pharmacother 2016;77:1-6.
+
+19. Xiao H, Liu Y, Liang P, et al. TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis. Cell Biosci 2018;8:23.
+
+20. Yu Y, Zhang P, Yao R, et al. Prognostic value of log odds of positive lymph nodes in node-positive lung squamous cell carcinoma patients after surgery: a SEER populationbased study. Transl Lung Cancer Res 2020;9:1285-301.
+
+21. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010;17:1471-4. 
+
+22. Kelly K, Crowley J, Bunn PA Jr, et al. Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine Cite this article as: Chen H, Wang W, Xiao C, Xia D, Li F, Liu S. ACY1 regulating PTEN/PI3K/AKT signaling in the promotion of non-small cell lung cancer progression. Ann Transl Med 2021;9(17):1378. doi: 10.21037/atm-21-3127 plus cisplatin in the treatment of patients with advanced non--small-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol 2001;19:3210-8.
+
+23. Yu B, Liu X, Cao X, et al. Study of the expression and function of ACY1 in patients with colorectal cancer. Oncol Lett 2017;13:2459-64.
+
+24. Smith T, Ghandour MS, Wood PL. Detection of N-acetyl methionine in human and murine brain and neuronal and glial derived cell lines. J Neurochem 2011;118:187-94.
+
+25. Tylki-Szymanska A, Gradowska W, Sommer A, et al. Aminoacylase 1 deficiency associated with autistic behavior. J Inherit Metab Dis 2010;33 Suppl 3:S211-4.
+
+26. Stocker P, Brunel JM, de Rezende L, et al. Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxinderived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides. Biochimie 2012;94:1668-75.
+
+27. Jin G, Kim MJ, Jeon HS, et al. PTEN mutations and relationship to EGFR, ERBB2, KRAS, and TP53 mutations in non-small cell lung cancers. Lung Cancer 2010;69:279-83.
+
+28. Spoerke JM, O'Brien C, Huw L, et al. Phosphoinositide 3-kinase (PI3K) pathway alterations are associated with histologic subtypes and are predictive of sensitivity to PI3K inhibitors in lung cancer preclinical models. Clin Cancer Res 2012;18:6771-83.
+
+29. Endoh H, Yatabe Y, Kosaka T, et al. PTEN and PIK3CA 
+expression is associated with prolonged survival after gefitinib treatment in EGFR-mutated lung cancer patients. J Thorac Oncol 2006;1:629-34.
+
+30. Yijing L, Liu H, Yuan C, et al. The effects of qindancapsule-containing serum on the TGF-β1/ERK 
+signaling pathway, matrix metalloproteinase synthesis and cell function in adventitial fibroblasts. Pharm Biol 2013;51:712-21.
+
+(English Language Editors: G. Stone and J. Gray)

medical/md/PMC8690856.md

@@ -0,0 +1,237 @@
+# Color Change After Tooth Bleaching With Ozone And 10% Ozonized Carbamide Peroxide For In-Office Use
+
+Thalyta dos Reis Furlani Zouain Ferreira Neves Dias, Francisco Ubiratan Ferreira de Campos, Cecilia Pedroso Turssi, Flávia Lucisano Botelho do Amaral, Fabiana Mantovani Gomes França, Roberta Tarkany Basting* Faculdade São Leopoldo Mandic, Campinas, São Paulo, Brazil
+*Correspondence to: Roberta Tarkany Basting, DDS, PhD, rbasting@yahoo.com.
+
+orcid: 0000-0002-0888-7342 (Thalyta dos Reis Furlani Zouain Ferreira Neves Dias); 0000-0003-0990-2425 (Francisco Ubiratan Ferreira de Campos); 0000-0002-0078-9895 (Cecilia Pedroso Turssi); 0000-0002-8934-6678 (Flávia Lucisano Botelho do Amaral); 0000-0002-2877-6797 (Fabiana Mantovani Gomes França); 0000-0002-5345-5776 (Roberta Tarkany Basting)
+
+## Abstract
+
+There is a constant search for bleaching treatments that can offer greater safety with fewer adverse effects, especially in the techniques performed in the office, which usually employ hydrogen peroxide in high concentrations (35% to 40%) that are not recommended by some international control agencies. This *in vitro* study evaluated the color change after tooth bleaching with the use of ozone and a 10% ozonized carbamide peroxide bleaching treatment for in-office use. Thirty molars were allocated (n = 10): three applications of ozone (1 hour every 3 days); three applications of 10% ozonized carbamide peroxide (1 hour every 3 days); 10% carbamide peroxide agent (8 hours a day for 7 days). The teeth were mounted on a plaster model to simulate the dental arch, and trays made of silicone were used for the application of the bleaching agents and to allow ozone to enter through. The ozone concentration used was 60 μg/mL, with an oxygen flow of 0.25 L/min. The values of color change showed no significant differences among treatments. The variations in the parameters over time, as well as the values of ΔEab, ΔE00, and WID , showed that there was no significant difference among the three treatments. The use of ozone and 10% ozon-ized carbamide peroxide for in-office use was effective for tooth bleaching with clinically perceptible and acceptable color alterations. The study was approved on September 10, 2019 by the São Leopoldo Mandic Ethics Research Committee (CAAE No. 17711719.4.0000.5374). Key words: carbamide peroxide; color; color change; gas; hydrogen peroxide; ozone; ozone therapy; oxygen; tooth bleaching; whitening doi: 10.4103/2045-9912.330693 How to cite this article: Dias TdRFZFN, de Campos FUF, Turssi CP, do Amaral FLB, França FMG, Basting RT. Color change after tooth bleaching with ozone and 10% ozonized carbamide peroxide for in-office use. Med Gas Res. 2022;12(3):100-106.
+
+## I**Ntroduction**
+
+Tooth bleaching techniques use agents with different concentrations of hydrogen peroxide or carbamide for in-office or at-home treatments.1 Higher concentrations of hydrogen peroxide (between 35% and 40%) are used for in-office techniques, whereas lower concentrations (up to 10% hydrogen peroxide and between 10% and 20% carbamide peroxide, which releases between 3% and 6% hydrogen peroxide), are used for techniques performed at-home by the use of trays.1 According to the American Dental Association,2 products with hydrogen peroxide concentrations up to 3.5% are considered safe, whereas the Committee for Scientific Consumer Products3 of the European Union approves products with up to 6.0% hydrogen peroxide. Thus, there is a need to develop safe bleaching treatments that can effectively deliver desired dental chromatic changes, especially for techniques that use concentrations higher than those recommended by control agencies.
+
+In this respect, at-home bleaching technique using 10% 
+carbamide peroxide can be considered a safe and effective procedure.1,4,5 However, some patients do not adapt well to the use of trays, or they may still require an in-office procedure.4 In case of the recommendation about using agents with low peroxide concentration, it may be useful to increase their oxidative capacity for possible use in-office for achieving effective results within a short duration after application.
+
+Ozone is an agent that has been studied as a powerful oxidizer alone or in association with high concentrations of hydrogen peroxide in.6-10 It has a short half-life and is unstable and dependent on system conditions such as temperature and pressure; therefore, it easily decomposes into pure oxygen.11 In addition to its use as a medical antimicrobial and therapeutic agent,11 its use in in-office bleaching techniques with agents with high concentrations of hydrogen peroxide, at 37.5%7 and 38%,8-10 has shown significant bleaching effects; additionally, it does not affect the mineral content of the enamel.12 Furthermore, ozone bleaching has been shown to result in reduced dental sensitivity compared with the conventional in-office technique.10 By using ozone with low concentrations of bleaching agents, Manton et al.13 found that applying ozone for 40 seconds did not potentiate the bleaching effect of an 8% carbamide peroxide aqueous solution, possibly because it was not used in gel form; the thickener leads to prolonged contact with the tooth surface, slowing the release of free radicals and reducing the duration of application.14,15 The concentration of hydrogen peroxide released by 10% 
+carbamide peroxide is 10 times less than that of hydrogen peroxide used in in-office techniques.4 Thus, because of the possible benefit of reduced sensitivity9,10 and the potential for oxidation, the use of ozone with a bleaching gel containing 10% carbamide peroxide for in-office techniques should also be evaluated.
+
+Therefore, the objective of this study was to assess *in vitro* the effects of color change after ozone and a 10% ozonized carbamide peroxide bleaching treatments for in-office use. The null hypothesis for this study was that there are no differences between the changes in the color of teeth subjected to bleaching treatments using ozone and ozonized carbamide peroxide for in-office use compared to 10% carbamide peroxide for at-home use.
+
+## Materials And M**Ethods** Ethical Aspects And Tooth Selection
+
+Thirty healthy human third molars were selected from a dental research biorepository after the project was approved on September 10, 2019 by the São Leopoldo Mandic Ethics Research Committee (CAAE No. 17711719.4.0000.5374) and stored in 0.1% thymol solution for a maximum of 6 months.12 Written informed consent (**Additional file 1**) was obtained from the patients to provide the teeth for the experiment.
+
+## Bleaching Protocols
+
+Thirty molars were allocated by randomization into three groups (n = 10): CP group: bleaching with 10% carbamide peroxide agent (Opalescence PF 10%/Ultradent); O group: bleaching with three applications of ozone; OCP group: bleaching with three applications of 10% ozonized carbamide peroxide. The groups and bleaching protocols are presented in **Table 1**. The ozone concentration followed the protocol of Santana et al.7 and Tessier et al.16 adapted for use herein. The pH values of the 10% carbamide peroxide and the 10% ozonized carbamide peroxide bleaching agents were measured at different application times (15, 30, 45, and 60 minutes) using a pH meter (MS Tecnopon Equipamentos Especiais Ltda., Piracicaba, SP, Brazil).
+
+Table 1: Groups, bleaching protocols, materials, and 
+
+manufacturers
+
+| manufacturers Bleaching   | Bleaching                          | Commercial name and                                                                                                                                                        |                                                           |
+|---------------------------|------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|
+| Groups                    | treatment                          | protocol                                                                                                                                                                   | manufacturer                                              |
+| CP                        | 10%  carbamide  peroxide           | 8 h/d for 7 d                                                                                                                                                              | Opalescence PF 10% -  Ultradent (Indaiatuba, SP,  Brazil) |
+| O                         | Ozone                              | 1 h every  3 d, for a  total of three  applications Medplus MX Philozon® (Balneário Camboriú,  SC, Brazil)                                                                 |                                                           |
+| OCP                       | 10%  ozonized  carbamide  peroxide | 1 h every  3 d, for a  total of three  applications Opalescence PF 10% -  Ultradent (Indaiatuba,  SP, Brazil) and Medplus  MX Philozon®  (Balneário  Camboriú, SC, Brazil) |                                                           |
+
+## Tooth Preparation For Initial Color Evaluation And Plaster Model Assembly
+
+The allocation of the teeth to the respective groups was performed based on the initial color evaluation by the Vita Classical criterion using a spectrophotometer (VITA EasyShade Advance 4.0, Vita Zahnfabrik, BadSackingen, Germany) in order to distribute the tooth colors equally among the groups, 
+   ¦  101 and to avoid the risk of bias. For this, the teeth remained im-mersed in an artificial saliva solution (7 mL for each tooth) (1.5 mM Ca; 50 mM KCl; 0.9 mM PO4
+; 20 mM Tris buffer; pH = 
+7.0), as recommended by Featherstone et al.17 and modified by Serra and Cury,18 for 7 days at 37°C in a bacteriological oven. They were removed from the solution, dried with gauze, and evaluated using a closed white box to prevent the effects of external luminosity and standardization of light during the readings. The color measurements were obtained in duplicate to ensure accuracy.19 The region for the assessment of color was the middle third of the buccal surface of the teeth. Based on the initial evaluations according to Vita Classical (the selected teeth had colors B3, A3.5, and A4), they were equally distributed among the three groups. Therefore, all the groups presented an equal amount of teeth with shades B3, A 3.5 and A4. At the same time, the color data for L*, a*, and b* were recorded by the CIELa*b* system for each tooth.
+
+After the initial color analysis, the teeth were mounted on a plaster model to simulate the dental arch for the bleaching techniques. The lower arch of a dental mannequin (Prodens - Pronew, São Gonçalo, RJ, Brazil) without the artificial teeth was duplicated with special silicone (RTV-2 silicone rubber, HB FLEX 5528 - HBQUIMICA Representações, Comércio e Serviços Ltda., Custóias, Portugal). After duplication, the natural teeth were positioned in the empty alveoli. To correctly fit the teeth, the regularization of these alveoli was performed with a carbide drill 060 (Horico, Berlin, Germany). A new duplication with special silicone and casting with a special micro-granulated IV plaster (Durone, Dentsply Sirona, São Paulo, SP, Brazil) was carried out with a vacuum spatula to obtain the models. For this, each group of teeth (respective to each bleaching treatment to be evaluated) was mounted on each cast model, using 7 wax for fixing the entire root of each tooth after sealing along the dental arch of the model. This step was carried out to position and fit the teeth in the models to complete the subsequent steps.
+
+## Manufacturing The Trays
+
+Three trays made of silicone (Crystal PVC, Bioart, São Carlos, SP, Brazil) (thickness = 3 mm) were used for the application of the bleaching agents (one tray was used for each bleaching treatment). The trays were obtained after molding the plaster models in which the teeth were fixed with the use of alginate and a tray. The molds were cast with a special type IV dental plaster. For the group that would receive the 10% carbamide peroxide treatment (PC group), the tray was cut 2 mm above the gingival sulcus. For the experimental groups that were treated with ozone (O group) or 10% ozonized carbamide peroxide gel (PCO group), wearing was carried out with a number 6 spherical bur coupled to the micromotor in the end region of the vestibule, both on the outer face and the face inside the respective plaster models. A dense condensation silicone (Speedex - Coltene, Rio de Janeiro, RJ, Brazil) paste was used, and it was manipulated for the application using a thickness of 0.5 mm above the occlusal surface of the cast to make room for ozone circulation inside the tray. After the aforementioned processes, the trays were created in the vacuum plasticizer (P7, Bio-art, São Carlos, SP, Brazil) by cutting each tray at the location where wearing was previously carried out in the backend region of the vestibule. Later, four holes were drilled in the vestibular side and one hole was drilled in the lingual surface of the trays. These holes were made for installing the plastic cannulas (Macrobots Descarpack Luer Slip, San Paulo, SP, Brazil) to allow ozone to enter through the vestibular side of the trays and exit from the lingual face of the respective trays.
+
+## Bleaching Treatment With 10% Carbamide Peroxide - Cp Group
+
+A disposable syringe (Biotecmed, São Paulo, SP, Brazil) was used to apply 0.02 mL of the bleaching gel on the vestibular surface of the enamel using trays with 10% carbamide peroxide. The bleaching agent was kept on the enamel surface for 8 hours a day in an artificial saliva solution and a bacteriological oven at 37°C, according to the manufacturer's recommendations. Subsequently, the gel was removed from the enamel surface with deionized water for 10 seconds, and the tooth was dried with gauze. Next, the model was immersed in an artificial saliva solution and kept in a bacteriological oven at 37°C. This cycle was repeated for 7 days. After the end of the applications, the enamel surface was washed with distilled water and stored again in the artificial saliva solution for 12 hours in a bacteriological greenhouse at 37°C until the final color assessments are made.
+
+## Ozone Gas Treatment - O Group
+
+An ozone gas generator (Medplus MX Philozon, Camboriú, SC, Brazil) was connected to an oxygen cylinder by a flowmeter standard20 to the experiment. The dose adjustment was applied according to the ozone concentration and the acting time. The ozone concentration used was 60 μg/mL, with an oxygen flow of 0.25 L/min, as per the manufacturer's specifications. The application time was 1 hour every 3 days for a total of three applications. For the ozone application, one of the ends of the cannulas was adapted to the four holes in the vestibular region of the tray, whereas the other end of the cannula was adapted to the ozone generator equipment. For the ozone output of the tray, one of the ends of the cannulas was adapted to the tray in the hole in the lingual region, whereas the other end was adapted to the ozone generator vacuum suction system (**Figure 1**). Between the bleaching sessions, the specimens were kept in a humid environment in a bacteriological oven at 37°C.
+
+![2_image_0.png](2_image_0.png)
+
+102    ¦ 
+
+![2_image_1.png](2_image_1.png)
+
+10% ozonized carbamide peroxide treatment - OCP group A disposable syringe (Biotecmed, São Paulo, SP, Brazil) was used to apply 0.02 mL of the 10% carbamide peroxide bleaching agent over the surface of the vestibular enamel of the teeth, and the tray was positioned with the previously prepared cannulas. The tray with plastic cannulas was connected to the ozone generator, as previously presented. The 10% carbamide peroxide, together with the ozone to be applied, remained on the specimens for 1 hour. This application was repeated every 3 days three times. Between the bleaching sessions, the specimens were kept in a humid environment in a bacteriological oven at 37°C.
+
+## Final Color Analysis
+
+The final color evaluations were performed 24 hours after completion of treatments using the Vita Classical scale and CIEL*a*b* system, as described for the initial color evaluation.
+
+The tooth color obtained using the Vital Classical scale was converted to numerical values (1 to 16) as previously reported.4,19 Lower numerical values represented brighter teeth. The color change (ΔEab) obtained by the CIELab system was calculated using the following formula21: ΔEab* = [(ΔL*)2 + (Δa*)2
++ (Δb*)2 ]1/2, where ΔL = Lfinal - Linitial; Δa = afinal - ainitial; Δb = 
+bfinal - binitial. The color change was also assessed by CIEDE2000 
+(ΔE00), which is based on hue and chroma values,22 and the Whiteness Index for Dentistry (WID), in which the parameters L*, a*, and b* are used in transmittance analysis according to the following equation: WID = 0.511L* - 2.324a* - 1.100b*.23
+
+## Statistical Analysis
+
+Since the data did not meet the assumptions of the one-way analysis of variance, they were analyzed by generalized linear models, considering the design of repeated measures for L*, a*, b*, and the color values using the Vita Classical scale. 
+
+The data from WID were not adjusted to a known distribu-tion. The non-parametric Kruskal-Wallis tests were used to compare the groups, and the Wilcoxon test was used for time comparison (WID). The data for ΔL, Δa, and Δb were not adjusted to a known distribution, and they were analyzed using the non-parametric Kruskal-Wallis test. Generalized linear models were also used, considering the treatment factor, to analyze ΔEab, ΔE00, and ΔWID. R (R Core Development Team 2020, Vienna, Austria) and SAS (SAS Institute Inc., Cary, NC, USA) were used for the analyses, assuming the level of significance of 5%.
+
+The values for ΔEab above 2.7 were considered clinically acceptable.24,25 The values for ΔE00 of 0.8 and 1.8 were adopted as thresholds for the perceptibility and acceptability criteria.24 The thresholds for ΔWID were 0.72 for perceptibility and 2.60 for acceptability.26
+
+## R**Esults** Ph Values
+
+The pHs of the bleaching agents evaluated at different times 
+(**Table 2**) were similar and close to neutrality within 60 minutes. There was a slight decrease in pH values for the 10% 
+ozonized carbamide peroxide throughout the period evaluation (from 7.43 to 7.21) while the 10% carbamide peroxide remained at 7.43 during this time. 
+
+## Ciel*A*B*, Vita Classical And Wi**D Results**
+
+For the color variable (**Table 3**), there was no significant association between the groups and the times (P > 0.05); there were also no significant differences among the three groups (P > 0.05). For all treatments, there was a significant increase in the parameters L* (teeth became lighter) and WID (teeth became whiter) and a significant decrease in a* (teeth became less red), b* (teeth became less yellow), and in the Vita Classical scale (an average change of 10 shade colors). 
+
+| Table 2: pH values according to treatments and  evaluation times Bleaching  treatment Initial 15 min 30 min 45 min   | 60 min   |      |      |      |      |
+|----------------------------------------------------------------------------------------------------------------------|----------|------|------|------|------|
+| 10% carbamide  peroxide (CP  group)                                                                                  | 7.43     | 7.43 | 7.43 | 7.43 | 7.43 |
+| 10% ozonized  carbamide peroxide  (OCP group) 7.43                                                                   | 7.38     | 7.38 | 7.29 | 7.21 |      |
+| Note: The analysis of pH was performed in triplicate (n = 3) at 25°C.                                                |          |      |      |      |      |
+
+Color change (ΔL*, Δa*, Δb*, ΔEab, ΔE00 and ΔWI**D) results**
+The variation of parameters L*, a*, and b* over time (Table 4), as well as the values of ΔEab, ΔE00 and ΔWID show that there was no significant difference between the groups (P > 0.05). 
+
+## D**Iscussion**
+
+The data from this study shows that all the evaluated treatments delivered effective tooth bleaching, thus accepting the null hypothesis. Carbamide peroxide (10%) in tray is recognized for its bleaching effectiveness due to its potential for color change4,5 and its safety.4 The results showed that it had significant effects on the brightness parameters (L*), the reduction of reddish (a*) and yellowish (b*) hues, an average change of 10 shade colors based on the Vita Classical scale, and a clinically perceptible color change by CIEL*a*b*, CIEDE2000, and WID, even with use for 7 days, which was less than what is usually employed clinically (about 14 days of use1, depending on the initial color of the teeth (longer protocol for darker teeth, and shorter for lighter teeth). The values of ΔEab were > 
+2.7,24,25 and they were considered clinically perceptible. This was also observed for the values of ΔE00 and WID, which were above the limits of perceptibility and acceptability,23,24 denoting the teeth bleaching effectiveness during 7 days of treatment. Some authors reported that the isolated use of ozone did not promote effective bleaching6,13,27; however, this study showed that ozone promoted tooth bleaching with the same effective-
+
+| Table 3: Color parameters, Vita Classical scale and Whiteness Index for Dentistry depending on the treatments or  groups and the evaluation times Variable Before treatment 24 h after application P-value L* P(group)=0.4113; P(time)=0.0075;  P(group × time)=0.7529 10% carbamide peroxide 79.15±4.37 84.77±6.46* Ozone 78.04±4.86 81.15±6.67* 10% ozonized carbamide peroxide 77.67±6.92 81.82±6.15* a* P(group)=0.4892; P(time)=0.0184;  P(group × time)=0.3499 10% carbamide peroxide 1.52±1.38 –0.69±0.75* Ozone 1.91±1.30 0.20±0.49* 10% ozonized carbamide peroxide 1.33±1.33 –0.24±0.83* b* P(group)=0.5875; P(time) < 0,0001;  P(group × time)=0.5732 10% carbamide peroxide 31.52±4.71 12.34±4.93* Ozone 31.55±4.84 11.02±4.05* 10% ozonized carbamide peroxide 31.47±3.56 13.10±4.63* Vita Classical scale P(group)=0.5290; P(time) < 0.0001;  P(group × time)=0.2749 10% carbamide peroxide 11.90±1.20 1.80±0.63* Ozone 12.50±1.78 1.60±0.52* 10% ozonized carbamide peroxide 12.10±1.60 2.10±1.10* Whiteness index for dentistry P(time)=0.0051 10% carbamide peroxide 3.13 (–10.71–12.79) 29.00 (24.61–42.61)* Ozone 0.44 (–9.02 to 13.52) 27.43 (23.23–41.89)* 10% ozonized carbamide peroxide 3.09 (–7.34 to 11.06) 27.82 (20.21–36.19)* Note: Data in color parameters and Vita Classical scale are expressed as mean ± SD, and data in whiteness index for dentistry are expressed mean (minimum-maximum  values) (n =10). Generalized linear models applied for L*, a*, b* and Vita Classical Scale analysis, and Kruskal-Wallis and Wilcoxon tests for the Whiteness index for  dentistry analysis. *P < 0.05, vs. before treatment.   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+www.medgasres.com
+
+| Table 4: Variation in color based on the parameters L*,  a*, and b* by CIELab, CIEDE 2000, and WID systems  according to different treatments or groups Variable Value P-value ΔL P=0.7472 10% carbamide peroxide 5.62±5.62 Ozone 3.11±9.51 10% ozonized carbamide peroxide 4.15±8.42 Δa P=0.6099 10% carbamide peroxide –2.21±1.39 Ozone –1.71±1.58 10% ozonized carbamide peroxide –1.57±1.25 Δb P=0.5226 10% carbamide peroxide –19.18±5.53 Ozone –20.53±6.81 10% ozonized carbamide peroxide –18.37±5.03 ΔEab P=0.7168 10% carbamide peroxide 20.93±5.15 Ozone 22.66±7.16 10% ozonized carbamide peroxide 20.68±4.43 ΔE00 P=0.6439 10% carbamide peroxide 11.00±2.76 Ozone 12.35±4.17 10% ozonized carbamide peroxide 11.39±2.60 ΔWID P=0.6960 10% carbamide peroxide 29.10±7.42 Ozone 28.15±11.13 10% ozonized carbamide peroxide 25.98±5.60 Note: Data are expressed as mean ± SD (n = 10). Kruskal-Wallis tests applied for  ΔL, Δa, and Δb analyses, and ggeneralized linear models applied for ΔEab, ΔE00,  and ΔWID analyses.   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+ness as 10% carbamide peroxide bleaching treatment in tray.
+
+The differences in results reported by different studies can be justified by factors that influence the oxidative effect of ozone. Factors such as reduced ozone application time - from only 40 seconds,13 4 minutes in the group where only ozone was applied, and 80 seconds repeated three times in the group where ozone and a bleaching gel were used6 - can justify such results. Another factor that may contribute to the ineffectiveness of ozone in bleaching with the use of trays is the design of the trays used. Adjusting the trays to the dental structures (as a "well-fitted" tray) restricts the free flow of ozone, preventing the direct contact of ozone with the vestibular surface of the anterior teeth.27 Thus, there is the need to ensure internal relief for effective ozone circulation inside the trays, as performed in this study.
+
+In addition to these factors, the ozone concentration of 140 ppm used in the Ozotop unit in the study by Zanjani et al.6 can be cited; the lack of suction capacity limited the use of a higher concentration of ozone. It was also found that the isolated application of ozone was effective in bleaching tetracyclinestained mouse teeth by using it for 3 and 5 minutes.16 Ozone is produced naturally from oxygen via molecular photodissociation in activated oxygen atoms, which react with other oxygen molecules. This radical transitional anion quickly becomes protonated and generates hydrogen trioxide, which, in turn, decomposes into an even more powerful oxidizer, the hydroxyl radical, giving ozone gas a higher oxidizing potential.28 This effect may justify the minimal reduction in the pH of ozonized carbamide peroxide after 60 minutes (from 7.43 to 7.21). In addition, chromophore groups present in the tooth structure can be broken down by ozone to form smaller molecules, resulting in tooth bleaching via one of the following three mechanisms: a) bonding mechanism, which occurs by the double bonding of ozone to form ozonide in the presence of non-aqueous solvents; however, if water is present, ozone is hydrolyzed to other products by cleaving the double bond that would have occurred; (b) a replacement mechanism, where an atom or functional group is replaced by another; (c) a cleavage mechanism, in which a carbon-carbon link is separated to produce organic composite fragments.11,20 Aqueous ozone facilitates a high level of biocompatibility in oral epithelial human cells, gingival fibroblast cells, and periodontal cells,11,20 while gaseous ozone makes the inhalation toxic to the lungs and other organs28; thus, a suitable system for application of ozone as a bleaching agent is required.20 Administration in gaseous form requires a sealing and suction system to avoid inhalation and adverse effects,29 like the trays that were developed and adapted for this study. In this respect, it was ensured that ozone remained in constant flux on the enamel structure with direct application, which may also have contributed to the significant results of color change. In contrast with other studies that used other types of systems, such as machines, in which the lack of suction capacity limited the use of a higher ozone concentration,6 or the use of a rectangular box with side openings for input and output7 and trays with an unsuitable design that did not allow the continuous circulation of ozone over the teeth.27 Because of the significant difference between the proposed treatments, the use of ozone appears to have advantages for shorter application durations to the dental structure and the absence of cytotoxic effects on the pulp over the use of agents containing peroxide.10 This type of tray could have led to dehydration of the teeth during the application of ozone (which enables a "false bleaching" at the end of the procedure), but the evaluation was performed after damping the teeth in artificial saliva for 24 hours. Clinically, dehydration during in-office bleaching may also be related to sensitivity,30 but some clinical studies report that the application of ozone or its combination with bleaching agents can minimize this effect.9,31 The combination of ozone and 10% carbamide peroxide gel applied in the in-office treatment has proven effective in teeth bleaching, although not better than the results of the other evaluated bleaching treatments. Only ozone may have been effective for bleaching since it was applied using the tray, and it remained in contact with the occlusal and lingual surfaces of the teeth, where the effect of carbamide peroxide applied to the vestibular faces was minor or absent. Some studies have shown that the use of low concentrations of hydrogen peroxide 
+(between 6% and 20%)32,33 or carbamide (between 15% and 40%)34 for in-office use is unsatisfactory given the borderline or minimal color change, unless they were associated with the use of light.35 On the other hand, the use of low peroxide concentrations has been a safety recommendation for bleaching procedures1; and control agencies have recommended gels in lower concentrations.36 Ozone appeared to satisfactorily facilitate tooth bleaching when combined with 10% carbamide peroxide for in-office application; clinically perceptible values in the ΔEab, ΔE00, and WID parameters were obtained.
+
+Other studies that combined ozone with bleaching agents did not show the enhancement of bleaching compared with ozonefree bleaching treatments,13 not even after employing different ozone application times (4 or 19 minutes, respectively) in combination with 35%10 or 37.5%11 hydrogen peroxide. Al-Omiri et al.8 suggested that ozone enhanced the bleaching response after application for 60 seconds immediately after the application of 38% hydrogen peroxide, in addition to keeping this mix for another 20 minutes on the teeth. In another study, Al-Omiri et al.9 described this potentiation in their study that applied 38% 
+hydrogen for 20 minutes and ozone for 60 seconds.
+
+This study suggests that ozone may have potentialized the 10% carbamide peroxide bleaching because it was applied for a shorter duration (total 3 hours of application) than in case of the conventionally applied carbamide peroxide for the 8 hours every day group (which totaled 56 hours of application). However, the benefits and costs of applying each treatment should be considered.
+
+According to the pH value analysis, neutrality was maintained within 60 minutes; however, there was a slight decrease in values, which was possibly associated with the free radical release. Although the use of ozone alone or in combination with low concentration carbamide peroxide for in-office dental bleaching following the adopted protocols facilitated favorable changes in color, they must be evaluated in clinical trials to not only prove their effectiveness but also their safety of use.28 It is important to consider the potential for soft tissue irritation, sensitivity of teeth, and acceptance of treatment by patients.
+
+It can be concluded that ozone and 10% ozonized carbamide peroxide for in-office use could promote tooth bleaching with clinically perceptible and acceptable altered chromaticity.
+
+Author contributions Study conception and design: RTB; data acquisition: TRFZFND, FUFC, CPT, FLBA, FMGF; data interpretation: TRFZFND, FUFC, RTB; data analysis: CPT, FLBA; manuscript writing: TRFZFND, FUFC, CPT, RTB; manuscript revision: CPT, FLBA, FMGF. All authors approved the final version of the manuscript. Conflicts of interest The authors have no financial, economic, commercial, and/or professional interests related to the topics presented in this manuscript.
+
+Open access statement This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. ©Article authors (unless otherwise stated in the text of the article) 2022. All rights reserved. No commercial use is permitted unless otherwise expressly granted. Additional file Additional file 1: Informed consent form (template).
+
+## References
+
+![5_Image_0.Png](5_Image_0.Png)
+
+1. de Geus JL, Wambier LM, Kossatz S, Loguercio AD, Reis A. Athome vs in-office bleaching: a systematic review and meta-analysis. *Oper Dent*. 2016;41:341-356.
+
+2. American Dental Association. Dentist dispensed home-use teeth bleaching products [Internet]. http://www.ada.org/ada/seal/standards/guide_home. Accessed by December 9, 2020.
+
+3. Committee for Scientific Consumer Products. Public consultation on a preliminary opinion on hydrogen peroxide in tooth whiten-ing products. https://ec.europa.eu/health/scientific_committees/
+consultations/public_consultations/sccp_cons_01_en. Accessed by December 9, 2020.
+
+4. Basting RT, Amaral FL, França FM, Flório FM. Clinical comparative study of the effectiveness of and tooth sensitivity to 10% and 20% carbamide peroxide home-use and 35% and 38% hydrogen peroxide in-office bleaching materials containing desensitizing agents. *Oper Dent.* 2012;37:464-473.
+
+5. Almeida AF, Torre Edo N, Selayaran Mdos S, et al. Genotoxic potential of 10% and 16% carbamide peroxide in dental bleaching.
+
+Braz Oral Res. 2015;29:S1806-83242015000100217.
+
+6. Zanjani VA, Ghasemi A, Torabzadeh H, Jamali M, Razmavar S,
+Baghban AA. Bleaching effect of ozone on pigmented teeth. *Dent* Res J (Isfahan). 2015;12:20-24.
+
+7. Santana MS, Bridi EC, Navarro RS, et al. Dental bleaching with ozone: effects on color and enamel microhardness. *Acta Odontol* Latinoam. 2016;29:68-75.
+
+8. Al-Omiri MK, Abul Hassan RS, AlZarea BK, Lynch E. Improved tooth bleaching combining ozone and hydrogen peroxide--A blinded study. *J Dent.* 2016;46:30-35.
+
+9. Al-Omiri MK, Al Nazeh AA, Kielbassa AM, Lynch E. Randomized controlled clinical trial on bleaching sensitivity and whitening efficacy of hydrogen peroxide versus combinations of hydrogen peroxide and ozone. *Sci Rep.* 2018;8:2407.
+
+10. Al-Omiri MK, Lamfon HA, Al Nazeh AA, Kielbassa AM, Lynch E. Randomized clinical trial on the comparison of bleaching outcomes using either ozone or hydrogen peroxide. *Quintessence Int.*
+2018;49:625-634.
+
+11. Saini R. Ozone therapy in dentistry: A strategic review. *J Nat Sci* Biol Med. 2011;2:151-153.
+
+12. Tahmassebi JF, Chrysafi N, Duggal MS. The effect of ozone on progression or regression of artificial caries-like enamel lesions in vitro. *J Dent.* 2014;42:167-174.
+
+13. Manton DJ, Bhide R, Hopcraft MS, Reynolds EC. Effect of ozone and Tooth Mousse on the efficacy of peroxide bleaching. *Aust Dent* J. 2008;53:128-132.
+
+14. Públio JD, D'Arce MB, Catelan A, et al. Influence of enamel thick-ness on bleaching efficacy: an in-depth color analysis. Open Dent J. 2016;10:438-445.
+
+15. do Carmo Públio J, Zeczkowski M, Burga-Sánchez J, et al. Influence of different thickeners in at-home tooth bleaching: a randomized clinical trial study. *Clin Oral Investig.* 2019;23:2187-2198.
+
+16. Tessier J, Rodriguez PN, Lifshitz F, Friedman SM, Lanata EJ. The use of ozone to lighten teeth. An experimental study. Acta Odontol Latinoam. 2010;23:84-89.
+
+17. Featherstone JDB, O'Really MM, Shariati M, Brugler S. Enhancement of remineralization in vitro and in vivo. In: Leach AS, ed.
+
+Factors relating to demineralization and remineralization of the teeth. Oxford: IRL; 1986.
+
+18. Serra MC, Cury JA. The in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model. *Quintessence Int.* 1992;23:143-147.
+
+![6_image_0.png](6_image_0.png)
+
+19. Carlos NR, Bridi EC, Amaral F, França F, Turssi CP, Basting RT.
+
+Efficacy of home-use bleaching agents delivered in customized or prefilled disposable trays: a randomized clinical trial. *Oper Dent.* 2017;42:30-40.
+
+20. Nogales CG, Ferrari PH, Kantorovich EO, Lage-Marques JL.
+
+Ozone therapy in medicine and dentistry. *J Contemp Dent Pract.* 2008;9:75-84.
+
+21. Commission Internationale del Éclairage. Commission Internationale del Éclairage. Vienna, Austria: CIE Central Bureau. 2004.
