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+# Astragaloside Iv Relieves Il-1Β**-Induced Human** Nucleus Pulposus Cells Degeneration Through Modulating Pi3K/Akt Signaling Pathway Lu Zhang, Bma, Junsheng Gao, Mda, Zhentao Li, Mma, Jun Liu, Mma, Chong Zhang, Mma, Jie Liu, Mma, Hui Dong, Mma, Wei Mei, Mma,*
+
+Abstract Background: Intervertebral disc degeneration (IDD) is a multifactorial disease that is associated with nucleus pulposus (NP) apoptosis and extracellular matrix (ECM) degeneration and inflammation. Astragaloside IV (AS IV) has antioxidant, free radical scavenging, anti-inflammatory and anti-apoptosis effects. This study was to investigate whether AS IV could inhibit IL-1β-mediated apoptosis of HNP cells and its possible signal transduction pathway. Methods: Human nucleus pulposus cells (HNPCs) were stimulated with AS IV or LY294002 (PI3K inhibitor), followed by exposure to IL-1β for 24 hours. CCK8, TUNEL analysis and flow cytometry, ELISA and Western blotting were used to analyze the effects of AS IV on cell proliferation, apoptosis, inflammation, ECM and PI3K/Akt pathway signaling path-related proteins in IL-1β-induced HNPCs.
+
+Results: Compared with IL-1β-induced HNPCs, AS IV could improve the proliferation activity and the expressions of Collagen II, Aggrecan and Bcl-2 proteins, inhibit the apoptosis rate, inflammation and Bax and cleaved caspase-3 protein expression, and increase the activity of PI3K/Akt pathway. LY294002 attenuated the protective effect of AS IV against IL-1β-induced HNPCs degeneration.
+
+Conclusion: AS IV can inhibit IL-1β-induced HNPCs apoptosis inflammation and ECM degeneration by activating PI3K/Akt signaling pathway, which can be an effective drug to reduce disc degeneration. Abbreviations: AS IV = Astragaloside IV, ECM = extracellular matrix, HNPCs = human nucleus pulposus cells, IDD = intervertebral disc degeneration, NP = nucleus pulposus, NPCs = nucleus pulposus cells. Keywords: apoptosis, Astragaloside IV, degeneration, extracellular matrix, human nucleus pulposus cells, inflammation
+
+## 1. Introduction
+
+Intervertebral disc degeneration (IDD) is the pathological basis of a series of spinal degenerative diseases and is one of the common orthopedic diseases leading to decreased quality of life. 
+
+However, there is no definitive drug available to address this condition.[1] The main structures of the intervertebral disc are the innermost nucleus pulposus (NP), the annulus fibrosus wrapped around the outer part of the NP, and the endplates, which are in contact with the upper and lower vertebral bodies.[2] Nucleus pulposus cells (NPCs) are the primary constituent cells of NP, which belong to the chondrocyte. They can secrete aggrecan and collagenII to synthesize extracellular matrix (ECM) to maintain the homeostasis of NP.[3] Under the stimulation of various factors in vivo and in vitro, proteoglycan depolymerization in NP increases, collagenII and ECM degradation, and the biological function of NPCs changes irreverently, leading to IDD 
+diseases.[4] At the same time, apoptosis and inflammation of NPCs are also closely related to IDD.[5,6] Therefore, inhibition of inflammation, cell apoptosis and degradation of ECM of human nucleus pulposus cells (HNPCs) may be therapeutic targets for delaying IDD.
+
+Astragaloside IV (AS IV, the chemical structure is shown in Fig. 1A) is one of the main active components extracted from Astragalus, and the molecular formula C41H68O14 and many pharmacological effects. Studies have shown that AS IV has antioxidant, free radical scavenging, anti-inflammatory and anti-apoptosis effects.[7,8] Currently, AS IV is used in the treatment This research was supported by Science and Technology Project of Henan Provincial Health Department (LHGJ20220790). The authors declare no conflicts of interest. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Not applicable. This article does not involve clinical or animal testing.
+
+a Orthopedics, The Fifth Clinical Medical College of Henan University of Chinese Medicine (Zhengzhou People's Hospital), Zhengzhou, Henan, China. *Correspondence: Wei Mei, Orthopedics, The Fifth Clinical Medical College of Henan University of Chinese Medicine (Zhengzhou People's Hospital), 33 Huanghe Road, Jinshui District, Zhengzhou 453003, Henan Province, China (e-mail: zngols@163.com).
+
+Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal. How to cite this article: Zhang L, Gao J, Li Z, Liu J, Zhang C, Liu J, Dong H, Mei W. Astragaloside IV relieves IL-1β-induced human nucleus pulposus cells degeneration through modulating PI3K/Akt signaling pathway. Medicine 2023;102:33(e34815). Received: 6 May 2023 / Received in final form: 3 July 2023 / Accepted: 27 July 2023 http://dx.doi.org/10.1097/MD.0000000000034815
+
+![1_image_0.png](1_image_0.png)
+
+of orthopedic diseases. AS IV can promote the differentiation of bone marrow mesenchymal stem cells into chondrocytes, and induce the proliferation of articular cartilage nuclear bone marrow mechanism cells.[9] Recent studies have shown that AS IV 
+attenuates IDD,[10] but the mechanism of action is unclear.
+
+PI3K/AKT signaling pathway is an important intracellular signaling (including the release of inflammatory cytokines) 
+pathway, which can regulate apoptosis, inflammatory response and ECM degradation.[11,12] AS IV can protect nerve and myocardial cells from damage by activating the PI3K/AKT signaling pathway.[13,14] However, it is unclear whether the mechanism of AS IV prevention against IDD is related to its activation of the PI3K/AKT signaling pathway. Therefore, in this study, IL-1β was used to induce the degeneration of HNPCs to prepare IDD models in vitro, To explore whether the effect of AS IV on HNPCs degeneration and its mechanism is related to the PI3K/ AKT signaling pathway.
+
+## 2. Materials And Methods 2.1. Hnpcs Isolation And Treatments
+
+HNPCs (ScienCell Inc.) were continuously cultured in DMEM 
+medium supplemented with 10% fetal bovine serum in an incubator containing 5% CO2 at 37°C. When the cells were grown and fused to about 80% of the bottom area of the dish, they were digested by trypsin and then cultured by passage. The second-generation cells were applied for subsequent experiments.
+
+HNPCs were treated with IL-1β (10ng/mL) for 24 hours as an IDD model in vitro. To investigate the effects of HNPCs on AS IV on IDD, HNPCs were pretreated with 100 μM AS IV (Shanghai Yuanye Biotechnology Co. Ltd, China) for 2 hours, followed by exposure to IL-1β (10ng/mL) for 24 hours. The concentration of AS IV used here was based on our previous study.[10] Meanwhile, HNPCs were stimulated with 10 μM LY294002 (Sigma-Aldrich) to block the PI3K/Akt pathway. HNPCs treated only with DMEM/ F12 medium with 15% FBS containing DMSO were a control.
+
+## 2.2. Cck-8 Assay
+
+HNPCs were inoculated in 96-well plates (1×105 cells/well) with 5 duplicated wells. Culture for 24 hours, added 10 μL 
+CCK-8 to each well following incubation at 37°C for 2 hours. 
+
+The absorbance of each pore sample was measured by a multifunctional enzyme labeler at 450nm. Cell viability was calculated with normal cells as control.
+
+## 2.3. Apoptosis Detection By Tunel Analysis And Flow Cytometry
+
+HNPCs apoptosis was determined with the TUNEL apoptosis detection kit (Roche Applied Science, Switzerland) following the procedures. The cells in each group were treated with 0.2% Triton X-100 (Solarbio) for 10 minutes, cells were incubated with the TdT labeling reaction buffer for 60 minutes. The counterstain for cell nuclear was performed by interacting with 4ʹ,6-diamidino-2-phenylindole (DAPI) 
+(Solarbio) for 5 minutes. Cells were examined under fluorescence microscopy (Leica, Wetzlar, Germany), and the apoptosis rate was presented as the ratio of TUNEL-positive cells.
+
+HNPCs apoptosis was also determined by flow cytometry. The cells in each group were digested by 0.25% trypsin, washed and resuspended with pre-cooling PBS buffer to adjust the cell density to 1 × 105 cells/mL. 5 μL Annexin V-FITC was added and incubated in darkness for 15 minutes at room temperature. Then propidium iodide was added and incubated for 5 minutes in darkness before the operation. The apoptosis rate of cells was detected by CytoFLEX flow cytometry.
+
+## 2.4. Elisa
+
+After digestion, cleavage and centrifugation, the supernatant was collected. ELISA kit (Solarbio) was used to detect IL-6 and TNF-α levels. The specific operations were conducted with the instructions of the ELISA kit.
+
+## 2.5. Western Blot
+
+The total protein extraction kit was used to extract the total protein. The BCA method was used to detect the protein concentration. SDS-PAGE electrophoresis was used to transfer the membrane and block. The first antibody was incubated overnight at 4°C. The primary antibodies (Abcam) including anti-Bax (1:1000), anti-cleaved caspase-3, anti-Bcl-2 (1:1000), 
+anti-Collagen II (1:5000), anti-Aggrecan (1:1000), antiMMP-13 (1:1000), anti-Akt(1:1000), anti-p-Akt (1:500) and anti-GAPDH (1:1000) antibodies. After washing the membrane, HRP-labeled IgG secondary antibodies (1:5000) were incubated at room temperature for 1 hour. ECL was used as a developer. The software Image J was used for image analysis. GAPDH was used as an internal parameter to calculate the relative protein expression according to the gray value of the band.
+
+## 2.6. Statistical Analysis
+
+All data were analyzed by SPSS19.0. The one-way ANOVA analysis was used for the comparison among the groups, and the LSD-t test was used for the pairwise comparison among the groups. P < .05 means the difference was statistically significant.
+
+## 3. Results
+
+3.1. Effects of AS IV on IL-1β*-induced HNPCs' viability* To estimate whether AS IV can alleviate the toxic effects of IL-1β on HNPCs, we determined the cell viability of IL-1β-induced HNPCs cells after 100 μM AS IV treatment by CCK-8 assay. As shown in Figure 1B, IL-1β treatment significantly decreased the cell viability, compared with the control HNPCs (P < .05). But AS IV treatment significantly increased the cell viability, compared with the IL-1β-induced HNPCs (P < .05). Additionally, the inhibitor LY294002 significantly decreased the cell viability, compared with the AS IV-treated HNPCs (P < .05).
+
+## 3.2. Effects Of As Iv On Apoptosis Of Il-1Β-Induced Hnpcs
+
+Abnormal apoptosis of HNPCs contributes to the progression of IDD. To elucidate the effect of AS IV on the apoptosis of IL-1β-induced HNPCs, we performed the TUNEL and FCM 
+analysis. As shown in Figure 2A and B, IL-1β treatment significantly increased the apoptosis rate, compared with the control HNPCs (P < .05). AS IV treatment significantly decreased the cell apoptosis rate, compared with the IL-1β-induced HNPCs 
+(P < .05). Moreover, the inhibitor LY294002 treatment significantly increased the apoptosis rate, compared with the AS 
+IV-treated HNPCs (P < .05).
+
+To further evaluate the effect of AS IV on IL-1β-induced apoptosis, we also examined the anti-apoptosis proteins Bcl-2 and pro-apoptosis proteins Bax and cleaved caspase-3 by Western blot analysis. As shown in Figure  2C, IL-1β treatment significantly up-regulated the expression of Bax and cleaved caspase-3, and down-regulated the expression of Bcl-2, compared with ![2_image_0.png](2_image_0.png)
+
+down-regulated the expression of Bax and cleaved caspase-3, and up-regulated the expression of Bcl-2 compared with the IL-1 β - induced HNPCs (P < .05). Moreover, the inhibitor LY294002 markedly reversed the effects treated by As IV (P < .05).
+
+## 3.3. Effects Of As Iv On Il-1Β-Induced Inflammation In Hnpcs
+
+To evaluate the anti-inflammatory properties of AS IV, we measured the expression levels of TNF-α and IL-6 by ELISA test. As shown in Figure 3A, IL-1β treatment significantly increased the concentrations of TNF-α and IL-6, compared with the control HNPCs (P < .05). While AS IV treatment significantly decreased the concentrations of TNF-α and IL-6, compared with the IL-1βinduced HNPCs (P < .05). Moreover, the inhibitor LY294002 significantly increased the concentrations of TNF-α and IL-6, compared with the AS IV-treated HNPCs (P < .05).
+
+## 3.4. Effects Of As Iv On Ecm In Il-1 Β -Induced Hnpcs
+
+To elucidate the effects of AS IV on the ECM produced by HNPCs, we examined the effects of AS IV on the major proteins
+
+![3_image_0.png](3_image_0.png)
+
+pulposus cells.
+
+4 by Western blot analysis Aggrecan, Collagen II and MMP-13. As shown in Figure 3B, IL-1β treatment significantly up-regulated the expression of MMP-13 and down-regulated the expression of Aggrecan and Collagen II, compared with the control HNPCs (P < .05). However, AS IV treatment significantly down-regulated MMP-13 expression, and up-regulated Aggrecan and Collagen II expression, compared with the IL-1β-induced HNPCs (P < 
+.05). Moreover, the inhibitor LY294002 significantly reversed changes in Aggrecan, Collagen II and MMP-13 proteins treated by AS IV (P < .05).
+
+## 3.5. Effects Of As Iv On Pi3K/Akt Pathway In Il-1Β-Induced Hnpcs
+
+To reveal the mechanisms of ECM degradation and apoptosis of HNPCs during IDD, we examined the proteins involved in the PI3K/Akt signaling pathway by Western blot analysis. As shown in Figure 4, IL-1β treatment significantly decreased the levels of phosphorylation of Akt, compared with the control HNPCs (P
+< .05). Additionally, AS IV treatment significantly upregulated the levels of phosphorylation of Akt, compared with the IL-1βinduced HNPCs (P < .05). Moreover, the inhibitor LY294002 markedly reversed the effects treated by AS IV (P < .05).
+
+## 4. Discussion
+
+IDD can cause spinal stenosis, spinal instability and lumbar disc herniation in patients. IDD leads to low back pain and related complications, seriously damaging the labor force and bringing a heavy economic burden to society.[15] Current treatments for IDD only temporarily relieve pain, failing to eradicate the causes.[10] Therefore, it is of great significance to find a safer and more effective treatment for IDD. Studies have shown that during IDD, inflammatory cytokines such as IL-1β and TNF-α can induce NPCs apoptosis by stimulating NP, increase the expression of MMP13, decrease the expression of collagen II 
+and aggrecan, and lead to the progressive deterioration of intervertebral disc.[16,17] Furthermore, Excessive apoptosis of NPCs leads to a decrease in the synthesis of ECM, and ultimately to IDD.[6,18] Therefore, inhibition of inflammation, apoptosis and ECM degradation may be therapeutic targets for delaying IDD.
+
+Chinese medicine is well known to play a significant role in health in Chinese. In recent years, some Chinese herbs proved to be effective in treating a variety of diseases, which have been used as an additional and alternative therapy for IDD patients.[19] Many recent studies have partially established the usefulness of Chinese medicine in treating IDD, with the primary mechanisms including reduction of oxidative stress, regulation of inflammatory response, and decrease of myeloid cell death, among others.[20,21] AS IV is a natural saponin purified from the traditional Chinese medicine *Astragalus membranaceus*, which has anti-inflammatory, antioxidant, anti-apoptosis and anti-cancer pharmacological activities.[22] Previous studies have shown that AS 
+IV inhibits IL-1β-induced chondrocyte inflammatory mediators, apoptosis and ECM degradation, and alleviates the progression of osteoarthritis.[23] Since NP and articular cartilage have similar histocytological composition and biomechanical function. 
+
+NPCs and chondrocytes have similar phenotypes. Therefore, it is speculated that AS IV may be effective in treating IDD. In this study, IL-1β was used to stimulate HNPCs to simulate IDDs in vitro, and treated with AS IV. The results showed that AS IV significantly reduced the levels of inflammatory cytokines TNF-α and IL-6 in IL-1β-induced HNPCs, indicating that AS IV effectively inhibited IL-1β-induced inflammatory response in vitro. 
+
+Meanwhile, AS IV significantly down-regulated the apoptosis rate and the expression of Bax and cleaved caspase-3 protein, and up-regulated the expression of Bcl-2 protein in IL-1βinduced HNPCs, suggesting that AS IV could alleviate IL-1βinduced apoptosis. Next, we evaluated the effect of AS IV on the ECM degradation of HNPCs. AS IV significantly down-regulated the expression of MMP-13, and up-regulated the expression of Aggrecan and Collagen II in IL-1β-induced HNPCs, suggesting that AS IV treatment alleviated IL-1β-induced ECM 
+degradation. Taken together, these results suggest that AS IV 
+alleviates IL-1β-induced inflammatory response, apoptosis, and ECM degradation in HNPCs, similar to previous research.
+
+PI3K/Akt pathway is a major intracellular pathway that regulates cell proliferation, apoptosis, differentiation, autophagy and other processes under physiological and pathological conditions by interacting with multiple downstream target proteins.[24] Currently, studies on PI3K/Akt pathway play a very important role in the pathological development of IDD.[25] In this study, the results showed that IL-1β significantly inhibited activation of the PI3K/Akt pathway whereas in HNPCs. It is further proved that the PI3K/Akt pathway plays an important role in IDD, which is consistent with the results of previous studies.[26,27] More and more evidence shows that the targeted regulation of the PI3K/Akt pathway has become one of the effective methods to treat IDD.[26,28] Jiang et al[29] found that Resveratrol partly suppressed IL-1β-induced HNPCs apoptosis by activating the PI3K/Akt pathway. Zhu et al[30] found that higenamine suppressed cell apoptosis in IL-1β-induced HNPCs via activating ROS-mediated PI3K/Akt pathway. At the same time, studies have found that AS IV effectively alleviated IDD in vivo and in vitro.[10,31] In addition, AS IV ability to stimulate the PI3K/
+
+![4_image_0.png](4_image_0.png)
+
+AKT pathway has been pointed out many times and is related to antiapoptosis, antioxidative stress and the protective effect of ischemia-reperfusion.[32,33] However, it is not clear whether AS IV can alleviate disc degeneration by interfering with PI3K/ Akt pathway. Thus, we detected the levels of phosphorylation of Akt, and the results showed that AS IV significantly upregulated the levels of phosphorylation of Akt. LY294002, a PI3K-specific protein kinase inhibitor, has been widely used in the study of the PI3K/Akt pathway. In this study, LY294002 treatment significantly inhibited the levels of phosphorylation of Akt, reversed the protective effect treated by AS IV on degraded HNPCs. LY294002 treatment promoted the inflammatory response and apoptosis, markedly reversed the effects treated by AS IV. All of the results supported that AS IV had a strong protective effect on IL-1β-induced HNPCs, and that the PI3K/AKT pathway was, at least partly, involved in this protective effect. However, it remains unknown whether AS IV alleviates IDD by activating PI3K/Akt pathway in vivo. In our future experiments, we will evaluate the protective properties of AS IV using the vivo model. Moreover, we only explored the involvement of the PI3K/Akt pathway, while other pathways were not evaluated. This issue could be addressed in future studies to fully elucidate the mechanism of AS IV involvement in IDD treatment.
+
+In conclusion, AS IV alleviates IL-1β-induced inflammation, apoptosis and ECM degeneration by activating PI3K/Akt pathway, suggesting that AS IV has therapeutic potential for treating IDD.
+
+## Author Contributions
+
+Conceptualization: Junsheng Gao, Jun Liu. Data curation: Jie Liu. Methodology: Chong Zhang. Software: Zhentao Li. Validation: Hui Dong. Writing - original draft: Lu Zhang. Writing - review & editing: Wei Mei.
+
+## References
+
+[1] Li Z, Chen X, Xu D, et al. Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration. Cell Prolif. 2019;52:e12704.
+
+[2] Sun Z, Liu B, Luo ZJ. The immune privilege of the intervertebral disc: 
+implications for intervertebral disc degeneration treatment. Int J Med Sci. 2020;17:685–92.
+
+[3] Lu XD, Liu YR, Zhang ZY. Matrilin-3 alleviates extracellular matrix degradation of nucleus pulposus cells via induction of IL-1 receptor antagonist. Eur Rev Med Pharmacol Sci. 2020;24:5231–41.
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+[4] Yang Z, Gao XJ, Zhao X. CDMP1 promotes type II collagen and aggrecan synthesis of nucleus pulposus cell via the mediation of ALK6. Eur Rev Med Pharmacol Sci. 2020;24:10975–83.
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+[5] Chen S, Lei L, Li Z, et al. Grem1 accelerates nucleus pulposus cell apoptosis and intervertebral disc degeneration by inhibiting TGF-betamediated Smad2/3 phosphorylation. Exp Mol Med. 2022;54:518–30.
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+[6] Cazzanelli P, Wuertz-Kozak K. MicroRNAs in intervertebral disc degeneration, apoptosis, inflammation, and mechanobiology. Int J Mol Sci . 2020;21:3601.
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+[7] Zhang J, Wu C, Gao L, et al. Astragaloside IV derived from astragalus membranaceus: a research review on the pharmacological effects. Adv Pharmacol. 2020;87:89–112.
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+[8] Liang XY, Hong FF, Yang SL. Astragaloside IV alleviates liver inflammation, oxidative stress and apoptosis to protect against experimental non-alcoholic fatty liver disease. Diabet Metab Synd Ob. 2021;14:1871–83.
+
+[9] Jin H, Du J, Ren H, et al. Astragaloside IV protects against iron loading-induced abnormal differentiation of bone marrow mesenchymal stem cells (BMSCs). FEBS Open Bio. 2021;11:1223–36.
+
+[10] Tian Y, Chu X, Huang Q, et al. Astragaloside IV attenuates IL-1betainduced intervertebral disc degeneration through inhibition of the NF-kappaB pathway. J Orthop Surg Res. 2022;17:545.
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+[11] Sun X, Chen L, He Z. PI3K/Akt-Nrf2 and anti-inflammation effect of macrolides in chronic obstructive pulmonary disease. Curr Drug Metab. 2019;20:301–4.
+
+[12] Sun K, Luo J, Guo J, et al. The PI3K/AKT/mTOR signaling pathway in osteoarthritis: a narrative review. Osteoarthrw Cartil. 2020;28:400–9.
+
+[13] Cheng S, Zhang X, Feng Q, et al. Astragaloside IV exerts angiogenesis and cardioprotection after myocardial infarction via regulating PTEN/ PI3K/Akt signaling pathway. Life Sci. 2019;227:82–93.
+
+[14] Yang L, Dong X, Zhang W. Astragaloside IV alleviates the brain damage induced by subarachnoid hemorrhage via PI3K/Akt signaling pathway. Neurosci Lett. 2020;735:135227.
+
+[15] Oichi T, Taniguchi Y, Oshima Y, et al. Pathomechanism of intervertebral disc degeneration. JOR Spine. 2020;3:e1076. [16] Hamamura K, Lin CC, Yokota H. Salubrinal reduces expression and activity of MMP13 in chondrocytes. Osteoarthr Cartil. 
+
+2013;21:764–72.
+
+[17] Lian C, Gao B, Wu Z, et al. Collagen type II is downregulated in the degenerative nucleus pulposus and contributes to the degeneration and apoptosis of human nucleus pulposus cells. Mol Med Rep. 
+
+2017;16:4730–6.
+
+[18] Tan Y, Yao X, Dai Z, et al. Bone morphogenetic protein 2 alleviated intervertebral disc degeneration through mediating the degradation of ECM and apoptosis of nucleus pulposus cells via the PI3K/Akt pathway. Int J Mol Med. 2019;43:583–92.
+
+[19] Zhang B, Xu H, Wang J, et al. A narrative review of non-operative treatment, especially traditional Chinese medicine therapy, for lumbar intervertebral disc herniation. Biosci Trends. 2017;11:406–17.
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+[20] Zhang YH, Shangguan WJ, Zhao ZJ, et al. Naringin inhibits apoptosis induced by cyclic stretch in rat annular cells and partially attenuates disc degeneration by inhibiting the ROS/NF-kappaB Pathway. Oxid Med Cell Longev. 2022;2022:6179444.
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+[21] Song C, Chen R, Cheng K, et al. Exploring the pharmacological mechanism of Duhuo Jisheng decoction in treating intervertebral disc degeneration based on network pharmacology. Medicine (Baltim). 2023;102:e33917.
+
+[22] Chen T, Yang P, Jia Y. Molecular mechanisms of astragalosideIV in cancer therapy (Review). Int J Mol Med. 2021;47:13.
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+[23] Wang B, Chen MZ. Astragaloside IV possesses antiarthritic effect by preventing interleukin 1beta-induced joint inflammation and cartilage damage. Arch Pharm Res. 2014;37:793–802.
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+[24] Khezri MR, Jafari R, Yousefi K, et al. The PI3K/AKT signaling pathway in cancer: Molecular mechanisms and possible therapeutic interventions. Exp Mol Pathol. 2022;127:104787.
+
+[25] Ouyang ZH, Wang WJ, Yan YG, et al. The PI3K/Akt pathway: 
+a critical player in intervertebral disc degeneration. Oncotarget. 2017;8:57870–81.
+
+[26] Wu B, Guo X, Yan X, et al. TSG-6 inhibits IL-1beta-induced inflammatory responses and extracellular matrix degradation in nucleus pulposus cells by activating the PI3K/Akt signaling pathway. J Orthop Surg Res. 2022;17:572.
+
+[27] Qi W, Ren D, Wang P, et al. Upregulation of Sirt1 by tyrosol suppresses apoptosis and inflammation and modulates extracellular matrix remodeling in interleukin-1beta-stimulated human nucleus pulposus cells through activation of PI3K/Akt pathway. Int Immunopharmacol. 2020;88:106904.
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+[28] Zhang C, Li Z, Li L, et al. Achyranthoside D (AD) improve intervertebral disc degeneration through affect the autophagy and the activation of PI3K/Akt/mTOR pathway. J Orthop Surg (Hong Kong). 
+
+2022;30:10225536221135474.
+
+[29] Jiang Y, Xie Z, Yu J, et al. Resveratrol inhibits IL-1beta-mediated nucleus pulposus cell apoptosis through regulating the PI3K/Akt pathway. Biosci Rep. 2019;39:BSR20190043.
+
+[30] Zhu X, Liu S, Cao Z, et al. Higenamine mitigates interleukin-1beta-induced human nucleus pulposus cell apoptosis by ROS-mediated PI3K/
+Akt signaling. Mol Cell Biochem. 2021;476:3889–97.
+
+[31] Hong H, Xiao J, Guo Q, et al. Cycloastragenol and Astragaloside IV 
+activate telomerase and protect nucleus pulposus cells against high glucose-induced senescence and apoptosis. Exp Ther Med. 2021;22:1326.
+
+[32] Wei D, Xu H, Gai X, et al. Astragaloside IV alleviates myocardial ischemia-reperfusion injury in rats through regulating PI3K/AKT/GSK3beta signaling pathways. Acta Cir Bras. 2019;34:e201900708.
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+From biomimetics to smart materials and 3D technology: Applications in orthodontic bonding, debonding, and appliance design or fabrication Theodore Eliades a,*, Nearchos Panayi a,b, Spyridon N. Papageorgiou a a Clinic of Orthodontics and Pediatric Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland b *European University Cyprus, School of Dentistry, Nicosia, Cyprus* 
+
+| ARTICLE INFO  Keywords:  Orthodontic  Biomaterials  Biomimetic  Self-healing  Shape-memory  3D printed   |
+|----------------------------------------------------------------------------------------------------------|
+
+| ABSTRACT   |
+|------------|
+
+This review covers aspects of orthodontic materials, appliance fabrication and bonding, crossing scientific fields and presenting recent advances in science and technology. Its purpose is to familiarize the reader with developments on these issues, indicate possible future applications of such pioneering approaches, and report the current status in orthodontics. The first section of this review covers shape-memory polymer wires, several misconceptions arising from the recent introduction of novel three-dimensional (3D)-printed aligners (mistakenly termed shape-memory polymers only because they present a certain degree of rebound capacity, as most non-stiff alloys or polymers do), frictionless surfaces enabling resistance-less sliding, self-healing materials for effective handling of fractured plastic/ceramic brackets, self-cleaning materials to minimize microbial attachment or plaque build-up on orthodontic appliances, elastomers with reduced force relaxation and extended stretching capacity to address the problem of inadequate force application during wire-engagement in the bracket slot, biomimetic (non-etching mediated) adhesive attachment to surfaces based on the model of the gecko and the mussel, and command-debond adhesives as options for an atraumatic debonding. This review's second section deals with the recent and largely unsubstantiated application of 3D-printed alloys and polymers in orthodontics and aspects of planning, material fabrication, and appliance design. 
+
+## 1. **Introduction**
+
+Advances in the field of biomimetics, termed from the greek βιο-bio meaning living and μίμησις-mimetic (adjective, meaning imitating/ 
+copying), have brought a significant growth in material development and applications. Despite numerous patents and substantial breakthroughs in biomedical or engineering materials, the orthodontic profession has yet to explore the feasibility of introducing such biomimetic materials in clinical practice. 
+
+The purpose of this comprehensive review is to familiarize the reader with these developments, indicate possible applications of such pioneering approaches in the introduction of materials/processes in the broader field of biomedical or engineering/industrial materials, and report the status of applications in orthodontics. 
+
+The first section of this review covers: 
+a.shape-memory polymer wires, aiming to clarifying several misconceptions arising from the recent introduction of three-dimensional 
+(3D)-printed aligners, mistakenly termed shape-memory polymers only because they present a certain degree of rebound capacity, as most 
+* Correspondence to: Clinic of Orthodontics and Pediatric Dentistry, Center of Dental Medicine, University of Zurich, Plattenstrasse 11, 8032 Zurich, Switzerland. 
+
+![0_image_2.png](0_image_2.png)
+
+E-mail address: theodore.eliades@zzm.uzh.ch (T. Eliades). 
+
+Contents lists available at ScienceDirect non-stiff alloys (multistrand wires) or polymers do. 
+
+b.frictionless surfaces enabling resistance-less sliding. c.self-healing materials for effective handling of fractured plastic/ 
+ceramic brackets. 
+
+d.self-cleaning materials to minimize microbial attachment and plaque build-up on bracket, aligner, wire, and elastomers. 
+
+e.elastomers with reduced force relaxation and extended stretching capacity to address the problem of inadequate force application during wire-engagement in the bracket slot. 
+
+f.biomimetic, non-etching mediated, adhesion based on the gecko or mussel model. 
+
+g.command-debond adhesives as options for an atraumatic debonding. 
+
+The clinical challenges for each issue will be discussed followed by corresponding advances in material manufacturing and the introduction of a relevant application in technology and industry. 
+
+The second section of this review deals with the recent and largely unsubstantiated application of 3D-printed alloys and polymers in orthodontics and aspects of planning, material fabrication, appliance https://doi.org/10.1016/j.jdsr.2023.10.005 Received 6 September 2023; Received in revised form 24 October 2023; Accepted 26 October 2023 1882-7616/© 2023 Published by Elsevier Ltd on behalf of The Japanese Association for Dental Science. This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+## 2. **Biomimetics And Smart Materials** 2.1. Shape-Memory Polymers
+
+2.1.1. *Clinical challenge* The clinical problem that led to the development of polymeric or surface-altered wires is the aesthetic problems from the use of metallic wires. 
+
+Earlier attempts to introduce solid plastic polymeric wires such as Optiflex (Ormco Co., Glendora, CA) for the initial treatment stages resulted in the application of polymers with reduced modulus of elasticity, stiffness, but also low resilience and thus low elastic energy stored in the wire (which in the stress-strain curve is provided by the area under the curve until the yield point, or the so called 'range')—precluding their use in most cases with average crowding. Moreover, their low bending stiffness adversely affected their levelling capacity [1]. This is different from the aligning properties of the wire as the bent wire in the buccal segment of deep curve of Spee cases needs have high bending stiffness and cross sections filling the edgewise size of the bracket, i.e., 0.018- or 0.022-inch. This contradicts the false notion that Nickel-Titanium (NiTi) wires were introduced exactly because with their low modulus can effectively level the curve of Spee. A round 0.016-inch stainless steel wire has much higher stiffness than all NiTi or Titanium-Molybdenum-Alloy (TMA) wires of any cross-section or shape. 
+
+Similarly, rectangular 0.016 × 0.022-inch NiTi and TMA wires to level the occlusion in the straightwire technique also face the problem of reduced bending stiffness owing to the reduced contribution to this effect of the flatwise dimension of the wire, in addition to the fact that they show play in the edgewise dimension (in the 0.018-inch system). 
+
+Other plastic wires were developed in the late 1990′s-early 2000's by BioMers Products, LLC (Jacksonville, Fla) to address these concerns and efficiently align / derotate teeth, but their hydrolytic degradation after extended periods of time precluded their widespread use. 
+
+Developed in the mid 2000′s, shape-memory polymers—which have nothing to do with the claimed memory properties of recently introduced 3D-printed aligners—were introduced initially for biomedical applications. Exposure of initially developed polymer threads to heat or to a specific wavelength of light [2,3] resulted in modification of their shape to their pre-engineered shape. Therefore, thrombectomies and removals of vessel clots were facilitated through the catheterization of the vessel with a shape-memory thread, which after being navigated through the thrombus, had its external to the patient's body part exposed to a specific stimulus that reverted the thread to its initial shape 
+(in that case of a cork), thereby facilitating clot removal. 
+
+However, this application has since 2005 not yet received any consideration for use in orthodontic wires or orthodontic aligner applications. 
+
+## 2.2. Frictionless Movement 2.2.1. Clinical Challenge
+
+Friction, although often overemphasized, plays a significant role in determining the magnitude of force applied to the tooth crown through an activated wire or a prescription bracket. It has also been shown to be an impeding factor against effective tooth movement and efforts to overcome this included the use of wires with smoother surfaces or alternative ligation modes such as self-ligation, which however have never associated with improved tooth movement rates in vivo. The problem lies with factors traditionally ignored in oversimplified in vitro studies, such as the specific tooth movement pattern (which deviates from the straight line path and includes several tilting spatial alterations) and the in vivo ageing of the involved materials (bracket, wire, 
+
+## Ligature) That Greatly Affect Exerted Forces. 2.2.2. Available Materials
+
+A possible solution to the abovementioned problems might come from broader industrial metallurgical applications mimicking the flaked surface texture of snakes. These reptiles show remarkably smooth movement across various terrains, owing not to the smooth surface of their skin, but to the textured pattern and the specific arrangement of their external flake layers [4]. 
+
+Relevant research has demonstrated that the friction coefficient of textured surfaces of snakes with a characteristic orientation of the superimposed skin flakes was almost 50% reduced compared to the modeled untextured one. 
+
+Steel surfaces used in industry or manufacturing made to resemble the snakes' external flake layer were built at the end of the first decade of 2000 and were met with success but have yet to be introduced in orthodontics. 
+
+## 2.3. Self-Healing Materials 2.3.1. Clinical Challenge
+
+Ceramic, and to less extent plastic, brackets were introduced as an appliance with superior aesthetics, but several issues arose from their large-scale clinical use. Most research on this topic is focused on their fracture strength and debonding characteristics, due to their unfavorable debonding pattern that was reported in the early 1990 s [5]. The observation that ceramic brackets fracture frequently, usually at the wings and most often during debonding, is universal knowledge for most readers. These materials are composed of atoms bound together with such strong forces that their flexibility is notably impaired. As a result, force application on them leads to a minimum elastic deformation and no permanent deformation. It follows that these materials maintain their dimensions / shape after fracture, as no deformation has set in, due to their bonding energy and strong directional characteristics. Also, the atomic packing factor of such materials is high, implying that their dense atomic distribution in 3D arrays results in high crystal density. Therefore, no plastic deformation of the wings is possible and, when the force exceeds a certain value, the wing fractures. 
+
+These issues led to interest being attracted to the production of selfhealing polymeric materials, owing to the potential flexibility in synthesizing polymers with desirable properties. One of the first such attempts involved including monomer-filled spheres, which upon crack propagation would empty their content to the fracture vicinity to facilitate filling of the fracture path with an auto-polymerized material. Later applications, which have already been adopted by the industry, include the development of self-healing of oxetane-chitosan compounds in the form of coatings that, upon exposure to ultraviolet sunlight, initiate a cascade of events bringing these two molecules together to form a coating that closes potential damage of the initially formed coating. This found applications in the automobile industry, with protective car coatings already being marketed. 
+
+In the area of ceramics, development of such materials is more challenging as the necessity of altering ceramic structural defects usually requires high temperatures, which exceed the intraoral spectrum. In as much, additional processes such as application of combustion chambers [6] or electric field-induced colloidal aggregation [7] to fill cracks and inhibit propagation, thereby avoiding a catastrophic failure, have no feasible application mode in orthodontics. Therefore, despite the advances in that field, the potential application to orthodontic materials involves only polymeric products (brackets and aligners), which on the other hand, are ductile and present a much lower clinical failure incidence. 
+
+## 2.4. Self-Cleaning Materials 2.4.1. Clinical Challenge
+
+Plaque accumulation on a biomaterial exposed to a biological system is accompanied by the organization of a non-cellular biofilm by spontaneous adsorption of extracellular macromolecules, composed mostly of glycoproteins and proteoglycans. These films induce a conditioning effect that modifies the biomaterial's surface properties and alters both the response of the subsequently attached cells and the interactions occurring at the biomaterial-host interface. This conditioning effect is based on the differing capacities of artificial surfaces to fractionate proteins from biological fluids, such as saliva or blood, and the ability to induce conformation and orientation changes of adsorbed proteins. 
+
+The outcome of biofilm adsorption is dependent on the biological fluid flow rate at the site of contact, the type of interfacial interactions involved, and the attachment strength with the substrate. Under static conditions or low flow rates, the biomaterial surface chemistry is the fundamental factor affecting the composition and organization of acquired biofilms, whereas in environments with high flow rates, substrate surface molecular motion and roughness are also important factors. In addition, with long exposure periods, material properties such as porosity, sorption, corrosion, and biodegradation further modify biomaterial-host interactions. Finally, the material's wettability is modulated by its critical surface tension and plays a significant role in the development of adverse effects either on the tooth's enamel (in the form of white spot lesions) or periodontal inflammation. 
+
+Most efforts in this field have concentrated around the alteration of the wetting characteristics of orthodontic materials such as brackets adhesives and elastomers. The problem with adhesives lies with the fact that a reduced wettability to disrupt the developed biofilm on the microbia-attracting adhesive margins, would also result in reduced wetting of the enamel etching-induced tags and projection of the resin in the enamel, thus adversely affecting the bond integrity of the bracket to enamel. 
+
+Potential solutions for brackets or elastomers could be their manufacturing with reduced wettability modelled by the lotus effect [8], which is based on the example of the synonymous plant that, through a dense network of hairs developed on its surface, allows the formation of a large contact angle, which is indicative of reduced wetting (Fig. 1). 
+
+The formation of superhydrophobic surfaces has been adopted in medicine [9], dentistry [10], and everyday applications ranging from utility material surfaces, dishes, handles, car surface coatings of cars to decrease dirt, and fabric repellant surfaces, among others. 
+
+In orthodontics, the fabrication of self-healing brackets [11] has Fig. 1. A sign of reduced wettability. Note the high contact angle arising from 
+
+![2_image_0.png](2_image_0.png)
+
+the formation of a high meniscus of liquid on the substrate. This wetting reduces the formation of biofilm, which is the first step for the adherence of plaque and colonization of the surface by microbia. This model is pursued to be applied to brackets and elastomers to reduce plaque accumulation on appliances. 
+
+involved the development of Titanium photocatalysis [12], which involves the coating of the bracket with a layer of Ti oxide that upon sunlight exposure releases reactive oxygen species or hydroxyl radicals, which have antimicrobial, odor-removing effects. However, such applications have yet to be widely employed in orthodontics. 
+
+## 2.5. New Elastomers 2.5.1. Clinical Challenge
+
+The use of polyurethane-based elastomeric ligatures and modules in engaging archwires and closing spaces in orthodontics is accompanied by a notable force relaxation that accounts for up to 40% of the initially applied load. This effect is accentuated by the laborious and multiperspective intraoral ageing pattern of these polymers that comprises of hydrolytic degradation, swelling, and softening, which further degrade the mechanical properties and decrease the exerted forces from the material. At the same time, they favor plaque build-up and contribute to the microbial colonization of the bracket-wire-elastomer complex. 
+
+Elastomeric force relaxation derives from the material's macroscopic degradation in the form of tearing of the structural surface and bulk structure, presenting discontinuities because of sustained load. Microscopically, the extension of the molecular chains that, in some cases fracture, leads the load to be exerted by fewer number of bearing units, and as a result presenting higher deformation. 
+
+Efforts to address this problem in the broader biomedical literature have focused on increasing the crosslinking of chains or their length to provide more area for load-distribution and longer chains. The problems arising from these approaches have to do with the increased initial stiffness in the cases of fortifying the crosslinking (which would result in increased exerted forces) and the entanglement in the scenario of incorporating much longer chains which cause the noodle effect–i.e. the increase in stiffness due to the perplexed structure and the entanglement of chains. 
+
+Thus, ideally elastomers should maintain the applied load for at least 4–6 weeks, while they should be hydrophobic to reduce the attraction of species and be water tolerant. 
+
+Elastomers used in industrial applications are structures with chains forming multi-dimensional networks [13,14]. As a result, these elastomers have much higher toughness and strength, producing extension ranges in the order of 50 times the original length (in general rubber elasticity refers to a property of a material to be capable of being extended 10 times its size). 
+
+An alternative approach could be engaging the wire tying a silk fiber around the bracket wings, as silk has remarkable properties [15] and has recently been produced synthetically [16], leading to a product stronger than Kevlar and more elastic than nylon. 
+
+## 2.6. Biomimetic Adhesives 2.6.1. Clinical Challenge
+
+Etching-mediated orthodontic bonding and consequently debonding is basically an interventional procedure that irreversibly alters enamel structure, roughness, and composition by introducing resin tags into the enamel structure. These formations remain after debonding and constitute a substrate where a series of effects such as decalcification and color alteration take place. The latter might also be attributed to the polymerization shrinkage that leads to detachment of resin-tags from the enamel walls, thereby allowing the penetration of colorants from the oral environment or corrosion products of the welded mesh of the bracket base, leaving characteristic black spots on the adhesive. 
+
+Along with glass-ionomer bonding that is associated with reduced adhesive penetration depths, alternative methods have been developed in the biomedical literature for bonding of tissues and materials. 
+
+2.6.2. *Available materials* In the mid-2000′s, efforts to replicate the ability of some animals to sustain their bodies against gravity were developed, using the lizard gecko as a model (Fig. 2). Analysis of the gecko's feet identified a dense hair-network, which increased substantially the effective contact area of the foot to the underlying surface providing a mechanism such as Velcro materials [17]. These surfaces were used as a model to fabricate carbon nanotube networks embedded into a polymer surface [18]. In addition to this mechanism, and with the objective of facilitating bonding to wet surfaces, the mussel model was adopted [19], as the latter have the ability to bond chemically to a number of dissimilar surfaces ranging from rock to wood or metal. The resulting adhesive synthesized was patented as geckel [20], a biomedical product which can be applied as substitute of deep layer stitching instead of sutural materials. 
+
+These biomimetic adhesives could become a standard, introducing a bonding method without irreversible effects on enamel and, ideally, without the necessity of applying rotary instruments for the removal of adhesive remnant. 
+
+## 2.7. Command-Debond Adhesives 2.7.1. Clinical Challenge
+
+Debonding of orthodontic appliances and attachments involves several traumatic procedures, which include the use of rotary instruments to remove the polymerized adhesive that was transformed into a crystalline structure from its initially viscous pre-polymerized form. This exposes enamel to alteration risks in the form of groove formation from the rotating bur (Fig. 3), potentially affecting the enamel's color parameters and gloss. The aforementioned process induces irreversible damage to enamel regardless of the etching-mediated process, i.e., the presence or resin tags after debonding and resin removal. Therefore, introducing a method that enables atraumatic appliance removal constitutes an independent variable offering the possibility to minimize some of these enamel adverse effects. 
+
+Polymeric adhesives have been experimentally synthesized for industrial applications by adding iron fillers. Orthodontic adhesives usually contain fillers in a ratio of 0.6–0.7 (% wt) in the form of silica particles or barium glasses, which possess a refractive index of 1.55 at the wavelength of the photoinitiator. Evidence available on the field of resin composites suggests that maximum light scattering occurs at particle sizes equivalent to the half of the wavelength of the polymerization photoinitiator, which for camphorquinone systems is 468 nm– hence a favorable filler size would be about 230 nm. However, the typical microscopy picture of an adhesive includes a large size variation of filler particles. The reflectance (r) of a composite material consisting of two different phases of indices n1 and n2 is given by the equation: 
+r= (n1-n2)
+2/(n1+n2)
+2 
+
+![3_image_0.png](3_image_0.png)
+
+and therefore, the deleterious effect of large index differences is well substantiated and should be avoided. 
+
+Iron fillers have the advantage of being of the preferred size and, most importantly, can be distributed within the bulk adhesive with a preferred orientation, which would vary among different layers and thus provide optimum mechanical properties to specific loading directions 
+[21]. Moreover, after completion of the adhesive's intended service, alternating-polarity magnets can be used to force the iron fillers to move inside the polymer structure and induce internal cracks. This internal induction of catastrophic failure as a method of facilitating composite debonding can be also achieved with the use of thermally expandable fillers [22]. In this instance, heat can induce an increase in the volume of fillers by 50–100 times, thus causing cracks and fragmentation of the polymerized resin composite. 
+
+In addition, research in this field has been directed towards using photooxidation-initiated, hydrolytic, or other forms of degradation to induce a reversal of the adhesive from the crystalline status to the viscous state [23]. However, this method is only available for industrial applications of adhesives, as this mode of debonding is non-compatible with biomedical applications. 
+
+The use of thermal treatment on the adhesive could also be applied to achieve a reversal of the condition of the adhesive phase. Glass transition temperature (Tg) in polymers indicates the temperature above which the polymer structure is transformed from the crystalline to the viscous state. In relevant research, 80% of the Tg initiates this transformation to a viscous state. Thus, heating the adhesive could transform it to a paste-like phase which could be removed without any rotary instruments. The problem with this approach lies in the relatively high Tg of currently available adhesives, which range above 100–110 degrees Celsius. Therefore, an 80%-Tg temperature could be well over 80 degrees, which could have adverse effects in the physiology of the pulp. 
+
+From the opposite side, a recent approach with conventional adhesives has demonstrated that freezing the bracket-adhesive complex with the use of freezing probe, resulted in an alteration of the remnant adhesive index showing reduced adhesive remaining onto enamel after debonding (Fig. 4). 
+
+2.8. Concluding remarks on the application of already existing technological advances on orthodontic materials and processes The application of many advantageous material applications seems to be impeded by: 
+
+a. lack of an overall knowledge on the level of scientific advances and 
+
+![3_image_1.png](3_image_1.png)
+
+related armamentaria of materials / tools that could potentially be used to optimize orthodontic materials / processes—as in the case of self-healing polymeric brackets or tribological considerations of metals; Fig. 2. A photograph of the edge of the feet of the lizard gecko, which is able to sustain its weight vertically . Note the dense network of hair, which increase the contact area with the substrate and provide mechanical retention. 
+
+![4_image_0.png](4_image_0.png)
+
+b. lack of interest, perhaps due to the projected reduced market share of orthodontic materials, as in the case of self-cleaning brackets, elastomers, or aligners; and c. limitations arising from the biomedical character and necessity to apply these materials in the human body, as in the case of photooxidation of composites to facilitate debonding. 
+
+## 2.9. 3D Technology And Orthodontics
+
+During the last years, digital technology changed the way dentistry and orthodontics is practiced, transforming many of its aspects from analogue to digital. It is not an exaggeration to say that digitization of the oral cavity using 3D scanners is the most important part of this evolution and is responsible for all digital changes that we are observing in the profession. The ability to digitally capture in detail the structures of the oral cavity is the trigger that allows designing and manufacturing of customized appliances. On the other hand, a 3D object without the proper tools to design, edit, and modify the 3D object would be useless. 
+
+For this reason, computer-aided-designing (CAD) software is an essential tool that can be used to virtually design the orthodontic appliances in a 
+"tailor-made" manner. CAD software has existed for more than 40 years for engineering, aerospace and other fields. In orthodontics, CAD software started to appear at the beginning of our century mainly for handling and creating dental models, or more recently, for the purpose of manufacturing thermoformed clear aligners from manipulated dental models. In the course of time, and as technology was advancing, other software was developed for the designing of more complex appliances such as maxillary expanders, lingual arches, molar-distalizers, etc. The last years, and as new materials appeared in the market, CAD software has been used to design directly printed aligners or even customized brackets manufactured in the orthodontic office, creating a digital selfsufficient environment. 
+
+The stage of bringing virtual appliances to real life is called undigitization and refers to the process of transforming a virtual 3D object into a real world object [24]. 3D manufacturing is the actual term pertaining to machines being used to perform this process [25] and is divided into subtractive and additive manufacturing. Subtractive manufacturing is the procedure where material is removed from a mass 
+(disc) to create 3D objects. This procedure is rarely used in orthodontics. 
+
+On the other hand, additive manufacturing is the process, where an object is developed by laying down successive layers of a material to build up an object. This procedure is commonly called 3D printing and is used in orthodontics for manufacturing dental models, occlusal splints, metallic appliances, direct printed aligners, brackets etc. Finally, following 3D printing, other machine units are used to clean and completely polymerize the 3D object. 
+
+In essence, a fully digital orthodontic office consists of software that gathers and analyses all digital input taken by cone beam computed tomography, an intraoral scanner, if possible, a face scanner, CAD software, and 3D printers. Intraoral and face records are also input into the software to create a "virtual patient" in contrast to the traditional way, where all the records (study models, cephalograms, panoramic radiographs, etc.) cannot be gathered and evaluated as a single patient file [24]. 
+
+## 2.10. Orthodontic Appliance Designing And Fabrication
+
+In order to be able to design and manufacture customized orthodontic appliances we need to be able to 3D scan the oral cavity, design the appliance in special CAD software, use a material (resin, slurry, metallic powder, etc.), use a 3D printer to fabricate the object, and finally proceed with the final steps of cleaning, curing, and debondingsintering. Most of these procedures can be performed in the orthodontic office, while printing metallic appliances (made by Cobalt-Chromium [CoCr], stainless steel, or titanium) can only be performed in special laboratories. 
+
+2.10.1. *Dental models and aligners* Printing of dental models using special resins is the first digital procedure that was performed in orthodontics by Invisalign (Align Technology, Santa Clara, Calif) at the beginning of our century, while up to that point dental models were only made by pouring plaster into alginate or silicon impressions. The intraoral cavity was digitized using a scanner or by scanning dental impressions and subsequently the 3D dental arches were imported in a CAD software where a setup was performed resulting in exporting multiple dental models. Those models were used to create aligners using the thermoforming procedure, which is not a new technique, but was developed by Sheridan back in the early 90′s [26]. Nowadays, the same workflow can be performed in the orthodontic office where thermoformed aligners are manufactured on dental models that are printed following a setup in commercially available CAD software. CAD software can be found online with the option of buying orthodontic cases (according to the number of dental models that are exported) or can be bought and installed in the orthodontic office. 
+
+Aligners that would be directly printed without the intermediate step of model-printing and thermoforming have always been in the mind of clinicians and companies (Fig. 5). Nevertheless, the obstacle of creating a proper resin that would adequately create an active appliance could not be easily overcome. Essentially, an aligner is the only customized orthodontic appliance that is printed and directly exerts force to move 
+
+![4_image_1.png](4_image_1.png)
+
+teeth. Anything else is not an active, but rather a passive appliance, which is not meant to move teeth of its own (occlusal splints, metallic appliances, customized brackets). For this reason, a directly printed aligner is an appliance that needs to possess properties, which will enable adequate and efficient tooth movement in the desired direction. On one side, the material properties are limiting the efficacy of the movement, if inadequate, and on the other side the multistep workflow, which is not consistent, creates a non-stable and repeatable environment that might pose threats in the demand to have every time the same aligner quality. Briefly, the workflow of printed aligner manufacturing includes 3D scanning, importing of the scans in a CAD software, where setup and designing of the virtual aligners is performed, 3D printing using aligner resin, removing of the excess resin, and finally post printing curing of the aligners in order to give the aligner its final properties. 
+
+The last two years studies have been conducted to investigate many aspects of directly printed aligners. One of the first available resins for printed aligners that appeared in the market was made by Graphy 
+(Seoul, Korea), followed by 3Dresyns (Barcelona, Spain), Luxcreo (Luxmark, Belmont, USA) and Clear A (Senertek, Ismir, Turkey) . The first study concerning Graphy's aligner resin investigated the properties of printed aligners aged for one week of use in the mouth of patients [27]. The results showed a non-significant decrease in all the mechanical properties of the aligners at the end of the week. Cytotoxicity and estrogenicity are terms that are discussed in the community when it comes to dental materials and units. In another study, no signs of cytotoxicity and estrogenicity were found in specimens of directly printed aligners [28]. Leaching is similarly an important factor that can lead to problems with the appiance's integrity and also to patient health hazards. In a recent study, urethane was detected in sets of printed aligners [29]—even though the impact of urethane to humans is not precisely known. Nevertheless, since aligners are the only appliances that are renewed every week, possible leaching phenomena of any substance will be kept in constantly high levels in the patient's mouth, thereby creating potential health hazards to the patient. Roughness is another property that should not be overlooked, especially in appliances that are made of polymers. A study comparing Invisalign aligners and directly printed aligners revealed higher roughness values after one week of wearing for the latter [30]. This could lead to easier aligner microfractures, loss of transparency, material leaching, and overall deterioration of their mechanical properties. Nevertheless, the study was done using the initial printed aligner manufacturing workflow and this could have affected the final outcome. A new study with optimized protocols might have shown more favorable results for printed aligners. 
+
+Comparison of thermoformed and in-office directly printed aligners in terms of dimensional accuracy revealed higher accuracy for the latter, while there was also a 12% increase of the thickness and significant thickness decrease of thermoformed aligners. Due to the inconsistent multistep procedure, which is prone to errors, a different workflow configuration could present different results [31]. However, in another study, printed aligners were found to apply a constant light force to the teeth owing to their flexibility and viscoelastic properties [32]. The force profile of printed aligners versus thermoformed was also studied from another research team concluding that the forces delivered by printed aligners in the vertical dimension were more consistent and of lower magnitude compared to forces exerted from thermoformed ones [33]. Furthermore, a comparison of the mechanical properties between printed and thermoformed aligners revealed a significant difference in elastic modulus, ultimate tensile strength, and stress relaxation. In addition, moisture of the simulated oral environment showed to have a greater effect on the mechanical properties of the printed aligners compared to the thermoformed ones, and this might affect printed aligners' ability to generate and maintain force levels appropriate for tooth movement throughout their use [34]. 
+
+Increases in thickness of the directly printed aligners and their effect on tooth movement was investigated in a recent vitro study [35], which concluded that the ability to change the thickness of printed aligners and the change in force and moments, could be used to optimize the prescribed orthodontic movement while minimizing unwanted tooth movements. This opens new possibilities in the provision completely individualized mechanotherapy for each patient, according to the specific needs of each treatment phase. 
+
+As can be shown from the abovementioned studies, each step of the 3D printed aligners workflow, if not performed correctly, could alter the final result. On the other hand, different printers, software, or curing units currently available in the market could have a different effect on the finally printed aligner outcome in terms of their mechanical properties, transparency, leaching, roughness, etc. For this reason, it is essential to investigate what is the effect of using different printers on the final outcome. A recent study comparing the mechanical properties of directly printed aligners using five different 3D printers showed that mechanical properties of 3D-printed orthodontic aligners are directly dependent on the 3D printer used [36]. 
+
+As is well known, uncured resin (monomer/ oligomer) might exert possibly toxic and allergic side effects on human cells. It is possible that incomplete post-printing cure can increase the chances of toxic or allergic reactions to the patient and therefore, relevant studies on this issue should be carried out before such appliances see widespread use. 
+
+Software for direct aligner printing resembles the ones for manufacturing of thermoformed aligners. The only difference is that the operator must design the virtual aligner on the 3D model that will be later printed (Fig. 6). In addition, the operator must define the proper thickness and offset of the aligner, while in Deltaface (Coruo, Limoges, France) software there is an option, where the software detects the tooth movement prescribed in setup and adds more material on the opposite side of the direction of tooth movement (from 0.1 mm to 0.9 mm according to the operator's wish) (Fig. 6). However, the usefulness of this feature in achieving more predictable tooth movement needs to be clinically proven. Artificial intelligence (AI) is another feature that can be incorporated in such software to facilitate faster designing workflow at the steps of teeth segmentation and setup. In addition, a central server, where all the data will be gathered from multiple offices, can be the center where AI analyzes and gives feedback to the orthodontist for future aligner orthodontic treatment. Thus, more accurate and predictable results might be obtained with the help of big data analysis through the use of AI. 
+
+2.10.2. *Customized metallic appliances* 
+
+![5_image_0.png](5_image_0.png)
+
+CoCr is an alloy used for decades in dentistry for casting removable or fixed partial dentures. During the last decades, a novel 3D printing technology, called selective laser sintering, was introduced, allowing the 3D printing of various metallic appliances in orthodontics. CoCr alloys are used most of the time, while stainless steel, and titanium ones are also available but not used that often. Several companies have released in the market machines for metallic printing with different names and technology variations to manufacture metallic objects. However, their big plethora and potentially hazardous materials used in the printing procedure do not allow their installation in the average orthodontic office. The development of this technology allowed the shift from the manual designing and manufacturing of orthodontic appliances to a digital one. Both techniques, analogue and digital, possess advantages and disadvantages which should be taken into consideration when manufacturing orthodontic appliances. Designing orthodontic appliances can be performed using dedicated CAD software such as 3Shape ortho system software (3Shape, Copenhagen, Denmark), OnyxCeph (Chemnitz, Germany), Deltaface (Coruo, Limoges, France), etc. For the skilled designing personnel, professional engineering CAD software 
+(Meshmixer, Blender, etc) can also be used to design appliances. However, the bigger disadvantage of CoCr alloys is the almost complete lack of flexibility of the appliances. 3D-printed bands are rigid compared to commercially-available bands and cannot pass the maximum circumference of the molars, thereby creating retention problems. Several orthodontic appliances can be designed and printed such as maxillaryexpanders, lingual arches, and distalizers (Fig. 7). Nevertheless, many times an orthodontic technician must manually add pre-fabricated parts such as expander-screws, springs, or other parts for molar distalization, because these cannot be printed. The behavior of CoCr alloy was evaluated in a study examining CoCr-based orthodontic appliances placed for 6 months in the oral environment. The results showed that intraoral ageing did not influence the mechanical properties of the appliances, but the appliance showed degradation in the breakdown potential of the protective oxide layer, which results in pitting corrosion. Thus, it is possible that Cobalt may be released in the patient's mouth [37] and this might be potentially detrimental to the patient's health. 
+
+2.10.3. *Customized orthodontic brackets* 
+
+![6_image_0.png](6_image_0.png)
+
+After introduction of the original Edgewise appliance by E.H. Angle 
+[38], the development of the straight-wire appliance [39] was in fact the first attempt to create customized orthodontic appliance (even if it was tailored simply to the average patient). A real need for completely customized brackets appeared when lingual orthodontic appliances were introduced, due to the unique nature of the lingual surfaces of the teeth. The following years, customized lingual appliances played a big role in orthodontic treatment, offering adequately predictable results, even for difficult cases. 
+
+It wasn't until a few years ago that Ormco (Orange, Calif, USA) 
+created its own series of labial customized brackets, while LightForce (Burlington, Massach, USA) introduced its polycrystalline 3D-printed customized orthodontic brackets. In both systems a 3D scanning is performed, which is then sent to the company to produce the customized brackets. Such online CAD software enables the orthodontist to create a virtual setup, where customized brackets are designed, approved by the orthodontist, and then printed by the company. 
+
+The evolution of 3D-technologies and competition between manufacturers enabled the development of faster, more accurate, and cheaper 3D printers. New materials were invented and introduced to the market, with CAD software being an integral part in the appliances' design. The last years, many orthodontic offices have installed all the necessary units for designing and 3D printing, thus creating small digital laboratories of their own and thermoformed or directly printed aligners, occlusal splints, indirect bonding trays, and dental models are nowadays often manufactured within the orthodontic office. 
+
+Nevertheless, orthodontic treatment is mainly based on fixed appliances, the orthodontic brackets, which up to now could not be adequately manufactured in the orthodontic office. 3D technology advancements enable the orthodontic office to become a small lab that can print customized orthodontic brackets. Novel software called Ubrackets (Coruo, Limoges, France) enables the orthodontist to perform a digital setup of imported dental scans and automatically design customized orthodontic brackets together with their customized archwires [24]. The workflow of manufacturing customized brackets can be divided into the designing and the printing part. At the designing part the operator separates the teeth from the gingiva in a stage called segmentation to perform the setup of the dental arches. At the next step the orthodontist chooses to design labial or lingual customized brackets which will be later printed. Following that, the brackets are automatically positioned on a flat rectangular archwire opposite the teeth's surfaces and with special manipulators the operator positions the brackets on the desired place creating customized brackets where their bases are adapted to the tooth surfaces (Fig. 8). The next step is to design indirect bonding trays or positioning keys for each bracket which will help the orthodontist bond them in an accurate way, and which should be easily removed after bonding. Customized archwires can also be exported as 3D files for an archwire bending robot or in a electronic drawing for manual plier bending. Maybe the most important issue to be solved is the material that will be used to create the customized brackets. In the orthodontist's armamentarium several printers can be found together with specialized software to solve the problem of designing and printing. Nevertheless, the key to creating good-quality brackets for orthodontic treatment is the material. Attempts have been made to print customized brackets using hybrid ceramic permanent crown resins (Fig. 9), while the first study to compare two resins, normally used for temporary and permanent crown resins, to print brackets was published a few years ago [40]. 
+
+The study concluded that there was no significant difference between 
+
+![6_image_1.png](6_image_1.png)
+
+![7_image_0.png](7_image_0.png)
+
+the two resins in terms of mechanical properties. In addition, hardness, a property that is very important for brackets, as it presents the resistance to wear off, was relatively low (though almost double compared to commercial plastic brackets available in the market). In another study, 3D printed zirconia brackets (Fig. 10) made in a ZiproD (AON, Seoul, Korea) zirconia printer were compared to commonly-available Clarity 
+(3 M, Monrovia, USA) and LightForce (Burlington, Massach, USA) brackets [41]. The study revealed high hardness values for Clarity brackets followed by LightForce and zirconia brackets. Despite that, hardness was above the ideal value for brackets making them all suitable for orthodontic treatment. In the same study, zirconia exhibited the higher fracture toughness values followed by Clarity and LightForce. A disadvantage of zirconia brackets was the color, which is white, while a solution to that could be the use of special zirconia paintings that are used to color the brackets the same color with the patient's teeth. Lately, AON (Seoul, Korea) released a translucent zirconia that can be used for brackets printing. 
+
+Designing and printing brackets in the orthodontic office is definitely a big step towards creating digital, self-sufficient orthodontic offices. In addition, it seems that the cost of customized brackets is much lower compared to the commercial pre-fabricated ones, while the ability to reprint brackets, in cases of accidental debonds, must not be underestimated. Nevertheless, the advantages of customized brackets are not limited only to the above. Brackets can be designed in bigger mesiodistal widths when great rotations must be corrected or can be smaller when severe crowding does not allow the use of regular sized brackets. In addition, in cases where increased torque must be used when retracting upper incisors, additional torque can be incorporated in the prescription. Overcorrection can be included to the canine brackets to counteract the tipping and rotational side effects movements when they are moved distally into extraction spaces. In the case of customized brackets, no single one prescription exists that will optimally move the teeth in a specific way. On the contrary, the operator must create a unique prescription for each specific patient following existing diagnostics and treatment-plan in a tailor-made fashion. The orthodontist can choose from a big amount of brackets variation to fulfill the orthodontic treatment in an efficient and predictable way. 
+
+Lastly, AI is already a reality in our lives and penetrates more and more medicine, dentistry, and orthodontics. In the case of customized brackets, AI can play the role of decreasing the designing time through automation of steps such as teeth segmentation, setup and most important customized brackets designing and positioning [42,43]. A 
+centralized server gathering all data from different offices could use AI analysis to provide feedback for the creation of optimized appliances. AI can be a reliable assistant to our orthodontic treatment, assisting in digital setups, tooth segmentation, predicting teeth movement and growth, tracing cephalograms, etc. Nevertheless, 3D technologies and AI will never substitute the orthodontist but might prove a valuable 
+
+![7_image_1.png](7_image_1.png)
+
+assistant in our efforts to create better smiles in a more efficient and predictable matter. 
+
+## Funding None.
+
+Declaration of Generative AI and AI-assisted technologies in the writing process 
+
+## None. Conflict Of Interest
+
+TE and SNP have no conflict of interest to declare. NP discloses a financial interest with the company Coruo (Limoges, France) concerning the orthodontic computer-aided design software UBrackets. 
+
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medical/md/PMC10761123.md