+
+22. Sharma G, Wu W, Dalal EN. The CIEDE2000 color-difference formula: Implementation notes, supplementary test data, and mathematical observations. *Color Res Appl*. 2005;30:21-30.
+
+23. Pérez Mdel M, Ghinea R, Rivas MJ, et al. Development of a customized whiteness index for dentistry based on CIELAB color space. *Dent Mater.* 2016;32:461-467.
+
+24. Paravina RD, Ghinea R, Herrera LJ, et al. Color difference thresholds in dentistry. *J Esthet Restor Dent.* 2015;27 Suppl 1:S1-9.
+
+25. International Organization for Standardization. ISO/TR 28642:
+2016. Technical Report (E): Dentistry - Guidance on colour measurements.
+
+26. Pérez MM, Herrera LJ, Carrillo F, et al. Whiteness difference thresholds in dentistry. *Dent Mater.* 2019;35:292-297.
+
+27. Aykut-Yetkiner A, Ertuğrul F, Eden E, Aladağ A, Ergin E, Özcan M. Color assessment after bleaching with hydrogen peroxide versus ozone: a randomized controlled clinical trial. *Gen Dent.* 2017;65:e12-e17.
+
+28. Naik SV, K R, Kohli S, Zohabhasan S, Bhatia S. Ozone- a biological therapy in dentistry- reality or myth????? *Open Dent J.*
+2016;10:196-206.
+
+29. Loncar B, Mravak Stipetic M, Matosevic D, Tarle Z. Ozone application in dentistry. *Arch Med Res.* 2009;40:136-137.
+
+30. Mounika A, Mandava J, Roopesh B, Karri G. Clinical evaluation of color change and tooth sensitivity with in-office and home bleaching treatments. *Indian J Dent Res.* 2018;29:423-427.
+
+31. Suh Y, Patel S, Kaitlyn R, et al. Clinical utility of ozone therapy in dental and oral medicine. *Med Gas Res*. 2019;9:163-167.
+
+32. Torres CR, Souza CS, Borges AB, Huhtala MF, Caneppele TM.
+
+Influence of concentration and activation on hydrogen peroxide diffusion through dental tissues in vitro. ScientificWorldJournal. 2013;2013:193241.
+
+33. Lilaj B, Dauti R, Agis H, et al. Comparison of bleaching products with up to 6% and with more than 6% hydrogen peroxide: whiten-ing efficacy using BI and WI (D) and side effects - an in vitro study.
+
+Front Physiol. 2019;10:919.
+
+34. Perdigão J, Baratieri LN, Arcari GM. Contemporary trends and techniques in tooth whitening: a review. *Pract Proced Aesthet* Dent. 2004;16:185-192; quiz 194.
+
+35. Loiola AB, Souza-Gabriel AE, Scatolin RS, Corona SA. Impact of hydrogen peroxide activated by lighting-emitting diode/laser system on enamel color and microhardness: an in situ design. *Contemp Clin Dent.* 2016;7:312-316.
+
+36. American Dental Association Council on Scientific Affairs. Tooth whitening/bleaching: Treatment considerations for dentists and their patients; September 2009, Revised November 2010 [cited 2019 June 30]. Available at: "https://www.ada.org/~/ media/ADA/
+About%20the%20ADA/Files/ada_house_of_ delegates_whiten-ing_report.ashx effectiveness". Accessed by December 9, 2020.
+
+Date of submission: December 6, 2020 Date of decision: January 5, 2021 Date of acceptance: February 7, 2021 Date of web publication: *November 29,* 2021

medical/md/PMC8820049.md

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+Contents lists available at ScienceDirect
+
+![0_image_0.png](0_image_0.png)
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+![0_image_2.png](0_image_2.png)
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+![0_image_5.png](0_image_5.png)
+
+North American Spine Society Journal (NASSJ)
+
+![0_image_3.png](0_image_3.png)
+
+![0_image_4.png](0_image_4.png)
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+![0_image_6.png](0_image_6.png)
+
+journal homepage: www.elsevier.com/locate/xnsj
+
+![0_image_7.png](0_image_7.png)
+
+Clinical Studies
+
+![0_image_1.png](0_image_1.png)
+
+Spine coding transition from ICD-9 to ICD-10: Not taking advantage of the specificity of a more granular system✩
+Matthew J. Sabatino∗, Patrick J. Burroughs, Harold G. Moore, Jonathan N. Grauer Department of Orthopaedics and Rehabilitation, Yale School of Medicine, 47 College Street, New Haven, CT 06510, United *States*
+
+| a r t i c l e                                                                                                                                                                                     | i n f o                                                   | a b s t r a c t   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|-------------------|
+| Keywords: Spine Clinical coding International classification of diseases Codes ICD ICD-9-CM ICD-10-CM Claim reporting insurance Health care costs Data collection Data interpretation statistical | care and research possibilities. Kolmogorov-Smirnov test. |                   |
+
+Background: The transition from International Classification of Diseases, 9th Edition (ICD-9) to the 10th edition (ICD-10) in 2015 increased the number and specificity of diagnostic codes with the goal of facilitating clinical care and research possibilities.
+
+Considering the potential to default to less specified ICD-10 codes, the current study evaluated the number of codes utilized for spine-related conditions before versus after the transition to ICD-10.
+
+Methods: The numbers of patients with an index encounter for a primary spine-related non-deformity diagnosis codes indexed as "dorsopathies" were abstracted from the Humana PearlDiver dataset. As the transition from ICD-9 to ICD-10 occurred in 2015, the current study compared the year prior (ICD-9) to the year after (ICD-10). The number of ICD-9 and ICD-10 codes was assessed, and distribution of utilization was compared using the Kolmogorov-Smirnov test. Results:: In 2014, 848,623 patients were assigned one of the 100 unique ICD-9 dorsopathy codes, of which 17 codes (17% of available codes) were used for more than 1% of the patients. In 2016, 840,310 patients were assigned one of the 504 unique ICD-10 dorsopathy codes, of which 21 (4% of available codes) were used for more than 1% of the patients. The top 20 codes in 2014 (ICD-9) and the top 20 codes in 2016 (ICD-10) both represented the majority of the patient population and were not statistically differently represented (p = 0.819). Further, analysis of ICD-10 codes demonstrated a clear bias toward utilizing less specified codes.
+
+Conclusions: Despite a five-fold increase in available diagnostic codes for spine conditions in ICD-10, in the year after implementation providers continued to select a small proportion of less specific diagnostic codes when treating spine patients.
+
+## Introduction
+
+In 2015, the Centers for Medicare & Medicaid Services (CMS) of the United States Department of Health and Human Services implemented the International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) as the official diagnostic tool for health care.1,2 This marked a conversion from the previous ICD-9-CM, which had served as the primary diagnostic system since 1979.1 The conversion from ICD-9-CM to ICD-10-CM involved increasing the number and complexity of diagnostic codes to provide health caregivers with a greater degree of detail. While the ICD-9-CM codebook had approximately 14,000 distinct diagnostic codes, ICD-10-CM has more than 69,000 diagnostic codes.1 The Center for Disease Control and Prevention (CDC) highlighted the benefits of more enhanced data collection with the transition to ICD-10-CM. These improvements included more specific data for tracking public health conditions, conducting epidemiological research, as well as the potential for enhancing clinical decision-making and payment/reimbursement systems.1,3 Although the CDC stated the intended advantages of the new classification system, only a few authors have published results assessing the improvements in data specificity and diagnostic coding since the conversion.4-7 In 2017, researchers using Veterans Affairs data analyzed the impact the conversion to ICD-10-CM had on the recording of the overall epidemiology of chronic conditions and mortality statistics. 8 Additionally, one group has evaluated the change in reimbursements and insurance denials associated with the conversion in an ophthalmology practice.5 Both of these studies highlighted a tendency for coding to capture similar prevalence of conditions diagnosed; however, the ophthalmology study found a trend in which providers used a higher frequency of less
+✩ Given his role as Editor in Chief, Jonathan Grauer, MD had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Tobias Mattei, MD.
+
+DOI of original article: 10.1016/j.xnsj.2020.100032 ∗ Corresponding author.
+
+E-mail *address:* Matthew.Sabatino@yale.edu (M.J. Sabatino).
+
+https://doi.org/10.1016/j.xnsj.2020.100035 Received 23 July 2020; Received in revised form 24 October 2020; Accepted 24 October 2020 Available online 31 October 2020 2666-5484/Published by Elsevier Ltd on behalf of North American Spine Society. This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/)
+
+| Table 1 Comparison of ICD-9 and ICD-10 diagnoses in study cohort. ICD-9 codes ICD-10 codes Categories 720.XX - 721.XX M45.XXX—M49.XXX Spondylopathies 722.X1 - 723.XX M50.XXX Cervical Disc Disorders 722.X2, 722.X3, M51.XXX Thoracic, Lumbar, Sacral Disc Disorders 724.X2, 722.X3 722.X0 - 724.X0 M53.XXX Other and Unspecified Dorsopathies 724.XX M54.XXX Dorsalgia Cross-mapping of spinal conditions that comprise the category "Dorsopathies"; note that deformity codes are excluded from ICD-9 or ICD-10 groups. ICD - International Classification of Diseases.   |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+specific or unspecified codes upon conversion to the ICD-10 coding system.
+
+Spine-related diagnosis codes are typically captured under a general heading of "dorsopathies". The diagnostic category of "dorsopathies" comprises all manner of pathologies originating from the back or neck. The category includes spondylopathies, deformities, disc disorders, radiculopathies as well as back and neck pain. The usage of different ICD-9 versus ICD-10 dorsopathy codes has not previously been reported in the literature.
+
+The current study was thus performed to assess the hypothesis that, despite ICD-10 offering more diagnostic coding options/granularity for dorsopathy conditions than ICD-9, providers would continue to use a limited subset of available codes and would tend to default to less specific codes. The large national, administrative Humana dataset available from PearlDiver was used for these analyses.
+
+## Materials And Methods Data Source
+
+Data were extracted from the Humana patient subset from PearlDiver Patient Records Database (PearlDiver Inc., Colorado Springs, CO, USA),
+which captured diagnostic and reimbursement data from 5,497,415 patients in the years 2014 and 2016 cumulatively. This a national subscription database made available by PearlDiver Inc. for the purpose of academic orthopaedic research. The database is stored on a passwordprotected server maintained by PearlDiver Inc.
+
+The database is searchable using ICD-9 or ICD-10 diagnoses. Our institutional Investigative Review Board has given exemption to studies utilizing this database.
+
+## Study Cohorts
+
+Using the ICD-9-CM diagnostic tables, codes were identified under the category "dorsopathies" containing spine (back or neck) as the primary origin or site of diagnosis. This encompassed every ICD-9 code from 720.XX to 724.XX (Table 1).
+
+Corresponding ICD-10 codes were identified using the publicly available general equivalency mapping (GEM) files provided by CMS.9 This encompassed codes M45.XXX - M54.XXX (Table 1).
+
+All deformity codes were excluded from the current study cohort
+(ICD-9: 737.XX and ICD-10: M40.XXX - M43.XXX). Because the spine deformity coding has evolved to reflect additional stratifications based on age of onset and underlying disease state, the decision was made to exclude them from this analysis.
+
+Using the PearlDiver Research Program, assessment was performed to identify patients who had one of the spine dorsopathy codes as the primary diagnosis in a clinical encounter in the respective years. Each diagnostic code analyzed was the primary diagnosis for an individual clinical encounter recorded in the insurance database. If a patient had multiple encounters during the assessed year, the earliest visit for the respective year was used as the encounter of record.
+
+The study cohorts were also analyzed based on service encounter location. For service encounter location, four distinct locations were assessed as designated in PearlDiver - 1) Hospital & Surgical center, 2)
+Outpatient Clinic, 3) Skilled Nursing or Rehab Facility, 4) Emergency Room or Urgent Care. Analysis to determine the percentage of patients seen in each type of encounter location was performed to compare between years 2014 and 2016.
+
+In 2014, the number of patients with the defined ICD-9 codes was assessed. In 2016, the number of patients with the ICD-10 codes was assessed. Demographic data of the two cohorts are listed in Table 2.
+
+## Subset Analysis
+
+A subset analysis was completed to illustrate the use of ICD-10 specificity. From the overall category of "dorsopathy" (M45-M54, 504 codes),
+the commonly used subset diagnoses that fall under "dorsalgia" (M54, 38 codes) were selected for in-depth analysis.
+
+This subset analysis is to see whether the providers used specified codes when available. Diagnostic codes that were "unspecified" (denoting an unspecific entity when more specific codes are available) were compared to "specified" codes (denoting a specific anatomic region or sidedness). For example, the diagnostic code of M54.1 is "radiculopathy, site unspecified", and the diagnostic code of M54.16 is "radiculopathy, lumbar region." In this case, "radiculopathy, site unspecified" would be considered unspecified because there are more specific classifications available that clarify location, whereas "radiculopathy, lumbar region" would be considered specified because it captures the most specific description of radiculopathy diagnosis available. Within the category of
+"dorsalgia" there are 10 unspecified codes and 28 specified codes.
+
+## Statistical Analysis
+
+The utilization of ICD-9 and ICD-10 codes was determined. First, the overall number of patients with primary spine diagnoses was tallied. The number of diagnostic codes that were used for more than 1% of the total cases was then determined for the respective years.
+
+Comparisons between the datasets to determine differences in distribution patterns were done using the two-sample Kolmogorov–Smirnov
+(K–S) test. This is a test that determines whether there is a statistically significant difference in distribution between the two samples. In comparing the two samples, a K–S Test p-value of < 0.05 would indicate a statistically significant difference in distribution between the samples, where a K–S Test p-value of > 0.05 would indicate no statistically significant difference.
+
+Comparisons between the 2014 and 2016 cohorts with regards to encounter location were done using a Chi-squared test.
+
+All summary statistics, distributions and calculations were performed using R, version 2.12.2 (R Development Core Team, Vienna, Austria).
+
+| Table 2 Cohort demographics. Category 2014 ICD-9 Dorsopathy Cohort                                                                                         | 2016 ICD-10 Dorsopathy Cohort   |                  |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------|------------------|
+| Total patients                                                                                                                                             | 848,623                         | 840,310          |
+| % Female                                                                                                                                                   | 59.50% (504,932)                | 59.94% (503,640) |
+| Age Under 45 years                                                                                                                                         | 11.58% (98,271)                 | 9.68% (81,342)   |
+| 45–64 years                                                                                                                                                | 29.75% (252,465)                | 30.48% (256,126) |
+| 65+ years                                                                                                                                                  | 58.67% (497,887)                | 59.84% (502,842) |
+| Race White                                                                                                                                                 | 56.50% (479,472)                | 61.84% (519,648) |
+| Non-White                                                                                                                                                  | 11.72% (99,459)                 | 14.45% (121,425) |
+| Unknown                                                                                                                                                    | 31.78% (269,692)                | 23.71% (199,237) |
+| Overview of cohort characteristics as reported in PearlDiver Patient Record Database reported as% (count). ICD - International Classification of Diseases. |                                 |                  |
+
+![2_image_0.png](2_image_0.png)
+
+## Results Study Cohorts
+
+With ICD-9, there were 100 unique codes for "dorsopathy" (720.XX–
+724.XX). Within the 2014 Humana dataset, 848,623 patients had one of these codes as their primary diagnosis. Of the 100 unique ICD-9 codes, 17 codes (17% of available codes) were each utilized for more than 1%
+of the primary diagnoses (Fig. 1).
+
+With ICD-10, there were 504 unique codes for "dorsopathy"
+(M45.XXX–M54.XXX). Within the 2016 Humana dataset, 840,310 patients had one of these codes as their primary diagnosis. Of the 504 unique ICD-10 codes, 21 codes (4% of available codes) were each utilized for more than 1% of primary diagnoses (Fig. 1).
+
+## Code Distributions
+
+For both ICD-9 and ICD-10 codes the majority of patients were assigned a small percentage of the available codes (Fig. 2). It can be noted in the distributions that the overwhelming majority of diagnoses were captured by a small number of codes.
+
+To highlight the more commonly used codes, the 20 most-utilized codes from ICD-9 were compared to the 20 most-utilized codes from ICD-10 (Fig. 3). Use of the two- sample Kolmogorov-Smirnov test demonstrated that the distributions of these codes were not statistically different (K-S Test p-value = 0.819).
+
+Table 3 lists the 20 most-utilized codes from ICD-9 and ICD-10, respectively, with their percentage of use in the overall cohort.
+
+The service encounter comparison from 2014 to 2016 demonstrated a similar proportion of visits to each of the designated location types.
+
+Although the Chi-squared test produced a statistically significant difference in each of the four location types from 2014 to 2016, this was likely due to a small difference in a large dataset comparison, and did not represent a meaningful difference between groups (Fig. 4).
+
+## Subset Analysis
+
+The most frequent ICD-10 coding prefix (M54.XXX - Dorsalgia) has 38 codes, of which there are 10 unspecified codes and 28 specified codes. The unspecified codes were 26.3% of the dorsalgia codes but were used for 71.8% of the dorsalgia encounters. The specified codes were 73.7%
+of the dorsalgia codes but were 28.2% of the dorsalgia encounters.
+
+## Discussion
+
+The transition from ICD-9 to ICD-10 gave providers more coding options to facilitate clinical care and research. Specifically, the category
+
+| Top 20 frequently used diagnostic dorsopathy codes, ICD-9 (2014) vs. ICD-10 (2016). Rank ICD-9 Diagnoses (2014) % of Total ICD-10 Diagnoses (2016)   | % of Total                                            |                          |                                                        |        |
+|------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|--------------------------|--------------------------------------------------------|--------|
+| All Dorsopathy (720–724)                                                                                                                             | 100%                                                  | All Dorsopathy (M45-M54) | 100%                                                   |        |
+| 1                                                                                                                                                    | Lumbago (724.2)                                       | 20.35%                   | Low back pain (M54.5)                                  | 19.58% |
+| 2                                                                                                                                                    | Backache, unspecified (724.5)                         | 9.60%                    | Cervicalgia (M54.2)                                    | 8.50%  |
+| 3                                                                                                                                                    | Cervicalgia (723.1)                                   | 9.00%                    | Radiculopathy, lumbar region (M54.16)                  | 5.70%  |
+| 4                                                                                                                                                    | Degeneration of lumbar or lumbosacral disc (722.52)   | 7.74%                    | Other disc degeneration, lumbar region (M51.36)        | 5.56%  |
+| 5                                                                                                                                                    | Thoracic or lumbosacral radiculitis, unspecified      | 7.13%                    | Dorsalgia, unspecified (M54.9)                         | 5.43%  |
+| (724.4)                                                                                                                                              |                                                       |                          |                                                        |        |
+| 6                                                                                                                                                    | Lumbosacral spondylosis without myelopathy (721.3)    | 6.94%                    | Spinal stenosis, lumbar region (M48.06)                | 4.53%  |
+| 7                                                                                                                                                    | Displacement of lumbar disc without myelopathy        | 4.52%                    | Spondylosis w/out myelopathy, radiculopathy, lumbar    | 4.24%  |
+| (722.10)                                                                                                                                             | (M47.816)                                             |                          |                                                        |        |
+| 8                                                                                                                                                    | Spinal stenosis, lumbar, w/out neurogen. claudication | 4.21%                    | Other disc displacement, lumbar region (M51.26)        | 2.77%  |
+| (724.02)                                                                                                                                             |                                                       |                          |                                                        |        |
+| 9                                                                                                                                                    | Cervical spondylosis without myelopathy (721.0)       | 3.41%                    | Spondylosis w/out myelopathy, radiculopathy, cervical  | 2.76%  |
+| (M47.812)                                                                                                                                            |                                                       |                          |                                                        |        |
+| 10                                                                                                                                                   | Degeneration of cervical intervertebral disc (722.4)  | 3.00%                    | Radiculopathy, cervical region (M54.12)                | 2.46%  |
+| 11                                                                                                                                                   | Sciatica (724.3)                                      | 2.99%                    | Pain in thoracic spine (M54.6)                         | 2.17%  |
+| 12                                                                                                                                                   | Brachial neuritis/radiculitis, unspecified (723.4)    | 2.68%                    | Spondylosis w/out myelopathy, radiculopathy,           | 2.03%  |
+| lumbosacral (M47.817)                                                                                                                                |                                                       |                          |                                                        |        |
+| 13                                                                                                                                                   | Pain in thoracic spine (724.1)                        | 2.13%                    | Radiculopathy, lumbosacral (M54.17)                    | 1.55%  |
+| 14                                                                                                                                                   | Displacement of cervical disc w/out myelopathy        | 1.54%                    | Intervertebral disc disorders w/radiculopathy, lumbar  | 1.50%  |
+| (722.0)                                                                                                                                              | (M51.16)                                              |                          |                                                        |        |
+| 15                                                                                                                                                   | Spinal stenosis in cervical region (723.0)            | 1.31%                    | Spinal stenosis, cervical region (M48.02)              | 1.48%  |
+| 16                                                                                                                                                   | Sacroiliitis, not elsewhere classified (720.2)        | 1.27%                    | Other disc degeneration, lumbosacral (M51.37)          | 1.38%  |
+| 17                                                                                                                                                   | Postlaminectomy syndrome, lumbar (722.83)             | 1.22%                    | Other cervical disc degeneration, unspecified cervical | 1.35%  |
+| region (M50.30)                                                                                                                                      |                                                       |                          |                                                        |        |
+| 18                                                                                                                                                   | Degeneration of disc, site unspecified (722.6)        | 0.80%                    | Sacroiliitis, not elsewhere classified (M46.1)         | 1.27%  |
+| 19                                                                                                                                                   | Disorders of sacrum (724.6)                           | 0.80%                    | Lumbago with sciatica, right side (M54.41)             | 1.13%  |
+| 20                                                                                                                                                   | Spinal stenosis, unspecified region (724.00)          | 0.71%                    | Lumbago with sciatica, left side (M54.42)              | 1.06%  |
+| ICD - International Classification of Diseases.                                                                                                      |                                                       |                          |                                                        |        |
+
+Fig. 3. ICD-9 vs. ICD-10 Code Utilization (Top 20 Codes).
+
+![3_image_0.png](3_image_0.png) A comparison plot of the top 20, highest volume diagnostic codes of ICD-9 and ICD-10. Analysis of empirical distribution (Kolmogorov–Smirnov Test) is applied to the two samples, a p-value of 0.819 demonstrates that the distributions are not statistically different. This means the overall pattern of common diagnostic code utilization is similar between ICD-9 and ICD-10 despite the availability of hundreds of more specific codes in ICD-10. ICD- International Classification of *Diseases*.
+
+of dorsopathies increased in complexity from 100 possible diagnostic codes in ICD-9 to 504 possible diagnostic codes in ICD-10.
+
+The current study found that the majority of diagnoses for dorsopathy spine conditions were captured by a small percentage of available codes, despite a five-fold increase in coding options with the transition from ICD-9 to ICD-10. In 2014, 17 diagnoses each accounted for utilization of more than 1% of the cumulative ICD-9 coding, while in 2016, 21 diagnoses each accounted for utilization of more than 1% of the cumulative ICD-10 coding.
+
+Fig. 4. ICD-9 vs. ICD-10 diagnostic code comparison by service encounter location.
+
+A graphical representation of the percentage of unique patient visits attributed to each of the four designated service encounter locations provided in PearlDiver between 2014 (ICD-9) and 2016 (ICD-10). While the Chi-squared test showed a statistically significant difference between the two groups in each location, the overall proportions were similar and represent no meaningful difference between groups.
+
+ICD- International Classification of *Diseases*.
+
+Comparison of the top 20 diagnostic code volumes from ICD-9 and ICD-10 demonstrated two distributions that were not statistically significantly different. This showed that providers tended to use the same few codes with similar frequency both in ICD-9 and after the transition to ICD-10.
+
+Not only did providers use a small fraction of available codes to account for the vast majority of diagnoses, they also tended to select nonspecific diagnostic codes despite having more complex coding choices available. This pattern highlighted by the example of the highest frequency category of dorsalgia (M54.XXX) codes, in which the 26.3% unspecified codes were used 71.8% of the time.
+
+The current study is the first to evaluate utilization patterns of spine codes in ICD-10 versus ICD-9. Similar to the currently presented results, in radiology Fleming et al. demonstrated that radiology used a small percentage (less than 3%) of available ICD-10 codes for the overwhelming majority (90%) of radiology claim coding.6 Also, in ophthalmology, Hellman et al. found that in an academic ophthalmology practice, the conversion to ICD-10 was associated with a bias toward unspecified codes.5 Consistent with the study hypothesis, the lack of usage of the full breadth of codes (number of codes and specificity of codes) is clearly a limitation to the objectives of introducing ICD-10. Clinical treatment algorithms cannot take advantages of the granular nature of ICD-10 if the specific codes are not used. Clinical research would have the similar limitations and lack of planned ICD-10 advantages.
+
+There are several possible reasons that the breadth of codes afforded by ICD-10 was not taken advantage of. First and foremost, clinicians could be expected to default to codes that are similar to what they had used in the past. Although new coding options were made available, it is understandable that providers continue to rely on the same habit of coding they have been using for years of practice.
+
+Electronic medical record systems may further facilitate physicians choosing pre-selected diagnoses or templates. Taking the time to change templated notes or poring through available codes may serve as barriers to change in the area of coding specificity. Without a targeted training
+/ incentive, it is hard to imagine a significantly different pattern of code utilization for spine care going forward.
+
+One opportunity to incentivize improvement in spine coding specificity exists with the new initiatives on reimbursements and bundled payments for spine care. Multiple studies outline the challenges in designing a bundled payment for spine surgery. 10-12 Kahn et al. note there is significant variation in spine surgeon preference and technique for the same coded procedure. However, this variation in provider preference may not account for as much difference in resource utilization as patient-level effects (comorbidities, demographics). 10 In another study, Malik et al. highlighted the limitations of a bundled payment system demonstrating a wide range of costs for the same cervical fusion procedure.
+
+In both of these studies, differences in patient diagnoses had a stronger association with cost of care than surgeon technique.11 A bundled payment system that reimbursed based on preoperative diagnostic accuracy would likely result in providers utilizing more specific codes.
+
+A strength of this study is its evaluation of a large private insurer database with millions of patient encounters. This allows for a robust depiction of diagnostic code usage for spine conditions. The samples used from the PearlDiver dataset are equitable in size and scope and therefore offer a valid comparison between ICD-9 in 2014 and ICD-10 in 2016.
+
+There are limitations to the current study. First, the study is based on a single insurer (Humana), which may not be fully representative of coding seen by other insurers. Secondly, the study assessed the most recent data available for the current work, but it is possible that code usage has further evolved over the most recent few years. Additionally, as the study only evaluated primary diagnosis codes for billed encounters, it is possible that further granularity of secondary diagnoses was not appreciated. Nonetheless, given that the primary codes showed little change, it could be postulated similar trends occurred for secondary diagnoses.
+
+## Conclusion
+
+Despite a five-fold increase in available diagnostic codes for spine conditions in ICD-10, immediately after implementation providers continue to select a small proportion of less specific diagnostic codes when caring for spine patients.
+
+The ongoing initiatives in data registries and bundled care systems for spine care demonstrate an association between diagnostic accuracy and outcomes. Providers should take the opportunity to code for spine care in a manner that is accurate and specific in order to benefit from the additional clarity and granularity provided by ICD-10.
+
+## 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.
+
+The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
+
+## Funding Disclosure Statement
+
+No financial support was provided for this study, and there was no study-specific conflict of interest for any of the authors of this paper.
+
+## Supplementary Materials
+
+Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.xnsj.2020.100035.
+
+## References
+
+[1] Centers for Disease Control and Prevention. International classification of diseases, (ICD-10-CM/PCS) transition - background. 2015; https://www.cdc.gov/
+nchs/icd/icd10cm_pcs_background.htm. Accessed August 14, 2019.
+
+[2] Centers for Medicare and Medicaid Services. The ICD-10 transition: an introduction. 2014; https://www.cms.gov/Medicare/Coding/ICD10/Downloads/ ICD10Introduction20140819.pdf. Accessed 9/2/2019, 2019.
+
+[3] Centers for Disease Control and Prevention. The ICD-10 transition and public health surveilance. 2013; https://www.cdc.gov/nchs/icd/data/CDC_ICD-10_
+Transition_FactSheet_12_2013.pdf. Accessed 8/12/2019, 2019.
+
+[4] Boyd AD, Li JJ, Burton MD, et al. The discriminatory cost of ICD-10-CM transition between clinical specialties: metrics, case study, and mitigating tools. J Am Med Inform Assoc 2013;20(4):708–17.
+
+[5] Hellman JB, Lim MC, Leung KY, Blount CM, Yiu G. The impact of conversion to international classification of diseases, 10th revision (ICD-10) on an academic ophthalmology practice. Clin Ophthalmol 2018;12:949–56.
+
+[6] Fleming M, MacFarlane D, Torres WE, Duszak R Jr. Magnitude of impact, overall and on subspecialties, of transitioning in radiology from ICD-9 to ICD-10 codes. J
+Am Coll Radiol 2015;12(11):1155–61.
+
+[7] Li B, Evans D, Faris P, Dean S, Quan H. Risk adjustment performance of Charlson and Elixhauser comorbidities in ICD-9 and ICD-10 administrative databases. BMC
+Health Serv Res 2008;8:12.
+
+[8] Yoon J, Chow A. Comparing chronic condition rates using ICD-9 and ICD-10 in VA
+patients FY2014-2016. BMC Health Serv Res 2017;17(1):572.
+
+[9] Centers for Medicare and Medicaid Services. ICD-10-CM and ICD-10 PCS
+and GEMs archive. 2018; https://www.cms.gov/Medicare/Coding/ICD10/ArchiveICD-10-CM-ICD-10-PCS-GEMs.html. Accessed 8/14/2019, 2019.
+
+[10] Kahn EN, Ellimoottil C, Dupree JM, Park P, Ryan AM. Variation in payments for spine surgery episodes of care: implications for episode-based bundled payment. J
+Neurosurg Spine 2018;29(2):214–19.
+
+[11] Malik AT, Yu E, Kim J, Khan SN. Posterior cervical fusion for fracture vs degenerative cervical spine disease: implications for a bundled payment model. Spine J
+2019;19(9):S20.
+
+[12] Ugiliweneza B, Kong M, Nosova K, et al. Spinal surgery: variations in health care costs and implications for episode-based bundled payments. Spine 2014;39(15):1235–42.

medical/md/PMC8913344.md

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+Contents lists available at ScienceDirect
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_2.png](0_image_2.png)
+
+# Saudi Journal Of Biological Sciences
+
+journal homepage: www.sciencedirect.com Original article
+
+![0_image_1.png](0_image_1.png)
+
+The prevalence and intensity of external parasites in domestic pigeons
+(Columba livia domestica) in Egypt with special reference to the role of deltamethrin as insecticidal agent Heba M. Salem a,⇑, Nahed Yehia b, Saad Al-Otaibi c, Ahmed M. El-Shehawi c, Alia A.M.E. Elrys d, Mohamed T. El-Saadony e, Marwa M. Attia f aDepartment of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt b Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Giza 12618, Egypt cDepartment of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia d School of Life Science, Nanjing Normal University, Nanjing, Jiangsu, PR China eDepartment of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt fDepartment of Parasitology, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt article info
+
+| Article history: Received 23 August 2021 Revised 4 October 2021 Accepted 15 October 2021 Available online 22 October 2021   |
+|-----------------------------------------------------------------------------------------------------------------------------|
+
+Keywords:
+Dermanyssus gallinae Deltamethrin, Hippobosca equine Menopon gallinae Knemidocoptes spp., Pseudolynchia canariensis
+
+## Abstract
+
+This study aimed to investigate the prevalence and intensity of external parasites in domestic pigeons in Giza, Egypt, from January 2020 to December 2020. A total of 300 domestic pigeons (25 pigeons per month) were examined. The birds were divided into groups based on their age. The oxidative stress parameters; serum zinc concentration, serum malondialdehyde (MDA), and serum Nitric oxide were evaluated in single and mixed external parasitic infestations. The prevalence of external parasites in examined pigeons was 80.3%. The detected parasites were Pseudolynchia canariensis (P. canariensis), Hippobosca equina (H. equina), Columbicola columbae (C. columbae), Menopon gallinae (M. gallinae), Knemidocoptes species (spp.) and Dermanyssus gallinae (D. gallinae); their incidences were 41.6, 26, 7, 5,0.33 and 0.33%, respectively. The highest infestation was recorded in both spring and summer. The incidence of disease was higher in squabs and young birds than in adults. The mixed external parasitic infestation was recorded in this study. The infected birds showed decreased serum zinc concentration and elevated MDA and serum Nitric oxide levels. In conclusion, regular monthly treatment with deltamethrin is recommended as an effective drug in the treatment of the infested birds and succeeded in reducing the incidence of externalparasites in the treated birds; in addition, pigeon management measures must be implemented to reduce the risk of external parasites.
+
+ 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+Abbreviations: MDA, serum malondialdehyde; P. canariensis, Pseudolynchia canariensis; H. equine, Hippobosca equine; C. columbae, Columbicola columbae; M.
+
+gallinae, Menopon gallinae; D. gallinae, Dermanyssus gallinae; spp., Species.
+
+⇑ Corresponding author.
+
+E-mail addresses: dr.hebasalem@cu.edu.eg (H.M. Salem), elshehawi@hotmail.
+
+com (A.M. El-Shehawi).
+
+Peer review under responsibility of King Saud University.
+
+| Production and hosting by Elsevier   |
+|--------------------------------------|
+
+## 1. Introduction
+
+Pigeons coexist with different animal and poultry species as well as humans all over the world (Alkharigy et al., 2018). In Egypt, domestic pigeons are a valuable source of essential protein for people as they gain more weight quickly than other birds, its meat is palatable, easy to breed, and require slight management (ElDakhly et al., 2016). Pigeons can be infected with a wide range of pathogens and serve as a reservoir for parasitic diseases. The proximity of pigeons to other domestic birds heightens the risk of parasitic infestation in poultry (Alkharigy et al., 2018). Pigeons harbor both external and/or internal parasites (Attia and Salem 2021).
+
+Poultry ectoparasites are abundant in the tropics, where poor husbandry practices and favorable climatic circumstances allow https://doi.org/10.1016/j.sjbs.2021.10.042
+
+![0_image_3.png](0_image_3.png)
+
+1319-562X/ 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
+
+This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+them to thrive (Imura et al., 2012; Badparva et al., 2015; Salem and Attia 2021). These parasites induce several direct and indirect effects on the pigeon, including weight loss, irritability, decreased productivity, malnutrition, low growth, decreased egg production and the development of a variety of clinical symptoms as; ruffled broken feathers, feathers loss, itching, pruritis, dermatitis, emaciation, inconvenience, death especially in squabs as well as indirect harm by transmitting other diseases (Radfar et al., 2012, Attia and Salem, 2021). The most common external parasites infesting pigeons are P. canariensis, Argas persicus, D. gallinae, M. gallinae, C.
+
+columbae, and Menocanthus stramineus (Atkinson et al.,2008).
+
+Insects can harbor a lot of pathogens and convey them to the host from these pathogens ''blood parasites" as Haemoproteus columbae which transmitted by the bite of ectoparasitic hippoboscid flies (Hussein and Abdelrahim, 2016); Plasmodium relictum, which is one of the most common species of avian malaria (Beadell et al., 2006) and Leucocytozoon marchouxi (Swinnerton et al., 2005, Martinsen et al., 2006, Hussein and Abdelrahim 2016). Deltamethrin (a pyrethroid derivative) has become a popular pesticide in many nations due to its low toxicity and wide safety margin for poultry (Eladl et al., 2018). Deltamethrin affects the nervous system of insects that touch or ingest them, which leads to the rapid paralysis and death of these insects (Elyazar et al., 2011). In the same way, deltamethrin considered as an effective insecticide for the control of insects on pigeons and the surrounding environment (Attia and Salem, 2021). Accordingly, the aim of this study is to understand the biodiversity and determine the prevalence of pigeon external parasitic infestation with evaluation of oxidative stress markers and zinc levels which will aid in the planning of actions to improve the health of these birds in Egypt.