@@ -0,0 +1,264 @@
+RESEARCH PAPER
+
+# Structure And Function Of The Pseudouridine 5'-Monophosphate Glycosylase Pumy From **Arabidopsis Thaliana**
+
+Jeongyun Leea, Sang-Hoon Kim a, and Sangkee Rhee a,b aDepartment of Agricultural Biotechnology, Seoul National University, Seoul, Korea; bResearch Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea ABSTRACT
+Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5'-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5'-monophosphate (ΨMP) into uridine and ribose 5'-phosphate, which are recycled via other metabolic pathways. Structural features of *Escherichia coli* PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5'-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.
+
+ARTICLE HISTORY
+
+![0_image_0.png](0_image_0.png)
+
+Revised 10 October 2023 Accepted 5 December 2023 KEYWORDS
+Crystal structure; C-nucleoside; metalloenzyme; conformational changes; substrate fidelity
+
+## Introduction
+
+Pseudouridine is one of the most prevalent RNA modifications [1,2] and is a noncanonical C-nucleoside containing a C-C glycosidic linkage between the C5 atom of the nucleobase uracil and the C1' atom of ribose (Figure 1). In transfer RNAs (tRNAs), as well as noncoding and coding RNAs, pseudouridine has a role in tRNA stability maintenance and messenger RNA translational regulation, together with diverse biological effects [1–3]. Pseudouridine degradation was first reported in *Escherichia* coli [4,5] and more recently in *Arabidopsis thaliana* [6]. In A. 
+
+thaliana, metabolic linkage of pseudouridine degradation and its products to other pathways were proposed (see Fig. 14 in [6]). Pseudouridine produced from RNA breakdown in the vacuole is exported to the cytosol and then into the peroxisome, in which pseudouridine degradation occurs via a two-step reaction (Figure 1). In the first reaction, pseudouridine is converted into pseudouridine 5'-monophosphate (ΨMP) by pseudouridine kinase (PUKI; YeiC in *E. coli*) and subsequently ΨMP is cleaved into uracil and ribose 5'-phosphate (R5P) by pseudouridine 5'- monophosphate glycosylase (PUMY; YeiN in *E. coli*) [5,6]. The products, uracil and R5P, are recycled via other metabolic pathways. Uracil enters a salvage reaction or pyrimidine ring catabolism, and R5P a phosphoribosyl transfer reaction or a pentose phosphate pathway. A bioinformatics search identified the genes for PUKI and PUMY in many bacteria and some eukaryotes [7], but not in mammals, consistent with the observation that pseudouridine is not metabolized in mammals. In *A. thaliana*, 
+malfunction of pseudouridine catabolism caused adverse physiological effects on plant development and growth [6]. Structural and biochemical studies of PUKI from *A. thaliana* [8] and *E. coli* [9] have shown that a threonine or serine residue contributes to the active site of the adjacent subunit in a dimeric enzyme and mediates interactions with the pseudouridine-specific N1 atom. These interactions trigger the conformational changes required for catalysis, providing mechanistic insight into the substrate specificity of PUKI, 
+which has greatly enhanced catalytic efficiency towards pseudouridine over a structurally homologous uridine, despite the similar Km values for the two nucleosides. Unlike PUKI, 
+which lacks sequence conservation from *E. coli* to eukaryotes [6,7,9], PUMY exhibited high sequence homology among many bacteria and some eukaryotes (Supplementary Fig. S1) 
+[7], with ~50% sequence identity between *E. coli* and *A. thaliana*. PUMY is therefore a distinct enzyme family and catalyses a reversible reaction for the cleavage and formation of the CC glycosidic bond in ΨMP to and from uridine and R5P, 
+respectively (Figure 1) [7]. *In vitro*, PUMY strongly favours the reaction for ΨMP synthesis, not ΨMP degradation, with equilibrium constants for the degradation reaction of 8.9 × 
+10−5 M [10] and 2.3 × 10−4 M [5].
+
+The structure of *E. coli* PUMY (EcPUMY; YeiN) [11] 
+showed the features of the enzyme and a novel catalytic CONTACT Sangkee Rhee srheesnu@snu.ac.kr Department of Agricultural Biotechnology, Seoul National University, Seoul, KOREA
+Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2023.2293340
+© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 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. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
+
+![1_image_0.png](1_image_0.png)
+
+mechanism for the C-C bond cleavage between the nucleobase (hereafter referred to as uracil-Ψ) and R5P. Here, we present three crystal structures of *A. thaliana* PUMY (AtPUMY), each in a different ligation state. We have also conducted a kinetic analysis of the putative active site residues for the ΨMP degradation reaction. Our findings provide structural and functional insights into the active site residues of PUMY.
+
+## Results And Discussion Structure Of Atpumy–Mn2+ Holoenzyme
+
+For the crystallographic analysis, a C-terminal His-tagged AtPUMY was crystallized by removing the N-terminal 20 residues (Glu21–Met330; Supplementary Fig. S1). In the crystal structure of the AtPUMY–Mn holoenzyme, the asymmetric unit in the space group *P213* contains a monomeric AtPUMY (Table 1). Crystallographic symmetry operation of those asymmetric units generated a trimeric structure of AtPUMY, consistent with the results of sizeexclusion chromatography (Supplementary Fig. S2A).
+
+The 1.45 Å resolution crystal structure of the AtPUMY– 
+Mn holoenzyme displayed structural features from Ser28 to Met330, with three additional Leu, Glu, and His residues at the C-terminus, which were introduced for gene cloning. Monomeric AtPUMY adopted an α/β-fold with a twisted central β-sheet (Figure 2A and Supplementary Fig. S1), 
+which exhibited convex and concave sides, with two βstrands (i.e., β5 and β6) and 13 α-helices flanking the βsheet. Among the flanking elements, two antiparallel helices at the C-terminal region, α11 and α12, protruded from the β-sheet as much as ~34 Å and ~21 Å, respectively, and were highly bent by ~90° towards the concave side of the central β-sheet. The flanking structural elements were topologically dissected into two regions, one with the N-terminal region of Ser28–Gln145 (i.e., α1–α6) and the other with the Cterminal region of Val147–Met330. Between the two regions was a 44 Å-long C-terminal α13. On the concave side of the β-sheet, there is a vacancy between the two flanking regions. The vacancy, which had a wide opening, was surrounded by three flanking clusters, comprising Ndomain antiparallel α2 and α3; a protruding loop (magenta in Figure 2A) connecting β7, α7, and α8; and C-domain α11, α12, and α13 (Figure 2A). C-domain α11 and α12 likely serve as a lid for the vacancy. A metal-binding site lies in the ~10 Å inside of the vacancy and along the loop after α7 (Figure 2B). The putative Mn2+ ion is of square bipyramidal geometry, with Asp164 and a water molecule across Asp164 in the axial ligand, and four water molecules at the equatorial ligands. Within 2.4 Å of the axial water molecule, one molecule of sulphate ion, which is from the crystallization solution, was identified farther inside the active site, without forming any possible direct coordination to the Mn2+ ion. The binding of sulphate was stabilized by interactions at distance less than 3.0 Å with Arg116, Ser166, and Ser167 and with the axial and equatorial water molecules in the metal-binding site. Based on these structural features, the vacancy could serve as an active site of AtPUMY, but the opening remained wide in the monomer (Figure 2B and Supplementary Fig. S3A).
+
+In trimeric AtPUMY, intersubunit interactions transformed the putative, open active site in a monomer into a pocket-shaped site. Three subunits were arranged with threefold symmetry, and each was oriented with the convex side outwards (Figure 2C). In the trimer, α9* (herein, an asterisk represents residues or elements from the nearby subunit) on the concave sides of β-sheet mediated intersubunit interactions with the flanking elements on the concave sides of the nearby subunits (Figure 2D), which provides an additional wall to the vacancy, thus generating a pocket-shaped active site in the trimer (Supplementary Fig. S3B). Protein interfaces, surfaces, and assemblies analysis [12] indicated that trimerization buried a surface area of 7770 Å2.
+
+## Structural Features Of Atpumy(K185A) In Complex With Citrate Or Ψmp
+
+We employed an AtPUMY(K185A) mutant to identify the ligand-binding modes. Lys185 in AtPUMY is equivalent to
+
+| AtPUMY                                              | AtPUMY(K185A)       | AtPUMY(K185A)      |                   |
+|-----------------------------------------------------|---------------------|--------------------|-------------------|
+| (Mn2+)                                              | (Mn2+, citrate)     | (Mn2+, ΨMP, R5P)   |                   |
+| Data set PDB ID                                     | 8K05                | 8K07               | 8K06              |
+| Data collection Crystal                             | Native              | Native             | Native            |
+| Wavelength (Å)                                      | 0.97933             | 0.97934            | 0.97932           |
+| Resolution (Å)                                      | 50.0–1.45           | 50.0–1.12          | 50.0–1.86         |
+| (1.50–1.45)a                                        | (1.16–1.12)         | (1.93–1.86)        |                   |
+| Unique reflections                                  | 104,528             | 316,141            | 81,648            |
+| Multiplicity                                        | 21.9 (21.9)         | 1.9 (1.9)          | 3.5 (3.5)         |
+| Completeness (%)                                    | 99.9 (100.0)        | 92.4 (82.1)        | 99.0 (99.9)       |
+| Mean I/sigma(I)                                     | 37.2 (1.3)          | 16.9 (1.0)         | 13.0 (2.6)        |
+| Wilson B-factors (Å2 )                              | 20.7                | 12.6               | 29.0              |
+| R-mergeb                                            | 0.07 (2.74)         | 0.05 (0.47)        | 0.14 (1.11)       |
+| CC1/2c                                              | 0.99 (0.46)         | 0.99 (0.59)        | 0.97 (0.49)       |
+| Space group                                         | P213                | P1                 | P21               |
+| Unit cell a, b, c (Å)                               | 121.1, 121.1, 121.1 | 57.3, 68.5, 73.1   | 72.6, 105.8, 73.6 |
+| α, β, γ (º)                                         | 90, 90, 90          | 117.9, 94.3, 109.4 | 90, 118.9, 90     |
+| Refinement R-workd (%)                              | 20.6                | 20.5               | 18.3              |
+| R-freee (%)                                         | 21.7                | 21.9               | 20.8              |
+| No. of atoms Macromolecules                         | 2,260               | 6,623              | 6,740             |
+| Ligands                                             | 11                  | 42                 | 17                |
+| Water                                               | 300                 | 809                | 646               |
+| RMS(bonds) (Å)                                      | 0.006               | 0.006              | 0.009             |
+| RMS(angles) (º)                                     | 1.059               | 1.153              | 1.197             |
+| Ramachandran Favored (%)                            | 99.01               | 98.76              | 98.45             |
+| Outliers (%)                                        | 0.00                | 0.34               | 0.11              |
+| Average B-factor (Å2 )                              | 30.7                | 19.7               | 33.5              |
+| Macromolecules                                      | 29.6                | 18.9               | 32.7              |
+| Ligands                                             | 44.5                | 16.1               | 45.4              |
+| Water                                               | 38.9                | 26.6               | 42.0              |
+| Table 1. Data collection and refinement statistics. |                     |                    |                   |
+
+Lys166 in EcPUMY (Supplementary Fig. S1); it was responsible for the cleavage of the C-C glycosidic bond in ΨMP (see Comparisons with EcPUMY and recognition of ΨMP) [11]. The 1.12 Å resolution crystal structure of the AtPUMY 
+(K185A)–Mn2+–citrate complex contained a trimeric structure in the asymmetric unit of the P1 space group (Table 1). 
+
+The complex showed binding of one molecule of citrate to each subunit, using a crystallization solution containing 0.1 M sodium citrate. Citrate is directly bound to the metal-binding site, with its binding mode differing from that of sulphate in the holoenzyme (Figure 3A and Supplementary Fig. S4A). The C2 hydroxyl group and C2 carboxylate group of citrate directly coordinated the metal ion by replacing an axial and one of the equatorial water molecules, respectively. The distal C1 and C3 carboxyl groups of citrate interacted with Thr289 in α12 and Ser167 in a loop following α7, respectively, and the C2 hydroxyl group mediated an intersubunit interaction with Glu198* in α9×. The C3 carboxyl group was located farther inside the active site at a position coinciding with that of sulphate in the holoenzyme.
+
+Co-crystallization of AtPUMY(K185A) with ΨMP produced a crystal of the *P21* space group with a trimer in the asymmetric unit (Table 1) and showed two different ligands at 1.86 Å resolution. Substrate ΨMP was present at two subunits (Figure 3B and Supplementary Fig. S4B) and the reaction product R5P was present in one subunit (Figure 3C and Supplementary Fig. S4C), possibly due to residual glycosylase activity of the K185A mutant or a non-enzymatic reaction towards ΨMP. The binding modes of ΨMP and R5P might represent unproductive ones for catalysis due to mutation K185A. We later noticed that ΨMP position is identical to that of a possible reaction intermediate of EcPUMY [11], but that R5P displayed a binding mode that differed from those of the other ligands (see *Comparisons with EcPUMY and recognition of ΨMP*). The complexes thus provide structural information that facilitates functional analysis. ΨMP and R5P were found in the active site with an orientation almost parallel to α12 (Supplementary Fig. S4B,C). The phosphate groups of the ligands were located near the metal-binding site, forming interactions with water molecules in the metal-coordinating shell. The ribosyl moiety and nucleobase uracil-Ψ in ΨMP were aligned towards the C-terminal α13, the ceiling element for the active-site pocket. There are several notable features, including the binding mode for ligands and conformational changes. First, there were no direct interactions of the phosphate group of ΨMP and R5P to the metal ion. In contrast to the citrate-binding mode, the metal-coordinating environments in this complex are identical to those in the holoenzyme. The 5'-phosphate group in R5P, which was within ~4 Å 
+of Arg116, Ser166, and Ser167, occupied the site for the sulphate ion in the holoenzyme, and its ribosyl moiety was further stabilized by interacting with Lys112 and Thr289
+
+![3_image_0.png](3_image_0.png)
+
+within ~4 Å (Figure 3C). Second, ΨMP showed a binding mode distinct from that of R5P (Figure 3B). The 5'-phosphate group in a ribosyl moiety of ΨMP occupied a site corresponding to the binding site for the ribosyl C4' atom of R5P, not the phosphate group, but maintained interactions with water molecules in the metal-coordinating shell. The phosphorus atoms in ΨMP and R5P showed ~3.3 Å displacement (Figure 3B,C). Thus, the uracil-Ψ moiety of ΨMP was located in the ceiling of the active site. The side chains of the following residues were within ~5 Å of each moiety in ΨMP: Lys112, His156, Ser166, Ser167, Glu198*, and Thr289 from the 5'- phosphate group; Glu50, Thr131, and Asp168 from the ribosyl moiety; and Thr52, Ile53, Phe215, Leu293, and Asn308 from uracil-Ψ. In particular, hydrophobic features, except for Asn308 at the ceiling element α13, were notable in the active site around uracil-Ψ, with one or two water molecules between uracil-Ψ and Thr149 (Figure 3B). We later noted that Thr149 likely plays a role in recognizing the uracil-Ψ 
+moiety in ΨMP (see *Comparisons with EcPUMY and recognition of ΨMP*).
+
+The structures of these ternary complexes exhibited high similarities with that of the holoenzyme, with a root-meansquare deviation of 0.89–1.27 Å for the 267 Cα atoms of AtPUMY(K185A)–Mn2+–citrate and 0.83–1.13 Å for the 272 Cα atoms of AtPUMY(K185A)–Mn2+–ΨMP and R5P. Despite the high homology in the overall structures, we observed ligand-dependent structural changes of the lid, which consisted of α11 and α12, to the active site pocket. We adopted the Cα-interatomic distance between Arg116 at α5 and Asn286 at α12, which indicated the movement of the lid
+
+![4_image_0.png](4_image_0.png)
+
+(Figure 2D and 3D). The Cα-interatomic distance was ~20 Å in the holoenzyme, ~15 Å in the complex with citrate, and 
+~14 and ~12 Å in the complexes with R5P and ΨMP, respectively, indicating that the lid closed the pocket upon binding of ligands, and the phosphate moiety and a uracil-Ψ base in the ligands contribute to the lid closure. There is a direct interaction of Thr289 at the lid element α12 with the citrate C1 carboxyl group in the complex with citrate (Figure 3A), to a ribosyl moiety of R5P and the 5'-phosphate group of ΨMP 
+in the complexes with R5P and ΨMP (Figure 3B,C), respectively, together with hydrophobic interactions between ΨMP 
+and residues in α12. These interactions possibly stabilize the closed conformation of the active site of AtPUMY.
+
+## Kinetic Analysis
+
+We conducted a steady-state kinetic assay of PUMY activity with full-length AtPUMY (Figure 4A); the Km and *kcat* values for ΨMP degradation were 2.3 µM and 62.8 min−1. Subsequent assay indicated that AtPUMY requires the Mn2+ 
+ion for full activity, with the Km value of 2.3 µM 
+(Supplementary Fig. S5A,B). Consistent with the C-nucleoside glycosylase activity of EcPUMY [11], AtPUMY was also specific for ΨMP thus exhibiting, essentially, no measurable activity on other purine and pyrimidine N-nucleoside monophosphates (Figure 4B). We next performed activity assays and kinetic measurements to identify the structural determinants of ΨMP degradation. Seventeen residues within ~5 Å of each moiety in ΨMP (Figure 3B) were mutated, and the mutants subjected to functional assays. Specific activity measurements indicated that most residues contributed to activity, with 0–17% of the wild-type enzyme activity (Figure 4C), except for H156A (74%) and S167A (99%). Subsequent kinetic analysis of the mutants also revealed that mutation of most residues significantly reduced catalytic efficiency (i.e., *kcat/* Km). The resulting catalytic efficiencies of mutants showing measurable activity were 0.5 ~ 4.5% relative to the wild-type enzyme, except for T52A (19.8%), S133A (31.8%), H156A (26.6%), and S167A (55.3%) (Table 2); all exhibited 0.5 ~ 27fold increases in Km values and 0.7 ~ 38.5-fold reductions in kcat values. We also found that certain mutants (i.e., E50A, 
+K112A, D164A, S166A, K185A, L293G, and T289A) were inactive even at enzyme concentrations up to 100-fold (i.e., 8 µM) of other mutants. Such inactivities closely reflected the functional roles. Glu50 corresponds to Glu31 of EcPUMY, which may function as a proton donor in catalysis (see Comparisons with EcPUMY and recognition of ΨMP) [11]. 
+
+Lys112 and Ser166 are phosphate-binding sites of the substrate ΨMP, and Leu293 serves as a binding site for the uracilΨ moiety in ΨMP. Asp164 is a metal-coordinating residue 
+(Figure 2B), suggesting that the metal-binding site anchors the phosphate group of the substrate, even in the absence of any
+
+![5_image_0.png](5_image_0.png)
+
+direct interactions between them. The role of Thr289 in α12 is to mediate interactions with the phosphate group in ligands and to accompany movement of the lid helix α12 in response to the binding of ligands. The inactive T289A mutant suggests that ligand-dependent movement of the lid region is required for catalysis.
+
+Interpreting the kinetic parameters of the mutants is difficult, given that PUMY-mediated catalysis features multiple steps [11]. The locations of the residues (Figure 3B,C) and the catalytic efficiencies of the mutants (Table 2) suggest possible functional roles of the residues of interest. In the vicinity of the uracil-Ψ moiety in ΨMP, 
+Thr149 and Asn308 may play an essential role in recognizing the uracil-Ψ moiety and catalysis, via hydrogen bonding, and Ile53 and Phe215 create a hydrophobic environment for uracil-Ψ moiety. Relative to the catalytic efficiency of the wild-type enzyme, the figures for the mutants were; T149A (0.8%), T149V (0.49%), N308A (0.52%), I53A (1.50%) and F215A (1.78%). Thr131 and Ser133 likely serve as the binding sites for the ribosyl moiety and His156 and Ser167 as phosphate-binding sites. Glu198 is unique in that it originates from an adjacent subunit, but nonetheless plays an important role as a phosphate-binding site of ΨMP.
+
+## Comparisons With Ecpumy And Recognition Of Ψmp
+
+The structural features reported here are similar to those of EcPUMY [11]. A previous structural analysis of EcPUMY identified two possible reaction intermediates: a ring-opened ribose ΨMP adduct (Supplementary Fig. S4D) and a ringopened R5P adduct, but ΨMP was identified as a free form in the EcPUMY(K166A) mutant. The binding modes of the two adducts and ΨMP were identical, particularly for the phosphate group. Each adduct was covalently attached to Lys166EcPUMY (superscript EcPUMY and AtPUMY represent those of EcPUMY and AtPUMY, respectively), providing a structural clue to the ribose ring-opening mechanism for the cleavage of the C-C glycosidic linkage in ΨMP. In the proposed mechanism, a ribose ring-opening reaction involves the proton donor Glu31EcPUMY and is followed by glycosyl bond cleavage, which is facilitated by the formation of a covalent linkage between ring-opened ribose and Lys166EcPUMY. During C-C 
+glycosidic bond cleavage, Thr130EcPUMY, which corresponds to Thr149AtPUMY, was suggested to stabilize the leaving uracil by forming a hydrogen bond to the uracil-Ψ N1 atom. In a ring-opened ribose ΨMP adduct, a uracil-Ψ repositioned its orientation by rotating ~90° from that of ΨMP and was located farther inside, where the pseudouridine-specific N1
+
+| Table 2. Kinetic parameters of AtPUMY mutants.                                                                                                | Km (μM)a     | kcat (min−1)   | kcat/Km (μM−1 min−1)   |       |       |
+|-----------------------------------------------------------------------------------------------------------------------------------------------|--------------|----------------|------------------------|-------|-------|
+| Wild type AtPUMY                                                                                                                              | 2.30 (0.29)b | 62.8 (2.4)     | 27.3                   | 100%  |       |
+| Mutants in the vicinity of the uracil-Ψ                                                                                                       | T52A         | 0.964 (0.30)   | 5.22 (0.27)            | 5.41  | 19.8% |
+| I53A                                                                                                                                          | 3.96 (3.2)   | 1.63 (0.32)    | 4.11 × 10−1            | 1.51% |       |
+| T149A                                                                                                                                         | 34.9 (12)    | 7.67 (0.79)    | 2.20 × 10−1            | 0.81% |       |
+| T149V                                                                                                                                         | 61.1 (22)    | 8.25 (1.1)     | 1.35 × 10−1            | 0.50% |       |
+| F215A                                                                                                                                         | 37.4 (16)    | 18.2 (0.05)    | 4.86 × 10−1            | 1.78% |       |
+| L293G                                                                                                                                         | n.d c        | n.d.           | n.d.                   | n.d.  |       |
+| N308A                                                                                                                                         | 15.9 (5.7)   | 2.27 (0.29)    | 1.43 × 10−1            | 0.52% |       |
+| Mutants in the vicinity of the ribosyl moiety                                                                                                 | E50A         | n.d.           | n.d.                   | n.d.  | n.d.  |
+| T131A                                                                                                                                         | 14.8 (3.3)   | 18.2 (0.91)    | 1.23                   | 4.51% |       |
+| S133A                                                                                                                                         | 1.98 (1.1)   | 17.2 (1.9)     | 8.68                   | 31.8% |       |
+| K185A                                                                                                                                         | n.d.         | n.d.           | n.d.                   | n.d.  |       |
+| Mutant in the vicinity of the 5'-phosphate ΨMP                                                                                                | K112A        | n.d.           | n.d.                   | n.d.  | n.d.  |
+| H156A                                                                                                                                         | 12.4 (1.8)   | 90.0 (3.9)     | 7.26                   | 26.6% |       |
+| S166A                                                                                                                                         | n.d.         | n.d.           | n.d.                   | n.d.  |       |
+| S167A                                                                                                                                         | 5.62 (0.83)  | 84.9 (2.8)     | 15.1                   | 55.3% |       |
+| E198A                                                                                                                                         | 62.5 (11)    | 51.1 (2.6)     | 8.18 × 10−1            | 3.00% |       |
+| T289A                                                                                                                                         | n.d.         | n.d.           | n.d.                   | n.d.  |       |
+| Mutant for the metal coordinating residue                                                                                                     | D164A        | n.d.           | n.d                    | n.d.  | n.d.  |
+| a The Km values were measured towards ΨMP. b The numbers in parentheses represent the standard errors. c n.d., not detected with 8 µM AtPUMY. |              |                |                        |       |       |
+
+atom is within a hydrogen-bonding distance of ~3.5 Å from Thr130EcPUMY (Supplementary Fig. S4D) [11]. Further kinetic analysis validates the proposed functional roles of these active site residues for ΨMP synthesis [11].
+
+In this study, we characterized that the binding mode of free ΨMP to AtPUMY was identical to those of EcPUMY including a ring-opened ribose ΨMP adduct (Supplementary Fig. S4D) and a free ΨMP in the K166A 
+mutant, except for the orientation of uracil-Ψ. The pyranosyl R5P of AtPUMY exhibits a binding mode distinct from those of other ligands (Figure 3B,C), indicating that the reaction product R5P readily departs from the active site. Our structure-based mutational analysis of AtPUMY affords further insights into the functional roles of residues around ΨMP. The AtPUMY-mediated catalysis exhibits several features of interest. First, Thr149 and Asn308 near uracil-Ψ are likely major determinants for recognizing the uracil-Ψ of ΨMP. It is known in EcPUMY that Thr130EcPUMY recognizes the uracil-Ψ N1 atom in a ringopened ribose ΨMP adduct [11], strongly supporting our kinetic analyses of T149A and T149V mutants (Table 2). Second, the sequence conservation (Supplementary Fig. S1) between EcPUMY [11] and AtPUMY and their structural features both indicate that hydrophobic properties and a spacious active site to accommodate water molecules around uracil-Ψ are general features conserved in the active site of PUMY (Supplementary Fig. S4D). These characteristics allow the nucleobase uracil-Ψ to reorient during catalysis. If there were specific or strong interactions around uracil-Ψ at the beginning of catalysis, uracil-Ψ repositioning could not be achieved. For example, a rotation of ~180° 
+around the NE–CZ bond in arginine is required for catalysis in arginine dihydrolase [13,14], which consecutively liberates two ammonia molecules from the guanidinium group of arginine. In arginine dihydrolase, asparagine is essential for orienting the susceptible guanidinium group of arginine to a catalytic residue. Mutation of asparagine to aspartate eliminated activity, indicating that relatively weak interactions with the substrate allow the proposed rotation of a reaction intermediate. Third, the binding modes of ΨMP and R5P phosphate groups to AtPUMY present additional features (Figure 3B,C). It is unusual in metalloenzymes that the phosphate group of ligands mediates interactions only with the metal-coordinating water molecules, which was commonly identified in both EcPUMY [11] and AtPUMY. Apart from the unusual indirect bindings of phosphate groups to metal ion in AtPUMY, the different binding locations of the ΨMP and R5P phosphates are also unique. Such results suggest that ligand phosphate groups do not serve as major anchors in terms of substratebinding to PUMY; rather, a nucleobase in the substrate dictates the productive binding mode for catalysis. This explains why the binding site of the reaction product R5P of the AtPUMY (K185A) mutant differed from that of the phosphate group in ΨMP (Figure 3B,C). However, the phosphate group-binding site of a ring-opened R5P adduct in EcPUMY was identical to those of a ring-opened ribose ΨMP adduct and a free ΨMP, given the covalent linkage to Lys166EcPUMY. Conformational changes in the lid region are an additional feature in catalysis (Figure 3D). Conformational changes, including the repositioning of uracil-Ψ in the substrate and lid movement in the enzyme, are required for catalysis, as suggested by the catalytically impaired T149A and T149V mutants, and the inactive T289A mutant, respectively.
+
+## Conclusions
+
+Structural and functional analyses of AtPUMY suggest that a nucleobase uracil-Ψ is required for ΨMP to bind to the active site in a productive mode for catalysis, and a concurrent transition of the active site into a closed state by lid movement is also essential for catalysis. Recognition of the uracil-Ψ N1 atom and the nucleobase release are likely facilitated by Thr149. Therefore, mutation of Thr149 rendered AtPUMY an inefficient enzyme, with <1% of the catalytic efficiency of wild-type AtPUMY. Our findings provide the structural and biochemical bases of AtPUMY catalysis towards ΨMP.
+
+## Materials And Methods Cloning And Purification
+
+A gene for a full-length AtPUMY (Met1–Met330; GenBank: AT1G50510) was cloned from the cDNA library of A. thaliana. For the structural and biochemical studies, genes for various AtPUMYs were produced by PCR with the respective primers (Supplementary Table s1).
+
+For crystallographic analysis, crystallization of C-terminal His-tagged AtPUMY was achieved by truncating the Nterminal 20 residues. The resulting variant of C-terminal His-tagged AtPUMY (Glu21–Met330) was expressed and purified as follows: The PCR product of AtPUMY was cloned into the pET-41b expression vector (Merck, Kenilworth, NJ, USA) containing a C-terminal (His)5-tag. 
+
+E. coli Rosetta2 (DE3) cells (Merck) transformed with constructs for AtPUMY were cultured at 37°C in Luria - 
+Bertani medium until the absorbance at 600 nm reached 0.5–0.7. Enzyme expression was induced by adding 0.5 mM IPTG, followed by incubation at 20°C for ~15 h. Cells were collected by centrifugation and sonicated in buffer A (50 mM Tris at pH 8.0, 300 mM NaCl) and 1 mM MnCl2, and the supernatant was obtained by centrifugation at 30,000 × g for 30 min at 4°C. The C-terminal Histagged enzyme was purified using a HisTrap HP column 
+(GE Healthcare, Chicago, IL, USA) with buffer A and eluted using buffer B (buffer A plus 500 mM imidazole) 
+and subsequently subjected to size-exclusion chromatography using a HiLoad 16/60 Superdex 200 pg column (GE 
+Healthcare) with buffer A.
+
+For biochemical analysis, the wild type and various mutants of full-length AtPUMY, with a C-terminal His-tag, were constructed. The AtPUMYs were purified using a HisTrap HP 
+column as described above followed by further purification with a HiPrep 26/10 Desalting column (GE Healthcare) with buffer A. All the enzymes were stored at −20°C.
+
+## Crystallization And Structure Determination
+
+Purified AtPUMY and AtPUMY(K185A) mutants in 50 mM Tris at pH 8.0 and 300 mM NaCl were concentrated to ~16 and ~44 mg mL−1 and subjected to crystallization using the sitting drop vapour diffusion method at 22°C. Crystallization of AtPUMY in the presence of Mn2+ was achieved, first by crystallizing an apo-AtPUMY, with a solution containing 0.1 M cacodylate (pH 6.5) and 1.4 M ammonium sulphate, and then by soaking a crystal of apo-AtPUMY for 1 min with 8 mM MnCl2. A crystal of AtPUMY(K185A) complexed with Mn2+ and citrate was obtained by crystallization of AtPUMY 
+(K185A) in the presence of 2 mM Mn2+, 2 mM R5P, and 2 mM uracil, with 0.1 M sodium citrate pH 6.5 and 16% PEG 
+10,000. Later, we noticed under these crystallization conditions that the structure of AtPUMY(K185A) contained Mn2+ 
+and citrate from the crystallization solution, not a reaction product. Crystallization of the AtPUMY(K185A) mutant in a complex with ΨMP was carried out in the presence of 100 µM 
+ΨMP and 2 mM MnCl2 using a solution of 0.2 M lithium sulphate, 0.1 M MES pH 6 and 20% (w/v) PEG 4000. The substrate ΨMP was enzymatically synthesized (see *Enzymedependent synthesis of ΨMP*). In all cases, 20% ethylene glycol was used as a cryoprotectant for data collection at 100 K. Diffraction data were collected with a 0.5° oscillation angle at beamline 7A and 11C of the Pohang Accelerator Laboratory (Pohang, Republic of Korea). Program HKL2000 suite was used for diffraction data processing [15] and a CC1/2 statistical value of approximately 0.4–0.5 was used as the high-resolution cut-off [16,17].
+
+Three structures had a high resolution of 1.12–1.86 Å. 
+
+Crystals of the AtPUMY–Mn2+ complex belonged to the space group *P213*, with a monomer in the asymmetric unit, and two complexes of AtPUMY(K185A)–Mn2+–citrate and AtPUMY 
+(K185A)–Mn2+–ΨMP/R5P in the space groups P1 and P21, respectively, with a trimer in the asymmetric unit. Structural determination of AtPUMY was carried out by molecular replacement using PHENIX AutoMR [18], with EcPUMY (PDB id, 4GIK) [11] as the search model. The refined model of the AtPUMY holoenzyme was used as a search model for molecular replacement of other complexes. The models were manually rebuilt by COOT [19,20] and refined with PHENIX. Details on data collection and refinement statistics are shown in Table 1.
+
+## Enzyme-Dependent Synthesis Of Ψmp
+
+ΨMP was synthesized via a PUKI-dependent reaction, with pseudouridine as the substrate (Figure 1). PUKI from *E. coli* was prepared as described previously [9]. The ΨMP synthesis reaction was carried out at 25°C for 20 min in 50 mM Tris-HCl 
+(pH 7.5), 10 mM pseudouridine, and 1 µM PUKI, with 20 mM 
+MgCl2, 50 mM KCl, and 15 mM ATP. After 20 min, PUKI was filtered out using an Amicon centrifugal filter (EMD Millipore, Burlington, MA, USA) with a molecular weight cut-off of 30 kDa 
+(Merck). The formation of ΨMP was assessed by high-performance liquid chromatography (HPLC) as described below (Supplementary Fig. S2B). The ΨMP-containing reaction mixture was stored as aliquots at −80°C.
+
+The Thermo Scientific™ UltiMate™ 3000 HPLC system 
+(Thermo Fisher Scientific, Waltham, MA, USA) was used to characterize ΨMP from PUKI-dependent reaction mixtures. For HPLC, the enzymatic reaction was carried out for the PUKI-dependent reaction described above. After 20 min, instead of removing PUKI with a filter, we quenched ΨMP synthesis by adding an equal volume of methanol. After thorough mixing, the reactants and products were collected for HPLC. The HPLC analysis was performed as described previously [10]. Analytes were separated using a reverse-phase ion-pairing INNO C18 column (5 µm, 4.6 × 150 mm; YoungJin Biochrom, Gyeonggi-do, Korea), at an oven temperature of 30°C. 
+
+The flow rate was 1.5 mL min−1 and the injection volume was 2 μL. Isocratic elution for 10 min was performed with a mobile phase containing 50 mM phosphate buffer (pH 5.9), 40 mM tetrabutylammonium bromide and 12.5% acetonitrile. The analytes were detected using a diode array detector at 260 nm.
+
+## Enzyme-Coupled Assay For Atpumy
+
+R5P, a reaction product of AtPUMY, was continuously monitored using an enzyme-coupled assay [5]. In the assay, ribose-5'-phosphate isomerase (RpiA) and an NADPH-dependent ribulose 5'-phosphate reductase (Acs1) catalysed two consecutive reactions - R5P into ribulose 5'-phosphate, and further into ribitol 5'-phosphate - with concurrent changes of absorbance at 340 nm. The steady-state kinetic assay was carried out at 30°C using a UV–Vis spectrophotometer (Jasco, Tokyo, Japan).
+
+The pre-reaction mixture consisted of 50 mM HEPES 
+(pH 7.5), 2 mM MnCl2, and a given concentration of ΨMP, with coupling enzymes of 5 µM C-terminal Histagged RpiA and 4 µM N-terminal His-tagged Acs1, and 180 μM NADPH. For maximal Acs1 activity, 2 mM dithiothreitol and 50 μM cytidine triphosphate were added to the pre-reaction mixture. After incubation at 30°C for 120 s, an enzyme-dependent reaction was triggered by adding 80 nM AtPUMY and decreases in absorbance at 340 nm were recorded. We verified in preliminary experiments that the concentrations of the coupling enzymes RpiA and Acs1 and the divalent cation Mn2+ were saturating in the assay. The initial velocity was between 15 and 30 s and was converted to the level of R5P using a standard curve. SigmaPlot (Systat Software, San Jose, CA, USA) was used to calculate the values of Km and *Vmax*.
+
+## Acknowledgments
+
+We thank Minsoo Kim for her comments on a PUKI-dependent ΨMP synthesis and Dr. Ah-Reum Lee for providing us with the cDNA library of *A. thaliana*.
+
+## Disclosure Statement
+
+No potential conflict of interest was reported by the author(s).
+
+## Funding
+
+This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [2021R1A2C2092118 and RS-2023-00207820].
+
+## Author Contributions
+
+SR supervised the project; SHK performed crystallographic experiments of holoenzyme and the complex with citrate, and JL determined the crystal structure of the complex with ΨMP/R5P; JL carried out biochemical and kinetic assays; SR and JL analysed the data and wrote the manuscript with contributions from SHK.
+
+## Data Availability Statement
+
+The atomic coordinates and structural factors have been deposited in the Protein Data Bank (http://www.rcsb.org) under ID code 8K05, 8K06, and 8K07.
+
+## Orcid
+
+Sang-Hoon Kim http://orcid.org/0000-0002-9186-2276 Sangkee Rhee http://orcid.org/0000-0002-8779-9737
+
+## References
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medical/md/PMC10828703.md

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+Research article 
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+Tractor acquisition and agricultural performance among Nigerian farmers: Evidence from Co-integration Modeling technique E.A. Alhassan a,*, J.A. Asaleye b,e, J.K. Biniyat a, T.R. Alhassan c, J.O. Olaoye d a Department of Agricultural and Biosystems Engineering, Landmark University, Omu Aran, Kwara State, Nigeria b *Department of Economics, Bowen University, Iwo 232101, Nigeria & Faculty of Business Sciences, Walter Sisulu University, Eastern Cape, South* Africa c Department of Economics, Kwara State University, Molete, Kwara State, Nigeria d Department of Agricultural and Biosystems Engineering, University of Ilorin, Ilorin, Kwara State, Nigeria e *Faculty of Business Sciences, Walter Sisulu University, Easter Cape, South Africa* ARTICLE INFO 
+
+## Abstract
+
+| Farm machinery  Agricultural output  Employment  Short and long run impact   |
+|------------------------------------------------------------------------------|
+
+Given the envisioned significance of the agricultural sector in Nigeria, which scholars have argued has the potential to promote pro-poor growth and poverty alleviation. However, questions have been raised about using and accessing modern agricultural machinery for farm cultivation. 
+
+In this regard, this study uses a co-integration modelling technique to investigate the impact of tractor acquisition on agricultural performance. Two equations were established through the normalisation processes to proxy agricultural performance: the agricultural output and employment equations. Firstly, the analysis procedure requires a preliminary test to determine the statistical properties of the series used in this study; evidence from the outcome indicates that the series are not stationarity at level form and are integrated of order one. Secondly, based on the outcome of the preliminary result, the error correction model approach to co-integration was adopted, which helps to capture the short-term dynamics and long-term equilibrium of the effect of tractor acquisition on agricultural output and employment. The findings from the agricultural output equation show that credit to the agricultural sector, land cultivated for agricultural purposes, and tractor acquisition are statistically significant contributors to agricultural output. The agricultural employment equation shows that credit to agricultural purposes, land cultivated for agriculture and rainfall have a positive long-term relationship with employment. At the same time, tractor acquisition has a negative impact on agricultural employment in the long run. In the short run, exchange rate, rainfall, and tractor acquisition positively correlate with agricultural employment. In contrast, credit for agricultural purposes and land cultivated for agriculture negatively affects employment. Based on the findings, there is a need to strengthen policies that support access to credit, promote sustainable land use practices, and enhance water management. Also, policymakers should consider implementing measures that encourage the adoption of tractor technology while simultaneously addressing potential challenges related to job displacement. 
+
+https://doi.org/10.1016/j.heliyon.2024.e24485 Received 29 October 2022; Received in revised form 8 January 2024; Accepted 9 January 2024 Available online 11 January 2024 2405-8440/© 2024 The Authors. 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/).
+
+## 1. **Introduction**
+
+Agriculture is fundamental to the socio-cultural and economic well-being of Nigerians. A vehicle for reducing hunger, alleviating poverty, economic emancipation, and restoration of human dignity. The hub services agro-based industries are the second-largest source of foreign exchange revenue after the oil sector. However, scholars have stressed that for Nigerian farmers to reap these benefits, agriculture must be transformed from a subsistence venture to a profitable commercial enterprise; this is critical and will serve as a potent force that stimulates development and long-term economic growth [1–3]. Nigeria's diverse range of agroecological zones helps to produce different agricultural products. Despite this rich endowment of agro-based resources, the sector has witnessed stunted growth; this is evident as less than half of the cultivable or arable lands are engaged for agriculture, mostly at the subsistence level using primitive tools with attendant drudgery and low yield [4–7]. Likewise, the issue of food insecurity persists, compounded by the enduring challenge of low economic capacity among farmers; the contribution of agriculture to aggregate welfare has recorded little effect [4,8–12]. With the agricultural sector's substantial contributions to gross domestic product (GDP) and its potential for employment generation, it is imperative to emphasise that adequate investment can boost growth and development in the long run. 
+
+Infusing necessary financial resources into agriculture may also propel sectoral development and overall economic prosperity [2,13]. 
+
+The advent of farm tractors has revolutionised agricultural practices as their applications cut across all facets of farm production, such as land clearing and tillage operations, to the movement of finished products to the market. It has helped to minimise food wastage and farm production costs by supplying the cheap farm power required along production processes [10]; this has led to the emergence of allied machinery that has assisted in value chain addition to agricultural products [14]. Access to or ownership of this vital machinery is one of the indices defining an economic level of mechanisation. Agricultural production has many problems, such as increasing energy costs and shrinking access to capital for farm machinery acquisition [15]. In addition to the rising environmental concerns due to land degradation, climate change effects, and other socio-economic factors that affect economic performance, the stakeholders in agribusiness have been striving to subdue these challenges to spur agri-productivity. Agricultural machines as a dynamic industry will continue to evolve to meet the changing needs of production agriculture [9,16]; this is significant as a robust agro-based power source is germane to the sustainability of the modern-day farming ecosystem. 
+
+The Nigerian farming community is burdened with continuous toilage and indignity, characterised by drudgery, physical stress, and an ageing farming population [17]. This is solely due to the low level of mechanisation as farm power is mostly supplied by human and animal-using crude implements thereby limiting the cultivable arable land acreage and overall productivity of the farmers [11,18]. The results are not far-fetched as, despite more than 70 % of the Nigerian labour force being engaged in agriculture, output is not commensurate with invested efforts into production. The introduction of farm machinery has made farming more appealing and acceptable, providing prospects for using higher intelligence, skills, experience, and initiative that enlightened men often possess, thereby promoting the dignity of the farming community [19]. 
+
+Farm tractor availability and ease of access by the farmers are fundamental to achieving mechanisation benefits. Still, available information shows that Nigeria is far behind in attaining the needed satisfaction regarding the numbers of tractor power density and service life available to serve the vast farming community [20]. Due to some factors, there has been a decline in farmers' capacity to acquire tractors and other farm machinery. Key among them is the capital base of the farming community to acquire these machinery. 
+
+Identifying and isolating these influencing success factors and building indicators that reflect the performance of established indices and their interactions with the principal or response variable is necessary, as lucid mechanisation policies can be based on this empirical information. This is evident as the access to viable and useable data has always been a limiting factor to making an informed decision and farm machinery policy. 
+
+In crop production, for a booty harvest, the land must be tilled, crop sown and tendered to maturity, harvested, processed, stored, and transported. These required energy inputs in which the tractor is at the centre, and hence its accessibility to the farmers must be supported for a robust, lucrative, and sustained agripreneurship [21]. Access to mechanical farm power such as tractors has always been a challenge to Nigerian farmers, especially at peak periods, because of the limited numbers available to serve or service the vast community of farmers. There have been concerted efforts over the years by various stakeholders towards easing the farmers' burden with little success recorded as the current number available is far below the required that can propel the country towards achieving the sustainable development goals of zero hunger, no poverty, responsible consumption and sustainable forms of environmental management. The need to examine indicators that govern investment in tractor acquisitions, access to its usage among Nigerian farmers and productivity in agricultural performance or output is imperative because of the huge socio-economic impacts. Many studies have established a direct link between agricultural performance and agribusiness development, economic growth, and sustainability 
+[19–24]. Therefore, the main objective of this study is to investigate the impact of tractor acquisition on agri-performance in Nigeria. 
+
+The specific objectives are as follows. 
+
+i. To examine the short and long-run impact of tractor acquisition on agricultural output. 
+
+ii. To investigate the short and long-run impact of tractor acquisition on agricultural employment. 
+
+The study uses the co-integration approach outlined by Engle and Granger to achieve these objectives. Use of this approach allows the study to examine the dynamic relationship between tractor acquisition and agricultural productivity in Nigeria. 
+
+## 2. **Research Methodology**
+
+This study investigates the impact of tractor acquisition on agricultural performance in Nigeria. For this study to achieve its objective, two equations are established through the normalisation processes, which will be referred to as the agricultural output and employment equations. Firstly, the analysis procedure requires a preliminary test to determine the statistical properties of the series used in this study (the correlation test, descriptive statistics, Johansen co-integration and unit root test). Secondly, using the error correction model approach to co-integration to investigate the short-term dynamics and long-term equilibrium of the effect of tractor acquisition on agricultural output and employment. To better understand the concept, model specification, estimation techniques, data sources and measurements were elucidated sequentially in this section. 
+
+## 2.1. Model Specification
+
+The study uses the output theory as the theoretical framework. The strength of this framework lies in the capability to show the relationship between inputs used in producing output. Also, it reveals how changes in input variables impact overall output levels; this has been the standard approach used in output and productivity studies [4,23,24]. 
+
+This theory assumes that inputs are needed to produce a given output (Eqn. (1)). 
+
+Y = f(x) (1) 
+In equation (1), Y is the output, and x is the input. The explicit function of equation (1) can be written as: 
+
+## Y = Α0 + Α1X (2)
+
+In equation (2), α0 is the constant term or the intercept while α1 shows the slope or gradient of the equation. To achieve the specific objectives of this study, which are to examine the short-run and long-run impacts of tractor acquisition on agricultural performance in Nigeria. The agricultural performance is proxy by aggregate employment in the agricultural sector or farming population and total output produced by the agricultural sector. The output model stated that capital and employment are essential to production in agriculture. So, equation (2) was modified as follows: 
+Yt = α0 + α1x1 + α2x2 (3) 
+From Equation (3), x1 is the capital input while x2 is the labour or employment inputs. Y in equation (3), according to the growth model, shows the aggregate output in a given country. This study focuses on the agricultural sector, so the study re-defines the variables as follows: 
+
+## Aot = Α0 + Α1Ca + Α2La (4)
+
+In equation (4), AO, CA and LA represent agricultural output, capital in the agricultural sector and labour in the agricultural sector, respectively. Also, factors such as land and rainfall are important variables to be considered in producing agricultural products. Likewise, tractor acquisition is important in large-scale or mechanised production. Therefore, with the inclusion of other inputs, equation (4) becomes: 
+AOt = α0 + α1CA + α2LA + α3RF + α4LD + *α5TQ* (5) 
+In equation (5), RF is the total amount of rainfall, LD is the land used for agricultural production, and TQ is tractor acquisition. Due to the effect of external factors from international trade, since most agricultural machinery is imported into the country, the exchange rate is also added to the variables, then equation (5) is modified and presented as equation (6): 
+AOt = α0 + α1CA + α2LA + α3RF + α4LD + *α5TQ* + α6EX (6) 
+Where EX is the exchange rate and other variables as earlier defined, to estimate the above equation, the error term is included to capture the effects of other factors that cannot be generalised due to minimised impacts on the model. In addition, minimising the error term will help to estimate the values for the parameters of the respective variables. 
+
+AOt = α0 + α1CA + α2LA + α3RF + α4LD + α5TQ + *α6EX* + μt (7) 
+The error term μt is included in equation (7), which makes it an econometric model. The study carried out preliminary analyses such as the unit root and co-integration. Further explanation of these was given in the following subsections. The outcome of the cointegration result presented in the results and discussion section shows that the series has two co-integrating vectors. Because of this, two equations were generated to represent the output and employment in the agricultural section, while the tractor acquisition effect is captured as an independent or response variable in both equations. 
+
+This study uses the error correction model approach to co-integration; this estimation technique benefits the study by allowing it to capture the short-term dynamics of the effect of tractor acquisition on agricultural productivity in Nigeria. Also, it shows the long-term equilibrium between the dependent and independent variables. The ECM gives the error correction term, which measures the speed of adjustment from the short-run deviation to the long-run equilibrium. This is particularly useful in understanding the interplay between tractor acquisition and agricultural performance, and the procedure outlined by ECM gives it an advantage over other co-integration approaches by its ability to allow this study assess how farmers' actions in response to deviations influence the long-term equilibrium. 
+
+Finally, employing ECM in this study, due to its flexibility, gives it a more nuanced perspective on the causal relationships between the dependent variable, which is agricultural output and other independent variables, which include tractor acquisition, and this enhances 3
+
+$$({\mathfrak{s}})$$
+$$(11)$$
+
+the accuracy of the analysis and gives valuable insights which make by crucial for policymakers and stakeholders in Nigerian agriculture. 2.2. *Techniques of estimation* Naturally, unit root is a common problem in time series analysis. Using series that are not stationary will give spurious results, which can typify a wrong signal. Therefore, carrying out the unit root is imperative before estimating time series data. More so, it helps to determine the appropriate approach to use in estimation. There are several ways to test for the stationarity property of the time series. However, this study uses the Augmented Dickey-Fuller approach (ADF) that helped to establish a technique for determining the order of integration of the series [22]. The ADF equation is given in Eqn. (8) as: 
+
+$\Delta M_{t}=\beta_{0}+\beta_{1}M_{t-1}+\varphi+\sum_{i=1}^{n}K_{1}\Delta M_{t-k}+\varepsilon_{t}$
+K1ΔMt− k + εt (8) 
+$\eqref{eq:walpha}$ . 
+4
+In Equation (8), Mt represents the respective time series used in this study, φ is the linear trend while β0 is the intercept and εt is the error term. Δ shows the differenced form. The null hypothesis is that the series is not stationary, while the alternative hypothesis is that the series is stationary using a 5 % significance level. If the null hypothesis is not rejected, the difference form will be considered, which is integrated of order one (1). If all the series are integrated of the same order, let's assume integrated of order one; then there is a tendency for the series to be co-integrated. The Johansen approach of co-integration was used in this study; this is used to test cointegrating relationships between several non-stationary time series data, allowing for more than one co-integrating relationship. 
+
+The Johansen approach is only used to determine the number(s) of co-integrating vectors; two statistics were presented: the trace and the maximum Eigenvalue statistics. The Error Correction Model (ECM), also known as the Engel Granger approach to co-integration, was used to estimate tractor acquisition's short- and long-term effects on agricultural performance in Nigeria. The models for the shortrun effects are given in Equations (9) and (10): 
+
+$\Delta AO_{t}=\delta_{0}+\delta_{1}\Delta CA_{t}+\delta_{2}\Delta RF_{t}+\delta_{3}\Delta LD_{t}+\delta_{4}\Delta TQ_{t}+\delta_{5}\Delta EX_{t}+ECT_{1}+v_{1t}$
+ΔAOt = δ0 + δ1ΔCAt + δ2ΔRFt + δ3ΔLDt + δ4ΔTQt + δ5ΔEXt + ECT1 + v1t (9) 
+$\Delta LA_{t}=\gamma_{0}+\gamma_{1}\Delta CA_{t}+\gamma_{2}\Delta RF_{t}+\gamma_{3}\Delta LD_{t}+\gamma_{4}\Delta TQ_{t}+\gamma_{5}\Delta EX_{t}+ECT_{2}+v_{2t}$
+ΔLAt = γ0 + γ1ΔCAt + γ2ΔRFt + γ3ΔLDt + γ4ΔTQt + γ5ΔEXt + ECT2 + v2t (10) 
+These equations show the short-run behaviour. ECT is the error correction term, which must be significant, and the coefficient must be less than one and negative to show the speed of adjustment to initial equilibrium. All the variables are in 'differenced form', 
+indicated by Δ. 
+
+The long-run equations for the two dependent variables are given as (Equations (11) and (12)): 
+AOt = ς0 + ς1CAt + ς2RFt + ς3LDt + ς4TQt + ς5EXt + ω1t (11) 
+
+$_i T Q_t+\xi_5E X_t+\omega_{1t}$. 
+$\rho_t-\phi_0+\phi_1\mathbb{C}\mathbb{A}_t$
+$\square$
+$\frac{1}{2}$ . 
+$LA_{t}=\kappa_{0}+\kappa_{1}CA_{t}+\kappa_{2}RF_{t}+\kappa_{3}LD_{t}+\kappa_{4}TQ_{t}+\kappa_{5}EX_{t}+\omega_{2t}$.  
+LAt = κ0 + κ1CAt + κ2RFt + κ3LDt + κ4TQt + κ5EXt + ω2t (12) 
+The ECM involves three stages. The first stage is to estimate the long-run relationship using the least square; this was followed by generating the residual from the long-run relationship and testing for stationarity. If the residual is stationary at 5 %, this validates the long-run relationship among the series. Based on the outcome, the third stage estimates the variable's first difference form with the residual lag as the error correction term. Afterwards, the models in this research are subject to diagnostic checks to determine if they are correctly specified. 
+
+Given the outcome of the time series properties, since all the series are integrated of order one, the error correction is the most appropriate and suitable for the study. Therefore, it was preferred over the autoregressive distribution (which is more suitable for mixed order of co-integration, that is integrated of order zero and one) and more than the Johansen co-integration equation because the ECM gives a unique equation [24,25]. In contrast, the former gives multiple equations and sometimes makes interpretations not straightforward. In addition, other rationales for this choice of estimation used in this study are as follows: Firstly, it offers a robust analytical approach, particularly in this study, which needs to capture both short-term fluctuations and long-term equilibrium relationships in a system. For example, in Nigeria, there is prolonged high unemployment and poverty rate, and the effect of time perspective implications are important in the agricultural sector. Using ECM enables the study to assess if tractor acquisition can have immediate effects and, likewise, if it can contribute to sustainable changes over time. Secondly, the ECM accounts for dynamic adjustments and measures the speed at which the system returns to equilibrium after the shocks; this is important for policy implications. 
+
+One limitation of the ECM is the assumption of linear relationships and stationary data, which might not hold in complex real-world systems. Additionally, the ECM assumes that the variables in the model are co-integrated, which might not always be the case, leading to unreliable results. However, this current study overcame the limitations by conducting a preliminary analysis, in which it was observed that the series used in this study are not stationary and are integrated of order one (as depicted in Table 1). Also, the cointegration test was carried out using the residual lag; evidence from the result shows the presence of co-integration (as depicted in Table 5). 
+
+## 2.3. Data Source And Measurement
+
+Time series data used for this investigation were from the secondary source obtained from the Central Bank of Nigeria (CBN) (www. 
+
+cbn.gov.ng), Nigeria Meteorological Agency (NMA) (www.nimet.gov.ng), National Bureau of Statistics, Nigeria (NBS-NG) (www. 
+
+nigerianstat.gov.ng) and government agricultural agencies (www.fmard.gov.ng) [26–29]. The time series data spanned from 1993 to 2018 for tractor price, annual rainfall, land area cultivated, farmers' population, exchange rate, agricultural output, and credit given to farmers. Data analyses were done using EViews version software to affirm the relationship between the response variable and the predictors. 
+
+## 3. **Results And Discussion** 3.1. Presentation Of Results Output From Analysed Data
+
+This section presents the unit root test, Johansen co-integration test, agricultural output equation concerning tractor acquisition 
+(Model 1), employment equation concerning tractor acquisition (Model 2), residual unit test, and implications of the findings and evaluation of hypotheses. 
+
+## 3.2. Presentation Of Unit Root Test
+
+Table 1 shows the outcome of the stationary test using the Augmented Dickey-Fuller (ADF) approach. The null hypothesis is that the series has a unit root, while the alternative hypothesis is that no unit root exists. The null hypothesis is rejected if the Augmented Dickey-Fuller (ADF) test statistic (absolute values) exceeds the critical values. Otherwise, probability can also be used; significance at any level shows the rejection of the null hypothesis. Using the absolute values, evidence from the result shows that AO, CA, EX, LA, LD, 
+RF and TQ with ADF test statistic values of − 0.476108, − 1.774045, − 1.371228, − 0.732250, − 2.195118, − 2.756186 and 1.087935 respectively is less than the critical value of − 2.892536, using 5 % significance level indicates that the series are not stationary. 
+
+However, considering only the absolute values, RF is stationary at the level of 10 % significance since the absolute value of the ADF test statistics − 2.756186 is greater than the critical value of − 2.583371. 
+
+All the series are integrated of order one I(1) since the series are stationary at the first differencing form. The ADF test statistic values of AO, CA, EX, LA, LD, RF and TQ with − 2.948583, − 2.940844, − 2.948416, − 3.045899, − 3.483188, − 4.919975 and − 2.943080 respectively are greater in absolute term than the critical values of − 2.892536 at 5 %. Only RF with the value of − 4.919975 is significant at 1 %. The study uses a 5 % significance level. 
+
+## 3.3. Presentation Of Johansen Co-Integration Test
+
+The Johansen co-integration test result is presented in Table 2. The test has two statistics: the Unrestricted co-integration rank test 
+(Trace) and the Unrestricted co-integration rank test (Maximum Eigenvalue). The two statistics accept the hypothesis of the presence of co-integrating vectors, showing the presence of two co-integrating vectors. Given the outcome of the result, the short and long-run implications of tractor acquisition on the agricultural sector were analysed by generating two equations to represent the agricultural output and employment, which were used to proxy the performance in the agricultural sector. 
+
+## 3.4. Presentation Of Output Equations Concerning Tractor Acquisition (Model 1)
+
+The agricultural output equation in relation to tractor acquisition is referred to as model 1, while the employment equation in relation to tractor acquisition is referred to as model 2. Table 3 presents the short and long-run impact of tractor acquisition using output in the agricultural sector as the dependent variable. In the long-run equation result, capital proxy by credit to the agricultural sector (CA), exchange rate (EX), land cultivated for agricultural purposes (LD) and tractor acquisition (TQ) are statistically significant since the probability values are less than 0.05. At the same time, rainfall (RF) is not statistically significant. CA, LD and TQ have a positive relationship with output in the long run; this implies holding all the variables constant; a percentage increase in CA, LD and TQ 
+
+| Table 1  Result of Stationary Test using Augmented Dickey-Fuller (ADF) Approach.  Series @ Level form @ First Difference Form   | Order of Integration   |               |       |
+|---------------------------------------------------------------------------------------------------------------------------------|------------------------|---------------|-------|
+| AO                                                                                                                              | − 0.476108             | − 2.948583**  | I (1) |
+| CA                                                                                                                              | − 1.774045             | − 2.940844**  | I (1) |
+| EX                                                                                                                              | − 1.371228             | − 2.948416**  | I (1) |
+| LA                                                                                                                              | − 0.732250             | − 3.045899**  | I (1) |
+| LD                                                                                                                              | − 2.195118             | − 3.483188**  | I (1) |
+| RF                                                                                                                              | − 2.756186*            | − 4.919975*** | I (1) |
+| TQ                                                                                                                              | 1.087935               | − 2.943080**  | I (1) |
+| Critical Values  1 %                                                                                                            | − 3.501445             | − 3.501445    |       |
+| 5 %                                                                                                                             | − 2.892536             | − 2.892536    |       |
+| 10 %                                                                                                                            | − 2.583371             | − 2.583371    |       |
+| ***, ** and * show significance at the level of 1 %, 5 %, and 10 % respectively                                                 |                        |               |       |
+
+5
+
+| Johansen Co-integration test results.  Series: AO CA EX LD RF LA TQ  Unrestricted Co-integration Rank Test (Trace)  Hypothesised no. of CE(s) Eigen Value   | Trace Statistic     | 0.05 Critical Value   | Prob **   |        |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|-----------------------|-----------|--------|
+| None*                                                                                                                                                       | 0.788598            | 276.6346              | 117.7082  | 0.0000 |
+| At most 1*                                                                                                                                                  | 0.525874            | 122.7892              | 88.80380  | 0.0000 |
+| At most 2                                                                                                                                                   | 0.220894            | 48.90739              | 63.87610  | 0.4633 |
+| At most 3                                                                                                                                                   | 0.107769            | 24.19621              | 42.91525  | 0.8281 |
+| At most 4                                                                                                                                                   | 0.086164            | 12.90720              | 25.87211  | 0.7454 |
+| At most 5                                                                                                                                                   | 0.039472            | 3.986896              | 12.51798  | 0.7440 |
+| Unrestricted Co-integration Rank Test (Maximum Eigenvalue)  Hypothesised No. of CE(s) Eigenvalue                                                            | Max-Eigen Statistic | 0.05 Critical value   | Prob. **  |        |
+| None *                                                                                                                                                      | 0.788598            | 153.8454              | 44.49720  | 0.0000 |
+| At most 1*                                                                                                                                                  | 0.525874            | 73.88184              | 38.33101  | 0.0000 |
+| At most 2                                                                                                                                                   | 0.220894            | 24.71118              | 32.11832  | 0.3035 |
+| At most 3                                                                                                                                                   | 0.107769            | 11.28901              | 25.82321  | 0.9126 |
+| At most 4                                                                                                                                                   | 0.086164            | 8.920301              | 19.38704  | 0.7326 |
+| At most 5                                                                                                                                                   | 0.039472            | 3.986896              | 12.51798  | 0.7440 |
+| Trace test indicates 2 co-integrating Eqn.(s) at the 0.05 level. * denotes rejection of the hypothesis at the 0.05 level. ** MacKinnon-Haug-Michelis        |                     |                       |           |        |
+
+| Long-run Result  Variable   | Coefficient   | Std. Error       | t-Statistic       | Prob.   |
+|-----------------------------|---------------|------------------|-------------------|---------|
+| CA                          | 0.141851***   | 0.014632         | 9.694668          | 0.0000  |
+| EX                          | − 0.140647*** | 0.024594         | − 5.718665        | 0.0000  |
+| LD                          | 1.282670***   | 0.315627         | − .063881         | 0.0001  |
+| RF                          | 0.038602      | 0.027532         | 1.402059          | 0.1641  |
+| TQ                          | 1.468651***   | 0.056576         | 25.95887          | 0.0000  |
+| C                           | − 1.317988**  | 0.677223         | − 1.946165        | 0.0545  |
+| Short-run Result  Variable  | Coefficient   | Std. Error       | t-Statistic       | Prob.   |
+| D(CA)                       | 0.110299***   | 0.013597         | 8.112010          | 0.0000  |
+| D(EX)                       | − 0.012513    | 0.018047         | − 0.693325        | 0.4898  |
+| D(LD)                       | 0.461242      | 0.309395         | 1.490789          | 0.1393  |
+| D(RF)                       | − 0.014054    | 0.017428         | − 0.806412        | 0.4220  |
+| D(TQ)                       | 0.734551***   | 0.182095         | 4.033900          | 0.0001  |
+| U (− 1)                     | − 0.067288**  | 0.027865         | − 2.414761        | 0.0176  |
+| C                           | 0.000711      | 0.001228         | 0.579218          | 0.5638  |
+| R-squared                   | 0.574025      | F-stat.; Prob.   | 21.56093; 0.00000 |         |
+| Adj. R-squared              | 0.547402      | Durbin-Wat. stat | 1.896455          |         |
+
+Short-run Result 
+
+Variable Coefficient Std. Error t-Statistic **Prob.** D(CA) 0.110299*** 0.013597 8.112010 0.0000 
+
+D(EX) − 0.012513 0.018047 − 0.693325 0.4898 
+
+D(LD) 0.461242 0.309395 1.490789 0.1393 
+
+D(RF) − 0.014054 0.017428 − 0.806412 0.4220 
+
+D(TQ) 0.734551*** 0.182095 4.033900 0.0001 
+
+U (− 1) − 0.067288** 0.027865 − 2.414761 0.0176 
+
+C 0.000711 0.001228 0.579218 0.5638 
+
+R-squared 0.574025 F-stat.; Prob. 21.56093; 0.00000 Adj. R-squared 0.547402 Durbin-Wat. stat 1.896455 
+
+***, ** and * show significance at the level of 1 %, 5 % and 10 % respectively. 
+
+The Durbin-Watson statistics value of 1.896455 is closer to 2, signifying the absence of autocorrelation in the model. The significance of the Fstatistics shows how the jointly independent variables explained the dependent variable. The value of the F-statistics is 21.56093, with a probability 
+
+value of less than 5 %; this indicates that the independent variables jointly explain the dependent variable. R-squared and adjusted R-squared are the 
+
+coefficient of determination, which measures the goodness of fit. The R-squared and adjusted R-squared values in Table 3 are 0.574025 and 0.547402, 
+
+respectively, since the values are more than 0.50. It indicates that more than 50 % variation of the independent variables can be explained in the 
+
+dependent variable. The U (− 1) is the error correction term (ECT). Theoretically, the value must be less than one, negative and statistically significant. 
+
+The value of the ECT is − 0.067288 with a probability value of 0.0176; this shows that the model will adjust about 6 % to the initial equilibrium. 
+
+will result to about 0.141851 %, 1.282670 % and 1.468651 % increase, respectively, in output. In a similar study by Zhang et al., 2021 [30] on market-oriented agriculture and farm performance: Evidence from rural China, it was affirmed that land endowment, fixed agricultural assets, access to formal credit, and land certification are part of the critical factors affecting farmers' decisions as it relates to agricultural performance. 
+
+Arisukwu et al., 2019 [31] stressed the need to increase agricultural production in Nigeria. Takeshima, 2016 [10] emphasised a significant direct correlation between tractor adoption and land development with 0.2 % points in the probit models. Similarly, Asaleye et al., 2020 [23] established that long-term benefits could be maximised by appropriate cotton and groundnut production funding to promote sustainable growth. The study affirmed the importance of finance as a key factor for employment and agricultural output. The exchange rate (EX) is negatively related to agricultural output; an increase in the exchange rate will affect farmers' capacity to acquire tractors and, hence, reduce agricultural output. The result shows that holding other variables constant, a 1 % change in the exchange rate will lead to about 0.140647 % reduction in agricultural output in the long run. The short-run behaviour shows that CA and TQ are statistically significant since the probability values are less than 5 %, while EX, LD and RF were not statistically significant. Likewise, CA and TQ positively impact agricultural output in the short run. A 1 % increase in CA and TQ will promote agricultural output by 0.11 % and 0.73 % respectively. The outcome reveals that access to capital and tractor usage is an impetus for increased agricultural output. 
+
+## 3.5. Presentation Of Employment Equation In Relation To Tractor Acquisition (Model 2)
+
+Table 4 presents tractor acquisition's short and long-run impact on agricultural employment. Agricultural employment is used as the dependent variable. In the long-run behaviour, capital proxy by credit to the agricultural sector (CA), exchange rate (EX), land cultivated for agricultural purpose (LD), rainfall (RF) and tractor acquisition (TQ) are statistically significant since the probability values are less than 0.05. CA, LD, and RF have a positive long-term relationship with employment; this implies holding all the variables constant; a 1 % increase in CA, LD and RF will result in about 0.034056 %, 0.443985 %, and 0.046359 % increase in employment, respectively. 
+
+The short-run behaviour shows that CA, EX, LD, RF and TQ are statistically significant since the probability values are less than 5 %. 
+
+EX, RF and TQ have a positive relationship with agricultural employment; this shows that holding all other variables constant, a percentage change in EX, RF and TQ will lead to about 0.093964 %, 0.025056 % and 0.612343 % increment in employment in the short-run. CA and LA have a negative relationship with employment; holding all variables constant, a 1 % change in CA and LA will result in about 0.225099 and 0.769325 reductions in employment in the short run. 
+
+The Durbin-Watson statistics value is 2.006025, which is close to 2, affirming no autocorrelation in the model. The F-statistics measures the joint significance of independent variables in explaining the dependent variable. The value of the F-statistics is 106.8320, with a probability value of less than 5 %; this indicates that the independent variables jointly explain the dependent variable. Rsquared and adjusted R-squared are the coefficient of determination, with values of 0.872686 and 0.863581, respectively, which measures the goodness of fit since the values are more than 0.50. It indicates that more than 50 % variation of the predictors can be explained in the dependent variable. 
+
+The U (− 1) is the error correction term (ECT). Theoretically, the value must be less than one, negative and statistically significant. 
+
+The value of the ECT is − 0.056612 with a probability value less than 0.05, which shows that the model will adjust about 5 % to the initial equilibrium. 
+
+## 3.6. Residual Unit Root Test
+
+Table 5 shows the stationary result for the two models examined in this investigation. Since the null hypothesis of the unit is rejected at the level of 5 % for both models, this validates the presence of co-integration in the study. Likewise, the models were subjected to various diagnostic checks; the outcome shows that the models are specified correctly. 
+
+| Short and long-run impacts of tractor acquisition on agricultural employment.  Short and long-run Impact of Tractor Acquisition on Employment  Long-run Result  Variable Coefficient Std. Error   | t-Statistic   | Prob.            |                   |        |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|------------------|-------------------|--------|
+| CA                                                                                                                                                                                                | 0.034056***   | 0.007852         | 4.337163          | 0.0000 |
+| EX                                                                                                                                                                                                | − 0.039084*** | 0.013198         | − 2.961272        | 0.0038 |
+| LD                                                                                                                                                                                                | 0.443985***   | 0.169380         | 2.621238          | 0.0102 |
+| RF                                                                                                                                                                                                | 0.046359***   | 0.014775         | 3.137665          | 0.0022 |
+| TQ                                                                                                                                                                                                | − 0.696756*** | 0.030361         | − 22.94883        | 0.0000 |
+| C                                                                                                                                                                                                 | 3.155791***   | 0.363429         | 8.683382          | 0.0000 |
+| Short-run Result  Variable                                                                                                                                                                        | Coefficient   | Std. Error       | t-Statistic       | Prob.  |
+| D(CA)                                                                                                                                                                                             | − 0.225099*** | 0.011864         | − 18.97364        | 0.0000 |
+| D(EX)                                                                                                                                                                                             | 0.093964***   | 0.011359         | 8.271866          | 0.0000 |
+| D(LD)                                                                                                                                                                                             | − 0.769325*** | 0.144312         | − 5.331002        | 0.0000 |
+| D(RF)                                                                                                                                                                                             | 0.025056***   | 0.006628         | 3.780056          | 0.0014 |
+| D(TQ)                                                                                                                                                                                             | 0.612343***   | 0.067988         | 9.006604          | 0.0000 |
+| U (− 1)                                                                                                                                                                                           | − 0.056612*** | 0.005084         | − 11.13533        | 0.0000 |
+| C                                                                                                                                                                                                 | 0.003086***   | 0.000247         | 12.49760          | 0.0000 |
+| R-squared                                                                                                                                                                                         | 0.872686      | F-stati.; Prob.  | 106.8320; 0.00000 |        |
+| Adj. R-squared                                                                                                                                                                                    | 0.863581      | Durbin-Wat. stat | 2.006025          |        |
+| ***, ** and * show significance at the level of 1 %, 5 %, and 10 % respectively.                                                                                                                  |               |                  |                   |        |
+
+| Table 5  Residual stationary test results.  Residual Stationary Test for Model 1   | t-Statistic   | Prob.*     |
+|------------------------------------------------------------------------------------|---------------|------------|
+| Augmented Dickey-Fuller test statistic                                             | − 2.656339    | 0.0083     |
+| Test critical values:                                                              | 1 % level     | − 2.589795 |
+| 5 % level                                                                          | − 1.944286    |            |
+| 10 % level                                                                         | − 1.614487    |            |
+| Residual Stationary Test for Model 2                                               | t-Statistic   | Prob.*     |
+| Augmented Dickey-Fuller test statistic                                             | 5.480648      | 0.0000     |
+| Test critical values:                                                              | 1 % level     | − 2.589795 |
+| 5 % level                                                                          | − 1.944286    |            |
+| 10 % level                                                                         | − 1.614487    |            |
+
+3.7. *Implications of findings and evaluation of hypotheses* The study investigates the effect of tractor acquisition on agricultural performance in terms of output and employment in the short and long run. The hypotheses are stated in null form as follows. 
+
+i. There is no significant short or long-run impact of tractor acquisition on agricultural output. 
+
+ii. There is no significant short or long-run impact of tractor acquisition on agricultural employment. 
+
+The two hypotheses were rejected at the significance level of 5 %; it was depicted from the result in Table 3 that tractor acquisition has a positive effect on agricultural output in the short and long run. This result implies that in the short run, investment in tractor acquisition can increase agricultural output, improving food security and reducing the dependency on imports, moving the economy towards self-sufficiency. Also, this can contribute to lower food prices and enhanced economic stability. In the long run, the positive relationship between tractor acquisition and agricultural output may help sustain agricultural productivity growth. And go beyond promoting aggregate welfare and overall economic development, generating employment opportunities, stimulating rural economies, and encouraging diversification of Nigeria's revenue base. 
+
+Likewise, Table 4 shows that tractor acquisition positively affects employment in the short run but negatively in the long run. The implication of tractor acquisition having a positive impact on employment in the short run may be related to the technological advancements and labour dynamics in the Nigerian economy [32,33]. For example, introducing tractors may generate employment opportunities through increased demand for skilled operators and maintenance personnel. However, the negative long-run effect suggests a potential displacement of labour as mechanisation increases; this makes the need to carefully manage the transition to modern agricultural practices, perhaps through targeted policies that promote skill development and diversification of employment opportunities to ensure sustainable and inclusive economic development in Nigeria. 
+
+Tractor acquisition and adoption for agriculture are based on economic and environmental factors. The study shows that despite the positive effect of tractor acquisition on agricultural output, the coefficient is still less than 1 %. In this regard, much still needs to be done for the agricultural sector to promote economic transformation, which can increase pro-poor growth. From the investigation, it can be inferred that investment in tractor acquisition and availability to farmers will enhance productivity as a positive relationship was established between agricultural output and employment. Affirmatively, there should be policy interventions towards a clear understanding of the issues connecting to advancing increased engagement of tractors in Nigeria's farming setting. These issues include determining the most appropriate mechanisms to improve farmers' access to tractors, defining the best geographical areas for the government to improve farmers' access to tractor services, and assessing soil characteristics to predict tractor demand [10]. 
+
+## 4. **Conclusion And Policy Recommendations**
+
+Access to mechanical farm power such as tractors has always been a challenge to Nigerian farmers, especially at peak periods, because of the limited numbers available to serve or service the vast community of farmers. On the other hand, the increased unemployment rate remained unresolved in Nigeria despite various policies and programmes the Nigerian government has introduced to improve the situation. However, arguments support the agriculture sector's development in the general economic transformation to promote aggregate welfare and development. Given this, the study aimed to examine the short and long-run impacts of tractor acquisition on agricultural output and employment, considering other controlling variables based on their relevance from empirical studies, such as credit to the agricultural sector, exchange rates, land cultivation, and rainfall. The findings from the agricultural output equation show the significance of tractor acquisition alongside other key variables. Credit to the agricultural sector, land cultivated for agricultural purposes, and tractor acquisition are statistically significant contributors to agricultural output. The positive effects of credit and land cultivation persist in the short and long run, indicating their sustained influence on enhancing productivity. The positive relationship observed in the short and long term implies that the advantage from the process can be used to boost aggregate output, Turning attention to the agricultural employment equation, credit to the agricultural sector, exchange rate, land cultivated for agricultural purposes, rainfall and tractor acquisition are statistically significant. Credit to agricultural purposes, land cultivated for agriculture and rainfall have a positive long-term relationship with employment. In contrast, tractor acquisition has a negative impact on agricultural employment in the long run. In the short run, exchange rate, rainfall, and tractor acquisition positively correlate with agricultural employment. While credit for agricultural purposes and land cultivated for agriculture have a negative relationship with employment. Emphasising the role of these factors in sustaining and fostering employment opportunities. However, the impact of tractor acquisition on employment takes an unexpected turn. While the short-term analysis shows a positive relationship, the longterm perspective reveals a negative impact; this suggests that while tractor acquisition may initially create jobs, concurrent strategies might be needed to address potential job displacement in the long run due to increased mechanisation. 
+
+Based on the findings, given the positive influence of credit, land cultivation, and rainfall on both output and employment, this shows that strengthening policies that support access to credit, promote sustainable land use practices, and enhance water management is important. Moreover, the impact of tractor acquisition calls for a balanced approach. Policymakers should consider implementing measures that encourage the adoption of tractor technology while simultaneously addressing potential challenges related to job displacement. Also, the study affirmed the importance of finance as a key factor for employment and agricultural output. The outcome reveals that access to capital and tractor usage is an impetus for increased agricultural output. The exchange rate is negatively related to agricultural output; an increase in the exchange rate will affect farmers' capacity to acquire tractors and, hence, reduce agricultural output. 
+
+This study examines the macro impact of tractor acquisition on Nigerian agriculture, emphasising the short and long-term effects. 
+
+While a macro study is important to understand the broader effect, future research may focus on the micro-level to understand individual farm dynamics; this will help by informing targeted interventions for inclusive and sustainable agricultural development. 
+
+Likewise, bridging macro and micro analyses is crucial for a comprehensive understanding of the growth process and effective policy formulation in Nigeria's evolving agricultural development. 
+
+## Availability Of Data And Materials
+
+The information and source of data used in this study are detailed in Section 2.3 under the heading of 'Data Source and Measurement'; the links are also provided. In case of further request, the corresponding author can be contacted through the e-mail address: alhassan.elijah@lmu.edu.ng. 
+
+## Credit Authorship Contribution Statement
+
+E.A. Alhassan: Writing - review & editing, Writing - original draft, Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Conceptualization. **J.A. Asaleye:** Writing - review & editing, Writing - original draft, Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **J.K. Biniyat:** Resources, Project administration, Investigation, Conceptualization. **T.R. Alhassan:** Writing - original draft, Resources, Formal analysis. **J.O. Olaoye:** 
+Writing - review & editing, Writing - original draft, Validation, Resources, Methodology, Investigation, Conceptualization. 
+
+## 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. 
+
+We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. 
+
+## References
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+
+[12] N.C. Eli-Chukwu, Applications of artificial intelligence in agriculture: a review, Eng. Technol. Appl. Sci. Res. 9 (4) (2019). 
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+[13] E.A. Alhassan, A.D. Adewumi, B. Okpodjah, Development of a self-propelled multi-crop two rows precision planter: A new design concept for the metering mechanism, International Journal of Mechanical Engineering and Technology 9 (10) (2018) 349–358. 
+
+[14] A.M. Olaniyan, B.K. Abdulkareem, M.M. Odewole, E.O. Ariyo, A.I. Oyebanji, E.A. Alhassan, Development of a Fermentation Vat for value chain addition in Locust bean processing, International Conference on Science, Engineering and Business for Sustainable Development Goals (SEB-SDG) 1 (2023) 1–6, 2023, April) IEEE. 
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+[15] C.E. Okonkwo, O. I Moses, E.A. Alhassan, M. Ogbevire, S.A. Alake, F.O. Ajao, A.T. Olayanju, Design and fabrication of a fish feed mixing cum pelleting machine for small-medium scale aquaculture industry." Open Agriculture 8 (1) (2023) 20220124. 
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+(2010) 83–89. 
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+489–500. 
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medical/md/PMC10901679.md