+
+## 2. Material And Methods 2.1 Vvv. Ethical Approval
+
+This study was designed according to the Ethical Committee, Faculty of Veterinary Medicine regulations. Birds were humanly handled and safely released after clinical investigations.
+
+## 2.2. Pigeon Sampling
+
+During the observation period from January to December 2020, 300 pigeons (25 bird per month) were investigated. Three hundred Birds were collected from different poultry clinics, poultry market and pigeon rearing houses from Giza, Egypt which lies in 29.9870N 31.2118E with a hot desert climate. The average ambient temperature in Giza in winter ranged from 21.1 C (70.0)F:
+24.2 C (75.6)F with relative humidity 46:57%, in spring, 28.4 C
+(83.1)F : 34.9 C (94.8)F with relative humidity 37:44%, in summer 34.5 C (94.1)F : 41 C (106)F with relative humidity 39:53% and in autumn 25 C (77.7)F : 32.4 C (90.3)F with relative humidity 47: 57%. The examined birds were divided according to age as 80 squabs (day old to one month), 100 young (form one month to 7 months old) and 120 adults (above 7 months of age). Every bird was physically examined to detect the age and presence of external parasites (data were retrieved from the owners).
+
+## 2.3. History And Clinical Examination Of Pigeons 2.3.1. External Parasites
+
+The feathers and skin were carefully examined for the presence of any macroscopic external parasites by naked eye then parasites were picked up and/ or skin scraping was adopted to identify the parasites using an Olympus Stereoscopic microscope (SZX16; Japan (Soulsby 1986; Attia and Salem, 2021).
+
+## 2.4. Identification Of The Parasites
+
+All the collected parasites from the birds and their premises were preserved in 70% ethyl alcohol. Then, these parasites were identified using the keys recorded by Soulsby (1986); Attia and Salem, (2021).
+
+## 2.5. Blood Samples
+
+Blood was collected from jugular vein of externally parasitized birds on plain tubes for sera separation. Sera were kept on 20 C for further investigations according to Attia et al., (2020).
+
+## 2.6. Biochemical Analysis
+
+Serum zinc concentration were analysed using ionized coupled plasma by mass spectrometry method as described by Page et al.,
+(2018); Attia et al., (2020).
+
+## 2.7. Evaluation Of Oxidative Stress Markers
+
+The level of serum malondialdehyde (MDA) using the reaction of thiobarbituric acid and then separation occurred on HPLC. The detection was performed using UV at 532 nm. Serum Nitric oxide
+(NO) level were analysed as mentioned by Khazaei and Nematbakhsh, 2012; briefly; colorimetric NO assay kit was used
+(Calbiochem-Novabiochem Corporation, San Diego, Calif), that measures the total nitrate and nitrite on serum based on Griess reaction and measured using wavelength 450 nm as mentioned by Aktas et al., (2017) and Aytekin and Unubol Aypak (2011).
+
+2.8. Recommendation for external parasites control Deltamethrin (Butox, 12.5 % solution, 1 mL / 4 L of water for birds & 1 mL /L for surrounding environment), is recommended for monthly spraying. Precautions have been taken during deltamethrin spraying, and the remains have been disposed of in a sanitary manner.
+
+## 2.9. Statistical Analysis
+
+Data were statistically analyzed by using SPSS Version 18.0 software (Inc., Chicago, IL, USA). Blood parameters in infested and control non infested group were compared by independent T-test following the normality of data. A p- value consider significant when (p < 0.05).
+
+## 3. Results 3.1. History And Clinical Investigations
+
+Investigated pigeons were exhibited indications of illness such as general weakness, decreased egg production in layers, unthriftiness, ruffled feathers, decreased weight gain, emaciation with protrusion of keel bone (Fig. 1A) and in some cases; pigeon flies were observed on birds' body, under/or on the wings or on tail (Fig. 1B) and Fig. 2.
+
+Most of the examined pigeons were positive with at least with one of external parasitic species. The overall prevalence rate was 80.3%.
+
+## 3.2. External Parasites
+
+External parasites usually observed on living birds. In case of lice infestation, eggs were attached with feathers roots, while in
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+red mite; the red spots were observed surround the bird vent, unilateral arthritis was observed in birds mange with presence of dull raised scales tinged with grayish to brown color exudate on shank of pigeon leg (Fig. 1C).
+
+Two hundred and forty-one; out of 300 examined pigeons, were positive for external parasites infestations. Six different types of external parasites were detected as follow: P. canariensis, H. equina, C. columbae, M. gallinae, Knemidocoptes spp. and D. gallinae with prevalence 41.6, 26, 7, 5, 0.33 and 0.33%, respectively. The intensity of external parasites on pigeon's body were ranged from (1–6), (1–
+4), (8–25) and (5–20) in case of P. canariensis, H. equina, C. columbae, and M. gallinae, respectively.
+
+The prevalence and intensity of ectoparasites in domestic pigeons based on their age were summarized in (Fig. 3 & Table 1) and Fig. 4.
+
+Single infestation of pigeons with P. canariensis, H. equina, C.
+
+columbae, M. gallinae, Knemidocoptes spp. and D. gallinae were recorded also; mixed external parasites infestation between
+(P. canariensis and H. equina); (P. canariensis and C. columbae);
+(C. columbae and M. gallinae) and (P. canariensis; C. columbae and M. gallinae) were observed and summarized in; Table 2.
+
+The highest prevalence of external parasites was detected in spring and summer seasons as seen in (Table 3).
+
+Infested pigeon with H. equina showed significantly higher in Nitric oxide (120.56 ± 4.56, ±95% C.I.) while in mixed infestation with P. canariensis it reaches to 145.89 ± 9.43; the Nitric oxide levels was low in M. gallinae reach to 56.89 ± 3.76. Serum MDA
+reach to 100.30 ± 6.48, 95% C.I.; in mixed infestation with P. canariensis, H. equina, while it was low in C. columbae infestation.
+
+zinc levels were decreased with parasitic infestation as it reaches to low levels in P. canariensis, H. equina mixed infestation; all data were compared to control levels of oxidative stress markers and zinc levels (Table 4).
+
+Periodical, monthly usage of deltamethrin in the infested flocks revealed a marked decrease in external parasites numbers after two days of treatment with relieve of the clinical signs.
+
+## 4. Discussion
+
+Pigeons are a point of anxiety since they can spread zoonoses to humans and are a reservoir for numerous parasite diseases affecting poultry (Sari et al., 2008). Most of examined pigeons showed 1827
+
+![3_image_0.png](3_image_0.png)
+
+| Parasites          | Positive cases (%)   | Parasite's intensity   | Age Squabs   | Young        |                |
+|--------------------|----------------------|------------------------|--------------|--------------|----------------|
+| (80)               | (1 00)               |                        |              |              |                |
+| P. canariensis     | 125 (41.6)           | 1–6                    | 50 (62.5%)   | 65 (65%)     | 10 (8.33%)     |
+| H. equina          | 78 (26)              | 1–4                    | 12 (15%)     | 22 (28%)     | 44 (36.6%)     |
+| C. columbae        | 21 (7)               | 8–25                   | 10 (12.5%)   | 8 (8%)       | 3 (2.5%)       |
+| M. gallinae        | 15 (5)               | 5–20                   | 6 (0.75%)    | 4 (4%)       | 5 (4.16%)      |
+| D. gallinae        | 1 (0.33)             | -                      | 0            | 0            | 1 (0.33%)      |
+| Knemidocoptes spp. | 1(0.33)              | -                      | 0            | 0            | 1 (0.33%)      |
+| Total              | 241 (80.3%)          | -                      | 78/80(97.5%) | 99/100 (99%) | 63/120 (52.5%) |
+
+![3_image_1.png](3_image_1.png)
+
+signs of ruffled feathers, severe emaciation. Similar signs were observed in case of parasitic infestation as recorded by Abd ElRahman et al., (2008); Mohamed et al., (2009) and Abdel Rahman et al., (2019).
+
+Ectoparasites live on feathers and skin of the host, using as a shelter and food provider, they also have a considerable negative influence on host health and productivity. From our results, the overall prevalence of external parasites was 80.3% (241/300) either single or mixed infestations, six different arthropods species were identified as follow: P. canariensis (41.6%), H. equina (26%), C.
+
+columbae (7%), M. gallinae (5%), D. gallinae (0.33 %), and Knemidocoptes spp (0.33%). Higher prevalence 89% (89/100) was recorded in Tripoli, Libya by Alkharigy et al., (2018) as they found the prevalence of C. columbae (82%), Goniodes gallinae (18%), M. gallinae (3%)
+and P. canariensis (1%). Also, Radfar et al., (2011) recorded higher prevalence of external parasites in pigeon, C. columbae (79.41%), M. gallinae (44.11%) and P. canariensis (63.72%) in south khorasan; Iran while, in Colombia, Pérez-García et al., (2015) found that the prevalence of C. columbae (64%), P. canariensis (52%), M. gallinae
+(24%). Borji et al., (2012) reported that the prevalence of C. columbae (42.8%), P. canariensis (16.1%) and M. gallinae (7.1%) in Mashhad city Iran. Also, Ali et al., (2020) recorded two external parasites on
+(Harami) pigeons, including the shaft louse M. gallinae and P.
+
+canariensis with 100 and 88.90% prevalence, respectively in Medina, Saudi Arabia.
+
+In another study, the incidence of H. equina was 26% while Sokół and Michalski, (2015) noticed the low host specificity of H. equina which could attack cattle, dogs, hares, birds, and humans.
+
+The prevalence of C. columbae was 21% however, Jahantigh et al., (2016), Dranzoa et al., (1999) and Foronda et al., (2004)
+recorded higher prevalence of C. columbae 78.40%, 94.1 and 100% in Iran, Uganda, and Tenerife, respectively.
+
+From our findings, Knemidocoptes spp. was observed in one case only with incidence of 0.33% during summer season and its lesion was found as unilateral arthritis and presence of severe dermatitis that appeared as raised dull color scales with presence of grayish color exudate between scales. Similar findings were observed in
+
+| Prevalence of single and mixed infestation of external parasites in domestic pigeons. Parasite Type of infestation   | Prevalence (Positive cases/300 total examined birds) No. %   |       |      |
+|----------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------|-------|------|
+| P. canariensis                                                                                                       | Single                                                       | 90    | 30   |
+| H. equina                                                                                                            | 66                                                           | 22    |      |
+| C. columbae                                                                                                          | 9                                                            | 3     |      |
+| M. gallinae                                                                                                          | 3                                                            | 1     |      |
+| D. gallinae                                                                                                          | 1                                                            | 0.33  |      |
+| Knemidocoptes spp.                                                                                                   | 1                                                            | 0.33  |      |
+| P. canariensis + H. equina                                                                                           | mixed                                                        | 22    | 7.33 |
+| P. canariensis + C. columbae                                                                                         | 31                                                           | 10.33 |      |
+| C. columbae + M. gallinae                                                                                            | 12                                                           | 4     |      |
+| P. canariensis + C. columbae + M. gallinae                                                                           | 6                                                            | 2     |      |
+| Total No. of positive pigeons                                                                                        | 241                                                          | 80.3  |      |
+
+| Table.3 Seasonal prevalence of external and internal parasites in domestic pigeons. External Parasite Spring   | Summer   | Autumn   | Winter   |       |    |      |    |      |
+|----------------------------------------------------------------------------------------------------------------|----------|----------|----------|-------|----|------|----|------|
+| No.                                                                                                            | %        | No.      | %        | No.   | %  | No.  | %  |      |
+| Pseudolynchia canariensis                                                                                      | 57       | 76       | 60       | 80    | 5  | 6.66 | 3  | 4    |
+| Hippobosca equina                                                                                              | 18       | 24       | 48       | 66.6  | 12 | 16   | 0  | 0    |
+| Columbicola columbae                                                                                           | 8        | 10.66    | 10       | 13.33 | 2  | 2.66 | 1  | 1.33 |
+| Menopon gallinae                                                                                               | 6        | 8        | 9        | 12    | 0  | 0    | 0  | 0    |
+| Dermanyssus gallinae                                                                                           | 0        | 0        | 1        | 1.33  | 0  | 0    | 0  | 0    |
+| Knemidocoptes spp.                                                                                             | 0        | 0        | 1        | 1.33  | 0  | 0    | 0  | 0    |
+| No. = number of positive cases.                                                                                |          |          |          |       |    |      |    |      |
+
+| Oxidative stress markers in relation to single and mixed infection of external parasites. Parasite Oxidative stress markers Nitric oxide MDA serum Zinc P. canariensis 95.57 ± 3.67 78.38 ± 5.17 54.04 ± 2.85 H. equina 120.56 ± 4.56 88.38 ± 4.49 36.77 ± 1.32 C. columbae 67.98 ± 7.00 30.33 ± 3.35 100.24 ± 1.00 M. gallinae 56.89 ± 3.76 33.56 ± 3.46 110.52 ± 2.96 D. gallinae 79.89 ± 5.99 50.33 ± 1.03 33.57 ± 2.82 Knemidocoptes spp. 80.14 ± 6.33 52.35 ± 1.67 34.66 ± 3.62 P. canariensis + H. equina 145.89 ± 9.43 100.30 ± 6.48 28.89 ± 1.52 P. canariensis + C. columbae 110.00 ± 3.59 98.33 ± 2.45 59.97 ± 3.95 C. columbae + M. gallinae 74.49 ± 9.32 60.38 ± 2.98 100.67 ± 2.96 P. canariensis + C. columbae + M. gallinae Control 49.78 ± 0.95 27.89 ± 0.69 118.00 ± 0.94   |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+a parallel study conducted by Abou-Alsoud and Karrouf, (2016) who detected Knemidocoptes Pilae with prevalence of (11.5 %) in Budgerigars in Mansoura public park, Egypt.
+
+The mixed infestation was reported as follow: P. canariensis + H.
+
+equina with prevalence 7.33%. P. canariensis + C. columbae with 10.33 % prevalence; C. columbae + M. gallinae (4%) and P. canariensis + C. columbae + M. gallinae (2%). Also, Alkharigy et al., (2018)
+recorded mixed infestation with external parasites in Libya. The mixed infestation of pigeon with external parasites could be contributed to that ectoparasites can live together without causing any hurtful effects on each other (Radfar et al., 2011).
+
+The occurrence of ectoparasitic infestations in squabs (82.5%)
+was higher than young (77%) and lowest prevalence was recorded in adults (18%). Same observations were recorded by da Cunha Amaral et al., (2013) in case of P. canariensis infestation but, Msoffe et al., (2010) and Radfar et al., (2012) noticed that the prevalence of P. canariensis were higher in adult pigeons than squabs. Adult pigeons are predicted to have a lower incidence of ectoparasites since they have a higher level of parasite immunity
+(Merila et al.,1995) and adult birds use their claws and the bill to get rid of ectoparasites (Clayton et al., 2010; Waite et al., 2012).
+
+From our results, the intensity (number of external parasites that are found per bird) of external parasites on pigeon's boy ranged from (1:6), (1:4), (8:25) and (5:20) in case of P. canariensis, H.
+
+equina, C. columbae and M. gallinae, respectively. This result is agreed with Attia and Salem (2021) as they noticed that the intensity of P. canariensis flies was 1: 6 (2.00 ± 1.0) on pigeons' body in El- Gharbia, Egypt.
+
+The ambient temperature is the key determinant of the frequency, quantity, and diversity of external parasites allover year
+(Al-Barwari and Saeed 2012; Attia et al. 2017). In our study the highest prevalence of external parasites infestations was recorded in both spring and summer. The prevalence of P. canariensis was 76% and 80%, C. columbae was 10.66% and 13.33%, M. gallinae was 8 % and 12% in spring and summer, respectively. D. gallinae only detected in summer season only 1.33%. The lowest prevalence of external parasites infestation in our study was recorded in autumn and winter season, P. canariensis was 6.66% and 4%, C. columbae was 2.66% and 1.33% in autumn and winter, respectively. on the other hand, no positive cases of M. gallinae and D. gallinae were recorded in autumn and winter. The differences in the seasonal prevalence may be contributed to the ambient temperature in Egypt exceed 25 Co in both spring and summer which considered a favorable condition for insects' population.
+
+Continuous exposure of poultry to ectoparasites that act as intermediate hosts for different parasitic worms increases the potential for the spread of parasitic diseases among birds
+(Ashenafi and Eshetu, 2004). So, monthly spray of deltamethrin is recommended and provoke acceptable result and succeeded in limitation of external parasites load. This result is agreed with (Attia and Salem, 2021) as they found deltamethrin is effective in P. canariensis control. Deltamethrin has proven its efficiency in controlling insects and we recommend in the future to load the active substance on biological nanoparticles to increase its efficiency.
+
+Also, recommend the use of some additives on pigeon food and drink as prebiotics (Abd El-Hack et al., 2021b; Yaqoob et al., 2021) probiotics (Abd El-Hack et al., 2021; Alagawany et al.,
+2021a; El-Saadony et al., 2021a), bioactive plants compounds (ElSaadony et al., 2021b), bioactive peptides (El-Saadony et al.,
+2021c,e), green synthesized nanoparticles (El-Saadony et al., 2021d,f), herbal extracts (Reda et al., 2021; Abou-Kassem et al., 2021), phytogenic compounds (Abd El-Hack et al., 2021c; Ashour et al., 2020), and essential oils (Alagawany et al., 2021b; ElTarabily et al., 2021; Abd El-Hack et al., 2020) to strengthen their immune system and increase disease resistance.
+
+The oxidative stress markers as (Nitric oxide and MDA levels)
+were raised in parasitic infestation either in single parasites or in mixed infestation due to rapid erythrocyte destruction and secretion of oxygen radicles (Mousa and Soliman, 2016); as recorded by Nazifi et al., (2011) and Razavi et al., (2011) found that the antioxidant enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase, were raised during parasitic infestation.
+
+## 5. Conclusion
+
+When we spoke with pigeon owners, we discovered that most of them were unaware of dealing with a parasitic infestation in domestic pigeons, and some of them do not remedy them at all.
+
+From our findings, pigeons have a significant prevalence of external parasites, with single or mixed infestation. Oxidative stress markers were significantly elevated during either single or mixed external parasites infestations. Pathogenicity studies, regular monitoring, treatment, and control strategies must be put in place to reduce pigeon parasitic infestation considering that these birds are in close contact with other poultry and human. Deltamethrin could be considered as an effective insecticide for controlling of external parasites in the pigeon and would be recommended for regular use hand by hand with biosecurity measures.
+
+## 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.
+
+## Acknowledgement
+
+The current work was funded by Taif University Researchers Supporting Project number (TURSP - 2020/221), Taif University, Taif, Saudi Arabia.
+
+## References
+
+Abd El-Hack, M.E., El-Saadony, M.T., Swelum, A.A., Arif, M., Abo Ghanima, M.M.,
+Shukry, M., El-Tarabily, K.A., 2021c. Curcumin, the active substance of turmeric: its effects on health and ways to improve its bioavailability. J. Sci. Food Agric.in press.
+
+Abd El-Hack, M.E., Alaidaroos, B.A., Farsi, R.M., Abou-Kassem, D.E., El-Saadony, M.T.,
+Ashour, E.A., 2021. Impacts of supplementing broiler diets with biological curcumin, zinc nanoparticles and Bacillus licheniformis on growth, carcass traits, blood indices, meat quality and cecal microbial load. Animals 11 (7),
+1878.
+
+Abd El-Hack, M.E., El-Saadony, M.T., Shafi, M.E., Alshahrani, O.A., Saghir, S.A., AlWajeeh, A.S., Al-Shargi, O.Y., Taha, A.E., Mesalam, N.M., Abdel-Moneim, A.-M.-E.,
+2021b. Prebiotics can restrict Salmonella populations in poultry: a review. Anim. Biotechnol. 19, 1–10.
+
+Abd El-Hack, M.E., El-Saadony, M.T., Shehata, A.M., Arif, M., Paswan, V.K., Batiha, G.-
+E.-S., Khafaga, A.F., Elbestawy, A.R., 2020. Approaches to prevent and control Campylobacter spp. colonization in broiler chickens: a review. Environ. Sci.
+
+Pollut. Res. 28 (5), 4989–5004.
+
+Abd El-Rahman, M.A.M., Seddiek, S.A., Soliman, A.S., 2008. Some studies on trichomoniasis of pigeons at Qualiobia governorate. Egypt. J. Comp. Pathol.
+
+ 
+Clin. Pathol. 21 (2), 123–141 http://erepository.cu.edu.eg/index.php/EJCPCP/ article/view/177.
+
+Abdel Rahman, M.M.I.A., Tolba, H.M.N., Abdel-Ghany, H.M., 2019. Ultrastructure, morphological differentiation, and pathological changes of Ascaridia species in pigeons. Adv. Anim. Vet. Sci. 7 (2), 66–72. https://doi.org/10.17582/journal.
+
+aavs/2019/7.2.66.72.
+
+Abou-Alsoud, M.E., Karrouf, G., 2016. Diagnosis and Management of Knemidocoptes Pilae in Budgerigars (Melopsittacus Undulates): Case Reports in Egypt. M J Vete. 1 (1), 007.
+
+Abou-Kassem, D.E., Mahrose, K.M., El-Samahy, R.A., Shafi, M.E., El-Saadony, M.T.,
+Abd El-Hack, M.E., Emam, M., El-Sharnouby, M., Taha, A.E., Ashour, E.A., 2021. Influences of dietary herbal blend and feed restriction on growth, carcass characteristics and gut microbiota of growing rabbits. Ital. J. Anim. Sci. 20, 896– 910.
+
+Aktas, M.S., Kandemir, F.M., Kirbas, A., Hanedan, B., Aydin, M.A., 2017. Evaluation of oxidative stress in pigeon infected with Psoroptes ovis using total antioxidant capacity, total oxidant status, and malondialdehyde level. Vet. Res. 61(2):197–
+201.https://doi. 10.1515/jvetres-2017-0025.
+
+Alagawany, M., El-Saadony, M., Elnesr, S., Farahat, M., Attia, G., Madkour, M., Reda, F., 2021a. Use of lemongrass essential oil as a feed additive in quail's nutrition: its effect on growth, carcass, blood biochemistry, antioxidant and immunological indices, digestive enzymes and intestinal microbiota. Poult. Sci. 100, (6) 101172.
+
+Alagawany, M., Madkour, M., El-Saadony, M.T., Reda, F.M., 2021b. Paenibacillus polymyxa (LM31) as a new feed additive: Antioxidant and antimicrobial activity and its effects on growth, blood biochemistry, and intestinal bacterial populations of growing Japanese quail. Anim. Feed Sci. Technol. 276, 114920.
+
+Al-Barwari, S.H., Saeed, I., 2012. The Parasitic Communities of the rock pigeon Columba livia from Iraq: Component and Importance. Turkiye Parazitol Derg 36, 232–239. https://doi.org/10.5152/tpd.2012.
+
+Ali, M., Ibrahim, R., Alahmadi, S., Elshazly, H., 2020. Ectoparasites and intestinal helminths of pigeons in Medina, Saudi Arabia. J Parasitol. 12, 106(6):721–729. https://doi.10.1645/20-64. DOI: 10.1645/20-64.
+
+Alkharigy, F.A., El Naas, A.S., EL Maghrbi, A.A., 2018. Survey of parasites in domestic pigeons (Columba livia) in Tripoli, Libya. Open Vet. J. 8(4): 360–366. http:// dx.doi.org/10.4314/ovj.v8i4.
+
+Ashenafi, H., Eshutu, Y., 2004. Study on Gastro-intestinal helminths of local chickens in Central Ethiopia. Rev. Med. Vet. 155(10): 504–507. https://www.
+
+revmedvet.com › RMV155_504_507.
+
+Ashour, E.A., El-Hack, M.E.A., Shafi, M.E., Alghamdi, W.Y., Taha, A.E., Swelum, A.A.,
+Tufarelli, V., Mulla, Z.S., El-Ghareeb, W.R., El-Saadony, M.T., 2020. Impacts of green coffee powder supplementation on growth performance, carcass characteristics, blood indices, meat quality and gut microbial load in broilers.
+
+Agriculture 10, 457.
+
+Atkinson, C.T., Thomas, N.J., Hunter, D.B., 2008. Parasitic diseases of wild birds. John Wiley & Sons, Inc. ISBN: 978-0-813-82081-1. U.S. Library of Congress. https://doi.10.1002/9780813804620.
+
+Attia, M.M., El-Gameel, S.M., Ismael, E., 2020. Evaluation of tumor necrosis factoralpha (TNF-a); gamma interferon (IFN-c) genes and oxidative stress in sheep:
+immunological responses induced by Oestrus ovis (Diptera: Oestridae)
+infestation. J. Parasit. Dis.44(2): 332–337.https://doi.org/10.1007/s12639-02001220-w.
+
+Attia, M.M., Salem, H.M., 2021. Morphological and molecular characterization of Pseudolynchia canariensis (Diptera: Hippoboscidae) infesting domestic pigeons. Int. J. Trop. Insect Sci. https://doi.org/10.1007/s42690-021-00597-2.
+
+Attia, M.M., Soliman, S.M., Khalf, M.A., 2017. Hydrophilic nanosilica as a new larvicidal and molluscicidal agent for controlling of major infectious diseases in Egypt. Vet World. 10 (9), 1046–1051.
+
+Aytekin, I., Unubol Aypak, S., 2011. Levels of selected minerals, nitric oxide, and vitamins in aborted Sakis pigeon raised under semitropical conditions. Trop.
+
+Anim. Health Prod. 43:511–514.https://doi.10.1007/s11250-010-9724-x.
+
+Badparva, E., Ezatpour, B., Azami, M., Badparva, M., 2015. First report of bird's infection by intestinal parasites in Khorramabad, west Iran. J. Parasit. Dis. 39 (4), 720–724. https://doi.org/10.1007/s12639-014-0427-5.
+
+Beadell, J.S., Ishtiaq, F., Covas, R., Melo, M., Warren, B.H., Atkinson, C.T., Bensch, S.,
+Graves, G.R., Jhala, Y.V., Peirce, M.A., Rahmani, A.R., Fonseca, D.M., Fleischer, R.C., 2006. Global phylogeographic limits of Hawaii's avian malaria. Proc R Soc Lond.
+
+Series B. 273 (2935–2944). https://doi.org/10.1098/rspb.2006.3671.
+
+Borji, H., Moghaddas, E., Razmi, G.R., Azad, M., 2012. A survey of ecto- and endoparasites of domestic pigeons (Columba livia) in Mashhad, Iran. Iran. J. Vet. Sci.
+
+Technol. 4(2):37–42. https://doi: 10.4314/ovj.v8i4.2.
+
+Clayton, D.H., Koo, J., Harbiso, C., Moye, B., Bush, S., 2010. How birds combat ectoparasites. J. Ornithol. 3: 41–71. https//doi: 10.2174/ 1874453201003010041.
+
+da Cunha Amarala, H., Bergmannb, F.B., Silveirac, T., Silveira dos Santosd, P.R.,
+Krügera, R.F., 2013. Pseudolynchia canariensis (Diptera: Hippoboscidae): distribution pattern and phoretic association with skin mites and chewing lice of Columba livia (Aves: Columbidae). J. Nat. Hist. 47 (47–48), 2927–2936. https://doi.org/10.1080/00222933.2013.791939.
+
+Dranzoa, C., Ocaido, M., Katete, P., 1999. The ecto,gastro-intestinal and haemoparasites of live pigeons (Columba livia) in Kampala, Uganda. Avian Pathol. 28, 119–124. https://doi: 10.1080/03079459994830.
+
+Eladl, A., Hamed, H.R., El-Shafei, R.A., 2018. Prevalence of mites and their impact on laying hen (Gallus gallus domesticus) farm facilities in Egypt, with an analysis of deltamethrin residues in eggs and tissue. Avian Pathol. 47 (2), 161–171. https:// doi.org/10.1080/03079457.2017.1388500.
+
+ 
+El-Dakhly, K., Mahrous, L.N., Mabrou, G.A., 2016. Distribution pattern of intestinal helminths in domestic pigeons (Columba livia domestica) and turkeys (Meleagris gallopavo) in Beni-Suef province Egypt. J. Vet. Med. Res. 23(1):112–120. https//doi: 10.21608/JVMR.2016.43226.
+
+El-Saadony, M.T., Alagawany, M., Patra, A.K., Kar, I., Tiwari, R., Dawood, M.A., AbdelLatif, H.M., 2021a. The functionality of probiotics in aquaculture: An overview.
+
+Fish Shellfish Immunol. 117, 36–52.
+
+El-Saadony, M.T., Zabermawi, N.M., Zabermawi, N.M., Burollus, M.A., Shafi, M.E.,
+Alagawany, M., Abd El-Hack, M.E., 2021b. Nutritional aspects and health benefits of bioactive plant compounds against infectious diseases: A review. Food Rev. Int., 1–23 El-Saadony, M.T., Abd El-Hack, M.E., Swelum, A.A., Al-Sultan, Sa.ad., El-Ghareeb, W.
+
+R., Hussein, E.O.S., Ba-Awadh, H.A., Akl, B.A., Nader, M.M., 2021c. Enhancing quality and safety of raw buffalo meat using the bioactive peptides of pea and red kidney bean under refrigeration conditions. Ital. J. Anim. Sci. 20 (1), 762– 776.
+
+El-Saadony, M.T., Alkhatib, F.M., Alzahrani, S.O., Shafi, M.E., Abdel-Hamid, S.E., Taha, T.F., Aboelenin, S.M., Soliman, M.M., Ahmed, N.H., 2021d. Impact of mycogenic zinc nanoparticles on performance, behavior, immune response, and microbial load in Oreochromis niloticus. Saudi J. Biol. Sci. 28 (8), 4592–4604.
+
+El-Saadony, M.T., Khalil, O.S., Osman, A., Alshilawi, M.S., Taha, A.E., Aboelenin, S.M.,
+Shukry, M., Saad, A.M., 2021e. Bioactive peptides supplemented raw buffalo milk: biological activity, shelf life and quality properties during cold preservation. Saudi J. Biol. Sci. 28 (8), 4581–4591.
+
+El-Saadony, M.T., Saad, A.M., Najjar, A.A., Alzahrani, S.O., Alkhatib, F.M., Shafi, M.E.,
+Selem, E., Desoky, E.-S.M., Fouda, S.E.-S.E.-S., El-Tahan, A.M., 2021f. The use of biological selenium nanoparticles in controlling Triticum aestivum L. crown root and rot diseases induced by Fusarium species and improve yield under drought and heat stress. Saudi. J. Biol. Sci. 28 (8), 4461–4471.
+
+El-Tarabily, K.A., El-Saadony, M.T., Alagawany, M., Arif, M., Batiha, G.E., Khafaga, A.F.,
+Elwan, H.A., Elnesr, S.S., Abd El-Hack, M.E., 2021. Using essential oils to overcome bacterial biofilm formation and their antimicrobial resistance. Saudi J. Biol. Sci. 28 (9), 5145–5156.
+
+Elyazar, I. R.F., Hay, S. I., Baird, J. K., 2011. Chapter 2 - Malaria distribution, prevalence, drug resistance and control in Indonesia. ADV PARASIT .74: 41–175. https://doi.org/10.1016/B978-0-12-385897-9.00002-1.
+
+Foronda, P., Valladares, B., Rivera-Medina, J.A., 2004. Parasites of Columba livia
+(Aves: Columbiformes) in Tenerife (Canary Islands) and their role in the conservation biology of the Laurel pigeons. Parasite. 1: 311-316. https//doi:
+10.1051/parasite/2004113311.
+
+Hussein, N.M., Abdelrahim, E.A., 2016. Haemoproteus Columbae infection and its histopathological effects on pigeons in Qena Governorate, Egypt. IOSR-JPBS. 11 (1): 79-90. https//doi: 10.9790/3008-11117990.
+
+Imura, T., Suzuki, Y., Ejiri, H., Sato, Y., Ishida, K., Sumiyama, D., Murata, K., Yukawa, M., 2012. Prevalence of avian haematozoa in wild birds in ahigh-altitude forest in Japan. Vet. Parasitol. 183: 244–248. https//doi: 10.1016/j.vetpar.2011.07.027.
+
+Jahantigh, M., Esmailzade Dizaji, R., Teymoori. Y., 2016. Prevalence of external parasites of pigeon in Zabol, southeast of Iran. J. Parasit. Dis. 40(4):1548-1551.
+
+https//doi: 10.1007/s12639-015-0725-6.
+
+Khazaei, M., Nematbakhsh, M., 2012. Effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide concentration in normal and high-cholesterol fed rabbits. Arch. Med. Sci. 8 (4), 719–723. https://doi.org/10.5114/aoms.2012.30296.
+
+Martinsen, E.S., Paperna, I. Schall, J., 2006. Morphological versus molecular identification of avian Haemosporidia: an exploration of three species concepts. Parasitol. 13(3):279–288. https//doi: 10.1017/S0031182006000424.
+
+Merila, J., Bjorklund, M., Bennett, G.F., 1995. Geographic and individual variation in haematozoan infections in the greenfinch, Carduelis chloris. Can. J. Zool. 73:1798–1804. https//doi: 10.1139/z95-212.
+
+Mohamed, I.E., El-Sakkar, G.H., Moursi, M.M., 2009. Pathological studies on pigeon trichomoniasis with reference to the associated bacteria. J. Comp. Pathol. Clin.
+
+Pathol. 22 (2), 67–87 http://www.pathologyeg.com/publication2009_2/ Publication3.pdf.
+
+Mousa, S.A., Soliman, S.M., 2016. Oxidant and Antioxidant Status in Pneumonic Goats with Special Reference to Bacterial Etiology. Int. J. Livestock Res. 6 (5), 15– 23. https://doi.org/10.5455/ijlr.20160417045508.
+
+Msoffe, P.L.M., Muhairwa, A.P., Chiwanga, G.H., Kassuku, A.A., 2010. A study of ectoand endo-parasites of domestic pigeons in Morogoro unicipality. Tanzania. Afr.
+
+J. Agric. Res. 5 (3), 264–2672.
+
+Nazifi, S., Razavi, S.M., Kianiamin, P., Rakhshandehroo, E., 2011. Evaluation of erythrocyte antioxidant mechanisms: antioxidant enzymes, lipid peroxidation and serum trace elements associated with progressive anemia in ovine malignant theileriosis. Parasitol. Res. 109 (275–281). https://doi.org/10.1007/
+s00436-010-2248-5.
+
+Page, C.M., Murphy, T.W., Van, M.L., Emon, J.G., Bowman, P., Wyffels, S.A., Stewart, W.C., 2018. Blood serum mineral element concentrations of weaned Montana ram lambs and their relationship with water quality characteristics. Prof. Anim. Sci. 34 (5), 410–420. https://doi.org/10.15232/pas.2018-01747.
+
+Pérez-García, J., Monsalve-Arcila, D., MárquezVillegas, C., 2015. Presencia de parásitos y enterobacterias en palomas ferales (Columba livia) en áreas urbanas en Envigado Colombia. Rev. Fac. Na.c Salud Pública. 33 (3), 370–376.
+
+Radfar, M.H, Asl, E.N., Seghinsara, H.R., Dehaghi, M.M., Faith, S., 2012. Biodiversity and prevalence of parasites of domestic pigeons (Columba livia domestica) in a selected semiarid zone of South Khorasan, Iran. TROP. ANIM. HEALTH PRO. 44 (2):225–229. https//doi: 10.1007/s11250-011-0002-3.
+
+Radfar, M.H., Fathi, S., Asl, E.N., Dehaghi, M.M., Seghinsara, H.R., 2011. A survey of parasites of domestic pigeons (Columba livia domestica) in South Khorasan, Iran. J. Vet. Res. 4(1):18–23. https//doi: 10.3923/vr.2011.18.23.
+
+Razavi, S., Nazifi, S., Bateni, M., Rakhshandehroo, E., 2011. Alterations of erythrocyte antioxidant mechanisms: antioxidant enzymes, lipid peroxidation and serum trace elements associated with anemia in bovine tropical theileriosis. Vet. Parasitol. 180(3):209–214. https//doi: 10.1016/j.vetpar.2011.03.011.