@@ -0,0 +1,242 @@
+Research Article Periodontal Science Bacterial cellulose matrix and acellular 
+
+![0_image_0.png](0_image_0.png)
+
+dermal matrix seeded with fibroblasts grown in platelet-rich plasma supplemented medium, compared to free gingival grafts: a randomized animal study Abraão Moratelli Prado 1, Cimara Fortes Ferreira 2,*, Luismar Marques Porto 3, Elena Riet Correa Rivero 4, Ricardo de Souza Magini 5, Cesar Augusto Magalhães Benfatti 5**, Jair Rodriguez-Ivich 6**
+1Implant Dentistry, Private Practice, Florianópolis, SC, Brazil 2Department of Periodontics, University of Tennessee Health Science Center (UTHSC) College of Dentistry, Memphis, TN, USA
+3Chemical and Food Engineering Department, Federal University of Santa Catarina, Florianópolis, SC, Brazil 4Pathology Department, Federal University of Santa Catarina, Florianópolis, SC, Brazil 5Dentistry Department, Federal University of Santa Catarina, Florianópolis, SC, Brazil 6Implant Dentistry, Private Practice, Guadalajara, Mexico
+
+## Abstract
+
+Purpose: Mucogingival defects (MGDs), such as dental root recessions, decreased vestibular depth, and absence of keratinized tissues, are commonly seen in dental clinics. MGDs may result in functional, aesthetic, and hygienic concerns. In these situations, autogenous soft tissue grafts are considered the gold-standard treatment. This study compares the healing process of free gingival grafts (FGGs) to bacterial cellulose matrix (BCM) and human acellular dermal matrix (ADM) seeded with fibroblasts from culture supplemented with platelet-rich plasma in a rat model.
+
+Methods: Surgical defects were made in rats, which received the following treatments in a randomized manner: group I, negative control (defect creation only); group II, positive control (FGG); group III, BCM; group IV, BCM + fibroblasts; group V, ADM; and group VI, ADM + fibroblasts. Clinical, histological, and immunological analyses were performed 15 days after grafting. Clinical examinations recorded epithelium regularity and the presence of ulcers, erythema, and/or edema.
+
+Results: The histological analysis revealed the degree of reepithelization, width, regularity, and presence of keratin. The Fisher exact statistical test was applied to the results (P<0.05). No groups showed ulcers except for group I. All groups had regular epithelium without erythema and without edema. Histologically, all groups exhibited regular epithelium with keratinization, and myofibroblasts were present in the connective tissue. The groups that received engineered grafts showed similar clinical and histological results to the FGG group.
+
+Conclusions: Within the limitations of this study, it was concluded that BCM and ADM can be used as cell scaffolds, with ADM yielding the best results. This study supports the use of this technical protocol in humans.
+
+Keywords: Cellulose; Cultured cells; Fibroblasts, animal study Copyright © 2024. Korean Academy of Periodontology 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/).
+
+| Received: Feb 10, 2022 Revised: Feb 25, 2023 Accepted: Mar 7, 2023 Published online: May 30, 2023 *Correspondence: Cimara Fortes Ferreira Department of Periodontics, University of  Tennessee Health Science Center (UTHSC)  College of Dentistry Dunn Dental Building,  5th Floor S503, 875 Union Ave, Memphis, TN  38163, Brazil.  Email: cimarafortes@hotmail.com Tel: +55-901-448-4494 Fax: +55-901-448-1294   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+ORCID iDs Abraão Moratelli Prado https://orcid.org/0000-0002-8658-0338 Cimara Fortes Ferreira https://orcid.org/0000-0002-0209-814X Luismar Marques Porto https://orcid.org/0000-0002-5777-5144 Elena Riet Correa Rivero https://orcid.org/0000-0002-8516-8771 Ricardo de Souza Magini https://orcid.org/0000-0003-0760-5611 Cesar Augusto Magalhães Benfatti https://orcid.org/0000-0002-4209-0054 Jair Rodriguez-Ivich https://orcid.org/0000-0002-7966-9262 Conflict of Interest No potential conflict of interest relevant to this article was reported. Author Contributions Conceptualization: Abraão Moratelli Prado, Elena Riet Correa Rivero, Ricardo de Souza Magini; Data curation: Abraão Moratelli Prado; Formal analysis: Abraão Moratelli Prado, Luismar Marques Porto; Investigation: Cimara Fortes Ferreira, Luismar Marques Porto, Elena Riet Correa Rivero; Methodology: Abraão Moratelli Prado, Cimara Fortes Ferreira, Luismar Marques Porto, Elena Riet Correa Rivero, Cesar Augusto Magalhães Benfatti, Jair Rodriguez-Ivich; Project administration: 
+Ricardo de Souza Magini, Cesar Augusto Magalhães Benfatti; Visualization: Cesar Augusto Magalhães Benfatti; Writing - original draft: Abraão Moratelli Prado, Luismar Marques Porto, Elena Riet Correa Rivero, Jair Rodriguez-Ivich; Writing - review & editing: Cimara Fortes Ferreira, Ricardo de Souza Magini, Cesar Augusto Magalhães Benfatti, Jair Rodriguez-Ivich.
+
+## Introduction
+
+![1_Image_0.Png](1_Image_0.Png)
+
+Mucogingival defects (MGDs), such as dental root recessions, decreased vestibular depth, and absence of keratinized tissues, are commonly seen in the dental clinic. MGDs may result in functional, aesthetic, and hygienic concerns. In these situations, autogenous soft tissue grafts are considered the gold-standard treatment [1]. The use of autogenous grafts (AGs) results in increased morbidity and discomfort to the patient due to the need for an additional surgical site to harvest the graft. There is also an increased risk of bleeding from the donor site, difficulty in standardizing tissue thickness, and color/thickness differences, which may result in an aesthetic challenge and limit graft quantity [2-8]. Thus, a less invasive reconstructive technique has been sought. To this end, tissue engineering aims to create a scaffold of absorbable or non-absorbable material, capable of storing cells and growth factors that may assist in the development of the lost tissues [9]. This technique can be used in the fields of periodontics and implant dentistry [10-15]. When using allografts for the purpose of restoring periodontal soft tissues (gingiva and periodontal ligament), it is necessary to acquire adequate cells. Gingival fibroblasts are fundamental in the fabrication/maintenance of the periodontal tissues [16]. Prato et al. [17] seeded fibroblasts on a matrix of hyaluronic acid and used this combination graft in a human clinical case series. This experimental technique was successful, as demonstrated by the clinical observation of restored keratinized mucosa and histological findings [17]. Fibroblasts are responsible for the fabrication and maintenance of extracellular matrix (ECM) and the production of periodontal collagen fibers. The manipulation and cultivation of these cells allow the establishment of a suitable and viable culture method for the development of a cellular scaffold. It is typical to use fetal bovine serum (FBS) in cell culture. However, aiming at the use of this technique in humans, in this study FBS was replaced by platelet-rich plasma (PRP) for the purpose of medium supplementation. The influence of progressively high concentrations of PRP on human osteoblast proliferation was evaluated [18]. PRPsupplemented culture medium showed higher osteoblast proliferation when stimulated by 50% PRP dilution, with or without FBS. The conclusion of that study was that PRP promotes osteoblast proliferation, suggesting that it could be applied clinically to bone graft procedures in implant dentistry. Bacterial cellulose matrix (BCM) (Jadam Co., Jeju, Korea) and human acellular dermal matrix (ADM) (SureDerm™; Hans Biomed Corp., Seoul, Korea) have been used for wound dressings and temporary skin replacement in the treatment of burns and ulcers, at graft sites, and as an aid in cases of dermal abrasion [19-22]. When the matrix is applied subcutaneously on the dorsum of wild-type mice, it supports cell infiltration and undergoes progressive remodeling, forming functional tissue without activation of the immune response [23]. Wound closure may occur by means of regeneration or repair. Regeneration results in newly formed tissue, fully reproducing the tissue architecture and function after an injury. Repair is a process that results in a scar, which is a disorganized cluster of ECM that, despite closing the lesion, does not reconstruct the original tissue characteristics. The repair process is divided into 3 phases: inflammation, proliferation, and remodeling. In the inflammation phase, coagulation factors are present. In the proliferation phase, wound reepithelization, granulation tissue formation, and angiogenesis occur, and fibroblasts differentiate into 
+
+![2_image_0.png](2_image_0.png) myofibroblasts (contractile cells that tend to close the wound) at the edges of the lesion. In the remodeling phase, tissue organization and keratin production take place [24]. In light of the above information, and since there are very few studies in this field, the aim of this study was to compare the healing process of free gingival grafts (FGGs) to BCM and ADM seeded with fibroblasts grown in PRP-supplemented medium in a rat model. The null hypothesis was that all 3 procedures would show similar healing patterns.
+
+## Materials And Methods Prp Medium Supplementation
+
+Whole blood was collected from 10 rats to fabricate the PRP used to supplement the culture medium. Venipuncture was conducted using minimal trauma to avoid triggering platelet factors, facilitating platelet adhesion, agglutination, and aggregation. Next, the blood was centrifuged [25]. The total blood collected from each rat was contained in a receptacle with citrate phosphate dextrose adenine, resulting in an approximate volume of 20 mL from each rat.
+
+## Fibroblast Primary Culture
+
+Tissue harvesting was conducted with the animals placed in the prone position, with their heads immobilized at 2 fixed points on a stereotactic table. A 5 mm2 (2.5×2.0 mm) biopsy of keratinized mucosa (epithelium and connective tissue) of the palatal region was performed under ketamine sedation after 0.12% chlorhexidine disinfection. The samples were used for primary fibroblast culture. Samples were processed under a laminar flow hood (VECO, Campinas, SP, Brazil) at room temperature, between 25°C and 28°C, in a type P2 safety laboratory. The rat oral mucosa tissue samples were washed twice with phosphate-buffered saline (PBS), and subsequently the epithelial tissue was removed and fragmented into 5 explants of approximately 1 mm2.
+
+The explants were incubated at 37°C, 5% CO2 with 2 mL of Dulbecco's modified Eagle medium (DMEM) culture medium supplemented with PRP. Cell growth was checked daily using an inverted phase microscope (×40 to ×200; Olympus, Tokyo, Japan). When approximately 70% confluence was found, the cells were trypsinized to dissociate them, and this primary culture yielded a new 1:1 subculture. In this first passage, the explants were removed. The trypsin was inactivated by adding supplemented DMEM culture medium. Next, the flasks were re-incubated until the new sub-confluence was reached.
+
+## Cell Viability Test
+
+An MTS calorimetric assay was used to assess fibroblast metabolic activity after 48 hours of incubation. inverted phase microscopy was used to observe fibroblasts growth. Absorbance was determined at 490 nm in a Spectramax spectrophotometer (Molecular Devices, San Jose, CA, USA). The positive control group consisted of cells cultured in a tissue culture flask 
+(TPP®; Sigma-Aldrich, St. Louis, MO, USA). Cell counting was performed using.
+
+## Ethical Considerations
+
+The research was approved by the Animal Ethics Committee (PP00908) and followed the ARRIVE guidelines. Twenty rats were selected for blood collection (for PRP preparation) and gingival tissue biopsy. Graft surgery was performed in the other 50 rats (**Figure 1**).
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+## Grafting Procedures
+
+The grafting procedure was conducted in the buccal mucosa; therefore, the grafting technique was adapted to the site. The surgical procedure is described as follows. The animals were anesthetized with 25 mg/kg ketamine hydrochloride (Dopalen, Sespo Indústria e Comércio, Paulínia, Brazil) via an intra-muscular injection. Next, intra-oral asepsis was conducted using 0.2% chlorhexidine digluconate. The incisions were made using BardParker 15C blades. A circular blade measuring 8 mm in diameter was used to delimit the recipient site starting 3 mm from the labial commissure of the rat's buccal mucosa. After preparing the recipient site, a partial-thickness flap was raised, exposing the connective tissue. Standardized circular defects were made bilaterally in the jugular mucosa, and group allocation was performed randomly. Group I only received the defects without any coverage. 
+
+Group II received an FGG from the palate. In groups III, IV, V, and VI, scaffolds were sutured over the surgical wound. The following technique was used to obtain the FGG. At the donor site, the same circular blade was used to harvest the graft. A Bard-Parker 15C blade was used with an approximate depth of 1.5 mm over the lines marked by the circular blade. The graft was dissected from the donor site. Irregularities and adipose tissues were removed from the graft, adjusting the graft's thickness. The suture technique was the same as used for the FGG and the remaining groups. The graft was positioned in the recipient site and stabilized using 5-0 thread to make an anterior and a posterior simple interrupted suture. Due to the rat's limited mouth opening, all grafts were stabilized with 2 simple interrupted sutures using 5-0 nylon thread 
+(**Figure 2**). Finally, digital compression was performed over the graft for 3 minutes.
+
+The animals were kept in cages, with 4 animals in each cage. The cages were stored in the University of Santa Catarina's Biological Unit facility, where the temperature and humidity were controlled. The animals were fed daily with food and water *ad libitum*. In the first 3 post-
+
+![4_image_0.png](4_image_0.png)
+
+![4_image_1.png](4_image_1.png)
+
+operative days, they were fed a softer diet. The hardwood chip bedding in the animal cages was changed every 2 days.
+
+## Randomization
+
+All animals were anesthetized and numbered with a tail tag. Tag numbers from 1 to 10 were placed inside a box. These tags were removed from the box by the operator and randomly distributed to each animal, which received three procedures. The randomization process followed a given sequence generated by an online website (Sealed Envelope Ltd., London, UK; 2017). The first randomized procedure was to be performed on the right and left sides of each rat. The sequence of animals operated started in group I and followed the rats' tag numbers, from 1 to 10. The rats were randomly divided into 6 groups: group I: negative control (n=12); group II: positive control (n=14); group III: BCM (n=16); group IV: fibroblasts in BCM (n=10); group V: ADM (n=8); and group VI: fibroblasts in ADM (n=6). Due to the deaths of some rats, some groups showed a different total number.
+
+## Clinical Evaluation
+
+Fifteen days after graft surgery, the animals were sedated for clinical and photographic evaluation. The clinical evaluation of the operated area considered the regularity of the epithelium and the presence or absence of ulcers, edema, and flushing. All information was tabulated and the predominant characteristics in each group were analyzed.
+
+## Histological Processing
+
+![5_Image_0.Png](5_Image_0.Png)
+
+At 5 days post-operatively, a biopsy was performed, removing a margin of untouched host tissue, and the explants were fixed in 10% buffered formalin for 48 hours. Next, the pieces were evaluated macroscopically, the explants were trimmed, and the specimens were removed, and a longitudinal cut was made to establish the inclusion profile of the grafted site. Afterward, the explants underwent histological processing and analysis, and 3-µmthick sections were obtained for hematoxylin and eosin staining. Images were taken of the operated site.
+
+## Immunohistochemical Processing
+
+At 5 days post-operatively, immunohistochemical processing was conducted with the biopsy samples. The manufacturer's guidelines were followed for each antibody used, and positive/negative controls were used for each reaction. The antibodies used were alphasmooth muscle actin (α-SMA) (Dako North America, Carpinteria, CA, USA), for identifying myofibroblasts in the sample's connective tissue, and pan-cytokeratin AE1/AE3 (Santa Cruz Biotechnology, Dallas, TX, USA), for identifying epithelial cells at the repair site if keratin was present. Reaction amplification was conducted using the EasyLink One kit (EasyPath, São Paulo, SP, Brazil). After mounting, the slides were kept in an oven (40°C) for at least 24 hours before being examined under a light microscope. The histology and immunohistochemistry were analyzed.
+
+## Histological/Immunohistochemical Analysis
+
+The slides were scanned with an AXIO SCAN Z.1 slide scanner (Zeiss, Köln, NRW, Germany) and analyzed by a previously calibrated observer using the AxionVision image analysis program (Zeiss). Next, the grafted site was identified and analyzed for the presence of complete reepithelization or ulcerations. The epithelium was classified as thin (1 to 3 cell layers) or regular (4 or more epithelial cell layers). The presence or absence of epithelial ridges and keratin (pan-cytokeratin) were evaluated. Blood vessels, inflammatory cells, and myofibroblasts (α-SMA) were counted within the same area (×20 magnification), and their presence was classified as mild (less than 20 cells), regular (20 to 60 cells) and intense (60 or more cells) (**Figure 2**). The Fisher exact test was used to verify statistical significance within each group (P<0.05). The kappa test was performed for examiner calibration.
+
+## Results
+
+The results of this study are described in the tables, organized by the relevant criteria. Clinical evaluations are presented in **Table 1**. Immunohistochemical and histological evaluations are presented in **Table 2**. All the data obtained can be seen and compared among the groups using the tables. The main comparisons are described in the Discussion section.
+
+## Statistical Analysis
+
+The sample size was calculated using the formula:
+n0 = Z2 × p × (1-p)/d n0 ≠ n Z ≠ z, where n = the sample size at time 0, z = the z value (also called the standard value), p = standard deviation, and d = the margin of error. Seventy healthy young-adult (20–24 weeks) male rats (*Rattus norvegicus albinus*; Wistar, Philadelphia, PA, USA) weighing 180 g on average, were used 
+
+Table 1. Results of the clinical evaluation of the groups
+
+![6_image_0.png](6_image_0.png) Groups Epithelium Erythema Edema
+
+Absent Present Irregular Regular Absent Present Absent Present
+
+Group I 10 - - - - 10 - 10 Group II - 10 1a) 9b) 9a) 1b) 9a) 1b)
+
+Group III - 10 5a) 5a) 5a) 5a) 5a) 5a)
+
+Group IV - 10 4a) 6a) 6a) 4a) 6a) 4a)
+
+Group V - 10 3a) 7a) 10 - 7a) 3a)
+
+Group VI - 10 - 10 10 - 10 - The evaluated criteria were presence of ulcer (absence of epithelium), regularity of the epithelium, presence of erythema, and presence of edema. The numbers represented quantity of the samples with the same characteristics. The results were statistically analyzed using the exact Fisher's test. All the analyzed criteria showed statistically significant values (P<0.05). Statistical difference was expressed within the groups. Subjects were randomly divided into 6 groups: group I, negative control; group II, positive control; group III, BCM; group IV, BCM + fibroblasts; group V, ADM; and group VI, ADM + fibroblasts. BCM: bacterial cellulose matrix, ADM: acellular dermal matrix.
+
+a,b)Groups that showed different letters (a, b) presented a predominant characteristic, the one present in the 
+
+majority of each of the groups. Groups that showed the same letters (a, a), did not present statistical significance. The groups that did not have letters, presented only one characteristic that was predominant in that group.
+
+Table 2. Histological results
+
+Groups Epithelium Epithelium width Epithelium ridges Inflammatory infiltrate
+
+Absent Present Thin Regular Absent Present Regular Intense
+
+Group I 10 - - - 10 - - 10
+
+Group II - 10 5a) 5a) 5a) 5a) 5a) 5a)
+
+Group III - 10 9b) 1a) 4a) 6a) 5a) 5a) Group IV - 10 7a) 3a) 2a) 8a) 9a) 1a)
+
+Group V - 10 3a) 7a) 1a) 9a) 9a) 1a)
+
+Group VI - 10 - 10 - 10 10 - The evaluated criteria were the presence of epithelium, thickness of the epithelium (thin or regular), presence of epithelial crests and quantity of inflammatory infiltrate (regular or intense). The numbers represent the quantity of the samples with the same characteristic. The results were statistically analyzed using the exact Fisher's test. 
+
+All the analyzed criteria showed statistically significant values (P<0.05). Statistical difference was expressed 
+
+within the groups. Subjects were randomly divided into 6 groups: group I, negative control; group II, positive control; group III, BCM; group IV, BCM + fibroblasts; group V, ADM; and group VI, ADM + fibroblasts. BCM: bacterial cellulose matrix, ADM: acellular dermal matrix. a,b)Groups that showed different letters (a, b) presented a predominant characteristic, the one present in the majority of each of the groups. Groups that showed the same letters (a, a), did not present statistical significance. The groups that did not have letters, presented only one characteristic that was predominant in that group.
+
+in the *in vivo* portion of this study. In the *in vitro* analysis, the kappa test result was 0.845. 
+
+Statistically significant intragroup results were obtained for all characteristics evaluated 
+(P<0.05). Different letters are used to show the presence of a significantly predominant characteristic in certain groups, while the same letters are used to indicate an even distribution of characteristics, with no predominance in specific groups (**Tables 1** and 2). As shown in **Table 1**, which presents details on clinical parameters, ulcers were only found in group I. Groups II, IV, V, and VI did not present ulcers. Groups II and VI presented regular epithelium at the operated site. Groups IV and V showed a non-statistically significant tendency for a regular epithelium. Erythema was present in group I and absent in groups II, V, and VI. Edema was present in group I and absent in groups II and VI. Group I presented ulcers, erythema, and edema clinically. Group II presented regular epithelium and an absence of ulcers, erythema, and edema. Ulcers were absent in group III. Group IV did not present ulcers. Group V presented an absence of ulcers and erythema. Group VI presented clinically regular epithelium and an absence of ulcers, erythema, and edema (**Table 1**). As presented in **Table 2**, the histological analysis revealed that ulcers were only present in Group I, which did not show epithelial ridges or keratin, but there was a large amount of 
+
+Table 3. Results of the immunohistochemical evaluation
+
+![7_image_0.png](7_image_0.png) Group Keratin Blood vessels Myofibroblasts
+
+Absent Present Regular Intense Regular Intense
+
+Group I 10 - - 10 10 - Group II 2a) 8a) 5a) 5a) 2a) 8a)
+
+Group III 4a) 6a) 3a) 7a) 3a) 7a)
+
+Group IV 2a) 8a) 7a) 3a) - 10
+
+Group V 1a) 9a) 7a) 3a) - 10
+
+Group VI - 10 10 - - 10
+
+The evaluated criteria were presence of keratin, expressed by the α-SMA marker; quantity of blood vessels and 
+
+myofibroblasts (regular or intense), expressed by the cytokeratin marker. The numbers represent the quantity of the samples with the same characteristic. The results were statistically analyzed using the exact Fisher's test. All the analyzed criteria showed statistically significant values (P<0.05). Statistical difference was expressed within the groups. Subjects were randomly divided into 6 groups: group I, negative control; group II, positive control; group III, BCM; group IV, BCM + fibroblasts; group V, ADM; and group VI, ADM + fibroblasts.
+
+BCM: bacterial cellulose matrix, ADM: acellular dermal matrix, α-SMA: alpha-smooth muscle actin. a)Groups that showed different letters (a, b) presented a predominant characteristic, the one present in the majority 
+
+of each of the groups. Groups that showed the same letters (a, a), did not present statistical significance. The groups that did not have letters, presented only one characteristic that was predominant in that group.
+
+inflammatory infiltrate and blood vessels, as well as a regular amount of myofibroblasts. Group II presented keratinized epithelium and a tendency for a high concentration of myofibroblasts. Group III presented thin epithelium, inflammatory infiltrate, and blood vessels. Group IV presented epithelium with keratin and inflammatory infiltrate and blood vessels in the connective tissues, as well as a large amount of myofibroblasts. Group VI showed regular epithelium. Epithelial ridges were absent in group I and present in group VI. Group II presented split results (50% with epithelial ridges). Groups III, IV, and V showed a tendency for epithelial ridges, although it did not reach statistical significance. Inflammatory infiltrate was present in all groups, with a high concentration in group I and a regular concentration in group VI. Groups II and III presented split results (50% with regular inflammatory infiltrate). Groups IV and V had a tendency for regular inflammatory infiltrate, but without statistical significance. Group V presented epithelium with keratin and myofibroblasts in large quantity, as well as epithelial ridges and a regular quantity of inflammatory infiltrate. Group VI presented regular epithelium with keratin and epithelial ridges, as well as a regular concentration of inflammatory infiltrate and blood vessels. In group VI, myofibroblasts were found in large quantities (**Table 2**).
+
+As detailed in **Table 3**, the immunohistochemical analysis showed the presence of keratin in groups II, IV, V, and VI. Group I did not exhibit keratin. Blood vessels were present in all groups, with a high concentration in group I and a regular concentration in group VI. A nonstatistically significant tendency was observed for groups II and III to have a large amount of blood vessels and for groups IV and V to have a regular amount. Myofibroblasts were present in all groups, with a regular amount in group I and an intense amount in groups IV, V, and VI. Groups II and III tended to have a large amount of myofibroblasts, albeit without statistical significance (**Table 3**).
+
+## Discussion
+
+For many years, AGs have been successfully employed, but certain limitations have forced researchers to seek alternative grafting procedures to enhance patient comfort, site acceptability and compatibility. In the FGG technique, a second surgical site is required to harvest the donor tissue. This secondary site heals by secondary intention, which may increase post-operative pain and morbidity. FGGs often result in an unaesthetic appearance 
+
+![8_image_0.png](8_image_0.png) due to the behavior of the graft. In addition, AGs cannot be used to increase the gingival width in many cases due to the limitations of the donor sites [26]. The main outcome of this study was that all groups, except for group I, did not have ulcers. All groups had regular epithelium without erythema and without edema. Histologically, all groups exhibited regular epithelium with keratinization, and myofibroblasts were present in the connective tissue. The groups that received engineered grafts showed similar clinical and histological results to those of the FGG group. No inflammatory responses were identified in any of the experimental or control groups, despite using blood in a homogenous manner to fabricate PRP. A possible reason for this finding may be the use of animals from an established and genetically mapped lineage, where the genetic variations between the animals are very minute. In the present study, the negative control group presented unfavorable clinical results, such as the presence of ulcers, erythema, and edema. It also presented unfavorable histological results, with no epithelial ridges or keratin. It had a high concentration of inflammatory infiltrate and blood vessels, as well as a regular amount of myofibroblasts. These results corroborate previously reported findings in the literature [1], since in the negative group, no procedure was conducted to cover the surgical site; for this reason, it was expected that this group would show the longest delay before healing in this region. Therefore, it is ideal to cover oral lesions. In the present study, the positive control group showed no ulcers, erythema, or edema. Histologically, it presented epithelium with a tendency to express keratin. A histological evaluation of the FGG healing process was detailed by Caffesse et al. [26] in rhesus monkeys. In that study, FGGs were placed at recipient sites with or without periosteum. The histological analysis of the FGGs on the periosteum at 14 days showed complete epithelialization with the development of the morphological characteristics of the attached gingiva. The findings of the present study agree with those of the 14-day histological analysis in the study of Caffesse et al. [26]. However, they did not show the presence of keratinization in the 14-day analysis, whereas the present study did show the presence of keratin in the samples collected at a 15-day time interval. Therefore, this is an inconsistency between the present study and the previous study. Some possible reasons are that 1) the samples in the present study were collected at 15 days post-operatively, whereas those in the previous study were collected a day earlier; and 2) the present study used a different animal model (namely, rats as compared to rhesus monkeys), and rats have a higher metabolic rate. Group III (BCM), in the present study, did not show the presence of ulcers. However, it presented thin epithelium, inflammatory infiltrate, and blood vessels. BCM has several indications and is a relevant tool for various purposes, such as burn therapy and peri-implant tissue grafting. Our results concur with the literature that healing was predictable when BCM was used [4]. Clinically, group IV exhibited regular tissue without ulceration. Histologically, it presented keratinized epithelium, inflammatory infiltrate, and blood vessels in the connective tissue. It also contained a large amount of myofibroblasts. The presence of keratin is an important factor that should be highlighted, as it demonstrates that the BCM seeded with fibroblasts favored the formation of epithelial tissue. The large amount of myofibroblasts also indicates 
+
+![9_image_0.png](9_image_0.png) that the healing process for this group was more advanced than that of group III. There are no previous reports in the literature describing the use of this material with this specific cell culture technique, indicating a need for further research. Group V showed similar results to the positive control group. Group V did not present ulcers or erythema. There was keratinized epithelium with a tendency for the presence of epithelial ridges, a regular amount of inflammatory infiltrate, and a high concentration of myofibroblasts. The literature also indicates the importance of permeability, with the presence of pores in the membranes in order to promote regeneration [27], which is a characteristic of the ADM. Studies have shown that ADM provides uniform thickness, easy handling, favorable adaptation, and a short rehydration time before its use [28]. ADM is not as effective as FGG in increasing the width of the grafted tissue, but its aesthetic results are more predictable, and it blends in with adjacent tissue [28]. The amount of grafted tissue acquired with ADM is clinically sufficient to prevent persistent inflammation [29]. ADM acts as a scaffold to allow the restocking of fibroblasts and blood vessels from the surrounding tissue epithelium, with later replacement by the host tissues [30]. Group VI presented promising results. In addition to its excellent clinical qualities, including a regular surface and absence of ulcers, edema, and erythema, this group had advanced histological healing, such as a regular epithelium, with an acceptable amount of keratinized tissue and well-defined and constant epithelial ridges, inflammatory infiltrate and blood vessels, and a high concentration of myofibroblasts. Group VI presented better clinical and histological results than the positive control group and the other groups. This could have been due to the presence of fibroblasts, which favor the healing process, especially when a direct comparison is made with group V (ADM without the presence of fibroblasts). There are no studies in the literature that would allow a direct comparison of this result. However, this finding shows great potential. A limitation of this study is that 5 rats died during anesthesia and 4 died in the post-operative period. Three rats chewed off their tags and had to be excluded from the study. Due to the operation dates being different, the authors were able to add animals to some groups, but not to others. In addition, blood collection for the fabrication of autogenous PRP, as would be performed in a clinical scenario, was not possible due to the low amount of blood that we were permitted to collect from each animal. The proposed techniques used for gingival grafting with PRP-supplemented primary fibroblast culture showed similar clinical and histological results to the gold-standard FGG technique. This study established a protocol to research the performance of soft tissue grafts in a rat model. The two matrices tested can be used as a scaffold for gingival tissue cells, with ADM being the most indicated matrix for FGGs in rat jugal mucosa. The use of fibroblasts obtained by primary culture positively influenced the repair process. Supplementation of the medium with PRP favored the healing process and could be used in humans. The technical use of tissue engineering without culture medium supplementation from a different species avoids the risk of immunological responses or cross-contamination. These factors facilitate the clinical application of this method by using primary culture originating from the recipient. Therefore, genetic testing and the application of this technique to medium-size animals (e.g., minipigs) should be conducted prior to tests in humans. In conclusion, within the limitations of this study, the histological analysis showed that 
+
+![10_image_0.png](10_image_0.png) the groups exhibited very similar results, with a slight difference favoring group VI, which exhibited the characteristics of more mature tissue. This study shows alternative treatment options for contemporary clinical challenges. However, future studies should be performed with different healing periods, at other anatomical sites, and in other types of organisms to derive more concrete conclusions regarding the clinical and histological performance of these materials, combined with cell culture or not.
+
+## Acknowledgements
+
+This research study was conducted in the Laboratory of Pain and Inflammation Neurobiology (LPIN) and the Laboratory of Integrated Technologies (LIT), and Laboratories of the Federal University of Santa Catarina. The Brazilian Ministry of Education funded a PhD student with the Coordination and Refinement of Superior Level Personnel (CAPES) scholarship. The authors would like to thank Mr. Raí Heidenreich for assistance with the animal surgery and Mr. Guilherme Colla for assistance with the cell culture.
+
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+Growth of human keratinocytes and fibroblasts on bacterial cellulose film. Biotechnol Prog 2006;22:1194-9. 
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+PUBMED | **CROSSREF**
+21. Zhang X, Deng Z, Wang H, Yang Z, Guo W, Li Y, et al. Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration. Biomaterials 2009;30:2666-74. PUBMED | **CROSSREF**
+22. Feng Y, Wang J, Ling S, Li Z, Li M, Li Q, et al. Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold. Neural Regen Res 2014;9:1968-78. PUBMED | **CROSSREF**
+23. Rennert RC, Sorkin M, Garg RK, Januszyk M, Gurtner GC. Cellular response to a novel fetal acellular collagen matrix: implications for tissue regeneration. Int J Biomater 2013;2013:527957. PUBMED | **CROSSREF**
+24. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity 2016;44:450-62. 
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+PUBMED | **CROSSREF**
+25. Devereaux J, Nurgali K, Kiatos D, Sakkal S, Apostolopoulos V. Effects of platelet-rich plasma and plateletpoor plasma on human dermal fibroblasts. Maturitas 2018;117:34-44. PUBMED | **CROSSREF**
+26. Caffesse RG, Burgett FG, Nasjleti CE, Castelli WA. Healing of free gingival grafts with and without periosteum. Part I. Histologic evaluation. J Periodontol 1979;50:586-94. PUBMED | **CROSSREF**
+27. Rambo CR, Carminatti CA, Pitlovanciv AK, Antônio RV, Porto LM. Template assisted synthesis of nanofibrous cellulose membranes for tissue engineering. Mater Sci Eng C 2008;28:549-54. **CROSSREF**
+28. Shulman J. Clinical evaluation of an acellular dermal allograft for increasing the zone of attached gingiva. 
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+30. Wei PC, Laurell L, Geivelis M, Lingen MW, Maddalozzo D. Acellular dermal matrix allografts to achieve increased attached gingiva. Part 1. A clinical study. J Periodontol 2000;71:1297-305. PUBMED | **CROSSREF**

medical/md/PMC10913121.md

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+Content of this journal is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
+
+# Socs3, Transcriptionally Activated By Nr4A1**, Induces Apoptosis And** Extracellular Matrix Degradation Of Vaginal Fibroblasts In Pelvic Organ Prolapse Xin Jin, Qing Hu, Meiying Qin, Yitong Yin, Zhijun Xia
+
+Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, China Background: Pelvic organ prolapse (POP) is a common gynecological chronic disorder. Human vaginal fibroblasts (HVFs) that maintain the integrity of vaginal wall tissues are essential for keeping pelvic organs in place. Apoptosis and the degradation of the extracellular matrix in HVFs contribute to the progression of POP. The cytokine signal transduction inhibitor 3 (*SOCS3*) exerts significant regulatory effects on cell signal transduction pathways, thereby affecting various pathological processes.
+
+Aims: To explore the role and mechanism of *SOCS3* on HVFs in the context of POP.
+
+Study Design: In vitro cell lines and human-sample study.
+
+Methods: Anterior vaginal wall tissues were obtained from POP or nonPOP patients for the analysis of *SOCS3* expression. HVFs were isolated from the vaginal tissues of POP patients, and *SOCS3* was either overexpressed 
+
+## Introduction
+
+Pelvic organ prolapse (POP), a common gynecological disease among females, is characterized by the protrusion of the pelvic organs (vagina, uterus, bladder, and/or rectum) into or out of the vaginal canal.1 Treatment options for POP include pelvic floor muscle training, vaginal pessaries, and reconstructive pelvic surgery. However, the therapeutic outcomes are often unsatisfactory and relapse is a common occurrence.2,3 Therefore, it is imperative to elucidate the pathogenesis of POP to develop effective treatments for this condition. The most common form of POP is anterior vaginal wall prolapse, 
+
+![0_image_0.png](0_image_0.png) commonly known as cystocele. In the vaginal wall, connective tissues or knocked down in HVFs via lentivirus infection. Subsequently, the biological function and mechanism of *SOCS3* in HVFs were investigated. Results: *SOCS3* was highly expressed in the vaginal tissues of POP patients compared to non-POP patients. Functionally, the overexpression of *SOCS3* suppressed cell viability while promoting cell apoptosis in HVFs. The overexpression of *SOCS3* also accelerated extracellular matrix degradation 
+(decreasing collagen I, collagen III, and elastin, and increasing *MMP2* and MMP9). In terms of mechanism, *NR4A1* transcriptionally activated *SOCS3* by binding to its promoter. Furthermore, rescue experiments revealed that *SOCS3* knockdown hindered *NR4A1* overexpression-induced cell apoptosis and extracellular matrix degradation in HVFs. Conclusion: *SOCS3* mediated the apoptotic and extracellular matrix degradation effects of *NR4A1* on HVFs, underlining that the restraining of the *SOCS3* expression may be a promising strategy for POP treatment.
+
+provide the supportive strength for the pelvic floor.4 The connective tissue layer consists of an extracellular matrix (ECM), which obtains its strength from collagen I, collagen III, and elastin.5 Vaginal fibroblasts control ECM production and remodeling, by generating anabolic proteins, like collagens, and activating catabolic enzymes, such as matrix metalloproteinases (*MMPs*), thus modifying their surrounding matrix.6 In patients with POP, fibroblast dysfunction leads to damaged collagen metabolism and an unbalanced disturbed ECM production.7,8 Additionally, some studies have confirmed increased fibroblast apoptosis in POP.9 However, there is a limited number of studies that focus on the underlying mechanism. Hence, enhancing the functions of fibroblasts emerges as a viable approach to impede the progression of POP.
+
+Corresponding author: Zhijun Xia, Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, China e-mail: xiaz123321@sina.com Received: October 19, 2023 **Accepted:** December 07, 2023 **Available Online Date:** February 29, 2024 - **DOI:** 10.4274/balkanmedj.galenos.2023.2023-10-60 Available at www.balkanmedicaljournal.org ORCID iDs of the authors: X.J. 0009-0000-8599-9033; Q.H. 0009-0001-8022-763X; M.Q. 0000-0001-6493-7976; Y.Y. 0009-0008-7283-5820; Z.X. 0009-0007-4352-6693.
+
+Cite this article as: Jin X, Hu Q, Qin M, Yin Y, Xia Z. *SOCS3*, Transcriptionally Activated by *NR4A1*, Induces Apoptosis and Extracellular Matrix Degradation of Vaginal Fibroblasts in Pelvic Organ Prolapse. Balkan Med J.; 2024; 41(2):105-12. Copyright@Author(s) - Available online at http://balkanmedicaljournal.org/
+The cytokine signal transduction inhibitor 3 (*SOCS3*), one of the SOCS family proteins, interacts with various activated proteins of the tyrosine kinase signaling pathway.10 *SOCS3* suppresses cell proliferation and its deletion inhibits myocardial apoptosis and fibrosis.11,12 Moreover, the downregulation of *SOCS3* promotes collagen release and aggravates fibrosis in murine models.13 Database analysis showed an upregulation in *SOCS3* expression in the anterior vaginal wall tissue of patients with POP. Therefore, SOCS3 may participate in the development of POP. Human nuclear receptor 4A 1 (*NR4A1*) is an orphan nuclear receptor associated with fibrosis of multiple organs. *NR4A1* limits fibrotic diseases by regulating transforming growth factor-β (*TGF-*β) 
+signaling.14 Hiwatashi et al.15 reported that *NR4A1* inhibits vocal fold fibrosis, while *NR4A1* knockdown promotes collagen I expression in fibroblasts. *NR4A1* also participates in the pathological progression of POP by promoting the apoptosis of fibroblasts.16 Furthermore, 9-AA, an agonist of *NR4A1*, enhances the expression levels of SOCS3.
+
+17 Based on these published results, we hypothesize that the NR4A1/SOCS3 axis may play a role in the progression of POP. In this study, we investigated the function and mechanism of SOCS3 in POP. We found that *SOCS3* expression was higher in the vaginal tissues of POP patients than that in non-POP patients. SOCS3 overexpression induced cell apoptosis and ECM degradation in human vaginal fibroblasts (HVFs) from patients with POP. 
+
+Additionally, we detected that *SOCS3* was transcriptionally activated by *NR4A1*, and the effect of *SOCS3* on HVFs was regulated by *NR4A1*. 
+
+This study provides evidence for the role of *SOCS3* in POP.
+
+## Materials And Methods Tissue Samples Collection
+
+Anterior vaginal wall tissues were collected from POP patients (n = 36, age range: 47-74) and non-POP patients (n = 18, age range: 39-57). All patients participating in the study signed the informed consent. This study was conducted following the Declaration of Helsinki, and approved by the Shengjing Hospital of China Medical University Ethics Committee (2022PS040K).
+
+## Fibroblast Culture And Identification
+
+Primary HVFs were isolated from anterior vaginal wall tissues and cultured as described previously.18,19 The cells were cultured in Dulbecco's modified Eagle's medium (Servicebio, China) supplemented with 10% fetal bovine serum (Sijiqing, China) at 37 
+°C and 5% CO2
+. Cells were passaged and expanded until reaching 60% confluence, and those up to the fourth passage were used for subsequent experiments. Immunofluorescence was performed to identify cells at the fourth passage using markers for vimentin (a marker for fibroblasts), cytokeratin (a marker for epithelial cells), and desmin (a marker for smooth muscle cells). 
+
+## Lentivirus Infection
+
+The coding sequences of SOCS3 or *NR4A1* were inserted into pLVXIRES-puro (Fenghbio, China) to generate *SOCS3* overexpression lentivirus (LV-*SOCS3*) or *NR4A1* overexpression lentivirus (LV-*NR4A1*). 
+
+The map of pLVX-IRES-puro is shown in Figure S2a. Additionally, SOCS3-targeted shRNA was inserted into pLVX-shRNA1 (Fenghbio, China) to establish sh*SOCS3* lentivirus (LV-sh*SOCS3*). The map of pLVX-shRNA1 is shown in Figure S2b. The *SOCS3* shRNA sequence is as follows: 5′-GCTTCGACTGCGTGCTCAAGC-3′, LV-SOCS3, LV-NR4A1, LV-shSOCS3, and their negative controls (NC) were subsequently packaged in HEK293T cells. The lentiviral-containing supernatant was harvested and filtered. HVFs were then infected with lentiviruscontaining supernatants at a multiplicity of infection of 100 and incubated at 37 °C with 5% CO2
+.
+
+## Statistical Analysis
+
+The data were analyzed using GraphPad Prism 8.0 (GraphPad Software, USA). All data were presented as the mean ± standard deviation. T-tests were employed to compare data between two groups, while one-way ANOVA with Bonferroni post-hoc test was used for comparing data involving more than two groups. P < 0.05 was deemed statistically significant. For detailed methods, please see the Supplementary material.
+
+## Results Socs3 Is Highly Expressed In The Vaginal Tissues Of Pop Patients
+
+Built on the differential expression analysis of the gene expression omnibus database, we found 265 upregulated and 13 downregulated genes in patients with POP. Subsequently, GO enrichment analysis of differential expression genes (*DEGs*) revealed an association among POP, ECM, and cell apoptosis (Figure 1a). In these *DEGs, SOCS3* knockdown inhibits cell apoptosis and promotes collagen release,12,13 suggesting that SOCS3 may be involved in the progression of POP. In addition to the GSE12852 dataset, the high expression of *SOCS3* in POP tissues was observed in the GSE53868 dataset (Figure 1b). To verify our conjecture, we collected vaginal tissues from POP and non-POP patients. Then, Masson trichrome staining and TUNEL staining were performed to explore the histomorphological differences. 
+
+The results of Masson trichrome staining showed decreased collagen (blue) in tissues of POP patients compared with nonPOP patients (Figure 1c). TUNEL staining revealed a higher incidence of apoptosis in tissues from POP patients (Figure 1d). These results indicated more prominent collagen degradation and apoptosis in patients with POP, consistent with previous reports7 and our bioinformatics analysis. To further investigate the role of *SOCS3* in POP, we detected *SOCS3* mRNA expression in vaginal tissues. The results showed that S*OCS3* was significantly upregulated in patients with POP (Figure 1e). Western blot analysis further verified the upregulation of *SOCS3* at the protein level (Figure 1f). Moreover, IHC and the histologic score (H-score) were performed to further verify the expression pattern of *SOCS3* in POP. The results showed higher *SOCS3* expression levels in the POP group compared with the non-POP group (Figure 1gh). Furthermore, we analyzed the association between the expression levels of  and patient characteristics in Table S1; however, no correlation was observed. These results suggested that *SOCS3* was upregulated in the vaginal tissues of patients with POP.
+
+## Morphological Identification Of Hvfs
+
+Primary HVFs obtained from the vaginal tissues of POP patients were 
+
+![2_image_0.png](2_image_0.png) cultured and collected for microscopic observation (Figure S1a). Then, the fourth passage of HVFs was subjected to immunofluorescence staining to identify the phenotype using specific biomarkers (Figure S1b). The results revealed high cytoplasmic expression of vimentin (a marker of fibroblasts) in the fourth passage of HVFs. In contrast, no significant increase in the expression of cytokeratin (a marker of epithelial cells) and desmin (a marker of smooth muscle cells) 
+was observed. The above results illustrated that cells isolated from vaginal tissues were mainly fibroblasts without the contamination of epithelial and smooth muscle cells. 
+
+## Socs3 Overexpression Inhibits Viability And Induces Apoptosis In Hvfs
+
+Considering the increased expression of *SOCS3* in POP, the function 
+
+![2_image_1.png](2_image_1.png) of *SOCS3* in POP was further investigated. *SOCS3* was overexpressed in the fourth passage of HVFs through lentivirus infection, and the gene overexpression was confirmed with RT-qPCR and western blot (Figure 2a, b). Subsequently, the results from the CCK-8 assay showed that *SOCS3* overexpression inhibited the cell viability of HVFs (Figure 2c). Additionally, a significantly higher apoptosis rate was observed in *SOCS3* overexpression groups compared to the control groups (Figure 2d). The TUNEL assay further verified the above results, with more TUNEL-positive cells observed in *SOCS3* overexpression HVFs (Figure 2e). In addition, pro-apoptotic protein Bax and cleaved-caspase-3, as well as anti-apoptotic protein Bcl-2, were detected by western blot. The expression levels of Bax and cleaved-caspase 3 increased, whereas that of Bcl-2 decreased, as observed after *SOCS3* overexpression (Figure 2f, g). These results suggested that *SOCS3* overexpression inhibited viability and induced apoptosis of HVFs.
+
+## Socs3 Overexpression Induces Ecm Degradation In Hvfs
+
+Previous experiments validated the significance of collagen degradation as a prominent characteristic in patients with POP.20 Thus, to explore whether *SOCS3* participates in the regulation of ECM components, the protein expression of collagen I, collagen III, and elastin was analyzed. The results revealed that *SOCS3* overexpression reduced the expression of collagen I, collagen III, and elastin (Figure 3a). Besides, we detected the protein expression of *MMP2* and *MMP9*. Collagen-degrading *MMPs* are involved in ECM remodeling processes, as shown in Figure 3b, increased expression of *MMP2* and *MMP9* was observed after *SOCS3* overexpression. 
+
+Moreover, the expression of components related to collagen synthesis pathways, including *TGF-*β, Smad2/3, and phosphorylated Smad2/3 (p-Smad2/3) was detected.21 We found that *TGF-*β1 and p-Smad2/3 showed low expression in the *SOCS3* overexpression HVFs, and the expression of Smad2/3 was almost unchanged (Figure 3c, d). Together, all the signs indicated that the upregulation of SOCS3 caused ECM degradation in HVFs.
+
+## Socs3 Is Transcriptionally Activated By Nr4A1 Via Binding The 
+
+![3_Image_1.Png](3_Image_1.Png) Socs3 Promoter
+
+NR4A1 accelerates the pathological process of POP by inducing fibroblast apoptosis.16 To investigate the relationship between SOCS3 and *NR4A1*, we constructed *NR4A1*-overexpressing lentivirus (LV-*NR4A1*) to infect HVFs. A clear rise of *NR4A1* mRNA and protein expression was shown in HVFs (Figure 4a, b). *NR4A1* overexpression was accompanied by increased *SOCS3* mRNA and protein levels 
+(Figure 4c, d), suggesting that *SOCS3* expression might be regulated by *NR4A1*. Based on the above results, we predicted *NR4A1*-binding sites on the *SOCS3* promoter (Figure 4e), and the interaction between NR4A1 and *SOCS3* was further confirmed by the dual luciferase reporter assay. As shown in Figure 4f, when fragment A (-1948, -1585 bp, containing four sites) or fragment B (-1585, -643 bp, containing two sites) was truncated, the luciferase activity was decreased. Moreover, when there was only fragment C (-643 to +36, containing one site), the promoter activity was increased compared with the control vector. These results indicated that the regions from -1948 to -1585 bp, -1585, -643 bp, and -643 to +36 bp contained binding sites of *NR4A1*. The above results suggest that *NR4A1* transcriptionally activates *SOCS3* by binding to its promoter.
+
+## Nr4A1 Overexpression Induces Cell Apoptosis And Ecm 
+
+![3_Image_0.Png](3_Image_0.Png) Degradation In Hvfs
+
+To further verify the effects of *NR4A1* on POP, we assessed *NR4A1* expression in vaginal tissues from POP and non-POP patients. Higher *NR4A1* expression at the mRNA level was observed in patients with POP (Figure 5a). IHC staining and H-score further confirmed that *NR4A1* expression was higher in patients with POP (Figure 5b, c). However, the expression level of *NR4A1* had no association with the characteristics of POP (Table S2). Subsequently, NR4A1 was overexpressed in the HVFs via lentivirus infection. 
+
+NR4A1 overexpression suppressed cell viability (Figure 5d) and promoted cell apoptosis in HVFs (Figure 5e, f). In addition, the protein expression levels of collagen I, collagen III, and elastin were attenuated in *NR4A1*-overexpression HVFs (Figure 5g), indicating that *NR4A1* promoted ECM degradation.
+
+## Socs3 Mediates The Effect Of Nr4A1 On Hvfs
+
+To determine whether *SOCS3* mediates the effect of *NR4A1* on HVFs, HVFs were co-infected with *NR4A1* overexpression lentivirus and sh*SOCS3* lentivirus. The results showed that *SOCS3* knockdown increased cell viability and decreased cell apoptosis in *NR4A1* overexpression HVFs (Figure 6a, b). Furthermore, upregulated protein expression of collagen I, collagen III, and elastin was observed when *SOCS3* was knocked down (Figure 6c). Together, these results revealed that the function of *NR4A1* on HVFs was blocked via SOCS3 knockdown, indicating that *NR4A1* affected HVFs by *SOCS3*- mediated apoptosis and ECM degradation in HVFs.
+
+## The Screening Of Potential Genes Regulated By Socs3
+
+RNA‐seq analysis was further performed to detect the possible 
+
+![4_image_0.png](4_image_0.png)
+
+mechanism of *SOCS3* on HVFs. Initially, principal component analysis exhibited significantly different component profiles between *SOCS3* overexpression (LV-*SOCS3*) and control (LV-NC) groups (Figure 7a). *DEGs* were identified by volcano plot filtering (Figure 7b). We identified 203 downregulated and 914 upregulated DEGs. Then, *DEGs* were significantly enriched for POP-related GO or KEGG terms, such as ECM organization, programmed cell death, and collagen catabolic process (Figure 7c). Further analysis identified DEGs involved in these processes, presented in a heatmap (Figure 7d). Gene Set Enrichment Analysis (GSEA) confirmed significant enrichment of *DEGs* involved in the regulation of ECM organization and collagen catabolic process (Figure 7e). These findings suggested that *SOCS3* may regulate ECM and collagen-related downstream signaling.
+
+## Discussion
+
+POP, characterized by a descent of pelvic organs into the vaginal 
+
+![4_image_1.png](4_image_1.png) cavity, significantly impairs the quality of life for many women.1,22 However, the etiopathogenesis of POP remains poorly understood. Anterior vaginal wall prolapse is the most common form of POP. Vaginal fibroblasts maintain the integrity of vaginal wall tissues, which is important for supporting pelvic organs and preventing POP.23 Numerous studies have demonstrated that fibroblast apoptosis and ECM loss play critical roles in the progression of POP.7,8 In our study, we selected vaginal tissues as experimental subjects to explore the pathogenesis of POP. As expected, the vaginal tissues showed decreased collagen deposition and enhanced apoptosis in patients with POP. Subsequently, through bioinformatics analysis, we observed an elevated expression of *SOCS3* in patients with 
+
+![5_image_0.png](5_image_0.png)
+
+![5_image_1.png](5_image_1.png)
+
+POP, suggesting a potential association between *SOCS3* and the progression of POP. 
+
+SOCS3, as a member of the suppressor of the cytokine signaling family, is a potent inhibitor of Janus kinase/signal transducer and activator of the transcription (Jak/STAT) pathway.10 *SOCS3* is implicated in various human diseases, including cancers, inflammatory diseases, cardiovascular diseases, diabetes, asthma, and neuronal diseases.24 However, thus far, these studies provide limited information on the relationship between *SOCS3* and POP, and the mechanism of action of POP remains unclear. In our study, we confirmed that *SOCS3* expression was higher in the vaginal tissues of patients with POP, suggesting that increased *SOCS3* expression may participate in the progression of POP. Recent studies have reported that *SOCS3* inhibits cell proliferation and promotes cell apoptosis.11,12 In our study, we also observed that *SOCS3* overexpression inhibited viability and induced apoptosis in HVFs. Additionally, collagen I, collagen III, and elastin are the main ingredients of ECM. The strength and resilience of vaginal tissues are determined by collagens and elastin, respectively.5 Dees et al.13 reported that the deletion of *SOCS3* promotes collagen release. In light of this finding, we sought to investigate whether *SOCS3* regulates the ECM in HVFs. Our results demonstrated that *SOCS3* overexpression led to decreased expression levels of collagen I, collagen III, and elastin. Moreover, MMPs are involved in the degradation of ECM and suppression of collagen deposition in POP.6 Some studies have reported that SOCS3 knockdown results in the downregulation of *MMP2* and MMP9.
+
+25 Consistent with previous research, our study showed that upregulation of *SOCS3* elevated the expression of *MMP2* and *MMP9*. Besides, our studies further found that *SOCS3* also downregulated TGF-β1 and p-Smad2/3 protein expression. *TGF-*β1 is a crucial regulator in fibrosis-related diseases, which induces myofibroblast transformation and regulates ECM production.26 It promotes ECM 
+deposition by enhancing collagen synthesis and suppressing MMPs.
+
+27 *TGF-*β1 exerts its diverse biological activities mainly through activating transcriptional regulators of the Smad pathway. The p-Smad2/3 produced upon *TGF-*β1 activation is responsible for regulating collagen and fibronectin gene expression.28 A previous study has reported that increased *SOCS3* expression inhibits *TGF-*β1 expression.29 *TGF-*β1 blocks the loss of ECM by suppressing *MMP2* and *MMP9* expression via the TGF-β1/Smad3 signaling pathway in the uterosacral ligament of patients with POP.30 Our findings revealed that *SOCS3* decreased the expression of p-Smad3 and p-Smad2. Therefore, we hypothesized that *SOCS3* may enhance the expression of *MMP2* and *MMP9* by inhibiting *TGF-*β1/Smad2/3, thereby promoting ECM degradation. Nevertheless, these possible mechanisms and detailed interactions among them require further exploration. In summary, *SOCS3* overexpression induced apoptosis and ECM degradation of HVFs.
+
+The abnormal expression of *SOCS3* is often attributed to the transcriptional activation of its promoter. For example, the transcriptional activation of the farnesoid X receptor enhances *SOCS3* expression.31 In this study, we confirmed that *NR4A1* overexpression enhanced the levels of *SOCS3*, and we identified the *NR4A1* binding sites on the *SOCS3* promoter. Thus, *SOCS3* might be transcriptionally regulated by NR4A1. *NR4A1*, belonging to the orphan nuclear hormone receptors of the Nur77 family, is a transcription factor involved in the cell cycle, apoptosis, and tumorigenesis.32 Similarly, Zeng et al.16 demonstrated that *NR4A1* induces apoptosis by linking the actin cytoskeleton in parametrial ligament fibroblasts of POP. Here, we found that *NR4A1* was highly expressed in the vaginal tissues of POP 
+patients. *NR4A1* overexpression promoted cell apoptosis and ECM degradation in HVFs. More importantly, *SOCS3* knockdown reversed the negative effects of *NR4A1* on HVFs, suggesting that *NR4A1* affected the function of HVFs by transcriptionally activating *SOCS3*
+(Figure 8). Furthermore, the RNA-seq analysis was performed to investigate the potential downstream genes that may be regulated by *SOCS3*. When *SOCS3* was overexpressed, *DEGs* were significantly enriched in ECM organization, programmed cell death, and collagen catabolic process, which was consistent with our findings. *SOCS3* overexpression upregulated the expression of various genes, such as *HTRA1* and *CTSS*. The upregulated expression of *HTRA1* has been reported to inhibit cell proliferation and induce cell apoptosis.33 Additionally, reducing the expression of *CTSS* increases ECM deposition.34 These downstream factors may play a role in the POP process. However, follow-up experiments are needed to further study the functions of these factors. The dysfunction of *SOCS3* may contribute to several disorders. The activation of *SOCS3* leads to insulin resistance and retinal endothelial cell apoptosis in the diabetic retina.35 *SOCS3* deletion suppresses myocardial apoptosis, which results in the block of left ventricular remodeling after myocardial infarction.36 Moreover, hepatocyte steatosis was accelerated by promoting *SOCS3* expression.37 Selective inhibition of *SOCS3* may attenuate some of these consequences, suggesting the potential role of *SOCS3* inhibitors as a promising therapy for disease treatment. For example, PARγ agonist ROZ 
+alleviated steatosis by decreasing *SOCS3* expression.37 Similarly, the inhibition of *SOCS3* expression by ruxolitinib attenuates atherosclerosis.38 Besides, microRNAs have been identified as regulators of *SOCS3* expression. Repression of microRNA-122 reduces SOCS3 expression by promoter methylation.39 These methods of inhibiting *SOCS3* expression may offer new clinical avenues for the treatment of POP. In addition, we found no significant association between the expression of SOCS3 or *NR4A1* and the characteristics of patients with POP. We speculated that this may be due to the insufficient number of samples. In the future, we will continue to collect clinical samples to clear the relationship between the expression levels of SOCS3 or NR4A1 and the characteristics of patients with POP. In conclusion, this study provides evidence that *SOCS3*, transcriptionally activated by *NR4A1*, promotes the progression of POP by inducing HVF apoptosis and ECM degradation. Thus, the NR4A1/SOCS3 axis emerges as a promising therapeutic target for the treatment of POP.
+
+Ethics Committee Approval: This study was conducted following the Declaration of Helsinki, and approved by the Shengjing Hospital of China Medical University Ethics Committee (2022PS040K).
+
+Data Sharing Statement: The datasets analyzed during the current study are available from the corresponding author upon reasonable request. Authorship Contributions: Concept- Z.X.; Collection and Processing- X.J., Q.H., M.Q., Y.Y.; Analysis or Interpretation- X.J., Q.H.; Literature Search- X.J.; Writing- X.J., Z.X.; Supervision- Z.X. Conflict of Interest: The authors declare that they have no conflict of interest. Funding: This study was supported by the Natural Science Foundation of Liaoning Province (under Grant 2021JH2/10300054).
+
+Supplementary: http://balkanmedicaljournal.org/uploads/pdf/suplement_2023-10-60.pdf
+
+## References
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+15. Hiwatashi N, Bing R, Kraja I, Branski RC. NR4A1 is an endogenous inhibitor of vocal fold fibrosis. *Laryngoscope*. 2017;127:E317-323. [CrossRef]
+16. Zeng W, Li Y, Li B, et al. Mechanical Stretching induces the apoptosis of parametrial ligament Fibroblasts via the Actin Cytoskeleton/Nr4a1 signalling pathway. *Int J Med Sci*. 2020;17:1491-1498. [CrossRef]
+17. Wang H, Xu X, Guan X, et al. Liposomal 9-Aminoacridine for Treatment of Ischemic Stroke: From Drug Discovery to Drug Delivery. *Nano Lett*. 2020;20:1542-1551. [CrossRef]
+18. Wen Y, Wani P, Zhou L, et al. Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age. *Stem Cells* Transl Med. 2013;2:118-128. [CrossRef]
+19. Ruiz-Zapata AM, Kerkhof MH, Zandieh-Doulabi B, Brölmann HA, Smit TH, Helder MN. 
+
+Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates. *Int Urogynecol J*. 2013;24:1567-1575. [CrossRef]
+20. Li Y, Nie N, Gong L, et al. Structural, functional and molecular pathogenesis of pelvic organ prolapse in patient and  *Loxl1*  deficient mice. *Aging (Albany NY).* 2021;13:25886902. [CrossRef]
+21. Min J, Li B, Liu C, et al. Therapeutic Effect and Mechanism of Electrical Stimulation in Female Stress Urinary Incontinence. *Urology*. 2017;104:45-51. [CrossRef]
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+23. Ruiz-Zapata AM, Heinz A, Kerkhof MH, et al. Extracellular Matrix Stiffness and Composition Regulate the Myofibroblast Differentiation of Vaginal Fibroblasts*. Int J* Mol Sci. 2020;21:4762. [CrossRef]
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+MAPK-mediated production of pro-inflammatory mediators by up-regulation of SOCS3 in Trypanosoma cruzi-infected cardiomyocytes. *PLoS One*. 2013;8:e79445. [CrossRef]
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+1994;331:1286-1292. [CrossRef]
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+28. Massagué J. TGF-beta signal transduction. *Annu Rev Biochem*. 1998;67:753-91. [CrossRef] 29. Dong X, Wang J, Tang B, et al. The role and gene expression profile of SOCS3 in colorectal carcinoma. *Oncotarget*. 2017:15984-15996. [CrossRef]
+30. Liu C, Wang Y, Li BS, et al. Role of transforming growth factor β1 in the pathogenesis of pelvic organ prolapse: A potential therapeutic target. *Int J Mol Med*. 2017;40:347-356. [CrossRef]
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medical/md/PMC3226428.md

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+SHORT COMMUNICATION
+NANO REVIEWS
+Single-molecule photochemical reactions of Auger-ionized quantum dots Morihiko Hamada1,2, Edakkattuparambil Sidharth Shibu1, Tamitake Itoh1, Manikantan Syamala Kiran1, Shunsuke Nakanishi2, Mitsuru Ishikawa1 and Vasudevanpillai Biju1,3* 1Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa, Japan; 2Department of Advanced Materials Science, Kagawa University, Takamatsu, Kagawa, Japan; 3Precursory Research for Embryonic Science and Technology
+(PRESTO), Japan Science and Technology Agency (JST), Tokyo, Japan Received: 14 February 2011; Revised: 3 July 2011; Accepted: 11 July 2011; Published: 13 October 2011 Abstract Photoinduced electron transfer in donor-acceptor systems composed of quantum dots (QDs) and electron donors or acceptors is a subject of considerable recent research interest due to the potential applications of such systems in both solar energy harvesting and degradation of organic pollutants. Herein, we employed single-molecule imaging and spectroscopy techniques for the detection of photochemical reactions between 1,4-diaminobutane (DAB) and CdSe/ZnS single QDs. We investigated the reactions by analyzing photoluminescence (PL) intensity and lifetime of QDs at ensemble and single-molecule levels. While DAB
+was applied to single QDs tethered on a cover slip or QDs dispersed in a solution, PL intensity of QD
+continuously decreased with a concomitant increase in the PL lifetime. Interestingly, these changes in the PL
+properties of QD were predominant under high-intensity photoactivation. We hypothesize that the above changes in the PL properties surface due to the transfer of an electron from DAB to Auger-ionized QD
+followed by elimination of a proton from DAB and the formation of a QD-DAB adduct. Thus, a continuous decrease in the PL intensity of QDs under high-intensity photoactivation is attributed to continuous photochemical reactions of DAB with single QDs and the formation of QD-(DAB)n adducts. We believe that detection and analysis of such photochemical reactions of single QDs with amines will be of considerable broad interest due to the significant impact of photoinduced electron transfer reactions in energy management and environmental remediation.
+
+Keywords: quantum dots; single-molecule; electron transfer; Auger ionization; CdSe/ZnS; photoluminescence; photochemical reaction Colloidal quantum dots (QDs) show unique optical properties such as broad absorption and narrow emission bands as well as bright, stable, and sizedependent tunable photoluminescence (PL) (1, 2). Also, the large surface area of QDs is promising for the conjugation of various organic and biomolecules (3).
+
+The unique optical and structural properties formulate QDs to be ideal donors or acceptors of electrons in the construction of donor-acceptor systems for modern photovoltaic devices (414).
+
+Given the significance of QDs in solar energy harvesting, investigation of photoinduced electron transfer in QD-based donor-acceptor systems such as QD-TiO2
+(59), QD-Indium Tin Oxide (ITO) (10), QD-inorganic complexes (11), QD-C60 (12), and QD-polymer (13) is a subject of considerable current research interest. In these systems, transfer of an electron from a photoactivated QD to the corresponding acceptor results in the formation of charge-separated states. Solar cells based on donor-acceptor systems are designed by directing the charge carrier recombination through an external electrical circuit. Although photoinduced electron transfer processes in QD-based donor-acceptor systems are greatly investigated, photochemical reactions between organic molecules and photoactivated QDs as well as potentials of such reactions for environmental remediation remain mostly unattended.
+
+Recently, Sharma et al. found that p-phenylenediamine lowers the PL quantum efficiency of CdSe QD by accepting an electron from it (14). On the other hand, n-butylamine is known to passivate surface defects in CdSe QD, suppress non-radiative carrier recombination in the defects, and enhance the PL quantum efficiency of QD (14). Nonetheless, controversies exist in the amineinduced variations of QD's PL quantum efficiency. For example, n-butylamine itself at higher concentrations activates non-radiative carrier recombination in CdSe QD and lowers its PL quantum efficiency (15). To-date electron transfer interactions in the above QD-based donor-acceptor systems are found to be reversible by energy wasting back electron transfer. Although reversible electron transfer interactions of QDs with donors or acceptors of electron are greatly investigated, photochemical reactions between hole-acceptors and QDs remain mostly unattended. Importantly, photochemical reactions of highly photostable QDs with dyes, amines, and other organic pollutants would be of great value for environmental waste management. Here we found that photoactivation of CdSe/ZnS QDs in the presence of 1,4-diaminobutane (DAB) results in a gradual but irreversible decrease in the PL intensity of single QDs.
+
+This irreversible decrease in the PL intensity is apparent only under high-intensity photoactivation of QDs, which gives us the notion that Auger-ionized QDs accept electrons from DAB molecules and subsequent elimination of a proton from the QD-DAB- complex results in the formation of a QD-DAB adduct. Thus, we propose that the gradual but irreversible decrease in the PL intensity of single QDs is due to successive photochemical reactions of Auger-ionized QDs with a large number of DAB molecules and the formation of QD-(DAB)n adducts.
+
+## Results And Discussion
+
+We have investigated photochemical reactions of DAB
+with QDs at ensemble and single-molecule levels by recording and analyzing PL spectra of QD solutions and PL intensity trajectories of single QDs both in the presence of DAB and under high-intensity photoactivation. Fig. 1A shows the effect of concentration of DAB
+on the PL spectra of a QD solution. As the concentration
+
+![1_image_0.png](1_image_0.png)
+
+of DAB was increased, PL intensity of QDs decreased.
+
+Also, we have examined temporal changes in the PL
+intensity of QDs under continuous photoactivation.
+
+Fig. 1B shows PL spectra of a QD solution in the presence of DAB and under continuous photoactivation. Here, PL intensity of QDs continuously decreased with time but without any changes in the concentration of DAB.
+
+A decrease in the PL intensity of QDs with an increase in the concentration of DAB (Fig. 1A) and a temporal decrease in the PL intensity of QDs under continuous photoactivation (Fig. 1B) are attributed to photochemical reactions of DAB with QDs and the formation of QD-DAB adducts having lower PL quantum efficiencies than pristine QDs.
+
+To validate the photochemical reaction of DAB with QDs, we have recorded and analyzed the PL intensity trajectories of single QDs in the presence and absence of DAB and under low- (0.22 kW/cm2) and high-
+(2.2 kW/cm2) intensity photoactivation. Fig. 2 shows PL intensity trajectories of single QDs tethered on a cover slip, immersed in water, and recorded in the presence or absence of DAB. Interestingly, in the presence of DAB and under high-intensity photoactivation, we detected a continuous decrease in the PL intensity of single QDs (Fig. 1BF). On the other hand, PL intensities of single QDs under high-intensity photoactivation but without DAB remained intact even after several minutes
+(Fig. 2A). The excitation power density (2.2 kW/cm2)
+applied in these experiments was high enough to activate multiple exciton pairs and induce Auger ionization in QDs, which is apparent from intermittent PL blinking of single QDs. Our hypothesis for the gradual decrease in the PL intensity of single QDs in the presence of DAB is that Auger-ionized QDs react with DAB and form QDDAB adducts having PL quantum efficiencies lower than that of QDs. The reaction of DAB with QD probably took place by the transfer of an electron from DAB to Auger-ionized QD followed by the elimination of a proton from QD-DAB- adduct. Steps involved in the photochemical reactions of DAB with photoactivated QD is schematically presented in Fig. 3. Under continuous photoactivation, a large number of DAB molecules can be added onto the surface of QDs. Thus, the continuous decrease in the PL intensity can be attributed to the formation of poorly luminescent QD-(DAB)n adducts.
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+![2_image_2.png](2_image_2.png)
+
+The above hypothesis was investigated by intermittent photoactivation of single QDs in the presence of DAB
+and under high excitation intensity (2.2 kW/cm2). Fig. 2C
+and D shows PL intensity trajectories of single QDs excited at 15 s and 60 s laser 'on'/'off' intervals. We found that the PL intensities of single QDs remained essentially unaffected before and after each laser 'off' period, which indicates that the reaction of DAB with QD should be absolutely photoinduced. In other words, DAB does not react with QDs in the dark. Our hypothesis was further investigated by photoactivating single QDs in the presence of DAB but under low excitation intensity
+(0.22 kW/cm2), which is insufficient to activate multiple excitons and induce Auger ionization. Under these conditions, we found that PL intensities of single QDs remained essentially intact even after 10 min. The relation between the concentration of DAB and PL intensities of single QDs is shown in Fig. 2B, E, and F. Here, as the concentration of a DAB solution in a single QD sample was increased (10 mM for Fig. 2B, 100 mM for Fig. 2E,
+and 1 mM for Fig. 2F), the rate at which PL intensities of single QDs decreased was also increased. These results and the gradual decrease in the PL intensity of single QDs support our hypothesis that Auger-ionized QDs react with DAB.
+
+The photochemical reaction between QD and DAB
+was further investigated by recording and analyzing PL decay profiles of QD solutions. Fig. 4 shows PL decay profiles of a QD solution recorded at 2 min intervals in the presence of DAB and under high-intensity
+(2.2kW/cm2) photoactivation. Auger ionization of QDs under such high-intensity photoactivation is apparent
+
+![3_image_0.png](3_image_0.png)
+
+from evolution of a fast component in the decay profiles
+(Fig. 4A and inset). Interestingly, we detected a gradual increase (from 5.9 ns to 9.8 ns, 66%) in the PL lifetime of QDs with time under photoactivation (Fig. 4A). On the other hand, PL lifetime of a QD solution was increased by only B20% when photoactivated without the addition of DAB (inset of Fig. 4A). The spontaneous increase in the PL lifetime of QDs is associated with an increase in the PL quantum efficiency, which originates from an increase in the contribution of radiative carrier recombination in the surface states (16, 17). However, an increase in the PL lifetime of QDs under high-intensity photoactivation in the presence of DAB is associated with a gradual but irreversible decrease in PL intensity. Because amines are known to passivate surface defects in QDs, we attribute the changes in the PL properties of QDs to surface passivation of QDs by photoinduced electron transfer reaction with DAB and the formation of QD-
+(DAB)n adducts. The contribution of DAB to an increase in the PL lifetime of QDs was further investigated by recording and analyzing PL decays of QDs under lowintensity photoactivation. Fig. 4B shows PL decay profiles of a QD solution recorded at 2 min intervals in the presence of DAB and under low (0.22 kW/cm2)
+excitation intensity, which is insufficient to activate multi-exciton pairs and induce Auger ionization. Therefore, photochemical reactions of DAB with QD takes place only under high-intensity photoactivation. In other words, the decrease in the PL intensity of QDs with time under photoactivation in the presence of DAB can be attributed to photoinduced electron transfer reactions between Auger-ionized QD and DAB and subsequent formation of poorly luminescent QD-(DAB)n adducts.
+
+In short, single-molecule photochemical reactions of an aliphatic amine on the surface of quantum dots were detected by analyzing photoluminescence intensity trajectories and lifetimes of quantum dots at ensemble and single particle levels. As QDs were photoactivated in the presence of DAB, we found continuous decrease in the photoluminescence intensity of quantum dots and concomitant increase in their photoluminescence lifetime values. These changes in the luminescence properties were apparent only under high-intensity photoactivation of quantum dots, during which Auger ionization is operational. Thus, the changes in the luminescence properties of single quantum dots are attributed to photoinduced electron transfer reaction of amine with Auger-ionized quantum dots and the formation of quantum dot-amine adducts. Such photochemical reactions of organic molecules on the surface of quantum dots and other nanoparticles would be of great value for energy management and environment remediation.
+
+## Experimental Procedure
+
+CdSe/ZnS (PL maximum 600/655 nm) QDs were obtained from Invitrogen Corporation (Japan) and DAB from Sigma Aldrich (Japan). Single-molecule samples were prepared by tethering QDs on cover slips. The procedure for preparation of single-molecule samples is reported elsewhere (9). In short, cover slips were cleaned by dipping in a nascent chlorine solution and silanized using a 0.5% solution of 3-aminopropyltriethoxysilane in acetone. Silanized cover slips were copiously washed with water and acetone, and biotinylated by dipping in a 10 mM aqueous solution of biotin 3-sulfo-Nhydroxysuccinimide ester (Sigma Aldrich, Japan) for 30 min. Biotinylated cover slips were copiously washed with deionized water and successively, CdSe/ZnS QDs were uniformly tethered on the cover slips by placing a 10 pM solution of QD655-streptavidin conjugate on one side of the cover slip. After incubation for 30 min at room temperature, unbound QDs were removed by washing with deionized water. Finally, QDs were dipped in deionized water and recorded single-molecule images, PL intensity trajectories, and PL lifetimes. Single-molecule experiments were carried out in an inverted optical microscope (Olympus IX 70) equipped with a 60 objective lens (Olympus, NA 0.98). Fluorescence signals collected using the objective lens were filtered through a 620 nm long pass filter, magnified using a 2.5telescopic lens, and recorded using an image intensifier, chargecoupled device assembly (Hamamatsu Photonics, Japan).
+
+For PL lifetime measurements, samples were excited with 400 nm fs laser pulses from an optical parametric amplifier (OPA). The OPA was pumped by 800 nm pulses (200 kHz) from a regenerative amplifier that was seeded by a mode-locked Ti:sapphire laser (76 MHz). Fluorescence decay profiles were recorded using an assembly of a polychromater and a streak-scope (Hamamatsu photonics, Japan).
+
+## Acknowledgement
+
+This work was supported by Precursory Research for Embryonic Science and Technology (to VB) from Japan Science and Technology Agency.
+
+Conflict of interest and funding There is no conflict of interest in the present study for any of the authors.
+
+## References
+
+1. Murray CB, Norris DJ, Bawendi MG. Synthesis and characterization of nearly monodisperse CdE (Esulfur, selenium, tellurium) semiconductor nanocrystallites. J Am Chem Soc 1993; 115: 870615.
+
+2. Biju V, Itoh T, Anas A, Sujith A, Ishikawa M. Semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications. Anal Bioanal Chem 2008; 391: 246995.
+
+3. Biju V, Itoh T, Ishikawa M. Delivering quantum dots to cells:
+bioconjugated quantum dots for targeted and nonspecific extracellular and intracellular imaging. Chem Soc Rev 2010; 39: 303156.
+
+4. Wood V, Bulovic´ V. Colloidal quantum dot light-emitting devices. Nano Rev 2010; 1: 52028.
+
+5. Robel I, Subramanian V, Kuno M, Kamat PV. Quantum dot solar cells. Harvesting light energy with CdSe nanocrystals molecularly linked to mesoscopic TiO2 films. J Am Chem Soc 2006; 128: 238593.
+
+6. Kamat PV. Quantum dot solar cells. Semiconductor nanocrystals as light harvesters. J Phys Chem C 2008; 112: 1873753.
+
+7. Robel I, Kuno M, Kamat PV. Size-dependent electron injection from excited CdSe quantum dots into TiO2 nanoparticles. J Am Chem Soc 2007; 129: 41367.
+
+8. Jin S, Lian T. Electron transfer dynamics from single CdSe/
+ZnS quantum dots to TiO2 nanoparticles. Nano Lett 2009; 9:
+244854.
+
+9. Hamada M, Nakanishi S, Itoh T, Ishikawa M, Biju V. Blinking suppression in CdSe/ZnS single quantum dots by TiO2 nanoparticles. ACS Nano 2010; 4: 444554.
+
+10. Jin S, Song N, Lian T. Suppressed blinking dynamics of single QDs on ITO. ACS Nano 2010; 4: 154552.
+
+11. Huang J, Stockwell D, Huang Z, Mohler DL, Lian T.
+
+Photoinduced ultrafast electron transfer from CdSe quantum dots to re-bipyridyl complexes. J Am Chem Soc 2008; 130:
+56323.
+
+12. Song N, Zhu H, Jin S, Zhan W, Lian T. Poisson-distributed electron-transfer dynamics from single quantum dots to C60 molecules. ACS Nano 2011; 5: 61321.
+
+13. Greenham NC, Peng XG, Alivisatos AP. Charge separation and transport in conjugated-polymers/semiconductor-nanocrystal composites studied by photoluminescence quenching and photoconductivity. Phys Rev B 1996; 54: 1762837.
+
+14. Sharma SN, Pillai ZS, Kamat PV. Photoinduced charge transfer between CdSe quantum dots and p-phenylenediamine. J Phys Chem B 2003; 107: 1008893.
+
+15. Landes C, Burda C, Braun M, El-Sayed MA. Photoluminescence of CdSe nanoparticles in the presence of a hole acceptor:
+n-butylamine. J Phys Chem B 2001; 105: 29816.
+
+16. Wang X, Qu L, Zhang J, Peng X, Xiao M. Surface-related emission in highly luminescent CdSe quantum dots. Nano Lett 2003; 3: 11036.
+
+17. Biju V, Kanemoto R, Matsumoto Y, Ishii S, Nakanishi S,
+Itoh T, et al. Photoinduced photoluminescence variations of CdSe quantum dots in polymer solutions. J Phys Chem C 2007; 111: 792432.
+
+## *Vasudevanpillai Biju
+
+Health Research Institute National Institute of Advanced Industrial Science and Technology 2217-14 Hayashi-Cho Takamatsu, Kagawa, Japan Tel: -81-878-693558 Fax: -81-878-694113 Email: v.biju@aist.go.jp

medical/md/PMC3633304.md

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+# Incidence Of Metabolic Syndrome And Its Characteristics Of Patients Attending A Diabetic Outpatient Clinic In A Tertiary Care Hospital
+
+Subash V. Kumar, A. Nagesh, M. Leena, G. Shravani, V. **Chandrasekar1**
+Departments of Pharmacy Practice, MGM Hospital, VCOP, 1*Department of Medicine, NTR University,* 
+Kakatiya Medical College/MGM Hospital, Warangal, Andhra *Pradesh, India* Address for correspondence: Dr. *Subash Vijaya Kumar, Head Department of Pharmacy Practice, Vaagdevi College of Pharmacy,* Hanamkonda, Warangal, Andhra Pradesh, India. E‑mail: *vijayvijay66@yahoo.co.in*
+
+## Abstract
+
+Objective: We sought to evaluate the incidence of metabolic syndrome and non-metabolic syndrome among type 2 diabetic patients attending the diabetic outpatient clinic at tertiary care hospital, Warangal. **Materials and Methods:** A cross-sectional study was conducted in a period of 6 months from January 2011 to June 2011. The study group consisted of 75 type 2 diabetic patients. They were screened for hypertension, hyperlipidemia, obesity, and clinical characteristics, and other co-morbidities were recorded. Metabolic syndrome diagnosis was made as per ATP III guidelines. **Results:** The prevalence of metabolic syndrome was significant in men (54.8%) compared to women (45.2%). Incidence of metabolic syndrome was found to be more in normal weight patients (43.56%). Low high density lipoprotein (HDL) levels were observed in both rural (90.63%) and urban (95.65%) patients with metabolic syndrome, followed by increase in waist circumference. The mean HDL level was found to be 23.77 mg/dl. Patients in the age group 51-60 years were found to be more affected with metabolic syndrome. Sedentary household female patients (58.3%) and illiterates (41.8%) were suffering from metabolic syndrome. Patients with metabolic syndrome had been suffering with diabetes (duration of diabetes) from 1 to 5 years. In summary, this cross-sectional study characterizes the metabolic and non-metabolic syndromes of type 2 diabetes patients living in Telangana regions, using ATP III guidelines, and generates a biological resource that enables further investigation of numerous hypotheses related to genetic exposure of both in a population. **Conclusion:**These results suggest that higher prevalence of metabolic syndrome was observed in non-obese male patients and was significantly associated with aging. Nevertheless, further studies are required to confirm the metabolic syndrome in larger population.
+
+Key words: Body mass index, diabetes, high density lipoprotein, metabolic syndrome, mortality
+
+## Introduction
+
+The metabolic syndrome (MetS), as defined by the National Cholesterol Education Program Adult Treatment Panel III (NCEP‑ATP III) criteria, is a constellation of fasting lipids and lipoproteins, waist circumference, glucose, and blood pressure abnormalities that have been associated 
+
+![0_image_0.png](0_image_0.png)
+
+| Access this article online Website: www.jnsbm.org DOI: 10.4103/0976-9668.107261   |
+|-----------------------------------------------------------------------------------|
+
+with increasing risk for developing cardiovascular disease 
+
+![0_image_1.png](0_image_1.png)
+
+(CVD) and diabetes.[1‑3] These definitions are based on expert opinion and not on evidence resulting from prospective epidemiologic studies. Therefore, it remains unclear whether single component features of the MetS or the thresholds at which each component is defined as present or absent are informative and optimal for predicting the risk of cardiovascular disease or early death.[4] Weiss and colleagues reported that the prevalence of the MetS increased with the severity of obesity and reached a proportion of 50% in severely obese youngsters. The prevalence of the MetS increased significantly with increasing insulin resistance after adjustment for the ethnic group factors and degree of obesity.[5] Type 2 diabetes and the MetS have been regarded as a disease of adults for a long time,[6] but it has clearly become evident that the disease can begin at different ages in all ethnic groups and can be already present in childhood.[7‑9]
+This observation also points toward the existence of a metabolic endocrine susceptibility factor determining the risk for clinically developing MetS already during childhood. An aggressive global approach to screening and management of the MetS is therefore recommended to slow the growth of the syndrome throughout the United States.[10] Therefore, our present study highlights the incidence of MetS and non‑metabolic syndrome (NMetS) in type 2 diabetic patients.
+
+## Materials And Methods
+
+We performed a cross‑sectional study from January 2011 to June 2011. This research was jointly conducted by Vaagdevi College of Pharmacy and Mahatma Gandhi Memorial (MGM) Hospital. Both are affiliated to Kakatiya University, Warangal, Andhra Pradesh, India.
+
+## Study Participants
+
+The inclusion criteria were: Age >18 years and diabetic. Exclusion criteria were: Endocrine diseases such as Cushing's disease, type 1 diabetes mellitus, acromegaly, hypothyroidism, hypogonadism, patient on prolong steroid use, and those who were on active drug treatment for obesity at the time of admission. The source of the data was the outpatients who were attending the MGM Hospital, Warangal. The Performa included co‑morbidities, characteristics, and details of physical and biochemical information and treatment, which were reviewed and recorded. The physical and biochemical measurements were made at the first visit. The independent variables of interest were age, gender, ethnicity, smoking, alcohol consumption, and history of diabetic types. All patients were interviewed regarding duration of diabetes and anti‑diabetic drugs. All subjects enrolled in the study underwent detailed clinical examinations, including measurement of height, weight, waist circumference, and blood pressure. Patients were considered hypertensive if these were already on antihypertensive therapy or with a systolic pressure ≥135 mm Hg and a diastolic pressure ≥85 mm Hg. Fasting venous blood was sampled from antecubital vein from all patients for the measurement of plasma glucose, total cholesterol, high density lipoprotein (HDL) cholesterol, and serum triglycerides.
+
+## Lifestyle Habits
+
+Patients were assessed for smoking habits, physical inactivity, and alcohol consumption. Briefly, patients were categorized based on their smoking habits into never smoker, former smoker, and current smoker
+
+## Data Collection
+
+In the first two weeks of January 2011, a workshop was conducted in cardiac unit at MGM Hospital, Warangal, which standardized the protocol and methodology (administering the structure questionnaires, drawing blood, and taking medications). Thus, the investigators (diabetologist, cardiologist) epidemiologist, PG students, Interns, students, training nurse, and Pharm. D students took part in the 2‑day training course on using NCEP‑ATP III guidelines. Patients were instructed and encouraged not to eat after 7:00 p.m. before the laboratory measurement. Baseline overnight fasting blood was drawn from 7:00 to 9:00 a.m. Fasting serum and plasma samples [preserved with ethylene diamine tetraaceticacid (EDTA) and sodium fluoride 
+(NaF)] were collected within 2 h of venous blood sampling and immediately frozen to −20°C until triglycerides, HDL, and cholesterol were analyzed and measured using standard methods.[11]
+Ethical approval was obtained from the Medical Review Board of Kakatiya Medical College (KMC), Andhra Pradesh, India. Eligible participants were invited to attend the diabetic unit on a specified date. They were free to participate and gave written consent according to Helsinki declaration. Descriptive strategies were calculated for each variable. All continuous variables were expressed as a mean with standard deviation; median, intraquartile range, frequency, and percentage were obtained for categorized variables. Univariables, multivariables, binary logistics, and regression analysis were used to identify factors significantly associated with MetS and NMetS classified based on NCEP‑ATP III criteria. All analyses were performed using the GraphPad Prism version 5.
+
+## Results
+
+There were 275 patients who attended the diabetic OP clinic from January 2011 to June 2011. However, only 75 (27.2%) patients were eligible for the study. Based on the inclusion and exclusion criteria, diabetic patients had complete data and comprised the study population. Mean duration of diabetes was 6.52 years in MetS and 4.8 years in NMetS. The relative proportion of alcoholic patients with MetS was 41.81%, while 50% of patients were non‑alcoholic with NMetS. Current smokers (20.8%) and past smokers (18.18%) with MetS were equally distributed. However, illiterate patients (41.8%) had higher incidence of MetS and reduced incidence (20%) of NMetS. The data are summarized in Table 1. Table 2 presents prevalence of MetS and NMetS according to NCEP‑ATP III guidelines. Sedentary lifestyle is more prone to MetS (64.18%) compared to NMetS (55%). Women's sedentary lifestyle (91.66%) resulted in higher incidence of MetS. Patients with high prevalence of MetS compared to those with NMetS were higher (34.88%) in 51-60 years age group, followed by 25.59% in 61-70 years age group [Table 3]. An individual component of the MetS and NMetS in gender and living area was also measured. Interestingly, urban people had higher incidence of developing MetS compared to rural people. Low HDL levels were consistently observed in rural and urban population (95.65% and 90.625%, respectively), followed by increase in waist circumference in rural and urban population (65.62% and 73.19%, respectively).
+
+Table 4 presents the physical characteristics of South Indian participants (Warangal adults) with type 2 diabetes. Interestingly, high incidence of MetS was observed in normal weight patients (43.56%) compared to obese (10.9%), overweight (40%), and underweight (5.45%) patients. Table 5 shows the gender wise Mean ± SD of the MeS 
+
+| according to socio-demographic details Category Metabolic syndrome   | Non-metabolic syndrome   |             |            |            |            |            |            |    |
+|----------------------------------------------------------------------|--------------------------|-------------|------------|------------|------------|------------|------------|----|
+| Male                                                                 | Female                   | Total       | Percentage | Male       | Female     | Total      | Percentage |    |
+| 43                                                                   | 12                       | 55          | 73.33      | 13         | 7          | 20         | 26.66      |    |
+| Place Rural                                                          | 16 (37.3%)               | 7 (58.3%)   | 23         | 41.88      | 7 (53.8%)  | 4 (57.1%)  | 11         | 55 |
+| Urban                                                                | 27 (62.7%)               | 5 (41.7%)   | 32         | 58.22      | 6 (46.2%)  | 3 (42.9%)  | 9          | 45 |
+| Education status 1–5 6 (13.95%)                                      | 0 (0%)                   | 6           | 11         | 2 (15.38%) | 0 (0%)     | 2          | 10         |    |
+| 6–10                                                                 | 12 (27.90%)              | 3 (25%)     | 15         | 27         | 3 23.07%)  | 4 (57.14%) | 7          | 35 |
+| Inter                                                                | 5 (11.62%)               | 1 (8.33%)   | 6          | 11         | 1 (7.69%)  | 1 (14.28%) | 2          | 10 |
+| Degree                                                               | 3 (6.97%)                | 0 (0%)      | 3          | 5.45       | 4 (3.76%)  | 1 (14.28%) | 5          | 25 |
+| PG                                                                   | 2 (2.32%)                | 0 (0%)      | 2          | 3.63       | 0 (0%)     | 0 (0%)     | 0          | 0  |
+| Religion Hindu                                                       | 33 (76.7%)               | 8 (66.6%)   | 41         | 74.5       | 8 (61.5%)  | 6 (85.7%)  | 14         | 70 |
+| Muslim                                                               | 6 (13.9%)                | 3 (25%)     | 9          | 16.3       | 3 (23%)    | 1 (14.2%)  | 4          | 20 |
+| Christian                                                            | 4 (9.4%)                 | 1 (8.4%)    | 5          | 9.2        | 2 (15.5%)  | 0 (0%)     | 2          | 10 |
+| Social habits smoking Current 1 (25.58%)                             | 0 (0%)                   | 11          | 20.8       | 1 (7.69%)  | 0 (0%)     | 1          | 5          |    |
+| Past                                                                 | 8 (18.60%)               | 2 (16.66%)  | 10         | 18.18      | 3 (23%)    | 0 (0%)     | 3          | 15 |
+| None                                                                 | 24 (55.81%)              | 10 (83.33%) | 34         | 61.8       | 9 (69.23%) | 7 (100%)   | 16         | 80 |
+| Drinking Current                                                     | 22 (51.1%)               | 1 (8.33%)   | 23         | 41.81      | 4 (30.7%)  | 0 (0%)     | 4          | 20 |
+| Past                                                                 | 4 (9.3%)                 | 2 (16.66%)  | 6          | 10.9       | 5 (38.4%)  | 1 (14.2%)  | 6          | 30 |
+| None                                                                 | 17 (39.5%)               | 9 (75%)     | 26         | 47.2       | 4 (30.7%)  | 6 (85.7%)  | 10         | 50 |
+
+| Table 2: Prevalence of metabolic and non-metabolic syndrome according to NCEP-ATP III guidelines Marital status Metabolic syndrome Non-metabolic syndrome Male Female Total Percentage Male Female Total Percentage Married 43 (100%) 10 (83.3%) 53 96.4 13 (100%) 7 (100%) 20 100 Unmarried 0 (0%) 2 2 3.6 0 (0%) 0 (0%) 0 0 Physical exercise Severe 1 (2.32%) 0 (0%) 1 2 1 (7.7%) 1 (14.28%) 2 10 Mild to moderate 18 (42%) 1 (8.33%) 19 35 4 (30.77%) 3 (42.86%) 7 10 Sedentary 24 (56%) 11 (91.66%) 35 64 8 (61.5%) 3 (42.8%) 11 55   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+| age, and group Age (years)   | Metabolic syndrome (55)   | Non‑metabolic syndrome (20)   |            |             |         |           |            |            |            |          |
+|------------------------------|---------------------------|-------------------------------|------------|-------------|---------|-----------|------------|------------|------------|----------|
+| Groups                       | Male (43)                 | Female (12)                   | Total (55) | Mean        | P value | Male (13) | Female (7) | Total (20) | Mean       | P value  |
+| ≤18                          | 0 (0%)                    | 0 (0%)                        | 0 (0%)     | ------      | ------- | 0 (0%)    | 0 (0%)     | 0 (0%)     | ------     | -------  |
+| 18-30                        | 2 (4.65%)                 | 0 (0%)                        | 2 (3.63%)  | 29±1.414    | 0.0219  | 0 (0%)    | 1 (14.2%)  | 1 (5%)     | ------     | -------  |
+| 31-40                        | 7 (16.27%)                | 1 (8.33%)                     | 8 (14.6%)  | 37.75±1.20  | 0.0001  | 2 15.3%)  | 1 (14.2%)  | 3 (15%)    | 35±0.10    | 0.0003   |
+| 41-50                        | 10 (23.25%)               | 3 (25%)                       | 13 (23.6%) | 46.77±2.74  | 0.0001  | 4 (30.7%) | 1 (14.2%)  | 5 (25%)    | 46.6±2.30  | 0.0001   |
+| 51-60                        | 13 (30.23%)               | 6 (50%)                       | 19 (34.8%) | 55.58±3.115 | 0.0001  | 5 (38.5%) | 1 (14.2%)  | 6 (30%)    | 56.33±2.94 | 0.0001   |
+| 61-70                        | 9 (20.9%)                 | 2 (16.6%)                     | 11 (20%)   | 65.45±1.8   | 0.0001  | 2 (15.5%) | 3 (42.8%)  | 5 (25%)    | 65.4±0.89  | 0.0001   |
+| 71-80                        | 2 (4.65%)                 | 0 (0%)                        | 2 (3.63%)  | 75±1.414    | 0.0085  | 0 (0%)    | 0 (0%)     | 0 (0%)     | ------     | -------- |
+
+| patients Body mass   | Metabolic syndrome   | Non‑metabolic syndrome   |            |           |         |           |           |          |            |         |
+|----------------------|----------------------|--------------------------|------------|-----------|---------|-----------|-----------|----------|------------|---------|
+| index                | Male                 | Female                   | Total      | Mean ± SD | P value | Male      | Female    | Total    | Mean ± SD  | P value |
+| Obese                | 3 (6.97%)            | 3 (25%)                  | 6 (10.9%)  | 33±2.15   | <0.201  | 0 (0%)    | 0 (0%)    | 0 (0%)   | ‑          | ‑‑      |
+| Overweight           | 19 (44.1%)           | 3 (25%)                  | 22 40%)    | 27±1.3    | <0.201  | 4 (30.7%) | 3 (42.8%) | 7 (35%)  | 26.41±0.99 | <0.0001 |
+| Normal weight        | 19 (44.1%)           | 5 (41.6%)                | 24 (43.6%) | 22.6±1.2  | <0.201  | 8 (61.5%) | 4 (57.4%) | 12 (60%) | 23±1.605   | <0.0001 |
+| Underweight          | 2 (4.65%)            | 1 (8.33%)                | 3 (5.45%)  | 15±1.9    | <0.005  | 1 (7.65%) | 0 (0%)    | 1 (5%)   | ‑          | ‑       |
+
+| according to gender Mean                                                                                | Metabolic syndrome   | Non‑metabolic syndrome   |            |         |        |           |             |         |
+|---------------------------------------------------------------------------------------------------------|----------------------|--------------------------|------------|---------|--------|-----------|-------------|---------|
+| Male                                                                                                    | Female               | Mean ±                   | P value    | Male    | Female | Mean ± SD | P value     |         |
+| SD                                                                                                      |                      |                          |            |         |        |           |             |         |
+| Waist circumference                                                                                     | 92.9                 | 92.95                    | 92.7±10.3  | <0.201  | 87.8   | 82        | 84.9±8.25   | <0.0001 |
+| BMI                                                                                                     | 25                   | 25.09                    | 25±4.004   | <0.0001 | 23.52  | 24.81     | 24.17±2.63  | <0.0001 |
+| Triglycerides                                                                                           | 186.2                | 186                      | 186±76.5   | <0.0001 | 126.94 | 185.82    | 156.8±75.43 | <0.0001 |
+| HDL                                                                                                     | 22.77                | 22.7                     | 23.7±10.1  | <0.0001 | 35.54  | 32.40     | 33.4±15.59  | <0.0001 |
+| SBP                                                                                                     | 130                  | 130                      | 130±13.71  | <0.0001 | 120    | 117.42    | 118±7.95    | <0.0001 |
+| DBP                                                                                                     | 83.93                | 85.83                    | 84.3±7.65  | <0.0001 | 80     | 75.71     | 77.86±5.53  | <0.0001 |
+| FBS                                                                                                     | 109                  | 140.6                    | 116.1±55.4 | <0.0001 | 116.02 | 107.14    | 111.58±34.6 | <0.0001 |
+| Age                                                                                                     | 52.6                 | 53.3                     | 52.8±11.16 | <0.0001 | 51.8   | 50.71     | 51.23±12    | <0.0001 |
+| Duration of diabetes                                                                                    | 64.7                 | 6.75                     | 6.52±97.5  | <0.0001 | 4.6    | 5         | 4.8±47.24   | <0.0001 |
+| BMI-Body mass index, SBP-Systolic blood pressure,DBP- Diastolic blood pressure,FBS- Fasting blood sugar |                      |                          |            |         |        |           |             |         |
+
+| Category                     | Generic drugs  No of drugs  % (Utilization/ prescribed prescribed MS patients   |    |       |
+|------------------------------|---------------------------------------------------------------------------------|----|-------|
+| Anti‑diabetics               | Metformin                                                                       | 80 | 33.2  |
+| Glibenclamide                |                                                                                 |    |       |
+| Vitamins                     | Vitamins                                                                        | 50 | 20.7  |
+| Anti‑ulcer                   | Ranitidine                                                                      | 43 | 17.84 |
+| Antihypertensives            | Enalapril                                                                       | 28 | 11.61 |
+| Amlodipine Atenelol Carvidol |                                                                                 |    |       |
+| Analgesics and               | Paracetamol                                                                     | 12 | 4.9   |
+| antipyretics                 | Aspirin                                                                         |    |       |
+| Anti‑inflammatory            | Ibuprofen                                                                       | 10 | 4.14  |
+| Antibiotics                  | Ciprofloxacin                                                                   | 06 | 2.5   |
+| Amoxicillin                  |                                                                                 |    |       |
+| Anti‑allergy                 | Cetrizine                                                                       | 04 | 1.7   |
+| Hyperlipidemic               | Atorvastatin                                                                    | 04 | 1.7   |
+| Drugs                        | Novostatin                                                                      |    |       |
+| Anti‑angina                  | Isosorbide                                                                      | 02 | 0.8   |
+| drugs                        | mononitrate Isosorbide  di‑nitrate                                              |    |       |
+| Anti‑diarrheal               | Metiranadozole                                                                  | 01 | 0.4   |
+| Anti‑platelet                | Clopidogrel                                                                     | 01 | 0.4   |
+| MS - metabolic syndrome      |                                                                                 |    |       |
+
+versus NMetS patients. Sixty percent of patients with NMetS were normal weight and 35% were overweight. Mean body mass index (BMI) was 25.01 kg/m2 in MetS and 24.165 in NMetS patients. The waist circumference mean was 92.7 ± 10.3 cm in MetS and 84.9 ± 8.25 cm in NMetS patients. However, triglycerides were 186.12 ± 76.5 mg/dl in MetS and 156.82 ± 75.43 mg/dl in NMets patients (P < 0.001). HDL levels were 23.77 ± 10.1 mg/dl in MetS, 
+compared to 33.47 ± 16 mg/dl in NMetS patients. The overall systolic blood pressure was 130 ± 13.7 mm Hg in MetS and 118.17 ± 7.95 mm Hg in NMetS, and diastolic blood pressure was 84.36 ± 7.65 mm Hg in MetS and 77.85 ± 5.53 in NMetS patients. Fasting blood glucose level was 116.12 ± 55.4 mg/dl in MetS and 111.58 ± 34.6 mm Hg in NMetS. Table 5 also depicts the prevalence of MetS and NMetS according to gender and age. Men and women aged between 51 and 60 years suffered more frjom MetS, followed by those from 41 to 50 years of age. 26.66% of total diabetic patients were not suffering from MetS. 73.33% of total diabetic patients were suffering from MetS. Mean age was 52.8 years in MetS and 51.3 years in NMetS. Table 6 presents the top 12 drugs utilized concomitantly in MetS patients with type 2 diabetes; anti‑diabetics were found to be highly used (33.2%), followed by vitamins (20.7%), anti‑ulcer drugs (17.84%), antihypertensives (11.61%), analgesics and antipyretics (4.9%), anti‑inflammatory drugs (4.14%), antibiotics (2.5%), anti‑allergic drugs (1.7%), hyperlipidemic drugs (1.7%), anti‑angina drugs (0.8%), anti‑diarrheal (0.4%), and anti‑platelet drugs (0.4%). Table 7 shows the Indian Diabetes Risk Score (IDRS) which was developed based on multiple logistic regression analysis. In 2007, Mohan et al. reported that IDRS was calculated using four simple variables, namely age, family history, regular exercise, and waist circumference. The individuals were classified as being at high risk (score > 60), moderate risk (score 30–50), and low risk (score < 30) out of total score. 55% of MetS patients had high score, 
+
+| Grades        | Metabolic syndrome (55)   | Non‑metabolic syndrome (20)   |               |                |                |         |
+|---------------|---------------------------|-------------------------------|---------------|----------------|----------------|---------|
+| Male (43) (%) | Female (12) (%)           | Total (55) (%)                | Male (13) (%) | Female (7) (%) | Total (20) (%) |         |
+| High          | 21 (48.9)                 | 09 (75)                       | 30 (54.54)    | 07 (53.8)      | 02 (28.7)      | 09 (45) |
+| Moderate      | 21 (48.9)                 | 03 (25)                       | 24 (43.63)    | 04 (30.76)     | 03 (42.8)      | 07 (35) |
+| Low           | 01 (2.32)                 | 0 (0)                         | 01 (1.8)      | 02 (15.3)      | 02 (28.57)     | 04 (20) |
+
+| Figure 1: Indian diabetes risk score (IDRS) Particulars            | Score   |
+|--------------------------------------------------------------------|---------|
+| Age (years) <35 (reference)                                        | 0       |
+| 35–49                                                              | 20      |
+| >50                                                                | 30      |
+| Abdominal obesity Waist <80 cm (female), <90 cm (male)             | 0       |
+| Waist ≥80–89 cm (female), ≥90–99 cm                                | 10      |
+| Waist ≥90 cm (female), ≥100 cm (male)                              | 20      |
+| Physical activity Vigorous exercise or strenuous (manual) labor at | 0       |
+| home/work Mild to moderate exercise at home/work                   | 20      |
+| No exercise or sedentary activities at home/work                   | 30      |
+| Family history No family history                                   | 0       |
+| Either parent                                                      | 10      |
+| Both parents                                                       | 20      |
+| Minimum score                                                      | 0       |
+| Maximum score                                                      | 100     |
+
+followed by 43.63% of patients with moderate score and 1.8% with low score. High score leads to the development of cardiovascular disease. Score was calculated on the basis of Figure 1. Even patients with NMetS had a high score (45%), followed by moderate 35% and low (20%) scores.
+
+## Discussion
+
+This study analyzed the incidence of the MetS and NMetS 
+among type 2 diabetic patients attending diabetic outpatient clinic at a tertiary care hospital, using the recently introduced definition of the MetS by the NCEP‑ATP III guidelines. The findings of the present study show that the incidence of MetS was significantly higher in males than in females. Contrary to this finding, a study in Iran found that MetS 
+was significantly higher in women than in men. Nizal et al. 
+
+highlighted the very low levels of physical activity in women than in men. Physical activity is a complex behavior which consists of different components, including leisure time transportation, hours of work, and household activities.[12]
+In our study, the MetS was more prevalent in urban areas. This finding is similar to the findings of recent surveys conducted in China and Greece, showing a higher prevalence of the MetS in urban compared to rural residents.[13,14]
+We found energy expenditure to be lower and the MetS to be considerably higher in women compared to men, in urban residents compared to rural residents, and in sedentary individuals compared to active individuals of both genders in urban and rural areas. This finding was similar to the previous reports.[15,16]
+Our findings highlight the very low levels of physical activity in women than in men. Physical activity is a complex behavior which consists of different components, including leisure time transportation, hours of work, and household activities. Physical inactivity could well be a major contributor for women being more prone to obesity problems than men. Hypertension is the most frequently occurring component of MetS when compared to NMetS in subjects with type 2 diabetes, and was found in 72.09% of men and 58.33% of women. This finding is in accordance with a previous report.[17]
+Abdominal obesity was common in South Asians and evident in non‑obese people. They have high percentage of body weight, thick subcutaneous adipose tissue, low muscular mass, insulin resistance, and hyperinsulinemia. This body composition is conducive to the development of MetS. According to a recent study on South Asians, the prevalence of MetS was estimated to be 25.8% in the general population as per international diabetes federation 
+(IDF) definitions.[18] A very recent study has reported the prevalence of MetS in Pakistan, according to a different definition, to be from 18 to 46%, comparable to the data from other South Asian nations.[19] Our finding was similar to that previously reported.[20]
+Moreover, the triglycerides were elevated and HDL levels reduced in patients with MetS when compared to NMetS patients. Hence, our results emphasize on prescription of hyperlipidemic drug to MetS patients to prevent further complications. Our data support the notion that obesity and central obesity in particular is a strong risk factor for type 2 diabetes, a finding that is consistent with the previous reports on various racial/ethnic populations.[21,22] Globally, it has been estimated that approximately 58% of type 2 diabetes is attributable to overweight and obesity, and 90% of type 2 diabetes in western countries is attributed to weight gain.[23] These findings also support that preventive lifestyle interventions should be targeted at lowering both BMI and central obesity in Indian adults. We found that high prevalence of MetS was primarily indicated by central obesity combined with dyslipidemia (low HDL, Hypertension and triglycerides). The high prevalence of hypertriglyceridemia, notably in men, might be due to the sedentary lifestyle. This finding was contrary to the previous report.[11]
+
+## Conclusion
+
+Our results suggest that prevalence of MetS was observed in non‑obese patients and was higher in male patients and positively correlated with aging. Nevertheless, further studies are required to validate the MetS in larger population.
+
+## Acknowledgments
+
+We thank the diabetic patients of MGM hospital who participated in this study. We also thank Dr. M. Satyadev, Superintendent, Mahatma Gandhi Memorial Hospital, for his valuable suggestion and guidance to complete this pilot study.
+
+## References
+
+1.	 Grundy S. 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.
+
+2.	 Manson JE, Skerrett PJ, Greenland P, Vanitallie TB. The escalating pandemics of obesity and sedentary life style. A Call to action for clinicians. Arch Intern Med 2004;164:249‑58.
+
+3.	 AlwanA, King H. Definition, diagnosis and Classification of Diabetes Mellitus and its Complications. Part I: Diagnosis and Classification of Diabetes Mellitus. Report of a WHO consultation; 1999.
+
+4.	 Franks PW, Olsson T. Metabolic syndrome and early death: Getting to the heart of the problem. Hypertension 2007;49:10‑2.
+
+5.	 Weiss R, Dziura J, Burgert TS, Tamborlane WV, Taksali SE,
+Yeckel CW, et al. Obesity and the metabolic syndrome in children and adolescents. N Engl J Med 2004;350:2362‑74.
+
+6.	 Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature 2001;414:782‑7.
+
+7.	 Sinha R, Fisch G, Teague B, Tamborlane WV, Banyas B, Allen K,
+et al. Prevalence of impaired glucose tolerance among children and adolescents with marked obesity. N Engl J Med 2002;346:802‑10.
+
+8.	 Sung RY, Tong PC, Yu CW, Lau PW, Mok GT, Yam MC, et al. 
+
+High prevalence of insulin resistance and metabolic syndrome in overweight/ obese preadolescent Hong Kong Chinese children aged 9-12 years. Diabetes Care 2003;26:250‑1.
+
+9.	 Wei JN, Sung FC, Lin CC, Lin RS, Chiang CC, Chuang LM. National surveillance for type 2 diabetes mellitus in Taiwanese children. JAMA 2003;290:1345‑50.
+
+10.	 Bestermann W, Houston MC, Basile J, Egan B, Ferrario CM,
+Lackland D, et al. Addressing the global cardiovascular risk of hypertension, dyslipidemia, diabetes mellitus, and the metabolic syndrome in the southeastern United States, part II: Treatment recommendations for management of the global cardiovascular risk of hypertension, dyslipidemia, diabetes mellitus, and the metabolic syndrome. Am J Med Sci 2005;329:292‑305.
+
+11.	 Faeh D, Bopp M. Proposed obesity body mass index correction for self‑reported data may not be appropriate. J Epidemiol Community Health 2009;63:863‑4.
+
+12.	 Sarrafzadegan N, Kelishadi R, Baghaei A, Hussein Sadri G,
+Malekafzali H, Mohammadifard N, et al. Metabolic Syndrome: An emerging public health problem in Iranian women: Isfahan healthy Heart Program. Int J Cardiol 2008;13:90‑6.
+
+13.	 Gu D, Reynolds K, WU X, Chen J, Duan X, Reynolds RF, et al. for the inter ASIA collaborate group prevalence of the MetS and overweight among adults in China. Lancet 2005;365:1398‑405.
+
+14.	 Athyros VG, Bouloukos VI, Pehlivanidis AN, Papageorgiou AA,
+Dionysopoulou SG, Symeonidis AN, et al. The prevalence of the MetS in Greece. The MetS Greece multicentre study. Diabetes Obes Metab 2005;7:397‑405.
+
+15.	 Laaksonen DE, Lakka HM, Salomen JT, Niskanen LK. Low levels of leisure‑time physical activity and cardio‑respiratory fitness predict development of the metabolic syndrome. Diabetes Care 2002;25:1612‑8.
+
+16.	 Ekelund U, Brag S, Franka PW. Hennings S, Emms S. Physical activity energy expenditure predicts progression towards the metabolic syndrome independently of aerobic fitness in middle aged healthy Caucasians; the medical research council Ely Study. Diabetes Care 2005;28:1195‑2000.
+
+17.	 Wokoma FS. Diabetes and hypertension in Africa‑an overview. Diab int 2002;12:36‑40.
+
+18.	 Deepa M, Farruq S, Datta M, Deepa R, Mohan V. Prevalence of metabolic syndrome using WHO, ATP III and IDF definitions in Asia Indians; the Chennai Urban Rural Epidemiology Study. Diabetes Metab Res Rev 2007;23:127‑34.
+
+19.	 Basit A, Shera AS. Prevalence of metabolic syndrome in Pakistan.
+
+Metab Syndr Relat Disord 2008;6:171‑5.
+
+20.	 Wang Y, Rimm EB, Stampfer MJ, Willett WC, Hu FB. Comparison of abdominal adiposity and overall obesity in predicting risk of type 2 diabetes among men. Am J Clin Nutr 2005;81:555‑63.
+
+21.	 Hu D, Xie J, FU P, Zhou J, Yu D, Whelton PK, et al. Central rather than overall obesity is related to diabetes in the Chinese population: The interASIA study. Obesity (Silver Spring) 2007;15:2809‑16.
+
+22.	 Visscher TL, Seidell JC. The public health impact of obesity. Annu Rev Public Health 2001;22:355‑75.
+
+23.	 Association CM‑aGoCOTFoILS. The recommendations of the body mass index categories for adult Chinese. Clin J Prev Med 2001;35:349‑50.
+
+How to cite this article: Kumar SV, Nagesh A, Leena M, Shravani G, 
+Chandrasekar V. Incidence of metabolic syndrome and its characteristics of patients attending a diabetic outpatient clinic in a tertiary care hospital. J Nat Sc Biol Med 2013;4:57-62.
+
+Source of Support: Nil. **Conflict of Interest:** None declared.

medical/md/PMC4147655.md

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+  of  Case  5  
+Contents lists available at ScienceDirect
+
+![0_image_1.png](0_image_1.png)
+
+# International Journal Of Surgery Case Reports
+
+journal homepage: www.casereports.com Simultaneous bilateral laparoscopic adrenalectomy for
+
+![0_image_0.png](0_image_0.png)
+
+pheochromocytoma in multiple endocrine neoplasia (MEN) syndrome: Case report with review literature Kaushal Yadava,∗, Ganesh Bakshi b, Gagan Prakashb, Anup Tamhankar a, Kamlesh Vermaa a Department of Surgical Oncology, Tata Memorial Hospital, Mumbai, India b Urooncology Services, Tata Memorial Hospital, Mumbai, India a r t i c l e i n f o Article history:
+Received 4 December 2013 Received in revised form 24 February 2014 Accepted 10 March 2014 Available online 6 June 2014 Keywords:
+Multiple endocrine neoplasia (MEN) 2 syndrome Pheochromocytoma Simultaneous bilateral adrenalectomy Laparoscopic adrenalectomy
+
+## 1. **Introduction**
+
+a b s t r a c t INTRODUCTION: Laparoscopic adrenalectomy has gained favour as a preferred surgical approach in the multiple endocrine neoplasia (MEN) type 2 patients. Currently, there is limited literature on bilateral simultaneous laparoscopic adrenalectomy in MEN 2 syndrome. We reported two cases of bilateral pheochromocytoma associated with MEN 2 syndrome cured by simultaneous bilateral laparoscopic adrenalectomy. PRESENTATION OF CASE: First patient presented with big lips since childhood and episodic abdominal pain. On investigations, he was diagnosed with features of MEN 2B syndrome. Second patient was hypertensive and presented with abdominal pain. On evaluation she had features of MEN 2A syndrome.
+
+DISCUSSION: Minimally invasive approach was preferred in both cases. Bilateral simultaneous adrenalectomies were uneventfully done with acceptable operative time and blood loss with rapid perioperative recovery. These cases highlighted the feasibility of laparoscopic simultaneous bilateral adrenalectomy for pheochromocytomas in MEN 2 syndrome.
+
+CONCLUSION: Laparoscopic simultaneous bilateral adrenalectomy is a safe feasible and preferable technique for pheochromocytomas associated with MEN 2 syndrome.
+
+© 2014 The Authors. Published by Elsevier Ltd. on behalf of Surgical Associates Ltd. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).
+
+experience has been reported in literature with simultaneous bilateral laparoscopic adrenalectomy.We describe two cases of bilateral pheochromocytoma in patients with MEN 2 syndrome treated with simultaneous laparoscopic bilateral adrenalectomy with a review of literature.
+
+## 2. Presentation Of **Case** 2.1. Case 1
+
+45 years, diabetic male with big lips as his identification mark since childhood, presented with episodic upper abdomen pain for 1 year. On workup, plasma free metanephrines and normetanephrines levels were 2905 pg/ml and 2240 pg/ml, respectively. Serum calcitonin was 6243 pg/ml. Contrast enhanced CT
+imaging showed bilateral enlarged adrenal,thyroid nodule and rest normal(Figs. 1–3). Thyroid nodule was diagnosed as medullary carcinoma thyroid on FNAC. His ophthalmologic evaluation detected eyelid neuroma and thickened corneal nerves. His anal manometric evaluation patient was suggestive of Hirschsprung's disease.
+
+He was diagnosed to have MEN 2B syndrome. Patient was optimized with alpha blockers and underwent surgery two weeks later.
+
+The patient was placed in supine position with a 30◦ tilt on the right
+∗ Corresponding author at: Room No. 1002, GJ Block, Tata Memorial Hospital, Mumbai 4000012, India. Tel.: +91 9167242802.
+
+E-mail addresses: kaushalyadavoo7@yahoo.com (K. Yadav),
+gkbakshi1973@gmail.com (G. Bakshi), gagan2311@gmail.com (G. Prakash), tamhankaranup@gmail.com (A. Tamhankar), kamlesh@gmail.com (K. Verma). http://dx.doi.org/10.1016/j.ijscr.2014.03.007 2210-2612/© 2014 The Authors. Published by Elsevier Ltd. on behalf of Surgical Associates Ltd. This is an open access article under the CC BY-NC-SA license
+(http://creativecommons.org/licenses/by-nc-sa/3.0/).
+
+Pheochromocytomas are neoplasms arising from chromaffin cells in the adrenal medulla and produce catecholamines.1 In patients of MEN 2 syndromes approximately 40–50% develop pheochromocytomas, at the age range of 30–40 years and rarely at 5–10 years.2 In MEN 2 syndrome pheochromocytomas are usually benign. They have multifocal medullary lesions and are bilateral in more than 50% of cases.3 Considering these features, minimal invasive surgical approach should be applied here.
+
+Since the first description of pheochromocytoma by Felix Frankel and its first successful surgical management by César Roux and Charles Mayo, the management of pheochromocytoma has evolved greatly.4 Today, laparoscopic adrenalectomy is preferred surgical approach for MEN 2 patients. The conversion rate to an open procedure is less than 10%.5 Although the benefits of unilateral laparoscopic adrenalectomy have been well documented, less
+
+![1_image_1.png](1_image_1.png)
+
+488 K.  et al. /   of  Case  5  
+
+![1_image_0.png](1_image_0.png)
+
+side and pneumoperitoneum was created. Four ports were used, one for camera and one for retracting liver. The peritoneum over the vena cava and in the Morrison space was incised by a hook. The adrenal gland was dissected from inferior vena cava with the help of harmonic ultrasound scalpel and appropriate use of clips. Climbing on this plane, adrenal vein was dissected, hemlock clips applied and cut. Right adrenal was then dissected off the superior pole of kidney and an endobag was used to remove the specimen through lower port. A drain was placed in the Morrison space and the right sided ports were closed. The patient was then tilted 30◦ on the left side. Three ports were used. After pneumoperitoneum was established again, the splenic flexure and colon was reflected to approach left adrenal. Left adrenal vein was identified, secured and cut. The gland was then dissected and specimen delivered through endobag from lower port. Operating time was 5 h, blood loss was 300 cm3. Postoperative period was uneventful. Patient was started on steroid replacement. The blood sugar also normalized. Patient was started on diet on postoperative day 1. Drains were removed on postoperative day 3 and patient discharged. Total thyroidectomy with bilateral neck dissection was done for medullary carcinoma thyroid after one month. The patient is doing well on follow-up.
+
+## 2.2. Case 2
+
+43 years, hypertensive female was having paroxysmal episodes of abdominal pain, headache, giddiness and palpitation since 15 years. Her imaging detected bilateral adrenal enlargement. Her family history revealed thyroid disorder in two of four siblings and early death of elder brother due to hypertension. Hypertension was recorded in both supine and standing position. She was having an interscapular 8 cm × 7 cm lichen amyloidosis which was later
+
+![1_image_2.png](1_image_2.png)
+
+![2_image_1.png](2_image_1.png)
+
+K.  et al. /   of  Case  5   489
+
+![2_image_0.png](2_image_0.png)
+
+Fig. 6. MIBG scan: B/L adrenal uptake.
+
+confirmed by skin biopsy. Her plasma free metanephrines, plasma free normetanephrines, serum calcitonin and serum parathyroid hormone were 923 pg/ml, 4289 pg/ml, 597 pg/ml and 346.7 pg/ml, respectively, all raised. PET-CECT showed bilaterally enlarged adrenals and thyroid nodule (Figs. 4 and 5). MIBG scan showed increased tracer uptake in bilateral adrenal masses and a left para aortic mass (Fig. 6).
+
+Clinical evaluation lead to the diagnosis of MEN 2A. Patient was optimized with alpha blockers. Laparoscopic bilateral adrenalectomy was done along with excision of left para aortic paraganglioma. Operative time was 4 h and blood loss was 300 cm3.
+
+Histopathologic examination confirmed bilateral pheochromocytoma with left paraganglioma. Total thyroidectomy with bilateral neck dissection and excision of enlarged right superior, right inferior and left inferior parathyroid glands was done after two months. Histopathology confirmed medullary carcinoma thyroid and parathyroid hyperplasia. Patient is doing well on followup.
+
+## 3. **Discussion**
+
+The reported frequency of bilateral pheochromocytoma is variable in published reports, ranging from 35% to 80%.6,7 It is also
+
+![2_image_2.png](2_image_2.png)
+
+known that the mutation in the codons 634 and 618 is related to bilaterality.7 Patients suspected to have MEN 2 syndromes should be screened for pheochromocytoma. North American Neuroendocrine Tumour Society (NANETS) recommends that initial testing must include measurements of fractionated metanephrines in plasma, urine, or both, as available.8 Anatomical imaging is most widely used in the initial evaluation of patients as it offers the advantage of low cost, universal availability and easy interpretation. CT scan can be used to localize adrenal tumours > 1 cm and extra adrenal tumours > 2 cm.9 Adrenal tumours should be imaged with either 123 I-MIBG scintigraphy, 18 F-DA PET or 18 F-DOPA PET to detect/exclude multifocal or metastatic disease. Patients are also screened for all associated MEN 2 abnormalities. Usually pheochromocytomas are dealt first. Thyroid medullary carcinoma and other conditions can be addressed later after excision of pheocytochromas.
+
+Although laparoscopic adrenalectomy is preferred therapy for pheochromocytoma, experience with simultaneous laparoscopic bilateral adrenalectomy is limited. Also open bilateral adrenalectomy is associated with a high rate of surgical complications including intraoperative and postoperative bleeding (22%), pancreatic fistula, incisional hernia (10%), and wound healing impairment
+(13.5%) as well as general complications. Bilateral laparoscopic adrenalectomy is feasible and seems to be associated with a lower operative risk.5,10 However the reported rate of complications is higher than for unilateral laparoscopic adrenalectomy.11 In laparoscopic approach as compared with the open one, there is less manipulation of the adrenal tumour.
+
+Case series has been reported for unilateral laparoscopic adrenalectomy in pheochromocytoma associated with MEN syndrome; but bilateral simultaneous laparoscopic adrenalectomy sparsely reported in literature. Absence of intraoperative and postoperative complications in our cases contributes to lower rate of complications for this surgical procedure. Surgical approaches described for laparoscopic synchronous bilateral adrenalectomy include; "anterior" transperitoneal approach, lateral transperitoneal or retroperitoneal approach and the "posterior" retroperitoneal approach.12,13 No randomized trial has been published yet to confirm superiority of one approach over another. We used transperitoneal laparoscopic approach as it allows proper identification of anatomic landmarks, especially early ligation of the suprarenal vein and thus avoid vena caval bleeding that constitutes the most dangerous complication of the entire procedure.14 This approach also saves time as it does not require a changing in the patient position but only a 30◦ lateral tilt achievable by operating table. Laparoscopic lateral approach has been frequently described 490 K.  et al. /   of  Case  5  
+for this kind of operation. It makes mobilization of abdominal organs less difficult due to effect of gravity upon organs and when performed with retroperitoneal approach provides direct access to the adrenal gland. Disadvantages of the lateral approach include the time spent in changing patient's position and longer learning curve. Even the posterior approach has been described for bilateral laparoscopic adrenalectomy. It does not require the time for repositioning of the patient as it allows a direct access to the adrenal glands vessels, and itis useful in case of patient with multiple intraabdominal adhesions. Disadvantages of this approach are the very narrow operation field, which allows operating only small tumour, a difficult vascular control and inability to explore intraperitoneal structures. Our operating time, similarly to those reported literature was fairly long. This time duration was probably due to the fact that our experience, like most of the cases reported in literature, was the initial experience dealing with such cases. Postoperatively our patients were given steroid replacement. These two cases highlight the safety, feasibility, early patient recovery, patient comfort and good outcome in doing simultaneous bilateral adrenalectomy.
+
+In future minimal invasive simultaneous bilateral adrenalectomy should be surgery of choice in MEN 2 patients.
+
+## Conflict Of **Interest**
+
+| None.   |
+|---------|
+
+| Funding   |
+|-----------|
+
+| None.   |
+|---------|
+
+## Consent
+
+Due consent was taken for video and photo recording and case publication for academic purpose. Consent can be produced at any time required.
+
+## Author **Contributions**
+
+DMG urooncology staff contributed for data collection and analysis.
+
+## References
+
+1. Zelinka T, Eisenhofer G, Pacak K. Pheochromocytoma as a catecholamine producing tumor: implications for clinical practice. Stress Int J Biol Stress 2007;10(2):195.
+
+2. Brandi ML, Gagel RF, Angeli A, et al. Guidelines for diagnosis and therapy of MEN
+type 1 and type 2. J Clin Endocrinol Metab 2001;86:5658–71.
+
+3. Lenders JW, Eisenhofer G, Mannelli M, et al. Pheochromocytoma. Lancet 2005;366:665–75.
+
+4. Welbourn RB. Early surgical history of phaeochromocytoma. Br J Surg 1987;74:594–6.
+
+5. Shen WT, Grogan R, Vriens M, et al. One hundred two patients with pheochromocytoma treated at a single institution since the introduction of laparoscopic adrenalectomy. Arch Surg 2010;145:893–7.
+
+6. Howe JR, Norton JA, Wells Jr SA. Prevalence of pheochromocytoma and hyperparathyroidism in multiple endocrine neoplasia type 2A: results of long-term follow-up. Surgery 1993;114:1070–7.
+
+7. Machens A, Brauckhoff M, Holzhausen H, et al. Codonspecific development of pheochromocytoma in multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 2005;90:3999–4003.
+
+8. The NANETS consensus guideline for the diagnosis and management of neuroendocrine tumors: pheochromocytoma; paraganglioma & medullary thyroid cancer. Pancreas 2010;39(6):775–83.
+
+9. Ilias I, Pacak K. Current approach and recommended algorithm for the diagosis and localization of pheochromocytoma. J Clin Endocrinol Metab 2004;89:479–91.
+
+10. Rodriguez JM, Balsalobre M, Ponce JL, Ríos A, Torregrosa NM, Tebar J, et al.
+
+Pheochromocytoma in MEN 2A syndrome. Study of 54 patients. World J Surg 2008;32:2520–6.
+
+11. O'Riordain DS, Farley DR, Young WF, Grant JS, Van Heerden JA. Long term outcome of bilateral adrenalectomy in patients with Cushing's syndrome. Surgery 1994;116:1088–93.
+
+12. Hsu TH, Gill IS. Bilateral laparoscopic adrenalectomy: retroperitoneal and transperitoneal approaches. Urology 2002;59(2):184–9.
+
+13. Bonjer HJ, Sorm V, Berends FJ, Kaziemer G, Steyerberg EW, de Herder WW, et al.
+
+Endoscopic retroperitoneal adrenalectomy: lessons learned from 111 consecutive cases. Ann Surg 2000;232:796–803.
+
+14. Fernandez-Cruz L, Saenz A, Benarroch G, Astudillo E, Taura P, Sabater L.
+
+Laparoscopic unilateral and bilateral adrenalectomy for Cushing's syndrome. Transperitoneal and retroperitoneal approaches. Ann Surg 1996;224(6):727–36.
+
+Open Access This article is published Open Access at sciencedirect.com. It is distributed under the IJSCR Supplemental terms and conditions, which permits unrestricted non commercial use, distribution, and reproduction in any medium, provided the original authors and source are credited.

medical/md/PMC4307663.md

@@ -0,0 +1,271 @@
+# Optogenetics For Retinal Disorders
+
+## Bradley S. Henriksen, Bs; Robert E. Marc, Phd; Paul S. Bernstein, Md
+
+Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, Salt Lake City, Utah, USA
+
+## Abstract
+
+Optogenetics is the use of genetic methods combined with optical technology to achieve gain or loss of function within neuronal circuits. The field of optogenetics has been rapidly expanding in efforts to restore visual function to blinding diseases such as retinitis pigmentosa (RP). Most work in the field includes a group of light‑sensitive retinaldehyde‑binding proteins known as opsins. Opsins couple photon absorption to molecular signaling chains that control cellular ion currents. Targeting of opsin genes to surviving retinal cells is fundamental to the success of optogenetic therapy. Viral delivery, primarily adeno‑associated virus, using intravitreal injection for inner retinal cells and subretinal injection for outer retinal cells, has proven successful in many models. Challenges in bioengineering remain for optogenetics including relative insensitivity of opsins to physiologic light levels of stimulation and difficulty with viral delivery in primate models. However, targeting optogenetic therapy may present an even greater challenge. Neural and glial remodeling seen in advanced stages of RP result in reorganization of remaining neural retina, and optogenetic therapy may not yield functional results. Remodeling also poses a challenge to the selection of cellular targets, with bipolar, amacrine and ganglion cells all playing distinct physiologic roles, and affected by remodeling differently. Although optogenetics has drawn closer to clinical utility, advances in opsin engineering, therapeutic targeting and ultimately in molecular inhibition of remodeling will play critical roles in the continued clinical advancement of optogenetic therapy.
+
+Keywords: Opsins; Optogenetics; Retinitis Pigmentosa J Ophthalmic Vis Res 2014; 9 (3): 374-382.
+
+## Introduction
+
+Optogenetics is the use of genetic methods combined with optical technology to achieve gain or loss of function within neuronal circuits.[1,2] The idea of activating selective neuronal circuits or cells with light was introduced in the 1970s with the discovery of bacteriorhodopsin, which functioned as an ion pump activated by visible light.[3‑5] Since then, advances in discovery of optically responsive proteins and viral delivery of genetic material in animal models have made optogenetics a rapidly evolving field. The validity of optogenetics was widely demonstrated in various organisms, but it was not until 2002 that successful studies were Correspondence to:
+Paul S. Bernstein, MD. Moran Eye Center, University of Utah School of Medicine, 65 Mario Capecchi Drive, Salt Lake City, Utah 84132, USA. E‑mail: paul.bernstein@hsc.utah.edu Received: 15-03-2014 Accepted: 10-05-2014 reported in vertebrate models. Zemelman et al have reported the simultaneous expression of *Drosophila* arrestin‑2, photoreversible *Drosophila* rhodopsin, and the alpha subunit of *Drosophila* photoreceptor G‑protein sensitized cultured hippocampal neurons to light stimulation.[6] However, this approach used a difficult triple transfection and yielded only moderate light‑sensitivity.[7] These drawbacks led to exploration for simpler and more effective signaling proteins, and the field of optogenetics has rapidly expanded. One major objective of optogenetics is the restoration of visual function in blinding diseases such as retinitis pigmentosa (RP).
+
+![0_image_0.png](0_image_0.png)
+
+www.jovr.org
+Access this article online
+
+| www.jovr.org DOI: 10.4103/2008-322X.143379   |
+|----------------------------------------------|
+
+## Defining Opsins
+
+Most of optogenetics is based on a group of light‑sensitive 
+
+![1_image_0.png](1_image_0.png) retinaldehyde‑binding proteins known generically as opsins. The strategy of using opsins as retinoid‑based light sensors for optogenetics technologies is schematized in Figure 1. The best known opsins are those of vertebrate retinal photoreceptors that couple photon absorption to molecular signaling chains that ultimately control cellular ion currents. Opsins are categorized as Type 1, used by prokaryotes for phototaxis or coupling light energy to cellular functions, and Type 2 found in metazoans involved in higher order processes such as vision and phase setting of circadian rhythms.[8] Types 1 and 2 opsins do not share significant homology,[9] but both use retinaldehyde variants as their chromophores. 
+
+In this setting, the retinaldehyde chromophore in vertebrate photoreceptors is the light sensor and upon photon absorption, isomerizes from the 11‑cis to the all‑*trans* configuration to induce conformational changes in opsin.[10]
+Rhodopsin is the visual pigment of rod photoreceptor cells in marine fishes and terrestrial vertebrates and is based on the 11‑cis isomer of retinaldehyde. The polyene chain 11‑cis retinaldehyde is covalently linked at C15 to the ε‑amino N of lysine 296 in the opsin chain. The insertion of retinaldehyde into the binding pocket shifts the absorption band of 11‑cis retinaldehyde from the ultraviolet (UV) to the visible blue-green spectrum. In mammals, the cone opsins expressed by the short‑wave system 1 (SWS1) gene generate blue‑shifted pigments, whereas the long‑wave system (LWS) gene generates a green shifted pigment in nonprimates, and both green and red shifted pigments in primates.
+
+Visual opsins are all G‑protein coupled receptors (GPCRs) characterized by 7 transmembrane domains (TMDs). The bound 11‑cis retinaldehyde acts as an inverse agonist that prevents activation of its cognate G‑protein transducin in the dark state.[11,12] In rods and cones, photon capture activates a multiprotein transduction cascade and effects a drop in outer segment cyclic guanosine monophosphate (cGMP), which leads to the closure of cyclic nucleotide‑gated cation channels as cGMP dissociates. This depresses an ongoing inward "dark current" and is observed macroscopically as a hyperpolarization of the photoreceptor. Like excitatory neurons in the brain, photoreceptors use glutamic acid as their neurotransmitter[13] via Ca2+‑dependent vesicle fusion, releasing high rates of glutamate when they are depolarized (dark) and reducing glutamate release upon hyperpolarization (light).
+
+As an optogenetics tool, rhodopsin is moderately fast and very sensitive, but it requires at least four transduction proteins to couple photon capture to membrane potential, as well as a ready supply of 11‑cis retinaldehyde, adenosine triphosphate and guanosine triphosphate to run transduction reactions. Further, rhodopsin opens channels in the dark and closes them in the light, which poses energetic problems in keeping channels closed without depleting these small molecules. It is still possible that the signaling cascade could be re‑engineered to exploit rhodopsin's high extinction coefficient and amplification.
+
+Melanopsin is an opsin expressed in a subset of mammalian ganglion cells known as intrinsically photosensitive ganglion cells (ipGCs). Signaling from ipGCs appears to influence circadian phase setting, pupillary constriction at high brightness, and sleep regulation.[14] ipGCs may also trigger photophobia in migraine.[15] ipGCs also have input from rod and cone pathways, and hence the differential role of melanopsin in their full range of functions remains unclear. Melanopsin also uses 11‑cis retinaldehyde as a primary chromophore, absorbs blue‑green light,[16] and acts as a GPCR to drive cell signaling likely through a Gq
+/11 G‑protein.[17] Unlike rhodopsin, photoactivated melanopsin triggers depolarization of ganglion cell transient receptor potential‑like cation channels, probably through phospholipase C signaling, although the exact mechanism remains a matter of debate.[18] Thus, melanopsin is appropriate for optogenetics by virtue of its polarity. The drawbacks to using this opsin for neural signaling include its very slow speed and requirement for native transducers. Even so, melanopsin has successfully been exploited to restore light responsivity in rd1 mice,[19] a model of human autosomal recessive RP. Thus ectopic expression of melanopsin in any ganglion cell appears effective in activating light responses, implying that all retinal neurons may have intrinsic access to 11‑cis retinaldehyde and possess the essential transduction chain used by this opsin. However, melanopsin triggers extremely sustained, persistent excitations making it ineffective for signaling at speeds necessary for simple tasks like walking.
+
+Halorhodopsin, first described by Matsuno‑Yagi and Mukohata in 1977, is a 7 TMD retinaldehyde‑binding protein with a λmax of 570 nm that acts as a light‑gated anion‑selective pump in a group of the *Archaea* known as halobacteria.[20] It is not a GPCR. A variant from Natronomonas phoraonis (NpHR) has been of particular interest due to its fast reactivity.[21] It has successfully been expressed in mouse retinal ganglion cells[22,23] and mouse and human cones ex *vivo*.
+
+[24] Limits to application of NpHR, include its lack of sensitivity, inability to excite cells and tendency to inactivate in mammalian cells.
+
+Channelrhodopsins (ChRs) are a family of cation‑channel forming algal 7 TMD opsins that bind all‑*trans* retinaldehyde via a Schiff's‑base linkage, as do vertebrate visual opsins. ChRs absorb blue‑green light (λmax 470 nm) and use photoisomerization of all‑*trans* retinaldehyde to the 13‑cis bent form to gate nonselective cation channels generating a depolarizing inward current in most cells. The rhodopsin‑like functions of ChR were initially discovered in a range of phototactic motile unicellular organisms.[25] And ultimately ChR1 and ChR2 (similar proteins with different activation behaviors) were described in *Chlamydomonasreinhardtii*[26]
+and expressed in nonneuronal mammalian cell lines by Nagel et al.[27] Later experiments reported activation of spike potentials in response to blue light stimulation in mammalian neurons expressing ChR2.[28,29] Similarly, in mouse models, control of action potential firing in response to blue light was achieved in hippocampal slices following stereotactic injection of ChR2‑encoding viruses.[30] Several successful studies have now exploited ChR2 expression in retinal bipolar cells to drive upstream signaling in mouse models of RP.[31,32] While ChR2 and several engineered variants represent strong proof of principle for optogenetics therapies, they lack sufficient photon capture efficacy to be used under normal lighting conditions.
+
+Finally, a novel fusion of photochemistry and genetics removes the necessity of using opsins altogether and instead exploits naturally expressed, high‑conductance ion channels charged with photosensitive azobenzenes. Caporale et al created a modified glutamate receptor LiGluR (based on ionotropic glutamate receptor iGluR6) modified with an exposed cysteine for cross linking to a maleimide‑azobenzene‑glutamate (MAG) 
+adduct.[33] After expression through adeno‑associated virus (AAV) delivery, intravitreal injection of MAG activates a photosensitive glutamate receptor. Photoactivation at 380 nm allowed the glutamate end‑link to occupy the binding site in LiGluR and activate the cation channel, whereas photoactivation at 500 nm withdrew the link. Like ChR2, expression of this protein after intravitreal injection of AAV‑LiGluR 
+followed MAG linking restored visual responsiveness as assayed by a number of physiological techniques. However, again, the world is not a set of UV‑green events, and some modification of this scheme is necessary to render it practical for human vision.
+
+## Viral Delivery
+
+Optogenetic therapy for degenerative retinal diseases hinges on successful targeting of opsin or other genes to surviving retinal cells. Viral vectors are the most widely validated method for opsin delivery to neuronal or other tissue cells, with vector administration via subretinal injection targeting outer retinal cells and intravitreal injection targeting inner retinal cells.[34] Over thirty papers in the past 5 years from the Hauswirth group have demonstrated the efficacy of AAV‑based gene delivery. Lentiviral and adenoviral models have been shown to express opsins in mouse, rat, and primate tissues.[35] The advantages to using viral vectors (as opposed to nonbiologic methods such as nanoparticles or gene‑gun approaches) include rapid delivery, high infectivity, and potential cell‑specificity based on pseudotyped AAV variants with different cell tropisms. The drawback to AAV is that promoter sequences must be relatively small and cell‑specific, as they must drive strong protein expression.[36] However, growing understanding of pseudotyped AAV efficacies[37] and coupling a wider‑range of cell‑specific transcriptional markers to high‑efficiency expression systems are rapidly easing these concerns.[38]
+A technical approach to overcome some of these challenges and optimize opsin performance in model systems includes viral delivery of opsins into Cre recombinase transgenic mice, allowing tighter control of gene expression in the research setting.[35,36] Indeed, there has been an explosion of studies that prove the efficacy of Cre‑driver lines for cell‑specific targeting.[39] Advances in viral delivery have allowed for light‑induced changes and activation of axons distant to cell bodies which have been virally infected to express opsin proteins. This method of distant activation has been described as projection‑based control.[40,41] However, these approaches are not viable clinical interventions, and direct AAV 
+targeting still remains the best proof‑of‑principle approach.
+
+Delivery of and signaling by opsins in primate models have proven to be more challenging. Lentiviral delivery of ChR2 to cortical neurons of macaques without observed behavioral response has previously been reported,[42,43] but recently Lee et al successfully delivered ChR2 to parvalbulin + inhibitory neurons in mouse V1 cortex and directly modulated behavior, suggesting that primate brain targeting is feasible.[44]
+Delivery of opsins with confirmed function has also been reported in primate stem cell neurons[45] and ex *vivo* retina.[24] However, targeting primate retina in *vivo* remains a challenge for AAV‑based methods, as intravitreal delivery is compromised by poor transport through the inner limiting membrane.[46] The cost of scaling viral production to deliver the large particle numbers necessary for the primate eye has also been part of the problem. While recent advances in animal models have proven valuable in understanding the role and function of opsins within neuronal circuits, challenges remain in clinical application of optogenetic therapy in humans.
+
+## Challenges In Clinical Application Of Optogentics: Remodeling
+
+As noted above, characteristics of the currently identified opsins make their use in clinical settings a challenge. 
+
+ChRs, halorhodopsins and azobenzene modified targets remain so insensitive that they require extreme, nonphysiological stimulus fluxes, and the dependence of some signaling regimes on UV stimulation is clearly problematic. Melanopsin is similar to invertebrate rhodopsins in chomophore associations and use of Gq/11‑protein signaling, but its response is so sustained as to prevent signaling of environmental cues. The use of rhodopsin itself is complicated by the requirement of intrinsic transduction pathways (ON bipolar cells), but new breakthroughs in this system are in the offing. In addition to these challenging properties of opsins, AAV 
+delivery to primates has proven difficult.[47]
+These are largely bioengineering challenges that should be rapidly soluble by designing higher sensitivity opsins, improving AAV targeting and designing optimized expression systems. Given this, does anything prevent rapidly deploying optogenetics in human retinal degenerations such a RP or even atrophic age‑related macular degeneration? The answer is yes: Retinal remodeling.
+
+Retinal remodeling is by far the most serious barrier to treating RP and other retinal degenerations with optogenetics, prosthetics, cell therapies or late‑stage gene therapies.[48‑52] RP is a collection of progressive diseases that continue to alter the neural retina long after photoreceptors have died. Every form of RP transitions through three phases that strongly influence the function of the surviving neural retina.[48]
+In Phase I, stressed photoreceptor cells rapidly change their synaptic associations with downstream retinal bipolar cells. In Phase II, photoreceptor cell death peaks, leading to the decimation of the outer nuclear layer truncation of bipolar cell dendrites, and the formation of the distal glial seal that completely separates the remnant neural retina from the surviving RP and choroid. This glial seal is formed by remnant Müller cell microvilli cross‑linked by homocellular intermediate junctions and is not a glial scar like those formed by GFAP + astrocytes in damaged neuropil of the central nervous system. In cone sparing forms of RP near the macula, surviving rod bipolar cells reprogram their signaling from their classical mGluR6‑based ON 
+polarity to an iGluR‑based OFF polarity.[50] Phase III is a persistent state of neural and glial remodeling, with progressive cell death, aberrant neuritogenesis, rewiring of all classes of neurons including the formation of pathologic synaptic microneuromas, pathologic neuronal migration of ganglion and amacrine cells to distal retina, and bipolar cell and amacrine cell migration to proximal retina. Further, retinal pigment epithelial cells begin to invade the neural retina, often descending completely to the inner limiting membrane. This generates the appearance of sharply delineated bone spicules. When this presentation is seen in ophthalmoscopy [Figure 2], it means that the retina is already in a state of advanced Phase III remodeling [Figure 3], and optogenetics or any other interventions will be largely ineffective. Figure 3b shows the effect of remodeling in a human retina, including absence of ganglion cells, virtually complete depletion of bipolar cells, and severe reorganization of the remnant neural retina through neuronal migration and glial hypertrophy. Figure 3c demonstrates rod bipolar cell reprogramming in human cone‑sparing RP. Importantly, the spared cones often lack opsin expression, but retain some synaptic connectivity. Finally, Phase III Müller cells begin to chaotically reprogram into varied states of altered glutamine metabolism, implying that glial support of the essential glutamate cycle may be compromised.[53]
+A major defect of virtually all animal research models on optogenetics is the use of early stages in retinal degeneration before significant remodeling begins. For example, Lagali et al show successful ChR2 expression in 
+
+![4_image_0.png](4_image_0.png)
+
+ON bipolar cells that still have dendrites.[31] This occurs only in Phase I animals. Ultimately, the expression of mGluR6 in ON bipolar cells in rd1 mice is strongly depressed in Phase III,[48] and Phase III interventions based on that promoter may be ineffective. The key question in the rd1 mouse is whether bipolar cell expression of ChR2 can be achieved in animals that are 200 postnatal day (PND) of age or older; or over 300 PND in P23H rhodopsin mutant models; or 600 PND in the RCS rat, as these are faithful mimics of human Phase III RP. No one has yet achieved visual function restoration in a Phase III retina.
+
+Finally, identifying proper cellular targets has also been challenging. Should light sensing genes be inserted into bipolar cells, amacrine cells, or ganglion cells, and if so, which ones? Figure 4 illustrates the global organization of a normal mammalian retina. The flow of signals in the retina can be thought of as a four‑layer model with rods, SWS1‑cones (blue cones) and LWS cones (red or green cones) as layer 1; bipolar cells as layer 2; amacrine cell networks that aggregate and tune bipolar cell signals as layer 3; and ganglion cells that collect the amacrine cell and bipolar cell signals as layer 4. Finally, Type AII amacrine cells are unique to mammals and form a hub that aggregates and distributes signals for all types of ganglion cells except primate midget pathways. We refer to this AII amacrine cell hub function as mimicking layer "2.5" as it is interspersed between bipolar cells (BCs) and all other neurons. In RP [Figure 5] the photoreceptor layer 1 is ablated. Assuming that all 
+
+![4_image_1.png](4_image_1.png)
+
+![5_image_0.png](5_image_0.png)
+
+networks in the neural retina remain normal (which is not true), picking a target network is difficult. If we choose to target ON bipolar cells [Figure 5a] through mGluR6 promoter or enhancer sequences, we must successfully target multiple varieties of ON cells because they have the best chance of appropriately driving retinal ganglion cells. The same is true of the AII amacrine cell, except it would be simpler to target if we knew the right promoter, which we do not. Targeting bipolar cells may be difficult in late‑stage because they remodel rapidly and die slowly in most retinal degenerations. While ganglion cells survive the longest, along with some gamma-aminobutyric-acid-ergic (GABA-ergic) 
+amacrine cells,[48,49] they require networks to generate appropriate visual signals and drive central pathways with appropriate timing. Thus, the expression of the same opsin in every ganglion cell may create a corrupted visual experience [Figure 5b] even if it restores crude light sensibility. If we can discover transcriptional controls for human foveal midget ganglion cells, it may be possible to target them specifically. Finally, the reality of remodeling is that many target neurons may die, and the remaining networks will become scrambled in ways that may render functional vision via optogenetics unlikely [Figure 6]. It appears that late‑stage remodeling is not slowed by interventions such as subretinal transplantation of normal fetal retina into transgenic rats early in life with advanced retinal degenerations.[54]
+Thus, it is unlikely that even optogenetics signaling will attenuate remodeling, since processes like pathologic de novo neuritogenesis in retinal degenerations depend on retinoic acid signaling rather than network activity.[55]
+
+![6_image_0.png](6_image_0.png)
+
+## Otlook
+
+The field of optogenetics has grown dramatically from its conception in the 1970s. With the identification of diverse opsins from unicellular organisms, the emergence of largely safe and effective viral vectors for delivery of genetic material and the development of numerous transgenic animal models, optogenetics has grown closer to clinical utility. Independent advances in opsin engineering; [56] viral packing, targeting and expression; [37] and ultimately, molecular control of remodeling [55] are all keys to eventual clinical application of optogenetic therapy for degenerative retinal diseases. It is possible that optogenetics therapy will have to be combined with some form of high-brightness video display to overcome intrinsic insensitivity of existing opsins. Direct comparisons of different cellular targets in large-eyed models of human retinal degenerations[51]
+and the possible development of nonhuman primate models of RP will also play critical roles in preclinical development of optogenetics. Ultimately, if successful, optogenetics therapies will represent one of the crowning achievements of modern genetics, neurobiology, and photobiology.
+
+## Acknowledgments
+
+This work was supported by an unrestricted grant to the Moran Eye Center, University of Utah School of Medicine by Research to Prevent Blindness.
+
+![7_image_0.png](7_image_0.png)
+
+Figure 6. Remodeling severely scrambles networks so that successful ChR2 expression may yield unexpected or fictive responses from surviving retinal cells.
+
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+Optogenetics for Retinal Disorders. J Ophthalmic Vis Res 2014;9:374-82.
+
+Source of Support: Nil. **Conflict of Interest**: None declared.

medical/md/PMC4468769.md

@@ -0,0 +1,218 @@
+RESEARCH ARTICLE
+
+![0_image_0.png](0_image_0.png)
+
+# Cx3 Chemokine Receptor 1 Deficiency Leads To Reduced Dendritic Complexity And Delayed Maturation Of Newborn Neurons In The Adult Mouse Hippocampus
+
+Feng Xiao, Jun-mei Xu, Xing-hua Jiang* Department of Anesthesiology, the Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
+
+| *Correspondence to: Xing-hua Jiang, M.D.,  Jiangxh1997@aliyun.com. doi:10.4103/1673-5374.156979  http://www.nrronline.org/ Accepted: 2015-02-16   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------|
+
+## Abstract
+
+Previous studies have shown that microglia impact the proliferation and differentiation of neurons during hippocampal neurogenesis via the fractalkine/CX3 chemokine receptor 1 (CX3CR1) 
+signaling pathway. However, whether microglia can influence the maturation and dendritic growth of newborn neurons during hippocampal neurogenesis remains unclear. In the present study, we found that the number of doublecortin-positive cells in the hippocampus was decreased, and the dendritic length and number of intersections in newborn neurons in the hippocampus were reduced in transgenic adult mice with CX3CR1 deficiency (CX3CR1*GFP/GFP*). Furthermore, after experimental seizures were induced with kainic acid in these CX3CR1-deficient mice, the expression of c-fos, a marker of neuronal activity, was reduced compared with wild-type mice. 
+
+Collectively, the experimental findings indicate that the functional maturation of newborn neurons during hippocampal neurogenesis in adult mice is delayed by CX3CR1 deficiency.
+
+Key Words: nerve regeneration; fractalkine; CX3 chemokine receptor 1; neuronal maturation; dendrites; doublecortin; synaptic maturation; newborn neurons; neural regeneration Xiao F, Xu JM, Jiang XH (2015) CX3 chemokine receptor 1 deficiency leads to reduced dendritic complexity and delayed maturation of newborn neurons in the adult mouse hippocampus. Neural Regen Res 10(5):772-777.
+
+## Introduction
+
+Microglia are the resident immune cells in the central nervous system and act as macrophages (Aloisi, 2001). Microglia can rapidly respond to homeostatic disturbances by secreting inflammatory molecules (Hu et al., 1995; Inoue, 2006) 
+and neurotrophic factors (Batchelor et al., 1999; O'Donnell et al., 2002; Nakajima and Kohsaka, 2004). Microglia are restrained by numerous microenvironmental factors, many of which are produced by neurons (Neumann, 2001). Fractalkine is a chemokine that is constitutively expressed by healthy neurons and functions as a neuroimmune regulatory protein. By binding to its receptor on microglia, CX3 chemokine receptor 1 (CX3CR1), fractalkine inhibits microglial activity under inflammatory conditions (Harrison et al., 1998; Ransohoff et al., 2007). In the brain, CX3CR1 is primarily expressed by microglia (Harrison et al., 1998). Only a few types of neurons express CX3CR1. Consequently, targeting fractalkine/CX3CR1 signaling has been used to modulate the neurotoxicity of microglia in diverse models of neurological disorders, such as neuropathic pain (Sun et al., 2007), 
+age-related macular degeneration (Combadière et al., 2007), peripheral lipopolysaccharide injection-induced intracranial inflammation, Parkinson's disease and amyotrophic lateral sclerosis (Cardona et al., 2006). Accumulating evidence indicates a major role of fractalkine/CX3CR1 signaling in the central nervous system. Bachstetter et al. (2011) showed that the functional disruption of CX3CR1 in CX3CR1*GFP/GFP* mice 772 results in the impaired proliferation and differentiation of neurons during hippocampal neurogenesis. Furthermore, another study from the same laboratory showed that the impaired hippocampal neurogenesis resulted in cognitive impairments, including behavioral deficits in the Morris water maze and contextual fear conditioning tests (Rogers et al., 2011). Interestingly, CX3CR1 signaling in microglial cells was shown to be necessary for the survival of layer V cortical neurons during development (Ueno et al., 2013). Additionally, microglia engulf synaptic material and play a major role in synaptic pruning. When not challenged by inflammatory factors, microglia play a critical role in synaptic maturation in the central nervous system (Paolicelli et al., 2011). These and other studies have shown that, in the intact central nervous system, microglia in the hippocampus impact the proliferation and differentiation of neurons during hippocampal neurogenesis, and consequently affect cognitive functioning. However, whether fractalkine/CX3CR1 signaling plays a role in the synaptic maturation of newborn neurons during hippocampal neurogenesis remains unclear. Therefore, in the present study, we investigated the role of fractalkine/CX3CR1 signaling during hippocampal neurogenesis.
+
+## Materials And Methods
+
+Animals CX3CR1-deficient (CX3CR1*GFP/GFP*) mice, backcrossed to the C57BL/6 background for greater than 10 generations were provided by JAX Laboratories (Bar Harbor, ME, 
+USA). Colonies of the CX3CR1+/GFP and CX3CR1*GFP/GFP*
+mice were maintained at Central South University (China). 
+
+In CX3CR1+/GFP and CX3CR1GFP/GFP mice, the receptor was knocked out and replaced with the GFP gene. Hence, in CX3CR1+/GFP mice, CX3CR1 was partially inactivated, while in CX3CR1*GFP/GFP* mice, the receptor was completely inactivated (Jung et al., 2000). Clean 12-week-old male CX3CR1+/GFP and CX3CR1*GFP/GFP* littermates and 12-weekold male C57BL/6 mice, of specific pathogen-free grade, were used in the experiments. Animals were correspondingly grouped into CX3CR1+/GFP, CX3CR1*GFP/GFP* and wildtype groups. Four animals in each group were used for the counting of doublecortin (DCX)-positive cells, and another four animals in each group were used for dendritic analysis. Three animals in each group were selected at each time point for cell maturation analysis. The protocols were approved by the Institutional Animal Care Committee of Central South University in China.
+
+## Sample Collection And Tissue Processing
+
+All animals were anesthetized by peritoneal injection of an overdose of 2% sodium pentobarbital and prefixed with 4% 
+paraformaldehyde transcardially. All the brains were postfixed for 48 hours in 4% paraformaldehyde and dehydrated in 30% sucrose solution until they sank to the bottom of the vial. Serial coronal frozen sections (40-µm thick) were cut on a freezing microtome (Norton, Trenton, NJ, USA) and prepared for immunohistochemistry.
+
+## Immunohistochemistry For Dcx-Positive Cells
+
+In the dentate gyrus, DCX is exclusively expressed in immature neurons from postnatal 1 day to 4 weeks (Couillard-Despres et al., 2005), and thus has been widely used as a marker of immature neurons, and it reliably reflects the level of dendritic growth in these cells. Brain sections were blocked with a solution containing 0.01 M PBS, 0.1% Triton X-100 and 10% normal goat serum for 30 minutes, and subsequently incubated at 4°C overnight with rabbit anti-DCX polyclonal antibody (1:500; Cell Signaling Technology, Beverly, MA, USA). After thorough washes, sections were then incubated with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA, USA) and avidin-biotin complex 
+(1:200; Vector Laboratories) at room temperature for 60 minutes. Sections were then reacted with diaminobenzidine 
+(Sigma, St. Louis, MO, USA) and 0.1% H2O2 for 10 minutes. 
+
+The sections were dehydrated in graded alcohol and cleared in toluene, and then coverslipped. Sections were observed using an Olympus CHBS light microscope (New York Microscope Company Inc., New York, NY, USA).
+
+## Immunohistochemistry For The Maturation Of Newborn Neurons
+
+The maturation and activation marker expression of newborn neurons were assessed using triple fluorescence labeling for BrdU, NeuN and c-fos (Deng et al., 2009). One of the six serial sections was treated with 2 M HCl for 30 minutes at 37°C and subsequently incubated simultaneously in three primary antibodies, diluted in PBS containing 0.5% Triton-X and 3% donkey serum, for 48 hours at 4°C. 
+
+Primary antibodies included rat anti-BrdU polyclonal antibody (1:1,000; Accurate Chemical & Scientific Corporation Company, Philadelphia, PA, USA), goat anti-NeuN antibody (1:400; Cell Signaling Technology Inc.) and rabbit anti-c-fos polyclonal antibody (1:500; Cell Signaling Technology Inc.). 
+
+After incubation of primary antibodies, donkey anti-rat Alexa 568, donkey anti-goat Alexa 647 and donkey anti-rabbit Alexa 488 antibodies (1:250; Invitrogen Life Technologies, Carlsbad, CA, USA) were added onto the slides for 90 minutes at room temperature. After three rinses, the slides were mounted with florescence mounting media (DAKO, Carpinteria, CA, USA) and coverslipped.
+
+## Analysis Of The Number Of Dcx-Positive Cells And Dendritic Complexity Of Newborn Neurons
+
+DCX-positive cells in the dentate gyrus in the dorsal part of the hippocampus (Yau et al., 2011) were counted using Stereo Investigator software (Micro-Bright Field Biotechnology, New York, NY, USA) by a skilled and blinded experimenter. The software randomly selected 30–50 visual fields under a 40× light lens, and the number of labeled cells was counted. The dendritic outline, dendritic length, and the number of intersections and branch points of DCX-positive cells in the granule cell layer of the hippocampal dentate gyrus were traced using Filament tracer software (Bitplane Inc., South Windsor, CT, USA) and then analyzed with ImarisTrack software (Bitplane Inc.). A qualified neuron for analysis displayed a comparatively independent dendritic tree with at least tertiary branches. Tracings were analyzed by Sholl analysis (Sholl and Uttley, 1953) in 10–200-µm concentric circles centered in the cell body. In brief, the dendritic length counted as the length of the trace between every two concentric circles. The intersection number was the number of traces crossing each circle. A higher dendritic length value or greater number of intersections indicates a neuron with more complex dendritic branching.
+
+## Seizures And C-Fos Analysis
+
+Kainic acid was used to induce seizures in mice and induce the expression of immediate early genes in the dentate gyrus (Deng et al., 2009). Although c-fos is upregulated in very few granule cells under physiological conditions, including novelty exposure or learning, all granule cells upregulate immediate early genes such as *c-fos* in response to electrical activity, such as seizures (Dragunow and Faull, 1989; Kempermann et al., 2003). c-fos expression in newborn neurons after seizure indicates that they are fully mature and integrated into the existing neuronal circuitry (Snyder et al., 2009). BrdU was injected intraperitoneally six times (50 mg/kg, twice per day; Sigma) to mice. Kainic acid (35 mg/kg; Tocris Bioscience, Minneapolis, MO, USA) was injected intraperitoneally 1, 2, 3, 4 and 10 weeks after the last injection of BrdU. Seizures were stopped by injection of the GABA agonist sodium pentobarbital (50 mg/kg, intraperitoneally; Sigma) 30 minutes after the onset of stage 5 seizure activity. One hour later, animals were sacrificed for the cell maturity analysis.
+
+## Statistical Analysis
+
+All data are presented as the mean ± SEM. Comparisons of multiple groups were done by one-way analysis of variance followed by Student-Newman-Keuls test for two-group comparisons within the multiple groups using SPSS 16.0 statistical software (SPSS, Chicago, IL, USA). P < 0.05 was considered to indicate a significant difference.
+
+## Results Fewer Neurons Were Produced In The Brain Of Cx3Cr1Gfp/Gfp Mice Than In Cx3Cr1+/Gfp **Mice**
+
+DCX is expressed in newly formed neurons, and is associated with the migration and differentiation of neuronal progenitor cells (Brown et al., 2003). Therefore, DCX can be used to label newborn neurons in the subgranular zone of the hippocampus. As shown in **Figure 1**, most of the DCX-positive cell bodies were located at or just beneath the bottom of the granular layer, and had short or long processes. The number of DCX-positive cells in the subgranular zone was substantially lower in CX3CR1*GFP/GFP* mice than in CX3CR1+/GFP mice (P
+< 0.01). A decrease in the number of DCX-positive cells in the subgranular zone was also observed in the CX3CR1+/GFP mice, compared with wild-type mice (P < 0.05; data not shown).
+
+## Cx3Cr1 Deficiency Reduced The Dendritic Complexity Of Newborn Hippocampal Neurons
+
+To evaluate the impact of CX3CR1 deficiency on the morphology of newborn neurons, we examined the morphology of DCX-positive cells in the dentate gyrus. DCX-positive cells were divided into class I and class II. Class II DCX-positive cells were characterized as cells with a primary dendrite that was oriented perpendicular to the subgranular zone 
+(Plümpe et al., 2006; Oomen et al., 2011). Class I DCX-positive cells were located in the subgranular zone, without a dendrite or with only a short dendrite. Every selected class II DCX-positive cell with at least tertiary branches was traced using Filament tracer software and analyzed by Sholl analysis. Compared with wild-type mice, the dendritic length of class II DCX-positive cells was greatly reduced in CX3CR1*GFP/GFP* mice (P < 0.05; **Figure 2A**). In addition, the number of intersections was reduced in CX3CR1*GFP/GFP*
+mice compared with wild-type mice (P < 0.05; **Figure 2B**). No significant dendritic defect in class II DCX-positive cells was observed in CX3CR1+/*GFP* mice (**Figure 2**). The dendritic length and the number of intersections are two parameters reflecting the complexity of dendritic trees in Sholl analysis. Our findings show that CX3CR1 deficiency reduces the dendritic complexity of newborn neurons.
+
+## Neuronal Functional Maturation Was Delayed In Cx3Cr1Gfp/Gfp **Mice**
+
+Mature granule cells functionally integrated into the neural circuitry can be activated by very strong stimuli, such as kainic acid. To assess the functional maturation of newborn neurons, changes in c-fos expression in BrdU+ cells in the dentate gyrus in response to kainate-induced seizures were measured in 1- to 10-week-old mice. Ten weeks after the final BrdU injection, more than 95% of BrdU+ cells in all groups strongly expressed c-fos. However, the expression of c-fos was significantly decreased in CX3CR1*GFP/GFP* mice with kainate-induced seizures 3 weeks after the last BrdU injection, compared with wild-type mice (P < 0.05; **Figure 3**). 
+
+This reduction in expression of c-fos indicates that CX3CR1 signaling contributes to the maturation of newborn neurons in the hippocampus.
+
+## Discussion
+
+In the present study, we found that the number of DCX-positive cells in the subgranular zone was dramatically lower in CX3CR1*GFP/GFP* mice compared with CX3CR1+/GFP mice. Furthermore, in CX3CR1*GFP/GFP* mice, the dendritic length and the number of intersections were greatly reduced. Notably, the disruption of fractalkine/CX3CR1 signaling resulted in a temporary reduction in c-fos expression after kainic acid-induced seizures. These findings indicate that the disruption of fractalkine/CX3CR1 signaling results in the impairment of dendritic morphology and a delayed maturation of newborn neurons during hippocampal neurogenesis.
+
+Recently, the role of microglia in the uninjured brain has attracted increasing attention (Lu et al., 2013). Microglia are highly dynamic cells with extremely motile processes and protrusions, and they continually survey their microenvironment (Nimmerjahn et al., 2005). In the current study, we found that disruption of the fractalkine/CX3CR1 signaling pathway caused a reduction in the number of DCX-positive cells in both homozygous and heterozygous mutant mice. These results are similar to previous reports (Bachstetter et al., 2011; Rogers et al., 2011). Impaired hippocampal neurogenesis eventually leads to cognitive decline. Hence, it is intriguing that CX3CR1+/GFP mice suffering from impaired hippocampal neurogenesis exhibit only mild cognitive deficits. The proliferation of neural progenitor cells and neuronal differentiation are only two stages of hippocampal neurogenesis. The successful migration and maturation of newborn neurons is necessary for learning and memory function in experimental animals. Based on this premise, we analyzed the changes in the dendritic morphology of immature neurons in the dentate gyrus. The dendritic length and the number of intersections in young neurons, as assessed by Sholl analysis, was significantly reduced in CX3CR1*GFP/GFP* mice, but not in CX3CR1+/GFP mice. 
+
+The findings show that the complexity of the dendritic tree in newborn neurons in CX3CR1+/GFP mice was similar to that in wild-type mice. This similarity might account for the observation that CX3CR1+/GFP mice displayed a comparatively normal performance in cognitive tasks.
+
+DCX is a protein that promotes microtubule polymerization, and is abundantly expressed in migrating neuroblasts and young neurons (Gleeson et al., 1998). DCX labeling reveals the processes of immature neurons and is useful for investigating the development of dendrites morphologically. Dendritic development in immature neurons is closely associated with the efficacy of antidepressant drugs (Wang et al., 2008), as well as seizures (Overstreet-Wadiche et al., 2006). Our data here show that in the CX3CR1*GFP/GFP* mice, CX3CR1 deficiency impairs cell proliferation and neuronal differentiation during hippocampal neurogenesis, and also affects the dendritic development of immature neurons Another critical stage in hippocampal neurogenesis is the maturation of young neurons and their subsequent integration into the existing hippocampal circuitry (Deng et al., 
+2009, 2010). This requires the ability to form synapses with hilar interneurons, mossy cells or CA3 pyramidal cells (Toni et al., 2008). A recent study showed that mice with a transient deficit in granular neuron maturation is unable to display robust, long-term spatial memory and exhibits impaired performance in extinction tasks (Deng et al., 2009). Therefore, we examined c-fos expression in newborn neurons following seizure. The expression of c-fos was reduced in 3-week-old, but not in 10-week-old, CX3CR1*GFP/GFP* mice. c-fos is an immediate early gene that is rapidly and transiently induced in response to certain stimuli in various types of cells (Hu et al., 1994). Acute expression of c-fos in neurons indirectly reflects the firing of action potentials (Dragunow and Faull, 1989). Here, the expression of c-fos in BrdU+/NeuN+ cells indicates that they are newborn neurons that have integrated into the existing neural circuitry (Kempermann et al., 2003; Snyder et al., 2009).
+
+To our knowledge, we provide the first evidence that fractalkine/CX3CR1 signaling participates in the maturation of newborn neurons during hippocampal neurogenesis. The finding that the expression level of c-fos was similar in wild-type and CX3CR1*GFP/GFP* mice is in keeping with the observation that the CX3CR1+/GFP mice displayed a comparatively normal cognitive phenotype in the previous study. 
+
+Moreover, a postponed maturation of newborn neurons in the CX3CR1*GFP/GFP* mice was observed. Thus, disruption of fractalkine/CX3CR1 signaling only delays the maturation of immature neurons in the brain.
+
+Previous studies have shown that microglia regulate the maturation of synapses during the embryonic stage, and the first wave of microglial ancestors enter neural tissue very early during embryonic development. This entry precedes both astrogliogenesis and oligodendrogliogenesis during the perinatal period (Ginhoux et al., 2010) and coincides with the first wave of embryonic synaptogenesis (Kettenmann et al., 2013). The promotion of synaptic maturation by microglia was also recently shown during the postnatal stage 
+(Paolicelli et al., 2011). Microglia engulf synaptic debris and actively monitor the surrounding environment. A transient increase in spine density was observed during development in mice with CX3CR1 deficiency, suggesting that microglia participate in synaptic pruning through fractalkine/CX3CR1 signaling. Our data suggest that CX3CR1 deficiency delays the maturation of neurons, and thereby postpones synaptic integration. Whether microglia function in synaptic pruning during hippocampal neurogenesis through fractalkine/CX3CR1 signaling is unknown and will require further study.
+
+In conclusion, disruption of fractalkine/CX3CR1 signaling results in dendritic defects and the delayed maturation of neurons in the subgranular zone. Our findings provide insight into the role of the fractalkine/CX3CR1 pathway during hippocampal neurogenesis in the normal brain.
+
+Author contributions: *FX designed and completed the experiment, as well as analyzed the data and wrote the paper.* 
+JMX and XHJ sponsored the experiment and revised the paper. XHJ assisted to design the experiment and perform part of the animal feeding. All authors approved the final version of the paper.
+
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+Copyedited by Patel B, Norman C, Yu J, Yang Y, Li CH, Song LP, Zhao M