+
+Reda, F., El-Saadony, M., El-Rayes, T., Farahat, M., Attia, G., Alagawany, M., 2021.
+
+Dietary effect of licorice (Glycyrrhiza glabra) on quail performance, carcass, blood metabolites and intestinal microbiota. Poult. Sci. 100, (1) 101266.
+
+Salem, M.H., Attia, M.M., 2021. Accidental intestinal myiasis caused by Musca domestica L. (Diptera: Muscidae) larvae in broiler chickens: a field study. Int. J.
+
+Trop. Insect. Sci. https://doi.org/10.1007/s42690-021-00492-w.
+
+Sari, B., Karatepe, B., Karatepe, M., Kara, M., 2008. parasites of domestic (Columba livia domestica) and wild (Columba livia livia) pigeons in nig˘de, turkey. Bull. Vet. Inst. Pulawy. 52 (4), 551–554.
+
+Sokół, R., Michalski, M.M., 2015. Occurrence of Hippobosca equina in Polish primitive horses during the grazing season. Ann. Parasitol. 61 (2), 118–122.
+
+Soulsby, E.J.L., 1986. Helminths, Arthropods and Protozoa of Domesticated Animals.
+
+Bailliere Tindall, London, pp. 167–174.
+
+Swinnerton, K.J., Peirce, M.A., Greenwood, A., Chapman, R.E., Jones, C.G., 2005.
+
+Prevalence of Leucocytozoon marchouxi in the endangered Pink Pigeon Columba mayeri. Ibis. 147(4):725–737. https/doi: 10.1111/j.1474-919X.2005. 00454.x Waite, J.L., Henry, A.R., Clayton, D.H., 2012. How effective is preening against mobile ectoparasites? An experimental test with pigeons and hippoboscid flies. Int. J. Parasitol. 42(5):463–467. https/doi: 10.1016/j.ijpara.2012.03.005.
+
+Yaqoob, M., Abd El-Hack, M., Hassan, F., El-Saadony, M., Khafaga, A., Batiha, G.,
+Yehia, N., Elnesr, S., Alagawany, M., El-Tarabily, K., 2021. The potential mechanistic insights and future implications for the effect of prebiotics on poultry performance, gut microbiome, and intestinal morphology. Poult. Sci. 100, (7) 101143.

medical/md/PMC9005259.md

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+# Environmental Epidemiology Original Research Article Does Early Life Phthalate Exposure Mediate Racial Disparities In Children'S Cognitive Abilities? Marisa A. Pattia,*, Melissa Eliota, Nan Lia, Kimberly Yoltonb, Bruce P. Lanphearc,D, Aimin Chene, Joseph M. Brauna
+
+Background: Early life exposure to phthalates may be associated with reduced cognition. However, it is unknown if disproportionate exposure to phthalates contributes to racial disparities in children's intellectual abilities.
+
+Methods: We used data from 253 mother-child pairs in Cincinnati, OH (the Health Outcomes and Measures of the Environment study, 2003–2006). We measured urinary concentrations of 11 phthalate metabolites twice during pregnancy and up to six times in childhood. We evaluated children's cognitive abilities at ages 5 and 8 years. Using mediation models, we quantified covariate-adjusted direct and indirect effects of race on children's Full-Scale Intelligence Quotient (IQ) scores for individual phthalate metabolite concentrations during gestation and childhood.
+
+Results: Average IQ scores among Black children (n = 90) were 7.0 points lower (95% confidence interval [CI] = −12, −1.8) than among White children (n = 145) after adjustment for socioeconomic factors. Urinary monobenzyl phthalate and monoethyl phthalate (MEP) concentrations during gestation and childhood were higher among Black than White children. We did not observe evidence that phthalate concentrations mediated the race-IQ association, with the exception of MEP. Childhood MEP concentrations partially mediated the race-IQ association. For instance, each 10-fold increase in MEP concentrations at age 2 years contributed to a 1.9point disparity in IQ scores between Black and White children (95% CI = −4.7, 0.7). Other phthalate metabolite concentrations during pregnancy or childhood did not mediate the race-IQ association. Conclusions: Despite observing racial disparities in exposure to some phthalates and IQ, we found little evidence that phthalates contribute to IQ disparities. Keywords: Racial disparities; Child intelligence; IQ; Phthalates; Gestational exposure; Childhood exposure a
+*Department of Epidemiology, Brown University, Providence, RI; bDepartment of* Pediatrics, Division of General and Community Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH; cChild and Family Research Institute, British Columbia Children's Hospital, Vancouver, British Columbia, Canada; dFaculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada; and e*Department of Biostatistics,* Epidemiology and Informatics, University of Pennsylvania, Philadelphia, PA. J.M.B. served as an expert witness in litigation related to perfluorooctanonic acid contamination in drinking water in New Hampshire. Any funds he received from this arrangement were/are paid to the Brown University and cannot be used for his direct benefit (e.g., salary/fringe, travel). The other authors have no conflicts to report. The results reported herein correspond to specific aims of grant R01 ES024381 to investigator J.M.B.; R01 ES020349 to investigators A.C., B.P.L., and K.Y.; R01 ES011261 to investigator B.P.L.; and R01 ES014575 to investigators B.P.L. and K.Y. from the National Institutes of Health.
+
+Data and Code Acquisition: Computing code can be obtained from the corresponding author (M.A.P.) upon request. Data from the Health Outcomes and Measures of the Environment (HOME) study are available upon request. The HOME study principal investigators have actively engaged in collaborative data-sharing projects. We welcome new collaborations with other investigators. Interested investigators in the HOME study data can explore data options at the following location and use the available link to contact the investigators to discuss collaborative opportunities: https:// homestudy.research.cchmc.org/. The Data Sharing Committee meets regularly to review proposed research projects and ensure that they do not overlap with extant projects and are an efficient use of scare resources (e.g., cord blood).
+
+SDC Supplemental digital content is available through direct URL citations in the HTML and PDF versions of this article (www.environepidem.com).
+
+*Corresponding Author. Address: Brown University, School of Public Health, 121 S Main Street, Box G-S121-4, Providence, RI 02912. E-mail: marisa_patti@ brown.edu (M.A.P.). Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The Environmental Epidemiology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Environmental Epidemiology (2022) 6:e205 Received: 5 November 2021; Accepted 1 March 2022 Published online 15 March 2022 DOI: 10.1097/EE9.0000000000000205
+
+## Isee Introduction 1
+
+Phthalates are a class of chemicals used in personal care and consumer products, including cosmetics, pharmaceuticals, vinyl flooring, medical supplies, food packaging, and plastic toys.1–7 Human exposure can occur through dermal absorption, inhalation, or ingestion.1,4,8 Phthalates are quickly excreted from the body (<24 hours), but exposure is chronic and ubiquitous in the US population, including among vulnerable subpopulations like pregnant women, infants, and children.3,9 Exposure may be higher in infants and children than adults due to differences in physiology, diet, and behaviors.9–14 Phthalates are suspected developmental neurotoxicants.15–23 Some epidemiologic studies24,25 report an inverse association of gestational17–22 or childhood phthalate exposure16,18–20 with cognitive abilities (i.e., Intelligence Quotient [IQ]) in children. While measures of intelligence are imperfect, they are strong predictors of educational achievement and work performance.26–30 Racial disparities in child IQ have been documented in the United States and abroad, such that individuals who identify as Black or have darker skin colors have lower measured IQ relative to their White peers.31–33 Parental cognition and genetics alone do not explain the full heterogeneity in child IQ,34,35 emphasizing 
+
+## What This Study Adds
+
+Phthalates are neurotoxicants associated with diminished cognitive abilities in children. Racial disparities in children's phthalate exposure and cognitive abilities have been documented previously, but it is unclear if disproportionate exposure to phthalates contributes to disparities in child cognition. Using mediation models, urinary concentrations of monoethyl phthalate but not other phthalates partly mediated racial disparities in child cognitive abilities. While racial disparities in exposure to some phthalates and child cognition exist, we found little evidence to suggest phthalates contribute to disparities in child cognition.
+
+the need to consider external factors such as environmental exposures and socioeconomic features.36 Further, race is a social construct, not a biological variable. No genetic basis exists to explain racial disparities in IQ, indicating that disparities in cognitive abilities are most likely related to environmental factors.31,33,37 Phthalate exposure is ubiquitous in the United States (US), 
+but urinary concentrations of some phthalates are consistently higher in the Black population.13,38 Thus, highly exposed populations may be more likely to experience phthalate-related health consequences than those with lower levels of exposure. Moreover, prior work suggests that racial and ethnic disparities in reproductive, neonatal, and cardiometabolic health outcomes may be related to racial and ethnic differences in phthalate exposure.13,38–42 For example, some phthalate-containing products, particularly hair products, are more frequently marketed to and used by the Black communities.43 This highlights how certain subpopulations may be more exposed and thus vulnerable to the adverse effects of phthalates.43 Despite the recognition that racial and ethnic differences exist in phthalate exposure and child IQ, no study has evaluated whether phthalate exposure mediates the relation between race and child IQ.
+
+To evaluate the potential for phthalate metabolite concentrations during gestation and childhood to mediate the race–cognitive abilities relation, we employed previously defined methods for evaluating how environmental exposures contribute to racial disparities.44 The framework outlined by Bellavia et al utilizes multiple mediation analyses to evaluate the contribution of an environmental exposure of interest on the association between race and health outcomes in cases where there is an unequal distribution of both the exposure and outcome. The goal of this approach is to identify modifiable sources of environmental exposures to reduce (and eliminate) unequal distributions of exposures by developing interventions to reduce exposure-related health disparities.
+
+Using data from the Health Outcomes and Measures of the Environment (HOME) study, we quantified racial disparities in gestational and childhood phthalate exposure and assessed the extent that phthalates mediate the relation between race and child IQ. We hypothesized that racial disparities in phthalate exposure would partly explain the disparity in cognitive abilities between Black and White children. While this study focused on phthalates, it also serves a proof-of-principle analysis of other environmental toxicants as potential explanatory factors of racial disparities in child IQ.
+
+## Methods Study Participants
+
+The HOME study is a general population, prospective pregnancy and birth cohort study. Information regarding recruitment and data collection details have been previously published.45 Briefly, we recruited 468 pregnant women from 1263 eligible women from nine prenatal clinics affiliated with three delivery hospitals in the greater Cincinnati, OH area between 2003 and 2006. 
+
+Inclusion criteria specified women be at least 18 years old, 16±3 weeks of gestation, HIV negative, not taking medications for seizures and/or thyroid disorders, living in homes built before 1978, and not having a diagnosis of diabetes, bipolar disorder, schizophrenia, or cancer the resulted in radiation treatment or chemotherapy. The Institutional Review Board at the Cincinnati Children's Hospital Medical Center and all participating hospitals approved this study (protocol code 01-8-5, 2008-0022 and dates of approval: 29 May 2002, 6 August 2007, and 4 August 2011). All participating mothers provided their written, informed consent for themselves and their children.
+
+Of 468 women who enrolled, 67 dropped out before delivery. Among the 389 live-born, singleton infants, our analysis included mother-child pairs that had at least one measure of child IQ at ages 5 or 8 years, at least one urinary phthalate concentration during gestation and childhood, and no missing covariate information (n = 7). We further restricted the analytic sample to include children who identified as Black or White. We excluded those children who identified as other races due to small sample size. Our final sample included 253 mother-child pairs (eFigure 1; http://links.lww.com/EE/A183).
+
+## Urinary Phthalate Metabolite Concentration Assessment
+
+Study staff collected maternal urine samples at approximately 16 and 26 weeks of gestation in polypropylene specimen cups. We collected child urine samples at ages 1, 2, 3, 4, 5, and 8 years. Among children who were not toilet trained, we collected urine by placing surgical inserts in clean diapers. We lined training potties with inserts to collect urine from children who were in the process of being toilet trained. Urine from inserts was collected with a syringe and expressed into a collection cup in the laboratory. For children who were toilet trained, caregivers assisted in the collection of urine using polypropylene specimen cups. We followed previously described protocols to minimize external contamination of urine samples.46,47 All urine samples were refrigerated for less than 24 hours before processing and stored at ≤−20°C until chemical analysis at the Centers for Disease Control and Prevention (CDC).
+
+Using a modified method of online solid phase extraction coupled with isotope dilution high-performance liquid chromatography–tandem mass spectrometry, CDC staff measured the urinary phthalate metabolite concentrations.13 We measured urinary concentrations of mono(3-carboxypropyl) phthalate 
+(MCPP), mono-isobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono-n-butyl phthalate (MBP), monocarboxyoctyl phthalate (MCOP), monocarboxynonyl phthalate (MCNP), and monoethyl phthalate (MEP). We used three oxidative di(2-ethylhexyl) phthalate (DEHP) metabolites (mono-2-ethyl-5-hydroxyhexyl phthalate, mono-2-ethyl-5-oxohexyl phthalate, and mono-2-ethyl-5-carboxypentyl phthalate [MECPP]) to create a summary DEHP metabolite measure (i.e., ΣDEHP) for both gestational and childhood samples. To calculate concentrations of ΣDEHP (in ng/mL), we divided each DEHP metabolite concentration by its molar mass, summed the individual concentrations, and then multiplied the molar sum by the molar mass of MECPP.48 We did not measure MCOP or MCNP concentrations in maternal urine samples because the method for these biomarkers had not yet been developed. We could not measure concentrations of MBP, MiBP, or MEHP from child urine samples collected at ages 1 to 3 years, as these metabolites were detected in the diaper inserts used during collection.9 The limits of detection (LODs) for the phthalate metabolites ranged from ~0.1 to ~1ng/mL. Values below the LOD were given a value of the LOD/√2 (eTable 3; http://links.lww.com/EE/A183).49 We accounted for urine dilution using urinary creatinine concentrations as described previously.50 Phthalate metabolite concentrations (ng/mL) were first creatinine standardized and log10 transformed before we conducted measurement error correction. Briefly, we accounted for phthalate exposure measurement error using a previously described regression calibration approach.51 For gestational phthalate concentrations, we used a linear mixed model with a random intercept to estimate subject-specific geometric mean creatinine-standardized urinary phthalate concentrations across the two measurement occasions (i.e., 16 and 26 weeks).52 Our model for phthalate concentrations collected on the ith mother at the jth measurement occasion during gestation: Yij ij = + µ ε i , 
+where µ µ σ i~ ( N , ) Β
+2 and ε σ ij~ ( N 0, ) W
+2 . Under this model, the reliability of observed geometric mean phthalate concentration for the ith mother across gestation, yi is given R n i B B
+W
+i
+= + σ σ/ ( ) σ 2 2 2
+, 
+where σ B
+2 and σW
+2 are the between-subject and within-subject variance components, respectively, and ni = 1–2 is the number of available phthalate measures during gestation.
+
+While this calibration approach is appropriate when geometric mean phthalate concentrations show no time trends across measurement occasions, this was not the case for childhood concentrations, which varied quadratically with child age.47 Therefore, we used a more complex linear mixed-effects model to account for measurement error in childhood phthalate measures. Thus, we included subject-specific age and agesquared terms for the age of urine sample collection from age 1 to 8 years. We included subject-specific intercepts and linear age slopes, while age squared was fixed. For each subject, we used the estimated best linear unbiased predictor to calculate phthalate metabolite concentrations at each time point and averaged across childhood. The average childhood value was calculated by dividing the area under the estimated trajectories of phthalate metabolite concentrations curve by the length of time (age, 1–8 years). This method accounts for measurement error, individual variation in the number of samples ascertained, and exposure time trends in urinary phthalate metabolite concentrations. For the remainder of the manuscript, we refer to these measurement error corrected values as urinary phthalate metabolite concentrations.
+
+## Cognitive Outcomes
+
+Three trained examiners assessed children's cognitive abilities using the Wechsler Preschool and Primary Scale of IntelligenceIII (WPPSI-III) at age 5 years (n = 202) and the Wechsler Intelligence Scale for Children-IV (WISC-IV) at age 8 years (n 
+= 220).53–55 Following initial training and certification on these assessments, quality control checks were unannounced and completed (by K.Y.) every 6 months to ensure IQ measures were properly administered and accurately scored. Both instruments assess children's overall intellectual abilities (Full-Scale Intelligence Quotient [FSIQ]) based on subscales for verbal comprehension, working memory, processing speed, and others. In this analysis, we focused on FSIQ as our primary outcome. The FSIQ scores from the WPPSI-III are comparable to those from the WISC-IV, and thus we averaged them if more than one measurement was available to maximize sample size and accommodate our modeling approach.53,54 We used standardized FSIQ 
+scores (i.e., mean = 100 and SD = 15) based on the US normative data. Higher scores indicate better cognitive performance.
+
+## Covariate Assessment
+
+We used directed acyclic graphs to identify potential confounders that may be associated with both race and IQ, considering how confounders may bias the association between race and child IQ through exposure to phthalates during gestation and childhood (eFigure 2; http://links.lww.com/EE/A183 and eFigure 3; http://links.lww.com/EE/A183). Sociodemographic and maternal factors were collected using standardized questionnaires administered by trained interviewers. Child race was determined based on maternal report and assessed separately from maternal race. Child sex was abstracted from hospital medical charts.56 We derived maternal prepregnancy body mass index (BMI) using self-reported height and weight and imputed weight for mothers with missing data.57 Briefly, a SuperLearner algorithm was used to predict prepregnancy weight based on covariates found to be significantly associated with weight from HOME study participants with nonmissing data.58 These covariates included maternal education, marital status, maternal race, insurance status, parity, income, cotinine concentrations, height, and chart-abstracted weight between 13 and 19 weeks of gestation. We assessed secondhand and active tobacco smoke exposure during pregnancy by averaging cotinine concentrations from serum collected at 16 and 26 weeks of gestation and using previously established thresholds to determine active, secondhand, and nonsmoking (unexposed) status using serum cotinine were ≥0.3, <3.0 to ≥0.015, and <0.015ng/mL, respectively.59 Maternal cognitive abilities were assessed when children were ≥1 year using the Wechsler Abbreviated Scale of Intelligence.60 In primary analyses, we adjusted for maternal age, prepregnancy BMI, household income, log10-transformed cotinine concentration, and maternal FSIQ scores.
+
+## Statistical Analyses
+
+We calculated univariate summary statistics and characterized distributions of maternal sociodemographic and reproductive factors by child race. We further explored the distribution of urinary phthalate metabolite concentrations across visits by calculating univariate statistics and creating violin plots.61 We also calculated mean child FSIQ scores by strata of the key covariates stratified by race.
+
+## Mediation Analyses
+
+First, we quantified the racial disparities in child FSIQ scores. We used a linear mixed model to estimate unadjusted and covariate-adjusted differences in FSIQ scores between Black and White children. This represents the total effect. Second, we examined racial disparities in average gestational and childhood urinary phthalate metabolite concentrations by examining univariate statistics and percentage differences in concentrations of urinary phthalate metabolites between Black and White children. In cases where we identified racial differences in average child urinary phthalate metabolite concentrations, we investigated if differences in urinary phthalate concentrations were also present at individual time points at ages 1, 2, 3, 4, 5, and 8 years.
+
+Third, for phthalates that differed by race, we conducted mediation analyses following previously established methods for evaluating health disparities by Bellavia et al.44 We evaluated if associations between child race and average FSIQ score were mediated by gestational or childhood urinary phthalate metabolite concentrations using the R package, Mediation.44,62 This package estimates the average causal indirect effects, average direct effects, and total effects based on previously established methods for linear structural equation modeling.63 We estimated the covariate-adjusted, average direct (association between race and child FSIQ), and average indirect effects (association between race and child FSIQ through average gestational and childhood urinary phthalate concentrations). Note that in models of average phthalate metabolite concentrations for childhood, we only included urinary phthalate concentrations from ages 1, 2, 3, 4, and 5 years, as the outcome of childhood FSIQ score included values at ages 5 and 8 years.
+
+To assess mediation by average gestational and childhood urinary phthalate concentrations, we focused on the natural indirect effect, which is the effect of child race on FSIQ that operates only by changing the value of urinary phthalate metabolite concentrations, as this provides information on the pathways where disparities in child FSIQ originate. We also present the controlled direct effects, which are the effects of child race on child FSIQ score after fixing values of urinary phthalate concentrations (eFigure 4; http://links.lww.com/EE/A183).
+
+## Secondary And Sensitivity Analyses
+
+We conducted mediation analyses to quantify the total, direct, and indirect effects of select phthalate metabolites on the association between child race and child FSIQ using phthalate concentrations at individual time points during childhood. Note that for mediation analyses at age 8 years, child FSIQ at age 8 years was used as the outcome (as opposed to average child FSIQ, which includes FSIQ scores ascertained at ages 5 and 8 years).
+
+We conducted sensitivity analyses to assess the robustness of our results to various adjustments. In addition to previously specified covariates, we adjusted for child sex, quality, and quantity of the caregiving environment as measured using the Home Observation for Measurement of the Environment (HOME inventory),64 time-varying blood lead concentrations, neighborhood socioeconomic position, maternal educational attainment, and parity. HOME inventory measures were assessed by study staff during home visits when children were 1 year of age. The HOME inventory is designed to measure the quality and quantity of stimulation and support available to children in their home.64 In addition to previously identified covariates, we also adjusted for blood lead concentrations at each time point where the relation between phthalates and FSIQ were assessed. We collected whole blood samples from women at 16 and 26 weeks of gestation and children at ages 1, 2, 3, 4, 5, and 8 years using collection materials prescreened for lead contamination.65 Blood lead concentrations were quantified at the Centers for Disease Control and Prevention laboratories using inductively coupled plasma mass spectrometry.66 Neighborhood socioeconomic position among HOME study participants was previously assessed using data from 2000 US census records.67,68 For each participant's 2000 US census tract at 20 weeks of gestation, neighborhood socioeconomic position scores were calculated based on log median household income, percentage of households with interest, dividend, or rental income, the log median value of housing unit, percent of residents who completed high school or college, and percentage of residents with executive, managerial, or professional occupations. For each measure, Z scores were calculated, summed, and categorized into terciles where lower scores represent lower socioeconomic position. We conducted statistical analyses using R Studio (version 4.0.3; R Development Core Team).69
+
+## Results Univariate Statistics: Study Participants And Fsiq Scores
+
+Among 253 mother-child pairs, 90 (38%) were Black children and 145 (62%) were White children. Compared with White children, Black children were more likely to have mothers who were younger (<25 years at the time of gestation) and had acquired less education, with lower household incomes, who were more likely to smoke during pregnancy, be multiparous, and have higher prepregnancy BMI (Table 1; eTable 1; http://links.lww. com/EE/A183). For both Black and White children, FSIQ scores were positively associated with maternal age, maternal education, and household income (Table 2).
+
+In unadjusted analyses, FSIQ scores among Black children were 16 points lower (β = −16.0 [95% confidence interval 
+(CI) = −19.4, ���12.7]) than among White Children. After adjusting for covariates, Black children had FSIQ scores 7 points 
+(β = −7.0 [95% CI, −12.1, −1.8]) lower than White children.
+
+## Univariate Statistics: Mediators (Phthalate Metabolites)
+
+Urinary MCPP, MiBP, MBzP, MBP, and ΣDEHP concentrations were lower during gestation relative to childhood (Figure 1; eTable 2; http://links.lww.com/EE/A183). During gestation and childhood, MBzP (gestation percentage difference, 16 [95% CI = 13, 21]; childhood percentage difference, 13 [95% CI = 12, 15]) and MEP (gestation percentage difference, 15 [95% CI = 11, 19]; childhood percentage difference, 21 [95% CI = 19, 24]) concentrations were higher in Black compared with White children (eTable 2; http://links.lww.com/EE/A183). 
+
+During pregnancy, urinary ΣDEHP concentrations were higher 
+
+| 5 and/or 8 years by child race, according to covariates: the  HOME study (2003–2006) Variable Full sample n (%) Black n (%) White n (%) Overall 253 (100) 90 (36) 145 (57) Maternal age (years) <25 57 (23) 45 (50) 8 (5) 25 to <35 154 (61) 40 (44) 104 (72) 35+ 42 (17) 5 (6) 33 (23) Maternal education High school or less 62 (25) 51 (57) 7 (5) Some college 71 (28) 31 (34) 37 (26) Completed college 120 (47) 8 (9) 101 (70) Maternal race Non-Hispanic Black 83 (33) 82 (91) 1 (0) Non-Hispanic White 152 (60) 5 (6) 142 (98) Other 18 (7) 3 (3) 2 (1) Annual income <$30,000 82 (32) 70 (78) 9 (6) $30,000–$75,000 77 (30) 17 (19) 55 (38) ≥$75,000 94 (37) 3 (3) 81 (56) Maternal smokinga Unexposed 78 (31) 5 (6) 66 (56) Secondhand smoking 116 (46) 40 (44) 71 (49) Active smoking 59 (23) 45 (50) 8 (6) Parity 0 114 (45) 29 (32) 73 (50) 1 79 (31) 29 (32) 46 (32) 2+ 60 (24) 32 (36) 26 (18) Prepregnancy BMI (kg/m2 ) Normal/underweight: <25 125 (49) 32 (36) 83 (57) Overweight: ≥25 to <30 65 (26) 23 (26) 38 (26) Obese: ≥30 63 (25) 35 (39) 24 (17) Child sex Female 140 (55) 57 (63) 76 (52) Percentages reflect columns (e.g., race) within each study sample characteristic. a Maternal smoking during pregnancy estimated based on maternal serum cotinine concentrations   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+in White than Black mothers. All other phthalate metabolite concentrations were similar during gestation and childhood in Black and White participants (percentage differences for average gestation or average childhood, <13; Figure 2). Thus, for subsequent analyses, we focused on MBzP and MEP concentrations.
+
+## Mediation Analyses
+
+Gestational phthalate metabolite concentrations did not independently contribute to racial disparities in child FSIQ (Table  3). Average childhood MEP concentrations showed modest evidence for mediation, but not MBzP. Each 10-fold increase in average childhood MEP concentrations contributed to −1.1 point (95% CI = −3.0, 0.8) difference in the natural indirect effect between race and FSIQ, though the confidence interval includes the null. The direct effect of race on child FSIQ scores, controlling for average childhood MEP 
+concentration, was modestly diminished (β = −5.8 [95% 
+CI = −12, −0.4]).
+
+## Secondary Analyses
+
+The natural indirect effects of MEP on IQ appeared to be stronger during early childhood (Figure  3). The strongest natural indirect effect of MEP on child FSIQ scores was observed at age 2 years (β = −1.9 [95% CI = −4.7, 0.7]; eTable 4; http://links.
+
+| (2003–2006)                                                                                                                                                                                                                                                                                            | Non-Hispanic Black   | Non-Hispanic White   |     |           |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|----------------------|-----|-----------|
+| Variable                                                                                                                                                                                                                                                                                               | n                    | Mean ± SD            | n   | Mean ± SD |
+| Overall                                                                                                                                                                                                                                                                                                | 90                   | 91±13                | 145 | 107±12    |
+| Maternal age (years) <25                                                                                                                                                                                                                                                                               | 45                   | 89±15                | 8   | 101±14    |
+| 25 to <35                                                                                                                                                                                                                                                                                              | 40                   | 91±11                | 104 | 107±12    |
+| 35+                                                                                                                                                                                                                                                                                                    | 5                    | 103±13               | 33  | 108±13    |
+| Maternal education High school or less                                                                                                                                                                                                                                                                 | 51                   | 88±14                | 7   | 98±17     |
+| Some college                                                                                                                                                                                                                                                                                           | 31                   | 92±13                | 37  | 103±13    |
+| Completed college                                                                                                                                                                                                                                                                                      | 8                    | 102±5.9              | 101 | 109±11    |
+| Annual income <$30,000                                                                                                                                                                                                                                                                                 | 70                   | 89±14                | 9   | 98±19     |
+| $30,000 to $75,000                                                                                                                                                                                                                                                                                     | 17                   | 95±11                | 55  | 104±12    |
+| ≥$75,000                                                                                                                                                                                                                                                                                               | 3                    | 103±6.6              | 81  | 110±10    |
+| Maternal smokingb Unexposed                                                                                                                                                                                                                                                                            | 5                    | 96±7.2               | 66  | 106±12    |
+| Secondhand smoking                                                                                                                                                                                                                                                                                     | 40                   | 91±12                | 71  | 107±13    |
+| Active smoking                                                                                                                                                                                                                                                                                         | 45                   | 91±17                | 8   | 111±17    |
+| Parity 0                                                                                                                                                                                                                                                                                               | 29                   | 88±14                | 73  | 109±13    |
+| 1                                                                                                                                                                                                                                                                                                      | 29                   | 91±13                | 46  | 107±10    |
+| 2+                                                                                                                                                                                                                                                                                                     | 32                   | 93±13                | 26  | 103±12    |
+| Prepregnancy BMI (kg/m2 ) Normal/underweight: <25                                                                                                                                                                                                                                                      | 32                   | 88±15                | 83  | 108±12    |
+| Overweight: ≥25 to <30                                                                                                                                                                                                                                                                                 | 23                   | 95±11                | 38  | 107±13    |
+| Obese: ≥30                                                                                                                                                                                                                                                                                             | 35                   | 90±13                | 24  | 104±14    |
+| Child sex Male                                                                                                                                                                                                                                                                                         | 33                   | 89±13                | 69  | 105±13    |
+| Female                                                                                                                                                                                                                                                                                                 | 57                   | 92±14                | 76  | 109±12    |
+| a Average FSIQ values represent the arithmetic mean of repeated child FSIQ scores. In cases  where children only had one FSIQ measure, that value was used to represent the mean. Of the  full analytic sample (n = 253), n = 169 children had two measures of FSIQ ascertained at 5 and  8 yrs (67%). |                      |                      |     |           |
+
+lww.com/EE/A183). While the 95% CI of the indirect effect estimate included the null value, the residual disparity in FSIQ 
+after controlling for MEP was attenuated to less than 5 points 
+(β = −4.9 [95% CI = −11.5, 1.6]; Figure 4).
+
+## Sensitivity Analyses
+
+We conducted sensitivity analyses for MEP. Additional adjustment for child sex, caregiving environment (HOME scores), or parity did not substantially change the pattern of results (eTable 5; http://links.lww.com/EE/A183). However, the total effect of race diminished once we adjusted for additional socioeconomic and environmental factors. Specifically, adjustment for maternal neighborhood socioeconomic position during gestation reduced the racial disparity in child FSIQ scores from 7.0 points (95% CI = −12.1, −1.8) to 6.1 points (95% CI = −11.7, −0.6). Adjusting for time-varying blood lead concentrations also decreased the disparity in child FSIQ scores, with the strongest indirect effect of MEP on child FSIQ observed at age 1 year from 7.0 points to 6.4 points (95% CI = −12.4, −0.4). Adjusting for maternal educational attainment also reduced the racial disparity in child FSIQ from 7.0 points to 6.3 points (95% CI = −11.5, −1.1). In fully adjusted models, where we considered all primary covariates and those included in sensitivity analyses, the racial disparity in child FSIQ was further attenuated to 3.4 points (95% CI = −10.5, 3.7).
+
+## Discussion
+
+By applying mediation methods to a longitudinal birth cohort, we sought to determine if racial disparities in children's cognitive abilities could be explained by early life phthalate exposure. During gestation and early childhood, urinary concentrations of MBzP and MEP were higher among Black than White children. Furthermore, child FSIQ scores were on average 7 points lower among Black children than White children after adjusting for socioeconomic factors. We did not observe compelling evidence that phthalate concentrations mediated racial disparities in child FSIQ, with the exception of MEP. We speculate this could be, in part, due to the disparities in urinary MEP concentrations observed between Black and White children, whereas urinary concentrations of other phthalates did not differ substantially.
+
+There was some evidence that MEP weakly mediated the association between race and child FSIQ. Other investigators have documented racial disparities in child FSIQ31–33 and differences in phthalate exposure across racial groups.13,38,39,43,70 Moreover, some studies report associations between gestational and early life phthalate exposure with decreased child IQ.16–22,24,25 Direct and indirect effects of all phthalate metabolites showed an inverse association with child cognitive abilities, with the exception of MCPP during childhood (β = 1.5 points 
+[95% CI = 0.2, 3.2]). This finding was counterintuitive, as we did not expect phthalates to have a protective effect; however, these results could be spurious as most indirect coefficients were centered around the null. Further, MCPP is a nonspecific phthalate metabolite, as it represents a breakdown product of several other higher molecular weight phthalate diesters.71 Consistent with our prior work in the HOME study, higher urinary MEP concentrations during childhood were associated with lower FSIQ.16 The lack of stronger mediating effects might be due to the relatively modest association that MEP and other phthalates have with child cognitive abilities. Note, while prior work in the HOME study16 did not identify strong evidence for sex-specific associations, future work may want to consider this in analyses investigating health disparities.
+
+MEP is of particular interest for several reasons. First, urinary concentrations of MEP are substantially higher in Black populations than White ones.5,72,73 Independent of socioeconomic position, Black women have higher body burdens of some phthalates and other environmental chemicals than White women, partly due to consumer beauty product use.38,74–77 In addition, Black women who are pregnant reported more use and frequency of use of hair care products during pregnancy than White women.42,78 Second, diethyl phthalate (DEP), the parent compound of MEP, is commonly used in a variety of consumer products such as makeup, nail polish, perfume, deodorant, and various hair products (spray, dyes, mousse, crème rinse, nutrients, and conditioners).5,25–28,72,79 The primary route of exposure for DEP is dermal absorption and suggests that frequent use of DEP containing products may result in higher internal body burdens of MEP compared to phthalates with different routes of exposure.72 In fact, frequent use of hair products during pregnancy, more commonly used among Black than White women, may be associated with decreases in gestational age,42 which is a predictor for child neurodevelopmental deficits.80 Some of these products are heavily marketed to Black communities, highlighting how cosmetics can be an environmental injustice81 but also a potentially modifiable source of exposure.42 This is an important consideration for future work, as consumers generally are often unaware of harmful chemical exposures present in their personal care products.81–85 Differences in exposure 
+
+![5_image_0.png](5_image_0.png)
+
+the HOME study (2003–2006). The shaded area represents the density function of phthalate metabolite concentrations, and the dot represents the median.
+
+Repeated maternal urinary phthalate metabolites assessed at 16 and 26 weeks of gestation. Repeated child urinary phthalate metabolites assessed at ages 1, 2, 3, 4, and 5 years. Note, the average value of urinary phthalate concentrations does not include those collected at age 8 years since the primary outcome, average FSIQ consists of scores from ages 5 and 8 years. Concentrations of ΣDEHP (in ng/mL) were calculated using the following formula: ΣDEHP
+(ng/mL) = (MECPP [ng/mL]/308.33 g/mol + MEHHP [ng/mL]/294.347 g/mol + MEOHP [ng/mL]/292.331 g/mol) × 308.33 g/mol.