medical/md/PMC4510720.md

@@ -0,0 +1,367 @@
+# Review Article
+
+Indian J Med Res 141, April 2015, pp 408-416
+
+# Organ Transplant & The Psychiatrist: An Overview
+
+B.N. Anil Kumar* & Surendra Kumar Mattoo Department of Psychiatry, Postgraduate Institute of Medical Education & Research, Chandigarh, India Received April 25, 2013 Organ transplantation has emerged as the saving grace for those who are suffering from end organ disease. Advent of modern surgical procedures and immunosuppressants further decrease morbidity and mortality. Meta-analyses have shown that post-organ transplantation quality of life improves for social, physical and daily activity functioning, but not consistently for psychological health. Psychiatrists can play a useful role not only in selecting the best suitable candidate for the procedure by psychosocial screening but also to tackle post-operation psychological issues that trouble patients as well as caretakers and decrease their quality of life. Issues like selection of patients with psychiatric disorders and substance abuse for transplantation process and their treatment both pre- and post- operation, risky health behaviours, treatment adherence for immunosuppressants and psychological support for caretakers can be better addressed by a psychiatrist who is sensitive towards these issues. Prescribing various psychotropics and immunosuppressants in the background of impaired organ function and drug-drug interaction is further challenging. Thus, psychiatrists need to be knowledgeable about these issues and should be an integral part of organ transplantation team for overall better outcome.
+
+Key words Organ transplantation - psychiatrist - psychiatric disorders - quality of life - substance abuse
+
+## Introduction
+
+Organ transplant/transplantation (OT) has been defined as the therapeutic grafting of an organ from a living or dead body to a living body1. OT is a complex, invasive and major surgical procedure which carries tremendous expectations from the OT candidates and caregivers for saving life and improving the quality of life (QOL).Thus, starting from the onset of the end stage organ disease (ESOD), undergoing evaluation for candidacy, surgical procedure and related complications, and ending with death, all through these phases OT and organ donation are inherently enormously distressing. Also, OT is bound to ethical and legal issues. In the developing countries, the picture gets complicated by the psychosocial milieu being different in terms of poverty, underdeveloped protective medical institutional mechanisms, family structure and functioning, cultural norms and values related to organ donating/receiving. Emerging evidence shows that pre-operative psychosocial variables can predict post-transplant psychiatric adjustment for most organ types2, and meta-analyses have shown that post-transplant QOL improves for social, physical and daily activity functioning, but not 
+**Present address*: Department of Psychiatry, Shridevi Institute of Medical Sciences & Research Hospital, Tumkur, Karnataka, India consistently for psychological health3,4. This implies that while medico-surgical teams take better care of the physical health, they may leave out the psychological perspective. The medico-surgical teams may overlook psychological distress, especially if underplayed by the subjects. 
+
+This overview highlights the psychiatrist's perspective on psychosocial and medication issues related to the organ recipients, donors, and caregivers, and their effect on the OT outcome. The aim is to highlight the role of the psychiatrist and to sensitize the medical fraternity in general and the OT teams in particular. The literature search strategies included electronic databases and manual searches of relevant publications or cross-references. Electronic search included pubmed and other search engines (Embase, Science Direct, Google Scholar) for the years 20052014. Cross-searches often yielded additional relevant material. The search terms used in various combinations were: transplantation, psychiatrist, psychiatry, living donors, caregivers, psychotropics. The relevant articles were selected based on their importance for psychosocial and psychiatric aspects of OT. Accordingly, we selectively focussed on good and large scale relevant reviews and meta-analyses. Thus, this review attempts to give an overview of the area and is not a systematic review of the area5. Relevant Indian research is summarised separately.
+
+## Organ Transplant: A Heavy Burden
+
+Organ transplant is a continuum of different phases, each associated with its own individual, social and economic problems.
+
+(i) End stage organ disease (ESOD) is the patient's worst clinical state with failed organ function. As a matter of life and death, it marks the beginning of OT.
+
+(ii) OT evaluation involves various steps to achieve OT candidacy, at the cost of a performance anxiety and the fear of omission.
+
+(iii) Waiting for organ donation is the most distressing phase. Continued deterioration of health and critical medical care like dialysis is associated with1018 per cent patients dying, and 5 per cent becoming medically unsuitable; another 2 per cent refuse OT6. 
+
+The greatest obstacle is the shortage of donated organs; in the USA 40 per cent candidates wait for >2 years before they get an organ6.
+
+(iv) Transplantation and after, means dealing with a complex OT procedure, perioperative complications, and a post-OT life with constant threat to survival, and inevitable and often distressing dependence on caregivers for help on various counts.
+
+## Unique Dual Role Of Psychiatrist In Transplantation
+
+Comprehensive pre-transplant psychosocial evaluation helps to get a more complete understanding of mutual, and sometimes contradictory, expectations and needs of transplant candidates, donors, caretakers and the transplant team. the psychiatrist plays a unique dual role in serving both the needs of the transplant team as well as the transplant patient, donor and the carers7.
+
+The psychiatrist also has a major role in identifying problematic areas/issues both pre- and post-transplant and address those to seek improved outcomes.
+
+## Pre-Transplant Psychosocial Evaluation
+
+It is a stepping stone for OT candidacy. Limited availability of donor organs, need for psychological sophistication, good family support, considerable medical and financial resources for better care and outcome - all dictate the selection of only those candidates who have the best chance of post-OT outcome. To select the best candidate for transplantation, there is a need to screen the patient's coping skills for stressors, co-morbid psychiatric conditions and their pre- and post-transplant monitoring and treatment, capacity to understand the transplant process and to provide informed consent, ability to collaborate with the transplant team and to adhere to treatment, substance use/abuse history, recovery, and ability to maintain long-term abstinence, health behaviours that may influence post-transplant morbidity and mortality like eating or exercise habits, level of social support available to the candidate for pre- and post-transplant phases and psychosocial needs of the patient and family and plan for services during the waiting, recovery, and rehabilitation phases of the transplant process8.
+
+Some of the important aspects of organ transplant will be considered in the following sections which demand a psychiatrist's expertise:
+
+## Treatment Adherence
+
+Treatment adherence generally includes regular intake of medications, monitoring vitals, undergoing diagnostic tests, following dietary and exercise protocols, abstinence from substance abuse, and regular follow up. A meta-analysis of 147 studies across all organ types showed average non-adherence per 100 PPY (persons per year) to be 1-4 for substance use, 1925 for immunosuppressants, diet, exercise, and other requirements9.
+
+A review of 17 studies showed non-adherence to be associated with poor clinical outcome, contributing to 20 per cent of late acute rejection episodes, lower number of quality adjusted life years and lower lifetime costs because of shorter survival10.Thus, it is very essential to identify candidates with ambivalence about treatment and prior history of non-adherence, substance abuse, poor social support, and poor organisational skills as they are more prone for treatment non-adherence.
+
+## Risky Health Behaviours
+
+These include substance abuse, poor eating habits and sedentary lifestyle leading to obesity. Studies have shown that pre-transplantation BMI >30 kg/m2 or body weight >140 per cent of ideal, decrease survival rate among transplant recipients compared to controls among renal11 and heart12 cases, respectively. Another study showed that compared to controls nutritional counselling of OT subjects, once weekly for a month and once monthly for next three months, reduced posttransplant weight gain13. Presumably poor outcome may be reduced by identifying reasons and risk factors for non-adherence and risky health behaviours, educating patient and family members regarding the same, making plan for monitoring both pre- and post-OT, and written informed behavioural and contingency contracting making the patient responsible for own acts. Recently, efforts have been made to develop a programme based on prevailing evidence to promote self-management skills for behaviours such as undesired weight gain, exercising and smoking14.
+
+## Psychiatric Disorders Among Ot Cases
+
+ Like other chronically medically ill subjects, OT cases are also at higher risk for psychiatric disorders. It may be due to either the exacerbation of a pre-existing disorder or the development of new-onset disorder. With no large-scale prevalence rate comparisons yet available, no strong evidence exists that in OT the rates of psychiatric disorders vary by the organ.
+
+## Mood And Anxiety Disorders
+
+Anxiety or depressive disorders are experienced by 25 per cent of lung, 40 per cent of liver and 50 per cent of heart pre-OT cases15. In post-OT cases during the first several years, major depression was experienced by 20 per cent of kidney, 30 per cent of liver, and 63 per cent of heart cases. Compared to pre-OT/waiting period the risk was greatest in the first post-OT year; contributing factor being the stress associated with physical recovery, rehabilitation, adaptation to immunosuppressive medications, and psychosocial role change16,17. Studies have shown that while the patient is in waiting period depressive symptoms are associated with poor QOL, poor adaptive coping, and low functional status, and there is high mortality in patients with depressive symptoms both during pre- or post- transplant period18-20.
+
+Among anxiety disorders during OT, post-traumatic stress disorder (PTSD) is most important:14-44 per cent of ICU-treated cases experience PTSD, with >24 per cent having persistent symptoms years later21. PTSD
+may result from any medical crisis during waiting period, surgery itself, and hallucinations and delusions associated with post-surgical delirium22. Post-traumatic stress disorder or symptoms are shown to increase mortality, decrease QOL and life satisfaction23,24. With mood and anxiety disorders conclusively predicting higher morbidity and mortality, 45 per cent of OT centres in the USA consider current affective disorders as an absolute contraindication for OT25.
+
+## Psychotic Disorders
+
+Psychosis emerging beyond post-operative recovery period occurs exclusively in OT cases with past history of such illness. Among such cases, 37 per cent experienced manic or psychotic episodes in the first several post-OT years26. Hence, there is a need for aggressive pre-OT treatment as psychotic symptoms have a negative impact on OT outcome. However, it remains debatable as to how fair it is to deny organ to a psychotic patient; in the USA, 92.3 per cent OT centres consider active schizophrenia as an absolute contraindication for OT25.
+
+A survey across 12 OT centres in US, Canada and Australia showed non-compliance with immunosuppressant drugs among psychotic cases living alone to be 45.5 per cent, 9.5 per cent among psychotic cases living with someone, and 20-50 per cent among overall OT cases. Suicide attempts in the pre-OT year were recorded in 35.7 per cent cases with psychotic symptoms versus 5.9 per cent of those without psychotic symptoms26. Thus, it is evident that in cases with psychosis, good social support and adequate preOT symptom control mean a better outcome.
+
+## Personality Disorders Substance Abuse Cognitive Disorders Mental Retardation (Mr)
+
+The OT process demands a series of adaptations in physical and social functioning and ability to cooperate with caregivers and OT team. The persisting and inflexible maladaptive patterns of behaviour with personality disorder (PD) cases lead to impaired interpersonal relationship and social functioning.
+
+In both the USA and Europe the most common indications for liver transplantation are cirrhosis due to alcoholic liver disease (ALD) and hepatitis B and C (HBV, HCV), especially among intravenous drug abusers. The OT teams continue to debate whether or not to offer OT to cases with substance abuse. The major concern is post-OT substance abuse relapse resulting in direct damage to transplanted organ, decreased efficacy of ongoing medical treatment, increased risk for cancer and non-adherence to treatment; all eventually leading to graft failure and recipient death32.
+
+Incidence of personality disorders among OT cases has been reported to be 10-26 per cent, similar to that in general population27,28. A survey of all active OT 
+programmes in the USA showed that 14.1 per cent of heart, 8.7 per cent of liver, 5.2 per cent of kidney OT 
+programmes viewed personality disorder as an absolute contraindication to OT25. Another study showed that borderline personality disorder carries highest risk for post-OT non-compliance, less stable social support and strained working relation with OT team29. In a study in alcoholic liver disease cases with anti-social personality disorder, 50 per cent returned to either alcohol or narcotic addiction compared to 20 per cent with no personality disorder30. 
+
+Tobacco use in immunosuppressed patients increases risk of cancer33. Smoking increases vascular complications among post-OT liver cases (17.8% in smokers vs. 8% in controls)33. In ALD liver transplant recipients, risk of oropharyngeal and lung cancer was 25 and 3.7 times higher compared to controls34.
+
+Another study showed transplanted ALD cases had 1and 5-year survival rates of about 80 and 70 per cent, respectively, comparable to transplants for other causes of cirrhosis35. Though there is sufficient evidence to suggest post-OT substance use carries major adverse health consequences, a meta-analysis of 54 studies showed post liver OT relapse to any alcohol use twice more than relapse to heavy alcohol use (~6 vs<3/100 PPY) and non-adherence to immunosuppressant medication being 3.2/100 PPY. These figures are comparable to cases with no prior history of abuse36. 
+
+The pre-transplant history of illicit drug use has not been consistently shown to be associated with increased risk for post-transplant alcohol relapse in ALD recipients37-42, possibly because many ALD
+recipients had discontinued their drug use many years prior to transplantation37. Thus, a history of substance abuse should not be an absolute contraindication for OT. The available research shows cases with history of alcohol abuse that are treated effectively have relapse rate, non-adherence to medications, and survival rates comparable to cases with no such history38. More worrying is relapse to tobacco use and associated medical complications. Smoking, both by donors and by recipients, has a major negative impact on outcomes after organ transplantation43. The risk factors for relapse include poor social support, family history of alcohol dependence, pre-transplant abstinence of ≤ 6 months, past failed rehabilitation, and multiple substance abuse.
+
+Through pre- to post-OT phases, cases frequently experience reductions in cognitive functioning ranging from subclinical to frank delirium. Among OT recipients cognitive impairment remains relevant in terms of potential negative impact on treatment compliance due to patient's inability to learn old or new instructions/ regimens for medication, investigations and behaviours related to memory deficits, delusions and hallucinations associated with delirium. Perhaps due to these facts dementia is considered as an absolute contraindication among 72 per cent of the US OT centres25.
+
+	 There isnoscientific evidence suggestingcaseswith MR should not have access to organ transplantation. Yet, 25.6 and 74.4 per cent OT centres in the USA considered IQ 70-90 and IQ <70, respectively as an absolute contraindication for transplantation25. This automatic exclusion of cases with MR from OT continues to be a matter of debate as a clear form of discrimination. A review of cases with MR and renal OT showed post-OT survival rates of 100 per cent at one year and 90 per cent at three years31. These rates were similar to those of non-MR cases. This review highlights that adequate support from family and other caregivers rather than the recipient's IQ level predict post-OT medication compliance31.
+
+	 There are no efficacy studies on treatment. General advice is for frequent follow up with random toxicological screening, motivation enhancement therapy (MET), and relapse prevention counselling (RPC). Among medications, naltrexone being a direct hepatotoxin, is contraindicated in liver failure. Disulfiram is hepatically metabolized and may lead to immunosuppressant toxicity. Selective serotonin reuptake inhibitors (SSRI) and tricyclic antidepressants (TCA) improve abstinence rates in depressed relapsing alcoholics42.
+
+## Immunosuppressant Related Neuropsychiatric Manifestations
+
+Common immunosuppressants currently used in OT (tacrolimus, cyclosporine, corticosteroids, azathioprine and mycophenolate mofetil), are all well known for their neuropsychiatric side effects.
+
+Calcineurin-inhibiting immunosuppressants(CII):
+Tacrolimus and cyclosporine have a similar neurotoxic profile: 40-60 per cent recipients have mild symptoms like tremulousness, headache, restlessness, insomnia, vivid dreams, photophobia, dysasthesias, anxiety, and agitation, and 20-32 per cent have moderate to severe symptoms like cognitive impairment, coma, seizures, focal neurological deficits, dysarthria, cortical blindness, psychosis, and delirium. With discontinuation of CII, most of the side effects resolve spontaneously. Short term anticonvulsants can be used for CII-induced seizures, and benzodiazepines or betablockers for anxiety, tremor, or restlessness42.
+
+Corticosteroids: High dose corticosteroids are usually given immediately post-transplantation and for initial treatment of rejection. Steroid induced psychiatric syndromes are common, complex, unpredictable, and range from subtle mood changes to full blown affective syndromes and frank psychosis. Mild symptoms of agitation, anxiety, distractibility, fear, insomnia, lethargy, labile mood, and restlessness occur in 20-30 per cent and severe symptoms of affective syndromes, psychosis and cognitive impairment in six per cent cases44. Treatment includes steroid dose reduction or discontinuation. If steroid dose reduction is not feasible, symptomatic treatment with antipsychotics, mood stabilizer or anti-depressants is recommended44.
+
+Azathioprine: There are reports of depressive symptoms induced by azathioprine in combination, with cyclosporine and prednisone in particular42.
+
+Mycophenolate mofetil: CNS adverse effects- anxiety, depression, delirium, seizures, agitation, psychosis and somnolence - have been recorded in 3-20 per cent recipients of mycophenolate42.
+
+## Management Of Psychiatric Disorders
+
+The risk for psychiatric disorders rises during organ transplantation and it adversely affects the OT outcome, compliance, morbidity, and mortality. Patients with mental health disorders, when adequately controlled and socially supported, have outcomes similar to the general transplant population45. Hence, there is need for early recognition and management.
+
+## Psychotropics
+
+Organ dysfunction during pre-transplant period changes the pharmacokinetics of psychotropic agents, requiring proper selection of the drug and the dose. Similarly in post-OT period, psychotropics selection is influenced by drug pharmacokinetics, adverse effects, and tolerability and drug-drug interactions. Studies on the treatment of psychiatric disorders in OT cases being limited, the decisions need to be based on a review of clinical and pharmacokinetic data46-49.
+
+## Selection Of Psychotropics In Liver Failure
+
+Antipsychotics are mainly metabolized by liver except for sulpiride, amisulpride and paliperidone. In mild-moderate liver disease all antipsychotics can be tried with caution, but in severe liver disease it is better to avoid those metabolised by liver49. All antidepressants are also mainly metabolized by liver. TCAs can cause sedation and constipation, and SSRIs can prolong prothrombin time (PT). Escitalopram with minimal effects on hepatic enzymes and sertraline with minimal drug-drug interactions are SSRIs of choice, although clinical experience is limited. Serotonin norepinephrine reuptake inhibitors (SNRIs) can be used with dose reduction. Mirtazapine with its minimal effect on CYP enzymes system is a better choice for patients with insomnia and anorexia46,48,49.
+
+Mood stabilizers except for lithium and gabapentin are metabolized by liver, alter liver function test, especially during the first month but it is transient49. 
+
+Benzodiazepines (BZDs) lorazepam, oxazepam, temazepam have short half-life with no active metabolites. So these are the better choice among BZDs49.
+
+## Selection Of Psychotropics In Renal Failure
+
+Antipsychotics except for amisulpride and sulpiride can be used in standard doses but with caution. Drugs with anticholinergic properties may cause urinary retention, confusion, and hypotension and ziprasidone with a potential to cause prolonged QTc interval are better avoided49. Among antidepressants, there is no preferred agent. In severe renal impairment and anuria, dose adjustment is indicated46,48. Benzodiazepines are mainly metabolized in liver. In ESRD dose reduction is generally not necessary except for midazolam and chlordiazepoxide whose active metabolites are excreted through kidney49. Among mood stabilizers, lithium is excreted unchanged in urine up to 95 per cent and is a known nephrotoxic. Also, being small molecule lithium is readily dialyzed and entirely eliminated by dialysis. if lithium is to be used in ESRD because of non-response to other mood stabilizers, single dose (usually 600 mg) treatment is recommended after each dialysis session. Other mood stabilizers can be used with caution48,49.
+
+## Psychotherapy
+
+Empirical data regarding psychotherapeutic interventions among OT recipients are very limited. Procedures that are commonly employed in clinical settings are group therapy, educational groups, support groups, internet based regimen and mentorship programme50. A study has shown both individual and group psychotherapy to be beneficial and individual therapy to be better than group therapy. Both decrease the negative affect, increase happiness and QOL51. 
+
+Another study showed telephone-based emotional support and cognitive behaviour therapy to increase QOL and social network52. Internet based intervention improved compliance, QOL, follow up care and decreased anxiety, depressive symptoms in both patients and caregivers among heart transplant recipients53. 
+
+Mentorship by an already transplanted recipient augmented patient care by providing information and support from a peer perspective54.
+
+## Drug-Drug Interactions Between Immuno Suppressants And Psychotropics
+
+Commonly used immunosuppressants like steroids, tacrolimus, cyclosporine and mycophenolate have narrow therapeutic index and are mainly metabolized by cytochrome P450 3A4. Specific cytochrome P450 3A4 inhibitors in decreasing order are: fluvoxamine, nefazodone>fluoxetine>sertraline, TCAs, paroxetine >venlafaxine55. There are many case reports of tacrolimus and cyclosporine toxicity, when fluvoxamine and nefazodone were added to treat depression55. In contrast, many case reports favour safe use of other antidepressants in combination with immunosuppressants. Cyclosporine increases lithium resorption by proximal tubule, so it is mandatory to monitor lithium level in patients on cyclosporine. Disulfiram may interact with the alcohol vehicle of cyclosporine and its active metabolite is an inhibitor of CYP450 3A455.
+
+## Burden Among Caregivers
+
+ Dependency of transplant recipients on caregivers for everything including self-care, treatment adherence, finance, transportation, and emotional support, put them under stress. Heavy burden on caregivers and negative coping styles are shown to be associated with higher levels of depression56. In a study on caregivers of lung OT cases stress related depressive and anxiety symptoms have been reported in 82 per cent caregivers. Acute illness stage was rated more stressful than rehabilitation stage. Compared to spouses, mothers were particularly vulnerable as support persons57.
+
+Another study on heart transplantation group showed employment status and caregivers' physical health as strong predictors of post-OT distress. Increased distress was noted in early post-OT but not in later months58. 
+
+In a study on renal OT group, all caregivers perceived burden, more if the patient was a male59. There was no relationship between caregiver burden score and patients' marital status, education level, operation time, age, donor type (cadaveric or living)59. The data regarding intervention studies are lacking; the available data show group therapy, support group and web based interventions as useful strategies in alleviating distress among caregivers.
+
+## Issues Of Living Donor
+
+In view of the organ shortage living organ donation is an alternative to deceased organ donation. In the USA, related and unrelated living donors constitute 44 per cent of all organ donors. Of these, 95 per cent are kidney donors, four per cent are liver donors, and the remaining one per cent are pancreas, intestine and lung donors6. 
+
+Donors with altruistic motivation, no ambivalence, good understanding about risks and benefits of donation, no history of psychiatric illness or substance abuse, good family support and financial stability are known to have better outcome. In a systematic review of 51 studies, 5139 living kidney donors assessed after four years showed that the majority experienced no depression (77-95%) or anxiety (86-94%), reported unchanged or improved relationship with the recipient (86-100%), and reported psychological benefits from donation in terms of gratification of helping the needy and experiencing increase self-esteem. Though most scored high on QOL measures, a small proportion had adverse psychosocial outcomes60. Another study reported a reasonable QOL among lobar lung donors, despite a subjective decline in exercise tolerance61. 
+
+In a six month follow up study of 392 liver donors, 16 (4.1%) had one or multiple psychiatric complications 
+[12 depression, 2 anxiety, 2 substance abuse, and bipolar affective disorder (BPAD), 1 accidental overdose, insomnia, crying episodes, and worsening of obsessive compulsive disorder (OCD) each]62. It was not clearly known if the donors were inherently more prone to psychiatric problems and had concealed this during evaluation or hepatectomy; or the psychiatric problems were a consequence of hepatectomy which is a more invasive procedure with more post-operative medical complications62.
+
+## Indian Scenario
+
+There is very limited research from India on psychiatric issues related to OT. An early study reported negative emotions among kidney recipients and donors. Among the recipients the common emotions included anxiety-depression and resentment during dialysis and perioperative period, and anxiety, dependency-frustration, body image change and decreased libido on a long term basis63. Among the donors, hostility towards family members, ambivalence towards recipients, adjustment problem at work and with spouse, anxiety about surgical procedures were recorded pre- and during OT period and excessive worry about physical symptoms of recipients, depression and hypochondriacal symptoms post-OT64. A study with live kidney donors showed their morbidity, mortality and QOL remained unchanged65. Cognitive profile and depression were studied in 30 renal transplant patients which showed depression rate of 87 and 57 per cent one month before and three month after transplantation and mean IQ increased from 88.5 to 101 following transplantation especially with respect to concentration and visual organization66. In a six month to six year follow up of 50 male kidney recipients psychiatric illness was noted in 46 per cent of patients and it was more common among unmarried, high education group and high socio-economic status67.
+
+## Conclusion
+
+Transplantation is a challenging process for patients, caregivers, and medical professionals. It is associated with substantial emotional distress with tremendous lifestyle changes and psychological stresses. Psychosocial evaluation, considered to be a gate pass for transplantation, carries great significance.
+
+Depression and anxiety are the most common psychiatric disorders among the OT candidates and recipients. Currently, there is no general consensus on how to select/reject cases for transplantation with affective disorders, psychotic disorders, personality disorders and substance dependence. As the psychiatric disorders predict poor outcome both preand post-OT, it calls for an aggressive management. 
+
+Immunosuppressants related neuropsychiatric side effects and drug-drug interactions with psychotropics should be kept in mind while treating psychiatric disorders. Caregivers also carry significant distress and need support. A psychiatrist as a member of the OT team can be of help for better outcome of OT.
+
+## References
+
+1. T*he Transplantation of Human Organs Act 1994*. New Delhi: 
+Ministry of Law, Justice and Company Affairs (Legislative Department); 11th July 1994. No. 42.
+
+2. Dew MA, Switzer GE, DiMartini AF, Matukaitis J, Fitzgerald MG, Kormos RL. Psychosocial assessments and outcomes in organ transplantation. *Prog Transplant* 2000; 10 : 239-59.
+
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+
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+
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+Group. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. *PLoS Med* 2009; 6 : e1000097. 
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+Lippincott Williams and Wilkins; 2009. p. 2441-55.
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+8. Levenson J, Olbrisch ME. Psychosocial screening and selection of candidates for organ transplantation. In: the transplant patient. Cambridge, UK, Cambridge University Press; 2000, p. 23.
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+Steel J, Unruh M, *et al*. Rates and risk factors for non adherence to the medical regimen after adult solid organ transplantation. Transplantation 2007; 83 : 858-73.
+
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+Schäfer-Keller P, Schaub S, *et al*. Prevalence, consequences, and determinants of nonadherence in adult renal transplant patients: a literature review. *Transpl Int* 2005; 18 : 1121-33.
+
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+literature overview. *Transplantation* 2000; 70 : 711-6.
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+30. Coffman KL, Hoffman A, Sher L, Rojter S, Vierling J, 
+Makowka L. Treatment of the postoperative alcoholic liver transplant recipient with other addictions. *Liver Transpl Surg* 1997; 3 : 322-7.
+
+31. Panocchia N, Bossola M, Vivanti G. Transplantation and mental retardation: what is the meaning of a discrimination? 
+
+Am J Transplant 2010; 10 : 727-30.
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+32. Parker R, Armstrong MJ, Corbett C, Day EJ, Neuberger JM. Alcohol and substance abuse in solid-organ transplant recipients. *Transplantation* 2013; 96 : 1015-24.
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+33. Pungpapong S, Manzarbeitia C, Ortiz J, Reich DJ, Araya V, 
+Rothstein KD, *et al*. Cigarette smoking is associated with an increased incidence of vascular complications after liver transplantation. *Liver Transpl* 2002; 8 : 582-7.
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+34. Jain AB, Fung JJ. Alcoholic liver disease and transplantation.
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+Transplant Proc 2003; 35 : 358-60.
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+Myaskovsky L, Unruh M, *et al*. Meta-analysis of risk for relapse to substance use after transplantation of the liver or other solid organs. *Liver Transpl* 2008; 14 : 159-72.
+
+37. Coffman KL, Hoffman A, Sher L, Rojter S, Vierling J, 
+Makowka L, *et al*. Treatment of the postoperative alcoholic liver transplant recipient with other addictions. Liver Transpl Surg 1997; 3 : 322-7.
+
+38. DiMartini A, Weinrieb R, Fireman M. Liver transplantation in patients with alcohol and other substance use disorders. 
+
+Psychiatr Clin North Am 2002; 25 : 195-209.
+
+39. Fireman M. Outcome of liver transplantation in patients with alcohol and poly-substance dependence. Presented at Research Society on Alcoholism: Symposium on Liver Transplantation for the Alcohol Dependent Patient. Denver, CO, June 2000.
+
+40. Foster PF, Fabrega F, Karademir S, Sankary HN, Mital D, 
+Williams JW, *et al*. Prediction of abstinence from ethanol in alcoholic recipients following liver transplantation. 
+
+Hepatology 1997; 25 : 1469-77.
+
+41. Newton SE. Recidivism and return to work posttransplant. 
+
+J Subst Abuse Treat 1999; 17 : 103-8.
+
+42. DiMartini AF, Dew MA, Trzepacz PT. Organ transplantation. 
+
+In: Levenson JL, editor. *Textbook of psychosomatic medicine*. 
+
+Washington, DC: American Psychiatric Press; 2005. p. 675700.
+
+43. Corbett C, Armstrong MJ, Neuberger J. Tobacco smoking and solid organ transplantation. *Transplantation* 2012; 94 : 
+979-87.
+
+before receiving a kidney transplant? *Transplantation* 1997; 64 : 599-604.
+
+12. Grady K, White-Williams C, Naftel D, Costanzo MR, Pitts D, Rayburn B, *et al*. Are preoperative obesity and cachexia risk factors for post heart transplant morbidity and mortality: A multi-institutional study of preoperative weight-height indices. *J Heart Lung Transplant* 1999; 18 : 750-63.
+
+13. Patel M. The effect of dietary intervention on weight gains after renal transplantation. *J Ren Nutr* 1998; 8 : 137-41.
+
+14. Schmid-Mohler G, Fehr T, Witschi P, Albiez T, Biotti B, Spirig R. Development of an evidence-based self-management programme for patients in the first year after renal transplantation with a focus on prevention of weight gain, physical exercise and drug adherence. *Pflege* 2013; 26 : 191-205.
+
+15. Trumper A, Appleby L. Psychiatric morbidity in patients undergoing heart, heart and lung, or lung transplantation. J 
+Psychosom Res 2001; 50 : 103-5.
+
+16. Olbrisch ME, Benedict SM, Ashe K, Levenson JL.
+
+Psychological assessment and care of organ transplant patients. 
+
+J Consult Clin Psychol 2002; 70 : 771-83.
+
+17. Dew MA, DiMartini AF. Psychological disorders and distress after adult cardiothoracic transplantation. *J Cardiovasc Nurs* 2005; 20 : 51-66.
+
+18. Singh N, Gayowski T, Wagener MM, Marino IR. Depression in patients with cirrhosis. Impact on outcome. *Dig Dis Sci* 1997; 42 : 1421-7.
+
+19. Corruble E, Barry C, Varescon I, Falissard B, Castaing D, 
+Samuel D. Depressive symptoms predict long-term mortality after liver transplantation. *J Psychosom Res* 2011; 71 : 32-7.
+
+20. DiMartini A, Dew MA, Chaiffetz D, Fitzgerald MG, Devera ME, Fontes P. Early trajectories of depressive symptoms after liver transplantation for alcoholic liver disease predicts longterm survival. *Am J Transplant* 2011; 11 : 1287-95.
+
+21. Kapfhammer HP, Rothenhäusler HB, Krauseneck T, Stoll C,
+Schelling G. Posttraumatic stress disorder and health-related quality of life in long-term survivors of acute respiratory distress syndrome. *Am J Psychiatry* 2004; 161 : 45-52.
+
+22. DiMartini AF, Crone C, Fireman M, Dew MA. Psychiatric aspects of organ transplantation in critical care. *Crit Care Clin* 2008; 24 : 949-81.
+
+23. Dew MA, Kormos RL, Roth LH, Murali S, DiMartini A, 
+Griffith BP. Early post-transplant medical compliance and mental health predict physical morbidity and mortality one to three years after heart transplantation. *J Heart Lung Transplant* 1999; 18 : 549-62.
+
+24. Baranyi A, Krauseneck T, Rothenhäusler HB. Posttraumatic stress symptoms after solid-organ transplantation: preoperative risk factors and the impact on health-related quality of life and life satisfaction. *Health Qual Life Outcomes* 2013; 11 : 111.
+
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+
+26. Coffman KL, Crone C. Rational guidelines for transplantation in patients with psychotic disorders. *Curr Opin Organ* Transplant 2002; 7 : 385-8.
+
+56. Goetzinger AM, Blumenthal JA, O'Hayer CV, Babyak MA, 
+Hoffman BM, Ong L, *et al*. Stress and coping in caregivers of patients awaiting solid organ transplantation. *Clin Transplant* 2012; 26 : 97-104.
+
+57. Ullrich G, Jänsch H, Schmidt S, Strüber M, Niedermeyer J. The experience of the support person involved in a lung transplant programme: results of a pilot study. *Eur J Med Res* 2004; 9 : 555-652.
+
+58. Canning RD, Dew MA, Davidson S. Psychological distress among caregivers to heart transplant recipients. *Soc Sci Med* 1996; 42 : 599-608.
+
+59. Einollahi B, Taheri S, Nemati E, Abbaszadeh S, Pourfarziani V, Nourbala MH. Burden among care-givers of kidney transplant recipients and its associated factors. *Saudi J Kidney* Dis Transpl 2009; 20 : 30-4.
+
+60. Clemens KK, Thiessen-Philbrook H, Parikh CR, Yang RC,
+Karley ML, Boudville N, *et al.* Psychosocial health of living kidney donors: a systematic review. *Am J Transplant* 2006; 6 : 2965-77.
+
+61. Prager LM, Wain JC, Roberts DH, Ginns LC. Medical and psychologic outcome of living lobar lung transplant donors. 
+
+J Heart Lung Transplant 2006; 25 : 1206-12.
+
+62. Trotter JF, Hill-Callahan MM, Gillespie BW, Nielsen CA,
+Saab S, Shrestha R, *et al*. Severe psychiatric problems in right hepatic lobe donors for living donor liver transplantation. Transplantation 2007; 83 : 1506-8.
+
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+
+64. Kuruvilla K, Pandey AP, Shastry JCM. Psychiatric aspects of renal transplantation - observations on donors. Indian J 
+Psychiatry 1979; 21 : 155-8.
+
+65. Sahay M, Narayen G, Anuradha. Risk of live kidney donationIndian perspective. *J Assoc Physicians India* 2007; 55 : 
+267-70.
+
+66. Pawar AA, Rathod J, Chaudhury S, Saxena SK, Saldanha D, 
+Ryali VSSR, *et al*. Cognitive and emotional effects of renal transplantation. *Indian J Psychiatry* 2006; 48 : 21-6.
+
+67. Chaturvedi SK, Pant VLN. Psychiatric aspects of renal transplantation. *Indian J Psyciatry* 1985; 27 : 77-81.
+
+44. Warrington TP, Bostwick JM. Psychiatric adverse effects of corticosteroids. *Mayo Clin Proc* 2006; 81 : 1361-7.
+
+45. Corbett C, Armstrong MJ, Parker R, Webb K, Neuberger JM. 
+
+Mental health disorders and solid-organ transplant recipients. 
+
+Transplantation 2013; 96 : 593-600.
+
+46. Crone CC, Gabriel GM. Treatment of anxiety and depression in transplant patients: pharmacokinetic considerations. Clin Pharmacokinet 2004; 43 : 361-94.
+
+47. Trzepacz PT, DiMartini A, Tringali R. Psychopharmacologic issues in organ transplantation. Part I: Pharmacokinetics in organ failure and psychiatric aspects of immunosuppressants and anti-infectious agents. *Psychosomatics* 1993; 34 : 
+199-207.
+
+48. Trzepacz PT, DiMartini A, Tringali RD. Psychopharmacologic issues in organ transplantation. Part 2: Psychopharmacologic medications. *Psychosomatics* 1993; 34 : 290-8.
+
+49. Taylor D, Paton C, Kapur S. Use of psychotropics in special patient groups. In: *The Maudsley prescribing guidelines*, 
+10th ed. UK: MBG books; 2010. p. 335-463.
+
+50. Engle D. Psychosocial aspects of the organ transplant experience: What has been established and what we need for the future. *J Clin Psychol* 2001; 57 : 521-49.
+
+51. Abbey S, Farrow S. Group therapy and organ transplantation. 
+
+Int J Group Psychother 1998; 48 : 163-85.
+
+52. Napolitano MA, Babyak MA, Palmer S, Tapson V, Davis RD,
+BlumenthalJA, *et al*. Effects of a telephone-based psychosocial intervention for patients awaiting lung transplantation. *Chest* 2002; 122 : 1176-84. 
+
+53. Dew MA, Goycoolea JM, Harris RC, Lee A, Zomak R, 
+Dunbar-Jacob J, *et al*. An internet-based intervention to improve psychosocial outcomes in heart transplant recipients and family caregivers: development and evaluation. *J Heart* Lung Transplant 2004; 23 : 745-58.
+
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+
+55. Fireman M, DiMartini AF, Armstrong SC, Cozza KL. 
+
+Med-psych drug interactions update: immunosuppressants. 
+
+Psychosomatics 2004; 45 : 354-60.
+
+Reprint requests: Dr Surendra Kumar Matto, Department of Psychiatry, Postgraduate Institute of Medical Education & Research, Chandigarh 160 012, India e-mail: skmattoo@ymail.com

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+Journal of Cachexia, Sarcopenia and Muscle 2016; 7: 239–245 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12124
+
+# Cancer Cachexia—When Proteasomal Inhibition Is Not Enough
+
+Jens Fielitz1,2* 1Department of Molecular Cardiology, Experimental and Clinical Research Center (ECRC), Charité––Universitätsmedizin Berlin, Max Delbrück Center (MDC) for Molecular Medicine in the Helmholtz Association, Berlin, Germany; 2Department of Cardiology, Heart Center Brandenburg and Medical School Brandenburg (MHB), Bernau, Germany Keywords Cancer; cachexia; wasting; proteasome Received: 20 April 2016; Accepted: 29 April 2016 *Correspondence to: Jens Fielitz, Experimental and Clinical Research Center, Lindenberger Weg 80, 13125 Berlin, Germany. Phone: +49 30 450 540424; Fax: +49 30 450 540928, Email jens.fielitz@charite.de Cachexia is a life threatening syndrome associated with several diseases, such as end-stage heart failure, end-stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1,2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2,4–6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non-Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small-cell and non-small-cell lung cancer patients develop cachexia.7–9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi-organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7,12–15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18,19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.
+
+## Muscle Wasting Is A Major Constituent Of Cancer Cachexia
+
+Cancer cachexia involves similar losses of muscle and adipose tissue and on a simplistic way, one could assume that this assures survival of the general organism. However, this response differs from starvation induced cachexia where the majority of weight loss is from adipose rather than muscle tissue.5,20,21 In cancer cachexia, skeletal muscle wasting cannot be reversed by nutritional intervention arguing for cancerspecific signals that are involved in its pathogenesis. The loss of muscle mass is accompanied by decreased strength, which is responsible for most of the cancer cachexia-associated symptoms, and increased mortality and morbidity of patients.22,23 Wasting not only involves skeletal but also chest, diaphragm, and cardiac muscle leading to fatigue and respiratory complications.3 In fact, the majority of cancer deaths are related to respiratory24 or cardiac failure.25 Therefore, treatments capable to slow down, stop or even reverse muscle wasting in cancer cachexia may be beneficial for cancer patients in terms of physical independence, quality of life, tolerance to and sensitivity to anticancer treatments, and possibly reduce morbidity and mortality. However, despite its clinical importance and the foreseen impact for patients, the pathophysiology of cachexia-associated muscle wasting is still poorly understood preventing the development of specific therapies. Because maintenance of skeletal muscle mass and function is assured by a well-regulated balance of protein synthesis and degradation, a disturbed protein homeostasis with increased degradation and or decreased synthesis is a major contributor to muscle wasting in cancer.26 Increased protein degradation is caused by an elevated activity of muscular protein degrading systems predominantly the ubiquitinproteasome system (UPS), which plays an important role in cancer patients experiencing weight loss.8
+
+## Protein Degradation Is Important For Skeletal Muscle Wasting In Cancer
+
+The UPS is the main protein degrading system in eukaryotic cells.27 It mediates the degradation of misfolded and mutated proteins as well as many proteins involved in the regulation of development, differentiation, cell proliferation, signal transduction, and apoptosis.28–30 Activation of the UPS in skeletal muscle leads to degradation of structural and contractile proteins, most notably myosin heavy chain,31,32 resulting in atrophy and decreased muscle function.33 Proteins targeted for proteasomal degradation are ubiquitinated by a hierarchical-ordered series of enzymes, including the ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and the ubiquitin E3 ligases, which allow the targeted proteins to be recognized by the 26S proteasome, a multi-subunit protease complex composed of the 20S catalytic core and 19S regulatory particles.28,29,34 Substrate specificity of the UPS is assured by E3 ligases and substrate adaptor proteins.35,36 In many models of cachexia, including cancer, UPS activation is thought to mediate muscle atrophy.37 During tumour cachexia, the UPS is up-regulated in skeletal muscle with an increased expression of the E3 ligase muscle-specific RING-finger 1 (MuRF1) and the substrate adaptor protein FBXO32/Atrogin-1.38 In a rat model of cachexia, induced by the Yoshida ascites hepatoma cells, UPS
+up-regulation was associated with increased Fbxo32/
+Atrogin-1 gene expression39 and increased protein ubiquitination.39 This UPS activation in tumour-bearing mice is mediated by an increased activity of the transcription factor nuclear factor kB (NF-kB), which stimulates MuRF1 expression and muscle wasting.40
+
+## Communication Between Tumour And Host
+
+It remains uncertain how tumour cells communicate with the host to induce cancer cachexia. The often anatomically distant locations between the tumour and the wasting musculature are suggestive for a signal released into the hosts' circulation by the tumour to cause muscle wasting in cachexia. Especially, chronic inflammation with elevated levels of circulating inflammatory cytokines is consistently observed in cachectic cancer patients.40 Because chronic inflammation affects the function of several tissues (i.e. skeletal muscle, fat, brain, and liver) it is an important cause of cancer cachexia.41 Among the best studied inflammatory cytokines promoting cachexia are tumour necrosis factor alpha (TNFα), interleukin-6 (IL-6),42 interleukin-1 (IL-1), and interferon gamma.43 These cytokines are elevated in cancer44 and may together trigger muscle wasting, probably by increasing NF-kB or by causing the release of other cytokines.45,46 For example, TNFα, initially named cachectin,47 promotes anorexia48 and skeletal muscle wasting mainly through activation of the NF-kB pathway.49 Therefore, it has been tested if TNFα blockade is beneficial to prevent cancer cachexia. Indeed, TNFα blockade improved cachexia-associated fatigue in a small group of cancer patients.50 However, in recent randomized controlled clinical trials anti-TNFα therapies using either the TNFα receptor–blocker etanercept in patients with incurable malignancies51 or the TNFα-specific monoclonal antibody infliximab in patients with metastatic non-small-cell lung cancer52 did not prevent or palliate weight loss or muscle atrophy. Instead, infliximab increased fatigue and adversely affected patients' quality of life.52 These data suggest that targeting TNFα alone is not sufficient to prevent cachexia.
+
+IL-6 and IL-1 are up-regulated in animal models of cancer cachexia,53 and IL-6 levels correlate with weight loss in certain human cancers.54 Interestingly, cancer cachexia can be attenuated in an adenocarcinoma mouse model treated with antiIL-6 antibodies.55 However, if this strategy prevents muscle wasting in cancer patients needs to be shown.
+
+As mentioned earlier, NF-kB is a key regulator of inflammatory responses and involved in muscle atrophy.40,56,57 In the majority of cells, NF-kB exists in an inactive form in the cytoplasm bound to the inhibitory protein IkB. When stimulated with inflammatory cytokines (i.e. TNFα, IL-1, and IL-6),
+NF-kB is activated by degradation of IkB proteins. This occurs primarily via activation of IkB kinase, which phosphorylates IkB. IkB phosphorylation leads to its ubiquitination and degradation by the proteasome.58 This allows free NF-kB to translocate to the nucleus and to stimulate the expression of its target genes.59 Because NF-kB activation is important for muscle atrophy, many approaches were undertaken to reduce its activity. In mouse muscles, miss-expression of IkB
+to inhibit NF-kB reduced neurogenic atrophy in tumour-bearing mice.40 In mice lacking IkB kinase-β, neurogenic atrophy was also attenuated.56 Synthetic double-stranded oligodeoxynucleotides, which block NF-kB binding to promoter regions has been shown to inhibit cachexia in a mouse tumour model.60 Importantly, proteasome inhibitors interfere with the NF-kB pathway because they inhibit IkB degradation, which in turn prevents NF-kB activation.61 Because MuRF1 and FBXO32/Atrogin-1 are NF-kB target genes, proteasome inhibition is expected to prevent muscle atrophy by maintaining NF-kB in an inactive state, and thus preventing up-regulation of MuRF-1 and FBXO32/Atrogin-1. Indeed, the proteasome inhibitor MG132 was shown to attenuate immobilization-induced atrophy. Likewise, the proteasome inhibitor bortezomib has been shown to reduce neurogenic atrophy.62
+
+## Proteasome Inhibition As Strategy To Treat Cancer Cachexia
+
+Several groups investigated if UPS inhibition is beneficial to prevent muscle atrophy in different mouse models. For this approach, mainly two-specific, potent and reversible proteasome inhibitors, MG132 and bortezomib, were used. Both compounds inhibit degradation of ubiquitin-conjugated IkBα resulting in suppression of the NF-kB signalling pathway.63 In addition, bortezomib and MG132 reduce proteolysis in skeletal muscle in vitro64,65 and prevented muscle mass loss in an in vivo rat model of skeletal muscle wasting induced by denervation and cast immobilization of the hind limb.62,66 MG132 was shown to preserve muscle and myofiber crosssectional area by down-regulating MuRF-1 and Fbxo32/
+Atrogin-1 mRNA in a mouse model of hind limb-immobilization resulting in a diminished rehabilitation period.67 In addition, an increased proteasome activity was found in a mouse model for Laminin-deficient congenital muscular dystrophy type 1A (MDC1A). Administration of MG132 increased lifespan, enhanced locomotive activity and enlarged muscle fibre diameter in MDC1A mice.68 Bortezomib had also bene-ficial effects in the MDC1A mouse model and in MDC1A patient cells.69 To this end, it was feasible to assume that proteasomal inhibition could be useful to block muscle wasting in cancer cachexia. This hypothesis was subsequently tested by Penna et al. who report their findings in this issue of the Journal.70 Penna et al. investigated if bortezomib attenuates skeletal muscle wasting in two different and wellestablished animal models of tumour-induced muscle wasting.70 Cancer cachexia was induced by intraperitoneal injection of Yoshida AH-130 ascites hepatoma cells in rats and by subcutaneous inoculation of C26 carcinoma cells in mice. As expected, bortezomib reduced proteasome activity on day 7 after transplantation of AH-130 tumour cells in the skeletal muscle, which was accompanied by a decreased NF-kB
+DNA-binding activity indicating that animals were effectively treated. However, bortezomib administration did not prevent body weight loss and muscle wasting in the AH-130 host rats.
+
+It also did not affect MuRF1 and Fbxo32/Atrogin-1 expression. Likewise, bortezomib did not prevent body and muscle weight loss 12 days after tumour implantation in C26-bearing mice. These data together with the published body of evidence indicate that the pathophysiology of cancer cachexia possibly involves additional NF-kB- and proteasome-independent protein degrading systems, such as autophagy and calpain proteases. For example, the autophagy pathway is activated in atrophying muscle of cancer patients.71–73 In a small cohort, lung cancer patients presented increased levels of the autophagy mediators BCL2/adenovirus E1B 19 kDa interacting protein 3 and light chain 3B, and the transcription factor FOXO1, which promotes autophagy.71 Similarly, in another study performed on esophageal cancer patients vs.
+
+weight-stable non-cancerous control patients, autophagy was identified as the main promoter of skeletal muscle proteolysis.74 Also calpain proteases have been proposed to initiate protein degradation during cachexia;75,76 however, limited information concerning their role in muscle wasting is available.39 Of note, in contrast to the data of Penna et al.,
+the proteasome inhibitor MG132 was found to attenuate weight loss and muscle atrophy, and increased spontaneous activity and survival time in a C26-tumour-induced cancer cachexia mouse model.77 Together with other studies in which treatment with MG132 was shown to be effective in preventing muscle dystrophy,78 disuse-induced atrophy,67 and immobilization-mediated skeletal muscle atrophy,79 these data indicate that MG132 could be useful to prevent cancer cachexia. However, MG132 also inhibits cathepsin and calpain proteases,80 which might partially explain its favourable treatment effects and the differences between MG132 and bortezomib used by Penna et al.. Therefore, the individual contribution of specific protein degrading systems and proteases in cancer cachexia as well as the effect of their inhibition as treatment option needs to be defined. In addition, MG132 also reduced the tumour burden with a reduction in tumour volume and weight.77 Because tumours seem to interact with the host by factors that induce muscle wasting a reduction in tumour size will also lead to their decrease, which may result in less wasting. Therefore, it is difficult to decide if proteasome inhibition by MG132 in muscle or in the tumour or both together reduced the occurrence of cancer cachexia in this mouse model. A side-by-side comparison of bortezomib and MG132 in C26-bearing mice could show if this accounts for the differences of both studies.
+
+In addition, despite the large body of evidence supporting the UPS as a major driver of muscle atrophy in animal models investigations of UPS components or activity in patient material are non-conclusive. Studies including patients with critical illness, following bed rest, limb immobilization, COPD, and ageing have demonstrated both increased and decreased expression of MuRF1 and FBXO32/Atrogin-1.81–84 Of note, there is no convincing evidence for increased UPS-mediated proteolysis in skeletal muscle biopsies of cachectic cancer patients.71,85 Investigations of UPS activity in biopsies of the quadriceps muscle have shown similar levels to healthy controls in patients with lung cancer and weight loss below 10%.85 Another study in lung cancer patients with low weight loss found no changes in UPS components.86 In contrast, in gastric cancer patients with average weight loss of 5%, increased UPS activity was measured compared with controls.87 These data implicate that there are certain forms of muscle atrophy and forms of cancer cachexia, respectively, where UPS-mediated proteolysis is not the main pathway in the disease process and proteasome inhibition will therefore not be able to attenuate the atrophy response. This hypothesis is strengthened by data from non-cancer patients and animal models. For example, the UPS is activated in muscle of patients with intensive care unit-acquired weakness81 and mice with polymicrobial sepsis.88,89 Accordingly, mice exposed to endotoxin, a cell wall component of gram-negative bacteria, display enhanced proteolytic activity in the diaphragm leading to a reduction in muscle mass, force, and protein content. However, proteasome inhibition of endotoxin-treated mice did not prevent reductions in diaphragm-specific force generation indicating that inhibition of proteasome-mediated proteolysis alone does not prevent endotoxin-induced reductions in diaphragm force generation.90 These data indicate that proteasome inhibition is not effective in each and every model of muscle atrophy.
+
+In general, bortezomib is well tolerated by patients. Nevertheless, it is associated with some toxicity, such as nausea, diarrhoea, fatigue, and generalized weakness. Indeed, proteasome inhibition is a double-edged sword as the proteasome mediates degradation of a multitude of proteins involved in critical biological processes and its inhibition possibly promotes protein accumulation that may cause proteotoxic effects. Penna et al. described that bortezomib exerted a transient toxicity, which led to a reduced food intake in their animals.70 Because food deprivation increases MuRF1 and Fbxo32/Atrogin-1 expression and causes muscle atrophy,89 this side effect is important for data interpretation. Even if bortezomib would have inhibited cachexia, the treatment-associated reduced food intake could have counteracted this effect.
+
+Finally, caution is needed when interpreting animal models of cancer cachexia to the true cachexia phenotype in patients. Penna et al. used well-established models of cancer cachexia. However, these models do have their limitations; the young age and rapid progression of inoculated tumour cells are only few of them. Furthermore, analysis 7 days after injection of AH-130 cells and 12 days after C26 transplantation, respectively, argues for an acute cachexia model. These points do not reflect the clinical situation in tumour patients where cachexia develops over a longer period in mainly older patients.
+
+Further studies also need to consider that cancer cachexia is a continuum with at least three stages of clinical relevance including pre-cachexia, cachexia and refractory cachexia.91
+
+## Cancer Cachexia Is A Multifactorial Syndrome
+
+In conclusion, we need to realize that not a single cytokine or signalling pathway is responsible for cancer cachexia; it is rather caused by a multitude of factors and signalling pathways that we only begin to understand. Therefore, it is unlikely that treatments targeting only one aspect of the syndrome, such as the proteasome or individual cytokines, will effectively block its pathogenesis or progression. Cachexia itself is a multifactorial syndrome that might phenotypically appear similar. However, the appearance of a patient does not provide mechanistic information. The path towards the cachectic phenotype is most likely different for various tumour types. When the pathway responsible for the cachectic phenotype is uncertain, it is difficult to develop or apply the right treatment. The data from Penna et al. should encourage us to look into proteasome- and NF-kB-independent signalling pathways involved in cancer cachexia and identify novel targets to treat this syndrome. Further studies are needed to elucidate precise signalling pathways involved in cancer cachexia; and first steps towards this direction are already being taken.92,93 Some of the factors increased in cancer cachexia, such as angiotensin II,94 and the transforming growth factor beta family members myostatin95 and activin A,96 have already been identified. Especially, myostatin and activin A
+are up-regulated in patients with various types of malignancies (for a review, see97). Myostatin inhibits muscle growth and its overexpression promotes it.98 Therefore, inhibition of myostatin–activin A signalling is an attractive therapeutic target for treatment of cancer-associated muscle wasting. Indeed, blockade of activin receptor IIB (ActRIIB), the receptor for activin A and myostatin and other transforming growth factor beta family members, was sufficient to prevent cachexia, increased muscle function and even prolonged survival in several cancer cachexia mouse models.99,100 This study showed that inhibition of cachexia has direct impact on cancer death.99 If myostatin inhibitors are beneficial as therapies for cancer, associated muscle wasting is currently being tested in clinical trials. Further mechanisms, which are involved in the pathogenesis of cancer cachexia include growth differentiation factor-15, macrophage inhibitory cytokine-1,101,102 leukaemia inhibitory factor,103 Fn14,104 signal transducer and activator of transcription 3,105 parathyroid hormone-related protein,106 and histone deacetylases;107 and this list is steadily increasing.92,93 Future studies need to be performed in a tumour-specific and disease stage-dependent manner to answer the question why certain cancers are more prone to cause cachexia than others, and to identify tumour-specific differences in cachexia pathways. Information gained by those studies will be useful to develop target and disease stage-specific treatments. Although animal models are useful in this regard they may or may not reflect the situation in patients at advanced tumour stages, and we need to appreciate this limitation cautiously. Results from treatment studies on cachexia animal models that could not be successfully confirmed in patients should encourage us to intensify collaboration between clinicians and basic scientists to promote patient-based research and to tackle this life-threatening syndrome.
+
+Editorial 243
+
+## Acknowledgements Conflict Of Interest
+
+The author certifies that he complies with the ethical guidelines for publishing in the Journal of Cachexia, Sarcopenia and Muscle: update 2015.108 None declared.
+
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+89. Schmidt F, Kny M, Zhu X, Wollersheim T,
+Persicke K, Langhans C, et al. The E3 ubiquitin ligase TRIM62 and inflammation-induced skeletal muscle atrophy. Crit Care 2014;18:545.
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+90. Supinski GS, Vanags J, Callahan LA. Effect of proteasome inhibitors on endotoxin-induced diaphragm dysfunction. Am J Physiol Lung Cell Mol Physiol 2009;296:L994–
+L1001.
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+91. Fearon K, Strasser F, Anker SD, Bosaeus I,
+Bruera E, Fainsinger RL, et al. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol 2011;12:489–95.
+
+92. Schafer M, Oeing CU, Rohm M, BaysalTemel E, Lehmann LH, Bauer R, et al.
+
+Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia. Mol Metabol 2016;5:67–78.
+
+93. McLean JB, Moylan JS, Horrell EM,
+Andrade FH. Proteomic analysis of media from lung cancer cells reveals role of 143-3 proteins in cachexia. Frontiers Physiol 2015;6:136.
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+94. Yoshida T, Delafontaine P. Mechanisms of cachexia in chronic disease states. Am J Med Sci 2015;350:250–6.
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+Wei M, Sze SK, McFarlane C, et al. Myostatin is a novel tumoral factor that induces cancer cachexia. Biochem J 2012;446:23–36.
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+96. Loumaye A, de Barsy M, Nachit M, Lause P, Frateur L, van Maanen A, et al. Role of activin A and myostatin in human cancer cachexia. J Clin Endocrinol Metab 2015;100:2030–8.
+
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+Myostatin/activin pathway antagonism:
+molecular basis and therapeutic potential. Int J Biochem Cell Biol 2013;45:2333–47.
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+99. Zhou X, Wang JL, Lu J, Song Y, Kwak KS,
+Jiao Q, et al. Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 2010;142:531–43.
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+Porta M, Capdevila E, et al. Myostatin blockage using actRIIB antagonism in mice bearing the Lewis lung carcinoma results in the improvement of muscle wasting and physical performance. J Cachexia Sarcopenia Muscle 2012;3:37–43.
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+key role for leukemia inhibitory factor in C26 cancer cachexia. J Biol Chem 2015;290:19976–86.
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medical/md/PMC5000329.md

@@ -0,0 +1,121 @@
+
+## Article Yi.? On Angina Pectoris. By Feancis W. Moinet, M.D. Edin.
+
+We propose in this paper to consider the paroxysm of angina pec-toris. What is it ? Many different theories have been, and are still held on this subject; but to the four principal ones only will we direct our attention. These are?first, that it is a cardiac neu-ralgia or neurosis ; second, a gouty attack; third, a spasm; and fourth, a paralysis. Each of these theories we will examine in their order, and try to demonstrate that the only way to understand the paroxysm is to prove it a paralysis. The first theory, that it is a cardiac neuralgia or neurosis, is maintained by the French school?" 
+Desportes, Jurine, Leallies, Losnnec, Lartigue, Trousseau, etc.?and by some distinguished men in our own country. At first sight it does not appear so very unreasonable. The pain in angina is undoubtedly of a neuralgic character ; the error the supporters of this theory have fallen into, is in not distinguishing functional from organic angina? 
+
+and in attaching too much importance to the prascordial pain. ^ e can certainly call the former a neurosis, that term implying that both the lesion and the paroxysm are mere nervous derangements; but not so in the case of the latter. Then the pain is only one of the symptoms which naturally, from its severity, absorbs all the patient S 
+attention, and frequently the medical attendant's also, and so has led them rashly to name the disease from its most prominent symp-tom. Besides, a neuralgia would not account for the history and symptoms of a case of organic angina, with its inevitably fatal ter-mination, and the post-mortem appearances found. So, although in angina the pain be neuralgic, the paroxysm cannot for a moment be considered such. The second theory is that it is a gouty affection Butler was the first to advocate this opinion; but, since his Eisner, Schoffer, Schmidt, Bergius, Hesse, and most of the errnan writers, have agreed with him. Formerly, many affections eie attributed to gout, as a cloak for ignorance, especially in ob- 
+^QUle and difficult cases of heart disease; but, in angina pectoris, we i n?T See ^ow ^ can Pr^e itself as serving its purpose even decent- 
+-X" in Scotland, where the gout is rare, angina is as common as in gland, where gout is comparatively much more prevalent. In 
+?st of the cases of angina I have seen, there Avas no history of b?ut, while gout, we know, seldom affects the heart without having Prirnarily manifested itself in other parts ; besides, the symptoms cubed to gout attacking the heart are quite different from e symptoms of angina pectoris. Thus we see that, as a direct 0?Use'. gout can have nothing whatever to do with a paroxysm ag angma, but, indirectly, it may, by giving rise to dyspepsia; for, 
+^ 
+^Te know that functional angina is often due to dyspepsia, there is Reason why gouty dyspepsia may not be looked upon as the oc- 
+'Sioiial cause of these attacks. Many of the most powerful predis- P sing causes of gout, such as indulgence in fermented liquors and 
+' lrnal food, tend to produce structural changes and degenerations, ? atheroma, etc. The long-continued existence of chronic gout 
+, so leads to a depraved condition of the general nutrition of the 
+?dy. This tendency to degeneration of structure we have seen to e a cause of weakened heart, which is, in our opinion, the cause of arigina, so the gouty diathesis, by favouring the depraved constitut-ional condition which leads to degeneration and weakening of the art, may be looked upon as a predisposing cause of angina ; other-1Sej it can have no relation to the paroxysm. Having dismissed 
+, 
+ese two theories, we have now a more difficult task before us?to ecide whether the paroxysm is a spasm or paralysis, it being now generally admitted that it must be one of the two. There has been 
+' constant division on this point, especially among English writers, j many names of weight can be ranged on either side. Heberden, atham, etc., were convinced of its spasmodic nature, and brought onvard many arguments in its favour; while Stokes, Walshe, and nie 0f tjie more recent writers point to paralysis as the more proable cause of the paroxysm. 
+
+i Vr Heberden, writing on this subject, gives us seven reasons for 
+.eving it to be a spasm, and as most of his followers have but 
+^?pied them, we will take his list and see whether or not they con-lln his doctrine of spasm. They are :? 
+
+?st. It comes suddenly and goes suddenly. 
+
+It has long and complete intermissions. 
+
+3g?. Wine, spirits, and opium give great relief. 
+
+It is increased by mental agitation. 
+
+It exists for years without other injury to the health. 
+
+The pulse is not quickened in the very paroxysm. 
+
+<th. A paroxysm attacks some after their first sleep?a frequent event in diseases which proceed from spasm. 
+
+vol. xvi.?no. vii. 4 h We will examine these reasons separately, in order to find ho^ 
+far they support the spasm theory, and if they are in any respects incompatible with that of paralysis. 
+
+The first is?It comes suddenly and goes suddenly. Most cer-tainly the paroxysm comes suddenly, frequently without _a moment's warning, and so does spasm; while, although paralysis may come suddenly, it generally goes away gradually. Now, why does paralysis disappear slowly ? Is there no exception to this rule? 
+
+and if there is, may not the paroxysm of angina pectoris be one ? 
+
+Paralysis, when it does disappear, does so usually in a very gradual manner, because, as a rule, the lesions causing it, as extravasatea blood, effusion of serum, or inflammation and its products, can only; if at all, disappear slowly by re-absorption or resolution of the in-flammatory process, so, necessarily, the paralysis goes slowly. But if, on the other hand, the cause of the paralysis can be suddenly with-drawn, there is no reason why the paralysis should not as suddenly cease. In angina, the paroxysm is due to a sudden and temporary strain being put upon an organ in a condition so weakened as to be unable to resist it, but the cause being temporary, and ceas-ing the moment the paroxysm comes on, the recovery is sudden. 
+
+The history of the earlier attacks of the disease shows this to be the case. The patient is seized with a paroxysm when walking rapidly or going up hill, and almost the moment he stops, the paroxysm is over. Here the over-exertion is the direct exciting_ 
+cause, and when that is withdrawn, the heart in the commencement ot the disease immediately recovers itself and the paroxysm ceases,but as the disease advances the recovery is not so rapid, the heart being more enfeebled, and in not so favourable a condition as formerly for re-gaining its irritability, so that Dr Heberden's first argument does not favour the doctrine of spasm more than that of paralysis. 
+
+The second reason is?It has long and complete intermissions. 
+
+Has it? Not always ; and when it has, it is not unless at the very commencement of the disease, and when the patient is under strict surveillance as regards exercise and diet, for any indiscretion m either is apt to bring on a paroxysm, and as the disease advances the paroxysms become much more frequent, and the interval diminishes. Over-exertion, over-eating, and mental emotion are some of the most common exciting causes of the paroxysm; so, naturally, if a patient suffering from this disease finds what is m his case the exciting cause of an attack?as he soon does?he can, if possessing self-control, determine at first to a certain extent the length of the intermission, but, as the disease advances, the interval diminishes, because a much smaller amount of exertion, physical or mental, will now, than formerly, bring on a paroxysm, so that the completeness and gradually diminishing interval are the symptoms we would expect from a temporary paralysis, and not from spasm of the heart. 
+
+The third reason is, that wine, spirits, and opium give great t Tliis is perfectly true, and stimulants ought to be equally ettieacious, whether it is a spasm or paralysis; for, looking at the Symptoms of a paroxysm, face pale, pulse small and feeble, and ear ?f syncope, we ought to see that stimulants are strongly indi-cted. Opium we know produces stimulation of the vascular and Nervous systems, as shown by increased fulness and rapidity of 
+^e pulse, then relief to pain; so whether it acts as a stimulant or anodyne, it is equally well pointed to as a suitable and proper remedy for the alleviation of a paroxysm of angina, even if it were a paralysis, and by experience we know it is the only treatment can present rely upon. 
+
+^ 
+J-he fourth reason is, that it is increased by mental agitation, pasm is frequently increased by mental agitation, but only, as a 
+^e, when the seat of the disease is cerebral, as in epilepsy, chorea, tc., for we do not find that spasm is aggravated by mental agita-i?n when it occurs in an organ or part in which the lesion or Cuse of the spasm is also situated, as in colic, cramp in the limbs l0m over-exertion. So in angina, as the lesion is purely local, Cental excitement should not bring on or increase the paroxysm, yet it invariably does, so we ought to infer that it is not a spasm, an^t not a spasm it must be a paralysis. 
+
+. J-he fifth reason is, that it exists for years without other in-JUry to the health. It may, to a certain extent, in the functional eases, but not in the organic; for injury to the health must and 
+?es follow to a greater or less extent, for the suffering, mental 
+,nct bodily, the secondary derangements which so invariably follow, Ue to the cardiac lesions, impair the health sometimes to so great an extent that the patient succumbs, not to the paroxysm, but 
+,? s?me of these secondary lesions. But even if it did not injure le health so severely as we have said, there is no reason why a P^ysis similarly situated should cause more damage than a spasm. 
+
+feixth reason is, that the pulse is not quickened in the paroxysm, enerallv speaking, it is not; and this is just what we would expect it were due to a paralysis. In the few cases in which it is quickened, 116 beats are very feeble, the acceleration being due to a greater decree of weakness in the heart. As Dr Graves has shown that the Maximum of strength and minimum of frequency are attained to-gether, so these two conditions of the pulse, seemingly contradictory, 
+.are thus quite naturally reconciled. 
+
+Seventh reason is, that a paroxysm attacks some after their first 
+, eep, a frequent event in diseases which proceed from spasm. This ast argument is not very convincing. It says, it attacks some in their rst sleep, and that this is frequent in diseases due to spasm. Now, if 
+.,s frequent in diseases due to spasm, it ought to be frequent in an- 
+?lna; but no, it only attacks some. The attacks are liable to come 
+?n at any time, so why not occasionally at that time; and if a Patient suffering from angina should waken with a start, as those ttected with heart-disease often do, we have a direct exciting cause of the attack, so that a paroxysm attacking some after their first sleep, does not by any means imply that the paroxysm is a spasm. 
+
+Having thus taken each of Dr Ileberden's reasons for believing it to be a spasm and examined them separately, we have not found one which in the least degree excludes the possibility of its being a paralysis, but rather strengthens the probability. So we will noW 
+endeavour to turn that probability into a certainty. 
+
+When we see a patient during a paroxysm of angina, in what condition do we find him ? His face is pale, sometimes bedewed with a clammy sweat, his look is anxious, expressing great bodily and mental suffering, and he is afraid to lie down, not from dysp-noea so much as from the fear of his heart stopping to beat, as he describes it. On examining the circulatory system, the heart we find beats more feebly than natural, the impulse being weak and hardly if at all perceptible ; in a few cases it appears slightly tumul-tuous. These cases are, however, exceptional, and only occur where there is hypertrophy of the heart from valvular disease, dilatation or aneurism of the aorta on which degeneration or atrophy of the heart has supervened, or perhaps by interference with its nervous supply produced angina. In these cases, the action of the heart is observed over a larger space than natural, nearly the whole ventricular, and not the apex beat alone, being visible ; and this strikes us the more forcibly when we compare it with what we usually observe in an-gina, when the heart is merely softened or atrophied without any counterbalancing hypertrophy, and when this is accompanied with an occasional intermission or irregularity, it gives a still greater appear-ance of reality to this quasi-tumultuous action. The pulse is found to vaiy considerably in different cases. Sometimes it is regular, sometimes irregular?in one patient frequent, in another slow ; but as a rule, it is not increased in frequency more than four or five beats ; 
+still, an increase of twenty has been observed according to Walshe. 
+
+In fits destined to terminate fatally it becomes almost imperceptible, occasionally altogether suppressed, most commonly it is small and weak. Are these the symptoms we should expect in spasm? We think not. And why ? Because if it were a clonic spasm, the sudden contractions would give the pulse a quick jerk, then, in the relaxation, it would fall to or below its normal standard, thus caus-ing a great variation in the strength of the radial pulse, but this we never find. It would also act irregularly. Now we know that this last symptom is not by any means a constant accompaniment. 
+
+Then if the spasm were tonic, a heart so affected could not continue to beat, and continue to beat regularly, for the length of time some of the paroxysms last, especially when they are frequently repeated, and continue through a course of two or three years or longer. The result we would look for and expect would be almost instantaneous?- 
+death in the first seizure. Or, if it were a spasm of only one or more of the cavities of the heart, but not involving the whole organ, the action of the cavities not spasmodically affected would be 8'ieatly increased to compensate for the inaction of the others, and 
+^Ve would then have symptoms of venous congestions due to the jfiechanical obstruction to the passage of the blood ; but we never 
+*ave these symptoms, the face is always pale, never congested unless VeiT slightly, if the paroxysm is a very protracted one. Still, in a Paralysis of a portion of the heart, these last symptoms would also apply, but as we never have them, even in a modified degree, think that shows that during a paroxysm the whole heart is in a state of paralysis, to a greater or less degree. Thus we see that ie symptoms we should expect in spasm are? 
+
+J-f tonic spasm of the entire heart?death in first attack inevitable. 
+
+? of portion of the heart, irregular and greatly increased action of cavities not affected. Venous congestions, and tolerably strong impulse. 
+
+-It clonic spasm of the entire heart?quick sharp beat followed by a small feeble one, this continuing throughout the entire paroxysm. Irregular action. 
+
+,, of portion of the heart?constant irregularity and venous congestions. 
+
+So we see that not under one of these four conditions can a par-oxysm of angina be placed as agreeing with the symptoms. Then We look at the nature of spasm, I think we find some points that contra-indicate the paroxysm being of that nature. Local spasm, y which we mean a spasm occurring in a part in which the cause 
+^ the spasm is situated, as distinguished from spasm in a part aue to irritation or disease of distant organs or parts. Cramp in exhausted or over-strained muscles of a limb is an example ?i the former. In these cases the spasms occur in muscular sub- 
+.ubstance otherwise healthy and unimpaired in its structure, while, ln cases where there is degeneration of the muscular fibres, as in P^uscular atrophy or in wasting from paralysis, spasm is rare, but lri Weakened heart the paroxysms of angina are very common. Again, spasm is frequently excited in a part quite healthy by irritation or isease of a distant organ or part, as spasm of the glottis in disease 1 the larynx, spasm in epilepsy, etc.; but in cases of organic angina, ^e never have other organs or parts diseased, which, by irritation or therwise, have ever been proved to produce spasm of the heart, and we have shown that spasm depending on disease of a distant P.ai*t usually attacks healthy structure, while the heart, in angina, is always in a morbid condition. 
+
+Again, an attack of angina is ahvays aggravated and the pain greatly increased by muscular exertion, and muscular exertion not 
+?nly aggravates an attack, but is very often the exciting cause of 
+?ne, while spasm, often the result of undue muscular exertion, is 
+.leved by increased or forced motion of the part. Thus we find ln Cramp in a limb, the readiest method of affording relief is by forced 
+^tension and movement of the affected limb, also, in colic, friction of le abdominal walls gives relief by the movement of their contents. 
+
+In an organ already weakened, over-exertion is very liable to cause a temporary increase of weakness or paralysis, and, naturally, it *s increased if the organ is still put on the stretch by motion, but as soon as it is placed in as favourable a position for rest as possible, relief is given, and such we find is what takes place in a paroxysm of an-gina in the first attacks. Then, if we look at the progress of the attacks, in the organic cases we can do nothing to prevent the paroxysms gradually becoming more frequent, each one occurring si a shorter interval than its predecessor. This a paralysis naturally explains, but not so a spasm, which, when it does occur in an organ, does not follow this regular course, neither in the increasing severity of the attacks nor the gradually diminishing interval showing the slightest similarity to the paroxysm of angina pectoris. 
+
+Lastly, let us see what are the post-mortem appearances of the heart found in patients who have died during a paroxysm of this disease. In all the reported cases we do not find that in one was the heart, or a portion of it, contracted on itself after death, but the reverse, the heart being always found flabby and unusually flaccid, frequently with clots in the cavities, showing how feebly the heart must have been acting. Professor Smith relates a case of tetanus, in which, after death, the heart was found so firmly contracted on itself as to cause obliteration of the cavity of the left ventricle, and so twisted as to present a spiral condition from the extreme contraction of the oblique muscular fibres. Now, this is a case of spasmodic disease affecting the heart, in which, after death, we find it still remaining in a state of contraction. So it is surely but reasonable to expect that we should find the heart in some of those who have died in a paroxysm of this disease in a state of partial or complete contraction, but no such condition has ever been described, while the flabby and flaccid state is the condition always found. Nor is it at all likely that spasm could change at the moment of death to syncope, which is always the immediate cause of death. 
+
+Having thus stated my arguments, let me sum them up. Not one of Heberden's arguments for spasm exclude paralysis, but some rather favour it. While the arguments for paralysis exclude spasm, and although, perhaps, Ave have not given any one fa^ 
+that is by itself perfectly conclusive, it is because so little is known of spasm of the heart, that probable conjectures are all that can be made on some points in the present state of our knowledgeStill, by the study of spasm in other organs and parts, we are en-titled to judge what would, to a certain extent, be its effects and symptoms in another organ, so that the probabilities are very reasonable ones, and do not invalidate our argument as a whole; and as it must be either one or the other?spasm or paralysis^" 
+no other theory being able to account for the symptoms and his-tory of the disease, we think we are justified in saying it is & 
+paralysis occurring in, and depending on, a weakened heart. 
+
+. Treatment.?During the paroxysm the treatment most efficacious relieving the symptoms is that by diffusable stimulants and opium, hot brandy-and-water, sinapisms to the feet, and placing the hands lri hot water. In the functional cases, treatment must be directed mainly to the stomach and nervous system. Tonics, such as iron quinine, and regular exercise, will be found most beneficial, trousseau in his work mentions some good results he obtained by galvanism; but from the reports of the cases operated upon, they must have been functional, as it is only in these cases that cures could be effected. Still, we are inclined to think that galvanism carefully, and at first gently, applied, might be of some service even in or-ganic angina, by giving tone and stimulus to the heart's muscular hbre, and that, at least, it is worthy of a trial. Another remedy we 
+)yould suggest is bleeding, for by relieving the languid and stagnat-lr\g circulation, you ease the heart, and at the same time give it a stimulus and opportunity to recover itself, especially when _ 
+the paroxysm is a protracted one. Nitrite of amyl is also sometimes J?und useful; it acts as a stimulant?its speedy action being due to being administered by inhalation?and not by relieving tension as Dr Brunton affirms in his paper in the Lancet of oth March, in 
+^hicli lie relates a case of angina pectoris in a man, tet. 26, and argues that the arterial expansion is diminished, and the tension correspondingly increased during a paroxysm of angina, the smallness the pulse being due to its hardness, and that by the nitrite ox amyl the expansive movement is increased by relieving the muscular 
+^ralls of the arteries ; but in angina the pulse is not hard, tnerc being tension, only relaxation of the arterial coats, and the amyl by ex-iting the heart to increased work by its stimulating properties, the blood is thrown into the vessels with increased rapidity, so the ar-erial expansion is increased, but not by diminishing tension, the arteries being put to their full power of expansion, but not beyond it. 

medical/md/PMC5277247.md

@@ -0,0 +1,40 @@
+
+## The Late Thomas Elliot, Esq., Surgeon, Of Carlisle.
+
+TVe record with great regret the death of Mr Thomas Elliot, who for many years deservedly bore the reputation of being one of the most able surgeons in the north of England. 
+
+Mr Elliot received the greater part of his education in Edinburgh, where he attracted the attention of many of the Professors of the University, as a student of more than ordinary promise; he was in particular a favourite pupil of Pro-fessor Syme, and afterwards carried out in his practice the principles which had been impressed upon him in the clinical wards of the Royal Infirmary. Having completed his studies in Edinburgh, Mr Elliot repaired to Paris, where, for two seasons, he diligently availed himself of every opportunity of perfecting his professional knowledge. He devoted himself in particular to anatomical pur-suits, and to the acquirement of a thorough knowledge of operative surgery, availing himself assiduously of the unrivalled advantages which the French capital affords. In 1839, Mr Elliot obtained the diploma of the College of Surgeons of England, and at once settled down in practice in Carlisle, with his brother, the late Dr William Elliot. Mr Elliot from the first devoted himself especially to the practice of surgery ; and having directed especial attention to the subject of squinting, his published observations upon that subject excited much attention, and attracted to him many persons labouring under that deformity. From that time Mr Elliot took the place, which he maintained till his death, of the leading surgeon of the county. Nor was his reputation con-fined to Cumberland; Mr Elliot was highly esteemed and frequently consulted by practitioners in the adjoining counties of Northumberland and Westmore-land, and in the south of Scotland. 
+
+Mr Elliot was not so widely known as many surgeons of far less eminence, for he had contributed but little to medical literature ; though few were better furnished with materials, his published papers and cases were very few. 
+
+Mr Elliot was eminently a conservative surgeon. Though an accomplished and successful operator, his great aim was to cure and preserve diseased and injured parts; in this respect he was a warm admirer and close imitator of his former teacher, Professor Syme. 
+
+Some years ago Mr Elliot, while excising the elbow-joint of a man of a very depraved constitution, received an injury, by the results of which his health for a long time was seriously impaired. He was compelled to retire from practice for many months, and at this period visited Egypt and the East. On his return, his health appeared to be re-established, but he was never so strong as previously ; yet he laboured most assiduously, and we believe went through an amount of bodily and mental exertion which few could have maintained. Mr Elliot was not less beloved for his social and domestic qualities than admired and respected for his professional attainments. 
+
+In the course of last autumn Mr Elliot went into Yorkshire for the benefit of his health; while residing near Leeds, he was attacked with typhoid fever, of which he died on the 18th of October, at the early age of 42 years. His remains were brought to Carlisle, and the funeral took place on the 25th of the same month. It was attended by many of the principal inhabitants of the town, and the members of the medical profession resident in Carlisle preceded the hearse to the grave. Mr Elliot has left behind him a name, which will be long remembered in the north of England, as a distinguished surgeon, and an amiable and philanthropic citizen.
+
+## Death Of Professor Lawrie Of Glasgow.
+
+We have just received intelligence of the death of Dr Lawrie, Professor of Surgery in the University of Glasgow, which took place at Bridge of Allan on the 22d November.
+
+## Varieties.
+
+Appointment. - Cuthbert Collingwood, M.B. Oxon., has been appointed Physician to the Liverpool Northern Hospital.
+
+THE ASTLEY COOPER PRIZE OF L.300.—The physicans and surgeons of Guy's Hospital have awarded the above prize to Dr Crisp of Parliament Street, Westminster, for his essay on " The Structure and Use of the Thyroid Gland.
+
+EDINBURGH UNIVERSITY.—The number of students at the university is considerably larger than it was last year. At the present date (23d November),
+1356 students have been enrolled j last year the total number who matriculated medical students (486) shows an increase of 10. The theological students are enrolled separately, and are consequently not included in the above returns ;
+this year they number between 70 and 80.
+
+NEWS FROM DE LIVINGSTONE —Professor Sedgwick has received a communication from Dr Livingstone, dated " River Zambesi, East Africa, May 27." Dr habitants cultivate cotton very extensively, while he adds, " every one spins and weaves it."
+A NICE POINT. —The Cheltenham Board of Guardians are at loggerheads with Mr Walters, one of their medical officers. He has charged the " amputation fee" for cutting off a portion of a man's thumb, and the Board contend that this is not an "amputation." It was determined the clerk should write to the Poor-law Board to ask " whether the amputation of the first phalanx of the thumb was such an amputation as was contemplated by the Act of Parliament, the Board considering the same to be only a removal of a portion of the thumb."
+DEATH OF A " SAGE FEMNE."—There died recently at Darmstadt, according to by name Charlotte Heildanreich, was born at Stebol, and devoted her attention more particularly to the requirements of young mothers. It is averred that, in 19, she was summoned to England to assist at the birth of Queen Victoria, A great number of the princely and ducal families of Germany are also reported to have, in like manner, had recourse to her aid, as occasion required. " Requiescat in Pace."—Gazette Medicale, July 1859.
+
+QUACKERY IN THE OLDEN TIME (April 1684).—Cornelius a-Tilbourne, a German mountebank, craved from the Privy Council license to crect a stage in Edinburgh.
+
+It was granted, notwithstanding opposition from the College of Physicians.
+
+He had made a successful experiment on himself in London, in presence of the king, for counteracting some poisons which the physicians there had prescribed to him—the secret consisting in drinking a considerable quantity It appears that he expressly excluded mereury, aquafortis, and other of oil.
+
+corrosives, from the trial. The king, who had a curiosity about chemical experi¬

medical/md/PMC5428788.md

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+Open Access Full Text Article REVIEW
+open access to scientific and medical research
+
+# Infrainguinal Open Reconstruction: A Review Of Surgical Considerations And Expected Outcomes
+
+Sevan R Komshian Kimberly Lu Steven L Pike Jeffrey J Siracuse Division of Vascular and Endovascular Surgery, School of Medicine, Boston University Boston, MA, USA Correspondence: Jeffrey J Siracuse Boston Medical Center, School of Medicine, Boston University, 88 East Newton Street, Collamore 5, Boston, MA 02210, USA Email jeffrey.siracuse@bmc.org Abstract: Infrainguinal arterial occlusive disease can lead to potentially disabling and limbthreatening conditions. Revascularization may be indicated for claudication, rest pain, or tissue loss. Although endovascular interventions are becoming more prevalent, open surgeries such as endarterectomy and bypass are still needed and performed regularly. Open reconstruction has been associated with postoperative morbidity, both at the local and at the systemic levels. Local complications include surgical site infections (SSIs 0–5.3%), graft failure (12–60%), and amputation (5.7–27%), and more systemic issues include cardiac (2.6–18.4%), respiratory (2.5%), renal (4%), neurovascular (1.5%), and thromboembolic (0.2–1%) complications. While such outcomes present an additional challenge to the postoperative management of surgical patients, it may be possible to minimize their occurrence through careful risk stratification and preoperative assessment. Therefore, individualized selection of candidates for open repair requires weighing the need for intervention against the likelihood of adverse outcomes based on preoperative risk factors. This review provides an overview of open reconstruction, focusing on identifying the clinical indications for surgery and perioperative morbidity and mortality.
+
+Keywords: infrainguinal bypass, outcomes, risk factors
+
+## Introduction
+
+Peripheral arterial disease (PAD) is one of the leading causes of atherosclerotic morbidity in the US, affecting 8–10 million people.1 As this number rises due to the persistence of cigarette smoking and the aging population, revascularization continues to play an increasingly important role in PAD management when medical therapy is insufficient or when there is critical limb ischemia (CLI).2,3 Although endovascular intervention is becoming more common and is in many cases the first-line treatment, there is still a role for open surgical intervention.4 Anatomic considerations as well as overall patient health and influence of comorbidities provide an integral role in guiding treatment5,6 Deciding on the optimal strategy for revascularization for infrainguinal PAD in symptomatic patients begins with a choice between endovascular and open approaches. While endovascular therapy is less invasive and has fewer perioperative risks, it is generally thought to produce diminished patency when compared with open procedures such as endarterectomy and bypass.4,7–9 Without a definitive indication for one approach over the other, the choice depends on a consideration of patient- and disease-specific factors that stratify risk among operative techniques. In patients with major tissue loss, a good-quality venous conduit, and an anatomic pattern consistent with multilevel disease, defined as Trans-Atlantic Inter-Society Consensus Document on the Management of Peripheral Arterial Disease (TASC) C or D, open intervention 
+
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+| Vascular Health and Risk Management 2017:13 161–168 © 2017 Komshian et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work  you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For  permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).   |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+is often advocated but still remains an issue of contention.10 Nevertheless, patients chosen to undergo open operations experience perioperative mortality in excess of 2% and complication rates remain >20% in some cohorts.11,12 This review aims to explore the current practice of infrainguinal reconstruction by providing an overview of open procedures and their associated outcomes.
+
+## Clinical Indications For Open Revascularization
+
+The decision to perform open repair of occlusive disease often begins with a consideration of the clinical manifestations of PAD. Symptomatic PAD presents as intermittent claudication, rest pain, or tissue loss. Intermittent claudication classically manifests as reproducible lower extremity aching or fatigue on exertion that is relieved with rest. Vascular origin of these symptoms, as opposed to musculoskeletal or neuropathic etiologies, is confirmed by an ankle brachial index <0.9, although this may be falsely elevated if there are calcified vessels.13 Several studies have shown that outside of certain populations, claudication is unlikely to progress to more severe disease involving rest pain in the short term.14–16 Therefore, patient education, exercise therapy, and medical treatment are offered initially, while surgical repair is reserved for individuals who fail conservative and medical therapy or for patients with severe disability.6 Surgical management of patients with PAD is the preferred method of treatment upon the development of CLI, 
+which manifests as rest pain and, in more severe cases, tissue loss and gangrene. Although CLI presents in only 1–3% of PAD patients, its causation of restricted lower extremity tissue perfusion has a broad impact on local and global physiologic processes. This necessitates revascularization to avoid unremitting pain, limb amputation, debilitating infection, and death.17–19 The Bypass versus Angioplasty in Severe Ischemia of the Leg (BASIL) trial has compared the outcomes of the two methods of revascularization.20–22 Despite similar perioperative mortality and higher perioperative morbidity in the surgery group, data indicate improved outcome in open surgical intervention in patients expected to survive more than 2 years due to higher amputation-free survival and lower all-cause mortality when compared with endovascular methods. The BASIL trial was limited by a lack of advanced endovascular treatment and hemodynamic monitoring. The Best Endovascular versus Best Surgical Therapy in patients with Critical Limb Ischemia (BESTCLI) trial, which is currently enrolling, seeks to clarify which patients will benefit from open versus endovascular options.23 Open surgical options include endarterectomy, often the treatment of choice for the common femoral artery (CFA) and bypass.
+
+## Common Femoral Endarterectomy (Cfe)
+
+Endarterectomy is the most common modality to revascularize the CFA and the profunda femoris artery in the treatment of PAD. Although CFE was previously the gold standard for artherosclerotic lesions of the CFA, there has recently been a shift toward endovascular therapy for revascularization of the CFA.4,7 Endovascular therapy has shown decreased patency but similar perioperative morbidity and mortality for common femoral disease.8,9,24 However, endovascular therapies may compromise blood flow to the profunda femoris, which originates at the femoral bifurcation. Endovascular stents at the CFA are also susceptible to fracture and kinking due to frequent flexion and movement at the inguinal ligament.25,26 Stenting in the CFA has been associated with high risk of intervention in a recent study, although there remains a lack of consensus with conflicting published data.24,27,28 The American College of Surgeons' National Surgical Quality Improvement Project database demonstrated that CFE is well tolerated by patients with PAD, showing a 30-day mortality rate of 1.5% and at least one complication in 7.9% of patients. The most common complications were superficial surgical site infection (SSI; 
+6.3%), urinary tract infection (1.7%), and deep vein thrombosis (DVT; 0.5%).29 These rates were comparable to those seen in endovascular intervention, revealing similar perioperative outcomes but favorable long-term postoperative outcomes in patients undergoing CFE. When comparing CFE to hybrid procedures involving endovascular repair, no difference in patency was observed between the two groups.30 At 1 year postoperatively, freedom from intervention in the ipsilateral limb is reported as 82% and survival rate as 89% for CFE.
+
+## Bypass And Expected Outcomes
+
+Surgical bypass uses autologous or prosthetic vein grafts to reroute blood flow to poorly perfused distal sites, circumventing vessel occlusions. The efficacy of reconstruction is heavily influenced by the anatomic location of the occlusion, as different arteries face varying hemodynamic stresses dependent on vessel size. Definitions of patency and stenosis can vary depending on the study and the type of intervention (bypass or endovascular). This makes direct comparison of retrospective studies challenging.
+
+## Femoral–Popliteal Disease
+
+Bypass of the femoral and popliteal arteries has generally proven to be durable and successful in suitable patient populations. Maximal hemodynamic supply and minimal risks for infection are achieved by the use of a greater saphenous vein (GSV) graft, producing 5-year patency at 70%.31 However, studies have found that ~40% of patients do not have an ipsilateral vein of sufficient length to use for the procedure.32,33 In the absence of a history for PAD in the other limb, contralateral saphenous vein may be used instead. Arm vein, either single segment or composite, is also an option for conduit if there is not any GSV or if the contralateral leg has PAD and good vein.34 Prosthetic grafts are used in the absence of suitable autologous vessels, although they exhibit mildly lower long-term durability, especially with distal vessel location.35,36 Despite high overall patency, femoral–popliteal bypass surgery is associated with a number of complications including lower extremity lymphedema, wound infection, graft occlusion, respiratory failure, acute kidney injury (AKI), stroke, and cardiovascular accident, contributing to a 30-day morbidity rate of 37%.37,38 Identification and prevention of patient risk factors is essential to minimize such high rates of these perioperative complications.
+
+## Tibial Disease
+
+With high amputation and 1-year mortality rates, patients with infrapopliteal occlusive disease represent a particularly challenging cohort to manage.1,39 Much of this stems from the anatomically distal site of occlusion and necessitates a longer venous conduit when compared with femoral–popliteal disease. The use of autologous veins are preferred and have been shown to produce similar long-term outcomes compared to the more proximal surgeries with 5-year patency rates at 47%.6,40 Meanwhile, the use of spliced or prosthetic grafts has been linked to diminished durability and higher rates of complications, and they may not be ideal for distal reconstruction.41 Furthermore, tibial disease is often associated with diabetes, which may compromise revascularization efforts and negatively impact graft patency.42 Careful selection of patients, use of appropriate operative techniques, and vigilant postoperative management are especially important to ensure clinical success in this cohort.43
+
+## Conduit
+
+The type of conduit used for bypass can heavily influence surgical outcome and should be selected on a patient to patient basis. The GSV is an ideal conduit due to its ease of harvest, vessel length, and inconsequential removal.38 Primary patency rates at 1, 5, and 10 years are 85%, 72%, and 55%, respectively.44 In infrainguinal disease, such durability has proven to be higher than with other conduits. Furthermore, GSV grafts have been associated with superior amputationfree survival in comparison to prosthetic grafts.9,20 In the absence of suitable GSV, other autologous veins such as superficial arm veins may be considered. Although the cephalic and basilic veins may be used as a single segment, they are often combined into a composite conduit to provide sufficient length in the setting of infrainguinal surgery. Single-segment cephalic vein has been shown to have the best durability of the three options.45 One long-term study revealed 5-year patency and limb salvage rates of 55% and 72%, respectively.34 These rates are still above those for comparable prosthetic conduits.46 Prosthetic grafts are used when autologous conduits are not available or suitable. Of these, expanded polytetrafluoroethylene (ePTFE), polyethylene terephthalate (Dacron), and heparin-bonded ePTFE grafts are most common. Generally, their use in infrainguinal bypass has been shown to have inferior results compared to autologous GSV. Four-year patency rates for infrainguinal bypass with ePTFE and GSV are 54% and 76%, respectively.47 Prosthetic grafts to tibial vessels have particularly poor outcomes, with 36-month primary patency rates at 20% and limb salvage rates of 55%.41 Meanwhile, heparin-bonded grafts have demonstrated improved longterm outcomes and show promise as a prosthetic option.48,49
+
+## Surgical Site Complications
+
+While patency is the primary measure of long-term success for bypass revascularization, it can be affected by a number of perioperative complications at the site of surgical repair that compromise technical success and quality of life (Table 1). Although there are a number of local complications, SSI, graft occlusion, and limb loss will be reviewed here. Each complication discussed is associated with risk factors that should be considered prior to surgery to avoid poor outcomes (Table 2).
+
+SSIs occur frequently in infrainguinal bypass in the 30-day perioperative window, presenting in as many as 11% of cases regardless of bypass origin.50 The common pathogens involved include *Staphylococcus epidermidis* (37%), methicillinsensitive *Staphylococcus aureus* (26%), and *Enterococcus*
+(10%).51 SSI often presents as graft infection. Reported rates of graft infection after bypass involving the femoral artery are 3.8%. Independent risk factors, such as female sex, diabetes mellitus, active infection at the time of bypass, and redo bypass, are shown to be associated with graft infection. In patients with prosthetic grafts, the rates of graft infection Komshian et al
+
+| Table 1 Postoperative complications Complications Surgical site Infection*   | 0–5.3%37    |
+|------------------------------------------------------------------------------|-------------|
+| Graft failure**                                                              | 12–60%80    |
+| Amputation**                                                                 | 5.7–27%81   |
+| Systemic Cardiovascular*                                                     | 2.6–18.4%66 |
+| Respiratory failure*                                                         | 2.5%69      |
+| Renal failure*                                                               | 4%71        |
+| Cerebrovascular*                                                             | 1.5%11      |
+| Venous thromboembolism*                                                      | 0.2–1%76    |
+| Death*                                                                       | 0–6.3%82    |
+| Notes: *30-Day postoperative range. **1-Year postoperative range.            |             |
+
+associated with the above risk factors are 6.8%, 6.5%, 11.7%, 
+and 11.5%, respectively. As prosthetic grafts are susceptible to infection, autologous conduits for infrainguinal bypass or endovascular revascularization are recommended in highrisk patients.51 Although graft infections may not increase postoperative mortality, they often contribute to lower limb amputation and may require removal or attempts at rescue with vacuum-assisted devices and rotational muscle flaps.52–55 Bypass vessels are often monitored for occlusion at regular intervals postoperatively.56 Over time, venous conduits are prone to undergo neointimal hyperplasia and ultimately vessel restenosis caused by hemodynamic stresses of the physiological environment.57 This is particularly true for prosthetic grafts, as discussed previously. Other factors predisposing to neointimal thickening include patient hypertension, diabetes, and hyperlipidemia58 Meanwhile, infrainguinal bypass patients are susceptible to developing graft occlusions due to enhanced local hypercoagulability in the perioperative setting, with rates reaching up to 4.5%.59 Various patient- and surgery-specific predictors for perioperative graft hypercoagulability include age, preoperative ambulatory status, advanced PAD, long duration of surgery, and intraoperative local trauma. Technical errors such as small vein diameter, inappropriate conduit selection, and graft length >50 cm are also associated with early graft failure.33 Impending graft failure can be identified using clinical symptoms, segmental blood pressures, and particularly duplex ultrasound.60 When performed early postoperatively and assessed alongside aforementioned risk factors for graft occlusion, duplex ultrasound may be useful in identifying grafts at high risk of failure.61 Occlusion may necessitate graft revision, which can be accomplished through either endovascular methods or a second open intervention. Open reintervention has been shown to be more durable than endovascular repair in thrombosed grafts undergoing salvage, with 12-month amputation rates of 75% and 56%, respectively 
+(p=0.006).62 Furthermore, endovascular-revised grafts require higher rates of reintervention to maintain patency, although they are less invasive.8 After lower extremity bypass, due to graft failure, persistent infection, or necrosis, amputation may still be necessary. Overall, 1-year postoperative freedom from major amputation and amputation-free survival has been reported as 87% 
+and 76%, respectively.63 Despite patent bypass grafts, some patients do not achieve limb salvage, and studies have demonstrated similar 1-year amputation rates in patients with patent 
+(6.3%) and occluded (5.7%) grafts.64 Thus, identification of patient comorbidities contributing to amputation in patent grafts in addition to prevention of graft failure is essential in limiting limb loss. Patients with diabetes, end-stage renal disease, previous amputations, peripheral neuropathy, preoperative gangrene, postoperative SSI, and abnormal wound healing are at an elevated risk for amputation.64,65 For this 
+
+| Table 2 Preoperative risk factors for poor outcomes Surgical site complications Adverse systemic events Graft infection Female sex, diabetes mellitus, active infection at time of  bypass, and redo bypass.51 Graft failure Acute: age, preoperative ambulatory status, advanced PAD,  long duration of surgery, and intraoperative local trauma.59 Chronic: prosthetic graft, hypertension, diabetes, and  hyperlipidemia.58 Cardiovascular Dependent functional status, smoking history, diabetes, heart failure, prior  percutaneous coronary intervention or cardiac surgery, angina ≤1 month before  surgery or myocardial infarction ≤6 months, and emergent case status.12 Respiratory Smoking status, recent MI, CHF, COPD requiring oxygen, and low body weight.69,70 Renal failure Male sex, CHF, diabetes, and angiotensin-converting enzyme inhibitor or angiotensin  receptor blocker use.71 Cerebrovascular accident Preoperative ventilation, previous CVA, postoperative MI, and return to operating  room.73 Venous thromboembolism Dependent functional status, metastatic cancer, surgical site infection, postoperative  CVA, preoperative ventilation, and return to operating room.76 Abbreviations: PAD, peripheral arterial disease; MI, myocardial infarction; CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; CVA,  cerebrovascular accident.   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+cohort, management of the underlying conditions is vital to improved outcomes postoperatively.
+
+## Adverse Systemic Events
+
+Infrainguinal bypass is also associated with certain adverse systemic events including, but not limited to, cardiovascular injury, respiratory failure, and death (Table 1). As for local outcomes, a number of patient characteristics and comorbidities have been shown to predict increased global morbidity and mortality (Table 2).
+
+Of all global morbidity associated with infrainguinal bypass, cardiac complications are the most common, occurring in 6.3% of patients.66 Much of this is tied to the similarity in the pathophysiology of PAD and cardiac events, where patients undergoing lower extremity bypass are prone to the same risk factors for cardiac injury. Such factors include dependent functional status, smoking history, diabetes, heart failure, prior percutaneous coronary intervention or cardiac surgery, angina ≤1 month before surgery or myocardial infarction (MI) ≤6 months, and emergent case status.12 The predisposition for cardiac injury often manifests during the physiologic stress caused by surgery, resulting in cardiovascular accident such as MI, arrhythmia, and congestive heart failure (CHF). Prognostication tools such as the Revised Cardiac Risk Index can be used to assess probability for perioperative cardiac events and determine a patient's indication for surgery.67 This type of risk stratification allows for better informed consent, closer postoperative monitoring, and better assessment of outcome retrospectively. Antiplatelet and antilipid therapies have not been shown to decrease the risk for perioperative cardiovascular accident.68 Postoperative respiratory events, defined as pneumonia and reintubation after initial extubation, are also prevalent in the perioperative window following infrainguinal bypass. Respiratory events are present in 2.5% of patients and are more likely to occur in patients who smoke, had a recent MI, have symptomatic CHF, and have chronic obstructive pulmonary disease (COPD) requiring oxygen.69 Furthermore, respiratory adverse events are also tied to low body weight, indicating inadequate nutrition.69,70 Thus, patient diet may signify a possible point of intervention in prevention of postoperative respiratory events. If left uncompensated, respiratory events often progress to death and are a major cause of postoperative mortality.
+
+Infrainguinal bypass patients not on dialysis preoperatively have been shown to be at risk of developing AKI. 
+
+AKI has been reported at rates of 4% perioperatively and is especially prevalent in patients predisposed to perioperative prescription of nephrotoxic medications such as antibiotics, systemic hypotension, and renal vessel hypoperfusion as a result of atherosclerosis.71 Thus, management of patient volume status and renal perfusion is vital in preventing AKI and subsequent renal failure. Furthermore, development of AKI is associated with various preoperative demographics and comorbidities including male sex, CHF, diabetes, and angiotensin-converting enzyme inhibitor or angiotensin receptor blocker use.71 Patients with postoperative AKI have a 90% increased risk of developing chronic renal insufficiency requiring life-long dialysis dependence and are also more likely to experience cardiovascular accident and mortality.71,72 Cerebrovascular accident (CVA) including stroke and transient ischemic attack may occur perioperatively in 1.5% of patients.11 As for cardiac complications, many of the risk factors for CVA are the same as those that contribute to the development of infrainguinal occlusive disease. Independent predictors for CVA include preoperative ventilation, previous CVA, postoperative MI, and reoperation.73 CVA following infrainguinal bypass is generally embolic in etiology, and cardioprotective medications such as antiplatelet therapy and statins have been shown to reduce occurrence of CVA in this cohort.74,75 Appropriate medical prevention is critical because CVA results in markedly increased disability and mortality in bypass patients.73 Venous thromboembolism (VTE) occurs in 1% of infrainguinal bypass patients within the first 30 days postoperatively, with DVT shown to present at higher rates than pulmonary embolism (PE).76 Postoperative venous duplex scanning is only recommended in symptomatic patients.77 However, perioperative anticoagulation prophylaxis is normally offered in bypass patients and especially for those with increased predisposition for postoperative VTE. Risk factors include dependent functional status, metastatic cancer, SSI, postoperative CVA, preoperative ventilation, and return to operating room.76 The total rate of 30-day perioperative mortality following infrainguinal bypass is 2.9%, and the majority of deaths result from major organ system failure caused by sepsis (33%), cardiac complications (44%), and pulmonary complications (38%).12 Furthermore, perioperative mortality is exceedingly high in patients who experience postoperative cardiac arrest requiring cardiopulmonary resuscitation (CPR), with rates at 73%.78 These relatively high mortality rates in the perioperative setting, combined with the frequency of the aforementioned comorbidities, has increased the favorability of less invasive endovascular procedures over bypass in high-risk patients. Decreased perioperative mortality, adjusted for comorbidities, of endovascular procedures compared to bypass supports this movement.79 However, there are still concerns about durability.
+
+## Conclusion
+
+Open reconstruction remains an important tool in the treatment of infrainguinal occlusive disease. However, open repair can be associated with local and systemic complications, and the identification of patient- and disease-specific risk factors is critical in selecting appropriate methods of surgical management. High-risk patients may warrant an increased level of postoperative monitoring in the intensive care unit (ICU) as preoperative coronary and carotid interventions have not been shown to be of benefit. The use of less invasive percutaneous methods is an important consideration in high-risk patients if adequate results can be obtained.
+
+## Disclosure The Authors Report No Conflicts Of Interest In This Work. References
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+49. Heyligers JMM, Lisman T, Verhagen HJM, Weeterings C, de Groot PG, Moll FL. A heparin-bonded vascular graft generates no systemic effect on markers of hemostasis activation or detectable heparin-induced thrombocytopenia-associated antibodies in humans. *J Vasc Surg*. 2008;47(2):324–329; discussion 329.
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+53. Dosluoglu HH, Loghmanee C, Lall P, Cherr GS, Harris LM, Dryjski ML. Management of early (<30 day) vascular groin infections using vacuum-assisted closure alone without muscle flap coverage in a consecutive patient series. *J Vasc Surg*. 2010;51(5):1160–1166.
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+Stonebridge PA. Development of a decision tree to streamline infrainguinal vein graft surveillance. *Ann Vasc Surg*. 2016;36:182–189.
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+
+2012;43(4):420–425.
+
+## Vascular Health And Risk Management
+
+Publish your work in this journal Vascular Health and Risk Management is an international, peerreviewed journal of therapeutics and risk management, focusing on concise rapid reporting of clinical studies on the processes involved in the maintenance of vascular health; the monitoring, prevention and treatment of vascular disease and its sequelae; and the involvement of Submit your manuscript here: https://www.dovepress.com/vascular-health-and-risk-management-journal metabolic disorders, particularly diabetes. This journal is indexed on PubMed Central and MedLine. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.
+
+## Dovepress

medical/md/PMC5489838.md

@@ -0,0 +1,23 @@
+Meeting III.?January 9, 1901. 
+
+Dr Milne Murray, President, in the Chair. 
+
+II. Br J. W. Ballantyne showed diagrams to illustrate a scheme of foetal growth. 
+
+III. Br Haig Ferguson showed a fibkoid uterus removed by abdominal hysterectomy. The case was interesting from two circumstances?first, as regards the symptoms, and secondly, as regards the subsequent history. The patient was unmarried, and 38 years of age. She was exsanguine from severe haemorrhage which resisted all treatment, and she suffered in addition from pressure symptoms affecting chiefly the nerves of the lower extremities, and producing great pain, tingling, and slight paresis. The tumour grew from the posterior wall of the uterus, and though not very large it could not be raised off the sacral nerves so as to mitigate the pressure. The patient was rendered absolutely unfit for anything in consequence of all this, she could not sleep for pain, and she could walk or stand very little. Some fifteen years ago Dr Ferguson had seen a similar case where the pressure of a large fibroid in the pelvis first led to paralysis in the lower extremities, and the patient ultimately died of multiple cerebro-spinal sclerosis, apparently from upward extension of the nerve irritation. 
+
+Secondly, as regards the subsequent history, the operation was not a, difficult one and the patient did well to begin with. The bowels moved in due course and the patient began to enjoy food. The pulse and temperature were uneventful. The patient expressed herself as being free from pain and feeling better than she had done for months. Four days after the operation she suddenly complained of faintness, her pulse became almost imperceptible, and she died shortly afterwards. 
+
+No examination was made except of the abdomen; the abdominal wound was well on in the healing process, there was no peritonitis, and no hemorrhage or serum in the peritoneal cavity. Everything was as normal as could be. Death was therefore evidently due to heart failure, or embolism. Most probably it was heart failure, as she had a mitral systolic murmur, and was much exhausted generally before operation with hemorrhage and continued pain. Dr Ferguson showed the specimen as he thought it was well to bring forward cases which showed uncommon manifestations and did not present the usual and fortunately most frequent result of uneventful recovery.
+
+IV.—ON INTRA-PERITONEAL RUPTURE OF SIMPLE
+OVARIAN CYSTS, WITH SPECIAL REFERENCE TO
+OPERATIVE TREATMENT.
+
+By F. W. N. HAULTAIN, M.D., F.R.C.P.Ed., Lecturer on Midwifery and Diseases of Women, School of Medicine, Edinburgh.
+
+THE spontaneous rupture of simple ovarian tumours, though a comparatively uncommon accident, is a subject worthy of more consideration than has hitherto been devoted to it. In the course of the operations I have performed for these growths, I
+have met with three cases in which the cyst wall was torn and the contents evacuated into the peritoneal cavity, while in three other cases a distinct history of previous rupture was elicited.
+
+When considered in detail, individually and collectively, they present points of sufficient practical importance to warrant attention, I trust, worthy of the Fellows of this Society, and it is on this assumption I take the opportunity of bringing the subject before you. The cases are as follows:—
+CASE I.—Mrs C., set. 53, I saw in 1892 with Dr M'Donald Robertson. She had complained for over a year of an abdominal swelling, for which she had refused operative treatment. One dy on poking the fire she suddenly experienced an intense pain in the abdomen, which gradually increased. On examination twenty-four hours afterwards, the abdomen appeared diffusely distended, and was so tender that palpation could only be E

medical/md/PMC5525466.md

@@ -0,0 +1,106 @@
+# Diagnostic Yield And Safety Of Closed Needle Pleural Biopsy In Exudative Pleural Effusion
+
+Govind Singh Rajawat, Supreet Batra1, Rajendra Prasad Takhar2**, Lalit Rathi, Chand Bhandari, Manohar Lal Gupta**
+Department of Chest and Tuberculosis, SMS Medical College, Jaipur, 2*Department of Respiratory Medicine, Government Medical College,* Kota, Rajasthan, 1Department of Respiratory Medicine, VP Chest Institute, University of Delhi, New *Delhi, India*
+
+![0_image_0.png](0_image_0.png)
+
+## Abstract
+
+Background: Closed pleural biopsy was previously considered a procedure of choice in cases of undiagnosed pleural effusion with good efficacy. Currently, the closed pleural biopsy has been replaced by thoracoscopic biopsy but not easily available in resource‑limited setups. 
+
+Objective: The objective of this study was to analyze the diagnostic yield and safety of closed needle pleural biopsy in exudative pleural effusion and assessment of patients' characteristics with the yield of pleural biopsy. **Design:** This was a cross‑sectional study. **Settings:** This study was conducted at Institute of Respiratory Diseases, SMS Medical College, Jaipur, a tertiary care center of West India. **Patients and Methods:** A total of 250 cases of pleural effusion were evaluated with complete pleural fluid biochemical, microbiological, and cytological examination. Out of these 250 patients, 59 were excluded from the study as the diagnosis could be established on initial pleural fluid examination. The remaining (191) patients were considered for closed pleural biopsy with Abrams pleural biopsy needle. Main Outcome **Measures:** The main outcome measure was diagnostic yield in the form of confirming diagnosis. **Results:** Out of the 191 patients with exudative lymphocytic pleural effusion, 123 (64.40%) were diagnosed on the first pleural biopsy. Among the remaining 68 patients, 22 patients had repeat pleural biopsy with a diagnostic yield of 59.9%. The overall pleural biopsy could establish the diagnosis in 136 (71.20%) patients with pleural effusion. The most common diagnosis on pleural biopsy was malignancy followed by tuberculosis. **Conclusions:** Closed pleural biopsy provides diagnostic yield nearly comparative to thoracoscopy in properly selected patients of pleural effusions. In view of good yield, low cost, easy availability, and very low complication rate, it should be used routinely in all cases of undiagnosed exudative lymphocytic pleural effusion. 
+
+Limitations: There was no comparison with a similar group undergoing thoracoscopic pleural biopsy.
+
+Key words: Closed pleural biopsy, malignant pleural effusion, thoracoscopic pleural biopsy, tubercular pleural effusion
+
+## Introduction
+
+Pleural effusion is one of the most commonly encountered clinical conditions in day‑to‑day pulmonary practice. In spite of good history, thorough clinico‑radiological, and laboratory investigations of aspirated fluid, it is not possible to establish the diagnosis in all the cases. Many of the patients often receive treatment empirically without any confirmatory diagnostic documentation.
+
+Before the availability of thoracoscopy, closed pleural biopsy was used as a standard practice to diagnose such patients, and the diagnostic efficacy of pleural biopsy in such situation has been reported between 60% and 80%.[1‑4] With the easy This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms.
+
+For reprints contact: reprints@medknow.com Address for correspondence: Dr. Rajendra Prasad Takhar, Quarter No 1/4, Medical College Campus, Kota, Rajasthan, India. E‑mail: drrajtakhar@gmail.com Cite this article as: Rajawat GS, Batra S, Takhar RP, Rathi L, Bhandari C, 
+Gupta ML. Diagnostic yield and safety of closed needle pleural biopsy in exudative pleural effusion. Avicenna J Med 2017;7:121-4.
+
+© 2017 Avicenna Journal of Medicine | Published by Wolters Kluwer - Medknow 121 availability of thoracoscopy, currently, the thoracoscopic biopsy is recommended in patients with undiagnosed pleural effusion replacing the closed pleural biopsy.[5] It is true that thoracoscopy has better yield than pleural biopsy, but such recommendation is not tenable in resource‑limited settings like our country because of scarce infrastructure and available expertise for thoracoscopy. Few previous studies have shown that the diagnostic yield of closed pleural biopsy can be improved by repeating the procedure.[6,7] Therefore, we planned this study to know the combined yield of the first and, if required, a repeat pleural biopsy in undiagnosed exudative pleural effusion and factors associated with diagnostic yield.
+
+## Patients And Methods
+
+This was a yearlong cross‑sectional study conducted at Institute of Respiratory Diseases, SMS Medical College, Jaipur  (India). The study protocol was approved by the Research and Review Board of SMS Medical College, Jaipur. Two hundred and fifty patients of pleural effusion were evaluated. After giving full explanation regarding the study, written informed consent was obtained from the patients. The entire study participants were evaluated in detail with history taking, physical and radiological examinations, and routine investigations. When examinations and investigations indicated a clear cause of effusion, no further workup was done  (e.g.,  patients presented with bilateral effusion in a clinical setting strongly suggestive of transudates were not enrolled in our study [unless there were atypical features or they fail to respond to therapy]). If no clear cause of pleural effusion was found, a diagnostic thoracocentesis was done, and aspirated fluid was evaluated for cell count, biochemistry, acid‑fast bacilli smear, and cytopathology for malignant cells. When these investigations of pleural fluid failed to establish the diagnosis, it was labeled as undiagnosed pleural effusion, and patients were subjected to pleural biopsy with Abrams pleural biopsy needle. The Abrams needle was inserted after making a small scalpel incision in the properly anesthetized skin and subcutaneous tissue under all aseptic precautions. Once the tip of the needle is thought to be in the pleural space, the inner stylet was removed, and the biopsy needle is then slowly withdrawn with constant aspiration until it hooks onto the pleura. The outer trocar is held firmly with one hand while the inner cannula is rotated into the closed position with the other hand to cut off a small piece of parietal pleura. Exclusion criteria for pleural biopsy were age  <12  years, noncooperative and/or moribund patient, pleural fluid thickness <3 cm on ultrasonography at the infrascapular border, patients with bleeding diathesis, transudative effusion, empyema[8]/neutrophilic effusion, and local skin infection. Those patients who had negative first pleural biopsy were asked to undergo repeat pleural biopsy. After taking consent, repeat pleural biopsy was done by similar procedure.
+
+## Study Design
+
+This was a tertiary care hospital‑based, cross‑sectional study. Assuming sensitivity of diagnosis at level one and two, which is around 70%, the sample size was calculated at a level of 0.05 and study power of 80%, thus minimal sample size obtained was 172 patients of pleural effusion. Hence, finally, we decided to include 250 patients of pleural effusion in our study. Patients' demographic characteristics are shown in Table 1.
+
+## Results
+
+Out of the total 250 patients, 59 were excluded from the study as the diagnosis could be established before closed pleural biopsy [Table 2]. The remaining  (191) patients were considered as having undiagnosed pleural effusion on initial evaluation and were subjected to closed pleural biopsy. To obtain four 
+
+| Table 1: Patients' demographic characteristics Patients and  Total Male                                                                                                                                                                                                                                                                                                    | Female      |             |             |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|-------------|-------------|
+| characteristics Number                                                                                                                                                                                                                                                                                                                                                     | 191         | 138         | 53          |
+| Mean age±SD                                                                                                                                                                                                                                                                                                                                                                | 52.12±16.42 | 50.60±17.72 | 55.13±15.56 |
+| Smoker Yes/no                                                                                                                                                                                                                                                                                                                                                              | 97/94       | 86/52       | 11/42       |
+| Side of effusion Unilateral/bilateral                                                                                                                                                                                                                                                                                                                                      | 177/14      | 126/12      | 51/2        |
+| Color of effusion Straw/hemorrhagic                                                                                                                                                                                                                                                                                                                                        | 124/67      | 94/44       | 30/23       |
+| Extent of effusion Mild/moderate/massive                                                                                                                                                                                                                                                                                                                                   | 10/132/49   | 10/98/30    | 0/34/19     |
+| Position of mediastinum Central/opposite/ipsilateral                                                                                                                                                                                                                                                                                                                       | 109/76/6    | 81/52/5     | 28/24/1     |
+| SD: Standard deviation Table 2: Etiology of effusion established on initial workup Etiological diagnosis established Number of patients Sputum positive for AFB smear 5 Pleural fluid positive for AFB smear 6 Cytology positive for malignant cells 9 Chylothorax 2 Transudative effusion 18 Parapneumonic effusion 11 Empyema 8 Total patients 59 AFB: Acid-fast bacilli |             |             |             |
+
+122  / Issue 3 / July‑September 2017 satisfactory pleural biopsy samples, the average number of needle passes was 4.52 per patient. The first pleural biopsy yielded pleural tissue in 186 (97.38%) patients. The yield of the first pleural biopsy was 64.40%. In spite of pleural tissue obtained on biopsy procedure, diagnosis could not be established in 63 patients on histopathological examination, while pleural biopsy failed to provide pleural tissue in 5/191 (2.62%) patients. Sixty‑eight patients had negative first pleural biopsy, out of which 22 patients could be subjected for repeat pleural biopsy (35 patients did not give consent, 7 lost to follow‑up, and 4 had partial resolution of pleural effusion). Out of 22 patients who had repeat pleural biopsy, 13 had definitive histopathological diagnosis. The yield of repeat pleural biopsy was 13/22 (59.09%). Hence, after repeat pleural biopsy, combined yield of closed pleural biopsy was 136/191 (71.20%). Out of the total 191 patients who underwent closed pleural biopsy, 63  patients were diagnosed as having tuberculosis  (TB), 71  patients as metastatic carcinoma, and 2  patients as Non‑Hodgkin's lymphoma  [Table  3]. In those 68 nondiagnostic pleural biopsy reports, one patient's report showed eosinophilic infiltration of the pleural tissue. Moreover, with the help of this, this patient was finally diagnosed as a case of Churg‑Strauss syndrome (rare disease). TB and metastatic carcinoma were the two most common and nearly equally distributed etiological diagnoses on the first closed pleural biopsy. However, repeat closed pleural biopsy in those with negative first pleural biopsy showed proportionately more patients with metastatic carcinoma than TB.
+
+## Discussion
+
+Closed pleural biopsy has been considered a valuable tool for the diagnosis of exudative pleural effusion. However, after the availability of thoracoscopy, the value of closed pleural biopsy has been downgraded. The BTS guidelines[5] released in the year 2010 recommend that thoracoscopic biopsy should be the next procedure after initial inconclusive diagnostic pleural aspiration in suspicious cases of malignancy, and Abrams needle biopsies are only diagnostically useful in areas with a high incidence of TB. However, in their early guidelines[9] in 2003, they advised thoracoscopic pleural biopsy only after initial negative closed pleural biopsy.
+
+In a country like India, a large number of patients with both TB and malignancy present similarly with pleural effusion. As per the BTS guidelines,[5] half of these patients should be subjected for thoracoscopic pleural biopsy without considering closed pleural biopsy. Considering the number of patients and availability of infrastructure and expertise, there is a large gap between what is recommended and what is really available, not only in our country but also in most of the developing world. In this context, yield of the first pleural biopsy (64.40%) which further improved on repeat pleural biopsy to 71.20% in our study is meaningful. This figure would have gone higher if all patients with the first negative pleural biopsy could have been subjected to repeat pleural biopsy. Our results of repeat pleural biopsy are higher in comparison to the studies by Chakrabarti et al.
+
+[6] and Basu et al.
+
+[7] To the best of our knowledge, this is the largest single‑center study from India of pleural biopsy and a repeat pleural biopsy if the first attempt was unsuccessful. The yield of closed pleural biopsy was analyzed with the age, sex, smoking habits, and extent of effusion, color of effusion, duration of disease, and shift of the mediastinum. We did not find any positive association between the yield and the demographic and other characteristics of pleural fluid. However, unilateral effusion was found to be significantly associated with positive yield of pleural biopsy than bilateral pleural effusion (P < 0.05). Possible reasons for such variability could be that some of the patients with bilateral effusion might be having systemic disease or long‑standing transudative effusion. The etiology of effusion was correlated with patients' characteristics, and it was found that age above 50 years, smoking background, female gender, and hemorrhagic effusion were significantly associated with malignant etiology. We observed that chances of malignant etiology on pleural biopsy increased in incremental order when patients were having more than one of these factors. Chances of malignant etiology increased from 13.15% to 83.33% when the risk factor increased from 1 to 4 whereas only 1 out of 34 patients, who was below 50 years of age, male gender, never smoker, and with straw color effusion, had malignancy on pleural biopsy.
+
+| Table 3: Etiological diagnosis after pleural biopsy procedure Etiological diagnosis First pleural biopsy (n=191) Repeat pleural biopsy (n=22)   | Total patients, n=191 (100%)   |    |             |
+|-------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------|----|-------------|
+| Tuberculosis                                                                                                                                    | 60                             | 3  | 63 (32.98)  |
+| Metastatic carcinoma                                                                                                                            | 61                             | 10 | 71 (37.17)  |
+| Non-Hodgkin lymphoma                                                                                                                            | 2                              | 0  | 2 (1.04)    |
+| Total                                                                                                                                           | 123                            | 13 | 136 (71.20) |
+| Avicenna Journal of Medicine / Volume 7 / Issue 3 / July‑September 2017                                                                         | 123                            |    |             |
+
+Considering the workload of TB and malignant effusion to the institutions, it is difficult to offer pleural biopsy to all those eligible patients, leave aside the option of thoracoscopic pleural biopsy. Therefore, considering the constraints and workload, one can offer the pleural biopsy to these patients  (age above 50  years, smoker, female gender, and hemorrhagic effusion) and others can be put on empirical antitubercular treatment. In case of persistence, recurrence of effusion, or other signs favoring malignancy, pleural biopsy to establish the etiological diagnosis can be considered in follow‑up. In our study, four patients developed small pneumothorax and three had pain at biopsy site after closed pleural biopsy. No patient required intercostal drainage tube for pneumothorax. These seven complications  (3.28%) were occurred after 213 attempts of closed pleural biopsy (191 attempts for the first pleural biopsy and 22 attempts for repeat pleural biopsy). Viskum and Enk[10] reported complication rates of 7%–8% in a series of 566 thoracoscopy examinations. The results of our study clearly show that closed pleural biopsy has good diagnostic yield (near comparable to thoracoscopic pleural biopsy) in a selected population. Yield also increased after repeat pleural biopsy, hence considering the yields of repeat pleural biopsy in our study, closed pleural biopsy should also be considered before thoracoscopic pleural biopsy. The relative ease of performance, obvious advantage over open biopsy, and lack of any significant complication should prompt its more frequent use in Indian centers. Provided that adequate training is given, blind pleural biopsy appears to be well tolerated by a population who often have a poor performance status, short life expectancy, and comorbidities. Therefore, the BTS guidelines[5,9] are not perfectly tenable in our setup, and one should not forget the usefulness of the simple procedure which can be performed even in a sick patient on bedside.
+
+## Conclusions
+
+In the diagnostic workup of pleural effusion, closed pleural biopsy provides a high diagnostic yield in the diagnosis of pleural TB and malignancy. Given low cost, easy availability, and low complication rates, closed pleural biopsy should always be considered as an initial diagnostic tool in the workup of exudative pleural effusion. Considering the relative yields of repeat pleural biopsy in our study, closed pleural biopsy should also be considered before thoracoscopic pleural biopsy. A higher diagnostic yield of thoracoscopic pleural biopsy should always be weighted in the context of available resources, expertise, and morbidity of patients.
+
+Acknowledgment We would like to acknowledge all the support staff involved in the care of patients.
+
+Financial support and sponsorship Nil.
+
+Conflicts of interest There are no conflicts of interest.
+
+## References
+
+1. Poe RH, Israel RH, Utell MJ, Hall WJ, Greenblatt DW, Kallay MC. 
+
+Sensitivity, specificity, and predictive values of closed pleural biopsy. Arch Intern Med 1984;144:325‑8.
+
+2. Suri JC, Goel A, Gupta DK, Bhatia A. Role of serial pleural biopsies in the diagnosis of pleural effusions. Indian J Chest Dis Allied Sci 1991;33:63‑7.
+
+3. Kettel LJ, Cugell DW. Pleural biopsy. JAMA 1967;200:317‑20. 4. Tomlinson JR, Sahn SA. Invasive procedures in the diagnosis of pleural disease. Semin Respir Med 1987;9:30‑6.
+
+5. Hooper C, Lee YC, Maskell N; BTS Pleural Guideline Group. Investigation of a unilateral pleural effusion in adults: British Thoracic Society pleural disease guideline 2010. Thorax 2010;65 Suppl 2:ii4‑17.
+
+6. Chakrabarti B, Ryland I, Sheard J, Warburton CJ, Earis JE. The role of Abrams percutaneous pleural biopsy in the investigation of exudative pleural effusions. Chest 2006;129:1549‑55.
+
+7. Basu A, Chakrabarti I, Ghosh N, Chakraborty S. A clinicopathological study of tuberculous pleural effusion in a tertiary care hospital. Ann Trop Med Public Health 2012;5:168‑72.
+
+8. Levine H, Cugell DW. Blunt‑end needle biopsy of pleura and rib. Arch Intern Med 1962;109:516‑25.
+
+9. Antunes G, Neville E, Duffy J, Ali N; Pleural Diseases Group, Standards of Care Committee, British Thoracic Society. BTS guidelines for the management of malignant pleural effusions. Thorax 2003;58 Suppl 2:ii29‑38.
+
+10. Viskum K, Enk B. Complications of thoracoscopy. Poumon Coeur 1981;37:25‑8.
+
+124  / Issue 3 / July‑September 2017 

medical/md/PMC5859793.md

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+
+
+![0_image_2.png](0_image_2.png)
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_3.png](0_image_3.png)
+
+![0_image_5.png](0_image_5.png)
+
+## Introduction Material And Methods
+
+| Article history: Received 2 February 2017 Received in revised form 16 March 2017 Accepted 18 March 2017 Available online 20 July 2017   |
+|-----------------------------------------------------------------------------------------------------------------------------------------|
+
+| Keywords: Persistent wound drainage Total joint arthroplasty Benign wound drainage Nonoperative treatment   |
+|-------------------------------------------------------------------------------------------------------------|
+
+Brief communication Michael S. Reich, MD, Kace A. Ezzet, MD *
+Division of Orthopaedic Surgery, Scripps Clinic, La Jolla, CA, USA
+abstract http://dx.doi.org/10.1016/j.artd.2017.03.009
+
+![0_image_1.png](0_image_1.png)
+
+2352-3441/© 2017 The Authors. Published by Elsevier Inc. on behalf of The American Association of Hip and Knee Surgeons. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+Arthroplasty Today 4 (2018) 71e73 Persistent wound drainage after total joint arthroplasty can potentiate periprosthetic joint infection. Although current recommendations are to treat persistent wound drainage with surgical debridement, we believe nonoperative treatment may be successful in selected patients. We performed a retrospective analysis of 25 persistently draining hip and knee arthroplasty wounds treated with a protocol consisting of a combination of surgical site aspiration, closure of open wound edges, cessation of anticoagulants, activity modification, and antibiotics (in select patients). Wound drainage ceased in 24 of 25 wounds treated with this protocol. One patient who continued to drain for 3 more days was successfully treated with surgical debridement and evacuation of hematoma. No patient developed infection. We believe this protocol can be successful in many arthroplasty patients.
+
+© 2017 The Authors. Published by Elsevier Inc. on behalf of The American Association of Hip and Knee Surgeons. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
+licenses/by-nc-nd/4.0/).
+
+the index procedure. Although this represented the majority opinion of the expert panel, many participants did not agree with the consensus opinion, including 23% who did not agree that surgical intervention was required on wounds still draining for 5-7 days after surgery [5]. This lack of consensus suggests that even with the best available evidence, among experts within the field of arthroplasty there exists considerable variability in practices.
+
+Although halting persistent wound drainage is a necessity, it is well recognized that surgical debridement is not a minor procedure and exposes the patient to the morbidity and costs of one or more additional operations, including the risk of infecting a wound that was not already infected. Here, we describe a single surgeon's experience with a nonoperative treatment protocol for patients with persistent postoperative wound drainage after total hip (THA)
+and total knee arthroplasty (TKA).
+
+Persistent wound drainage has been shown to be a risk factor for development of periprosthetic joint infection (PJI) after total joint arthroplasty [1-4]. Although nonoperative treatment/passive observation of wound drainage may be successful in the early postoperative phase [4,5], persistent drainage is typically treated with surgical debridement. Although these generalities are well accepted, there lacks a consensus as to how long a draining wound can be safely observed. Additionally, data are lacking regarding the effectiveness of active nonoperative strategies for halting wound drainage in the early weeks following total joint arthroplasty.
+
+Despite lacking good data, algorithms are still needed for managing postoperative wound drainage. The International Consensus Meeting on Periprosthetic Joint Infection recommended that surgical irrigation and debridement with modular component exchange be considered for wounds still draining for 5-7 days after Institutional review board approval was obtained for this study, which aims to document the effectiveness of a nonsurgical protocol for managing draining THA and TKA wounds. Our protocol included surgical site aspiration, closure of open areas of the wound in the office, cessation of anticoagulants, activity reduction, and select use of antibiotics in some patients. All patients in this study underwent TKA or THA by the lead author between 2008 and 2016 and presented in the acute postoperative period with persistent wound One or more of the authors of this paper have disclosed potential or pertinent conflicts of interest, which may include receipt of payment, either direct or indirect, institutional support, or association with an entity in the biomedical field which may be perceived to have potential conflict of interest with this work. For full disclosure statements refer to http://dx.doi.org/10.1016/j.artd.2017.03.009.
+
+* Corresponding author. 10666 N. Torrey Pines Rd., MS 116, La Jolla, CA 92037, USA. Tel.: þ1 858 554 9068.
+
+E-mail address: Ezzet.Kace@scrippshealth.org
+
+# A Nonsurgical Protocol For Management Of Postarthroplasty Wound Drainage
+
+journal homepage: http://www.arthroplastytoday.org/
+
+![0_image_4.png](0_image_4.png)
+
+article info 72 drainage beyond postoperative day (POD) \#5. Wounds that appeared grossly infected on clinical examination were not entered into this protocol.
+
+The initial step in the protocol consisted of aspirating the wound to rule out infection and decompress the pressure of the underlying hematoma. If the aspirate was positive for infection, the patient was dropped from this protocol and taken to surgery for operative debridement. For draining knees, a standard arthrocentesis was performed. For draining hips, the patient was placed in a lateral decubitus position and the subcutaneous hematoma was aspirated under any palpable area of fullness, or at the distalposterior end of the incision if no areas of fullness were palpated, as this area tends to collect the most serosanguinous fluid. Small areas of wound dehiscence, if identified, were closed with nylon sutures and/or Steri-Strips (3M, St. Paul, MN). If no areas of wound dehiscence were identified, but a focal area of the incision appeared to be the source of drainage, we applied Dermabond (Ethicon, Somerville, NJ) to the area from which the drainage seemed to occur. In total, 17 patients had some method of facilitated wound closure as follows: Dermabond alone (4), Steri-Strips alone (3), sutures alone (2), sutures and Dermabond (2), sutures and Steri-Strips (1), Dermabond and Steri-Strips (4), sutures, Dermabond, and Steri-Strips (1).
+
+Antibiotic prophylaxis was administered in 16 patients with drainage who either had a cloudy aspirate or had slight erythema suggesting possible cellulitis. Anticoagulants for deep vein thrombosis prevention were discontinued until the wound was felt to be stable (4 patients). Physical therapy was temporarily put on hold until the wound was dry (8 patients). Knee immobilizers were used for some draining TKAs at the surgeon's discretion. All patients were contacted the following work day. If the drainage had not ceased or substantially improved by 24 hours after administration of the measures listed, the patient was considered to have failed the protocol and was taken to surgery for wound debridement. For wounds that had substantial improvement but not full cessation of drainage after 24 hours, we continued daily telephone follow-ups or office visits until the wound was completely dry, up to 7 days. Patients' charts, including office notes and laboratory data, were retrospectively reviewed.
+
+The patient cohort receiving this protocol consisted of 24 consecutive patients with 25 draining wounds. Mean age was 67.7 ± 10.4 years and 66.7% patients were women. Average body mass index was 34.3 ± 7.5 kg/m2 and 29.2% had a BMI 40.0 kg/m2.
+
+There were 12.5% diabetic or prediabetic patients, 12.5% with chronic kidney disease, and 8.3% current smokers. Surgeries included 11 THAs, 13 TKAs, and 1 revision THA.
+
+## Results
+
+Mean time from surgery to clinic presentation for drainage was 12.8±4.4 days postoperatively. The earliest presentation to our clinic for persistent drainage was on POD \#6 (N ¼ 2). No patient treated with this protocol developed an infection, and 24 of the 25 wounds had cessation of drainage with this protocol. One THA patient had persistent bloody drainage 3 days after the protocol and was treated with a surgical debridement and subsequently did well. Among the 24 wounds that did not require surgery to stop the drainage, followup has ranged from 6 months to 8 years and no patient demonstrated clinical evidence of PJI or wound complication.
+
+Although we sought to aspirate all wounds, due to logistical limitations and protocol violations, only 19 of the 25 wounds were aspirated. Although cell counts were obtained at the time of aspiration, because the patients are in the early postoperative period we did not find this test to be useful in guiding our management. Mean synovial white blood cell (WBC) count was 3383 WBC/mL (range, 234-20,600 WBC/mL). Polymorphonuclear leukocytes percentages ranged from 21% to 99%.
+
+## Discussion
+
+PJIs are a devastating complication and efforts aimed at minimizing their occurrence are warranted [5,6]. An area of controversy remains how best to manage persistent postoperative drainage so that it does not lead to infection. The current consensus indicates that surgical treatment be considered for drainage persisting more than 5-7 days [5,6], although the data set that generated this consensus opinion is sparse.
+
+Here, we present an alternative treatment regimen. Although we agree that persistent drainage cannot be allowed to continue indefinitely, we do not believe that all draining wounds mandate a surgical solution. The literature has shown that persistent wound drainage is associated with increased risk of PJI [1-4] and that each day of continued drainage increases the risk of PJI after THA and TKA [1]. However, Jaberi et al [4] demonstrated that the majority
+(72%) of 300 patients with wound drainage more than 48 hours postoperatively were successfully treated with local wound care and prophylactic antibiotics for 2 to 4 days. The authors also found that those who underwent more delayed surgical debridement (POD 22 vs POD 14) were more likely to require explant or chronic antibiotic suppression instead of retention of implants [4]. An important feature of our protocol which was not routinely adopted in the Jaberi study and others looking at postoperative drainage was the inclusion of joint aspiration. By aspirating the wound, we have attempted to rule out infection in conjunction with our nonoperative treatment protocol. In the studies referenced above, and in most other studies that have evaluated draining wounds, infection was not ruled out with aspiration. We contend that many of those draining wounds in these other studies were probably infected while they were being observed, and this may account for the high proportion of treatment failures and subsequent infections in those other series, whereas we did not encounter that problem. Additionally, in our protocol we actively try to arrest the drainage through wound closure techniques, rather than simply observing the wounds.
+
+The treatment protocol presented is admittedly controversial.
+
+Antibiotic use for draining wounds has been discouraged due to concerns of obscuring an indolent infection [5,6], as many draining wounds are draining precisely because they are already infected.
+
+However, by aspirating the site before starting antibiotics, we believe the risk of missing a true infection is low, especially if the protocol is only applied to wounds that clinically appear not infected. Additionally, there is data documenting a benefit in prescribing antibiotics to postoperative patients with superficial infections, in an effort to prevent deep infection [7]. We similarly contend that when evaluating a draining wound with mild to moderate erythema, it can be impossible to differentiate cellulitis from benign postsurgical reactive erythema, and antibiotics seem reasonable in such cases after aspiration.
+
+Our study does have limitations. Although we advocate aspiration before any antibiotics, this is not always feasible as some patients may have been evaluated by another physician and provided antibiotics before the surgeon has had a chance to aspirate the surgical site. Recent antibiotics can increase the risk of falsenegative cultures [8], and surgeons will need to take this into account. Although aspirating the knee joint is straightforward, aspirating the true hip joint requires fluoroscopy, which is generally not available in an outpatient setting. Therefore, when we aspirated the hips in this protocol, we aspirated the subcutaneous hematoma/seroma and make the assumption that all fluid collections under the skin are in confluence. Additionally synovial fluid WBC
+counts are generally not helpful in determining presence of infection as they are known to be high immediately postoperatively.
+
+Nonetheless, it has been proposed that a total neutrophil count
+(synovial fluid WBC multiplied by polymorphonuclear leukocytes percentage) >25,000 is highly suggestive of infection acutely after TKA [9].
+
+There are other limitations inherent to this study. Although this is one of the largest studies to evaluate treatment of noninfected draining arthroplasty wounds, the sample size is relatively small. Therefore, the success we report should be viewed accordingly. Additionally, the protocol evolved over the 8 years under investigation. In the early years of this protocol, closure of any open wound edges required sutures, which are cumbersome, or Steri-Strips, which are less effective. In later years, Dermabond became available. Therefore, all 3 of these adjuncts were used at different time points. Antibiotics were prescribed at the discretion of the surgeon on a case-by-case basis. Additionally, as this is a multifaceted protocol, it is not possible to determine the additive benefit of each step in the protocol. Lastly, although no clinically apparent infections developed in these patients, we recognize that some infections may present years after surgery.
+
+## Conclusions
+
+The draining arthroplasty wound remains a vexing problem.
+
+Careful clinical evaluation coupled with this protocol along with close clinical follow-up may be appropriate in select patients to help reduce the incidence of reoperation and the associated morbidity and cost.
+
+References
+
+[1] Patel VP, Walsh M, Sehgal B, et al. Factors associated with prolonged wound drainage after primary total hip and knee arthroplasty. J Bone Joint Surg Am 2007;89(1):33.
+
+[2] Saleh K, Olson M, Resig S, et al. Predictors of wound infection in hip and knee joint replacement: results from a 20 year surveillance program. J Orthop Res 2002;20(3):506.
+
+[3] Surin VV, Sundholm K, Backman L. Infection after total hip replacement. With special reference to a discharge from the wound. J Bone Joint Surg Br 1983;65(4):412.
+
+[4] Jaberi FM, Parvizi J, Haytmanek CT, Joshi A, Purtill J. Procrastination of wound drainage and malnutrition affect the outcome of joint arthroplasty. Clin Orthop Relat Res 2008;466(6):1368.
+
+[5] Ghanem E, Heppert V, Spangehl M, et al. Wound management. J Arthroplasty 2014;29(2 Suppl):84.
+
+[6] Parvizi J, Gehrke T, Chen AF. Proceedings of the International Consensus on Periprosthetic Joint Infection. Bone Joint J 2013;95-B(11):1450.
+
+[7] Guirro P, Hinarejos P, Pelfort X, et al. Long term follow-up of successfully treated superficial wound infections following TKA. J Arthroplasty 2015;30(1):
+101.
+
+[8] Malekzadeh D, Osmon DR, Lahr BD, Hanssen AD, Berbari EF. Prior use of antimicrobial therapy is a risk factor for culture-negative prosthetic joint infection.
+
+Clin Orthop Relat Res 2010;468(8):2039.
+
+[9] Christensen CP, Bedair H, Della Valle CJ, et al. The natural progression of synovial fluid white blood-cell counts and the percentage of polymorphonuclear cells after primary total knee arthroplasty: a multicenter study. J Bone Joint Surg Am 2013;95(23):2081.

medical/md/PMC5950068.md

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+# Gynaecology And Obstetrics. By E. H. Lawrence Oliphant, M.D.
+
+Sugar Treatment of Uterine Inertia.?Dr. Mosso records, in the Semaine Medicale, 1894, p. 41, eleven cases of uterine inertia during labour. 
+
+These were treated with sugar in doses of one ounce dissolved in water. In a few cases the dose had to be repeated in an hour. In ten cases the result was good, active contractions setting in in from twenty-five to forty-five minutes. 
+
+The sugar seems to excite natural pains, and is not open to the objection urged against ergot, which sets up a continuous tonic contraction. Mosso ascribes the result to the action of sugar in exciting unstriped muscular fibres. 
+
+Version in the Prone Posture.?Dr. Mensinga of Flensburg (quoted in Medizin. Novitaten, November, 1894, from Aerztl. Runsch.) advocates the prone posture in preference to the dorsal for the operation of version. In a case to which he was called Dr. Mensinga attempted version in the ordinary continental dorsal position, but was unable to grasp a knee. Keeping his hand in the uterus, he made the patient turn on to her face and lie on her abdomen. The pillows were drawn down to support the thorax down to the abdomen. He was then able to grasp the knee and complete the version. In a second case he was again able to turn in the prone position after failing with_the patient in the dorsal position. He explains this by stating that the pressure of the patient's weight on the uterus forces down that organ, at the same time pushing down the vagina. The genital canal, including the os uteri, becomes wider at the expense of its length, and the hand is more easily passed through it as it, so to speak, is passed over the hand, while the foetal parts are at the same time pushed down towards the hand. He further finds this posture a useful one for watching the perineum. 
+
+Gangrene of the Vagina from Tincture of the Chloride of Iron.?-Dr. Nammack of New York read a paper on this subject (reported in the New York Medical Journal, August, 1894). He warns the practitioner, against the dangers of expectant treatment in cases of incomplete abortion, advocating the use of the curette and intra-uterine irrigating tube. As a warning against the treatment of the mere symptom of haemorrhage, he relates the following case:?J. K., aged 30, 6-para. Last child two years ago. Since that time periods regular till eight months ago, when they ceased for four months. Haemorrhage then set in, lasting three months, till patient consulted a physician, who plugged the vagina with cotton saturated with the tincture of chloride of iron, using in all four ounces of the tincture. She came to hospital two days later complaining of pain, dysuria, inability to walk, and haemorrhage. On examination she was found anaemic, with rapid, feeble pulse; temperature fluctuating between 100? and 104?, with chills. 
+
+Locally the whole vaginal mucous membrane, including that covering the cervix uteri, was covered with thick blackish sloughs. Douches and boro-glyceride tampons were used, and the dead tissue removed as soon as possible. 
+
+The granulations were dusted with iodoform, and the vagina packed with iodoform gauze. Later, the use of the vaginal glass plug will be necessary to prevent stenosis. Dr. Nammack refers to similar cases recorded where iron chloride and persulphate had been used in strong solutions. 
+
+Palliative versus Surgical Treatment of Rupture of the Uterus.?Dr. Charles M. Green read a paper on this subject before the American Gynaecological Society (reported in the New York Medical Journal, September, 1894). By palliative treatment Dr. Green said he meant sustaining the patient by nourishment and stimulation, the use of antiseptics, and the arrest of haemorrhage by means of packing, and so forth. Laparotomy was attractive, but did not offer the good results that it seemed to do. He reported several cases where recovery had taken place after rupture under palliative treatment. Unfortunately, no details are given in the report before us. He thought it wrong to expose the patient, already in a dangerous condition, to the further shock of an abdominal section, and the fallacious attempt to stop the haemorrhage by suturing the uterus. The tear was usually a separation of the tissues in an uneven jagged manner impossible of apposition, and the tissues were generally so friable that sutures would not hold if inserted. The palliative treatment consisted of compression bandages, cold irrigation, tamponing the seat with wicking and gauze, and drainage. 
+
+Laparotomy should never be attempted unless the issue seemed sure to be fatal, and the child had escaped through the tear into the abdominal cavity, and could not be extracted through the natural passages. Under such circumstances the abdomen should be opened, the child removed, and the uterus sutured. Manipulation and bimanual pressure should be used to ensure uterine contraction. Serious haemorrhage was always present, but a tampon properly applied was usually sufficient. There was no need of irrigating the abdominal cavity through the rent unless the amniotic fluid had been emptied into it. What would be better was the injection of a hot physiological salt solution into the cavity, from which absorption would take place, and the volume of the blood be increased. The peritoneum. was tolerant of aseptic blood, so there was no need to get rid of it at once. 
+
+Laparotomy would be justified where the haemorrhage was uncontrollable. 
+
+In the discussion which followed, the President, Dr. Lusk, speaking from personal experience, said that in incomplete rupture situated in the posterior wall of the uterus there was 110 doubt palliative measures were the proper thing. He had always found that, if the tear was situated anteriorly, the case terminated fatally. Where the rupture was complete, he would feel better if he brought the peritoneum together and closed up the wound ; as for sewing up the uterus, it was a fallacy and a snare. Where the women recovered without having thus much done, they were invalided ; they would seem to do well for a time, but their existence would be shortened. He did not think much of Porro's operation in this class of cases. He advised complete removal of the uterus where the tear was considerable and the tissue much infiltrated, as offering the best results. 
+
+Dr. Polk thought it best to deliver by the natural passages if possible, but also to make a small opening above and cleanse the abdominal cavity. In cases where he had irrigated from the vagina, and subsequently opened the abdomen, he had found the irrigation had not cleansed the peritoneal cavity. 
+
+Dr. Murray related four cases of apparently incomplete rupture?that is, into the broad ligaments, not into the peritoneal cavity. Two had recovered under palliative treatment alone. 