+
+to phthalate-containing products could, in part, be due to discriminative advertising practices, 82,83 directed to maintain European, White beauty standards (i.e., hair relaxers and skin lighteners). 84,86 Our study has some limitations. First, the sample size was modest, and it varied during follow-up. We retained fewer Black than White families, which may result in selection bias. The reductions in sample size due to loss to follow-up also
+
+![5_image_1.png](5_image_1.png)
+
+6
+
+| through gestational and childhood urinary phthalate metabolite concentrationsc: the HOME study (2003–2006) Gestationc Childhoodd Metabolite Visit  β (95% CI) P β (95% CI)                                        | P                |                    |                  |                    |       |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|--------------------|------------------|--------------------|-------|
+| MCPP                                                                                                                                                                                                              | Direct effect    | −6.7 (−11.9, −1.6) | 0.01             | −8.6 (−13.8, −3.3) | <0.01 |
+| Indirect effect                                                                                                                                                                                                   | −0.2 (−1.7, 1.3) | 0.82               | 1.5 (0.2, 3.2)   | 0.02               |       |
+| MiBP                                                                                                                                                                                                              | Direct effect    | −7.0 (−12.3, 1.9)  | <0.01            | −7.1 (−12.2, −1.9) | 0.01  |
+| Indirect effect                                                                                                                                                                                                   | 0.1 (−0.3, 0.7)  | 0.72               | 0.0 (−0.7, 0.5)  | 0.86               |       |
+| MBzP                                                                                                                                                                                                              | Direct effect    | −7.2 (−12.6, −1.5) | 0.02             | −7.4 (−12.2, −2.3) | <0.01 |
+| Indirect effect                                                                                                                                                                                                   | 0.1 (−0.4, 0.7)  | 0.77               | 0.5 (−0.3, 1.7)  | 0.23               |       |
+| MBP                                                                                                                                                                                                               | Direct effect    | −7.0 (−11.5, −1.9) | <0.01            | −6.9 (−11.9, −1.9) | <0.01 |
+| Indirect effect                                                                                                                                                                                                   | −0.1 (−0.5, 0.3) | 0.81               | −0.1 (−0.9, 0.5) | 0.70               |       |
+| MCNPe                                                                                                                                                                                                             | Direct effect    | -                  | -                | −7.1 (−12.5, −1.7) | 0.01  |
+| Indirect effect                                                                                                                                                                                                   | -                | -                  | 0.0 (−0.7, 0.7)  | 0.91               |       |
+| MCOPe                                                                                                                                                                                                             | Direct effect    | -                  | -                | −7.1 (−12.2, −1.6) | <0.01 |
+| Indirect effect                                                                                                                                                                                                   | -                | -                  | 0.1 (−0.6, 1.0)  | 0.72               |       |
+| ΣDEHPf                                                                                                                                                                                                            | Direct effect    | −6.3 (−11.8, −1.0) | 0.01             | −7.0 (−12.3, −1.7) | 0.01  |
+| Indirect effect                                                                                                                                                                                                   | −0.6 (−1.9, 0.4) | 0.21               | 0.0 (−0.7, 0.7)  | 0.92               |       |
+| MEP                                                                                                                                                                                                               | Direct effect    | −7.0 (−12.2, −2.0) | <0.01            | −5.8 (−11.6, −0.4) | 0.03  |
+| Indirect effect                                                                                                                                                                                                   | 0.1 (−0.4, 0.8)  | 0.78               | −1.1 (−3.0, 0.8) | 0.24               |       |
+| a Adjusted for maternal age (continuous), prepregnancy BMI (continuous), income (continuous), log-10 transformed average gestational cotinine concentration (continuous), and maternal FSIQ scores  (continuous). |                  |                    |                  |                    |       |
+
+reduce statistical power and precision, limiting our ability to observe subtler associations. Second, measures of child cognition, including assessing IQ, are historically controversial.26–30 Despite efforts to reduce racial, ethnic, and socioeconomic biases in tools for assessing intelligence, some biases remain.31,33,37 Third, we were unable to consider multiple mediators in our analysis to consider both sources of phthalate exposure (i.e., daily personal care product use or fast-food consumption) in addition to phthalate biomarkers, as recommended by Bellavia et al.44 While assessing urinary concentrations of phthalate metabolites can provide an assessment of phthalate exposure from multiple sources, it remains difficult to directly translate these findings to personal recommendations or policy.44 For example, it may be more practical to intervene on behavior patterns related to phthalate-containing product use as a modifiable exposure, rather than any one specific phthalate metabolite.1 Future work could consider phthalate exposure sources, such as hair or beauty products, in addition to biomarkers of phthalates when investigating racial disparities in child FSIQ.
+
+Fourth, while we adjusted for many relevant covariates, residual confounding remains a possibility. For example, adjusting for income as a measure of socioeconomic status may not fully capture the cumulative effects of daily experiences for minority populations, such as racism or chronic stress.33,37 Further, given that adjustment for socioeconomic and environmental factors attenuated the racial differences in IQ by almost half of an SD in IQ scores, additional environmental factors are likely to play a major role in explaining racial disparities in IQ. This is further supported from sensitivity analyses, where additional adjustment for socioeconomic position, blood lead concentrations, and parity attenuated the effect of race on the association between phthalates and FSIQ. In fully adjusted models where we considered all covariates from primary and sensitivity analyses, we observed a large attenuation of racial Figure 3. Adjusted indirect associations between child race and average child FSIQ at ages 5 and/or 8 years mediated through gestational and childhood urinary MEP concentrations: the HOME study (2003–2006). Adjusted for maternal age (continuous), prepregnancy BMI (continuous), income (continuous), log-10 
+
+![6_image_0.png](6_image_0.png)
+
+transformed average gestational cotinine concentration (continuous), and maternal FSIQ scores (continuous). Repeated maternal urinary phthalate metabolites assessed at 16 and 26 weeks of gestation. Repeated child urinary phthalate metabolites assessed at ages 1, 2, 3, 4, and 5 years. Note, the average value of urinary phthalate concentrations does not include those collected at age 8 years since the primary outcome, average FSIQ consists of scores from ages 5 and 8 years.
+
+![7_image_0.png](7_image_0.png)
+
+differences in IQ, emphasizing the potential for multiple maternal, child, and community-level factors to be responsible for disparities in child IQ scores. It is also important to reiterate that race is a social construct, rather than a biological variable. As such, including race as a variable in our models does not capture the totality of minority and marginalized people's experience that may impact child cognitive development. Further, we were underpowered to examine other race or ethnicities in this study, and given the regional nature of the study location (Cincinnati, OH), our findings may not be generalizable to other major US cities. While we considered blood lead levels as an additional coexposure, we did not consider other known chemicals (i.e., organophosphates or polychlorinated biphenyls).17,87 Future work may consider other exposures, as well as chemical mixtures as potential mediators of the race-cognitive abilities relation.88 Fifth, it is possible our study findings are subject to type I 
+error (in the case of MEP), as we did not account for multiple comparisons. We attempted to mitigate this by conducting our statistical analyses in a stepwise fashion, as to reduce the overall number of statistical tests. Further, secondary and sensitivity analyses were only conducted for MEP, as this was the only phthalate metabolite that was found to partly mediate the relation between child race and cognitive abilities.
+
+This study had several strengths. First, we had extensive biomarkers of phthalate exposure and two measures of cognitive ability. Most published studies only had one phthalate biomarker, which is subject to substantial within-person variation due to the episodic nature of phthalate exposure and the short half-life of phthalates.3,9,89,90 Second, we used regression calibration to repeated urinary phthalate concentrations from gestation and early childhood to account for measurement error. 
+
+Third, we applied previously established epidemiological methods to evaluate phthalates as a source of racial disparities in child FSIQ.44
+
+## Conclusion
+
+We observed racial disparities in child cognition and exposure to some phthalates during gestation, infancy, and childhood. 
+
+Overall, we did not find that phthalates mediated racial disparities in child IQ. However, further studies are warranted to determine if efforts to reduce exposure to neurotoxicants may diminish some of the disparities in cognitive abilities observed between Black and White children.
+
+## References
+
+1. Braun JM, Just AC, Williams PL, Smith KW, Calafat AM, Hauser R. 
+
+Personal care product use and urinary phthalate metabolite and paraben concentrations during pregnancy among women from a fertility clinic. J Expo Sci Environ Epidemiol. 2014;24:459–466.
+
+2. Carlstedt F, Jönsson BA, Bornehag CG. PVC flooring is related to human uptake of phthalates in infants. *Indoor Air.* 2013;23:32–39.
+
+3. Hauser R, Calafat AM. Phthalates and human health. Occup Environ Med. 2005;62:806–818.
+
+4. Rudel RA, Gray JM, Engel CL, et al. Food packaging and bisphenol A 
+and bis(2-ethyhexyl) phthalate exposure: findings from a dietary intervention. *Environ Health Perspect.* 2011;119:914–920.
+
+5. Parlett LE, Calafat AM, Swan SH. Women's exposure to phthalates in relation to use of personal care products. *J Expo Sci Environ Epidemiol.* 2013;23:197–206.
+
+6. Hernández-Díaz S, Su YC, Mitchell AA, Kelley KE, Calafat AM, Hauser R. Medications as a potential source of exposure to phthalates among women of childbearing age. *Reprod Toxicol.* 2013;37:1–5.
+
+7. Agency for Toxic Substances and Disease Registry (ATSDR). 
+
+Toxicological profile for Di(2-Ethylhexyl)Phthalate (DEHP). Atlanta GA Agency Toxic Subst Dis Regist. Published online 2004:484.
+
+8. Bornehag CG, Lundgren B, Weschler CJ, Sigsgaard T, Hagerhed-Engman L, Sundell J. Phthalates in indoor dust and their association with building characteristics. *Environ Health Perspect.* 2005;113:1399–1404.
+
+9. Watkins DJ, Eliot M, Sathyanarayana S, et al. Variability and predictors of urinary concentrations of phthalate metabolites during early childhood. *Environ Sci Technol.* 2014;48:8881–8890.
+
+10. Woodruff TJ, Zota AR, Schwartz JM. Environmental chemicals in pregnant women in the United States: NHANES 2003-2004. Environ Health Perspect. 2011;119:878–885.
+
+11. Koch HM, Preuss R, Drexler H, Angerer J. Exposure of nursery school children and their parents and teachers to di-n-butylphthalate and butylbenzylphthalate. *Int Arch Occup Environ Health.* 2005;78:223–229.
+
+12. Koch HM, Drexler H, Angerer J. Internal exposure of nursery-school children and their parents and teachers to di(2-ethylhexyl)phthalate (DEHP). *Int J Hyg Environ Health.* 2004;207:15–22.
+
+13. Silva MJ, Barr DB, Reidy JA, et al. Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999-2000. Environ Health Perspect. 2004;112:331–338.
+
+14. Sørensen LK. Determination of phthalates in milk and milk products by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:1135–1143.
+
+15. Desvergne B, Feige JN, Casals-Casas C. PPAR-mediated activity of phthalates: a link to the obesity epidemic? *Mol Cell Endocrinol.* 2009;304:43–48.
+
+16. Li N, Papandonatos GD, Calafat AM, et al. Identifying periods of susceptibility to the impact of phthalates on children's cognitive abilities. 
+
+Environ Res. 2019;172:604–614.
+
+17. Doherty BT, Engel SM, Buckley JP, Silva MJ, Calafat AM, Wolff MS. 
+
+Prenatal phthalate biomarker concentrations and performance on the Bayley Scales of Infant Development-II in a population of young urban children. *Environ Res.* 2017;152:51–58.
+
+18. Factor-Litvak P, Insel B, Calafat AM, et al. Persistent associations between maternal prenatal exposure to phthalates on child IQ at age 7 years. *PLoS One.* 2014;9:e114003.
+
+19. Huang HB, Chen HY, Su PH, et al. Fetal and childhood exposure to phthalate diesters and cognitive function in children up to 12 years of age: Taiwanese maternal and infant cohort study. *PLoS One.* 2015;10:e0131910.
+
+20. Kim JI, Hong YC, Shin CH, Lee YA, Lim YH, Kim BN. The effects of maternal and children phthalate exposure on the neurocognitive function of 6-year-old children. *Environ Res.* 2017;156:519–525.
+
+21. Kim S, Eom S, Kim HJ, et al. Association between maternal exposure to major phthalates, heavy metals, and persistent organic pollutants, and the neurodevelopmental performances of their children at 1 to 2years of age- CHECK cohort study. *Sci Total Environ.* 2018;624:377–384.
+
+22. Polanska K, Ligocka D, Sobala W, Hanke W. Phthalate exposure and child development: the Polish Mother and Child Cohort Study. *Early* Hum Dev. 2014;90:477–485.
+
+23. Engel SM, Patisaul HB, Brody C, et al. Neurotoxicity of ortho-phthalates: recommendations for critical policy reforms to protect brain development in children. *Am J Public Health.* 2021;111:687–695.
+
+24. Ipapo KN, Factor-Litvak P, Whyatt RM, et al. Maternal prenatal urinary phthalate metabolite concentrations and visual recognition memory among infants at 27 weeks. *Environ Res.* 2017;155:7–14.
+
+25. Téllez-Rojo MM, Cantoral A, Cantonwine DE, et al. Prenatal urinary phthalate metabolites levels and neurodevelopment in children at two and three years of age. *Sci Total Environ.* 2013;461-462:386–390.
+
+26. Gottfredson LS. *Social Consequences of Group Differences in Cognitive* Ability. University of Delaware; 2004.
+
+27. Herrnstein RJ, Murray C. The Bell Curve: Intelligence and Class Structure in American Life. Free Press; 1994.
+
+28. Coyle TR, Pillow DR. SAT and ACT predict college GPA after removing g. *Intelligence*. 2008;36:719–729.
+
+29. Schmidt FL, Hunter JE. The validity and utility of selection methods in personnel psychology: practical and theoretical implications of 85 years of research findings. *Psychol Bull*. 1998;124:262–274.
+
+30. Schmidt FL, Hunter J. General mental ability in the world of work: 
+occupational attainment and job performance. *J Pers Soc Psychol.* 2004;86:162–173.
+
+31. Thomas D. Racial IQ differences among transracial adoptees: fact or artifact? *J Intell*. 2017;5:1.
+
+32. Templer DI, Arikawa H. Association of race and color with mean IQ 
+across nations. *Psychol Rep.* 2006;99:191–196.
+
+33. LeWinn KZ, Bush NR, Batra A, Tylavsky F, Rehkopf D. Identification of modifiable social and behavioral factors associated with childhood cognitive performance. *JAMA Pediatr.* 2020;174:1063–1072.
+
+34. Rushton JP, Jensen AR. Thirty years of research on race differences in cognitive ability. *Psychol Public Policy Law*. 2005;11:235–294.
+
+35. Lynn R. *Race Differences in Intelligence: An Evolutionary Analysis*. 
+
+Washington Summit Books; 2006.
+
+36. Nisbett RE, Aronson J, Blair C, et al. Intelligence: new findings and theoretical developments. *Am Psychol.* 2012;67:130–159.
+
+37. Breslau N, Chilcoat HD, Susser ES, Matte T, Liang KY, Peterson EL. 
+
+Stability and change in children's Intelligence Quotient scores: a comparison of two socioeconomically disparate communities. *Am J Epidemiol.*
+2001;154:711–717.
+
+38. Kobrosly RW, Evans S, Miodovnik A, et al. Prenatal phthalate exposures and neurobehavioral development scores in boys and girls at 6-10 years of age. *Environ Health Perspect.* 2014;122:521–528.
+
+39. Potischman N, Troisi R, Thadhani R, et al. Pregnancy hormone concentrations across ethnic groups: implications for later cancer risk. Cancer Epidemiol Biomarkers Prev. 2005;14:1514–1520.
+
+40. Faupel-Badger JM, Wang Y, Karumanchi SA, et al. Associations of pregnancy characteristics with maternal and cord steroid hormones, angiogenic factors, and insulin-like growth factor axis. *Cancer Causes* Control. 2011;22:1587–1595.
+
+41. Henderson BE, Bernstein L, Ross RK, Depue RH, Judd HL. The early in utero oestrogen and testosterone environment of blacks and whites: potential effects on male offspring. *Br J Cancer.* 1988;57:216–218.
+
+42. Preston EV, Fruh V, Quinn MR, et al. Endocrine disrupting chemical-associated hair product use during pregnancy and gestational age at delivery: a pilot study. *Environ Health*. 2021;20:86.
+
+43. Huang T, Saxena AR, Isganaitis E, James-Todd T. Gender and racial/ethnic differences in the associations of urinary phthalate metabolites with markers of diabetes risk: National Health and Nutrition Examination Survey 2001-2008. *Environ Health.* 2014;13:6.
+
+44. Bellavia A, Zota AR, Valeri L, James-Todd T. Multiple mediators approach to study environmental chemicals as determinants of health disparities. *Environ Epidemiol.* 2018;2:e015.
+
+45. Braun JM, Kalloo G, Chen A, et al. Cohort profile: the Health Outcomes and Measures of the Environment (HOME) study. *Int J Epidemiol.* 2017;46:24.
+
+46. Jackson-Browne MS, Papandonatos GD, Chen A, et al. Identifying vulnerable periods of neurotoxicity to triclosan exposure in children. 
+
+Environ Health Perspect. 2018;126:057001.
+
+47. Stacy SL, Eliot M, Etzel T, et al. Patterns, variability, and predictors of urinary triclosan concentrations during pregnancy and childhood. Environ Sci Technol. 2017;51:6404–6413.
+
+48. Meeker JD, Ferguson KK. Urinary phthalate metabolites are associated with decreased serum testosterone in men, women, and children from NHANES 2011-2012. *J Clin Endocrinol Metab.* 2014;99:4346–4352.
+
+49. Hornung RW, Reed LD. Estimation of average concentration in the presence of nondetectable values. *Appl Occup Environ Hyg*. 1990;5:46–51.
+
+50. Larsen K. Creatinine assay by a reaction-kinetic principle. *Clin Chim* Acta. 1972;41:209–217.
+
+51. Jackson-Browne MS, Papandonatos GD, Chen A, Yolton K, Lanphear BP, Braun JM. Early-life triclosan exposure and parent-reported behavior problems in 8-year-old children. *Environ Int.* 2019;128:446–456.
+
+52. Measurement Error in Nonlinear Models *: A Modern Perspective*, 
+Second Edition. Chapman and Hall/CRC; 2006.
+
+53. Wechsler D. *Manual for the Weschler Intelligence Scale For ChildrenThird Edition (WISC-III)*. The Psychological Corporation; 1991.
+
+54. Wechsler D. The Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III). The Psychological Corporation; 2002.
+
+55. Goldstein S, Naglieri JA (eds). *WISC-IV. In. Encyclopedia of Child* Behavior and Development, Springer; 2011.
+
+56. Werner EF, Braun JM, Yolton K, Khoury JC, Lanphear BP. The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: the HOME study. *Environ Health.* 2015;14:75.
+
+57. van der Laan MJ, Polley EC, Hubbard AE. Super learner. *Stat Appl* Genet Mol Biol. 2007;6:Article25.
+
+58. Braun JM, Kalkbrenner AE, Just AC, et al. Gestational exposure to endocrine-disrupting chemicals and reciprocal social, repetitive, and stereotypic behaviors in 4- and 5-year-old children: the HOME study. Environ Health Perspect. 2014;122:513–520.
+
+59. Benowitz NL, Bernert JT, Caraballo RS, Holiday DB, Wang J. Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004. *Am J Epidemiol.* 2009;169:236–248.
+
+60. David W. *Wechsler Abbreviated Scale of Intelligence* | Second Edition. The Psychological Corporation; 1999. Accessed June 9, 2021. https://www.
+
+pearsonassessments.com/store/usassessments/en/Store/ProfessionalAssessments/Cognition-%26-Neuro/Wechsler-Abbreviated-Scale-ofIntelligence-%7C-Second-Edition/p/100000593.html 61. Hintze JL, Nelson RD. Violin plots: a box plot-density trace synergism. 
+
+Am Stat. 1998;52:181.
+
+62. Tingley D, Yamamoto T, Hirose K, et al. Causal mediation analysis; 2019. https://imai.princeton.edu/projects/mechanisms.html 63. Baron RM, Kenny DA. The moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. *J Pers Soc Psychol.* 1986;51:1173–1182.
+
+64. Caldwell BM, Bradley RH. The home observation for measurement of the environment revisited. *Child Adolesc Ment Health*. 1978;9:25–35.
+
+65. Braun JM, Hornung R, Chen A, et al. Effect of residential lead-hazard interventions on childhood blood lead concentrations and neurobehavioral outcomes: a randomized clinical trial. *JAMA Pediatr.*
+2018;172:934–942.
+
+66. Jones DR, Jarrett JM, Tevis DS, et al. Analysis of whole human blood for Pb, Cd, Hg, Se, and Mn by ICP-DRC-MS for biomonitoring and acute exposures. *Talanta.* 2017;162:114–122.
+
+67. Sears CG, Braun JM, Ryan PH, et al. The association of traffic-related air and noise pollution with maternal blood pressure and hypertensive disorders of pregnancy in the HOME study cohort. *Environ Int.*
+2018;121(pt 1):574–581.
+
+68. Diez Roux AV, Merkin SS, Arnett D, et al. Neighborhood of residence and incidence of coronary heart disease. *N Engl J Med.* 2001;345:99–106.
+
+69. R Core Team. *R: A Language and Environment for Statistical* Computing. R Foundation for Statistical Computing; 2020. https:// www.R-project.org/.
+
+70. James-Todd TM, Meeker JD, Huang T, et al. Racial and ethnic variations in phthalate metabolite concentration changes across full-term pregnancies. *J Expo Sci Environ Epidemiol.* 2017;27:160–166.
+
+71. Calafat AM, Silva MJ, Reidy JA, et al. Mono-(3-carboxypropyl) phthalate, a metabolite of di-n-octyl phthalate. *J Toxicol Environ Health A.*
+2006;69:215–227.
+
+72. Wallner P, Kundi M, Hohenblum P, Scharf S, Hutter HP. Phthalate metabolites, consumer habits and health effects. *Int J Environ Res* Public Health. 2016;13:E717.
+
+73. Bloom MS, Wenzel AG, Brock JW, et al. Racial disparity in maternal phthalates exposure; association with racial disparity in fetal growth and birth outcomes. *Environ Int.* 2019;127:473–486.
+
+74. James-Todd TM, Chiu YH, Zota AR. Racial/ethnic disparities in environmental endocrine disrupting chemicals and women's reproductive health outcomes: epidemiological examples across the life course. *Curr* Epidemiol Rep. 2016;3:161–180.
+
+75. Varshavsky JR, Zota AR, Woodruff TJ. A novel method for calculating potency-weighted cumulative phthalates exposure with implications for identifying racial/ethnic disparities among U.S. reproductive-aged women in NHANES 2001-2012. *Environ Sci Technol.*
+2016;50:10616–10624.
+
+76. Branch F, Woodruff TJ, Mitro SD, Zota AR. Vaginal douching and racial/ethnic disparities in phthalates exposures among reproductive-aged women: National Health and Nutrition Examination Survey 2001-2004. *Environ Health.* 2015;14:57.
+
+77. James-Todd T, Connolly L, Preston EV, et al. Hormonal activity in commonly used Black hair care products: evaluating hormone disruption as a plausible contribution to health disparities. J Expo Sci Environ Epidemiol. 2021;31:476–486.
+
+78. Chan M, Mita C, Bellavia A, Parker M, James-Todd T. Racial/ethnic disparities in pregnancy and prenatal exposure to endocrine-disrupting chemicals commonly used in personal care products. *Curr Environ* Health Rep. 2021;8:98–112.
+
+79. Buckley JP, Palmieri RT, Matuszewski JM, et al. Consumer product exposures associated with urinary phthalate levels in pregnant women. J Expo Sci Environ Epidemiol. 2012;22:468–475.
+
+80. Twilhaar ES, Wade RM, de Kieviet JF, van Goudoever JB, van Elburg RM, Oosterlaan J. Cognitive outcomes of children born extremely or very preterm since the 1990s and associated risk factors: a meta-analysis and meta-regression. *JAMA Pediatr.* 2018;172:361–367.
+
+81. Zota AR, Shamasunder B. The environmental injustice of beauty: framing chemical exposures from beauty products as a health disparities concern. *Am J Obstet Gynecol.* 2017;217:418.e1–418.e6.
+
+82. Krieger N. Discrimination and health inequities. *Int J Health Serv.*
+2014;44:643–710.
+
+83. Bailey ZD, Krieger N, Agénor M, Graves J, Linos N, Bassett MT. 
+
+Structural racism and health inequities in the USA: evidence and interventions. *Lancet.* 2017;389:1453–1463.
+
+84. Parameswaran R, Cardoza K. Melanin on the margins: advertising and the cultural politics of fair/light/white beauty in India. *J Commun* Monogr. 2009;11:213–274.
+
+85. Bristor JM, Lee RG, Hunt MR. Race and ideology: African-American images in television advertising. *J Public Policy Marketing*. 1995;14:48–59.
+
+86. Bristor JM, Lee RG, Hunt MR. *Race and Ideology: African-American* Images in Television Advertising; 1995. Accessed February 22, 2021. https://journals.sagepub.com/doi/abs/10.1177/074391569501400105 87. Vreugdenhil HJ, Lanting CI, Mulder PG, Boersma ER, Weisglas-Kuperus N. Effects of prenatal PCB and dioxin background exposure on cognitive and motor abilities in Dutch children at school age. *J Pediatr.*
+2002;140:48–56.
+
+88. Kalloo G, Wellenius GA, McCandless L, et al. Chemical mixture exposures during pregnancy and cognitive abilities in school-aged children. 
+
+Environ Res. 2021;197:111027.
+
+89. Adibi JJ, Whyatt RM, Williams PL, et al. Characterization of phthalate exposure among pregnant women assessed by repeat air and urine samples. *Environ Health Perspect.* 2008;116:467–473.
+
+90. Braun JM, Smith KW, Williams PL, et al. Variability of urinary phthalate metabolite and bisphenol A concentrations before and during pregnancy. *Environ Health Perspect.* 2012;120:739–745.

medical/md/PMC9128869.md

@@ -0,0 +1,288 @@
+# Review Article
+
+![0_image_0.png](0_image_0.png)
+
+# Challenges In Clinical Research And Care In Pruritus
+
+Manuel P. PEREIRA1, Claudia ZEIDLER1, Michael STORCK2, Konstantin AGELOPOULOS1, Wolfgang G. PHILIPP-DORMSTON3, Alexander ZINK4 and Sonja STÄNDER1 1Department of Dermatology and Center for Chronic Pruritus, University Hospital Münster, 2Institute of Medical Informatics, University of Münster, Münster, 3Faculty of Health, University Witten/Herdecke, and 4Department of Dermatology and Allergy, School of Medicine, Technical University of Munich, Munich, Germany Acta DermatoVenereologica Advances in dermatology and venereology Chronic pruritus is a frequent global condition. The pathophysiology, underlying aetiology, clinical manifestation, associated burden and response to therapy of chronic pruritus varies from patient to patient, making clinical research and management of this condition challenging. There are still several unmet needs, such as the need to standardize translational research protocols, diagnostic and therapeutic procedures and to enhance the knowledge of the humanistic and economic burden associated with chronic pruritus. Basic and clinical research is of the utmost importance to target these matters. Clinical research has the potential to identify new relevant mechanisms in affected patients, which may lead to identification of novel therapy targets. This article discusses in depth current shortcomings in the daily care of patients with chronic pruritus and the challenges clinical researchers and physicians treating chronic pruritus face in addressing these matters. 
+
+Key words: itch; patient-reported outcome; guideline; clinical trials; clinical research. Accepted Oct 15, 2019; Published Jan 9, 2020 Acta Derm Venereol 2020; 100: adv00028.
+
+Corr: Sonja Ständer, Department of Dermatology and Center for Chronic Pruritus, University Hospital Münster, Von-Esmarch-Str. 58, DE-48149 Münster, Germany. E-mail: Sonja.Staender@ukmuenster.de Chronic pruritus (CP), defined as pruritus lasting for 6 weeks or longer, is highly prevalent, affecting approximately one-sixth of the population in Germany 
+(1). Population-based analyses of the nationwide search volume of pruritus on Google suggest an even larger number of individuals suffering from CP within Germany and the USA (2–4). Those affected often report a substantial burden and a relevant impairment of their quality of life (QoL) (5, 6). The aetiology, clinical manifestation, diagnostic measures and therapeutic response to CP vary from patient to patient, which contributes to the challenging management of CP (7). Although recent years have witnessed a marked increase in basic and clinical research efforts in this field, CP researchers and physicians treating CP currently face several challenges when targeting unmet needs that impact on clinical routine. The aim of this review is to highlight and discuss 
+
+## Significance
+
+Itch lasting for 6 weeks or more is considered chronic and represents a high burden for those affected. Various aspects of chronic itch, including the underlying origin of the itch, symptoms, skin manifestations, response to therapies and impairment of quality of life vary from patient to patient, constituting a challenge for clinicians and clinical researchers. Unmet needs, such as the standardization of experimental and clinical research protocols, diagnostic procedures and therapeutic regimens, as well as a better understanding of associated burdens and the development of novel effective therapies should be targeted by physicians and researchers dealing with chronic itch.
+
+methodologies used in CP research, as well as current challenges and unmet needs in clinical care.
+
+## Chronic Pruritus: A Multidimensional Condition
+
+CP is a heterogeneous condition in terms of demographics, clinical presentation and underlying origin. Although the prevalence of CP increases with age, patients of all age groups, including children, may be affected 
+(8). Moreover, patients of both sexes and all ethnic backgrounds may suffer from CP. According to the International Forum for the Study of Itch (IFSI), CP may present on inflamed skin (IFSI I), on normal appearing skin (IFSI II), or accompanied by chronic scratch lesions, such as chronic prurigo or lichen simplex chronicus (IFSI III) (9). As for the underlying aetiology, CP may arise from dermatological, systemic, neurological or psychiatric/ psychosomatic conditions. Some patients show multiple causes for the pruritus (multifactorial CP), while in few cases the origin of the pruritus remains unknown despite extensive diagnostic work-up (9). Recent studies have further suggested a substantial impact of climate and weather on the prevalence of pruritus, especially involving specific localizations on the body, but these issues have yet to be examined in epidemiological studies (2–4). Adding to these factors, patients with CP vary in terms of medical history, comorbidities, co-medication, socioeconomic backgrounds and therapy goals, contributing to the complexity of the management of these patients. 
+
+Acta Derm Venereol 2020; 100: adv00028 This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2020 Acta Dermato-Venereologica.
+
+doi: 10.2340/00015555-3348
+
+## Unmet Needs In Clinical Research
+
+Due to the complexity and multidimensional nature of CP, clinical research investigating several aspects of pruritic diseases and its management is needed. In particular, clinical research contributes to a better understanding of pathophysiological mechanisms involved in the development and chronicity of CP in humans. Moreover, clinical researchers should focus on various aspects of clinical management, such as standardizing diagnostic and therapeutic procedures, and assessing the humanistic burden and impact of CP on affected patients. Another important area is the performance of longitudinal translational studies, clinical trials investigating novel agents for the treatment of CP, and psychometric research. It should also be investigated how the care of patients with CP is integrated into the health system in order to identify shortcomings and to adopt innovative strategies, as, for example, digital tools to improve care. The next section of this article discusses the challenges and difficulties with regard to these issues. 
+
+## Challenges In Clinical Research And Care Understanding The Pathophysiology
+
+Basic and clinical research has led in recent decades to a better understanding of the mechanisms underlying CP. 
+
+In clinical-translational research, different methods investigating specific pathophysiological features that augment data obtained from morphological and molecular biological research have been established and validated. The collaboration with biostatisticians and experts of medical informatics is essential for the interpretation of data and for obtaining a comprehensive understanding of the results. An overview of these methods is given in **Table I**. 
+
+Several methodologies have been developed in order to investigate the functions of peripheral nerves. Skin stimulation with pruritogens (e.g. histamine, capsaicin, cowhage, β-alanine) has led to the characterization of several subpopulations of C-fibres. Histamine activates mechano-insensitive C-fibres (CMi-fibres), while cowhage and β-alanine activate heat and mechano-sensitive C-fibres (CMH-fibres) (10). Hyperknesis related to cowhage-induced pruritus, but not histaminergic pruritus, is present in several types of CP compared with controls, arguing for sensitization of CMH-fibres in CP (11). One limitation of this methodology is the high inter-individual variability, while intra-individual variability is low (12). Another method, transcutaneous electrical stimulation, allows the selective activation of subpopulations of peripheral nerve fibres. For example, using the Neurometer® C, Aδ and Aβ fibres are activated by electrical stimulation at a frequency of 5, 250 and 2,000 Hz, respectively (13). Higher itch intensities could be induced upon stimulation at 5 and 2,000 Hz in patients with CP of different origins compared with healthy individuals, arguing for hyperknesis in patients with CP (14). Of note, patients are often anxious when undergoing experimental procedures with electrical stimulation, which leads to biased ratings of evoked sensory symptoms, thus constituting a limitation of this method. Another functional well-established diagnostic tool that is used for clinical itch research is quantitative sensory testing (QST). It is a battery of psychophysical tests, in which response to graded thermal and mechanical stimuli is assessed (15). This procedure, originally developed for determination of neuropathic pain, informs about gain or loss of function of different peripheral nerve fibre subpopulations and hints at possible signs of central sensitization. In itch research it could be shown that patients with CP of different origins have an altered QST profile, arguing for disordered peripheral neuronal mechanisms 
+(11). Although QST is widely used in neurological routine care, it has some limitations. It is time-consuming, requires highly qualified personnel, as well as the collaboration of patients. Morphological methodologies for investigation of the anatomy of cutaneous cells and expression of receptors and mediators are essential in clinical itch research. In particular, determination of the intraepidermal nerve fibre density is useful in order to reveal potential epidermal neuroanatomical alterations (16). In this examination, a skin biopsy is obtained and intraepidermal nerves are stained with the axonal marker protein gene product 9.5 (17). This simple method, which is also used in routine care, has some limitations. 
+
+Table I. Clinical research methodologies used in itch research 
+
+| Methodology                                     | Mechanism                                                                                                                                                                                              |
+|-------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+| Cutaneous stimulation with histamine            | Chemical-evoked itch via activation of mechano-insensitive C-fibres (CMi-fibres), mast cell degranulation                                                                                              |
+| Cutaneous stimulation with cowhage or ß-alanine | Chemical-evoked itch via activation of mechano and heat-sensitive C-fibres (CMH-fibres) as well as A-fibres.                                                                                           |
+| Hyperknesis                                     | Increased itch in response to (chemical, electrical) pruritogens or pinprick stimulation                                                                                                               |
+| Alloknesis                                      | Itch response to non-pruritogenic stimuli: touch/brush strokes. Functional testing of itch sensitization  phenomena with punctuate or dynamic mechanical stimulation (von Frey filaments, cotton swab) |
+| Transcutaneous electrical stimulation           | Electrical-evoked itch via selective activation of peripheral nerve fibre classes                                                                                                                      |
+| Quantitative sensory testing                    | Psychophysical testing of subpopulations of peripheral nerve fibres (C-fibres, Aδ-fibres, Aβ-fibres) and signs  of central sensitization using graded thermal and mechanical stimuli                   |
+| Functional magnetic resonance imaging/positron  | Identification of activated and deactivated cerebral centres upon pruritic stimulation                                                                                                                 |
+| emission tomography                             |                                                                                                                                                                                                        |
+| Conditioned pruritic modulation                 | Central descending inhibition of pruritus                                                                                                                                                              |
+| Intraepidermal nerve fibre density              | Morphological determination of epidermal nerve fibre numbers (skin biopsy)                                                                                                                             |
+
+Acta DermatoVenereologica The skin probe is sensitive to the fixative and needs to be processed in a timely manner in the laboratory. In addition, reference values are available only for the distal lower leg (18, 19), thus interpretation of values from other body sites is difficult. It is a purely morphological examination, which does not inform on functional alterations of peripheral nerve fibres.
+
+Central processing of pruritus is very challenging. 
+
+Most current knowledge on spinal transmission of itch was obtained from animal studies (20). Functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) studies have been used to investigate transmission and processing of pruritus in upper centres. Most studies have been performed on healthy volunteers exposed to pruritic stimulation during the scans. Brain regions associated with sensory recognition, cognition, motor response and emotional states are activated in response to pruritus (21). Interestingly, both overlapping and distinct brain areas are activated upon histaminergic and non-histaminergic pruritic stimulation, pointing to differences in cerebral processing of distinct pruritus types (22). Patients with CP also show differences in brain activation upon stimulation with histamine compared with healthy individuals, arguing for central sensitization in CP (23). Also, structural changes in grey matter have been observed in patients with CP, showing the neuroplasticity in chronic states (24). Although functional imaging studies have enhanced our understanding of cerebral processing of pruritus, this methodology has some pitfalls. fMRI and PET are very expensive methodologies, which are available only in specialized centres. 
+
+In addition, patients with claustrophobia, ferromagnetic prostheses or implanted devices (e.g. defibrillator) cannot undergo an MRI examination, while others (e.g. children) have difficulty lying still during the procedure, leading to poor imaging quality. More imaging studies, enrolling patients with CP of different origins, are needed in order to establish similarities and differences in cerebral brain processing of pruritus across distinct CP conditions. 
+
+Upper centres exert a descending inhibitory modulation via a noradrenergic and a serotonergic pathway, as shown by imaging studies (21, 25). The paradigm of conditioned pain modulation (CPM), in which a noxious stimulus inhibits pain elsewhere in the body, can be used experimentally to assess descending inhibition (26). In patients with CP, as in those with chronic pain, CPM is impaired, which may contribute to the chronicity of the pruritus (11). An analogous paradigm of conditioned pruritic modulation, in which a pruritic stimulus inhibits pruritus elsewhere in the body, has also been investigated, with conflicting results regarding its effect (27, 28). 
+
+More studies are needed to enhance the understanding of the mechanisms of descending modulation in CP and its role in the chronicity of the disease. 
+
+## Standardization Of Assessment Instruments
+
+Since pruritus has a subjective dimension, patientreported outcomes (PRO) play a pivotal role. A plethora of standardized questionnaires and scales have been developed in order to gather information on several aspects of CP, including pruritus characteristics, course of the disease, QoL, reactive disorders, such as anxiety, depression and sleep impairment, and therapy goals. 
+
+Pruritus characteristics (e.g. onset, duration and distribution of pruritus, as well as accompanying sensory symptoms and scratching behaviour) can be assessed using general patient-oriented itch questionnaires (29, 30). Mono-dimensional scales, namely the visual analogue scale, the numerical rating scale and the verbal rating scale, have been validated to assess the intensity of pruritus (31). Intensity scales are also validated for the use in electronic diary applications (32). The interpretation of itch intensity scales is challenging, since scores vary from patient to patient according to external factors, such as sex or ethnic background (1, 33). Some efforts have been put into understanding the minimal clinical relevant difference of itch intensity change, e.g. after initiating a therapy (34). However, many factors (e.g. the aetiology of the pruritus, recall period, socioeconomic background) may influence the minimal clinical relevant difference, making the interpretation of results difficult. 
+
+An overview of other instruments for the assessment of itch is shown in **Table II**.
+
+As for secondary conditions arising due to CP, standardized questionnaires are used to screen for anxiety and 
+
+| Table II. Examples of instruments for itch assessment Instrument Description Visual analogue scale (VAS) Monodimensional scale for assessment of itch intensity; used in clinical trials and routine care (electronic and paper versions  available) Numerical rating scale (NRS) Monodimensional scale for assessment of itch intensity; used in clinical trials and routine care (electronic and paper versions  available) Verbal rating scale (VRS) Categorial scale for assessment of itch intensity; used in clinical trials and routine care (electronic and paper versions available) Labelled magnitude scale (LMS) Monodimensional scale for assessment of itch intensity; used in experimental research (paper version) Itch Severity Scale (ISS) Multidimensional scale for assessment of itch intensity; used in clinical trials (paper version) Dynamic Pruritus Score (DPS) Patient global impression of change for assessment of itch improvement; used in clinical trials and routine care (electronic and  paper versions available) 5-D itch scale (5 dimensions itch  Multidimensional questionnaire for assessment of itch characteristics; used in clinical trials (paper version) scale) ItchyQuant Monodimensional scale for assessment of itch intensity; combination of VAS and cartoon depictions of scratching; developed  for populations with cognitive limitations (elderly, children); used in routine care (paper version)   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Acta DermatoVenereologica Advances in dermatology and venereology depression (e.g. Hospital Anxiety and Depression Scale (35)), to assess sleep disorders (36, 37) and to measure the impairment of QoL (e.g. Dermatological Life Quality Index (38) and pruritus-specific ItchyQol (39)). 
+
+PRO measures are usually collected via paper and pencil questionnaires, but electronic PRO systems are emerging. Computerized collection of PRO measures offers a couple of advantages compared with paper questionnaires: reduction of errors emerging through typewriting, reduction of missing data by requiring completion, and reduction of invalid data by implementation of skip patterns (40). Furthermore, electronic assessment can increase the compliance of completing questionnaires at home by up to 70%, using, for example, computerized reminder functionality (40). To overcome the drawbacks of paper questionnaires, an electronic PRO system, enabling the patient to complete the PRO measures via digital survey, may be implemented (41–43). Since most PRO measures have been validated as paper tools, it cannot be assumed that the electronic version of the same PRO measures deliver the same results for a patient encounter. Gwaltney et al. (40) showed that "as long as substantial changes are not made to the item text or response scales, equivalence studies should not be necessary to demonstrate anew the equivalence or validity of a computerized measure". However, in order to ensure the validity of electronic measures, it would be preferable if the concrete implementations were examined using test–retest or alternate-forms reliability.
+
+Standardization of itch assessment instruments across centres treating patients with CP is of great importance, since it would allow the comparability of data. A first step was already taken by the Task Force Pruritus of the European Academy of Dermatology and Venereology. In a consensus conference, it was agreed that pruritus intensity and QoL should be regarded as the 2 most important parameters to be assessed in routine care. The visual analogue scale and the ItchyQol were regarded as the instruments of choice (44). Another important task is the validation of the assessment tools into various languages in order to be used in clinical trials and routine care across different countries. Future clinical research should focus on showing which instruments have better utility for the clinical practice and which tools are better accepted by patients and physicians. 
+
+## Understanding The Humanistic And Economic Burden
+
+CP, similarly to chronic pain, can lead to a severe impairment of QoL (45–47) and has negative effects on mood, ability to concentrate, quality of sleep, everyday life and work productivity (48, 49). Some patients have mental health problems, such as depression, in response to CP, and are at significantly higher risk of suicide (50, 51). QoL is a highly subjective construct and is influenced by various factors, such as pruritus intensity, but also duration, frequency and localization of pruritus. Another challenge in understanding the impact of pruritic conditions on affected patients is that cross-cultural factors influence the reported intensity of pruritus and QoL rather than the specific dermatological diagnosis. This could be found in a study in 9 European countries enrolling more than 500 patients with CP due to different dermatoses 
+(6). Clinical research should take the cultural background of patients with CP into account when investigating the humanistic burden of pruritic conditions.
+
+In addition to limiting QoL, there are often psychiatric complications. Patients with CP and with a psychosomatic/psychiatric comorbidity suffer more from CP and its consequences. Interestingly, even if their symptoms improve, psychological suffering is higher in these patients (52). In order to cope with these associated disorders, individual multimodal medical care is needed, not only for diagnosing the underlying disease of CP and for symptomatic antipruritic therapy, but also for adjunct therapy against, for example, sleep disorders or mental health conditions. An important challenge for clinical researchers is to detect risk factors for developing mental diseases in patients with CP and thus identify patients at risk. This would aid attending physicians in redirecting vulnerable patients to mental health professionals and ultimately to improve care. 
+
+## Definition Of Treatment Goals
+
+As a multifactorial chronic disease, CP requires complex and cost-intensive therapy. Knowledge of the treatment goals of patients with CP is therefore of great importance in order to plan an efficient therapy regimen. Therapy goals were recorded and evaluated in 2,474 patients with CP using the Patient Benefit Index - Pruritus (PBI-P), which was validated in 2009 for patients with CP and consists of 27 items, including various aspects, such as physical and mental well-being, professional and everyday performance, social and leisure activities, and QoL 
+in general. The relevance of these items is calculated on the basis of a 5-step Likert scale (0="not at all important" to 4="very important") (53). It was shown that, in addition to demographic data such as sex and age, the CP 
+IFSI group, pruritus intensity and QoL had a significant impact on different patient needs. Women considered the reduction in physical and psychological symptoms, such as depressive feeling, nervousness, or burning sensations, as more important (54). For men, an important goal was the improvement in aspects of social life, such as social contacts, partnership, and sex life. Patients with CP on inflamed skin (IFSI I) or pruritus with chronic scratch lesions (IFSI III) considered the healing of the skin lesions as a very important treatment goal (54). Overall, it has been shown that therapeutic goals relate to the diagnosis and medical therapy. Reduction in pruritus or confidence in therapy were considered important or very important Acta DermatoVenereologica Advances in dermatology and venereology by the majority of patients with CP. However, patients with CP focus not only on symptom relief, but also on trustworthy and efficient medical action. Their overall need level seems to be higher than in other dermatological patients as, for example, patients with atopic dermatitis (54). The complexity of patients' needs constitute a challenge for clinical researchers, since patients' needs and goals influence how they perceive their disease and the efficacy of a therapy. Patients' needs should thus be taken into consideration when planning studies involving patients with CP. 
+
+## Standardization Of Diagnostic And Therapeutic Procedures
+
+Owing to the heterogeneity of CP conditions, it is difficult to achieve a standardized diagnostic and therapeutic approach across specialized centres and physicians treating patients with CP. The first guideline addressing CP was published in Germany in 2006 (55) and constituted an important first step in this regard. Since then, the German guideline has been regularly updated (56), while a European guideline was also developed and recently updated (57). 
+
+There is still a lack of randomized controlled trials investigating anti-pruritic drugs, and therefore many of the recommendations of current guidelines are based on case series, case reports and expert opinion. Another challenge that guidelines face is the heterogeneity of CP aetiologies. For instance, while antihistamines are effective in the treatment of urticaria, they do not reduce pruritus in other diseases, such as atopic dermatitis or systemic pruritic conditions (58). Therefore specific therapeutic recommendations are needed for the various pruritic conditions. Clinical trials targeting different pruritic diseases (e.g. atopic dermatitis, uraemic pruritus, cholestatic pruritus, paraneoplastic pruritus) are of the utmost importance in order to generate quality data on which to base therapeutic recommendations. 
+
+Dermatologists are usually the first specialists consulted for CP and play a key role in the management of CP. 
+
+Their job is not only to treat the CP, but also to assign the patient to local physicians of other specialties for specific diagnostic procedures (e.g. medical imaging). However, due to time constraints, taking a comprehensive medical history in patients with CP is challenging in a dermatological practice. Reactive disorders of CP, such as depression and anxiety, need to be considered, as possible causative factors and skin findings, comorbidities and medication must be well documented. The consultation of patients with CP in specialized pruritus centres can offer an important advantage in the management of CP, especially when refractory to basic therapeutic measures, such as antihistamines, topical steroids and the use of emollients. However, the centres are scarce and more are needed for the current demand (7). Pruritus centres should work on an interdisciplinary basis together with medical specialties other than dermatology, such as internal medicine, neurology, pain medicine, psychosomatics, radiology, and medical informatics (59). In addition to outpatient care, specialized centres should also offer the possibility of inpatient care for highly complex patients who require extensive diagnostic procedures or who have relevant psychosocial factors, such as suicidal ideation or severe sleep impairment (59). 
+
+Guideline-based treatment of patients with CP at specialized centres can significantly reduce costs, both in the inpatient and outpatient sectors. This is the result of a recent study in which data from 300 patients with CP were analysed regarding their QoL, health economic burden and therapeutic benefits. Six months after the start of treatment at a specialized pruritus centre there was not only a significant improvement in the pruritus intensity, QoL and therapeutic benefit (PBI-P), but also a significant reduction in all costs (60).
+
+## Need For Novel Effective Therapies
+
+Owing to a better understanding of the mechanisms underlying CP, new promising agents with an anti-pruritic effect have been identified and are being tested in randomized controlled trials. These include monoclonal antibodies (e.g. nemolizumab, tralokinumab), neurokinin-1 receptor antagonists (e.g. serlopitant, aprepitant), opioid modulators (e.g. nalbuphine, nalfurafine), phosphodiesterase-4 inhibitors (e.g. apremilast, crisaborole), janus kinase inhibitors (e.g. tofacitinib) among other novel agents. 
+
+The development of innovative drugs faces important challenges. So far, clinical trials have been performed only for a few indications, especially atopic dermatitis, chronic prurigo, uraemic and cholestatic pruritus. For many other pruritic conditions clinical trials are lacking, and thus no novel drugs are available. 
+
+Since these new agents represent a high cost for the healthcare system, it is important to select the patients who can profit the most from them. Clinical trials and observations from routine care should inform which target population is suitable for each drug. 
+
+At present, innovative drugs being tested in clinical trials are available in only a few centres and thus not all patients have access to them. Licensing of these drugs is needed in order to extend the availability of these promising agents to patients in need. Hence, clinical research should focus on producing high-quality data on the safety and efficacy of novel anti-pruritic drugs, so that regulatory agencies can approve these medications.
+
+## Conclusion
+
+Clinical research efforts in CP have increased in recent years, leading to better understanding of this condition, and ultimately to better care. However, the multidimensional nature of this condition and the heterogeneous population affected by CP pose a challenge for clinical researchers and attending physicians. Unmet needs, such as the shortage of knowledge on chronicity mechanisms, insufficient standardization of a diagnostic and therapeutic approach to CP patients, and the development of novel promising drugs for refractory CP, ought to be targeted by researchers and physicians dealing with CP.
+
+## Acknowledgements
+
+This article was supported by the German Research Foundation 
+(DFG, No. FOR2690 to KA and SST) and by the European Academy of Dermatology and Venereology (EADV, No. 2016-012 to MP and SST). The authors have no conflicts of interest to declare.
+
+## References
+
+1. Matterne U, Apfelbacher CJ, Loerbroks A, Schwarzer T, 
+Buttner M, Ofenloch R, et al. Prevalence, correlates and characteristics of chronic pruritus: a population-based crosssectional study. Acta Derm Venereol 2011; 91: 674–679.
+
+2. Zink A, Schuster B, Ruth M, Pereira MP, Philipp-Dormston WG, Biedermann T, et al. Medical needs and major complaints related to pruritus in Germany: a 4-year retrospective analysis using Google AdWords Keyword Planner. J Eur Acad Dermatol Venereol 2019; 33: 151–156.
+
+3. Tizek L, Schielein M. Influence of climate on Google internet searches for pruritus across 16 German cities: retrospective analysis. J Med Internet Res 2019; 21: e13739.
+
+4. Wongvibulsin S, Khanna R, Kwatra SG. Anatomic localization and quantitative analysis of the burden of itch in the United States. J Am Acad Dermatol 2019. pii: S01909622(19)30993-4.
+
+5. Hay RJ, Johns NE, Williams HC, Bolliger IW, Dellavalle RP, 
+Margolis DJ, et al. The global burden of skin disease in 2010: an analysis of the prevalence and impact of skin conditions. 
+
+J Invest Dermatol 2014; 134: 1527–1534.
+
+6. Steinke S, Zeidler C, Riepe C, Bruland P, Soto-Rey I, Storck M, 
+et al. Humanistic burden of chronic pruritus in patients with inflammatory dermatoses: results of the European Academy of Dermatology and Venereology Network on Assessment of Severity and Burden of Pruritus (PruNet) cross-sectional trial. J Am Acad Dermatol 2018; 79: 457–463.e455.
+
+7. Pereira MP, Steinke S, Bruland P, Ständer H, Dugas M, Augustin M, et al. Management of chronic pruritus: from the dermatological office to the specialized itch center. a review. Itch 2017; 2: e06.
+
+8. Matterne U, Apfelbacher CJ, Vogelgsang L, Loerbroks A, 
+Weisshaar E. Incidence and determinants of chronic pruritus: a population-based cohort study. Acta Derm Venereol 2013; 93: 532–537.
+
+9. Ständer, Weisshaar E, Mettang T, Szepietowski JC, Carstens E, Ikoma A, et al. Clinical classification of itch: a position paper of the International Forum for the Study of Itch. Acta Derm Venereol 2007; 87: 291–294.
+
+10. Johanek LM, Meyer RA, Hartke T, Hobelmann JG, Maine DN, 
+LaMotte RH, et al. Psychophysical and physiological evidence for parallel afferent pathways mediating the sensation of itch. J Neurosci 2007; 27: 7490–7497.
+
+11. Pogatzki-Zahn EM, Pereira MP, Cremer A, Zeidler C, Dreyer T, 
+Riepe C, et al. Peripheral sensitisation and loss of descending inhibition is a hallmark of chronic pruritus. J Invest Dermatol 2019; pii: S0022-202X(19)31851-2.
+
+12. Andersen HH, Sorensen AR, Nielsen GA, Molgaard MS, Stilling P, Boudreau SA, et al. A Test-retest reliability study of human experimental models of histaminergic and non-histaminergic itch. Acta Derm Venereol 2017; 97: 198–207.
+
+13. AAEM Equipment and Computer Committee AAoEM. Technology review: the Neurometer Current Perception Threshold 
+(CPT). Muscle Nerve 1999; 22: 523–531.
+
+14. Pereira MP, Agelopoulos K, Kollner J, Neufang G, Schmelz M, Ständer. Selective nerve fibre activation in patients with generalized chronic pruritus: hint for central sensitization? 
+
+Acta Derm Venereol 2019; 99: 1009–1015.
+
+15. Rolke R, Baron R, Maier C, Tolle TR, Treede RD, Beyer A, 
+et al. Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): standardized protocol and reference values. Pain 2006; 123: 231–243.
+
+16. Lauria G, Hsieh ST, Johansson O, Kennedy WR, Leger JM, 
+Mellgren SI, et al. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society. Eur J Neurol 2010; 17: 903–912, e944–909.
+
+17. Lauria G, Cornblath DR, Johansson O, McArthur JC, Mellgren SI, Nolano M, et al. EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy. Eur J Neurol 2005; 12: 747–758.
+
+18. Lauria G, Bakkers M, Schmitz C, Lombardi R, Penza P, Devigili G, et al. Intraepidermal nerve fiber density at the distal leg: a worldwide normative reference study. J Peripher Nerv Syst 2010; 15: 202–207.
+
+19. Provitera V, Gibbons CH, Wendelschafer-Crabb G, Donadio V, 
+Vitale DF, Stancanelli A, et al. A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg. 
+
+Eur J Neurol 2016; 23: 333–338.
+
+20. Akiyama T, Carstens E. Spinal Coding of Itch and Pain. In: 
+Carstens E, Akiyama T, editors. Itch: mechanisms and treatment. Boca Raton, FL: CRC Press/Taylor & Francis Group, LLC.; 2014.
+
+21. Mochizuki H, Kakigi R. Central mechanisms of itch. Clin Neurophysiol 2015; 126: 1650–1660.
+
+22. Mochizuki H, Papoiu ADP, Yosipovitch G. Brain processing of itch and scratching. In: Carstens E, Akiyama T, editors. Itch: mechanisms and treatment. Boca Raton, FL: CRC Press/
+Taylor & Francis Group, LLC; 2014.
+
+23. Schneider G, Ständer, Burgmer M, Driesch G, Heuft G, 
+Weckesser M. Significant differences in central imaging of histamine-induced itch between atopic dermatitis and healthy subjects. Eur J Pain 2008; 12: 834–841.
+
+24. Papoiu AD, Emerson NM, Patel TS, Kraft RA, Valdes-Rodriguez R, Nattkemper LA, et al. Voxel-based morphometry and arterial spin labeling fMRI reveal neuropathic and neuroplastic features of brain processing of itch in end-stage renal disease. J Neurophysiol 2014; 112: 1729–1738.
+
+25. Mochizuki H, Tashiro M, Kano M, Sakurada Y, Itoh M, Yanai K. 
+
+Imaging of central itch modulation in the human brain using positron emission tomography. Pain 2003; 105: 339–346.
+
+26. Talbot JD, Duncan GH, Bushnell MC, Boyer M. Diffuse noxious inhibitory controls (DNICs): psychophysical evidence in man for intersegmental suppression of noxious heat perception by cold pressor pain. Pain 1987; 30: 221–232.
+
+27. van Laarhoven AI, Kraaimaat FW, Wilder-Smith OH, van de Kerkhof PC, Evers AW. Heterotopic pruritic conditioning and itch - analogous to DNIC in pain? Pain 2010; 149: 332–337.
+
+28. Andersen HH, van Laarhoven AIM, Elberling J, Arendt-Nielsen L. Modulation of itch by conditioning itch and pain stimulation in healthy humans. J Pain 2017; 18: 1437–1450.
+
+29. Yosipovitch G, Zucker I, Boner G, Gafter U, Shapira Y, David M. A questionnaire for the assessment of pruritus: validation in uremic patients. Acta Derm Venereol 2001; 81: 108–111.
+
+30. Weisshaar E, Ständer, Gieler U, Matterne U, Darsow U. Entwicklung eines deutschsprachigen Fragebogens zur Erfassung von chronischen Pruritus (AGP-Fragebogen). Hautarzt 2011; 62: 914–927.
+
+31. Phan NQ, Blome C, Fritz F, Gerss J, Reich A, Ebata T, et al. 
+
+Assessment of pruritus intensity: prospective study on validity and reliability of the visual analogue scale, numerical rating scale and verbal rating scale in 471 patients with chronic pruritus. Acta Derm Venereol 2012; 92: 502–507.
+
+32. Gernart M, Tsianakas A, Zeidler C, Riepe C, Osada N, Pihan D, et al. ItchApp(c): an app-based ediary for assessment of chronic pruritus in clinical trials. Acta Derm Venereol 2017; 97: 601–606.
+
+33. Ständer, Stumpf A, Osada N, Wilp S, Chatzigeorgakidis E, 
+Pfleiderer B. Gender differences in chronic pruritus: women present different morbidity, more scratch lesions and higher burden. Br J Dermatol 2013; 168: 1273–1280.
+
+34. Reich A, Riepe C, Anastasiadou Z, Medrek K, Augustin M, 
+Szepietowski JC, et al. Itch assessment with visual analogue scale and numerical rating scale: determination of minimal clinically important difference in chronic itch. Acta Derm Venereol 2016; 96: 978–980.
+
+35. Snaith RP. The Hospital Anxiety And Depression Scale. Health Qual Life Outcomes 2003; 1: 29.
+
+36. Glenville M, Broughton R. Reliability of the Stanford Sleepiness Scale compared to short duration performance tests and the Wilkinson Auditory Vigilance Task. Adv Biosci 1978; 21: 235–244.
+
+37. Johns MW. A new method for measuring daytime sleepiness: 
+the Epworth sleepiness scale. Sleep 1991; 14: 540–545.
+
+38. Finlay AY, Khan GK. Dermatology Life Quality Index (DLQI) - 
+a simple practical measure for routine clinical use. Clin Exp Dermatol 1994; 19: 210–216.
+
+39. Desai NS, Poindexter GB, Monthrope YM, Bendeck SE, 
+Swerlick RA, Chen SC. A pilot quality-of-life instrument for pruritus. J Am Acad Dermatol 2008; 59: 234–244.
+
+40. Gwaltney CJ, Shields AL, Shiffman S. Equivalence of electronic and paper-and-pencil administration of patient-reported outcome measures: a meta-analytic review. Value Health 2008; 11: 322–333.
+
+41. Rogausch A, Sigle J, Seibert A, Thuring S, Kochen MM, Himmel W. Feasibility and acceptance of electronic quality of life assessment in general practice: an implementation study. Health Qual Life Outcomes 2009; 7: 51.
+
+42. Storck M, Zeidler C, Rehr M, Riepe C, Dugas M, Ständer, et al. Validation of pruritus measures gathered with the electronic patient-reported outcome system MoPat. Acta Derm Venereol 2018; 98: 38–43.
+
+43. Wagner LI, Schink J, Bass M, Patel S, Diaz MV, Rothrock N, 
+et al. Bringing PROMIS to practice: brief and precise symptom screening in ambulatory cancer care. Cancer 2015; 121: 927–934.
+
+44. Ständer, Zeidler C, Riepe C, Steinke S, Fritz F, Bruland P, et al. European EADV network on assessment of severity and burden of Pruritus (PruNet): first meeting on outcome tools. J Eur Acad Dermatol Venereol 2016; 30: 1144–1147.
+
+45. Erturk IE, Arican O, Omurlu IK, Sut N. Effect of the pruritus on the quality of life: a preliminary study. Ann Dermatol 2012; 24: 406–412.
+
+46. Carr CW, Veledar E, Chen SC. Factors mediating the impact of chronic pruritus on quality of life. JAMA Dermatol 2014; 150: 613–620.
+
+47. Kini SP, DeLong LK, Veledar E, McKenzie-Brown AM, Schaufele M, Chen SC. The impact of pruritus on quality of life: the skin equivalent of pain. Arch Dermatol 2011; 147: 1153–1156.
+
+48. Kimball AB, Edson-Heredia E, Zhu B, Guo J, Maeda-Chubachi T, Shen W, et al. Understanding the relationship between pruritus severity and work productivity in patients with moderate-to-severe psoriasis: sleep problems are a mediating factor. J Drugs Dermatol 2016; 15: 183–188.
+
+49. Shutty BG, West C, Huang KE, Landis E, Dabade T, Browder B, et al. Sleep disturbances in psoriasis. Dermatol Online J 2013; 19: 1.
+
+50. Konda D, Chandrashekar L, Rajappa M, Kattimani S, Thappa DM, Ananthanarayanan PH. Serotonin and interleukin-6: 
+Association with pruritus severity, sleep quality and depression severity in Prurigo Nodularis. Asian J Psychiatr 2015; 17: 24–28.
+
+51. Halvorsen JA, Dalgard F, Thoresen M, Bjertness E, Lien L. 
+
+Itch and pain in adolescents are associated with suicidal ideation: a population-based cross-sectional study. Acta Derm Venereol 2012; 92: 543–546.
+
+52. Schneider G, Grebe A, Bruland P, Heuft G, Ständer. Chronic pruritus patients with psychiatric and psychosomatic comorbidity are highly burdened: a longitudinal study. J Eur Acad Dermatol Venereol 2019; 33: e288–e291.
+
+53. Blome C, Augustin M, Siepmann D, Phan NQ, Rustenbach SJ, Ständer. Measuring patient-relevant benefits in pruritus treatment: development and validation of a specific outcomes tool. Br J Dermatol 2009; 161: 1143–1148.
+
+54. Steinke S, Bruland P, Blome C, Osada N, Dugas M, Fritz F, 
+et al. Chronic pruritus: evaluation of patient needs and treatment goals with a special regard to differences according to pruritus classification and sex. Br J Dermatol 2017; 176: 363–370.
+
+55. Ständer, Streit M, Darsow U, Niemeier V, Vogelgsang M, 
+Ständer H, et al. Diagnostisches und therapeutisches Vorgehen bei chronischem Pruritus. J Dtsch Dermatol Ges 2006; 4: 350–370.
+
+56. Ständer, Zeidler C, Augustin M, Bayer G, Kremer AE, Legat FJ, et al. S2k-Leitlinie zur Diagnostik und Therapie des chronischen Pruritus - Update - Kurzversion. J Dtsch Dermatol Ges 2017; 15: 860–873.
+
+57. Weisshaar E, Szepietowski JC, Dalgard FJ, Garcovich S, Gieler U, Gimenez-Arnau AM, et al. European S2k Guideline on Chronic Pruritus. Acta Derm Venereol 2019; 99: 469–506.
+
+58. Klein PA, Clark RA. An evidence-based review of the efficacy of antihistamines in relieving pruritus in atopic dermatitis. Arch Dermatol 1999; 135: 1522–1525.
+
+59. Ständer, Ständer HF, Steinke S, Bruland P, Dugas M, Augustin M. Chronischer Pruritus: Versorgung in der Praxis. Hautarzt 2016; 67: 640–647.
+
+60. Steinke S, Müller S, Gutknecht M, Borowski M, Augustin M, Ständer S, editors. Versorgung von Patienten mit chronischem Pruritus: Kosten und Nutzen der spezialisierten Versorgung in einem Kompetenzzentrum. 50. Tagung der Deutschen Dermatologischen Gesellschaft; Berlin, 2019.
+
+Advances in dermatology and venereology

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+journal homepage: www.elsevier.com/locate/afjem Original article
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+Severe traumatic brain injuries secondary to motor vehicle crashes in two Namibian regions: A retrospective review Paulus Ambundaa,b,∗, Andrit Lourens c a Motor Vehicle Accident Fund of Namibia, Windhoek, Namibia b Department of Health Sciences, Faculty of Health, Applied Sciences and Natural Resources, Namibia University of Science and Technology, Windhoek, Namibia c Division of Emergency Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South *Africa* a r t i c l e i n f o
+
+## A B S T R A C T
+
+| Keywords: Traumatic brain injury MVCs Severe Head injury Head trauma TBI   |
+|----------------------------------------------------------------------------|
+
+Introduction: Traumatic Brain Injuries (TBIs) are a leading cause of morbidity and mortality among trauma patients globally, with motor vehicle crashes (MVCs) being a major contributor. Namibia had a World Health Organization (WHO) estimated MVC-related fatality rate of 30.4 per 100 000 population in 2016, higher than that of the African continent, while no epidemiological studies describing the distribution and determinants of TBIs exist in the country. The study aimed to describe the characteristics of adult patients (≥18 years) with severe TBI secondary to MVCs which occurred in two regions of Namibia between the years 2014–2018.
+
+Methods: A retrospective descriptive observational study was conducted in adult patients who sustained severe TBIs secondary to MVCs in two Namibian regions. The inclusion criteria were patients ≥18 years with a severe
+(as described on the Motor Vehicle Accident Fund system) MVC-related TBI who sustained an injury in the Otjozondjupa or Khomas regions between the years 2014–2018.
+
+Results: A total of 87 patients met the inclusion criteria, 65 (74.7%) from the Khomas region, and 22 (25.3%)
+from the Otjozondjupa region. The overall mean age of patients was 34 years (SD 11.79), most were male
+(n = 78. 89.7%) and 55.2% (n = 48) of all patients sustained an isolated TBI. The majority of the patients were admitted to a state healthcare facility (n = 52, 59.8%). Pedestrians were the most injured (n = 34, 52.3%) in the Khomas region while vehicle drivers were the most injured (n = 11, 50%) in the Otjozondjupa Region. A total of 34 (39.1%) patients died and 53 (60.9%) were discharged from hospital. Overall, there were no statistically significant relationships between patient outcomes and independent variables. Conclusions: The study was to our knowledge the first to describe the epidemiology of TBIs in Namibia. Young individuals are the main people who sustained TBIs, which may subsequently place a socio-economic burden on the country. There is however limited research in Namibia to guide healthcare planning.
+
+## African Relevance
+
+- Provide the first quantitative description of TBI epidemiological characteristics in Namibia
+- Develop a broader understanding of the impact of MVAs in the Namibian setting
+- Empower healthcare sector planning and possible intervention
+- Develop a platform for further research
+
+## Introduction
+
+Traumatic Brain Injuries (TBIs) have become a global health and socio-economic concern and a major contributor to morbidity and mortality amongst trauma patients [1]. The estimated incidence of TBIs, globally, was 10 million cases per annum and 150 cases per 100 000 population in the United States of America (USA) in 2007 [1]. About 4.8 million people died worldwide in 2013 secondary to a TBI and many more were left disabled [2]. The annual (direct and indirect) medical costs of TBIs in the USA in the year 2009 (USD 302 million and USD
+2.8 billion, respectively) demonstrate the significant financial burden caused by TBIs [3].
+
+Motor vehicle crash (MVC) related injuries have increased over the last 30 years with MVCs recognised as the leading cause of TBIs [1,4]. The highest number of MVCs emanates from Africa and South-East Asia
+[1]. Males sustain TBIs more frequently than females with the age of those injured ranging mainly between 20-40 years [5] and it is a major causative factor of morbidity and mortality amongst young adults Abbreviations: ECC, Emergency Communications Centre; LMICs, Low- and middle-income countries; MVCs, Motor Vehicle Crashes; TBIs, Trauma Brain Injuries; WHO, World Health Organization. ∗ Corresponding author.
+
+E-mail *address:* paul.ambunda@gmail.com (P. Ambunda).
+
+https://doi.org/10.1016/j.afjem.2022.04.009 Received 26 May 2021; Received in revised form 18 April 2022; Accepted 22 April 2022 2211-419X/© 2022 The Authors. Published by Elsevier B.V. on behalf of African Federation for Emergency Medicine. This is an open access article under the CC
+BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
+globally [6]. The 2018 World Health Organization (WHO) global status report on road safety states that low- and middle-income countries
+(LMICs) have 85% of the world's population and 60% of the world's registered vehicles while 93% (13% and 80%, respectively) of MVC-related deaths occurs here [7]. In 2013, MVC-related injuries were estimated to cost LMICs between 1% and 2% of their gross national product [8]. Although MVC-related injuries have increased over the last 3 decades, the mortality rates in South Africa and a few other African countries may be decreasing and likely attributed to the development of pre-hospital systems in these regions [4]. Severe TBI patients commonly require lengthy hospital admissions, and the survivors may require long-term rehabilitation for conditions secondary to the TBI, some from which patients never fully recover [6].
+
+Namibia is a sparsely populated, upper-middle-income country in Southern Africa with vast distances between towns. Tertiary level hospitals are mainly located in the capital city, Windhoek (Khomas Region)
+where patients can receive definitive care. These challenges are further compounded by the high rate of MVCs exerting more pressure on the already resource-limited facilities. The WHO (2018) states that MVCrelated death rates are highest in Africa at 26.6 per 100 000 population, however, Namibia had an estimated MVC-related death rate of 30.4 per 100 000 population in 2016 [7]. With no published data on the distribution and determinants of TBIs in Namibia and Namibia identified as a country with a high rate of MVC-related deaths [7], the results of this study will be valuable, allowing the first description of TBI characteristics in Namibia, broadening the understanding of the impact of MVCs, and develop a platform for further research. The study aimed to describe and compare the characteristics of adult patients with severe TBI
+secondary to MVCs which occurred in two Namibian regions between 2014 and 2018 as captured by the Motor Vehicle Accident (MVA) Fund of Namibia.
+
+## Methods Study Design
+
+A descriptive observational study in the form of a retrospective review was conducted to describe the characteristics of adult patients who sustained a severe TBI secondary to a MVC in 2 Namibian regions (Khomas & Otjozondjupa).
+
+## Study Setting And Population
+
+Namibia has a population of about 2.5 million [9] and is divided into 14 regions including the Khomas and Otjozondjupa regions. The Khomas region is centrally located with most of the population residing in Windhoek [10]. Otjozondjupa region, one of the biggest regions, has 4 district hospitals [11] providing emergency care but not definitive care, for which patients require inter-hospital transfers (road or air) to tertiary hospitals in Windhoek. The hospitals are located in Okahandja, Otjiwarongo, Okakarara and Grootfontein. Okahandja is closest to Windhoek at 72km while Grootfontein is the farthest at 458 km. The 2 regions contain four of the five top roads for crash location and severity identified in Namibia and recorded some of the highest injury and fatality rates in the country in the period under study, hence the selection as the study setting [12–17].
+
+Established in 1991, the MVA Fund of Namibia is an organization that designs, develops, promotes, and implements MVC injury prevention measures and aid persons injured in road crashes. As part of assisting MVC victims, the MVA Fund operates a national 24-hour emergency communications centre and emergency medical response services with bases in various towns across the country. The main source of data is the MVA Fund Emergency Communications Centre (ECC), where crashes are reported, through the Toll-free Accident Response Number. Information collected and recorded by the ECC is verified with the Namibian Police, Emergency Medical Rescue Services - Paramedics and Health Officials from public and private hospitals throughout the country. Only crashes which result in injuries are recorded by the MVA Fund ECC. Hence, crashes that result in property damage only, are duly excluded [13–17].
+
+The study population were adult patients (≥18 years old) who sustained a severe TBI (as classified in the MVA Fund system) secondary to an MVC in the Khomas region and Otjozondjupa region in Namibia between the 1st of January 2014 and the 31st of December 2018. The classification of severe TBI is recorded on the MVA Fund system as made by the clinical evaluation of the treating physician.
+
+## Inclusion And Exclusion Criteria The Inclusion Criteria Were As Follows:
+
+- Adult patients (≥18 years)
+- Severe TBI (as classified in the MVA Fund database)
+- Injury sustained in the Khomas or Otjozondjupa region between 1 January 2014 and 31 December 2018 The exclusion criteria were any patient that did not meet the stated inclusion criteria. Patients that died on scene or enroute to hospital were not included. It was hypothesized that the epidemiological characteristics of adult patients who sustained severe TBI in the Khomas region differ from those who sustained injuries in the Otjozondjupa region in terms of the outcome given the time taken to definitive care by patients from the Otjozondjupa region.