medical/md/PMC6194934.md

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+# Evaluating Alternative Designs Of A Multilevel Hiv Intervention In Maharashtra, India: The Impact Of Stakeholder Constraints
+
+Anik R. Patel, Kelly V. Ruggles, Kimberly Nucifora, Qinlian Zhou, Stephen Schensul, Jean Schensul, Kendall Bryant, and R. Scott Braithwaite MDM Policy & Practice
+
+![0_image_0.png](0_image_0.png) 1–11 - The Author(s) 2018 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/2381468318803940 journals.sagepub.com/home/mdm
+
+## Abstract
+
+Background. Multilevel interventions combine individual component interventions, and their design can be informed by decision analysis. Our objective was to identify the optimal combination of interventions for alcohol-using HIV+ individuals on antiretroviral drug therapy in Maharashtra, India, explicitly considering stakeholder constraints. Methods. Using an HIV simulation, we evaluated the expected net monetary benefit (ENMB), the probability of lying on the efficiency frontier (PEF), and annual program costs of 5,836 unique combinations of 15 singlefocused HIV risk-reduction interventions. We evaluated scenarios of 1) no constraints (i.e., maximize expected value), 2) short-term budget constraints (limits on annual programmatic costs of US$200,000 and $400,000), and 3)
+a constraint stemming from risk aversion (requiring that the strategy has .50% PEF). Results. With no constraints, the combination including long individual alcohol counseling, text-message adherence support, long group counseling for sex-risk, and long individual counseling for sex-risk (annual cost = $428,886; PEF ; 27%) maximized ENMB
+and would be the optimal design. With a cost constraint of $400,000, the combination including long individual alcohol counseling, text-message adherence support, brief group counseling for sex-risk, and long individual counseling for sex-risk (annual cost = $374,745; PEF ; 4%) maximized ENMB. With a cost constraint of $200,000, the combination including long individual alcohol counseling, text-message adherence support, and brief group counseling for sexrisk (annual cost = $187,335; PEF ; 54%) maximized ENMB. With the risk aversion constraint, the same configuration (long individual alcohol counseling, text-message support, and brief group counseling for sex-risk) maximized health benefit. Conclusion. Evaluating the costs, risks, and projected benefits of alternatives supports informed decision making prior to initiating study; however, stakeholder constraints should be explicitly included and discussed when using decision analyses to guide study design. Keywords constrained optimization, decision analytic modeling, economic evaluation, HIV, study design Date received: November 10, 2016; accepted: August 20, 2018 The World Health Organization (WHO) has announced the goal of achieving an AIDS-free generation by 2030.1 Multilevel interventions are particularly useful to manage multiple risk factors facing high-risk people living with HIV/AIDS (PLWHA).2 Through behavioral modification, risk behaviors associated with HIV progression and further transmission can be mitigated. There are an estimated 2.1 million PLWHA in India, and a multilevel intervention is being considered for implementation and/
+or further study.3 The goal of the multilevel intervention is to reduce risks among alcohol-using HIV+ individuals on antiretroviral therapy (ART).
+
+Corresponding Author Anik R. Patel, Department of Medicine, University of British Columbia, 828 West 10th Avenue, Vancouver, British Columbia V5Z IL8, Canada (anikpa@mail.ubc.ca).
+
+Multilevel interventions, which combine several singlefocused interventions, are increasingly utilized in HIV
+prevention and care.4–6 Interventions targeting risk factors such as drug nonadherence, sexual risk-taking, or depression have been developed and can reduce the risk of HIV progression and transmission through improved patient outcomes.7–12 Single-focused interventions may have been evaluated individually but not collectively, raising programmatic questions about their feasibility, effectiveness, scalability cost, and value, when combined.
+
+Ideally, implementation decisions would be based on evidence supported by randomized controlled trials or other studies. However, studying the vast array of permutations of a multilevel intervention is not financially or operationally possible. A decision analysis can project the health and economic outcomes of multilevel interventions under consideration for further study and/or implementation and therefore potentially identify optimal permutations of alternative multilevel intervention designs.13,14 However, in addition to traditional measures of expected value, stakeholders often have constraints that cannot be easily incorporated within a decision analysis framework, such as short-term budget constraints due to near-term financial shortfalls. Therefore, the option with the highest expected value may not be affordable to a health system, thus affecting the options under consideration. Furthermore, decision makers may be risk averse, preferring to have a high level of certainty regarding effectiveness and benefit-to-harm prior to dissemination. Organizational decision makers not only may want to maximize average expected value but may also prefer a higher likelihood that an option is efficient, even if the magnitude of health benefit is smaller than the average.15 Risk information can potentially be a valuable resource to decision makers that are investing in new research projects with many alternatives to choose from.
+
+Based on the stakeholder constraints described above, we sought to optimize a multilevel intervention design for HIV-infected persons in Maharashtra, India, under three scenarios: 1) no constraint (''maximize expected value''), 2) the combination must maximize expected value within short-term budget constraints (''maximize expected value with an annual intervention cost constraint''), and 3) the combinations must maximize expected value with a preference of a high likelihood that it is efficient relative to alternatives (''maximize expected value for a risk-averse stakeholder'').
+
+## Methods Overview
+
+For the purpose of this article, constituent intervention refers to a single-focused intervention addressing an HIV risk factor such as motivational interviewing targeting unhealthy alcohol use or cognitive behavioral therapy targeting depression. Multilevel intervention refers to two or more constituent interventions in combination, and different permutations of these constituent interventions that would result in different configurations of the multilevel intervention. We examined different ways the constituents could be configured to achieve optimal outcomes based on our three scenarios (maximize expected value, maximize expected value with an intervention cost constraint, and maximize expected value for a risk-averse stakeholder). To perform this constrained optimization, we utilized a decision analytic model and applied successive constraints (financial or risk) to evaluate the best choice in each scenario.
+
+## Decision Model
+
+We developed a decision analytic model for the Indian state of Maharashtra that was adapted from a previous HIV simulation model developed for East Africa (Figure 1). The simulation describes HIV progression and transmission dynamics at a population level. Further details describing the progenitor model, including development, parameterization, and calibration, can be found elsewhere.16,17 We revised the model with updated input data from Maharashtra that came from an electronic medical record (EMR) data analysis or published literature. We then calibrated the HIV progression component of the model (''progression module'') to patient-level survival, time to viral failure, and CD4 response data from medical records of Indian HIV patients or published literature.18 We calibrated the transmission component of the model (''transmission module'') to population-level HIV
+
+![2_image_0.png](2_image_0.png)
+
+incidence, prevalence, death, and number of people on treatment based on published data from UNAIDS estimates for India.19 Additional details of model development and calibration are listed in the technical appendix
+(available in the online version of the article). Outcomes of interest included number of HIV transmissions, costs of treatment and care, and quality-adjusted life years (QALYs) within a simulated population of Maharashtra in the next 20 years. We chose a 20-year time horizon because it allowed us to more fully evaluate the long-term implications of the interventions on secondary HIV transmission, and we have found that 20 years is the longest time horizon viewed as credible by most stakeholders. All results were presented at the population level, and we did not allow for partial funding of programs such that the entire target population was assumed to receive all interventions if that package was selected.
+
+## Input Parameters
+
+Model inputs and their uncertainty distribution are listed in Tables 1 and technical appendix Table S4. There were four categories of inputs in this simulation model:
+intervention-related inputs (costs and effectiveness), HIV progression, HIV transmission, and HIV risk behavior inputs. Two main sources were used to estimate inputs or to derive calibration data: state-level EMR data from Maharashtra collected from HIV clinics from 2007 to 2014 (n = 23,701) and literature reviews. The EMR data provided individual-level characteristics for the progression model including CD4 count trajectory over time, survival, and the distribution of individual-level covariates that altered HIV progression. Other risk factors relationships including sexual risk-taking and adherence to ART were derived from the published literature (technical appendix Table S4).
+
+## Constituent Interventions
+
+Constituent interventions were identified from the published literature. The two primary goals of the multilevel intervention are to reduce ART nonadherence and to reduce the risk of HIV transmission. The multilevel intervention could be composed using possible components of individual or group alcohol counseling, individual or group depression counseling, individual or group or
+
+| Table 1                                | Intervention Inputs   |               |           |        |           |           |
+|----------------------------------------|-----------------------|---------------|-----------|--------|-----------|-----------|
+| Intervention                           | Risk Mediator         | Relative Risk | Rangea    | Costb  | Range     | Reference |
+| Brief individual alcohol counseling    | Alcohol use           | 0.68          | 0.50–0.93 | $1.64  | 0.5x–1.5x | 7         |
+| Long individual alcohol counseling     | Alcohol use           | 0.36          | 0.15–0.82 | $6.56  | 0.5x–1.5x | 7         |
+| Brief group alcohol counseling         | Alcohol use           | 0.62          | 0.42–0.91 | $1.64  | 0.5x–1.5x | 7         |
+| Long group alcohol counseling          | Alcohol use           | 0.47          | 0.25–0.86 | $5.90  | 0.5x–1.5x | 7         |
+| Brief individual sex-risk counseling   | Condom use            | 1.15          | 1.03–1.26 | $1.64  | 0.5x–1.5x | 8         |
+| STI prevalence                         | 0.84                  | 0.73–0.96     |           |        |           |           |
+| Long individual sex-risk counseling    | Condom use            | 1.52          | 1.10–2.00 | $14.76 | 0.5x–1.5x | 8         |
+| STI prevalence                         | 0.64                  | 0.44–0.89     |           |        |           |           |
+| Brief group sex-risk counseling        | Condom use            | 1.23          | 1.05–1.41 | $1.64  | 0.5x–1.5x | 8         |
+| STI prevalence                         | 0.81                  | 0.68–0.95     |           |        |           |           |
+| Long group sex-risk counseling         | Condom use            | 1.38          | 1.08–1.70 | $5.90  | 0.5x–1.5x | 8         |
+| STI prevalence                         | 0.71                  | 0.54–0.92     |           |        |           |           |
+| Community sex-risk reduction           | Condom use            | 1.2           | 1.03–1.40 | $6.67  | 0.5x–1.5x | 9         |
+| STI prevalence                         | 0.78                  | 0.59–1.04     |           |        |           |           |
+| Brief individual depression counseling | Depression            | 0.84          | 0.43–1.33 | $13.12 | 0.5x–1.5x | 10        |
+| Long individual depression counseling  | Depression            | 0.62          | 0.28–1.10 | $36.08 | 0.5x–1.5x | 10        |
+| Brief group depression counseling      | Depression            | 0.81          | 0.65–0.97 | $3.61  | 0.5x–1.5x | 10        |
+| Long group depression counseling       | Depression            | 0.71          | 0.58–0.84 | $7.22  | 0.5x–1.5x | 10        |
+| Brief adherence counseling             | ART adherence         | 1.09          | 1.01–1.15 | $2.46  | 0.5x–1.5x | 11        |
+| Weekly SMS support                     | ART adherence         | 1.11          | 1.05–1.16 | $6.56  | 0.5x–1.5x | 11        |
+| STI, sexually transmitted infection.   |                       |               |           |        |           |           |
+
+STI, sexually transmitted infection.
+
+aRange based on 95% confidence interval reported in published reviews. bAnnual cost per person in 2016 US dollars based on labor and pragmatic costs estimates; average time to deliver intervention was derived from published reviews or assumed based on expert opinion.
+
+community sexual risk intervention, brief adherence counseling, or short-message service (SMS) adherence support (Table 1). Additional description of the constituent interventions is listed in the technical appendix.
+
+## Multilevel Intervention Costs And Effectiveness
+
+We abstracted constituent intervention effectiveness data from the individual studies reported in five systematic reviews of randomized trials. Table 1 lists cost and effectiveness of the constituent interventions, along with their sources. We estimated the effectiveness of longer or brief interventions by pooling the results of individual trials that studied long or brief versions of the same intervention. In the simulation, some constituent interventions predominantly addressed patient-level risks, while others predominantly addressed HIV transmission risks. We assumed that two interventions addressing the same risk did not have additive effects and used the higher effect size if they were both bundled together. However, we included the cost of both interventions to the total package cost. This particular assumption was made in order to deliberately bias the analysis toward being conservative (i.e., underestimating rather than overestimating the effect of interventions in combination).
+
+We estimated constituent intervention cost data from the same studies. If time data were published, we used the total time required for the behavioral interventions and hourly counselor wage in India to calculate the cost of labor. We increased the labor costs to account for program administration costs based on a WHO report of average HIV program costs.20 We derived uncertainty distributions from published data when available or used a uniform distribution over a range informed by expert opinion if data were not sufficient. Programmatic staff from India provided insights when assumptions regarding intervention costs were needed. The total time of the individual and group constituents was a function of the number of risk factors addressed. For example, the individual component could focus on alcohol use, depression, and nonadherence, but it would increase the time
+(and cost) to address all three. Additionally, the cost was dependent on whether an intervention was brief (e.g., alcohol motivational interview for 60 minutes) or long (e.g., alcohol motivational interview for 240 minutes).
+
+## Analytical Approach
+
+We compared multilevel interventions for a population of HIV+ males on ART that misuse alcohol in Maharashtra. Based on the combinations of 15 constituent interventions, we iterated 5,836 possible alternative configurations for the multilevel intervention, ranging from single interventions to nine interventions combined together. Long and brief versions of the same intervention would not logically be combined. We used Monte Carlo simulation to draw parameters from prespecified uncertainty distributions during probabilistic analyses
+(PA). We used a health system payer perspective and discounted cost and QALY outcomes at a 3% discount rate based on WHO recommendations.21
+
+## Identifying Optimal Choices
+
+Using probabilistic results, we constructed efficiency frontiers based on the expected value of each option and then calculated the probability of being on the efficiency frontier (PEF) using individual iterations of the PA. An efficiency frontier is plotted relative to the option with the lowest average incremental cost-effectiveness ratio (origin). From the origin, subsequent alternatives are categorized as efficient, dominated, or extendedly dominated until all options have been categorized. The options that are efficient form a frontier that reflects the most efficient options in order of increasing ICER. Options that are dominated or extendedly dominated would not be considered for investment based on economic attributes alone. Through this process, the most efficient interventions can be selected with diminished health benefits as the ICER increases. We used the efficiency frontier technique outlined in Hunink et al and encourage interested readers to reference this book for more details on its calculation.13 The PEF was calculated by deriving a new efficiency frontier for each iteration of a PA and tracking the number of times that a configuration was on the efficiency frontier, an approach that has been previously reported.22 Through this approach, we determined the probability that a given package was efficient relative to alternative packages.
+
+## Stakeholder Scenario 1: Maximize Expected Value
+
+Some stakeholders would make a choice based on efficiency and expected value alone. The most valuable configuration to study would maximize ENMB at a plausible value of willingness-to-pay (WTP) threshold.23,24 WHO guidance previously suggested approximately three times the GDP of a country as a threshold, which was US$4,746 for India in 2014.25 While this threshold rule has been challenged in recent years, the few alternative approaches to decision making have limitations of their own.12 Since the WTP threshold is unknown in India and cannot easily be estimated by looking at past budget-constrained decisions, the old WHO WTP threshold guidance served as a reasonable starting point for decision making. We used WTP thresholds of $5,000/QALY and $15,000/QALY (2016 US dollars) to reflect two potential values for the WTP threshold. The net monetary benefit was calculated using the formula below and the ENMB was the average NMB across the PA iterations.
+
+## Enmb= Ð Þ Qaly Ð Þ- Willingness To Pay Threshold Ð Þ Costs Stakeholder Scenario 2: Maximize Expected Value With A Program Cost Constraint
+
+While maximizing expected value is the ultimate health system goal and every budget should correspond to a societal WTP threshold, unexpected financial constraints in the short term may result in unplanned deviations from resource distributions made in accordance with that WTP threshold. Correspondingly, for the purpose of this article, the ''budget'' represents the real and often unanticipated constraint faced by program managers who seek to implement programs, and may be different from the stated ''WTP threshold,'' which under perfectly efficient circumstances and longer term horizons would be reflective of the overall health budget. Note that, in a perfectly efficient system, it would not be necessary to specify both budget and WTP threshold because the value of one would imply the value of the other. However, we choose deliberately to distinguish between the two, because changes in budget may occur over the short term in unpredictable ways, particularly in specific sectors (e.g., foreign aid might unexpectedly stop),
+whereas a change in the WTP threshold would lag behind. To consider the constraints ''on the ground'' including shorter term decision making and coexisting short-term budget limitations, the optimal options were evaluated when annual program costs were constrained at $200,000 and $400,000, estimated based on the sizes of other simultaneous programmatic budget constraints of the same ministry.
+
+## Stakeholder Scenario 3: Maximize Expected Value For A Risk-Averse Stakeholder
+
+Risk-averse organizational decision makers could choose to forego some expected value for a lower risk option over a higher risk option.15 We specified a heuristic that is a possible expression of risk-aversion: choosing a program with a high likelihood of being efficient is preferred to choosing a program with a lower likelihood of being efficient, even if the alternative program has greater expected value (i.e., greater expected health benefit per incremental cost, during PA). We chose this heuristic in full appreciation of its limitations: a risk-averse entity might prefer a program with 99% likelihood of some benefit (albeit a benefit not as great as one corresponding to the efficient frontier) to a program with \99% likelihood of being on the efficient frontier, even if it has greater benefit, as it is possible that the optimal program for a particular risk attitude is not on the efficient frontier. That being said, our heuristic has the desirable effect of using the efficient frontier as a ''lens'' through which to focus on a tractable subset of the 5000+ interventions with the capability of delivering the greatest value.
+
+To communicate risk and uncertainty, a PEF was used instead of the traditional CEAC (cost-effectiveness acceptability curve).23,26 While the CEAC plots the probability of an option being the most cost-effective option at a specific WTP threshold, it cannot distinguish between alternatives with different variance in their outcomes (i.e., different joint distributions of incremental costs and effectiveness).26 Two alternatives can have the same CEAC if they have the same proportion of their incremental joint distributions above and below a given WTP threshold even if they each have vastly different outcome uncertainty. The PEF uses the incremental joint distribution directly in its calculation, since a new frontier was constructed for each iteration of the PA. This approach reflects the probability that an option will be efficient relative to alternatives. While each organizational decision maker may have a different level of risk tolerance, we used an arbitrary threshold that an option must appear on the efficiency frontier .50% of the time to evaluate the impact of this constraint on the optimal choice. We additionally report the probability of efficiency for each option, so that decision makers could use other values if desired.
+
+## Results
+
+The analyses yielded configurations with contrasts in affordability, outcomes, and risks. The optimal choice depended on which stakeholder constraints were employed. Full results are listed in Table 2, but are discussed by the three scenarios. Of the initial 5,836 configurations, 22 were dominant over the remaining configurations because they were both more effective (higher population-level QALYs) at lower costs than the other configurations. The analytical procedures that yielded this result are described in greater detail in the technical appendix.
+
+## Stakeholder Scenario 1: Maximize Expected Value
+
+Five configurations were on the efficiency frontier and were cost-saving compared to standard care. Stakeholders may consider one these five configurations as possible candidates, but need a precise WTP threshold since the optimal option was dependent on it (Table 1). The optimal configuration of a multilevel intervention at a WTP threshold of $15,000/QALY consisted of long individual alcohol counseling, individual weekly textmessage support, long sex-risk group counseling, and long individual counseling for sex-risk (annual cost =
+$428,886; PEF ; 27%). The optimal configuration of a multilevel intervention at a WTP threshold of $5,000/ QALY consisted of long individual alcohol counseling, individual weekly text-message support, and long sex-risk group counseling (annual cost = $241,476; PEF ; 48%).
+
+## Stakeholder Scenario 2: Maximize Expected Value Within A Program Cost Constraint
+
+Assuming an annual intervention cost constraint of $200,000 (Figure 2A), the combination that maximized ENMB consisted of long individual alcohol counseling, individual weekly text-message support, and brief sex-risk group counseling (annual cost = $187,335; PEF ; 54%). Assuming an annual intervention cost constraint of $400,000 (Figure 2B), the combination that maximized ENMB included long individual alcohol counseling, individual weekly text-message adherence support, brief group counseling for sex-risk, and long individual counseling for sex-risk (annual cost = $374,745; PEF ; 4%), an option that was extendedly dominated in the unconstrained scenario (Figure 2C).
+
+## Stakeholder Scenario 3: Maximize Expected Value With Risk Aversion
+
+Decision makers could impose any level of risk and choose an alternative based on the expected value in conjunction with risk. In the last scenario, the risk tolerance of decision makers was considered (Figure 3A and B). The top choice, assuming the requirement that an option has .50% chance of being on the efficiency frontier, was a combination that included long individual alcohol counseling, individual weekly text-message support, and brief
+
+| Table 2                                            | Health and Economic Outcomes of Top Configurations Arranged in Ascending 1-Year Program Costs 20-Year Population-Level Costs (Thousands) 20-Year Population-Level QALY (Thousands) ENMBb at WTP ofc $5,000/QALY (Thousands) ENMBb at WTPc of $15,000/QALY (Thousands)   | Probability of Being on the Efficiency Frontier (%)   |                |                 |          |       |
+|----------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|----------------|-----------------|----------|-------|
+| One-Year Program Costs                             |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| Multilevel Intervention Compositiona Standard care | $880,835                                                                                                                                                                                                                                                                | 1,474,040                                             | $7,369,320,095 | $22,109,721,955 | $ 0      | 2.30% |
+| I-B-Adh                                            | $880,095                                                                                                                                                                                                                                                                | 1,474,047                                             | $7,369,359,050 | $22,109,837,340 | $31,226  | 1.3%  |
+| G-B-Sex and I-B-Adh                                | $879,742                                                                                                                                                                                                                                                                | 1,474,048                                             | $7,369,364,968 | $22,109,854,388 | $52,044  | 5.3%  |
+| I-SMS-Adh                                          | $880,108                                                                                                                                                                                                                                                                | 1,474,049                                             | $7,369,368,617 | $22,109,866,067 | $83,177  | 2.7%  |
+| I-L-Alc                                            | $878,302                                                                                                                                                                                                                                                                | 1,474,050                                             | $7,369,373,328 | $22,109,876,588 | $83,177  | 19.2% |
+| I-L-Alc and I-B-Adh                                | $877,967                                                                                                                                                                                                                                                                | 1,474,054                                             | $7,369,392,988 | $22,109,934,898 | $114,445 | 25.5% |
+| I-L-Alc, G-B-Sex, and                              | $877,887                                                                                                                                                                                                                                                                | 1,474,054                                             | $7,369,396,238 | $22,109,944,488 | $135,267 | 52.1% |
+| I-B-Adh                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-SMS-Adh and G-L-Sex                              | $880,096                                                                                                                                                                                                                                                                | 1,474,051                                             | $7,369,376,794 | $22,109,890,574 | $158,235 | 1.7%  |
+| I-L-Alc and I-SMS-Adh                              | $878,278                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,397,782 | $22,109,949,902 | $166,512 | 16.1% |
+| I-L-Alc, I-SMS-Adh, and                            | $878,217                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,400,843 | $22,109,958,963 | $187,335 | 53.9% |
+| G-B-Sex                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, G-L-Sex, and                              | $878,314                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,397,651 | $22,109,949,581 | $189,403 | 29.4% |
+| I-B-Adh                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, G-B-Sex, G-L-Sex,                         | $878,537                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,397,632 | $22,109,949,972 | $210,225 | 3.2%  |
+| and I-B-Adh                                        |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-SMS-Adh, and                            | $878,657                                                                                                                                                                                                                                                                | 1,474,056                                             | $7,369,402,142 | $22,109,963,742 | $241,476 | 48.0% |
+| G-L-Sex                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-SMS-Adh, G-B-Sex,                       | $878,883                                                                                                                                                                                                                                                                | 1,474,056                                             | $7,369,402,111 | $22,109,964,101 | $262,299 | 5.3%  |
+| and G-L-Sex                                        |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-SMS-Adh and I-L-Sex                              | $881,218                                                                                                                                                                                                                                                                | 1,474,051                                             | $7,369,377,607 | $22,109,895,257 | $270,664 | 0.7%  |
+| I-SMS-Adh, G-B-Sex, and                            | $881,438                                                                                                                                                                                                                                                                | 1,474,051                                             | $7,369,377,607 | $22,109,895,697 | $291,485 | 0.2%  |
+| I-L-Sex                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-L-Sex, and I-B-Adh                      | $879,521                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,397,629 | $22,109,951,929 | $301,840 | 20.1% |
+| I-SMS-Adh, I-L-Sex, and                            | $881,436                                                                                                                                                                                                                                                                | 1,474,052                                             | $7,369,379,579 | $22,109,901,609 | $301,895 | 0.1%  |
+| I-B-Adh                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-SMS-Adh, and                            | $879,751                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,397,538 | $22,109,952,118 | $322,662 | 1.8%  |
+| I-L-Sex                                            |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-SMS-Adh, G-B-Sex,                       | $879,875                                                                                                                                                                                                                                                                | 1,474,056                                             | $7,369,402,050 | $22,109,965,900 | $353,922 | 46.1% |
+| and I-L-Sex                                        |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, G-L-Sex, I-L-Sex,                         | $880,106                                                                                                                                                                                                                                                                | 1,474,056                                             | $7,369,401,953 | $22,109,966,073 | $374,745 | 4.4%  |
+| and I-B-Adh                                        |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-B-Adh, G-L-Sex,                         | $880,328                                                                                                                                                                                                                                                                | 1,474,055                                             | $7,369,397,342 | $22,109,952,682 | $376,798 | 11.1% |
+| and I-L-Sex                                        |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+| I-L-Alc, I-SMS-Adh, G-L-Sex,                       | $880,687                                                                                                                                                                                                                                                                | 1,474,056                                             | $7,369,401,727 | $22,109,966,557 | $428,886 | 27.1% |
+| and I-L-Sex                                        |                                                                                                                                                                                                                                                                         |                                                       |                |                 |          |       |
+
+aAdh, adherence; Alc, alcohol; B, brief; G, group; I, individual; L, long; QALY, quality-adjusted life year; Sex, sexual-risk; SMS, weekly textmessages.
+
+bExpected net monetary benefit (ENMB)—Option with the highest ENMB is in bold.
+
+cWillingness-to-pay threshold.
+
+sex-risk group counseling (annual cost = $187,335; PEF
+
+## Discussion
+
+; 54%). The risk of being off the efficiency frontier rose along with the number or intensity of constituents across our analyses (Figure 3B). This finding suggested that more intensive options had diminishing health returns at greater risk of inefficiency, thus requiring a tradeoff to be made between expected value and probability of efficiency.
+
+We evaluated alternative designs of a multilevel behavioral HIV intervention by systematically evaluating long-term health and economic outcomes, narrowing the number of permutations under consideration from 5,836 to 22 options. We estimated the annual program costs, PEF, and ENMB. Of the final choices, the configuration with the highest PEF differed from the most affordable
+
+![7_image_0.png](7_image_0.png)
+
+configuration by about $50,000 per year and differed from the configuration with the most health benefits by 671 QALYs.
+
+The efficiency frontier shows five alternatives that could be valuable to implement and/or designate for future study with the sole goal of maximized expected value (Figure 3B). As would be expected, the option that maximized ENMB changed between WTP thresholds of $5,000/QALY and $15,000/QALY. Thus, in cases where a WTP threshold is not well established, additional attributes of interventions should be considered to make appropriate funding decisions about further research or implementations. One consideration is the annual budget constraints of the health system to implement the interventions. Decision makers may choose to impose a limit on annual programmatic costs. Imposing a limit led to the optimal option to include long individual alcohol counseling, individual weekly text-message adherence support, brief group counseling for sex-risk, and long individual counseling for sex-risk, an option that was off the unconstrained efficiency frontier.
+
+A second consideration could be the risk of choosing an option that is not the efficient choice. A notable insight of our analyses is that the expected value could only be improved by allowing substantial uncertainty of efficiency relative to alternatives, a situation unlikely to
+
+![8_image_0.png](8_image_0.png)
+
+be acceptable to risk-averse decision makers. In particular, as the length or number of sexual risk reduction constituents increased, the uncertainty of efficiency also increased. This finding was consistent across our analyses, suggesting that a risk-averse research stakeholder could favor a multilevel intervention with a single brief sexual-risk counseling constituent rather than an intervention with multiple or long sexual-risk counseling constituents. The probability of efficiency can be displayed along with expected value to give research stakeholders a more transparent understanding of the decisions they are making. The presentation of this information emphasized the explicit tradeoff between incremental increases in expected value with increased risk of inefficiency to guide conversations about how thorough and exhaustive the final intervention combination should be.
+
+## Limitations
+
+As with any model, the input data and model structure can have unknown biases or imperfections. We extensively calibrated our simulation using epidemic and surveillance data and conducted a probabilistic sensitivity analysis to limit the bias introduced by uncertainties.
+
+Additionally, there could have been synergies or redundancies when two interventions targeting the same risk factor were combined. We made the simplifying assumption that two or more interventions targeting the same risk had the effect size of the most effective intervention, and we did not consider synergies or redundancies in the base case analysis. But it is important to note that our model may be used even if particular known synergies or redundancies are incorporated into the analysis. To demonstrate this fact, we conducted a deterministic sensitivity analysis where intervention effect sizes were added when two interventions targeting the same risk were paired. The results of this scenario analysis are in the technical appendix. In contrast to the MAX assumption when two interventions targeted the same risk factor, if two interventions targeted the same outcome through different risk factors, their effects were assumed to be additive, and we did not consider any synergies or redundancies. However, we also explored these possibilities in sensitivity analyses (technical appendix Figures S5 and S6).
+
+A third limitation was that evidence about intervention effectiveness came from outside of India, and the evidence had a varying degree of generalizability to the target population. The direction of bias introduced by lack of generalizability remains unknown. To mitigate this risk, we predominantly relied on randomized trial data to inform the intervention effectiveness inputs.
+
+Additionally, expert opinion was needed to make reasonable assumptions for some parameters. The uncertainty distributions were based on the best available information, but bias in the inputs could affect the final results in an unknown direction. Related to this point, while a value of information analysis would be a natural extension of our proposed work because it is unknown a priori whether additional research would be desired prior to investing in the optimal multilevel intervention, we decided not to perform this analysis because the organization would mandate new evidence prior to implementation regardless of our estimated value of information.
+
+This is heightened by concerns of the propriety of generalizing evidence to Maharashtra and/or of numerically representing the underlying uncertainty with sufficient accuracy. An important limitation of our study is that we do not consider a full array of possible constituents in our multifaceted intervention, such as PrEP, which may have substantial implications for HIV prevention.
+
+In conclusion, to aid multilevel intervention design decisions, we identified the optimal configuration of a multilevel intervention while considering potential stakeholder constraints. By considering intervention risks and benefit tradeoffs in advance of a study, the resulting intervention has a higher chance of health system adoption and acceptance after the trial. This type of systematic evaluation could improve the efficiency of the research process and provide insights for transparent discussion of the risks-benefits when research teams are faced with a staggering number of options.
+
+## Acknowledgments
+
+We thank Mark Roberts for feedback and input with this work.
+
+## Supplemental Material
+
+The online supplementary appendix for this article is available on the Medical Decision Making Policy & Practice website at http://journals.sagepub.com/home/mpp.
+
+## References
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+2015/hiv-treat-all-recommendation/en/
+2. World Health Organization. Consolidated Guidelines on the Use of Antiretroviral Drugs for Treating and Preventing HIV Infection. Recommendations for a Public Health Approach. 2nd ed. Geneva: World Health Organization; 2016.
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+Thresholds for the cost-effectiveness of interventions: alternative approaches. Bull World Health Organ. 2015,93(2):
+118–24.
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+13. Hunink MM, Weinstein MC, Wittenberg E, et al. Decision Making in Health and Medicine: Integrating Evidence and Values. 2nd ed. Cambridge: Cambridge University Press; 2014.
+
+14. Briggs A, Sculpher M, Claxton K. Decision Modelling for Health Economic Evaluation. Oxford: Oxford University Press; 2006.
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+15. Kahneman D, Tversky A. Prospect theory: an analysis of decision under risk. Econometrica. 1979;47(2):263–92.
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+16. Kessler J, Ruggles K, Patel A, et al. Targeting an alcohol intervention cost-effectively to persons living with HIV/ AIDS in East Africa. Alcohol Clin Exp Res. 2015;39(11): 2179–88.
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+17. Braithwaite RS, Nucifora KA, Yiannoutsos CT, et al.
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+Alternative antiretroviral monitoring strategies for HIVinfected patients in east Africa: opportunities to save more lives? J Int AIDS Soc. 2011;14(1):38.
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+Lim PL; TREAT Asia HIV Observational Database.
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+Impact of drug classes and treatment availability on the rate of antiretroviral treatment change in the TREAT Asia HIV Observational Database (TAHOD). AIDS Res Ther. 2007;4:18.
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+19. Jamison JC, Karlan D, Raffler P. Mixed-Method Evaluation of a Passive mHealth Sexual Information Texting Service in Uganda (Working Paper 19107). Cambridge:
+National Bureau of Economic Research; 2013.
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+20. Johns B, Baltussen R, Hutubessy R. Programme costs in the economic evaluation of health interventions. Cost Eff Resour Alloc. 2003;1(1):1.
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+21. World Health Organization. Choosing effectiveness and strategic planning (WHO-CHOICE) [cited July 11, 2016]. Available from: http://www.who.int/choice/en/
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+Open Access Full Text Article
+
+# Review Chromogranin A Is A Predictor Of Prognosis In Patients With Prostate Cancer: A Systematic Review And Meta-Analysis
+
+This article was published in the following Dove Press journal: Cancer Management and Research Zhenlang Guo1 Yuliang Wang2 Songtao Xiang3 Shusheng Wang3 Franky Leung Chan2 1The Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, People's Republic of China; 2School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China; 3Department of Urology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, People's Republic of China Correspondence: Shusheng Wang Department of Urology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Dade Road No. 111, Yuexiu District, Guangzhou, Guangzhou 510120, People's Republic of China Email shushengwanggzy@163.com Franky Leung Chan Lo Kwee-Seong Integrated Biomedical Sciences Building, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China Tel +8 523 943 6841 Fax +8 522 603 5031 Email franky-chan@cuhk.edu.hk Background: The prognostic value of chromogranin-A (CHGA) as a biomarker of prostate cancer (PCa) has been evaluated extensively. However, to date the results still remain controversial. This study aims to perform a meta-analysis on previous studies in order to determine whether CHGA would be a biomarker for survival in PCa patients.
+
+Methods: MEDLINE, Embase, Web of Science, and Cochrane Library databases were searched to identify eligible studies published before September 2018, regarding the association of CHGA gene expression with survival outcomes in patients with PCa. Multivariate adjusted HRs and associated 95% CIs were calculated using random effects models.
+
+Results: Ten cohort studies involving 3,172 patients were finally included. According to the included studies, circulating CHGA levels were tested in serum, plasma, and tissues. The results showed an association between high CHGA expression and worse overall survival
+(OS) (HR=1.24, 95% CI: 1.07–1.44; P=0.004; I
+2=77.6%) in PCa patients. However, no significant association was observed between increasing CHGA expression and shorter progression-free survival (HR=1.73, 95% CI: 0.92–3.28; P=0.090; I
+2=73.9%). The results of sensitivity analysis validated the rationality and reliability of our analysis.
+
+Conclusion: Current evidence indicates that high CHGA expression is a potential marker for poor OS in PCa. Future studies are needed to explore tailored treatments that directly target CHGA for the improvement of survival in men with PCa.
+
+Keywords: prostate cancer, chromogranin-A, prognosis, survival, meta-analysis
+
+## Background
+
+Prostate cancer (PCa) is the second leading cause of cancer death in men with a worldwide annual mortality of over 200,000.1–3 The first symptom occurs mostly during the progression of PCa when it cannot be treatable anymore. Therefore, early detection of PCa in asymptomatic men is the key to reducing mortality. Prostatespecific antigen (PSA)-based screening detects substantial clinically insignificant patients with PCa.4,5 However, most of these asymptomatic patients are treated unnecessarily and subject to side effects of treatment. Therefore, population-based PSA screening is not recommended by current guidelines.6 In order to distinguish between indolent and high-risk PCa, reliable biomarkers are needed for the better prognostication of PCa.
+
+Chromogranin-A (CHGA) is a common glycoprotein expressed in neuroendocrine cells. Neuroendocrine activity can also be detected in other tumors except for neuroendocrine tumors, such as prostate and breast tumors.7–10 One study demonstrated that CHGA levels are significantly elevated in patients with castration-resistant prostate cancer
+(CRPC) in immunohistochemical studies.11 As tumors develop in neuroendocrine tissues, they become the main source of circulating CHGA which can be detected in blood and tissues.12,13 Moreover, elevated circulating CHGA levels have been confirmed to be a helpful biomarker for the diagnosis of different types of neuroendocrine tumors.14–17 Some researches have shown that high serum CHGA levels are associated with advanced stage disease and poor prognosis,18,19 but its practicability and prognostic value in clinically localized PCa is still debatable.
+
+Several studies have suggested that the high mRNA
+expression of CHGA is related to worse survival, but other studies did not reach these conclusions.20–23 We thus performed a meta-analysis to reveal the prognostic significance of CHGA expression for overall survival (OS) and progression-free survival (PFS) in patients with PCa.
+
+## Materials And Methods
+
+This study was performed in accordance with the Cochrane Collaboration criterion.24 For reporting, we followed the PRISMA statement guidelines.25 Thus, no ethical approval and patient consent are required.
+
+## Literature Search
+
+To identify eligible studies, we performed a comprehensive literature search in the electronic databases of MEDLINE, Embase, Web of Science, and Cochrane Library databases for eligible studies regarding the association of CHGA expression with survival outcomes in PCa from database inception up to September 2018. Each database was searched without restrictions to languages, publication types, or regions using the following combination of Medical Subject Headings (MeSH) and non-MeSH search terms:
+("chromogranin A" OR "parathyroid secretory protein" OR "CHGA") AND ("prostate cancer" OR "prostatic cancer" OR "prostate neoplasm" OR "prostatic neoplasm"). The main search was completed by the senior author (ZLG). Any discrepancy was resolved through consultation of an investigator (SSW) not involved in the initial procedure (as shown in Supplementary Material).
+
+## Inclusion And Exclusion Criteria
+
+Two independent investigators (ZG and YW) selected eligible studies regarding the association of CHGA expression with survival outcomes in PCa without publication status or language restrictions in accordance with the following inclusion criteria: 1) diagnoses were confirmed as PCa by pathology; 2) original trials regarding the association of CHGA expression with survival outcomes (eg, OS, PFS, etc.) in PCa; and 3) studies reporting sufficient data of risk estimates with corresponding 95% CIs or enough data to calculate them directly were eligible for inclusion. If more than one study was identified from the duplicated database, we retained only the most recent or largest study to avoid duplication of information. The exclusion criteria were as follows: 1) reviews, letters, conference papers, case reports, case series, and expert opinion articles were not eligible for inclusion; 2) absence of detailed results; and 3) animal studies. Any disagreement was resolved through adjudication of senior authors.
+
+## Data Extraction And Methodological Quality Assessment
+
+Two reviewers (ZG and SX) extracted the data independently using a predefined data extraction form, and discrepancies were settled through a consensus discussion. The following data were extracted into a standardized evidence table: first author, year of publication, country, study design, baseline population characteristics (ie, mean age and sample size), duration, adjusted factors, and HRs with corresponding 95% CIs for survival in each comparisons. We also checked these data for accuracy. Moreover, we contacted, if possible, the primary authors of studies with insufficient information to acquire and verify the data.
+
+The quality and risk of bias of the included cohort studies were evaluated by two independent reviewers (FLC and SW) separately according to the Newcastle-Ottawa Scale
+(NOS).26 Disagreements were also settled through discussion among authors. The standards consist of 10 items that assess the representativeness of the included studies. Each item was evaluated as "yes," "no," or "unclear" corresponding, respectively, to "1," "0," or "0" according to the information provided by the studies. The total score ranged from 0 to 10 with the overall score categorized as follows: 7 to 10:
+"high quality," 4 to 6: "moderate quality," and 0 to 3: "low quality."
+
+## Statistical Analyses
+
+For meta-analysis, the total risk estimate of extracted data was pooled using HRs with associated 95% CIs through the STATA statistical package (version 14.0; serial number: 10699393; StataCorp Wyb) to determine the association of CHGA expression with survival outcomes in PCa.
+
+For consistent definitions, HRs with corresponding 95%
+CIs were used as a common measure in the included studies because CHGA expression in PCa was considered as a rare event. We could generally ignore the distinctions among the various measures of risk estimates. Thus, the ORs values in the observational studies were considered as approximations of HRs.27 The aggregated results and 95%
+CIs for effect size were calculated using inverse-variance weighted meta-analysis. The I-square (I
+2) test was performed to assess the effect of study heterogeneity on the meta-analysis results, with I
+2 values of 0%, 25%, 50%, and 75% representing no, low, moderate, and high heterogeneity, respectively. Based on the Cochrane review guidelines,24 a severe heterogeneity of I
+2≥50% warrants the use of random-effects models. Otherwise, a fixedeffects model is utilized. Statistical significance was set at P<0.05. Subgroup analyses were performed in accordance with different countries, study designs, and chromogranin-A level measurements. Sensitivity analysis was conducted by deleting each study individually to evaluate the quality and consistency of the results. A metaregression analysis was conducted to investigate possible sources of heterogeneity on certain variable. The restricted maximum likelihood method was used for analysis.
+
+Finally, Egger's and Begg's tests were performed to assess publication bias, and the funnel plot symmetry was examined.28,29
+
+## Results Study Selection Process
+
+A flow chart depicts the search process and study selection
+(as shown in Figure 1). A total of 665 studies were identified through our comprehensive search of several electronic databases. Only 594 studies were retrieved after removal of duplicates. After screen the titles and abstracts, only 28 studies remained. Finally, a total of 18 full-text articles were discarded for the following reasons:
+
+![2_image_0.png](2_image_0.png)
+
+survival was not the end point (6 studies), men did not match the PCa definition (3 studies), duplicated database (1 studies), and no sufficient data for extraction (8 studies).
+
+Therefore, 10 cohort studies20–23,30–35 comprising 3,172 participants were included in our meta-analysis in compliance with the inclusion criteria.
+
+## Study Characteristics And Methodological Quality
+
+The basic characteristics of the included studies20–23,30–35 are described in Table 1. These studies (7 retrospective cohort studies20,21,23,30–33 and 3 prospective cohort studies22,34,35) were published between 2005 and 2018 and involved 3,172 participants. In total, 10 eligible cohorts with 3,172 patients were analyzed for OS
+outcomes,20–23,30–35 and 3 qualified studies with 139 patients were analyzed for PFS outcomes.21,31,35 The sample sizes varied from 39 to 1,018. Among the included studies, five were conducted in Italy,20–22,30,31 two in Germany,33,35 two in USA,32,34 and one in UK.23 Furthermore, serum CHGA levels were measured in six studies,21–23,31,32 tissue CHGA levels were measured in one study20 and plasma CHGA levels were measured in three studies,30,33,34 respectively. All studies were published in English,20–23,30–35 and nine studies20,21,23,30–35 provided adjusted HRs and one22 reported ORs for survival, accounting for confounding factors. Moreover, the
+
+| Table 1 Characteristics of the included studies First author, year Study design (duration) Country Study characteristics                                                                                                                                                                                                                                                                                                                                                                                                              | Adjustments                                                                | CHGA levels                         | Diseases     |                                    |        |      |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------|-------------------------------------|--------------|------------------------------------|--------|------|
+| measurements                                                                                                                                                                                                                                                                                                                                                                                                 | stage                                                                      |                                     |              |                                    |        |      |
+| Berruti et al,                                                                                                                                                                                                                                                                                                                                                                                               | Retrospective                                                              |                                     |              |                                    |        |      |
+| 200530                                                                                                                                                                                                                                                                                                                                                                                                       | cohort (1998–2003)                                                         | Italy                               | 108 males,   | PSA, serum ALP, Hb, Gleason score, | Plasma | CRPC |
+| 74 (58–86) y                                                                                                                                                                                                                                                                                                                                                                                                 | serum ALB, and serum LDH                                                   |                                     |              |                                    |        |      |
+| Berruti et al,                                                                                                                                                                                                                                                                                                                                                                                               | Retrospective                                                              |                                     |              |                                    |        |      |
+| 201020                                                                                                                                                                                                                                                                                                                                                                                                       | cohort (1996–2003)                                                         | Italy                               | 414 males,   | Gleason score, serum PSA, disease  | Tissue | PC   |
+| 69 (43–91) y                                                                                                                                                                                                                                                                                                                                                                                                 | stage, and local treatments                                                |                                     |              |                                    |        |      |
+| Burgio et al,                                                                                                                                                                                                                                                                                                                                                                                                | Retrospective                                                              |                                     |              |                                    |        |      |
+| 201431                                                                                                                                                                                                                                                                                                                                                                                                       | cohort (2011–2012)                                                         | Italy                               | 48 males,    | Gleason score, serum PSA, and age  | Serum  | CRPC treated with      |
+| 73 (57–90) y                                                                                                                                                                                                                                                                                                                                                                                                 | abiraterone                                                                |                                     |              |                                    |        |      |
+| Conteduca                                                                                                                                                                                                                                                                                                                                                                                                    | Retrospective                                                              | Italy                               | 39 males,    | Gleason score, serum PSA, and age  | Serum  | CRPC treated with      |
+| et al, 201436                                                                                                                                                                                                                                                                                                                                                                                                | cohort (NA)                                                                | 75 (43–91) y                        | enzalutamide |                                    |        |      |
+| De Nunzio                                                                                                                                                                                                                                                                                                                                                                                                    | Prospective                                                                |                                     |              |                                    |        |      |
+| et al, 201422                                                                                                                                                                                                                                                                                                                                                                                                | cohort (2006–2012)                                                         | Italy                               | 1,018 males, | Age, PSA, DRE, and prostate volume | Serum  | PC   |
+| 68 (62–74) y                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                            |                                     |              |                                    |        |      |
+| Giridhar                                                                                                                                                                                                                                                                                                                                                                                                     | Retrospective                                                              |                                     |              |                                    |        |      |
+| et al, 201832                                                                                                                                                                                                                                                                                                                                                                                                | cohort (2002–2009)                                                         | USA                                 | 256 males,   | Age, Gleason score, serum PSA, disease stage, and local treatments                                    | Serum  | CRPC treated with      |
+| 72 (65–77) y                                                                                                                                                                                                                                                                                                                                                                                                 | abiraterone                                                                |                                     |              |                                    |        |      |
+| Jeetle et al,                                                                                                                                                                                                                                                                                                                                                                                                | Retrospective                                                              |                                     |              |                                    |        |      |
+| 201223                                                                                                                                                                                                                                                                                                                                                                                                       | cohort (1990–1996)                                                         | UK                                  | 806 males,   | Gleason score and serum PSA        | Tissue | PC   |
+| >76 y                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                            |                                     |              |                                    |        |      |
+| Niedworok                                                                                                                                                                                                                                                                                                                                                                                                    | Retrospective                                                              |                                     |              |                                    |        |      |
+| et al, 201733                                                                                                                                                                                                                                                                                                                                                                                                | cohort (2003–2004)                                                         | Germany                             | 110 males,   | Gleason score, serum PSA, disease  | Plasma | PC   |
+| 66 (49–86) y                                                                                                                                                                                                                                                                                                                                                                                                 | stage and, local treatments                                                |                                     |              |                                    |        |      |
+| Taplin et al,                                                                                                                                                                                                                                                                                                                                                                                                | Prospective                                                                |                                     |              |                                    |        |      |
+| 200534                                                                                                                                                                                                                                                                                                                                                                                                       | cohort (1996–1998)                                                         | USA                                 | 321 males,   | Demographics, metastases, Gleason  |        |      |
+| 70 (65–75) y                                                                                                                                                                                                                                                                                                                                                                                                 | score, performance status, disease assessment, prior therapy, suramin dose | Plasma                              | CRPC         |                                    |        |      |
+| Germany                                                                                                                                                                                                                                                                                                                                                                                                      | 52 males, 71.3±7.2                                                         | Gleason score, Charlson comorbidity | Serum        | CRPC treated with                                    |        |      |
+| y                                                                                                                                                                                                                                                                                                                                                                                                            | index, age, and local treatments                                           | docetaxel                           |              |                                    |        |      |
+| Abbreviations: ALB, albumin; ALP, alkane phosphatase; CHGA, chromogranin-A gene; CRPC, castration-resistant prostate cancer; DRE, digital rectal examination; Hb, hemoglobin; LDH, lactate dehydrogenase; OS, overall survival; PC, prostate cancer; PFS, progression-free survival; PSA, prostate-specific antigen; NA, not available; y, years. von Hardenberg et al 201735 Prospective cohort (2013–2015) |                                                                            |                                     |              |                                    |        |      |
+
+duration and the mean age of patients were also detailed in Table 1.
+
+In addition, the methodological quality was evaluated in accordance with NOS.23 It was found that eight studies20,23,31–35 got 8 points, respectively, one study22 got 6 points, and one study21 obtained 5 points. In all, the methodological quality of eight studies was considered of high quality,20,23,31–35 and two studies were evaluated as moderate quality.21,22 The main deficiency was the selection bias related to the insufficient adjustment of clinical tumor stage and initial treatment modality among the included studies.
+
+## Chga Expression And Os In Pca
+
+Ten cohort studies involving 3,172 patients provided the data on OS in PCa.20–23,30–35 The results showed an association between high CHGA expression and worse OS
+(HR=1.24, 95% CI: 1.07–1.44; P=0.004) in PCa.
+
+Significant heterogeneity was observed among studies
+(I
+2=77.6%, P<0.001), and a random effects model was used for pooled analysis (Figure 2). Sensitivity analysis indicated that none of the individual studies influenced the summary statistic by omitting single study from the pooled estimate substantially (Table 2). As summarized in Table 3, we conducted subgroup analyses regarding different study designs, showing that the summary HR was statistically significant in the retrospective cohort studies
+(HR=1.51, 95% CI: 1.14–1.99; P=0.004; I
+2=72.4%) rather than prospective cohort studies (HR=1.08, 95% CI:
+0.95–1.23; P=0.257; I
+2=69.1%). Additionally, as for subgroup analyses of the different countries and CHGA levels measurements, the results were inconsistent with overall analysis except for the plasma CHGA level measurement, USA, and Germany on account of the limited number of studies evaluated. Literature reports of potential impact on tissue CHGA levels in PCa are scarce and also with contrary results. Thus future studies regarding tissue CHGA
+levels in PCa would verify our results. Notably, when stratified by clinical tumor stage, the results showed that the summary HR was statistically significant in patients with CRPC (HR=1.29, 95% CI: 1.07–1.55; P=0.008; I
+2=57.9%) compared with patients with PCa (HR=1.23,
+
+![4_image_0.png](4_image_0.png)
+
+| Table 2 Results of sensitivity analyses Study omitted HR   | 95% CI   |      |      |
+|------------------------------------------------------------|----------|------|------|
+| Berruti et al, 200530                                      | 1.26     | 1.07 | 1.49 |
+| Berruti et al, 201020                                      | 1.20     | 1.04 | 1.39 |
+| Burgio et al, 201431                                       | 1.22     | 1.06 | 1.42 |
+| Conteduca et al, 201436                                    | 1.20     | 1.05 | 1.37 |
+| De Nunzio et al 201422                                     | 1.33     | 1.11 | 1.60 |
+| Giridhar et al 201832                                      | 1.23     | 1.06 | 1.44 |
+| Jeetle et al 201223                                        | 1.31     | 1.11 | 1.55 |
+| Niedworok et al 201733                                     | 1.19     | 1.04 | 1.36 |
+| Taplin et al 200534                                        | 1.23     | 1.05 | 1.44 |
+| von Hardenberg et al 201735                                | 1.32     | 1.09 | 1.59 |
+| Combined                                                   | 1.24     | 1.07 | 1.44 |
+
+95% CI: 0.93–1.63; P=0.148; I
+2=81.7%). However, the subgroups could not find the potential factors that may substantially affect the heterogeneity. Meta-regression analysis results shown that none of the covariates (country, P=0.826; study design, P=0.295; CHGA levels measurements, P=0.415; clinical tumor stage, P=0.762) resulted in heterogeneity among the included studies. Therefore, the adjusted R-squared of −57.20% to −27.62% what expresses is that the regressors are contributing little to the explanation of the response variables (Table 4). Finally, potential publication bias might exist because of the small number of studies evaluated (Egger's test, P=0.001; Begg's test, P=0.020) (Figure 4).
+
+## Chga Expression And Pfs In Pca
+
+Three cohort studies involving 139 patients provided data on PFS in PCa.22,34,35 The combined analysis of three studies suggested that high CHGA expression was not significantly associated with shorter PFS (Figure 3), with
+
+| Table 3 Results of subgroup analyses Overall survival Studies, N                                                                 | Participants, N   | HR (95% CI)      | P-value          | P of heterogeneity   | I 2 (%)   |      |
+|----------------------------------------------------------------------------------------------------------------------------------|-------------------|------------------|------------------|----------------------|-----------|------|
+| 10                                                                                                                               | 3,172             | 1.24 (1.07–1.44) | 0.004            | <0.001               | 77.6      |      |
+| Country Italy                                                                                                                    | 5                 | 1,627            | 1.36 (1.03–1.79) | 0.029                | <0.001    | 82   |
+| UK                                                                                                                               | 1                 | 806              | 0.97 (0.78–1.20) | 0.782                | NA        | NA   |
+| USA                                                                                                                              | 2                 | 577              | 1.34 (1.09–1.63) | 0.005                | 0.949     | 0    |
+| Germany                                                                                                                          | 2                 | 162              | 1.96 (0.52–7.42) | 0.322                | 0.004     | 87.9 |
+| Study design Retrospective cohort                                                                                                | 7                 | 1,781            | 1.51 (1.14–1.99) | 0.004                | 0.001     | 72.4 |
+| Prospective cohort                                                                                                               | 3                 | 1,391            | 1.08 (0.95–1.23) | 0.257                | 0.039     | 69.1 |
+| CHGA levels measurements Tissue                                                                                                  | 2                 | 1,220            | 1.21 (0.76–1.92) | 0.423                | 0.018     | 82.0 |
+| Serum                                                                                                                            | 5                 | 1,413            | 1.17 (0.96–1.43) | 0.113                | 0.005     | 72.8 |
+| Plasma                                                                                                                           | 3                 | 539              | 1.43 (1.03–2.01) | 0.035                | 0.031     | 71.3 |
+| Clinical tumor stage CRPC                                                                                                        | 6                 | 824              | 1.29 (1.07–1.55) | 0.008                | 0.036     | 57.9 |
+| PC                                                                                                                               | 4                 | 2,348            | 1.23 (0.93–1.63) | 0.148                | 0.001     | 81.7 |
+| Abbreviations: CHGA, chromogranin-A protein; CRPC, castration-resistant prostate cancer; NA, not available; PC, prostate cancer. |                   |                  |                  |                      |           |      |
+
+| Covariates                                                                                                                                                                                                                   | The exponent of b   | Standard error   | t     | P>|t|   | 95% CI    | R-squared   |         |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|------------------|-------|---------|-----------|-------------|---------|
+| Country                                                                                                                                                                                                                      | 0.9739739           | 0.1127419        | −0.23 | 0.826   | 0.457975  | 1.271961    | −57.20% |
+| Study design                                                                                                                                                                                                                 | 0.764022            | 0.1834384        | −1.12 | 0.295   | 0.4391912 | 1.329101    | −29.22% |
+| CHGA levels measurements                                                                                                                                                                                                     | 0.9011292           | 0.1091306        | −0.86 | 0.415   | 0.6815581 | 1.191437    | −27.62% |
+| Clinical tumor stage                                                                                                                                                                                                         | 0.9203951           | 0.2434193        | −0.31 | 0.762   | 0.5001601 | 1.693712    | −49.17% |
+| Notes: Country (1=Italy, 2=USA, 3=UK, 4=Germany); study design (1=retrospective cohort, 2=prospective cohort); CHGA levels measurements (1=plasma, 2=tissue, 3=serum); and clinical tumor stage (1=CRPC, 2=prostate cancer). |                     |                  |       |         |           |             |         |
+
+![6_image_0.png](6_image_0.png)
+
+![6_image_1.png](6_image_1.png)
+
+HR of 1.73 (95% CI: 0.92–3.28; P=0.090; I
+2=73.9%).
+
+However, the use of subgroup analysis and sensitivity analysis were limited because of the small number of studies evaluated.
+
+## Discussion Main Findings
+
+In this study, we analyzed the associations between CHGA
+expression and survival in patients with PCa using a metaanalysis of the 10 included studies.20–23,30–35 Overall, our results indicated a significant association of high CHGA
+expression with poor OS in PCa, although statistically significant difference was not observed in the PFS group. Also, it should be noted that significant heterogeneity was observed in the overall analysis. The meta-regression and subgroup analyses could not find the potential factors that may substantially affect the heterogeneity. The results of sensitivity analysis validated the rationality and reliability of our analysis.
+
+Five studies suggested a significant association between high CHGA expression with poor OS in PCa,20,21,30,33,34 whereas five studies reported conflicting results.22,23,31,32,35 The retrospective cohort conducted by Burgio et al31 reported that in the multivariate analysis, high CHGA expression was a predictor of PFS (HR=2.70, 95% CI: 1.30–5.20; P=0.0047), while CHGA levels remained of borderline significance of OS (HR=1.90, 95% CI: 0.90–4.10; P=0.0919). Moreover, De Nunzio et al22 focused on poorly differentiated PCa, their study indicated that high CHGA expression was not a significant predictor of OS on univariate (OR=1.00, 95% CI:
+0.99–1,01; P=0.66). Similarly, Giridhar et al32 Jeetle et al23 and von Hardenberg et al35 demonstrated that CHGA
+expression was not an independent predictor of survival
+(HR=1.35, 95% CI: 0.89–1.97; P=0.154; HR=0.97, 95% CI: 0.89–1.37, P=0.87; HR=1.07, 95% CI: 0.93–1.24, P=0.0919, respectively). When we excluded this study from the meta-analysis, the stability of the results showed no significant changes, validating the rationality and reliability of our analysis.
+
+## Implications For Clinical Practice
+
+CHGA is considered to be a useful predictor in PCa patients with lower PSA levels.36,37 However, Our results suggest that the lack of correlation between the low PSA levels and high CHGA value, which may be the expression of patients with advanced CRPC with neuroendocrine differentiation in the context of a heterogeneous tumor volume. Moreover, elevated serum CHGA levels are usually associated with neuroendocrine differentiation of CRPC, suggesting that patients with CRPC have a poor prognosis and may be related to the increasing degrees of differentiation.34,38 In recent years, several studies have suggested that serum levels of CHGA are significantly higher in metastatic PCa as compared to non-metastatic and primary PCas.39,40 Nevertheless, despite this interest in neuroendocrine serum markers, little is known about their sources in different growth patterns and PCa metastasis.
+
+Previous studies indicated that CHGA values were not substantially affected by either endocrine therapy or chemotherapy.30,41 Since preoperative treatment decisions based on the prognosis may be active surveillance rather than surgery, our results may have direct clinical relevance. In addition, our results show that those who are eligible for active surveillance may also benefit from the analysis of CHGA, as this may improve the accuracy of disease surveillance.
+
+Latest classification of neuroendocrine prostate tumors suggests that variants of neuroendocrine prostate cancer
+(NEPC) contain a mixed form between conventional adenocarcinoma and NEPC, which was often characterized by androgen receptor independence.42 However, neuroendocrine markers and androgen receptor are dually expressed in the mixed tumor cells due to clinical and pathological heterogeneity among patients.42 Moreover, a large proportion of the treatment-related recurrent prostate tumors have strong neuroendocrine characteristics, which was caused by pre-existing prostate adenocarcinoma.43 Several studies indicated that neuroendocrine differentiation cells are potentially derived from the common pluripotent stem cell populations, which play a crucial role in CRPC and might contribute to hormone therapy and chemoresistance.44,45 Notably, NEPC cells express high levels of chromogranin and synaptophysin.46 Hence, the overlapping clinical entities require more accurate clinical and molecular classification, and further research is needed to determine their prognostic impact.47
+
+## Strengths And Limitations
+
+In general, our meta-analysis exhibited several crucial strengths. First, the meta-analysis was the first to assess the association between CHGA expression and survival in patients with PCa through thorough systematic search and rigorous analytical approaches. Second, multivariable-adjusted risk estimates were applied to minimize the confounding factors that might influence the whole results. Third, the rationality and reliability of our metaanalysis was observably improved because the overall combined estimates were based on a large sample size.
+
+Furthermore, sufficient subgroup analyses and sensitivity analyses were also performed to ensure the reliability of this study.
+
+This study has several limitations, which may affect the interpretation of some of our results. Firstly, there was the problem of heterogeneity in the overall analysis, although we could not find the potential factors that may substantially affect the heterogeneity through the meta-regression and subgroup analyses. Moreover, clinical tumor stage and initial treatment modality should also be accounted for potential considerable factors, which possibly contribute to heterogeneity. However, our understanding of the effects of clinical tumor stage and initial treatment modality among the included studies on the overall results remains insufficient, although these factors have been investigated, albeit inadequately, in other studies. Further research is needed to verify the findings of this meta-analysis with regard to the factors affecting extensive consequences and common objectives as mentioned above. Second, CHGA expression has been associated with other molecular biomarkers for PCa prognosis. Inadequate adjustment for these biomarkers in several included studies might have resulted in spurious associations. Finally, potential publication bias might exist because of the small number of studies evaluated and the small effect size estimated.
+
+## Conclusion
+
+In summary, current evidence supported the viewpoint that high CHGA expression is significantly associated with poor OS in PCa. Future studies with large sample sizes and stratified analyses according to clinicopathological characteristics are needed to explore tailored treatments that directly target CHGA for the improvement of survival in men with PCa.
+
+## Acknowledgments
+
+This study was supported by grants from the National Natural Science Foundation of China (Grant No. 61301294). The supporting institution had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review or approval of the manuscript; and decision to submit the manuscript for publication.
+
+## Author Contributions
+
+All authors contributed toward data analysis, drafting and critically revising the paper, and agree to be accountable for all aspects of the work. All authors read and approved the final manuscript.
+
+## Disclosure
+
+The authors declare no conflicts of interest in this work.
+
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+doi:10.1016/S0022-5347(01)69075-X
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+40. Genitsch V, Zlobec I, Seiler R, et al. Neuroendocrine differentiation in metastatic conventional prostate cancer is significantly increased in lymph node metastases compared to the primary tumors. Int J Mol Sci. 2017;18(8):E1640.
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+
+## Supplementary Material Appendix 1: Detailed Search Strategy
+
+2. Embase search strategy
+
+| Appendix 1: Detailed search strategy    | Search                                                                                                                          | Search Terms   |
+|-----------------------------------------|---------------------------------------------------------------------------------------------------------------------------------|----------------|
+| ID#                                     |                                                                                                                                 |                |
+| 1. MEDLINE (via PubMed) search strategy | #29                                                                                                                             | #20 AND #28    |
+| #28                                     | #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27                                                                                   |                |
+| #27                                     | "CHGA":tw                                                                                                                       |                |
+| #26                                     | "Secretory Protein I, Parathyroid Gland":tw                                                                                     |                |
+| #25                                     | "Secretory Protein I, Parathyroid Gland":tw                                                                                     |                |
+| #24                                     | "CHGA Protein":tw                                                                                                               |                |
+| #23                                     | "Secretory Protein, Parathyroid":tw                                                                                             |                |
+| #22                                     | "Parathyroid Secretory Protein":tw                                                                                              |                |
+| #21                                     | "Chromogranin A"/exp                                                                                                            |                |
+| #20                                     | #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19        |                |
+| #19                                     | "prostatic neoplasms":tw                                                                                                        |                |
+| #18                                     | "cancer of prostate":tw                                                                                                         |                |
+| #17                                     | "prostatic cancers":tw                                                                                                          |                |
+| #16                                     | "cancers, prostatic":tw                                                                                                         |                |
+| #15                                     | "cancer, prostatic":tw                                                                                                          |                |
+| #14                                     | "prostatic cancer":tw                                                                                                           |                |
+| #13                                     | "cancer of the prostate":tw                                                                                                     |                |
+| #12                                     | "prostate cancers":tw                                                                                                           |                |
+| #11                                     | "cancers, prostate":tw                                                                                                          |                |
+| #10                                     | "cancer, prostate":tw                                                                                                           |                |
+| #9                                      | "prostatic neoplasm":tw                                                                                                         |                |
+| #8                                      | "neoplasm, prostatic":tw                                                                                                        |                |
+| #7                                      | "neoplasms, prostatic":tw                                                                                                       |                |
+| #6                                      | "prostate neoplasm":tw                                                                                                          |                |
+| #5                                      | "neoplasm, prostate":tw                                                                                                         |                |
+| #4                                      | "neoplasms, prostate":tw                                                                                                        |                |
+| #3                                      | "prostate neoplasms":tw                                                                                                         |                |
+| #2                                      | "prostate cancer":tw                                                                                                            |                |
+| #1                                      | "prostate cancer"/exp                                                                                                           |                |
+| Search                                  | Search Terms                                                                                                                    |                |
+| ID# #29                                 | Search #20 AND #28                                                                                                              |                |
+| #28                                     | Search #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27                                                                            |                |
+| #27                                     | Search "CHGA"[Text Word]                                                                                                        |                |
+| #26                                     | Search "Secretory Protein I, Parathyroid Gland"[Text Word]                                                                      |                |
+| #25                                     | Search "Secretory Protein I, Parathyroid Gland"[Text Word]                                                                      |                |
+| #24                                     | Search "CHGA Protein"[Text Word]                                                                                                |                |
+| #23                                     | Search "Secretory Protein, Parathyroid"[Text Word]                                                                              |                |
+| #22                                     | Search "Parathyroid Secretory Protein"[Text Word]                                                                               |                |
+| #21                                     | Search "Chromogranin A"[Mesh]                                                                                                   |                |
+| #20                                     | Search #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 |                |
+| #19                                     | Search Cancer of Prostate[Text Word]                                                                                            |                |
+| #18                                     | Search Prostatic Cancers[Text Word]                                                                                             |                |
+| #17                                     | Search "Cancers, Prostatic"[Text Word]                                                                                          |                |
+| #16                                     | Search "Cancer, Prostatic"[Text Word]                                                                                           |                |
+| #15                                     | Search Prostatic Cancer[Text Word]                                                                                              |                |
+| #14                                     | Search Cancer of the Prostate[Text Word]                                                                                        |                |
+| #13                                     | Search Prostate Cancers[Text Word]                                                                                              |                |
+| #12                                     | Search "Cancers, Prostate"[Text Word]                                                                                           |                |
+| #11                                     | Search "Cancer, Prostate"[Text Word]                                                                                            |                |
+| #10                                     | Search Prostate Cancer[Text Word]                                                                                               |                |
+| #9                                      | Search Prostatic Neoplasm[Text Word]                                                                                            |                |
+| #8                                      | Search "Neoplasm, Prostatic"[Text Word]                                                                                         |                |
+| #7                                      | Search "Neoplasms, Prostatic"[Text Word]                                                                                        |                |
+| #6                                      | Search Prostate Neoplasm[Text Word]                                                                                             |                |
+| #5                                      | Search "Neoplasm, Prostate"[Text Word]                                                                                          |                |
+| #4                                      | Search "Neoplasms, Prostate"[Text Word]                                                                                         |                |
+| #3                                      | Search Prostate Neoplasms[Text Word]                                                                                            |                |
+| #2                                      | Search Prostatic Neoplasms[Text Word]                                                                                           |                |
+| #1                                      | Search "Prostatic Neoplasms"[Mesh]                                                                                              |                |
+
+3. Cochrane Library search strategy
+
+| Search   | Search Terms                                                                                                             |              |             |
+|----------|--------------------------------------------------------------------------------------------------------------------------|--------------|-------------|
+| ID#      | Search                                                                                                                   | Search Terms |             |
+| ID#      |                                                                                                                          |              |             |
+| #29      | #20 and #28                                                                                                              |              |             |
+| #28      | #21 or #22 or #23 or #24 or #25 or #26 or #27                                                                            |              |             |
+| #27      | "CHGA":ti,ab,kw                                                                                                          |              |             |
+| #26      | "Secretory Protein I, Parathyroid Gland":ti,ab,kw                                                                        |              |             |
+| #25      | "Secretory Protein I, Parathyroid Gland":ti,ab,kw                                                                        |              |             |
+| #24      | "CHGA Protein":ti,ab,kw                                                                                                  |              |             |
+| #23      | "Secretory Protein, Parathyroid":ti,ab,kw                                                                                |              |             |
+| #22      | "Parathyroid Secretory Protein":ti,ab,kw                                                                                 |              |             |
+| #21      | MeSH descriptor: [Chromogranin A] explode all trees                                                                      |              |             |
+| #20      | #1 or #2 or #3 or#4 or #5 or #6 or #7 or #8 or #9 or #10 or #11 or #12 or #13 or #14 or #15 or #16 or #17 or #18 or #19  |              |             |
+| #19      | "prostatic neoplasms":ti,ab,kw                                                                                           |              |             |
+| #18      | "cancer of prostate":ti,ab,kw                                                                                            |              |             |
+| #17      | "prostatic cancers":ti,ab,kw                                                                                             |              |             |
+| #16      | "cancers, prostatic":ti,ab,kw                                                                                            |              |             |
+| #15      | "cancer, prostatic":ti,ab,kw                                                                                             |              |             |
+| #14      | "prostatic cancer":ti,ab,kw                                                                                              |              |             |
+| #13      | "cancer of the prostate":ti,ab,kw                                                                                        |              |             |
+| #12      | "prostate cancers":ti,ab,kw                                                                                              |              |             |
+| #11      | "cancers, prostate":ti,ab,kw                                                                                             |              |             |
+| #10      | "cancer, prostate":ti,ab,kw                                                                                              |              |             |
+| #9       | "prostatic neoplasm":ti,ab,kw                                                                                            |              |             |
+| #8       | "neoplasm, prostatic":ti,ab,kw                                                                                           |              |             |
+| #7       | "neoplasms, prostatic":ti,ab,kw                                                                                          |              |             |
+| #6       | "prostate neoplasm":ti,ab,kw                                                                                             |              |             |
+| #5       | "neoplasm, prostate":ti,ab,kw                                                                                            |              |             |
+| #4       | "neoplasms, prostate":ti,ab,kw                                                                                           |              |             |
+| #3       | "prostate neoplasms":ti,ab,kw                                                                                            |              |             |
+| #2       | "prostate cancer":ti,ab,kw                                                                                               |              |             |
+| #1       | MeSH descriptor: [Prostatic Neoplasms] explode all trees                                                                 | #29          | #20 AND #28 |
+| #28      | #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27                                                                            |              |             |
+| #27      | Topic=(CHGA)                                                                                                             |              |             |
+| #26      | Topic=(Secretory Protein I, Parathyroid Gland)                                                                           |              |             |
+| #25      | Topic=(Secretory Protein I, Parathyroid Gland)                                                                           |              |             |
+| #24      | Topic=(CHGA Protein)                                                                                                     |              |             |
+| #23      | Topic=(Secretory Protein, Parathyroid)                                                                                   |              |             |
+| #22      | Topic=(Parathyroid Secretory Protein)                                                                                    |              |             |
+| #21      | Topic=(Chromogranin A)                                                                                                   |              |             |
+| #20      | #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 |              |             |
+| #19      | Topic=(prostatic neoplasms)                                                                                              |              |             |
+| #18      | Topic=(cancer of prostate)                                                                                               |              |             |
+| #17      | Topic=(prostatic cancers)                                                                                                |              |             |
+| #16      | Topic=(cancers, prostatic)                                                                                               |              |             |
+| #15      | Topic=(cancer, prostatic)                                                                                                |              |             |
+| #14      | Topic=(prostatic cancer)                                                                                                 |              |             |
+| #13      | Topic=(cancer of the prostate)                                                                                           |              |             |
+| #12      | Topic=(prostate cancers)                                                                                                 |              |             |
+| #11      | Topic=(cancers, prostate)                                                                                                |              |             |
+| #10      | Topic=(cancer, prostate)                                                                                                 |              |             |
+| #9       | Topic=(prostatic neoplasm)                                                                                               |              |             |
+| #8       | Topic=(neoplasm, prostatic)                                                                                              |              |             |
+| #7       | Topic=(neoplasms, prostatic)                                                                                             |              |             |
+| #6       | Topic=(prostate neoplasm)                                                                                                |              |             |
+| #5       | Topic=(neoplasm, prostate)                                                                                               |              |             |
+| #4       | Topic=(neoplasms, prostate)                                                                                              |              |             |
+| #3       | Topic=(prostate neoplasms)                                                                                               |              |             |
+| #2       | Topic=(prostate cancer)                                                                                                  |              |             |
+| #1       | Topic=(prostate cancer)                                                                                                  |              |             |
+
+4. Web of Science search strategy
+
+## Cancer Management And Research Dovepress
+
+Publish your work in this journal The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.
+
+Submit your manuscript here: https://www.dovepress.com/cancer-management-and-research-journal Cancer Management and Research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient.

medical/md/PMC6735069.md

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+
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+![0_image_0.png](0_image_0.png)
+
+# An Evaluation Of Training In Brief Cognitive–Behavioural Therapy In A Non-English-Speaking Region: Experience From India
+
+A. K. Gupta1 and H. Aman2 1 Consultant Psychiatrist, Northumberland Tyne and Wear NHS Foundation Trust, Cherry Knowle Hospital, Ryhope, Sunderland, UK, email ArunKumar.Gupta@ntw.nhs.uk 2 Registrar in the Child and Adolescent Psychiatry, Prudhoe Hospital, Prudhoe, Northumberland, UK
+We acknowledge the support provided by Dr P. Psitholey and his colleagues at CSM Medial University, Lucknow. We also acknowledge the support of Dr R. Arjundas, Mr Thomas Reeves, Dr Steve Moorehead and Dr Suresh Joseph (Medical Director, NTW NHS Trust).
+
+The authors wanted to learn whether it was possible to deliver cognitive–behavioural therapy (CBT) in a low-income country where English is neither the first nor the preferred spoken language and to evaluate the effectiveness of the training in terms of skills acquisition. Twenty participants attended a 3-day workshop on the technique. All had experience of communicating in Hindi with patients, although their medical training was in English. There is no manual for CBT in Hindi. Role-plays focused on basic CBT skills such as Socratic dialogue, the 'five area' approach, the use of the 'downward arrow', developing an automatic negative thought record and devising behavioural experiments, in Hindi. The findings suggested that it is feasible to train mental health professionals in CBT where English is not the first language. 
+
+## Background
+
+Growing evidence for the efficacy of cognitive– behavioural therapy (CBT) in the treatment of mental illnesses (Williams & Garland, 2002) has stimulated the interest of psychiatrists in India. 
+
+Over the course of 3 years, a series of 3-day workshops was organised by the first author at the Department of Psychiatry, Chhatrapati Sahuji Maharaja Medical University (CSMMU), previously known as King George's Medical College, Lucknow, India. This article focuses on outcome data from the fourth of these workshops. The first two workshops were at an introductory level, while the third focused on the use of behavioural experiments within a CBT context. (The results of these initial workshops have been presented at annual conferences of the British Association for Behavioural and Cognitive Psychotherapies and the Indian Psychiatry Society's Central Zone.) 
+The authors wanted to learn whether it was possible to deliver CBT in a low-income country where English is neither the first nor the preferred spoken language and to evaluate the effectiveness of the training in terms of skills acquisition.
+
+## How Delivering Cbt Training Was Different In India
+
+A commonly encountered barrier to initiating psychological therapies in non-English-speaking countries is the focus on biological/medical models of treatment on the part of both medical professionals and their patients. Training doctors in approaches that are new to them and their patients can be difficult. We distributed a report of a randomised controlled trail of CBT (King, 2002) to suggest to our psychiatric colleagues that CBT is an appropriate treatment for common mental health problems. An initial comment from one influential member of the local faculty was that CBT is appropriate for Western culture but may not work with Indian patients. This comment was turned into a question for the training programme and was presented as a behavioural experiment. The presumption was that if trainees are able to learn CBT skills, to use these with their patients and to get some good results in the first instance, it will be worthwhile developing a randomised controlled trial at a later stage for gathering strong scientific evidence in favour of the efficacy of CBT delivered to Indian patients in the local language.
+
+Another barrier we faced was the hierarchical professional system within medicine in India (as is the case in many other low-income countries). When the workshop started, we noticed that there were four distinct groups: consultants, junior doctors, psychologists and medical students. We addressed this issue by ensuring a good mix of skills in each group when dividing them into smaller groups for skill practice, in order to give everyone a chance to participate fully in the training and to work as a team. There was naturally some apprehension among participants about this approach as it is difficult to break with tradition. But doing so helped the group to understand the concept of a collaborative approach. 
+
+The teaching environment and equipment were of a poorer standard than one would have in the UK and this necessitated a certain amount of improvisation. A 40-inch flat-screen television was attached to laptop via S-video cable and a home cinema surround-sound system was used for the audio output. We also utilised more roleplays in order to compensate for the lack of good presentation equipment. Role-plays focused on the demonstration of basic CBT skills by the first author, such as Socratic dialogue, discussing the 'five area' approach with the patient, the use of the 'downward arrow', developing an automatic negative thought record, devising behavioural experiments and so on, in Hindi.
+
+In order to help trainees consolidate and continue to practise skills learnt, some clinical supervision sessions were provided after the 3-day workshop, via telephone. Video-chat and email were also used, using the principles of distance learning. Trainees submitted therapy notes and case summaries for review. The supervision suggested that the trainees had been able to utilise CBT skills learnt during the workshop. 
+
+All participants had previous experience of communicating in Hindi with patients, although their medical training was in English. The first author worked with the participants during the workshop to agree Hindi equivalents (in the local dialect of the region) of common Socratic dialogue questions used in CBT, as there is no manual for CBT in Hindi. Unfortunately, the participants could not use rating scales when monitoring the results of CBT as there is a lack of standardised scales in Hindi. 
+
+## The Workshop
+
+Twenty participants attended the course. The agenda for all three training days was agreed at the beginning of each session based on the learning objectives of the participants. Eight participants had attended previous workshops (in 2008 and 2009) and rest were new to the training (denoted 'repeat' and 'fresh', below). The fresh participants received introductory lectures from CSMMU faculty members beforehand, based on two books covering the basic principles of CBT (Hawton *et al*, 1998; Simmons & Griffiths, 2009). 
+
+On day 1, the participants were asked to highlight their learning needs, which included understanding the 'five areas' model, assessment of patient suitability for CBT, the art of Socratic questioning and eliciting negative automatic thoughts. One of the participants volunteered to bring therapy notes (with the patient anonymous) 
+to facilitate a discussion of the practical difficulties. The same participant was asked to role-play the patient, while the trainer (the first author) role-played the therapist and demonstrated the technique of Socratic dialogue to elicit negative automatic thought. A thought record was completed, based on the 'seven columns' technique as used by Greenberger & Padesky (1995). The criteria used for the selection of patients for therapy were discussed (Blenkiron, 1999). 
+
+Day 2 started with a recap of day 1. Based on feedback, an agenda for the day was agreed, which was to develop the skills of explaining the CBT model to patients and the rationale behind the behavioural experiments. A simple method was developed of explaining the CBT model to the patients in Hindi using an interactive vignette and Socratic dialogue.
+
+Day 3 started with a recap of days 1 and 2. 
+
+An agenda was agreed to discuss supervision for trainee therapists and the practical aspects of behavioural experiments. Possible modifications to behavioural experiments based on cultural norms were also discussed and an experiment was devised based on a vignette brought by a trainee. The participants practised devising behavioural experiments in groups of three or four and the first author supervised the skill practice.
+
+## Assessment
+
+The baseline knowledge of all the participants was assessed before the workshop, using a questionnaire designed for participants to self-rate their core CBT skills. It covered seven areas: activity scheduling; the use of the positive data log; Socratic dialogue; core belief; negative automatic thought; formulation; and all-or-nothing thinking. 
+
+Participants were asked to complete the questionnaire again after the workshop. There was no formal measure of competency following the 
+
+![1_image_0.png](1_image_0.png)
+
+![2_image_0.png](2_image_0.png)
+
+course owing to a lack of facilities such as videoequipment and difficulty in gaining consent from patients because of issues of stigma.
+
+All the trainees were asked to provide feedback at the end of each day on four different aspects of the workshop: content relevant to work, length of workshop, presentation style and overall rating. These were scored on a scale of 0–4: 0, poor; 1, fair; 2, good; 3, very good; 4, excellent. 
+
+All the data collected were analysed using SPSS. 
+
+Means and standard errors (s.e.) were calculated and significance was determined using paired t-tests. 
+
+## Results And Discussion
+
+We found that all participants (both repeat and fresh) improved after training (mean score on the self-assessment questionnaire 75.29, s.e.=5.95) compared with baseline (mean 32.71, s.e.=5.92) 
+(t(13)=–6.899, P<0.001, r=0.886) (see Fig. 1).
+
+In the fresh participants group a skill level of 100% was noted for Socratic dialogue and formulation. There was no difference observed in either cohort of trainees in their understanding of core belief and negative automatic thoughts, which reached satisfactory levels (around 70%). Skills like activity scheduling, core belief, negative automatic thoughts and all-or-nothing thinking showed some improvement (to around 75%). There was poor understanding of the use of the positive data log (25%) among all the trainees and this area needs to be addressed in future workshops. 
+
+The trainees' feedback is summarised in Fig. 2. 
+
+## Areas For Improvement/Limitations Of The Study
+
+•	 There is a lack of standardised rating scales in Hindi for the measurement of mental illness. 
+
+The first author has started work on one such measure.
+
+•	 The questionnaire we used was from an unpublished small-scale research project and so its validity is questionable. Also, it is based on self-evaluation and lacks objective assessment.
+
+•	 Accuracy of measurement of skills development would have been improved if formal assessment measures were used and this needs further consideration for future training.
+
+•	 The lack of suitable teaching aids such as audioand video-equipment needs to be addressed.
+•	 As the participants had different levels of theoretical knowledge at the beginning of training it was difficult to compare the groups to measure the outcome of training.
+
+•	 Future work is now focusing on the development of rating scales in Hindi, video-recording of therapy sessions, the use of formal assessment measures and the development of randomised controlled trials for evaluating the efficacy of CBT in an Indian population.
+
+## Conclusion
+
+This initial study would suggest that it is feasible to train mental health professionals in CBT in India and possibly other countries where English is not the first language. As this was a small project with a number of methodological constraints, the evidence cannot be seen as conclusive. In the context of training mental health professionals, the central indicator of an effective training programme would be an improvement in client care and clinical outcomes (Kirkpatrick, 1979). There is therefore a need to carry out more robust work in recruiting more patients and carrying out systematised research.
+
+## References
+
+Blenkiron, P. (1999) Who is suitable for cognitive behaviour therapy? *Journal of the Royal Society of Medicine*, 92, 222–229.
+
+Greenberger, D. & Padesky, CA. (1995) *Mind over Mood*. Guilford Press. Hawton, K., Salkovskis P.M., Kirk, J., *et al* (1998) *Cognitive* Behaviour Therapy for Psychiatric Problems: A Practical Guide. Oxford University Press. King, M. (2002) Effectiveness of teaching general practitioners skills in brief cognitive behaviour therapy to treat patients with depression: randomised controlled trial. *BMJ*, 324, 947. Kirkpatrick, D. L. (1979) Techniques for evaluating training programs. *Training and Development Journal,* 33, 78–92. Simmons, J. & Griffiths, R. (2009) *CBT for Beginners*. Sage. Williams, C. & Garland, A. (2002) Identifying and challenging unhelpful thinking. *Advances in Psychiatric Treatment*, 8, 377–386.

medical/md/PMC6823801.md

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+
+
+![0_image_5.png](0_image_5.png)
+
+## A B S T R A C T Introduction
+
+| Received 15 August 2019 Revised 4 September 2019 Accepted 4 September 2019   |
+|------------------------------------------------------------------------------|
+
+| Keywords: Vertebral Metastases Atypical Hemangioma   |
+|------------------------------------------------------|
+
+Lucas Paul Paladino, BSa, Ana C. Belzarena, MDb,∗, Evita Henderson-Jackson, MDa, David M. Joyce, MDa a Sarcoma Department, Moffitt Cancer Center, 12902 Magnolia Dr., Tampa, FL, 33612, USA
+b Orthopaedic Oncology Department, Miami Cancer Institute, 8900 N Kendall Dr., Miami, FL, 33176, USA
+Atypical hemangiomas of the spine can mimic metastatic lesions on magnetic resonance imaging, therefore making this distinction is a diagnostic challenge. In most cases, this conundrum can usually be solved with positron emission tomography/computed tomography images, because hemangiomas do not usually present with increased uptake while metastatic lesions do. Here we present a case of a patient with a unique diagnosis, myxoid liposarcoma, in which the vertebral metastatic lesion did not present with increased uptake in positron emission tomography/computed tomography scans. While keeping the imaging particularity of this rare sarcoma in mind, proceeding with a biopsy when the suspicion of metastasis remains high will help elucidate the diagnosis and allow for proper management.
+
+© 2019 The Authors. Published by Elsevier Inc. on behalf of University of Washington.
+
+This is an open access article under the CC BY-NC-ND license.
+
+(http://creativecommons.org/licenses/by-nc-nd/4.0/)
+30% of cases [4,5]. IH have a classic radiologic appearance due to its histological content, which consists of a hamartomatous lesion with well-differentiated thin-walled vessels and a nonvascular component. The latter of which includes different percentages of fat, fibrous tissue, bone, hemosiderin, and smooth muscle [2,4]. The spatial arrangement of such components within the vertebral body translates into several classic radiologic findings. On radiographs, vertical striations and honeycomb appearance can be seen, while on computed tomography (CT) polka-dots are observed [6]. Magnetic resonance imaging visualizes IH as hyperintense T1-sequence lesions due to its lipomatous components and as hyperintense Intraosseous hemangiomas (IH) are benign vascular tumors which very rarely cause symptoms and are often found incidentally. Based on postmortem studies, it is estimated that they are found in 11% of the population [1]. IH most commonly occur between the fourth and sixth decade with a slight female predominance and 80% of cases will be localized to the skull or spine [2,3], where they are usually located in the vertebral body. Although they are most commonly found in the thoracic region, multiple locations can be present in up to
+∗ Corresponding author.
+
+E-mail address: ceciliabel@baptisthealth.net (A.C. Belzarena).
+
+https://doi.org/10.1016/j.radcr.2019.09.008 1930-0433/© 2019 The Authors. Published by Elsevier Inc. on behalf of University of Washington. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)
+Available online at www.sciencedirect.com
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_2.png](0_image_2.png)
+
+![0_image_3.png](0_image_3.png)
+
+![0_image_4.png](0_image_4.png)
+
+Case **Report**
+
+![0_image_1.png](0_image_1.png)
+
+Metastatic vertebral lesion mimicking an **atypical**
+hemangioma with negative 18F-FDG positron emission tomography-computed **tomography**
+a r t i c l e i n f o
+
+![1_image_0.png](1_image_0.png)
+
+T2-sequence lesions due to the vascular portion, which also causes these lesions to be seen as enhancing under contrast administration [7–9]. On 18F-FGD positron emission tomography/CT (PET/CT) scans IH are visualized as cold lesions with no increased uptake above background.There have been some reported cases of increased uptake of rib hemangiomas and spine hemangiomas, however this is usually not the case [10–
+13].
+
+An atypical hemangioma is a hemangioma that does not present with a classical imaging appearance and may resemble a more aggressive type of lesion [14]. Different distributions of tissue content within these lesions results in this idiosyncrasy. Atypical hemangiomas with low lipomatous percentage may present as hypointense on T1-sequence, but because of the lesion's vascular component, it can display T2-sequence hyperintensity with enhancement under contrast, thus resembling a metastatic lesion and clouding the difference between the 2 [15,16]. Adding to this diagnostic challenge, an atypical hemangioma may also present with aggressive features such as cortical destruction, bone expansion, and even invasion of the spinal canal. These are observations frequently associated with metastatic processes [2,17]. Prior studies have shown that new MRI techniques using diffusion weighted imaging combined with apparent diffusion coefficient maps and T1-weighted dynamic contrast-enhancing may help resolve this diagnostic problem, but unfortunately these techniques are not part of the standard of care [18,19].
+
+## Case **Report**
+
+A 31-year-old female with a history of a myxoid liposarcoma
+(MLS) excised from the left thigh, the primary location of the tumor, 2 years prior was under our care for a recurrent localized tumor with no evidence of other metastatic disease.
+
+The primary tumor presented on MRI as a soft tissue localized lesion hypointense in T1-weighted sequences, hyperintense in T2 with fat suppression ones and with heterogeneous enhancement with gadolinium contrast. She had received systemic treatment and awaited surgical resection and endoprosthetic reconstruction of this lesion. The patient presented to our clinic in May 2018, prior to her procedure, with acute back pain that developed during physical activity without any significant trauma. The pain was severe and included the lower back but did not have radiation to the lower extremities or any other neurologic findings. Radiographs of the lumbar spine obtained on that same date, showed an age indeterminate compression deformity of the L5 vertebral body involving the superior endplate, no signs of osteoporosis were noted. The radiographic image and the patient's symptoms prompted the acquisition of an MRI with and without gadolinium contrast, a week after the initial presentation, which confirmed a compression deformity and fracture of the L5 vertebral body, which could explain the patient's symptoms, and ruled out a neoplastic process at L5. Additional findings included a T1sequnce hypointense and T2-sequence hyperintense lesion, enhancing under contrast in the L3 vertebral body with sharp margins. The lesion measured 1.24 × 1.10 × 0.93 cm and was reported as potentially representing a metastatic lesion or an atypical vertebral body hemangioma (Fig. 1). A PET/CT
+scan was obtained 2 weeks after the initial presentation and showed increased uptake of the fractured L5 body but failed to show significant findings at the L3 vertebral body (Fig. 2). The case was presented at our sarcoma tumor board and we decided to delay the patient's femur resection and instead perform a CT-guided biopsy of the L3 vertebral body lesion as it would change surgical plan (Fig. 3). Histopathologic analysis of
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+the specimen obtained 3 weeks after the initial presentation, revealed a blood clot and medullary bone with no evidence of malignancy (Fig. 4). We later proceeded with her proximal femur resection and the preprocedure MRI showed no change in her L3 lesion at 2 months from the patient's presentation.
+
+The procedure was performed without complications and a margin-free resection was achieved.
+
+Four months from the patient's initial presentation for acute back pain, a new PET/CT showed increased activity in the left medial thigh, a new large hypermetabolic left axillary lymph node, continued visualization of the L5 compression fracture, and no evidence of increased activity at L3.Work up of her left axillary lymph node verified metastatic spread of her MLS. After removal of this node, follow up PET/CT revealed a new mass in her left ovary, but again did not appreciate any significant findings at the L3 vertebral body at 7 months from presentation (Fig. 5). Although a pelvic MRI also at 7 months from the initial back pain symptoms, showed the ovarian mass was benign, we found that the L3 vertebral lesion's diameter had nearly tripled from 1.1 to 3.1cm and occupied the entire vertebral body (Fig. 6).
+
+![2_image_2.png](2_image_2.png)
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+The sarcoma tumor board reviewed the patient's case again and now strongly suspected that the L3 lesion was a site of metastasis. Kyphoplasty and radiofrequency ablations were performed 7 months after presentation and a histopathologic sample obtained during the procedure confirmed this as a metastatic lesion of her primary MLS. A moderately cellular neoplasm with myxoid stroma, arborizing capillary vasculature, and spindled to round tumor cells with scant cytoplasm, and hyperchromatic nuclei were observed in the sample with involvement of the bone marrow (Fig. 7).
+
+The patient completed targeted lumbar radiation therapy.
+
+Follow up imaging of L3 almost a year from the initial symptoms has shown an increase in abnormal signal/marrow replacement, extending into the left pedicle and periphery of the vertebral body. Surveillance imaging has also discovered a new site in the left buttocks concerning for soft tissue
+
+![4_image_0.png](4_image_0.png)
+
+metastasis. Now 32 years old, our patient is being evaluated for cellular therapy and a new systemic chemotherapy.
+
+## Discussion
+
+Atypical hemangiomas and metastatic lesions, as previously mentioned, share common imaging features, such as signs of aggressiveness, similar patterns of intensity on T1 and T2 sequences, and enhancement seen on MRI [2,15–17]. They even present with similar age demographics: fourth to sixth decade for hemangiomas and fourth to fifth decades for MLS [2,26].
+
+Both diagnoses should be considered a part of the differential, especially in patients with a known primary malignancy presenting with a vertebral lesion. More than 50% of the primary cancers will develop secondary bone lesions [20]. For many of these primary cancer types, studies have shown that PET/CT
+scans have a high specificity and sensitivity for detecting bone metastases when compared to other imaging modalities. Most tumor cells have increased glucose metabolism that causes the tracer to accumulate inside of these cells [21].The majority of metastatic lesions analyzed with a PET/CT scan will display uptake, while typical and atypical hemangiomas usually have no increased signal. Therefore, PET/CT's are normally an important tool in differentiating metastases and hemangiomas.
+
+However, in the case presented, the patient had a unique primary cancer type, MLS, which has many atypical and peculiar attributes, especially when it comes to PET/CT. MLS undergoes a different pattern of metastasis than other soft tissue malignancies.While most soft tissue cancers primarily spread to the lungs, MLS spreads more often to bone and lipomatous soft tissue in the retroperitoneum, axilla and mesentery, and classically the paraspinal muscles [22,23]. A third of MLS patients will progress to stage IV disease, with 14% of these patients having bone secondary lesions in a spine location [24].
+
+The diagnosis of vertebral lesions secondary to MLS with 18FFGD PET/CT or bone scintigraphy have shown low sensitivity, 14% and 16%, respectively [25], even though PET/CT are effective in highlighting primary and metastatic soft tissue sites of MLS. Schwab et al suggested that, in the setting of MLS,
+myxoid stroma in the vertebral lesion potentially prevents labeled glucose from reaching cells in sufficient quantity to be detected by the scanner, thus accounting for such a low rate of detection by PET/CT [25]. The recommended method for detection of vertebral metastases, with the highest sensitivity, in the setting of MLS is the MRI [26].
+
+If suspicion of metastasis remains high, it is recommended to proceed with a biopsy of the vertebral lesion and may even require and open biopsy. A prior study has shown a 10% rate of samples with insufficient material for histopathologic diagnosis with CT-guided biopsies of the spine, so having a pathologist to confirm the adequacy of sample on site can help lower this rate [27]. The overall accuracy for the diagnosis of spinal metastatic lesions with CT-guided biopsies ranges from 90%
+to 95% with a complication rate of <5% [28].
+
+## Conclusion
+
+Atypical hemangiomas and MLS secondary spine lesions often present with many similar characteristics on MRI. While for most primary cancer types, PET/CT is useful in differentiating the 2, it is not helpful in the setting of MLS due to the tumor cell's reduced ability to absorb labeled glucose in the spine location. Here we presented this unusual scenario where an atypical hemangioma and metastatic process mimicked one another. Our patient had multiple negative PET/CT and no definitive MRI for over 6 months. It was not until an MRI,
+performed 2 weeks after a negative PET/CT, showed progression could we ultimately say there was a metastatic bone lesion. Ultimately, only an accurate biopsy confirmed metastatic spread to the vertebral body.
+
+Sarcoma multidisciplinary treatment teams could face similar diagnostic challenges, analogous to the case presented. Making a distinction between a localized and a stage IV sarcoma has several implications in terms of treatment options and prognosis. In this case based on a questionable MRI
+with a negative PET/CT and negative biopsy the decision was made to proceed forward with resection and megaprosthetic reconstruction in the setting of previous radiation which has significant risks associated. Consequently, it is paramount to arrive at a definitive diagnosis in the setting of MLS, utilizing MRI surveillance and biopsy when suspicion for metastasis is high.
+
+## Conflict Of **Interest**
+
+The authors declare no conflict of interest.
+
+## References
+
+[1] Junghanns H, Schmorl G. The human spine in health and disease. New York: Grune & Stratton; 1971.
+
+[2] McEvoy SH, Farrell M, Brett F, et al. Haemangioma, an uncommon cause of an extradural or intradural extramedullary mass: case series with radiological pathological correlation. Insights Imaging 2016;7:87–98.
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+radiologic and pathologic features. Skeletal Radiol 2000;29(2):63–74.
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+et al. Hemangioma from head to toe: MR imaging with pathologic correlation. Radiographics 2004;24:367e85.
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+''cold'' vertebrae on bone scintigraphy and fluordeoxyglucose positron emission tomography-computed tomography. Indian J Nucl Med 2011;26:49e51.
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+Abnormal appearance of spinal hemangioma mimicking metastasis Mani S, Shanthly N, Oommen R. Abnormal appearance of spinal hemangioma mimicking metastasis in bone scintigraphy and SPECT CT: a case report. J Nucl Med Radiat Ther. 2013;S6:16–18.
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+Nagoya S, Yamashita T. Hemangioma of the rib showing a relatively high 18F-FDG uptake: a case report with a literature review. Acta Radiol Open 2017;6(9):2058460117728416. doi:10.1177/2058460117728416.
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+et al. Osseous hemangioma of the seventh cervical vertebra with osteoid formation mimicking metastasis: a case report. J Med Case Rep 2009;3:92.
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+[18] Morales KA, Arevalo-Perez J, Peck KK, Holodny AI, Lis E,
+Karimi S. Differentiating atypical hemangiomas and metastatic vertebral lesions: the role of T1-weighted dynamic contrast-enhanced MRI. AJNR Am J Neuroradiol 2018;39(5):968–73.
+
+[19] Balliu E, Vilanova JC, Pelaez I, et al. Diagnostic value of apparent diffusion coefficients to differentiate benign from malignant vertebral bone marrow lesions. Eur J Radiol 2009;69:560e6.
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+[20] Du Y, Cullum I, Illidge TM, Ell PJ. Fusion of metabolic function and morphology:sequential [18F]fluorodeoxyglucose positron-emission tomography/ computed tomography studies yield new insights into the natural history of bone metastases in breast cancer. J Clin Oncol 2007;25:3440–7.
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+Taniike K, et al. Pitfalls of FDG-PET for the diagnosis of osteoblastic bone metastases in patients with breast cancer.
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+doi:10.1007/s00259-005-1842-8.
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+Brennan MF, Woodruff JM, Jhanwar SC, Ladanyi M. Specificity of TLS-CHOP rearrangement for classic myxoid/round cell liposarcoma: absence in predominantly myxoid well-differentiated liposarcomas. J Mol Diagn 2000;2:132–8.
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+[23] Estourgie SH, Nielsen GP, Ott MJ. Metastatic patterns of extremity myxoid liposarcoma and their outcome. J Surg Oncol 2002;80(2):89–93. doi:10.1002/jso.1009.
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+Spinal metastases from myxoid liposarcoma warrant screening with magnetic resonance imaging. Cancer 2007;110:1815–22. doi:10.1002/cncr.22992.
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+Antonescu CR. Skeletal metastases in myxoid liposarcoma:
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+Bredella MA. Metastatic myxoid liposarcomas: imaging and histopathologic findings. Skeletal Radiology 2008;37(3):251–8.
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medical/md/PMC6965039.md