+
+## Data Collection
+
+Data was collected from the MVA Fund case management google spreadsheet and SIEBEL system (MVA Fund in-house production system). Patients who sustained a severe TBI were extracted from the case management google spreadsheet and the MVA Fund's SIEBEL system was used to determine the costs, ages, and location of MVCs. Patients whose diagnosis was not clearly stipulated as severe TBI on the case management spreadsheet were excluded as the severity of the injury could not be established.
+
+## Data Analysis
+
+Data were analysed using SPSS statistical package, Version 25 [18].
+
+Descriptive statistics were used to report counts, percentages, mean and standard deviation and presented in tables. Pearson chi-square test for independence was done to determine whether a relationship existed between the dependent variable and independent variables. A p-value of 0.05 was deemed to be statistically significant.
+
+## Ethical Approval
+
+The study received ethical approval from the Ethics Committees of the Namibian University of Science and Technology and the Ministry of Health and Social Services of Namibia. In addition, written permission to conduct research was granted by the MVA Fund. To respect patient confidentiality and anonymity, no patient identifiable data was extracted.
+
+## Results
+
+During the study period, a total of 20 095 crashes were recorded by the MVA Fund of Namibia in which 33 099 occupants were injured. In the Khomas region, 7725 (38.4%) crashes occurred with 8869 (26.8%)
+injuries while 1848 (9.2%) crashes occurred in the Otjozondjupa region with 4372 (13.2%) injuries. A total of 352 TBIs were recorded. This is however not specific to severe TBIs.
+
+| Table 1 Patient characteristics per region. Khomas Region (n = 65)   | Otjozondjupa Region (n = 22)   | Total      |            |
+|----------------------------------------------------------------------|--------------------------------|------------|------------|
+| Characteristics                                                      | n (%)                          | n (%)      | n (%)      |
+| Gender: Male                                                         | 58 (89.2%)                     | 20 (90.9%) | 78 (89.7%) |
+| Female                                                               | 7 (10.8%)                      | 2 (9.1%)   | 9 (10.3%)  |
+| Age Groups: 18-28 Years                                              | 25 (38.5%)                     | 7 (31.8%)  | 32 (36.8%) |
+| 29-39 Years                                                          | 20 (30.8%)                     | 13 (59.1%) | 33 (37.9%) |
+| 40-50 Years                                                          | 11 (16.9%)                     | -          | 11 (12.6%) |
+| 51-61 Years                                                          | 8 (12.3%)                      | 2 (9.1%)   | 10 (11.5%) |
+| >61 Years                                                            | 1 (1.5%)                       | -          | 1 (1.2%)   |
+| Healthcare Facilities: Public Hospital: Katutura State Hospital      | 34 (52.3%)                     | 5 (22.7%)  | 39 (44.8%) |
+| Windhoek Central State Hospital                                      | 4 (6.2%)                       | -          | 4 (4.6%)   |
+| Okahandja District Hospital                                          | -                              | 5 (22.7%)  | 5 (5.7%)   |
+| Otjiwarongo District Hospital                                        | -                              | 3 (13.6%)  | 3 (3.4%)   |
+| Grootfontein District Hospital                                       | -                              | 1 (4.5%)   | 1 (1.1%)   |
+| Total                                                                | 38 (58.5%)                     | 14 (63.6%) | 52 (59.8%) |
+| Private Hospital: Mediclinic Windhoek                                | 16 (24.6%)                     | 5 (22.7%)  | 21 (24.1%) |
+| Roman Catholic Hospital                                              | 6 (9.2%)                       | 2 (9.1%)   | 8 (9.2%)   |
+| Lady Pohamba Private Hospital                                        | 4 (6.2%)                       | 1 (4.5%)   | 5 (5.7%)   |
+| Rehoboth St. Mary's Hospital                                         | 1 (1.5%)                       | -          | 1 (1.1%)   |
+| Total                                                                | 27 (41.5%)                     | 8 (36.4%)  | 35 (40.2%) |
+
+## Patient Characteristics
+
+Eighty-seven patients met the inclusion criteria, 22 (25.3%) from the Otjozondjupa region and 65 (74.7%) from the Khomas region. The mean age of patients in Khomas and Otjozondjupa regions was 34 (SD 12.79)
+years and 32 (SD 8.36) years respectively, while the mean overall age was 34 (SD 11.79) years. The age group, 29-39 years (n = 33, 37.9%),
+contained the highest proportion of patients while 74.7% (n = 65) of all patients were <40 years. Most patients were male (n = 78, 89.7%) while patients were mainly admitted at state healthcare facilities (n = 52, 59.8%) situated in the two regions (Table 1).
+
+## Mvc Characteristics
+
+Most patients in the Khomas region travelled by sedan (n = 42, 64.6%) while in the Otjozondjupa region most travelled by pick-up vehicle (n = 10, 45.5%). In the Khomas region, pedestrians (n = 34, 52.3%) were the most injured while in the Otjozondjupa region, the most common position of an injured occupant was the driver (n = 11, 50%) (Table 2).
+
+## Patient Outcomes
+
+The mortality rate among all included patients (n = 87) was 39.1%
+(n = 34). Amongst the male patients, the mortality rate was 37.1% (29/78) and 55.6% (5/9) amongst females (Table 3). The three age groups, 18-28 years (40.6%), 40-50 years (45.5%) and 51-61 years
+(40.0%), each had a mortality rate of 40.0% or higher while the age group 29-39 years had a 36.4% (12/33) mortality rate. The mortality rate amongst patients with an isolated TBI was 39.6% (19/48) and 38.5% (15/39) in the group of patients with compounding injuries. No statistically significant associations were found between patient outcomes (died or discharged from hospital) and the independent variables
+(age group, gender, type of MVC, vehicle type, occupant position) and facility (public versus private) for the two regions as well as for the overall data.
+
+## Medical Cost
+
+The total medical costs for managing the included patients from the Otjozondjupa region was USD 550 173.47 (N$ 8 423 155.86) and USD 1 315 077.00 (N$ 20 133 842.22) for included patients from the Khomas region. This totaled to USD 1 865 251.34 (N$ 28 556 998.08). The average cost per patient in the Otjozondjupa region was USD 25 007.89 (N$
+382 870.72) while the average cost per patient in the Khomas region was USD 20 231.95 (N$ 309 751.42). These costs include ambulance costs up to hospital expenses involved directly in the medical management of the patients and were extracted from invoices submitted to the MVA Fund for payment of services rendered by medical providers and ambulance service providers.
+
+## Discussion
+
+To our knowledge, this is the first research to describe the characteristics of adult patients who sustained a severe TBI secondary to an MVC
+in Namibia. Our study found that in the two Namibian regions, male patients, and patients 40 years or younger more commonly sustained severe TBIs secondary to MVCs. No statistically significant difference were found between the mortality rates of severe TBI patients in the 2 regions.
+
+## Characteristics Of Tbi Patients
+
+It is well documented in the literature that males sustain TBIs more commonly than females whether in low or high-resource settings [5,19]
+and this has been mainly attributed to behavioral factors, amongst others, such as driving under the influence of alcohol & other illicit substances and reckless driving [5,19].
+
+Pedestrians in the African region have been identified as vulnerable road users at high risk of injury and death [20,21]. Similarly, the current study found a high proportion of pedestrians sustaining a severe TBI and this may be indicative of unsafe pedestrian conditions requiring further investigation and interventions to prevent further injury in this class.
+
+Younger persons (20-40 years), similar as suggested in the current study, are the main population to sustain TBIs. Because this group is the economically active population, TBIs can be expected to place a major socioeconomic burden on countries. Additionally, the transport sector is an integral part of modern economies and MVCs cause a negative impact on a country's economy secondary to capital losses and the loss of the labour force [22].
+
+| Table 2 MVC characteristics per region. Characteristics Khomas Region (n = 65)                                  | Otjozondjupa Region (n = 22)   | Total        |               |
+|-----------------------------------------------------------------------------------------------------------------|--------------------------------|--------------|---------------|
+| n (%)                                                                                                           | n (%)                          | n (%)        |               |
+| Vehicle Type: Pick-up                                                                                           | 15 (23.1%)                     | 10 (45.5%)   | 25 (28.7%)    |
+| Sedan                                                                                                           | 42 (64.6%)                     | 6 (27.2%)    | 48 (55.1%)    |
+| Bus                                                                                                             | 3 (4.6%)                       | 5 (22.7%)    | 8 (9.2%)      |
+| Truck                                                                                                           | 3 (4.6%)                       | -            | 3 (3.4%)      |
+| Motorcycle                                                                                                      | 2 (3.1%)                       | -            | 2 (2.3%)      |
+| Sport Utility Vehicles (SUV)                                                                                    | -                              | 1 (4.5%)     | 1 (1.2%)      |
+| Type of MVC (as characterized by the Namibian police): Rollover 18 (27.7%)                                      | 10 (45.4%)                     | 28 (32.2%)   |               |
+| Pedestrian                                                                                                      | 34 (52.3%)                     | 3 (13.6%)    | 37 (42.5%)    |
+| Head-side                                                                                                       | 7 (10.8%)                      | 3 (13.6%)    | 10 (11.5%)    |
+| With animal                                                                                                     | 2 (3.1%)                       | 1 (0.04%)    | 3 (3.4%)      |
+| With fixed object                                                                                               | 2 (3.1%)                       | 1 (0.04%)    | 3 (3.4%)      |
+| Motorcycle                                                                                                      | 2 (3.1%)                       | -            | 2 (2.3%)      |
+| Fell from moving vehicle                                                                                        | -                              | 1 (0.04%)    | 1 (1.2%)      |
+| Head-on                                                                                                         | -                              | 3 (13.6%)    | 3 (3.4%)      |
+| Occupant Position: Driver                                                                                       | 11 (16.9%)                     | 11 (50%)     | 22 (25.3%)    |
+| Passenger                                                                                                       | 17 (26.2%)                     | 8 (36.4%)    | 25 (28.7%)    |
+| Pedestrian                                                                                                      | 34 (52.3%)                     | 3 (13.6%)    | 37 (42.5%)    |
+| Animal driver                                                                                                   | 1 (1.5%)                       | -            | 1 (1.2%)      |
+| Motorcyclist                                                                                                    | 2 (3.1%)                       | -            | 2 (2.3%)      |
+| Table 3 Mortality rate per age group, gender, and injuries. Khomas Region (n = 65) Otjozondjupa Region (n = 22) | Total                          |              |               |
+| Died n (%)                                                                                                      | Died n (%)                     | deaths       |               |
+| Age groups: 18-28 years                                                                                         | 10/25 (40%)                    | 3/7 (42.9%)  | 13/32 (40.6%) |
+| 29-39 years                                                                                                     | 8/20 (40%)                     | 4/13 (30.8%) | 12/33 (36.4%) |
+| 40-50 years                                                                                                     | 5/11 (45.5%)                   | -            | 5/11 (45.5%)  |
+| 51-61 years                                                                                                     | 3/8 (37.5%)                    | 1/2 (50%)    | 4/10 (40.0%)  |
+| >61 years                                                                                                       | -                              | -            | 0/1 (0%)      |
+| Total                                                                                                           | 26/65 (40.0%)                  | 8/22 (36.4%) | 34/87 (39.1%) |
+| Gender: Male                                                                                                    | 22/58 (37.9%)                  | 7/20 (35.0%) | 29/78 (37.1%) |
+| Female                                                                                                          | 4/7 (57.1%)                    | 1/2 (50%)    | 5/9 (55.6%)   |
+| Total                                                                                                           | 26/65 (40.0%)                  | 8/22 (36.4%) | 34/87 (39.1%) |
+| Injuries: Isolated TBI                                                                                          | 15/39 (38.5%)                  | 4/9 (44.4%)  | 19/48 (39.6%) |
+| Compounding injuries                                                                                            | 11/26 (42.3%)                  | 4/13 (30.8%) | 15/39 (38.5%) |
+| Total                                                                                                           | 26/65 (40.0%)                  | 8/22 (36.4%) | 34/87 (39.1%) |
+
+## Mortality
+
+From the data collected, we were unable to determine the mortality period of the patients and for this reason, patient outcomes were described as discharged from hospital or died. The MVA Fund since 2009 has however adopted the WHO definition of road death which is any death occurring immediately or within 30 days as a result of a road crash.
+
+Although no differences were found between patients that sustained an isolated TBI (39.6%) versus those with compounding injuries with a severe TBI (38.5%), evidence suggests TBI severity to be the major determining factor to patient outcomes while compounding injuries do not necessarily have a significant impact [23].
+
+We expected that mortality in the Otjozondjupa would be higher given the increased time to definitive care. This hypothesis was based on the cornerstone of the management of TBI, which is aimed at preventing secondary TBI, making the initial management vital as it can influence patient outcomes [24]. Pakkanen et al.[25] suggests that better patient survival outcomes and better neurological function post-TBI
+can likely be linked overall to effective pre-hospital care. The Namibian pre-hospital care system is relatively new and still under-developed.
+
+In comparison to the rest of the country, the Khomas region has the most developed pre-hospital care services. Services in the Otjozondjupa region are limited with an absence of ambulances in some areas. Finding no difference between the mortality rates of the two regions was therefore not expected due to the limited resources in the Otjozondjupa region and overall time to definitive care.
+
+Our findings did not suggest worse outcomes in any particular age group, however, other literature suggests older patients have poorer outcomes after sustaining TBIs. This is due to the physiological changes that occur with age which predisposes them to injury and resultant worse outcomes [26].
+
+The patients of the current study were admitted and managed at various facilities, some private facilities and others, public facilities. A cross-analysis found no statistically significant difference in the outcome of the patients in regard to whether they were managed at public or private facilities, however, the study sample was small and not powered to detect these differences.
+
+It is evident that further in-depth studies are required to fully understand and describe the extent of the TBI and more generally the trauma burden in Namibia. Investigating the impact of distance and time to definitive care as well as the impact of pre-hospital interventions on the outcome of severe TBI patients, may assist in focusing attention on the need for emergency care development in the country. Additionally, trauma registries need to be established in Namibia in order to streamline trauma research and care in the country.
+
+## Medical Costs
+
+International studies suggest that the financial burden associated with TBIs in high-resource settings are significant [3,27]. Less is known about the economic burden in Africa, however, the impact of TBIs in low-resource settings is believed to be far exceeding those in highresource settings [1]. Modelling suggests that the estimated total number of TBIs in Africa in 2050 will be almost 6 million cases with the highest occurrence in males between 15 and 34 years and the lowest amount in Southern Africa [28]. Although the medical cost from the current study (USD 1 865 251.34/N$ 28 556 998.08) might seem less significant, it only represents data from two Namibian regions, adults with severe MVC-related TBIs and excludes costs such as rehabilitation, medical benefits, injury grants, etc. Webster et al. [29] reported unpublished data from a 2009 internal audit at Groote Schuur Hospital in Cape Town, South Africa, finding that the direct costs of managing 654 TBI
+(moderate-severe) patients at this hospital were USD 1 139 696.67 (N$
+17 448 756.00) [29]. Although the South African figure is from 4 years prior to our study, the direct cost (USD 1 315 077.00/N$ 20 133 842.22) of managing the 65 severe TBI patients from the Khomas region alone exceeded that of managing patients in the South African study. This may be indicative of the high costs involved in managing severe TBI patients in the country. The true cost of TBIs in Namibia is likely much higher, potentially constraining the growth of the country. Dedicated studies evaluating and describing the financial burden and economic impact of TBIs of any cause in Namibia are vital.
+
+The retrospective nature of the current study may present various potential limitations like selection and information bias, issues with missing data and uncertainty about generalisability. The convenience sampling method employed may have subjected the study findings to selection bias. The study sample was small, affecting the ability to detect a statistically significant difference in patient outcomes even if they existed and raises concerns with generalisability.
+
+In addition, MVC-related TBIs in younger victims, mild and moderate MVC-related TBIs and TBIs secondary to causes other than MVCs was not described. Patients who sustained severe TBIs but were not captured on the tool used for data collection and those that died on scenes or prior to arrival at hospital were not included in the study, potentially resulting in underreporting. The classification of the TBI severity was at the discretion of the medical doctor and the criteria used is not known further restricting the generalisability of the study findings. Although anecdotal information, local neurosurgeons suggest that the Glasgow Coma Scale (GCS) and the Marshall score are the 2 systems used to classify the TBI severity in Namibia.
+
+Furthermore, the data was collected only from the MVA Fund thus further limiting the study, however, data capturing systems remain under-developed in the country which greatly hinders the conduction of research. Despite these limitations, we hope that this study highlights the need for further research aimed at describing the epidemiology of injuries in Namibia and aid in the development of more rigorous data capturing systems to facilitate research.
+
+## Conclusion
+
+The study, to our knowledge, was the first to describe the epidemiology of TBIs in Namibia. Young individuals who are the economically active population sustain more TBIs with potentially greater resultant socio-economic burdens that ensue post-injury. The two regions studied, carried a huge medical cost for managing these patients which is only a fraction of the total cost of managing TBI patients.
+
+A larger focus needs to be placed on trauma research in Namibia, particularly related to MVCs to curb the TBI issue and inform healthcare planning. This research is a step forward in understanding severe TBI's from MVC's in Namibia and can inform healthcare planning, particularly in the pre-hospital setting. More research is however required to study the effect of pre-hospital care, on the outcome of TBI patients in Namibia, as the literature suggests that adequate pre-hospital care may lead to improved outcomes in TBI patients.
+
+## Dissemination Of Results
+
+Results from this study were submitted and presented to the academic staff of the Namibia University of Science and Technology
+(NUST). The research report was also shared with the MVA Fund Chief:
+Human Capital.
+
+## Author'S Contributions
+
+Authors contributed as follows to the conception or design of the work; the acquisition, analysis, or interpretation of data for the work; and drafting the work or revising it critically for important intellectual content: PA contributed 50% and AL contributed 50%. All authors approved the version to be published and agreed to be accountable for all aspects of the work.
+
+## Declaration Of Competing Interest
+
+The corresponding author is an employee of the Motor Vehicle Accident Fund of Namibia. The authors declared no further conflict of interest.
+
+## Acknowledgements
+
+I would like to thank the MVA Fund staff for the assistance offered during the data collection phaseand Mr. Etuhole Mwahi, the statistician who assisted in the data analysis.
+
+## References
+
+[1] Hyder AA, Wunderlich CA, Puvanachandra P, Gururaj G. The impact of traumatic brain injuries: a global perspective. Neurorehabilitation 2007;22(5):341–53. [Internet]Available from https://pubmed.ncbi.nlm.nih.gov/18162698/.
+
+[2] Haagsma JA, Graetz N, Bolliger I, Naghavi M, Higashi H, Mullany EC, et al. The global burden of injury: incidence, mortality, disability-adjusted life years and time trends from the global burden of disease study 2013. Inj Prev 2016;22(1):3–18. [Internet]Available from http://dx.doi.org/10.1136/injuryprev-2015-041616.
+
+[3] Humphreys I, Wood R, Phillips C, Macey S. The costs of traumatic brain injury: a literature review. Clinicoecono Outcomes Res 2013;5:281–7. [Internet]Available from:
+https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699059/pdf/ceor-5-281.pdf.
+
+[4] Adeloye D, Thompson J, Ayo C. The burden of road traffic crashes, injuries and deaths in Africa: a systematic review and meta-analysis. Bull World Health Organ 2016;94(7):510–21. [Internet]Available from http://dx.doi.org/10.
+
+2471/BLT.15.163121.
+
+[5] Krebs E, Gerardo CJ, Park LP, Ricardo J, Vissoci N, Byiringiro JC, et al. Mortalityassociated characteristics of patients with traumatic brain injury at the university teaching hospital of Kigali, Rwanda. World Neurosurg 2017;102:571–82. [Internet]Available from: https://doi.org/10.1016/j.wneu.2017.03.001.
+
+[6] Stocchetti N, Zanier ER. Chronic impact of traumatic brain injury on outcome and quality of life: a narrative review. Crit Care 2016;20(148):1–10. [Internet]Available from: https://doi.org/10.1186/s13054-016-1318-1.
+
+[7] World Health Organisation. Global status report on road safety 2018. Geneva:
+World Health Organisation; 2018. p. 424. Available from https://www.who.int/
+publications/i/item/9789241565684.
+
+[8] World Health Organisation. Global status report on road safety: supporting a decade of action. Geneva: World Health Organisation;; 2013. p. 318. Available from https://apps.who.int/iris/handle/10665/78256.
+
+[9] Review W.P. Namibia population by year (historical). [Internet]. 2020. Available from: https://worldpopulationreview.com/countries/namibia-population/.
+
+[10] Kafidi L. Namibia 2011 population and housing census. Wind. Repub. Namib.
+
+2011:214.
+
+[11] MoHSS. Regional directorates [Internet]. 2019. Available from: http://www.
+
+mhss.gov.na/otjozondjupa
+[12] NRSC. Road accidents in Namibia: statistical report 2009. 2009. [13] MVA Fund. Road crash and claims report 2014. Windhoek: MVA Fund; 2014. p. 31. [14] MVA Fund. Road crash and claims report 2015. Windhoek: MVA Fund; 2015. p. 52.
+
+Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab.
+
+[15] MVA Fund. Road crash and claims report 2016. Windhoek: MVA Fund; 2016. p. 52.
+
+Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab.
+
+[16] MVA Fund. Road crash and claims report 2017. Windhoek: MVA Fund; 2017. p. 52.
+
+Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab.
+
+[17] MVA Fund. Road crash and claims report 2018. Windhoek: MVA Fund; 2018. p. 52.
+
+Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab.
+
+[18] IBM SPSS statistics for windows. Armonk, NY: IBM Corp; 2017.
+
+[19] Peeters S, Blaine C, Vycheth I, Nang S, Vuthy D, Park KB. Epidemiology of traumatic brain injuries at a major government hospital in Cambodia. World Neurosurg 2016;97:580–9. Available from: https://doi.org/10.1016/j.wneu.2016.09.127.
+
+[20] World Health Organisation. Global status report on road safety: time for action.
+
+Geneva: World Health Organisation; 2009. p. 301. Available from www.who.int/
+violence_injury_prevention/road_safety_status/2009.
+
+[21] Onywera VO, Blanchard C. Road accidents: a third burden of disease in subSaharan African. Glob Health Promot 2014;20(4):52–5. [Internet]Available from https://doi.org/10.1177/1757975913502688.
+
+[22] Enu P. Road traffic accidents and macroeconomic conditions in Ghana. Soc Basic Sci Res Rev 2014;2(9):374–93. [Internet]Available from https://www.researchgate. net/publication/276266936_Road_Traffic_Accidents_and_Macroeconomic_
+Conditions_in_Ghana.
+
+[23] Watanabe TW, Awai YK, Wamura AI, Aegawa NM, Ukushima HF, Kuchi KO.
+
+Outcomes after traumatic brain injury with concomitant severe extracranial injuries. Neurol Med Chir 2018;58(9):393–9. [Internet]Available from https://doi.
+
+org/10.2176/nmc.oa.2018-0116.
+
+[24] Wells AJ, Hutchinson PJ. The management of traumatic brain injury. Surgery 2018;36(11):613–20. [Internet]Available from https://doi.org/10.1016/j.mpsur.
+
+2018.09.007.
+
+P.  and A.    of   12  
+[25] Pakkanen T, Nurmi J, Huhtala H, Silfvast T. Prehospital on-scene anaesthetist treating severe traumatic brain injury patients is associated with lower mortality and better neurological outcome. Scand J Trauma, Resusc Emerg Med 2019;27(9):1–7. [Internet]Available from https://doi.org/10.1186/s13049-019-0590-x.
+
+[26] Nnanna N, Babatola B, Gbalipre F. Traumatic brain injury in adults: age, etiology, and treatment outcome. Med Res Chron. 2016;3(1):29–39. [Internet]Available from:
+https://www.academia.edu/22670864/TRAUMATIC_BRAIN_INJURY_IN_ADULTS_ AGE_ETIOLOGY_AND_TREATMENT_OUTCOME.
+
+[27] Scholten AC, Polinder S, Panneman MJM, Beeck EFVan, Haagsma JA. Incidence and costs of bicycle-related traumatic brain injuries in the Netherlands. Accid Anal Prev 2015;81:51–60. [Internet]Available from: https://doi.org/
+10.1016/j.aap.2015.04.022.
+
+[28] Wong JC, Linn KA, Shinohara RT, Mateen FJ. Traumatic brain injury in Africa in 2050: a modeling study. Eur J Neurol 2016;23(2):382–6. [Internet]Available from https://doi.org/10.1111/ene.12877.
+
+[29] Webster J, Balchin R. Traumatic brain injury, the hidden pandemic: a focused response to family and patient experience and needs. S Afr Med J 2015;105(3):194–8.
+
+doi:10.7196/samj.9014.

medical/md/PMC9380560.md

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+Cite this: RSC Adv., 2022, 12, 23263
+
+# Construction Of A Binary S-Scheme S-G-C3N4/Cozf Heterojunction With Enhanced Spatial Charge
+
+![0_Image_0.Png](0_Image_0.Png) Separation For Sunlight-Driven Photocatalytic Performance†
+
+Ali Bahadur,a Shahid Iqbal, *b Mohsin Javed,c Syeda Saba Hassan,c Sohail Nadeem,c Ali Akbar, d Rami M. Alzhrani,e Murefah Mana Al-Anazy, f Eslam B. Elkaeed, g Nasser S. Awwad,h Hala A. Ibrahiumij and Ayesha Mohyuddinc A step-scheme (S-scheme) photocatalyst made of sulfurized graphitic carbon nitride/cobalt doped zinc ferrite (S-g-C3N4/Co-ZF) was constructed using a hydrothermal process because the building of Sscheme systems might increase the lifespan of highly reactive charge carriers. Utilizing cutting-edge methods, the hybrid photocatalyst was evaluated by employing TEM, XPS, XRD, BET, FTIR, transient photo-response, UV-vis, EIS and ESR signals. In order to create a variety of binary nanocomposites (NCs),
+nanoparticles (NPs) of 6% cobalt doped zinc ferrite (Co-ZF) were mixed with S-g-C3N4 at various concentrations, ranging from 10 to 80 wt%. For photocatalytic dye removal, a particular binary NC
+constructed between S-g-C3N4 and Co-ZF produces a huge amount of catalytic active sites. The findings showed that loading of S-g-C3N4 on 6% Co-ZF NPs serves as a good heterointerface for e/h+
+separation and transportation through the S-scheme S-g-C3N4/Co-ZF heterojunction. By boosting the hybrid system's BET surface area for the photocatalytic process, the addition of 6% Co-ZF improves the system's ability to absorb more sunlight and boosts its photocatalytic activity. The highest photo-removal effectiveness (98%), which is around 2.45 times higher than that of its competitors, was achieved by the hybrid photocatalyst system with an ideal loading of 48% Co-ZF. Furthermore, the trapping studies showed that the primary species involved in the MB aqueous photo-degradation were cOH and h+.
+
+Received 21st November 2021 Accepted 8th August 2022 DOI: 10.1039/d1ra08525e rsc.li/rsc-advances aDepartment of Chemistry, College of Science and Technology, Wenzhou-Kean University, Wenzhou, China bDepartment of Chemistry, School of Natural Sciences (SNS), National University of Science and Technology (NUST), H-12, Islamabad, 46000, Pakistan. E-mail: shahidiqbal.chem@sns.nust.edu.pk cDepartment of Chemistry, School of Science, University of Management and Technology, Lahore, Pakistan dDepartment of Physics, University of Agriculture Faisalabad (UAF), Faisalabad, Punjab, 38000, Pakistan eDepartment of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia fDepartment of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia gDepartment of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Riyadh 13713, Saudi Arabia hChemistry Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia iBiology Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia jDepartment of Semi Pilot Plant, Nuclear Materials Authority, P.O. Box 530, El Maadi, Egypt
+† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d1ra08525e
+
+## Introduction
+
+The need for clean water sources has spurred interest on a global scale as a result of the world population's ongoing rise as well as the quickening pace of industrialization and urbanization.1–3 Several textile businesses release byproducts that include water during this fast growth, especially colors like methyl orange (MO) and methylene blue (MB), both of which are very lethal and toxic.4–6 Mixing dirty water with pure water results in considerable environmental pollution.7–9 Degrading incompatible organic molecules and purifying the water are therefore important to save the ecology.10–12 Organic dye molecules are challenging to spontaneously decay due to their bulky and complicated chemical structure.13–15 Organic pollutant elimination is a well-established potential of photon-initiated oxidation processes, notably photocatalysis.16–20 This method's notable benets include the utilization of inexpensive photons, mild working temperatures, nontoxic photocatalysts, and full mineralization.21–26 Light absorption, photocatalytic redox interactions with reactive radicals, and the separation and transport of the photogenerated electron–hole pair are generally the three key components of the photocatalytic process.27–30 Due to its distinctive qualities, such as environmental friendliness, affordability, a moderate working temperature, and high oxidation power, semiconductor-based photocatalysts have been extensively used to remove environmental contaminants from water.31–33 It is a drawback because TiO2 has a signicant bandgap (3.22 eV), which implies that only 4% of the solar spectrum is captured and the rest is lost. There have lately been several novel photocatalytic semiconducting materials that destroy organic complex compounds by reacting to visible light. Physiochemical characteristics, non-toxicity, and outstanding photocatalytic efficiency of spinel ferrites have made them popular photocatalysts in environmental cleanup.34–36 Zinc ferrite (ZnFe2O4) is a spinel-type (AB2O4)
+material that is stable because Zn(II) ions dominate the tetrahedral A-site and Fe(III) ions reside in the octahedral B-sites.34 It has been used in several applications because of its optoelectronic qualities, photochemical stability and narrow bandgap
+(1.9 eV), including magnetic materials, gas sensors, pigment, hydrogen production, solar energy active photocatalyst for the photo-removal of organic pollutants and as an anode material in batteries.6,37 There are several ways to make zinc ferrite (ZF), including solvothermal, co-precipitation, hydrothermal, microemulsion, sol–gel, the reverse micelle technique, solid-state processes and combustion.38,39 Due to its simplicity, ability to produce crystals with the required morphology, size, and form, high productivity, and improved solubility, the hydrothermal process is more oen used than other synthetic techniques.40–42 Additionally, a lot of crystals may be produced using this technique without altering the composition, and materials with high vapor pressure close to their melting point can also be formed.
+
+ZnFe2O4 nanocrystals were created by Fan et al. via a hydrothermal technique, and they were then utilized to degrade acid orange II.43 In addition, Sun et al. and Han et al. examined the photocatalytic efficiency of ZnFe2O4 nanoplates and octahedral ZnFe2O4 produced by hydrothermal techniques against RhB.44,45 However, because of insufficient e and h+ pair separation and substantial recombination of photoinduced e/h+, the photocatalytic efficiency of zinc ferrite is severely constrained.
+
+As a consequence, many areas are being concentrated on increasing the effectiveness of the photocatalytic procedure, involving expanding the light-harvesting spectrum range and boosting the transfer and separation of photoinduced e and h+
+sets.46–48 The use of 2D layered materials and metal-doping are typical methods for achieving these objectives. Numerous investigations have shown that non-local-conjugated structures in conductive polymers improve charge carrier transport and boost photocatalytic performance. Co-ZnFe2O4, 49 ZnFe2O4/gC3N4, 50 ZnFe2O4/graphene,51 and ZnFe2O4/Fe2O3 (ref. 52) are cited in earlier papers as materials that have shown exceptional performance in the elimination of contaminants.
+
+The cobalt-doped ZnFe2O4 (Co-ZF) NPs used in this study have varying percentages of cobalt (2, 4, 6, 8, and 10 wt%) and a 6% cobalt-doped ZnFe2O4 composite with varying percentages
+(12, 24, 48, 60, and 80 wt%) of S-g-C3N4 photodegraded using these synthetic photocatalysts and a suggested mechanism for MB degradation was made.
+
+## Experimental Materials
+
+Ferric nitrate nonahydrate (Fe(NO3)3$9H2O, 99% Alfa Aesar), distilled water, Zn(NO3)3$6H2O, 97% Sinopharm, sodium hydroxide (NaOH, 99% Alfa Aesar), polyethylene glycol (PEG,
+97% DAEJUNG), MB (C16H18ClN3S, 99% Simpsons), cobalt nitrate hexahydrate (Co(NO3)2$6H2O, 97% DAEJUNG) and thiourea (CH4N2S, 99% Sinopharm).
+
+## Synthesis Of Znfe2O4 Nps
+
+ZnFe2O4 NPs were created using the hydrothermal technique. In a nutshell, 2.98 g of Zn(NO3)3$6H2O and 8.09 g of Fe(NO3)3-
+$9H2O were dissolved in 31 mL of distilled water before being combined with 11 mL of polyethylene glycol (PEG) and rapidly agitated for 22 minutes. To keep the mixture's pH at 11, a dropwise addition of a 7 M NaOH solution was made with constant stirring. For hydrothermal treatment at 182 C for 17 hours, this suspension was vacuum sealed in a 100 mL Teon autoclave. The temperature was then raised to the normal range. The item underwent many rounds of ltering and distilled water washings before being dried in the oven for three hours at 85 C. A ne powder was created once the product was nished.
+
+## Synthesis Of Co-Zf Nps
+
+By adjusting the cobalt content of ZnFe2O4 (2, 4, 6, 8, and 10 wt%), cobalt doped ZnFe2O4 (Co-ZF) NPs were also produced hydrothermally. To make 2% Co-ZF, 0.08 g of Co(NO3)2$6H2O,
+8.09 g of Fe(NO3)3$9H2O and 2.91 g of Zn(NO3)3$6H2O were dispersed in 31 mL of distilled H2O. The mixture was then given a further addition of 11 mL of PEG and aggressively agitated for 22 minutes. When the combination's pH reached 11, a dropwise addition of a 7 M solution of NaOH was made while the mixture was continually stirred. For hydrothermal treatment at 182 C
+for 17 hours, this suspension was vacuum sealed in a 100 mL
+Teon autoclave. Then, it was let cool to room temperature. The precipitates underwent ltration, washing, and three hours of drying at 85 C. The substance was then milled into a ne powder aer drying. The same process was also used to create Co-ZF in a sequence with wt% of 4, 6, 8, and 10. Table 1 lists the different compositions and testing circumstances.
+
+## Construction Of S-G-C3N4
+
+Utilizing thiourea and the thermal polycondensation technique, S-g-C3N4 was created. This process included placing thiourea in the crucible and heating it for three hours at a temperature of 555 C at a pace of 6 C min1. Aer being milled into a ne powder, the resulting yellow color agglomerates were produced.
+
+## Designing Of S-G-C3N4/Co-Zf Heterostructures
+
+By combining different amounts (12, 24, 48, 60, and 80 wt%) of S-g-C3N4 with Co-ZF, a series of 6% S-g-C3N4/Co-ZF NCs were made. Solution A was created by dissolving 8.09 g of
+
+| Table 1   | Co-ZF NPs' chemical make-up and an evaluation of their photocatalytic potential Co(NO3)2$6H2O Zn(NO3)3$6H2O Fe(NO3)3$9H2O   | MB dye degradation   |      |      |    |     |
+|-----------|-----------------------------------------------------------------------------------------------------------------------------|----------------------|------|------|----|-----|
+| S. no.    | Doping in wt%                                                                                                               | (g)                  | (g)  | (g)  | pH | (%) |
+| 1         | Pure ZF                                                                                                                     | 0                    | 2.99 | 8.09 | 11 | 37  |
+| 2         | 2% Co-ZF                                                                                                                    | 0.08                 | 2.91 | 8.09 | 11 | 46  |
+| 3         | 4% Co-ZF                                                                                                                    | 0.16                 | 2.83 | 8.09 | 11 | 54  |
+| 4         | 6% Co-ZF                                                                                                                    | 0.24                 | 2.75 | 8.09 | 11 | 60  |
+| 5         | 8% Co-ZF                                                                                                                    | 0.32                 | 2.67 | 8.09 | 11 | 52  |
+| 6         | 10% Co-ZF                                                                                                                   | 0.40                 | 2.59 | 8.09 | 11 | 44  |
+
+Table 2 The progression of the photocatalytic efficacies of the synthesized heterostructures and their composition Fe(NO3)2$9H2O, 2.75 g of Zn(NO3)2$6H2O and 0.24 g of
+
+![2_image_0.png](2_image_0.png)
+
+Co(NO3)2$6H2O in 27 mL of distilled water for the synthesis of 12% NCs of S-g-C3N4 with 6% Co-ZF. To create solution B, 0.217 g of S-g-C3N4 were mixed with 16 mL of distilled water.
+
+Following that, solution A and solution B were combined, and 11 mL of PEG were added. This process took place while the mixture was vigorously stirred for 22 minutes. The pH was
+
+| Wt% of          | MB dye removal   |      |    |
+|-----------------|------------------|------|----|
+| 6% Co-ZF in NCs | S-g-C3N4 (g)     | (%)  |    |
+| 1               | 12%              | 0.26 | 69 |
+| 2               | 24%              | 0.53 | 86 |
+| 3               | 48%              | 1.06 | 98 |
+| 4               | 60%              | 1.32 | 86 |
+| 5               | 80%              | 1.76 | 75 |
+
+raised to 11 by adding the 7 M solution of NaOH dropwise while stirring the mixture continuously. In a Teon autoclave, the suspension was moved, and it was heated for 17 hours at 182 C.
+
+The temperature was then lowered to ambient. The sample underwent three hours of drying at 85 C aer being ltered and many times in distilled water. The item was then crushed into a ne powder when it had dried. The same process was also used to create a series of NCs of S-g-C3N4 with 6% Co-ZF (24, 48, 60, and 80 wt%). For their synthesis, Table 2 lists the experimental conditions and S-g-C3N4 dosage. On the other hand, the ESI† includes the material characterizations and photocatalytic activity data.
+
+## Results And Discussion
+
+Fig. 1 shows the crystallinity and structure of S-g-C3N4 NSs, 6%
+Co-ZF, ZF, and 48% S-g-C3N4/6% Co-ZF NCs as determined by XRD. The cubic spinal structure of 6% Co-ZF, ZF and 48% S-g-
+Table 1 Co-ZF NPs' chemical make-up and an evaluation of their photocatalytic potential C3N4/6% Co-ZF NCs is shown by their XRD patterns. Fig. 1
+
+![3_image_0.png](3_image_0.png)
+
+shows that the crystalline form of spinal ZF is signicantly associated with (JCPDS no. 22.1012) each of the diffraction peaks. The peaks at 2q of 35.3, 30.1, 42.1, 52.1, 62.3 and 56.5 are recognized in the (311), (220), (400), (422), (440) and
+(511) crystal facets of zinc ferrite. The XRD of 6% Co-ZF is also shown in Fig. 1 for your viewing pleasure. Given that cobalt has a smaller ionic radius (0.57 A) than zinc, the pattern shows that adding 6% of cobalt to ZF marginally enhances the broadness of the peaks (0.59 A). Therefore, a smaller crystallite results from the replacement of Co in ZF. The rise in peak widening is proof that Co was successfully doped in ZF. The S-g-C3N4 diffraction pattern is also seen in Fig. 1. The 002 facets of the crystal are assigned a separate peak in this pattern at 27.37, and this pattern closely resembles (JCPDS 00-087-1526) the pattern of Sg-C3N4 that has been described. The intense peaks in the XRD
+pattern of 48% S-g-C3N4/6% Co-ZF NCs are in strong agreement with the existence of pure ZF. This suggests that adding 48% Sg-C3N4 to 6% Co-ZF cannot cause a phase shi, and the fact that there is one peak of SCN at 27.37 indicates that S-g-C3N4 was successfully loaded into 6% Co-ZF.
+
+As shown in Fig. 2, TEM images were used to assess the surface morphologies of manufactured photocatalysts in addition to their crystal structures. Curiously, TEM investigation of S-g-C3N4 revealed that the produced S-g-C3N4 exhibit almost translucent and stretchable 2D homogeneous NSs constructions with bendy shapes (Fig. 2a). Spherical NPs are collected in erratic formations in the ZF TEM picture shown in Fig. 2b, with the size of the particles varying from 22 to 38 nm. This morphology is known as a 0D morphology. However, the Co-ZF with dimensions between 27 and 49 nm was also shown to have a 0D-like shape (Fig. 2c). The TEM picture (Fig. 2d) for the 48%
+S-g-C3N4/6% Co-ZF NCs demonstrates an equal distribution of S-g-C3N4 across the Co-ZF NPs. Co-ZF is evenly dispersed as NPs throughout the S-g-C3N4 segment, which is really a 2D layered structure. The manufacture of 6% Co-ZF NPs on S-g-C3N4 NSs produced the most clearly characterized heterojunction between these two materials.
+
+Additionally, XPS was used to identify the valence state and
+
+![4_image_0.png](4_image_0.png)
+
+elemental conformation and of NCs that were 48% S-g-C3N4/6%
+Co-ZF. According to Fig. S1a,† the Zn 2p1/2 and Zn 2p3/2 particles are responsible for the peaks that occurred at 1044.73 eV and 1021.72 eV in the Zn 2p spectra of S-g-C3N4/6% Co-ZF.53,54 Two unique peaks identied at 529.91 and 531.22 eV that may be linked with Fe–O and Zn–O, correspondingly, are validated by the deconvoluted O 1s observations (Fig. S1b†) of S-g-C3N4/6%
+Co-ZF.55 Two major peaks of the measurements of Fe 2p were used to determine the oxidation states of Fe3+ in the synthesized photocatalyst (Fig. S1c†), Fe 2p1/2 (722.13) and Fe 2p3/2
+(708.43).56 As shown in Fig. S1d,† the deconvoluted spectra of Co 2p exhibit two distinct peaks that may be assigned to the Co 2p1/2 and Co 2p3/2 at BEs of 793.94 and 778.93 eV. The acquired ndings are in line with the prior work for the Co-ZF NPs. Three unique peaks seen in the C 1s spectra of S-g-C3N4/6% Co-ZF NCs may be assigned to the C–C, C]O, and C–O, individually. These peaks are seen at 283.19, 287.16, and 284.71 eV.57,58 In Fig. S1e,†
+the C 1s spectrum is shown. Similarly, three distinct peaks at 397.89, 400.67, and 399.79 eV in the N 1s high-resolution spectra are attributed to the respective N–C–N, N–H, and C–
+(C)3 functions (Fig. S1f†).14 The successful synthesis of hybrid Sg-C3N4/6% Co-ZF NCs was shown by these XPS experimental results. The XPS ndings demonstrated the close contact between Co-ZF and S-g-C3N4, resulting in the formation of the nanoscale hybrid system with a content of 48% S-g-C3N4 and 6%
+Co-ZF.
+
+In order to identify the functional group in prepared samples, FTIR spectroscopy is a crucial method. In the 650–
+4000 cm1 range, FTIR spectroscopy was used to study samples of 6% Co-ZF, ZF, S-g-C3N4 and S-g-C3N4/6% Co-ZF. Their FTIR
+spectra are shown in Fig. 3a. The tetrahedral site's M–O bond is responsible for the distinctive peaks in all FTIR spectra that are in the 849–898 nm range. Our ability to observe the M–O bond's peaks at octahedral positions is limited by our instrumentation.
+
+Peaks at 842 and 782 cm1 are caused by triazine units, whereas peaks in the area of 1201–702 cm1 are caused by C–N and C]N
+stretching in the spectra for S-g-C3N4 and 48% S-g-C3N4/6% CoZF.
+
+As shown in Fig. 3b, the isotherms of N2 desorption–
+absorption for each of the generated samples of ZF, S-g-C3N4, 6% Co-ZF and a 48% S-g-C3N4/6% Co-ZF heterostructure are all simulated. The isotherm of a 48% S-g-C3N4/6% Co-ZF has been reported to be well suited to mesoporous structures and the standard isotherm pattern of the IUPAC.59 ZF, S-g-C3N4, 6% CoZF, and 48% S-g-C3N4/6% Co-ZF heterojunction all had BET
+surface areas computed as 7.98, 29.98, 13.74, and 68.73 m2 g1, correspondingly. In comparison to the ZF, S-g-C3N4, 6% Co-ZF,
+and the 48% S-g-C3N4/6% Co-ZF have a greater surface area.
+
+This is most likely the outcome of the phenomena of arrangement and building of numerous integrated components, which not only promotes the well-dened fabrication but also makes 48% S-g-C3N4/6% Co-ZF more active by generating additional active positions for photocatalysis. High photocatalytic activity is provided by photocatalysts made of 48% S-gC3N4/6% Co-ZF due to the well-dened manufacturing of heterointerface and enhanced surface area. However, the mesoporous 48% S-g-C3N4/6% Co-ZF exhibits an extraordinary capacity to suppress the photogenerated e/h+ pairs recombination processes, which subsequently helps to ne-tune the photocatalytic abilities of the 48% S-g-C3N4/6% Co-ZF
+photocatalyst.
+
+Following that, the light-absorption of the synthesized photocatalysts S-g-C3N4, Co-ZF NPs, ZF NPs and 48% S-g-C3N4/6%
+Co-ZF were explored by UV-vis spectra. Fig. 3c shows the aggregate collection of UV-vis evaluations with wavelengths between 255 and 755 nm. When the UV-vis measurements of Co-ZF and ZF NPs are compared, a consistent variation in absorption (redshi) is seen. Associating the 48% S-g-C3N4/6%
+Co-ZF light absorption spectrum to all other samples, including ZF, S-g-C3N4 and Co-ZF NPs spectra, it is notable that the light cultivation is enriched from 255 nm to 755 nm. This increase in absorption is mostly the result of the integration of the 6% CoZF with S-g-C3N4, which also contributes to enhancing the photocatalytic efficacies of the heterostructures made up of 48%
+S-g-C3N4/6% Co-ZF. Additionally, a signicant improvement has been made in the light-harvesting capacity in the 505–755 nm region, which is important for photocatalytic efficiency.
+
+By creating the Tauc's plot of the UV-vis measurements, shown in Fig. 3c, the energy bandgap values of these produced photocatalysts were evaluated. The computed bandgap values were found to be 2.02 eV, 1.94 eV, and 2.71 eV for the 6% Co-ZF,
+ZF and 48% S-g-C3N4/6% Co-ZF, correspondingly, as shown in Fig. 3c. Because cobalt is doped into the ZF NPs, which most likely leads to the creation of new energy regions below the conduction band, the energy bandgap of 6% Co-ZF may be lowered. The sample with a cobalt concentration of 6% had the lowest energy bandgap out of all the designed samples of Co-ZF (2, 4, 8 and 10%). Similar results were obtained when the energy bandgap of the Co-ZF equated to the S-g-C3N4 and the 48% S-gC3N4/6% Co-ZF fell from 2.71 eV for S-g-C3N4 to 2.23 eV for 48%
+S-g-C3N4/6% Co-ZF. The effective surface fusion of both components may be to blame for this drop in bandgap values, which signicantly increases the photocatalytic abilities of the binary photocatalyst.
+
+Fig. 4 compares the photocatalytic efficiency of a sequence of NCs and ZF with MB. The NCs had Co contents of 2, 4, 6, 8 and 10 wt%. Fig. 4a demonstrates that 6% Co-ZF may degrade MB at a faster rate than other samples, eliminating 60% of it in under 86 min. Later, a nanocomposite of 6% Co-ZF is made with various concentrations of S-g-C3N4 (12, 24, 48, 60 and 80 wt%),
+and MB degradation is conducted to further improve the photocatalytic efficacy of the nanocomposite. To examine the
+
+![5_image_0.png](5_image_0.png)
+
+![6_image_0.png](6_image_0.png)
+
+![6_image_1.png](6_image_1.png)
+
+photocatalytic characteristics of generated NCs with various CoZF (12, 24, 48, 60, and 80 wt%) NPs concentrations, visible light illumination of MB was utilized (Fig. 4b). Our investigation of the photodegradation of organic pollutants over a 48% S-gC3N4/6% Co-ZF revealed that MB degradation may be sped up by raising the Co-ZF content in the 48% S-g-C3N4/6% Co-ZF
+(Table 2). The highest photodegradation yield of 98% that was observed with this concentration under visible-light irradiation proves that the optimal amount of Co-ZF NPs is 50 wt%. For example, when S-g-C3N4/Co-ZF with 80 wt% of Co-ZF content were used as photocatalysts for MB degradation, the yield was reduced to 75%, suggesting that the yield would be lower with further increasing the Co-ZF NPs content. Even aer six-hour runs, the S-g-C3N4/Co-ZF exhibits the highest photodegradation efficiency and chemical stability (Fig. 5a).
+
+Fig. 4c and d illustrate the results of computing the photoremoval rate of dye by photocatalysts using the formula, lnCoC
+
+¼ Kt; where Co and C represent the dye content at zero and t minutes, correspondingly, and K represents the reaction rate. According to this gure, the photo-removal rate of MB is 8.99  104 min1. The photolysis rate of 6% Co-ZF (2.58 103 min1) is greater than the photocatalysis rate of MB utilizing ZF, which is 1.67 103 min1 then by creating a composite of 6% Co-ZF and 48% S-g-C3N4, the rate was further elevated and reached 0.42  103 min1 as a consequence of the synergistic interaction between Co-ZF and S-g-C3N4. Then, it is shown from estimated results that NCs of 48% S-g-C3N4/6%
+Co-ZF showed the greatest 98% photo-removal of dye aer 56 min, whereas with 6% Co-ZF and ZF the degradation of dye was 60% and 37%, correspondingly. Additionally, as demonstrated in Table 3, 48% S-g-C3N4/6% Co-ZF photocatalytic efficiency is practically greater than that of past studies.
+
+Since it is generally known that the chemical constancy is a key factor in a catalyst's ability to be extensively utilized, the 48% S-g-C3N4/6% Co-ZF heterostructures that were created were assessed for their potential to be employed up to six times in the reaction of dye photo-removal under solar light. The catalyst's impressive chemical stability and availability for routine experimental usage were shown by the fact that there was no visible decline in its efficiency even aer 6 runs (Fig. 5a).
+
+Photocurrent studies have been performed with all of the available catalysts to dive further into the underlying factors that contribute to the exceptional photocatalytic efficacy of 48%
+S-g-C3N4/6% Co-ZF for the MB mineralization. This is accomplished by creating a proportional association of photocurrent responses for 48% S-g-C3N4/6% Co-ZF, along with all other catalysts ZF NPs, S-g-C3N4, and 6% Co-ZF NPs, which provides a useful perspective on the transportation of photogenerated electron/hole pairs. The photocurrent response for 6% Co-ZF,
+ZF, S-g-C3N4, and 48% S-g-C3N4/6% Co-ZF was explored in a 0.5 M Na2SO4 solution under chopping radiance (Fig. 5b). The photocurrent response of the 48% S-g-C3N4/6% Co-ZF was by far the greatest when compared to all other produced catalysts,
+
+S. no. Photocatalyst Dyes Light source
+
+Irradiation time (min) % degradation Ref.
+
+1 Zn1xDyxFe2O4 MB Mercury lamp 75 97.3 34
+
+2 ZnFe2O4 MB Xenon lamp 141 76.2 60 3 CeO2/ZnFe2O4 MB Sunlight 60 90.48 31
+
+4 ZF–RGO Fulvic acid Solar light 220 80.1 61
+
+5 rGO/ZnFe2O4 MB Sunlight 180 99.8 62
+
+6 SnFe2O4/ZnFe2O4 MB Visible light 120 93.2 63 7 ZnFe2O4/rGO MB Visible light 121 99.99 64
+
+8 ZnFe2O4@rGO MB Xenon lamp 142 92.5 60
+
+9 ZnFe2O4/Ag/AgBr RhB Visible light 80 88 65
+
+10 S-g-C3N4/Co-ZF MB Sunlight 56 98 Present work
+
+demonstrating once again how effectively charges are transferred and consumed in the binary catalyst. On the other hand,
+
+![7_image_0.png](7_image_0.png) high photocurrent responses provided well-dened heterointerfaces, excellent electron–hole pair separation, and efficient transfer of electron charge carriers in the very advantageous self-assembled binary 48% S-g-C3N4/6% Co-ZF photocatalytic procedure for dye degradation.
+
+At the electrode–electrolyte junction, an EIS evaluation was recognized in the dark to assess the heterojunction charge transfer rate. The relationship between a shorter arc radius and lowered electron diffusion resistance, faster interfacial photogenerated charge transport, and better exit prociency is oen seen. According to our experimental results (Fig. 5c), the S-gC3N4/Co-ZF with a 48% S-g-C3N4/6% Co-ZF content had the lowest charge-transmission resistance of all the samples that were produced. According to this research, the heterointerface contact of the binary heterostructure S-g-C3N4/Co-ZF may greatly promote electron transport and increase electron consumption, enhancing photocatalytic performance. The transitory photocurrent responses and the EIS data accord quite well. According to the aforementioned experimental ndings, a well-built S-g-C3N4/Co-ZF might signicantly improve light harvesting, quick heterointerface electron transmission, and efficient separation of photoinduced e–h+ sets.
+
+Additionally, the 48% S-g-C3N4/6% Co-ZF heterostructures may produce a signicant number of effective oxygen species that are useful for removing MB from the 48% S-g-C3N4/6% CoZF under visible light illumination (Fig. 5d). p-Benzoquinone
+(BQ), ethylenediaminetetraacetic acid (EDTA), and isopropanol
+(IPA) were each employed to capture cOH, cO2 and h+ in the trapping experiment. This demonstrated that the primary energy species involved in the catalytic dye degradation process are cOH and cO2
+. Examining the EPR spectra of 48% S-g-C3N4/
+6% Co-ZF served as additional proof that functional species cOH
+and cO2 were present in the photodegradation process
+(Fig. S2a and b†). However, the signals are not discernible in the dark, indicating that both cO2 and cOH are constructed through the photolysis reaction practices. The apparent ESR
+signals are associated with DMPO$O2 and DMPO$OH
+adducts under sunlight enlightenment. The EPR results not only show that the 48% S-g-C3N4/6% Co-ZF heterojunction is produced, but they also show how effective the self-assembled method of S-g-C3N4/Co-ZF construction is for pollutant degradation. Fig. 6 shows a schematic representation of a photocatalytic process in the S-scheme under visible light. The MB
+dye degradation mechanism with the help of 48% S-g-C3N4/6%
+Co-ZF photocatalyst is shown in eqn (I)–(IV).
+
+The adsorption of MB dye on 48% S-g-C3N4/6% Co-ZF photocatalyst surface and the e/h+ pair contribute a signicant role in MB degradation. Firstly, the MB molecule was adsorbed on the 48% S-g-C3N4/6% Co-ZF photocatalyst surface. On the illumination of solar light on the S-g-C3N4/6% Co-ZF photocatalyst generate e/h+ pairs. Band gap alignment of Co-ZF with S-gC3N4 minimizes the e/h+ recombination. The holes in the lowest energy state in the conduction band of Co-ZF oxidized the MB dye directly and generate hydroxyl free radicals by reacting with water molecules which further interact with MB
+dye to oxidize into valuable products. Similarly, electrons in the highest energy state in the valence band of S-g-C3N4 reduce the adsorbed MB dye into CO2. The degradation of MB is started by either cOH radicals, cO2 radicals, or direct transfer of holes. A
+detailed illustration of the photocatalytic mechanism is written below.
+
+(I) Formation of electron–hole pair
+
+Co-ZF + $hv\to h^{+}$ (Co-ZF) + e${}^{-}$ (Co-ZF)
+S-g-C${}_{3}$N${}_{4}$ + $hv$ $\rightarrow$ h${}^{+}$ (S-g-C${}_{3}$N${}_{4}$) + e${}^{-}$ (S-g-C${}_{3}$N${}_{4}$)
+(II) Formation of cOH radical
+
+  **e${}^{-}$ (S-g-C${}_{3}$N${}_{4}$) + H${}_{2}$O${}_{2}$ - ${}^{\prime}$OH + OH${}^{-}$**
+ $\text{h}^{+}$ (Co-ZF) + H$_2$O $\rightarrow$ 'OH + H$^{+}$'  $\text{h}^{+}$ (Co-ZF) + OH$^{-}$ $\rightarrow$ 'OH
+(III) Generation of other reactive species
+
+$\mathbf{v}$
+e
+ (S-g-C3N4)+O2 / cO2
+
+$\mathbf{a}=\mathbf{a}\cdot\mathbf{a}=\mathbf{a}\cdot\mathbf{a}$. 
+
+${\bf H^{+}+O_{2}-O+OOH}$  ${\bf2H^{+}+2^{+}O_{2}-O+O_{2}+H_{2}O_{2}}$
+(IV) MB degradation mechanism MB + Rc / CO2 + H2O + other degradation products where Rc ¼ cOH, cOOH, h+ (Co-ZF), e (S-g-C3N4), cO2
+, cO2.
+
+## Conclusion
+
+In a nutshell, the goal of the current work was to create a better nanocomposite system by integrating S-g-C3N4 with a sunlightsensitive photocatalyst with narrow bandgap, Co-ZF, to enhance the photo-responsive window of the material and, in turn, the photo-removal prociency. The hydrothermal technique was successfully used to create the binary S-g-C3N4/Co-ZF photocatalysts. Additionally, the Co-ZF on S-g-C3N4 were consistently loaded aer their rst manufacture. Both binary and individual photocatalysts were used to break down MB in water. It was discovered that the binary S-g-C3N4/Co-ZF NCs considerably improved the photo-removal of MB and sunshine harvesting.
+
+The ideal loading is 48 wt% for the binary S-g-C3N4/Co-ZF NCs.
+
+The photolysis rate of dye is 0.42  103 min1, which is almost 2.45 times more than that of the individual photocatalysts (ZF,
+S-g-C3N4 and Co-ZF), and Co-ZF demonstrated the greatest photo-removal efficiency with a 98% efficiency. Additionally, the chemical stability experiment of the sample with a 48% S-gC3N4/6% Co-ZF content showed that the hybrid system is extremely robust and reusable. The current research made a ground-breaking suggestion for a brand-new synthesis method and a distinctive binary photo-catalyst that is active in the sunshine.
+
+## Author Contributions
+
+Ali Bahadur: conception, performed photocatalytic experiments, visualization of data, writing reviewing, and editing.
+
+Shahid Iqbal: design of study, performed major experimental works, writing-original dra preparation. Mohsin Javed: conception, design of study, writing-original dra preparation and critical revision, supervision. Syeda Saba Hassan: analysis and/or interpretation of data, performed FTIR analysis. Sohail Nadeem: material synthesis, visualization of data, writing reviewing, and editing. Ali Akbar: methodology, reviewed original manuscript, and critical revision. Rami M. Alzhrani:
+draing the revised manuscript, performed XRD analysis and critical revision. Murefah Mana Al-Anazy: reviewed original manuscript, and critical revision. Eslam B. Elkaeed: visualization of data, reviewed the original manuscript and critical revision. Hala A. Ibrahium: analysis and/or interpretation of data, performed FTIR analysis. Ayesha Mohyuddin: conducted XRD analysis, acquisition of data, writing-original dra preparation.
+
+## Conflicts Of Interest
+
+The authors declare no conict of interest.
+
+## Acknowledgements
+
+The authors express their appreciation to the Deanship of Scientic Research at King Khalid University, Saudi Arabia, for funding this work through research groups program under grant of number RGP.2/164/43. Rami M. Alzhrani would like to acknowledge Taif University Researchers Supporting Project number (TURSP-2020/209), Taif University, Taif, Saudi Arabia.
+
+This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number
+(PNURSP2022R7), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.
+
+## References
+
+1 T.-B. Nguyen, P.-N.-T. Ho, C.-W. Chen, C. P. Huang, R.-a. Doong and C.-D. Dong, Environ. Sci.: Nano, 2022, 9, 229–242.
+
+2 X. Bai, T. Jia, X. Wang, S. Hou, D. Hao and N. Bingjie, Catal.
+
+Sci. Technol., 2021, 11, 5432–5447.
+
+3 H. A. Abubshait, S. Iqbal, S. A. Abubshait, M. T. Alotaibi, N. Alwadai, N. Alfryyan, H. O. Alsaab, N. S. Awwad and H. A. Ibrahium, RSC Adv., 2022, 12, 3274–3286.
+
+4 H. Mao, Q. Zhang, F. Cheng, Z. Feng, Y. Hua, S. Zuo, A. Cui and C. Yao, Ind. Eng. Chem. Res., 2022, 61, 8895–8907.
+
+5 S. Iqbal, M. Javed, S. S. Hassan, S. Nadeem, A. Akbar, M. T. Alotaibi, R. M. Alzhrani, N. S. Awwad, H. A. Ibrahium and A. Mohyuddin, Colloids Surf., A, 2022, 636, 128177.
+
+6 K. Riaz, S. Nadeem, A. Chrouda, S. Iqbal, A. Mohyuddin, S. U. Hassan, M. Javed, A. BaQais, N. Tamam, K. Aroosh, A. Rauf, M. A. S. Abourehab, M. I. Jamil, E. B. Elkaeed, R. M. Alzhrani, N. S. Awwad and H. A. Ibrahium, Colloids Surf., A, 2022, 649, 129332.
+
+7 S. Nadeem, H. H. Khushi, M. Javed, S. Iqbal, H. O. Alsaab, N. S. Awwad, H. A. Ibrahium, T. Akhter, A. Rauf and H. Raza, J. Mol. Struct., 2022, 1252, 132191.
+
+8 S. Iqbal, M. Javed, M. A. Qamar, A. Bahadur, M. Fayyaz, A. Akbar, H. O. Alsaab, N. S. Awwad and H. A. Ibrahium, ChemistrySelect, 2022, 7, e202103694.
+
+9 M. Fan, Y. Cheng, X. Peng, H. Zhang, J. Wu, H. Tang, B. Li and Y. Zong, ACS Sustainable Chem. Eng., 2022, 10, 7664–
+7676.
+
+10 M. Javed, M. A. Qamar, S. Shahid, H. O. Alsaab and S. Asif, RSC Adv., 2021, 11, 37254–37267.
+
+11 G. Sarp and E. Yilmaz, ACS Omega, 2022, 7, 23223–23233.
+
+12 M. Chandra, U. Guharoy and D. Pradhan, ACS Appl. Mater.
+
+Interfaces, 2022, 14, 22122–22137.
+
+13 A. Bahadur, S. Iqbal, H. O. Alsaab, N. S. Awwad and H. A. Ibrahium, RSC Adv., 2021, 11, 36518–36527.
+
+14 S. Iqbal, A. Bahadur, M. Javed, O. Hakami, R. M. Irfan, Z. Ahmad, A. AlObaid, M. M. Al-Anazy, H. B. Baghdadi, H. S. M. Abd-Rabboh, T. I. Al-Muhimeed, G. Liu and M. Nawaz, Mater. Sci. Eng., B, 2021, 272, 115320.
+
+15 S. Iqbal, N. Ahmad, M. Javed, M. A. Qamar, A. Bahadur, S. Ali, Z. Ahmad, R. M. Irfan, G. Liu and M. B. Akbar, J. Environ. Chem. Eng., 2021, 9, 104919.
+
+16 J. Ding, L. Song, X. Li, L. Chen, X. Li, J. Sun, X. Zhang, Y. Wang and X. Tian, ACS Appl. Energy Mater., 2022, 5, 8800–8811.
+
+17 C. Wang, W. Shi, K. Zhu, X. Luan and P. Yang, Langmuir, 2022, 38, 5934–5942.
+
+18 U. Sahoo, S. Pattnayak, S. Choudhury, S. Padhiari, M. Tripathy and G. Hota, Ind. Eng. Chem. Res., 2022, 61, 9703–9716.
+
+19 F. Chen, T. Ma, T. Zhang, Y. Zhang and H. Huang, Adv.
+
+Mater., 2021, 33, 2005256.
+
+20 T. Chen, L. Liu, C. Hu and H. Huang, Chin. J. Catal., 2021, 42, 1413–1438.
+
+21 W. Liu, D. Zhang, R. Wang, Z. Zhang and S. Qiu, ACS Appl.
+
+Mater. Interfaces, 2022, 14, 31782–31791.
+
+22 M. Jafarzadeh, ACS Appl. Mater. Interfaces, 2022, 14, 24993–
+25024.
+
+23 S. Iqbal, A. Bahadur, M. Javed, G. Liu, T. I. Al-Muhimeed, A. A. AlObaid, Z. Ahmad, K. Feng and D. Xiao, J. Alloys Compd., 2022, 892, 162012.
+
+24 L.-Z. Wu, Acta Phys.-Chim. Sin., 2020, 36, 2004005.
+
+25 S. Shen, J. Pan, W. Zhou, J. Tang, H. Ding, J. Wang, L. Chen, C.-T. Au and S.-F. Yin, 2020, 36, 1905068.
+
+26 Z. Jin, Y. Li and X. Hao, Acta Phys.-Chim. Sin., 2021, 37, 1912033.
+
+27 L. Jia, X. Tan, T. Yu and J. Ye, Energy Fuels, 2022, DOI:
+10.1021/acs.energyfuels.2c01137.
+
+28 C. Coromelci, M. Neamtu, M. Ignat, P. Samoila, M. F. Zaltariov and M. Palamaru, Ceram. Int., 2022, 48, 4829–4840.
+
+29 A. Aridi, D. Naoufal, H. El-Rassy and R. Awad, Ceram. Int.,
+2022, DOI: 10.1016/j.ceramint.2022.07.046.
+
+30 S. Iqbal, Appl. Catal., B, 2020, 274, 119097.
+
+31 H. A. Al-Shwaiman, C. Akshhayya, A. Syed, A. H. Bahkali, A. M. Elgorban, A. Das, R. S. Varma and S. S. Khan, Mater.
+
+Chem. Phys., 2022, 279, 125759.
+
+32 S. Anwer, D. H. Anjum, S. Luo, Y. Abbas, B. Li, S. Iqbal and K. Liao, Chem. Eng. J., 2021, 406, 126827.
+
+33 C. Akshhayya, M. K. Okla, A. M. Thomas, A. A. Al-ghamdi, M. A. Abdel-Maksoud, B. Almunqedhi, H. AbdElgawad, L. L. Raju and S. S. Khan, Mater. Chem. Phys., 2022, 277, 125464.
+
+34 P. A. Vinosha, J. V. A. Vinsla, J. Madhavan, S. Devanesan, M. S. AlSalhi, M. Nicoletti and B. Xavier, Environ. Res., 2022, 203, 111913.
+
+35 R. Rajini and A. C. Ferdinand, Chem. Data Collect., 2022, 38, 100825.
+
+36 R. Muhammad Irfan, M. Hussain Tahir, M. Maqsood, Y. Lin, T. Bashir, S. Iqbal, J. Zhao, L. Gao and M. Haroon, J. Catal.,
+2020, 390, 196–205.
+
+37 K. Patil, K. Jangam, S. Patange, S. Balgude, A. G. Al-Sehemi, H. Pawar and P. More, J. Phys. Chem. Solids, 2022, 167, 110783.
+
+38 R. Belakehal, K. Atacan, N. Guy, A. Megriche and M. ¨ Ozacar, ¨
+Appl. Surf. Sci., 2022, 154315, DOI: 10.1016/
+j.apsusc.2022.154315.
+
+39 X. Jiang, Z. Wang, M. Zhang, M. Wang, R. Wu, X. Shi, B. Luo, D. Zhang, X. Pu and H. Li, J. Alloys Compd., 2022, 912, 165185.
+
+40 H. B. Truong, B. T. Huy, S. K. Ray, G. Gyawali, Y.-I. Lee, J. Cho and J. Hur, Chemosphere, 2022, 299, 134320.
+
+41 B. Al-Najar, A. Younis, L. Hazeem, S. Sehar, S. Rashdan, M. N. Shaikh, H. Albuasa and N. P. Hankins, Chemosphere, 2022, 288, 132525.
+
+42 S. Choudhary, D. Hasina, M. Saini, M. Ranjan and S. Mohapatra, J. Alloys Compd., 2022, 895, 162723.
+
+43 G. Fan, Z. Gu, L. Yang and F. Li, Chem. Eng. J., 2009, 155, 534–
+541.
+
+44 Y. Sun, W. Wang, L. Zhang, S. Sun and E. Gao, Mater. Lett.,
+2013, 98, 124–127.
+
+45 L. Han, X. Zhou, L. Wan, Y. Deng and S. Zhan, J. Environ.
+
+Chem. Eng., 2014, 2, 123–130.
+
+46 Y. Shi, L. Li, Z. Xu, H. Sun, S. Amin, F. Guo, W. Shi and Y. Li, Mater. Res. Bull., 2022, 150, 111789.
+
+47 Y. Fang, Q. Liang, Y. Li and H. Luo, Chemosphere, 2022, 302, 134832.
+
+48 S. Gao, D. Feng, F. Chen, H. Shi and Z. Chen, Colloids Surf., A,
+2022, 648, 129282.
+
+49 H.-Y. He, Y. Yan, J.-F. Huang and J. Lu, Sep. Purif. Technol.,
+2014, 136, 36–41.
+
+50 S. Renukadevi and A. P. Jeyakumari, Inorg. Chem. Commun.,
+2020, 118, 108047.
+
+51 J. Ai, L. Hu, Z. Zhou, L. Cheng, W. Liu, K. Su, R. Zhang, Z. Chen and W. Li, Ceram. Int., 2020, 46, 11786–11798.
+
+52 X. Zhang, B. Lin, X. Li, X. Wang, K. Huang and Z. Chen, Chem. Eng. J., 2022, 430, 132728.
+
+53 S. Khosravi-Gandomani, R. Youse, F. Jamali-Sheini and N. M. Huang, Ceram. Int., 2014, 40, 7957–7963.
+
+54 Z. Zhu, F. Guo, Z. Xu, X. Di and Q. Zhang, RSC Adv., 2020, 10, 11929–11938.
+
+55 S.-W. Zhao, M. Zheng, H.-L. Sun, S.-J. Li, Q.-J. Pan and Y.-R. Guo, Dalton Trans., 2020, 49, 3723–3734.
+
+56 M. F. Abdel Messih, M. A. Ahmed, A. Soltan and S. S. Anis, J.
+
+Phys. Chem. Solids, 2019, 135, 109086.
+
+57 S. Iqbal, N. Ahmad, M. Javed, M. A. Qamar, A. Bahadur, S. Ali, Z. Ahmad, R. M. Irfan, G. Liu, M. B. Akbar and M. A. Qayyum, J. Environ. Chem. Eng., 2021, 9, 104919.
+
+58 S. Iqbal, A. Bahadur, S. Ali, Z. Ahmad, M. Javed, R. M. Irfan, N. Ahmad, M. A. Qamar, G. Liu, M. B. Akbar and M. Nawaz, J. Alloys Compd., 2021, 858, 158338.
+
+59 F. Khurshid, M. Jeyavelan, M. S. L. Hudson and S. Nagarajan, R. Soc. Open Sci., 2019, 6, 181764.
+
+60 K. S. Riaz, M. S. Nadeem, V. V. Chrouda, P. A. Iqbal, M. Alsawalha, T. Alomayri and B. Yuan, Colloids Surf.,
+2021, 125835.
+
+61 J. Feng, ACS Appl. Mater. Interfaces, 2017, 9, 14103–14111.
+
+62 Q. Sun, K. Wu, J. Zhang and J. Sheng, Nanotechnology, 2019, 30, 315706.
+
+63 J. Wang, Q. Zhang, F. Deng, X. Luo and D. D. Dionysiou, Chem. Eng. J., 2020, 379, 122264.
+
+64 G. J. Rani, K. J. Babu, G. G. Kumar and M. A. J. Rajan, J. Alloys Compd., 2016, 688, 500–512.
+
+65 M. M. Sabzehmeidani, H. Karimi, M. Ghaedi and V. M. Avargani, Mater. Res. Bull., 2021, 143, 111449.