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+# Correction To: Cost–Utility And Budget Impact Analysis For Stopping The Inappropriate Use Of Proton Pump Inhibitors After Cessation Of Nsaid Or Low‑Dose Acetylsalicylic Acid Treatment
+
+Sek Hung Chau1  · Reinier Luuk Sluiter2  · Jacqueline Geertruida Hugtenburg1  · Michel Wensing3,4  · Wietske Kievit2  **· Martina Teichert5**
+Published online: 5 November 2019 © The Author(s) 2019 Correction to: Drugs & Aging 
+
+![0_image_0.png](0_image_0.png)
+
+https://doi.org/10.1007/s40266-019-00713-5 Errors were subsequently identifed in the article originally published online, and the following corrections should be noted:
+Correction to author name: Page 1, author listing and corresponding author details: The name of the frst and corresponding author was incorrectly formatted and should read:
+Sek Hung Chau.
+
+The original article can be found online at https://doi.org/10.1007/ s40266-019-00713-5.
+
+* Sek Hung Chau s.chau@amsterdamumc.nl 1 Department of Clinical Pharmacology and Pharmacy, Amsterdam Public Health Research Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1117, Amsterdam, The Netherlands 2 Department for Health Evidence, Radboud University Medical Center, Geert Grooteplein 21, Nijmegen, The Netherlands 3 Radboud University Medical Center, Scientifc Institute for Quality of Healthcare (IQ Healthcare), Geert Grooteplein 21, Nijmegen, The Netherlands 4 Department of General Practice and Health Services Research, Heidelberg University, Im Neuenheimer Feld 130.3, Heidelberg, Germany 5 Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Correction to Figure 1:
+Page 3, Fig. 1: An arrowhead was omitted between the 
+"Healthy" and "Hip fracture" health states in the fgure. The corrected fgure is shown here. **Open Access** This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License 
+(http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

medical/md/PMC7283996.md

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+W J C
+
+![0_image_0.png](0_image_0.png)
+
+# World Journal Of
+
+![0_Image_1.Png](0_Image_1.Png) Cardiology
+
+Submit a Manuscript: https://www.f6publishing.com *World J Cardiol* 2020 May 26; 12(5): 210-219 DOI: 10.4330/wjc.v12.i5.210 ISSN 1949-8462 (online)
+ORIGINAL ARTICLE
+Observational Study Preoperative nuclear stress testing in the very old patient population Amgad N Makaryus, John N Makaryus, Joseph A Diamond ORCID number: Amgad N Makaryus
+(0000-0003-2104-7230); John N Makaryus (0000-0002-3745-1054); Joseph A Diamond (0000-0002-4179-9547).
+
+Author contributions: Makaryus AN, Makaryus JN, Diamond JA designed the research, performed the research, analyzed the data, and wrote the paper.
+
+Institutional review board statement: This study was reviewed and approved by the Northwell Health System Institutional Review Board (IRB\# 04.08.141T).
+
+Informed consent statement: This retrospective study with anonymized data does not require informed consent due to nonidentifiable (anonymized) data as approved by the institutional review board.
+
+Conflict-of-interest statement:
+There is no conflict of interest associated with any of the senior author or other coauthors who contributed their efforts to this manuscript.
+
+Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at amakaryu@numc.edu. Consent was not obtained but the presented data are anonymized and risk of identification is low.
+
+STROBE statement: The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items.
+
+Amgad N Makaryus, John N Makaryus, Joseph A Diamond, Department of Cardiology, Northwell Health, New Hyde Park, NY, 11042, United States Amgad N Makaryus, Department of Cardiology, Nassau University Medical Center, East Meadow, NY 11554, United States Corresponding author: Amgad N Makaryus, MD, Associate Professor, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States, and Chairman, Department of Cardiology, Nassau University Medical Center, 2201 Hempstead Turnpike, East Meadow, NY 11554, United States. amakaryu@numc.edu
+
+## Abstract Background
+
+Elderly patients awaiting moderate to high-risk surgery may undergo nuclear stress testing (NST) in order to evaluate their cardiovascular risk. The prognostic utility of such testing in the very elderly (≥ 85 years) has yet to be fully evaluated. Octogenarians and nonogenarians frequently have a number of concurrent conditions including a high rate of coronary disease, and therefore the prognostic value of NST for their preoperative risk assessment has been questioned. Our evaluation assesses the ability of nuclear stress testing to predict peri-operative cardiac outcomes in this patient population. AIM To investigate the ability of NST to predict peri-operative cardiac outcomes in elderly patients awaiting moderate to high-risk surgery.
+
+## Methods
+
+Patients ≥ 85 years undergoing pre-operative NST were retrospectively evaluated. Patients undergoing low-risk surgery were excluded. Major adverse cardiac events (MACE) were considered any adverse event that occurred prior to discharge and included acute heart failure, arrhythmia, acute myocardial infarction, unstable angina, or death. Associations between patient risk factors, MACE, and the obtained results of the pre-operative stress testing, ejection fraction (< 40% or ≥ 40%), summed stress score (≤ 8, ≥ 9), and the summed difference score (≤ 0, > 0) were analyzed.
+
+## Results
+
+A total of 69 patients (mean age 88 ± 2.6 years, 31 males) underwent nuclear stress testing prior to surgery. There were 41 (60%) patients found to have an abnormal NST. Sixteen (23%) patients were noted to experience post-operative MACE. No significant associations between risk factors and MACE were noted. Patients with an abnormal NST and/or a summed stress score ≥ 9 were
+
+| 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/licen ses/by-nc/4.0/ Manuscript source: Invited Manuscript Received: November 8, 2019 Peer-review started: November 8, 2019 First decision: December 13, 2019 Revised: April 21, 2020 Accepted: May 12, 2020 Article in press: May 12, 2020 Published online: May 26, 2020 P-Reviewer: Basso N, Berezin AE, Nechifor G S-Editor: Wang J L-Editor: A E-Editor: Wu YXJ   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+significantly (P < 0.01) more likely to develop peri-operative MACE.
+
+CONCLUSION Indicated preoperative NST is useful to assess pre-operative risk in elderly patients ≥ 85 years undergoing moderate to high-risk surgery.
+
+Key words: Pharmacologic nuclear stress testing; Prognosis; Elderly; Preoperative assessment; Outcomes ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
+
+Core tip: It is common practice for elderly patients awaiting moderate to high-risk surgery to undergo nuclear stress testing in order to evaluate their cardiovascular risk. However, the prognostic utility of such testing in the very elderly (≥ 85 years) has yet to be evaluated. Octogenarians and nonogenarians frequently have a number of concurrent conditions in addition to a high rate of coronary disease and therefore the prognostic value of nuclear stress testing for their preoperative risk assessment has been questioned. We sought to assess the ability of nuclear stress testing to predict peri-operative cardiac outcomes in these patients. We found that there were no significant associations between risk factors and major adverse cardiac events. Patients with an abnormal nuclear perfusion result and/or a summed stress score ≥ 9 were significantly (P < 0.01) more likely to develop peri-operative major adverse cardiac events. Indicated nuclear stress testing, therefore, is useful to assess pre-operative risk in elderly patients ≥ 85 years undergoing moderate to high-risk surgery.
+
+Citation: Makaryus AN, Makaryus JN, Diamond JA. Preoperative nuclear stress testing in the very old patient population. *World J Cardiol* 2020; 12(5): 210-219 URL: https://www.wjgnet.com/1949-8462/full/v12/i5/210.htm DOI: https://dx.doi.org/10.4330/wjc.v12.i5.210
+
+## Introduction
+
+The overall prognostic utility of both treadmill and pharmacologic stress testing is well established[1-4]. However, the utility of stress testing in those ≥ 85 years old is not well established. Octogenarians and nonogenarians typically have an elevated rate of coronary artery disease. Furthermore, to our knowledge no prior study has specifically examined the utility of nuclear stress testing (NST) with respect to surgical outcomes in the ≥ 85 years old age group. Thus, the prognostic value of nuclear stress testing for preoperative risk assessment has been questioned in this patient population.
+
+In the present study, we sought to determine whether results of indicated preoperative nuclear stress tests (NST's) have prognostic value in predicting perioperative and post-operative cardiac events (one or more, none) in elderly patients undergoing moderate to high-risk surgery.
+
+## Materials And Methods Patient Selection
+
+We performed a retrospective descriptive study of 69 patients ≥ 85 years of age who underwent pre-operative nuclear stress testing (Figure 1). Patients undergoing lowrisk surgery as defined by the American College of Cardiology/ American Heart Association guidelines[5] were excluded. Other exclusion criteria were patients aged less than 85, and patients undergoing resting nuclear perfusion studies (i.e., viability studies).
+
+## Stress Testing
+
+Either pharmacologic (n = 67) or exercise treadmill stress testing (n = 2) was employed. An intravenous line was inserted prior to the start of the test. Exercise testing patients underwent symptom-limited treadmill exercise testing using a standard Bruce protocol. Heart rate, blood pressure, and 12-lead electrocardiograms
+
+![2_image_0.png](2_image_0.png)
+
+were recorded before (supine and upright positions) and during each minute of exercise and recovery. Patients exercised until the point of fatigue unless marked electrocardiographic abnormalities, hemodynamic instability, chronotropic incompetence, ventricular tachycardia or fibrillation, or disabling chest pain symptoms occurred. Exercise was discontinued if exertional hypotension, malignant ventricular arrhythmias, marked ST depression, or limiting chest pain occurred. An abnormal electrocardiogram was defined as ≥ 1 mm ST depression.
+
+In the pharmacologic testing patients, adenosine or dobutamine pharmacologic stress was performed. Adenosine was infused intravenously, at a dose of 140 mcg/kg per min over 6 min, with an administration of the radiopharmaceutical at the midpoint of the infusion. If patients exhibited persistent side effects from the adenosine, 75-125 mg of aminophylline were administered intravenously as needed.
+
+Dobutamine was infused using standard incremental dosing from 5-40 mcg/kg per min during which patients remained under continuous clinical, and electrocardiographic monitoring. Test endpoints included completion of the final stage of the protocol, severe ischemia (severe angina, > 2 mm ST depression), hypertension (systolic blood pressure > 220 mmHg), hypotension (drop in systolic blood pressure > 20 mmHg), arrhythmias, or side effects intolerable to the patient.
+
+Atropine (1 mg IV) was administered in patients who failed to attain 85% of agepredicted maximal heart rate at peak dose.
+
+## Myocardial Perfusion Imaging
+
+Single photon emission computed tomography (SPECT) was performed at one institution (Long Island Jewish Medical Center) using one or two-day single-isotope protocols, employing either 99mTc-sestamibi or 99mTc-tetrofosmin. At our other site (North Shore University Hospital), a dual isotope (201Tl and 99mTc-sestamibi) one-day protocol was employed. The myocardial single-photon emission computed tomography perfusion image acquisition was acquired at rest and after peak stress using a dual head Optima NX gamma camera at one site and a dual-head Millenium gamma camera (General Electric, ™ Milwaukee, WS, United States) at our other respective site. The images were processed using standard commercial software (Pegasys™, Milwaukee, WS, United States). All SPECT acquisitions were acquired at rest and after peak pharmacologic stress using a circular orbit (180°), in a 64 × 64 matrix. Image processing was performed in a standard manner. All image sets (horizontal and vertical long-axis and short-axis planes) were normalized to the maximal myocardial activity in that set and displayed in the standard American College of Cardiology orientation. An experienced observer blinded to clinical outcome data reviewed all scans. Stress images were compared with rest images. A
+20-segment scoring system that uses 3 short-axis slices [distal (apical), mid, and basal]
+of the left ventricle, with the apex represented by 2 segments visualized on a midvertical long-axis image was used. Each segment was scored as follows: 0 = normal, 1 = slight reduction of uptake, 2 = moderate reduction of uptake, 3 = severe reduction of uptake, and 4 = absence of radioactive uptake.
+
+## Adverse Cardiac Events
+
+A major adverse cardiac event (MACE) was considered for any adverse event that occurred prior to post-operative discharge. MACE included arrhythmia, myocardial infarction, unstable angina, heart failure, or death. The associations between patient risk factors, MACE, and the results of the pre-operative NST [normal (nl), abnormal (abnl)], the ejection fraction (EF < 40% or ≥ 40%), summed stress score (SSS ≤ 8, ≥ 9), and the summed difference score (SDS ≤ 0, > 0) were examined.
+
+## Statistical Analysis
+
+The use of logistic regression to examine the multivariate effects of nuclear stress test results and presence of cardiac risk factors prior to admission on intraoperative cardiac events (one or multiple) was undertaken. A Cox proportional hazards regression analysis was used to examine the multivariate effects of nuclear stress test results, presence of cardiac risk factors prior to admission and intraoperative cardiac events on time until first post-operative cardiac event.
+
+In the analysis of in-hospital adverse events, it is generally felt preferable to use survival methods; we therefore examined length of stay since patients staying longer are more likely to have events regardless of other risk factors. However, because this was a retrospective chart review, it was not possible to obtain the dates of all events with certainty. Therefore, rather than deleting patients from the analysis, the analysis was carried out without regard to time. In order to examine the association between length of stay and stress test result, we estimated length of stay (LOS) using the product limit method, and compared LOS stratified by stress test results using the log rank test only for those patients not having an event in hospital. Presumably, any difference in LOS would then be due to the test result and not the occurrence of an adverse event. Similar analyses were carried out for ejection fraction, summed stress score, and summed difference score.
+
+## Results Clinical Characteristics And Stress Test Results
+
+From the group of 69 patients (mean age 88 ± 2.6 years, 31 males) who underwent NST prior to surgery, Table 1 highlights the study population demographics. A majority of the patients had hypertension (82.6%) and 33.3% had hyperlipidemia. Abnormal NST was found in 60% (41/69) of the patients. A majority of the patients (37.7%) underwent orthopedic procedures followed by vascular surgeries (21.7%) and abdominal surgeries (21.7%).
+
+## Adverse Cardiac Events And In-Hospital Outcomes/Mortality
+
+Sixteen (23%) patients suffered post-operative MACE. Adverse events are detailed in Table 2. No significant associations were noted between risk factors and MACE (Table 3). Patients with an abnormal NST and/or an SSS ≥ 9 were significantly more likely (P < 0.02) to develop peri-operative MACE (Table 4). Patients with an abnormal stress test were 6.3 times more likely (P < 0.03) than those with a normal test to have a MACE prior to discharge (95% confidence interval: 1.3-30.5). Those with an EF < 40% or SDS > 0 (denoting ischemia) did not each show a statistical significance to developing peri-operative MACE, although in those with an EF < 40%, there was a trend toward significance with a P value of 0.065. Figure 2 shows the example of a patient who suffered post-operative heart failure after hip fracture repair. The images reveal an area of infarct in the proximal inferolateral wall with ischemia in the mid to distal inferolateral wall. A SSS of 22, and a SDS of 7 were noted in this patient.
+
+## Discussion
+
+Our investigation adds significant information to the body of evidence that shows prognositic utility of both treadmill and pharmacologic stress testing, but it adds this information in the population of octogenarians and nonagenarians who were underrepresented in many of the landmark prognostic evaluations of NST. Furthermore, no prior study has specifically examined the utility of NST with respect to surgical
+
+| Table 1 Patient demographics Characteristic   | Number of patients (%)   |
+|-----------------------------------------------|--------------------------|
+| Gender Male                                   | 31 (44.9)                |
+| Female                                        | 38 (55.1)                |
+| Mean age                                      | 88 yr                    |
+| Risk factors Hypertension                     | 57 (82.6)                |
+| Diabetes mellitus                             | 13 (18.8)                |
+| Hyperlipidemia                                | 23 (33.3)                |
+| Obesity                                       | 1 (1.4)                  |
+| Smoking                                       | 7 (10.1)                 |
+| Medications ASA                               | 13 (18.8)                |
+| Plavix                                        | 4 (5.8)                  |
+| Beta blocker                                  | 34 (49.3)                |
+| Statin                                        | 20 (29.0)                |
+| ACE-I/ARB                                     | 27 (39.1)                |
+| Digoxin                                       | 8 (11.6)                 |
+| Nitrates                                      | 9 (13.0)                 |
+| CC blocker                                    | 17 (24.6)                |
+| Diuretics                                     | 23 (33.3)                |
+| Procedures Orthopedic                         | 26 (37.7)                |
+| Vascular                                      | 15 (21.7)                |
+| Abdominal                                     | 15 (21.7)                |
+| Other                                         | 13 (18.8)                |
+
+ASA: Aspirin; ACE-I/ARB: Angiotensin converting enzyme inhibitor/angiotensin receptor blocker; CC blocker: Calcium channel blocker.
+
+outcomes in the ≥ 85 year old age group. This information is useful as it is not uncommon for these patients to undergo surgical operations, particularly orthopedic procedures. We found that clinical underlying risk factors did not predict outcomes. This is an important finding as many of these patients are sent to surgery without prior further stratification aside from risk factor assessment and modification. Also, although the SSS was a significant predictor of MACE, the SDS and the EF were not. This may be due to the small sample size as there is a trend in the P values towards significance. Prior studies[1,2] support the fact that the SSS is the major predictor of adverse events as opposed to the SDS. The NST, specifically an abnormal nuclear perfusion result and/or a SSS ≥ 9 (severely abnormal study), identified those patients significantly (P < 0.01) more likely to develop peri-operative MACE.
+
+Few prior investigations have examined the question of the prognostic utility of NST in the very elderly (≥ 85) age group[6-15]. Although these studies demonstrate the utility of NST in the elderly for predicting cardiac events, they do not focus on the specific situation of pre-operative risk assessment; our study does. Another discrepancy in these studies is the age cutoff for an "elderly" patient. Some authors ascribe an "elderly" patient to the ≥ 55 years range[13], while others use the cutoff of ≥ 65 years[6,11,12], ≥ 70 years[14,15], ≥ 75 years[9], or even ≥ 80 years[7,8,10]. Our study specifically assessed prognostic outcome after moderate-to high-risk surgery in patients ≥ 85 years, thus targeting a special population of very elderly individuals.
+
+In the evaluation by Steingart *et al*[6], the clinical, electrocardiographic (ECG) stress test, NST, and follow-up data for 626 patients aged ≥ 65 years (mean age 70 ± 4.4) with interpretable electrocardiograms undergoing symptom-limited exercise NST between 1992 and 1996 were evaluated with follow-up occurring after 4.4 ± 1.3 years. They found that the following factors predicted death or myocardial infarction: Male sex, increasing age, an abnormal rest ECG result, lower exercise tolerance and lower peak exercise heart rates, exercise ST-segment depression, left ventricular dilatation, and the number of ischemic regions. After multivariable analysis, only increasing patient
+
+| Table 2 Major adverse cardiac events Adverse event   | Number of subjects (%)   |
+|------------------------------------------------------|--------------------------|
+| Atrial fibrillation                                  | 3 (0.04%)                |
+| Congestive heart failure                             | 6 (0.09%)                |
+| Myocardial infarction                                | 1 (0.01%)                |
+| Supraventricular tachycardia                         | 1 (0.01%)                |
+| Angina                                               | 2 (0.03%)                |
+| Bradycardia                                          | 1 (0.01%)                |
+| Cardiogenic shock                                    | 1 (0.01%)                |
+| Trigeminy                                            | 1 (0.01%)                |
+| Death                                                | 3 (0.04%)                |
+| Total                                                | 16 (0.23%)               |
+
+age, male sex, limitation of exercise tolerance, and the number of ischemic segments by NST were predictive of death or myocardial infarction. They concluded that in elderly patients undergoing exercise NST, age, sex, exercise tolerance, and ischemia on NST provide significant prognostic information in this patient population. Nuclear imaging provided incremental prognostic information that was significant in comparison to clinical information and results of exercise testing[6].
+
+Goraya *et al*[11] assessed treadmill exercise testing in an elderly population (≥ 65 years of age) to evaluate the incremental value of the testing to clinical data. A comparison was undertaken with a median follow-up of 6 years with results of treadmill testing in younger patients (< 65 years). Elderly patients, as compared with younger patients, had more comorbidities, achieved a lower workload (6.0 vs 10.7 metabolic equivalents; P < 0.001), and had a greater likelihood of an abnormal exercise ECG (28% vs 9%; P < 0.001). Overall survival (63% vs 92%; P < 0.001) and cardiac event–free survival (66% vs 95%; P < 0.001) were worse among the elderly persons. The amount of workload achieved was the only treadmill exercise testing variable associated with all-cause mortality (P < 0.001) in both age groups. Workload was also the only additional treadmill exercise testing variable that was predictive of cardiac events (P < 0.05). Notably, each one metabolic equivalent rise in exercise capacity was associated with a 14% and 18% reduction in cardiac events among younger and elderly persons, respectively. They conclude that in elderly persons, treadmill exercise testing provided incremental prognostic information that was significant and incremental in comparison to clinical information. ECG results of stress testing (e.g., the presence of ST-segment depression) was likely not a significant contributor to prognosis, because in the elderly there is a higher occurrence of resting (baseline) STsegment changes and higher use of medications that may cause falsely positive ECG
+changes. Exercise workload on the treadmill was the only variable that was strongly associated with outcome, and its prognostic effect was equal in both the young and elderly age groups[11].
+
+A report by Zafrir *et al*[8] examined 162 consecutive patients (mean age 83 ± 3 years)
+who underwent NST with a duration of follow-up of 45 ± 12 months. Nineteen percent of patients (31) had major cardiac events recorded; including 26 cardiac deaths and 5 MI events, in 6 of the 61 women (10%) and 25 of the 101 men (25%) (P < 0.03). The univariate predictors of cardiac death or MI, aside from known coronary artery disease (CAD), included NST variables such as left ventricular dilatation, increased lung uptake, abnormal scan, and the presence and extent of myocardial ischemia. The only predictors for major cardiac events, however, were: left ventricular (LV) dilatation (OR = 6.9, 95%CI: 2.7-17.4, P < 0.0001) and ischemia by scan (OR = 2.75, 95%CI: 1.09-6.96, P < 0.03). Survival curves demonstrated significant differences in survival between patients with or without LV dilatation and patients with or without ischemia. They conclude that in octogenarian patients with CAD or with suspicion of CAD, LV dilatation and myocardial ischemia are useful predictors of cardiac death and MI[8].
+
+A report by De Winter *et al*[9] further used not only perfusion imaging in the prognostic assessment of elderly patients, but adds left ventricular function analysis as a further criterion in the evaluation. The clinical and gated SPECT predictors of cardiac and all-cause death in 294 patients aged ≥ 75 years with known or suspected CAD who were referred for tetrofosmin cardiac gated SPECT imaging were examined. During a median follow-up of 25.9 months, a total of 47 patients (16%) died. In their multivariate Cox proportional hazards regression analysis, the summed
+
+Figure 2
+
+![6_image_0.png](6_image_0.png)
+
+Figure 2 This image shows the nuclear stress testing result of one of our patients: An 88 year old man who presented with a fracture of his left hip. He was risk stratified by undergoing adenosine pharmacologic nuclear testing. The images reveal an area of infarct in the proximal inferolateral wall with reversibility (ischemia) in the mid to distal inferolateral wall.
+
+rest score, transient ischemic dilatation index, and resting left ventricular ejection fraction were independent predictors of all-cause death. The summed rest score and resting end-systolic volume were independent predictors of cardiac death. They conclude that NST left ventricular functional data assessed during myocardial gated SPECT provides independent and incremental information above clinical and perfusion SPECT data for the prediction of cardiac and all-cause death in patients aged ≥ 75 years[9].
+
+## Study Limitations
+
+The relatively small sample size of the study group limits our study findings. The small number of post-operative MACE is also a limitation, but may just be a credit to our advancing health-care techniques in the peri-operative period. Additional study of larger numbers of individuals would allow more specific analyses of NST's. For example, prognosis may be stratified by different categories of summed stress scores. Another limiting factor is that our analysis only included those patients who underwent surgery. It is not unreasonable to surmise that many patients who had abnormal NST might have had their surgeries cancelled due to their assumed high risk. This may introduce referral bias into the analysis, with potential for decreasing the correlation between abnormal studies and unfavorable surgical outcomes. It is also assumed that certain surgical techniques may have been modified as a result of the findings on NST.
+
+In the analysis of in-hospital events, it is generally preferable to use survival methods, so that length of stay is considered, since patients staying longer are more likely to have events regardless of other risk factors. Since this was a retrospective chart review, and it was not possible to obtain the dates of all events with certainty, rather than deleting patients from the analysis, we carried out the analysis without regard to time. However, if there were an association between risk factor and length of stay, then this analysis would be biased, since patients with a longer stay would be more likely to have experienced an event simply because they were in the hospital longer. On the other hand, patients experiencing an event may have longer stays as a result of the event. Therefore, in order to examine the association between length of stay and stress test result, we estimated length of stay (LOS) using the product limit method, and compared LOS stratified by stress test results using the log rank test only for those patients not having an event in hospital. Presumably, this way any difference in LOS would then be due to the test result and not the occurrence of an adverse event. Similar analyses were carried out for ejection fraction, summed stress score, and summed difference score. These analyses showed no associations between any of the test results and LOS for subjects not having an in hospital event.
+
+In conclusion, the use of nuclear stress testing is a useful tool to assess preoperative risk and post-opertive outcome stratification in elderly patients ≥ 85 years undergoing moderate to high-risk surgery in whom the test is indicated based on latest preoperative risk stratification guidelines. In a time when recommendations
+
+| Table 3 Major adverse cardiac events and risk factors Major adverse cardiac event Yes, n (%)   | No, n (%)   | P value   |         |
+|------------------------------------------------------------------------------------------------|-------------|-----------|---------|
+| Hypertension                                                                                   | Yes         | 13 (23)   | 44 (77) |
+| No                                                                                             | 2 (17)      | 10 (83)   | NS      |
+| Diabetes                                                                                       | Yes         | 3 (23)    | 10 (77) |
+| No                                                                                             | 12 (21)     | 44 (79)   | NS      |
+| Hypercholesterolemia                                                                           | Yes         | 2 (9)     | 21 (91) |
+| No                                                                                             | 13 (28)     | 33 (72)   | NS      |
+| Smoking                                                                                        | Yes         | 2 (29)    | 5 (79)  |
+| No                                                                                             | 13 (21)     | 49 (79)   | NS      |
+| Obesity                                                                                        | Yes         | 1 (100)   | 0 (0)   |
+| No                                                                                             | 14 (21)     | 54 (79)   | NS      |
+
+NS: Non-significant.
+
+now tend to less commonly recommend pre-operative testing, because the prediction of the development of post-operative complications using NST is possible, the use of NST in the appropriate patients should yield important and applicable information for it to benefit the patient and affect management. As our study demonstrates, an abnormal NST, and more specifically a significantly abnormal study with SSS ≥ 9, was noted to be highly predictive for the development of MACE in the immediate operative period in elderly patients ≥ 85 years undergoing moderate to high-risk surgery.
+
+| Table 4 Major adverse cardiac events and nuclear stress test variables Yes, n (%)   | No, n (%)   | P value   |         |
+|-------------------------------------------------------------------------------------|-------------|-----------|---------|
+| Nuclear stress test result                                                          | Abnormal    | 13(32)    | 28 (68) |
+| Normal                                                                              | 2 (7)       | 26 (93)   | < 0.02  |
+| Ejection fraction                                                                   | < 40%       | 6 (46)    | 7 (54)  |
+| ≥ 40%                                                                               | 9 (17)      | 45 (83)   | 0.065   |
+| Summed stress score                                                                 | ≥ 9         | 11 (37)   | 19 (63) |
+| ≤ 8                                                                                 | 4 (11)      | 34 (89)   | < 0.02  |
+| Summed difference score                                                             | > 0         | 10 (26)   | 28 (74) |
+| ≤ 0                                                                                 | 5 (17)      | 24 (83)   | 0.387   |
+
+## Article Highlights Research Background
+
+It is common for elderly patients awaiting moderate to high-risk surgery to undergo nuclear stress testing in order to evaluate their cardiovascular risk. However, the prognostic utility of such testing in the very elderly (≥ 85 years) has yet to be evaluated.
+
+## Research Motivation
+
+The very elderly (octogenarians and nonagenarians) frequently have a number of concurrent conditions in addition to a high rate of coronary disease and therefore the prognostic value of nuclear stress testing for their preoperative risk assessment has been questioned.
+
+## Research Objectives
+
+We sought to assess the ability of nuclear stress testing to predict peri-operative cardiac outcomes in these patients.
+
+## Research Methods
+
+We undertook a retrospective descriptive study of 69 patients ≥ 85 years of age who underwent pre-operative nuclear stress testing. Patients undergoing low-risk surgery as defined by guidelines were excluded. Other exclusion criteria were patients aged less than 85, and patients undergoing resting nuclear perfusion studies (i.e., viability studies).
+
+## Research Results
+
+We found that there were no significant associations between risk factors and major adverse cardiac events (MACE). Patients with an abnormal nuclear perfusion result and/or a summed stress score ≥ 9 were significantly (P < 0.01) more likely to develop peri-operative major adverse cardiac events.
+
+## Research Conclusions
+
+Indicated nuclear stress testing is useful to assess pre-operative risk in elderly patients ≥ 85 years undergoing moderate to high-risk surgery.
+
+## References
+
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+2 **Hachamovitch R**, Berman DS, Kiat H, Cohen I, Lewin H, Amanullah A, Kang X, Friedman J, Diamond GA. Incremental prognostic value of adenosine stress myocardial perfusion single-photon emission computed tomography and impact on subsequent management in patients with or suspected of having myocardial ischemia. *Am J Cardiol* 1997; 80: 426-433 [PMID: 9285653 DOI: 10.1016/s0002-9149(97)00390-1]
+3 **Hachamovitch R**, Berman DS, Kiat H, Cohen I, Cabico JA, Friedman J, Diamond GA. Exercise myocardial perfusion SPECT in patients without known coronary artery disease: incremental prognostic value and use in risk stratification. *Circulation* 1996; 93: 905-914 [PMID: 8598081 DOI: 10.1161/01.cir.93.5.905]
+4 **Banerjee A**, Newman DR, Van den Bruel A, Heneghan C. Diagnostic accuracy of exercise stress testing for coronary artery disease: a systematic review and meta-analysis of prospective studies. *Int J Clin Pract* 2012; 66: 477-492 [PMID: 22512607 DOI: 10.1111/j.1742-1241.2012.02900.x]
+5 **Fleisher LA**, Fleischmann KE, Auerbach AD, Barnason SA, Beckman JA, Bozkurt B, Davila-Roman VG,
+Gerhard-Herman MD, Holly TA, Kane GC, Marine JE, Nelson MT, Spencer CC, Thompson A, Ting HH, Uretsky BF, Wijeysundera DN. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. *Circulation* 2014; 130: e278e333 [PMID: 25085961 DOI: 10.1161/CIR.0000000000000106]
+6 **Steingart RM**, Hodnett P, Musso J, Feuerman M. Exercise myocardial perfusion imaging in elderly patients. *J Nucl Cardiol* 2002; 9: 573-580 [PMID: 12466780 DOI: 10.1067/mnc.2002.127013]
+7 **Katsikis A**, Theodorakos A, Papaioannou S, Tsapaki V, Kolovou G, Drosatos A, Koutelou M. Long-term prognostic value of myocardial perfusion imaging in octogenarians able to undergo treadmill exercise stress testing. *J Nucl Cardiol* 2014; 21: 1213-1222 [PMID: 25189145 DOI: 10.1007/s12350-014-9991-4]
+8 **Zafrir N**, Mats I, Solodky A, Ben-Gal T, Sulkes J, Battler A. Prognostic value of stress myocardial perfusion imaging in octogenarian population. *J Nucl Cardiol* 2005; 12: 671-675 [PMID: 16344229 DOI: 10.1016/j.nuclcard.2005.08.001]
+9 **De Winter O**, Velghe A, Van de Veire N, De Bondt P, De Buyzere M, Van De Wiele C, De Backer G,
+Gillebert TC, Dierckx RA, De Sutter J. Incremental prognostic value of combined perfusion and function assessment during myocardial gated SPECT in patients aged 75 years or older. *J Nucl Cardiol* 2005; 12: 662-670 [PMID: 16344228 DOI: 10.1016/j.nuclcard.2005.08.002]
+10 **Zafrir N**, Mats I, Solodky A, Ben-Gal T, Battler A. Characteristics and outcome of octogenarian population referred for myocardial perfusion imaging: comparison with non-octogenarian population with reference to gender. *Clin Cardiol* 2006; 29: 117-120 [PMID: 16596834 DOI: 10.1002/clc.4960290307]
+11 **Goraya TY**, Jacobsen SJ, Pellikka PA, Miller TD, Khan A, Weston SA, Gersh BJ, Roger VL. Prognostic value of treadmill exercise testing in elderly persons. *Ann Intern Med* 2000; 132: 862-870 [PMID: 10836912 DOI: 10.7326/0003-4819-132-11-200006060-00003]
+12 **Iskandrian AS**, Heo J, Decoskey D, Askenase A, Segal BL. Use of exercise thallium-201 imaging for risk stratification of elderly patients with coronary artery disease. *Am J Cardiol* 1988; 61: 269-272 [PMID: 3341202 DOI: 10.1016/0002-9149(88)90929-0]
+13 **Abidov A**, Hachamovitch R, Hayes SW, Ng CK, Cohen I, Friedman JD, Germano G, Berman DS.
+
+Prognostic impact of hemodynamic response to adenosine in patients older than age 55 years undergoing vasodilator stress myocardial perfusion study. *Circulation* 2003; 107: 2894-2899 [PMID: 12796141 DOI: 10.1161/01.CIR.0000072770.27332.75]
+14 **Shaw L**, Chaitman BR, Hilton TC, Stocke K, Younis LT, Caralis DG, Kong BA, Miller DD. Prognostic value of dipyridamole thallium-201 imaging in elderly patients. *J Am Coll Cardiol* 1992; 19: 1390-1398 [PMID: 1593029 DOI: 10.1016/0735-1097(92)90592-b]
+15 **Hilton TC**, Shaw LJ, Chaitman BR, Stocke KS, Goodgold HM, Miller DD. Prognostic significance of exercise thallium-201 testing in patients aged greater than or equal to 70 years with known or suspected coronary artery disease. *Am J Cardiol* 1992; 69: 45-50 [PMID: 1729866 DOI: 10.1016/0002-9149(92)90674-n]
+Published by Baishideng Publishing Group Inc
+
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+
+![10_image_1.png](10_image_1.png)
+
+7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA
+Telephone: +1-925-2238242 E-mail: bpgoffice@wjgnet.com Help Desk: https://www.f6publishing.com/helpdesk https://www.wjgnet.com

medical/md/PMC7981622.md

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+Portal venous gas after a failed endoscopic retrograde cholangiopancreatography attempt in a patient with a large hepatocellular carcinoma: A case report Gianluca Cassese1 | Francesco Maione1 | Mariantonietta Alagia1 | Luisa Ambrosio1 | Emanuele Caruso1 | Alessia Chini1 | Claudio Cimmino1 | **Riccardo Aurelio Nasto1** | Francesca Pegoraro1 | Mariano Cesare Giglio1 | **Giovanni Domenico De Palma1,2**
+1Department of Clinical Medicine and Surgery, University of Naples "Federico II", Napoli, Italy 2Center of Excellence for Technical Innovation in Surgery of Naples, "CEITC Unina", Napoli, Italy Correspondence Gianluca Cassese, Department of Clinical Medicine and Surgery, University of Naples "Federico II", Via S. Pansini, 5, Napoli, Italy. Email: gianluca.cassese91@gmail.com
+
+## Abstract
+
+The cause of hepatic portal vein gas (HPVG) is variable. Good knowledge of the possible causes, combined with the clinical assessment of the patient and a good quality imaging, is required to correctly identify the underlying cause of HPVG and to best predict the prognosis.
+
+KEYWORDS
+endoscopy, ERCP, hepatocellular carcinoma, portal vein gas
+
+## 1 | **Introduction**
+
+Portal vein gas is a rare radiologic finding, with an unclear pathogenesis. Different management strategies have been proposed, without unanimous consent. A patient with massive hepatocellular carcinoma (HCC) that compressed the biliary ducts, with poor performance status, underwent endoscopic biliary drainage attempt. The patient developed portal pneumatosis that lead to death.
+
+Portal vein gas or hepatic portal vein gas (HPVG) is a rare radiologic finding, with only <200 cases reported in the literature by 2001.1 It was first described in 1955 by Wolfe and Evans in pediatric patients with necrotizing enterocolitis.2 First reports showed a high mortality rate, with very rare case of patients that survived to such an event, suggesting a risk of imminent death in HPVG patients.3 More recent imaging modalities such as Doppler imaging and computed tomography 
+(CT) showed higher sensitivity,4 allowing to detect HPVG 
+as an incidental finding in many benign diseases.5 Different causes and conditions have been described to be associated HPVG in adults6-10 (Table  1), with intestinal ischemia that seem to be the most common (75%) and the worst one in term of prognosis.11 The underlying clinical events associated with HPVG seem to play the key role for patients' survival and prognosis that could be mainly related to the pathology itself and not to the presence of HPVG.12 The pathogenesis of HPVG is not well known. Different factors have been proposed: translocation of gas produced by gas-forming bacteria in bowel lumen or in an abscess which then somehow circulate into the liver, as well as the presence of mucosal lesions that allow a passage of gas-forming bacteria into the blood vessels, or a mechanical effect of a gaseous distension of the viscera that somehow overcomes an already weak wall due to underlying pathologies.5 A management algorithm has been proposed by Nelson et al,13 with the so-called "ABC strategy". Urgent laparotomy 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.
+
+© 2021 The Authors. *Clinical Case Reports* published by John Wiley & Sons Ltd.
+
+## 1340 |  Al.
+
+| TABLE 1                                                                                                                                                                                                       | Pathologic conditions associated with Portal Vein Gas   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------|
+| Intestinal ischemia Biliary infections Gastric ulcer Gastric dilatation Duodenal perforation Transgastric ultrasound-guided biopsy Inflammatory bowel disease Intra-abdominal abscess Retroperitoneal abscess |                                                         |
+
+("Aggressive management") is recommended for patients with signs of bowel necrosis or ischemia at CT scan, with a mortality approximated at 75%. Careful monitoring should be carried out in patients with a more nuanced clinical condition, leaving a threshold for surgical correction under appropriate conditions ("Be careful"). The risk for mortality in these cases is estimated between 20% and 30%. Finally, patients who present HPVG in nonurgent conditions or postoperatively should be treated conservatively ("Conservative management"), by acting a close observation.14 Portal venous gas after endoscopic retrograde cholangiopancreatography (ERCP) is a rare complication, described in one of 6-8 per 1000 procedures.15,16 It can occur because of a vascular laceration during precut sphincterotomy or because of a porto-biliary fistula, maybe associated with tumor infiltration or inflammatory-related conditions.17,18 To our knowledge, we report the first case of portal vein gas after a simple ERCP failed attempt, without large sphincterotomy or cannulation. Literature reports showed a low morbidity and mortality after ERCP, compared to other causes. The case reported below has had a very rapid course toward the death of the patient.
+
+## 2 | **Presentation Of Case**
+
+A 70-year-old male patient was referred to the gastrointestinal surgery department by his general practitioner with an 
+
+![1_image_0.png](1_image_0.png)
+
+history of progressive jaundice with itching, inappetence, weight loss, and asthenia. He was known to have a previous HCV infection eradicated for about six years, venous varices of the lower limbs. He had no current therapy. The clinical examination showed significant jaundice, palpable abdominal mass in right hypochondria, no abdominal tenderness, and some scratch injuries. Performance status of the patient was ECOG 3. A contrast-enhanced CT scan of the abdomen showed a central liver mass of about 131 millimeters, with arterial wash-in and portal wash-out features, typical for HCC. The neoplastic lesion constricted the biliary confluence, with a dilation of the intrahepatic biliary tract. Furthermore, there were no distant metastases, but some local and distant para-centimetric lymph nodes were reported. The case was discussed in a multidisciplinary oncology meeting, with an indication to perform systemic therapy after drainage of the biliary tract, to palliate jaundice. The patient was hospitalized to undergo to endoscopic biliary drainage. Preoperative blood tests showed bilirubinemia up to 279 µmol/L, CRP at 30 mg/L, normal blood cell count, normal INR, and albuminemia at 29  g/L. The endoscopic procedure was carried out under general anesthesia, but without success. The endoscopist found a marked ab extrinsic compression of the gastric cavity with associated distortion of the piloro-duodenal axis. The orifice of the papilla of Vater was dislocated. A 
+small precut of only 5 mm was carried out, but the numerous attempts to advance the guide-wire were unsuccessful. 
+
+It was then decided to proceed with a percutaneous biliary drainage CT-guided. However, the patient's clinical conditions showed a rapid worsening, with a deterioration of the respiratory function, an increase in inflammation blood tests and a mild fever the night after the procedure. An urgent contrast-enhanced thoraco-abdominal CT scan was carried out. It showed no signs of pulmonary embolism, but a slight bilateral pleural effusion. On the other hand, it revealed portal, splenic, and gastro-epiploic vessels pneumatosis (Figure 1). There was no associated intestinal ischemia or intestinal pneumatosis. A support therapy was set up with a careful and close monitoring of the patient. 
+
+FIGURE 1 Preoperative (A) and postoperative (B) abdominal contrastenhanced CT scan. Yellow arrow showing presence of gas in portal vein After an active debate among surgeons and anesthesiologists, it was decided to not proceed with surgical therapy because of the clinical conditions of the patients together with the absence of intestinal pneumatosis and intestinal ischemia. Subsequent blood tests showed leukocytosis at 18 × 103, increased blood creatinine level at 220 µmol/L, 
+increased PCR at 130  mg/L, reduced albuminemia at 25  g/L, increased AST levels at 500  U/L, and increased LDH and blood lactic acid levels. It was then set an antibiotic therapy and a support of a subintensive type. Despite a further intensive care, the patient developed a multi-organ failure that led to death in a few days.
+
+## 3 | **Discussion**
+
+Clinical case here reported seems to fall into one of the scenarios leading to HPVG: bowel distention/obstruction, ischemia, and idiopathic.19 In particular, we can speculate about the mechanical pathogenesis related to gas insufflation during the endoscopic procedure. Mechanical disruption of mucosal integrity may result in dissection of gas into the intestinal wall and eventually the portal system. The loss of mucosal integrity has been descripted also to be related to gastrointestinal neoplasms.20 The translocation of the intestinal wall by gas-forming bacteria may also have concurred, resulting in the production of gas within the portal system itself.
+
+Several therapeutic algorithms have been proposed over the years, leaving open the discussion on whether or not to perform urgent surgery. Various studies highlighted the importance of the clinical status of the patient, rather than the CT finding alone, including physical examination findings, vital signs, and laboratory values.19,21 Very interesting was the paper by Koami et al that in a retrospective court of 33 patients with HPVG, using a criteria of lower blood pressure (systolic BP <108 mm Hg), high lactate dehydrogenase circulating levels (LDH >387 U/L), and the presence of pneumatosis intestinalis led to 100% sensitivity and 78.9% specificity for necrotic bowel.22 A very detailed algorithm was suggested by Wayne et al by analyzing a retrospective series of 88 patients. The algorithm incorporates many clinical findings, including abdominal examination, lactate (>3 mg/dL), and radiologic findings of pneumatosis intestinalis, significant past medical history suggestive of vascular risk.23 In a larger retrospective study, Hani et al24 pooled data from four tertiary centers and evaluated 209 patient who had HPVG focusing on factors associated with the need for operative intervention. They found that older age, peritoneal signs, and elevated BUN are most likely associated with intestinal ischemia and need for urgent surgery. A very recent case report by Dibra et al25 also tried to suggest a set of interesting decision-making criteria in patients with portal vein gas, although with associated intestinal pneumatosis. The authors focused on the presence of clinical signs (age >60 years, hemodynamic instability, peritonitis, adynamic ileus) and laboratory/radiologic signs (elevated White Blood Cells, lactate ≥2 mmol/L, elevated INR, hepatic portal venous gas, small bowel location), according with the indications for intestinal pneumatosis of the American Association for the Surgery of Trauma.26 The presence of so many papers on the same subject testifies the absence of a single therapeutic conduct universally accepted.
+
+The case we reported is a perfect example of a patient in which it is difficult to choose the right conduct. CT scan showed no intestinal pneumatosis and signs of intestinal ischemia, but at the same time there was an increase in inflammatory indices at blood tests and a worsening of renal function. This case is a perfect example of the importance of underlying clinical conditions in determining the prognosis. The complicated postoperative course, along with the clinical conditions and the finding of HPVG at imaging without pneumatosis intestinalis, lead to an active debate among surgeons, anesthesiologists, and gastroenterologists regarding patient's management, as well as the high risk of surgery.
+
+Nonetheless, the clinical findings and outcomes of this case illustrate the inability to predict the outcomes based only on the presence of HPVG on CT imaging, suggesting that an aggressive approach may be appropriate when the prognosis appears adverse. The underlying advanced cancer disease, along with poor performance status, inexorably leads the patient to death.
+
+Based on the presented case, we think that the patient's performance status and underlying pathology should be considered as important additional criteria in the decision-making process of the patient with HPVG.
+
+## 4 | **Conclusions**
+
+The cause of HPVG without pneumatosis intestinalis is variable. Good knowledge of the possible causes combined with a good clinical assessment and an abdominal computed tomographic scan is required to correctly identify the underlying cause of HPVG and to decide whether urgent surgery must be performed.
+
+Our patient presented a case of fatal HPVG, after an ERCP 
+attempt without cannulation, fistulas, or perforations. We are convinced that each individual patient must be carefully discussed for risks and benefits of all possible procedures. 
+
+The outcome is strongly linked to the patient's underlying condition.
+
+ACKNOWLEDGMENTS Nothing to declare.
+
+## Conflict Of Interest None Declared. Author Contribution
+
+Giglio MC and De Palma GD: were responsible for study conception and design. Cassese G and Alagia M: made the drafting of manuscript. Nasto RA, Pegoraro F, Ambrosio L, and Cimmino C: participated to the acquisition of data. Maione F and Chini A: helped with the critical revision of the manuscript. ETHICAL STATEMENT Written informed consent was obtained from the patient for the use of data for anonymous research and publication purposes. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available on request from the authors. The data are not publicly available due to privacy and ethical restrictions.
+
+## Orcid
+
+Gianluca Cassese https://orcid.org/0000-0001-9185-2054
+
+## References
+
+1. Kinoshita H, Shinozaki M, Tanimura H, et al. Clinical features and management of hepatic portal venous gas: Four case reports and cumulative review of the literature. *Arch Surg*. 2001;136(12):1410. 
+
+https://doi.org/10.1001/archsurg.136.12.1410 2. Wolfe JN, Evans WA. Gas in the portal veins of the liver in infants; a roentgenographic demonstration with postmortem anatomical correlation. *Am J Roentgenol Radium Ther Nucl Med*. 
+
+1955;74(3):486–488.
+
+3. Liebman PR, Patten MT, Manny J, Benfield JR, Hechtman HB. 
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+Hepatic-portal venous gas in adults: etiology, pathophysiology and clinical significance. *Ann Surg*. 1978;187(3):281-287. https://doi.
+
+org/10.1097/00000658-197803000-00012 4. Ben PH, Huang JS, Yang TL, Liang HL. Hepatic portal venous gas in ultrasonogram-benign or noxious. *Ultrasound Med* Biol. 2007;33(8):1179-1183. https://doi.org/10.1016/j.ultrasmedb io.2007.01.014 5. Abboud B, El Hachem J, Yazbeck T, Doumit C. Hepatic portal venous gas: Physiopathology, etiology, prognosis and treatment. 
+
+World J Gastroenterol. 2009;15(29):3585. https://doi.org/10.3748/
+wjg.15.3585 6. Pfaffenbach B, Wegener M, Böhmeke T. Hepatic portal venous gas after transgastric EUS-guided fine-needle aspiration of an accessory spleen. *Gastrointest Endosc*. 1996;43(2):515-518. https://doi.
+
+org/10.1016/S0016-5107(96)81566-5 7. Speer CS, Carlson DH. Portal venous air with survival in ulcerative colitis: report of a case. *Dis Colon Rectum*. 1972;15(6):453-458. 
+
+https://doi.org/10.1007/BF02642654 8. Lee C-S, Kuo Y-C, Peng S-M, et al. Sonographic detection of hepatic portal venous gas associated with suppurative cholangitis. 
+
+J Clin Ultrasound. 1993;21(5):331-334. https://doi.org/10.1002/
+jcu.1870210507 9. Radin DR, Rosen RS, Halls JM. Acute gastric dilatation: a rare cause of portal venous gas. *AJR Am J Roentgenol*. 1987;148:279-280.
+
+10. Chang SG, Lee SC, Hong DH, Chai SE. Portal and superior mesenteric venous gas with retroperitoneal abscess–CT diagnosis (case report). *J Korean Med Sci*. 1992;7(1):62. https://doi.org/10.3346/
+jkms.1992.7.1.62 11. Peloponissios N. Hepatic portal gas in adults. *Arch Surg*. 
+
+2003;138(12):1367. https://doi.org/10.1001/archsurg.138.12.1367 12. Monneuse O, Pilleul F, Barth X, et al. Portal venous gas detected on computed tomography in emergency situations: Surgery is still necessary. *World J Surg*. 2007;31(5):1066-1072. https://doi.
+
+org/10.1007/s00268-006-0589-0 13. Nelson AL, Millington TM, Sahani D, et al. Hepatic portal venous gas: the ABCs of management. *Arch Surg*. 2009;144(6):575. 
+
+https://doi.org/10.1001/archsurg.2009.88 14. Griffiths DM, Gough MH. Gas in the hepatic portal veins. *Br J* 
+Surg. 1986;73(3):172-176. https://doi.org/10.1002/bjs.18007 30305 15. Espinel J, Pinedo ME, Calleja JL. Portal vein filling: an unusual complication of needle-knife sphincterotomy. *Endoscopy*. 
+
+2007;39(S 1):E245. https://doi.org/10.1055/s-2007-966156 16. Kurella R, Maple J. Portal vein cannulation: an unusual complication of ERCP. *Am J Gastroenterol*. 2010;105:S541. https://doi.
+
+org/10.14309/00000434-201010001-01456 17. Kalaitzakis E, Stern N, Sturgess R. Portal vein cannulation: an uncommon complication of endoscopic retrograde cholangiopancreatography. *World J Gastroenterol*. 2011;17(46):5131. https://doi.
+
+org/10.3748/wjg.v17.i46.5131 18. Kawakami H, Kuwatani M, Kudo T, Ehira N, Yamato H, Asaka M. Portobiliary fistula: Unusual complication of wire-guided cannulation during endoscopic retrograde cholangiopancreatography. *Endoscopy*. 2011;43(S 02):E98-E99. https://doi.
+
+org/10.1055/s-0030-1256150 19. Wayne E, Ough M, Wu A, et al. Management algorithm for pneumatosis intestinalis and portal venous gas: treatment and outcome of 88 consecutive cases. *J Gastrointest Surg*. 2010;14(3):437-448. 
+
+https://doi.org/10.1007/s11605-009-1143-9 20. Schatz TP, Nassif MO, Farma JM. Extensive portal venous gas: 
+Unlikely etiology and outcome. *Int J Surg Case Rep*. 2015;8:134136. https://doi.org/10.1016/j.ijscr.2014.10.058 21. Villa M, Ranade A, Jaimes-Vanegas N, et al. "STOP, LOOK, and LISTEN." Hepatic portal venous gas: time for clinical assessment!. Int J Angiol. 2013;22(2):123–126. https://doi.org/10.1055/s-0032-1333066 22. Koami H, Isa T, Ishimine T, et al. Risk factors for bowel necrosis in patients with hepatic portal venous gas. *Surg Today*. 
+
+2015;45(2):156-161. https://doi.org/10.1007/s00595-014-0941-1 23. Wayne E, Ough M, Wu A, et al. al. Management algorithm for pneumatosis intestinalis and portal venous gas treatment and outcome of 88 consecutive cases. *J Gastrointest Surg*. 2009;14(3):437-448.
+
+24. Hani MB, Kamangar F, Goldberg S, et al. Pneumatosis and portal venous gas: do CT findings reassure? *J Surg Res*. 2013;185(2):581586. https://doi.org/10.1016/j.jss.2013.06.006 25. Dibra R, Picciariello A, Trigiante G, et al. Pneumatosis intestinalis and hepatic portal venous gas: watch and wait or emergency surgery? A case report and literature review. *Am J Case Rep*. 2020;21: 
+e923831. https://doi.org/10.12659/AJCR.923831 26. Ferrada P, Callcut R, Bauza G, et al. Pneumatosis intestinalis predictive evaluation study: a multicenter epidemiologic study of the American association for the surgery of trauma. J Trauma Acute Care Surg. 2017;82(3):451-460. https://doi.org/10.1097/TA.00000 00000001360 How to cite this article: Cassese G, Maione F, 
+Alagia M, et al. Portal venous gas after a failed endoscopic retrograde cholangiopancreatography attempt in a patient with a large hepatocellular carcinoma: A case report. *Clin Case Rep*. 
+
+2021;9:1339–1343. https://doi.org/10.1002/ccr3.3766

medical/md/PMC8128203.md

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+AUTHOR'S VIEWS
+
+# New Link Between The Rna Polymerase Ii-Ctd And Replication Stress
+
+Helga B. Landsverk, Lise E. Sandquist, Lilli T. E. Bay, and Randi G. Syljuåsen Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway ABSTRACT
+
+![0_image_1.png](0_image_1.png)
+
+Conflicts between transcription and replication are a major source of replication stress. Our recent findings show that proper dephosphorylation of Serine 5 in the carboxy-terminal domain (CTD) of DNA-directed RNA polymerase II subunit RPB1 is needed to prevent such conflicts in human cells.
+
+Replication stress promotes genome instability and can be exploited in cancer treatment. Detailed knowledge of the causes and consequences of replication stress is therefore relevant both to understand the development and to improve the treatment of cancer. Transcription-replication conflicts (T-R conflicts) are a common source of replication stress, but how the transcription machinery is regulated to prevent such conflicts has until now remained poorly understood. In our recent work,1 we identify a regulatory pathway that promotes degradation of the largest core component of RNA polymerase II, DNA-directed RNA polymerase II subunit RPB1 (hereafter referred to as RNAPII), on chromatin and suppresses T-R conflicts in human cells. Since degradation of RNAPII on chromatin occurred both in the absence and presence of exogenous replication stress, our work suggests that continuous turnover of RNAPII on chromatin is required to prevent T-R conflicts even during normal cell growth.
+
+Notably, RNAPII degradation has previously been proposed to promote the overall efficiency of transcription by removing stalled RNAPII complexes.2 Our work further extends this by showing that RNAPII degradation is also required to prevent T-R conflicts. Furthermore, we identify an inhibitory role for Serine 5 phosphorylation (pS5) in the Carboxy-terminal domain (CTD) in RNAPII degradation of chromatin. A similar role for pS5 in RNAPII degradation was previously observed in yeast.2 At the time, pS5 was proposed to facilitate the specific degradation of elongating RNAPII, as pS5 was believed to be primarily associated with promoter proximal regions.2 However, as pS5 was later shown to be located throughout the gene body,3 the reason pS5 inhibits RNAPII 
+degradation is currently unclear. Interestingly, pS5 is particularly enriched at splice sites,3 which are sites of RNAPII 
+ARTICLE HISTORY 
+
+![0_image_0.png](0_image_0.png)
+
+Received 1 March 2021 Revised 23 March 2021 Accepted 24 March 2021 KEYWORDS 
+Replication stress; transcription-replication conflicts; PP1 phosphatase; RNA polymerase II; PP1 nuclear targeting subunit (PNUTS); carboxy-terminal domain (CTD); WD repeatcontaining protein 82 
+(WDR82)
+pausing.4 One possibility is therefore that pS5 may protect RNAPII from unwanted degradation during transcriptional pausing.
+
+Our study focuses on the protein phosphatase complex containing the serine/threonine-protein phosphatase 1 catalytic subunit (best known as PP1) and PP1 regulatory subunit 10 
+(best known as PP1 nuclear targeting subunit (PNUTS)). PP1 is involved in many cellular processes, and specific functions are mediated by regulatory proteins such as PNUTS.5 The only established substrate of PNUTS-PP1 is pS5 in RNAPII.6 Moreover, PNUTS is enriched in promoter-proximal regions, at splice sites and termination sites,7 which are frequently associated with RNAPII pausing.4 We show that a strong binding partner of PNUTS, WD repeat-containing protein 82 
+(WDR82), is also required for pS5 dephosphorylation in live cells.1 Moreover, depletion of either PNUTS or WDR82 caused less degradation and higher stability of RNAPII on chromatin, resulting in T-R conflicts. We hypothesized that reduced pS5 dephosphorylation was causing the effects on RNAPII stability and T-R conflicts. Supporting this, wild-type PNUTS, but not a PP1 binding deficient mutant, rescued the effects on DNA replication and RNAPII chromatin stability after depletion of endogenous PNUTS. Furthermore, depletion of a different pS5 RNAPII phosphatase, RNA polymerase II C-terminal domain phosphatase SSU72 (best known as SSU72), also stabilized RNAPII on chromatin and caused replication stress, thus supporting that pS5 is the relevant substrate that stabilizes RNAPII 
+on chromatin.1 Underscoring the intricate role of RNAPII pS5 phosphorylation is the observation that it also plays a role in signaling during the DNA damage response. Indeed, our previous work on PNUTS-PP1 suggests that RNAPII pS5 promotes signaling CONTACT Helga B. Landsverk helga.bjarnason.landsverk@rr-research.no; Randi G. Syljuåsen randi.syljuasen@rr-research.no Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Ullernchausseen 70, Oslo N-0379, Norway.
+
+via the apical DNA damage kinase, ataxia telangiectasia, and Rad3-related (ATR).8 At first glance the connection between our previous and current findings seems obvious. ATR is well known to respond to replication stress, and therefore, T-R conflicts caused by RNAPII pS5 could be leading to ATR signaling. Although the latter is likely a contributing factor, ATR signaling after depletion of PNUTS was also observed in G1 phase, arguing that ongoing DNA replication is not a strict requirement. In addition, ATR signaling after depletion of PNUTS was higher than expected compared to similar levels of replication stress caused by hydroxyurea.8 Furthermore, replication stress is believed to activate ATR by leading to single stranded DNA coated with replication protein A (RPA).9 However, the high ATR signaling after depletion of PNUTS was not reduced by co-depletion of replication protein A 70 kDa DNA-binding subunit (RPA70), an essential subunit of the RPA complex. The high ATR signaling after PNUTS depletion therefore appears to be non-canonical. Interestingly, ATR activity after depletion of PNUTS was strongly reduced by co-depletion of cell division cycle 73 (CDC73), a phospho-CTD binding protein and component of the polymerase associated factor 1 (PAF1) transcription complex.8 Furthermore, in our new work, we found that replication stress and RNAPII chromatin stability after depletion of PNUTS or its interacting partner WDR82 was also dependent on CDC73.1 CDC73 may therefore contribute to ATR activity by stabilizing S5 phosphorylated RNAPII on chromatin.
+
+Altogether, our results suggest that the phosphorylation status of the RNAPII CTD plays a key role in the regulation of both ATR activity8 and replication stress1 (Figure 1). In addition to its important roles in the regulation of transcription and mRNA processing,10 the RNAPII CTD thus also appears to act as a signaling platform in genome maintenance.
+
+Figure 1. Model for how the RNA polymerase II-CTD acts as a signaling platform in 
+
+![1_image_0.png](1_image_0.png) genome maintenance. Serine 5 phosphorylation (pS5) in the carboxy-terminal domain (CTD) of DNA-directed RNA polymerase II subunit RPB1 (best known as RNAPII) promotes replication stress and signaling via the apical DNA damage kinase ataxia telangiectasia and Rad3-related (ATR). The phosphatase complex, consisting of serine/threonine-protein phosphatase 1 catalytic subunit (best known as PP1), PP1 regulatory subunit 10 (best known as PP1 nuclear targeting subunit (PNUTS)) and WD repeat-containing protein 82 (WDR82), dephosphorylates pS5 in the RNAPII CTD and thereby suppresses ATR signaling and replication stress in human cells. Note that ATR signaling via pS5 in the RNAPII CTD does not strictly require replication. See main text for details.
+
+## Disclosure Of Potential Conflicts Of Interest
+
+No potential conflicts of interest were disclosed.
+
+## Funding
+
+This work was supported by funds from Norges Forskningsråd [275918], 
+Helse Sør-Øst RHF [2014035, 2013017] and Kreftforeningen [3367910].
+
+## Orcid
+
+Randi G. Syljuåsen http://orcid.org/0000-0003-3404-6069
+
+## References
+
+1. Landsverk HB, Sandquist LE, Bay LTE, Steurer B, Campsteijn C, 
+Landsverk OJB, Marteijn JA, Petermann E, Trinkle-Mulcahy L, Syljuåsen RG. WDR82/PNUTS-PP1 prevents transcription-replication conflicts by promoting RNA polymerase II degradation on chromatin. Cell Rep. 2020;33(9):108469. doi:10.1016/j.celrep.2020.108469.
+
+2. Somesh BP, Reid J, Liu WF, Sogaard TM, Erdjument-Bromage H, 
+Tempst P, Svejstrup JQ. Multiple mechanisms confining RNA 
+polymerase II ubiquitylation to polymerases undergoing transcriptional arrest. Cell. 2005;121(6):913–923. doi:10.1016/j. 
+
+cell.2005.04.010.
+
+3. Nojima T, Gomes T, Grosso ARF, Kimura H, Dye MJ, Dhir S, 
+Carmo-Fonseca M, Proudfoot NJ. Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing. Cell. 2015;161(3):526–540. doi:10.1016/j.cell.2015.03.027.
+
+4. Mayer A, Landry HM, Churchman LS. Pause & go: from the discovery of RNA polymerase pausing to its functional implications. Curr Opin Cell Biol. 2017;46:72–80. doi:10.1016/j. 
+
+ceb.2017.03.002.
+
+5. Kuntziger T, Landsverk HB, Collas P, Syljuåsen RG. Protein phosphatase 1 regulators in DNA damage signaling. Cell Cycle. 2011;10 
+(9):1356–1362. doi:10.4161/cc.10.9.15442.
+
+6. Ciurciu A, Duncalf L, Jonchere V, Lansdale N, Vasieva O, 
+Glenday P, Rudenko A, Vissi E, Cobbe N, Alphey L, et al. PNUTS/PP1 regulates RNAPII-mediated gene expression and is necessary for developmental growth. PLoS Genet. 2013;9(10): 
+e1003885. doi:10.1371/journal.pgen.1003885.
+
+7. Cossa G, Roeschert I, Prinz F, Baluapuri A, Silveira Vidal R, 
+Schulein-Volk C, Chang YC, Ade CP, Mastrobuoni G, Girard C, et al. Localized inhibition of protein phosphatase 1 by NUAK1 promotes spliceosome activity and reveals a MYC–sensitive feedback control of transcription. Mol Cell. 2020;77(6):1322–1339 e11. 
+
+doi:10.1016/j.molcel.2020.01.008.
+
+8. Landsverk HB, Sandquist LE, Sridhara SC, Rødland GE, Sabino JC, 
+De Almeida SF, Grallert B, Trinkle-Mulcahy L, Syljuåsen RG. Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1. Nucleic Acids Res. 2019;47 (4):1797–1813. doi:10.1093/nar/gky1233.
+
+9. Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: 
+ensuring faithful duplication of a challenging genome. Nat Rev Mol Cell Biol. 2017;18(10):622–636. doi:10.1038/nrm.2017.67.
+
+10. Harlen KM, Churchman LS. The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain. Nat Rev Mol Cell Biol. 2017;18(4):263–273. doi:10.1038/ nrm.2017.10.

medical/md/PMC8555337.md

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+![0_image_1.png](0_image_1.png)
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+Adaptations made to delivery of comprehensive medication management in the community pharmacy setting during COVID-19 Morgan K. Stoa a,⁎, Caitlin K. Frail a, Joel F. Farley a, Deborah L. Pestka a, Carrie M. Blanchard b a Department of Pharmaceutical Care & Health Systems, College of Pharmacy, University of Minnesota, Minneapolis, MN, United States of America b Center for Medication Optimization, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America
+
+ARTICLE INFO ABSTRACT
+
+| Article history: Received 1 June 2021 Received in revised form 3 September 2021 Accepted 27 October 2021 Keywords: Comprehensive medication management Community pharmacy Adaptations COVID-19   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Background: As a result of COVID-19, numerous adaptations were made to health care delivery, including comprehensive medication management (CMM) delivered in community pharmacies.
+
+Objective: Identify and describe the adaptations that have been made to the delivery of CMM among community pharmacies due to COVID-19.
+
+Methods: Community pharmacies participating in a CMM implementation and research initiative had regular coaching calls throughout COVID-19 and completed a survey of changes that occurred as a result of COVID-19. Coaching notes and survey results were summarized and mapped to the Framework for Reporting Adaptations and ModificationsEnhanced (FRAME) to systematically capture changes that occurred.
+
+Results: A number of reactive adaptations were made to CMM delivery as a result of COVID-19, including increased virtual or remote delivery of CMM, delaying CMM visits to allow pharmacies to provide care directly related to the pandemic including COVID-19 testing and vaccines, wearing personal protective equipment (PPE) in visits, new ways of obtaining clinical patient information, and shifting CMM staffing models.
+
+Conclusion: Adaptations that occurred to CMM during COVID-19 allowed pharmacists to continue to serve their patients and meet public health needs.
+
+## 1. Introduction
+
+Comprehensive medication management (CMM) has demonstrated substantial benefit in improving patient outcomes and mitigating avoidable public health costs that stem from suboptimal medication use.1–4 Despite this evidence of effectiveness, incomplete adoption and implementation has limited its ability to benefit vulnerable patients. Implementation of CMM in community pharmacies presents both unique opportunities to reach patients and challenges.5 Community pharmacies provide existing relationships, easy access points to care, and unique perspective on medication use behaviors and patient preferences. However, barriers to patient access to care include creating time for CMM, partnering with the care team, and connecting health information.
+
+Research within the field of implementation science has demonstrated that evidence-based interventions, such as CMM, are often modified to address differences between the context in which they were developed and studied, and the context into which they are ultimately implemented.6 Adaptations are critical to successfully implementing evidence-based practices in a specific context.7 Adaptations can be planned to improve fit in a particular setting or context, or reactive in response to challenges that arise.
+
+Recent literature has pointed to the opportunities and strategies for adaptations in implementing CMM in community pharmacy.8 The coronavirus disease (COVID-19) pandemic led to rapid and significant changes in health care delivery. During the pandemic, adaptations to overcome challenges within the community pharmacy setting provide a unique perspective on prevention, preparedness, response, and recovery during the pandemic.9 Although much has been written about adaptations to medication dispensing activities in community pharmacies, there is less documentation of adaptions to the delivery of CMM in this setting. Understanding adaptations implemented in the pandemic may offer lessons for sustained innovations in implementing CMM in community pharmacies beyond the duration of the pandemic to improve patient care. The objective of this study was to identify and describe the adaptations made to the delivery of CMM to patients as a result of the COVID-19 pandemic among a group of community pharmacies.
+
+## 2. Methods
+
+This study took place from March to December of 2020 and was part of a larger study to measure the adoption, implementation, and impact of
+⁎ Corresponding author.
+
+E-mail addresses: hoeft049@umn.edu (M.K. Stoa), ckfrail@umn.edu (C.K. Frail), farl0032@umn.edu (J.F. Farley), pestk003@umn.edu (D.L. Pestka), carriebm@email.unc.edu
+(C.M. Blanchard).
+
+Contents lists available at ScienceDirect
+
+![0_image_0.png](0_image_0.png)
+
+## 3. Results 2.1. Data Collection 2.2. Design
+
+| Table 1 Characteristics of Slice of PIE pharmacies. Characteristic (N = 11)                                                                   | Frequency, n (%)   |
+|-----------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
+| Average pharmacist FTE dedicated to the delivery of CMM                                                                                       | 1.18 ± 1.52        |
+| through the PIE program (mean ± SD)                                                                                                           |                    |
+| Engagement in the delivery of CMM for the PIE Program: Pharmacy students                                                                      | 6 (55)             |
+| Pharmacy residents                                                                                                                            | 2 (18)             |
+| Pharmacy technicians                                                                                                                          | 2 (18)             |
+| Other medication management services being provided in addition to the PIE Programa Medicare Part D Vendors                                   | 10 (91)            |
+| eMTM Initiatives                                                                                                                              | 5 (45)             |
+| MN Medicaid                                                                                                                                   | 4 (36)             |
+| Other                                                                                                                                         | 1 (9)              |
+| Objective clinical patient data for remote CMM is collected bya Accessing the patient's electronic medical record                             | 3 (27)             |
+| Contacting the provider's office                                                                                                              | 10 (91)            |
+| Patient reported values                                                                                                                       | 9 (82)             |
+| Data are not able to be collected                                                                                                             | 2 (18)             |
+| Blood pressure data from patients that monitor their blood pressure at home is obtained bya Accessing the patient's electronic medical record | 1 (9)              |
+| Contacting the provider's office                                                                                                              | 5 (45)             |
+| Patient reported values                                                                                                                       | 11 (100)           |
+| Data are not able to be collected                                                                                                             | 2 (18)             |
+| Pharmacy monitoring                                                                                                                           | 1 (9)              |
+| Blood glucose data from patients that monitor their blood glucose at home is obtained bya Accessing the patient's electronic medical record   | 0 (0)              |
+| Contacting the provider's office                                                                                                              | 5 (45)             |
+| Patient reported values                                                                                                                       | 11 (100)           |
+| Data are not able to be collected                                                                                                             | 2 (18)             |
+| Pharmacy monitoring                                                                                                                           | 1 (9)              |
+| a Indicates a question where sites were allowed to enter free text describing information or select all that applied.                         |                    |
+
+CMM in a value-based care model in community pharmacies. Value-based care models focus on the quality of care provided, rather than the volume of patient services. HealthPartners, an integrated health care organization in Minnesota, launched an innovative program supporting CMM services through their pharmacy Partners in Excellence (PIE) program in 2015.
+
+The parent study, an 18-month implementation and research initiative named Slice of PIE, was initiated in November 2019. Slice of PIE was developed to build community pharmacist engagement in the PIE program through education and collaboration in an overall effort to improve patient care and help pharmacists achieve clinical performance and patient engagement goals. All pharmacies within the HealthPartners CMM network were invited to participate in Slice of PIE. Of all community pharmacy organizations that were participating in PIE at the beginning of the study period, all but one organization participated in Slice of PIE. A total of 12 organizations and 45 pharmacists within those organizations chose to participate. Six organizations were chain pharmacies (chain defined as having three or more pharmacy locations) and six were independent pharmacies. Slice of PIE provided participating pharmacies an implementation structure and educational support, such as regular webinars to connect with other organizations, coaching calls with an implementation coach, and data and feedback to guide their CMM development and implementation.
+
+A total of 11 organizations responded to the survey and field notes were reviewed for all 12 organizations. Table 1 illustrates the characteristics of the pharmacies participating in Slice of PIE, including the staffing model, strategies for collecting clinical data, and extent of other medication management services provided by the organization. Most of the pharmacies participating were independently owned; however, the study did include three large chain grocery establishments that operated multiple sites. Nearly all of the responding organizations also provided services through Medicare Part D MTM vendors, including DocStation, Outcomes, Medwise, and Tabula Rasa. Fig. 1 displays changes that occurred before and after the emergence of COVID-19, including proportion of initial and follow-up visits delivered remotely, and proportion of CMM patients monitoring blood pressure at home. Each aspect of the FRAME is outlined in Table 2 describing the adaptations made to CMM among community pharmacies during COVID-19.
+
+The first three components of the FRAME address when adaptations were made, whether the adaptations were planned, and the person responsible for determining which modifications were made. Adaptations that were made by pharmacies were reactive to the COVID-19 pandemic and organizations were at various phases in their implementation of CMM when these adaptations occurred. Some organizations had been providing CMM
+for several years, whereas others were earlier in implementation. As a result, modifications occurred during the implementation, scale up, and maintenance phases of implementation, depending on the organization.
+
+Adaptations were also determined at many different levels. For example, adaptations were made at the sociopolitical level when the Minnesota Department of Human Services eased restrictions on reimbursement for telemedicine for a number of providers, including pharmacists providing CMM, which was supported by the Minnesota Board of Pharmacy.10 Adaptations were also determined by pharmacy managers, pharmacists, as well as patients.
+
+Adaptations were collected through field notes documented by implementation coaches during their regular coaching calls, which occurred approximately every one to two months. Coaches worked with sites during the time of the collaborative by providing additional support surrounding implementing the Slice of PIE program into their site, including troubleshooting issues, coordinating collaboration between sites, and assisting with clarifying outstanding questions. The implementation coaches worked closely together, as they worked for the same organization and used a consistent coaching model when interacting with sites during coaching calls.
+
+To gather information from sites regarding adaptations, these questions were integrated into their regular coaching calls.
+
+A survey was administered to the lead pharmacist at all participating Slice of PIE pharmacies via Qualtrics to gather changes to CMM as a result of COVID-19, which addressed staffing and delivery of CMM, other medication management services provided at the site, and strategies used to collect clinical data for CMM delivery. This survey included 23 items that varied from yes or no questions to free response/select all that apply questions to allow sites to describe their adaptations. Sites were asked to reflect and respond on the adaptations pre- and post-COVID-19 in a single survey.
+
+In 2013, Stirman and colleagues created a system for classifying the types of adaptations that are made when interventions or services are implemented.6 In 2019, this framework was expanded upon to create the Framework for Reporting Adaptations and Modifications-Enhanced
+(FRAME).7 The FRAME not only characterizes adaptations and modifications, but captures the complex factors that contribute to them as well. According to the FRAME, the following eight aspects should be reported,
+(1) when in the implementation process the modification was made,
+(2) whether the modification was planned/proactive or unplanned/reactive, (3) who determined that the modification should be made, (4) what is modified, (5) at what level of care delivery the modification is made, (6) type or nature of context or content-level modifications, (7) the extent to which the modification is fidelity-consistent, and (8) the reasons for the modification.7 The survey previously described was not structured by the frame, so survey data and field notes were mapped to FRAME by two researchers (CMB and DLP) and discussed with the rest of the research team to systematically capture changes to CMM delivery that occurred as a result of COVID-19.
+
+The University of Minnesota Institutional Review Board determined that this project did not require review.
+
+# Percent Of Cmm Visits Ulizing Each Modality Pre- And Post-Covid (N=11)
+
+![2_image_0.png](2_image_0.png)
+
+The next components of the FRAME specify what was modified and the nature of the modifications. Five broad categories of adaptations were made to the delivery of CMM as a result of COVID-19. Most modifications were contextual in nature changing the setting or mode of delivery, but one modification (i.e., increased time between CMM follow-up visits) was to the CMM intervention content.
+
+## 3.1. Increased Time Between Cmm Follow-Up Visits
+
+Pharmacists often followed up with patients when they would come into the pharmacy for a medication refill. However, during COVID-19, many patients sought 90-day supply fills of their medications to minimize outside exposure or utilized drive-through windows to pick up medications.
+
+As a result, the length of time between CMM follow-up visits was often extended or did not occur.
+
+## 3.2. Increased Virtual Or Remote Delivery Of Cmm
+
+The setting of CMM visits changed as face-to-face visits were not an option in the early phases of the pandemic, so all visits needed to occur virtually or telephonically. This adaptation is illustrated in Fig. 1, where the percent of CMM encounters and follow-up visits that occurred remote or telephonically increased dramatically after the emergence of COVID-19.
+
+## 3.3. Delaying Cmm Visits
+
+Given competing demands, many pharmacies focused their efforts on other urgent tasks, which resulted in delaying CMM delivery in some instances. Early in the pandemic, payers reduced limits on early refills and 90-day fills, and many patients rushed to fill prescriptions in anticipation of supply chain disruption or pharmacy closures. Later in the pandemic year, influenza season and immunization efforts also required a shift in staff resources and demands.
+
+## 3.4. Need For Personal Protective Equipment (Ppe)
+
+Another contextual change to the delivery of CMM was the necessity of safety mechanisms for in-person CMM delivery. To protect themselves and patients, pharmacists began to wear PPE, such as face masks, face shields, and gowns, if a face-to-face CMM encounter did occur with a patient.
+
+## 3.5. Obtaining Clinical Patient Information
+
+Prior to COVID-19, some pharmacies relied on point-of-care testing to gather clinical information within the pharmacy, such as A1c and blood pressure. However, this was not a feasible option as many pharmacies suspended these services as a result of the pandemic. Complicating things further, many provider offices were closed or had limited hours and services during earlier stages of the pandemic. Therefore, the mechanism to obtain necessary clinical information had to be modified to either gather it from the patient or rely on previously obtained data. As shown in Fig. 1, sites increased their reliance on using home blood pressure monitoring to obtain clinical blood pressure data from patients where previously other means were used to obtain this data.
+
+## 3.6. Cmm Staff Changes
+
+Lastly, personnel for CMM support staff fluctuated during the pandemic as dispensing disruptions (e.g., patients seeking early fills of medications, staff needing to carry out prescriptions to patients' cars) affected CMM operations along with staff quarantining if a pharmacy staff person was exposed to or contracted COVID-19.
+
+The final components of the FRAME include for whom the modification was made, if the adaptations still allowed CMM to be carried out with fidelity, and the reasons for the adaptations. Adaptations were made at the patient level, as well as at the organizational and providerlevel. There were a number of different goals driving the modifications that occurred. These include ensuring patient and provider safety, improving patient access to CMM services, and improving the feasibility of delivering CMM given the environment of COVID-19. Contextual factors that influenced these adaptations were local and national public health recommendations (e.g., social distancing) as well as emergent policies (e.g., stay-at-home orders). Despite the adaptations that occurred, this did not significantly affect pharmacists' ability to follow the CMM patient care process.11 Therefore, most modifications were fidelity-consistent with the CMM model.
+
+## 4. Discussion
+
+This study highlighted some of the key adaptations that were made to the delivery of CMM in community pharmacies during COVID-19. A number of adaptations that were made in the community pharmacy setting are temporary, such as short-term staffing changes and the need for PPE,
+Table 2 Adaptations made to CMM delivery in community pharmacies during COVID-19 using the FRAME.
+
+1. When in the implementation process did the modification occur?
+
+Modifications occurred within the implementation, scale up, and maintenance phases of CMM
+2. Were adaptations planned?
+
+Modifications were reactive to COVID-19 3. Who determined that the modification should be made?
+
+- Pharmacy managers - CMM practitioners - Patients - State organizations (i.e., Minnesota Department of Human Services and the Minnesota Board of Pharmacy)
+4/5. What was modified and what was the nature of the modification?
+
+- Some pharmacists preferred to follow up with CMM patients when they came in to the pharmacy for a medication refill. However, with many patients pursuing 90-day fills of their medications, CMM follow ups did not occur as frequently.
+
+Therefore, the nature of the adaptation was extending the time between CMM follow-ups in some cases.
+
+- As face-to-face visits were not an option in the early phases of COVID-19. As a result, the setting of CMM visits changed to virtual or telephonic.
+
+- Delivery of CMM was also delayed as some pharmacies prioritized completing medication fills and providing influenza immunizations during flu season.
+
+- The format of CMM visits had to be adapted due to pharmacists' sometimes limited ability to obtain patient clinical data. For example, prior to COVID-19, some pharmacies relied on point of care testing to gather clinical information within the pharmacy, such as A1c and blood pressure. In the early phases of COVID-19, many provider offices were closed or had limited hours. Therefore, pharmacists had to modify their approach to collecting clinical data and obtain patient self-reported values or rely of previously documented values.
+
+- The format of CMM was also modified in that if a face-to-face visit did occur with a patient, personal protective equipment (PPE) was required.
+
+- CMM personnel also faced adaptations due to COVID-19. For example, pharmacy staff busy filling 90-day orders, taking prescriptions out to patients' cars, etc.
+
+Personnel was also limited if pharmacy staff had to quarantine if there was a COVID-19 exposure.
+
+6. At what level of delivery (for whom/what was the modification made)?
+
+Adaptations were made at the patient-level and organizational-level 7. Was the modification fidelity-consistent?
+
+Fidelity to CMM was maintained despite the adaptations 8. What were the reasons for the modification?
+
+Goals
+- Improve provider safety - Improve feasibility of delivering CMM during COVID-19 restrictions - Improve acceptability from patient perspective - Improve patient access - Improve patient comfort/safety Contextual factors that influenced the decision
+- Emergent policies - Local and national public health recommendations but other adaptations may be long-term and continue beyond the COVID19 pandemic. For example, although telemedicine has existed for years, several barriers, such as payment and provider uptake had limited its use to expand access to care in the past.12–14 Yet, with the rapid rise of telehealth delivery of care over the past year, this may be an adaptation that both patients and pharmacists choose to maintain. In addition, pharmacists indicated there was an increase use of home blood pressure monitoring among CMM patients after COVID-19. It is unknown if this will be sustained beyond the pandemic, as well as the effect this has on CMM delivery.
+
+The FRAME was developed based on adaptations presented in a variety of studies in the literature.6 Given the broad nature of the framework, applying the FRAME to CMM delivery during such unique times required frequent meetings with the research team to ensure that we were consistent in our understanding and application of the various pieces of the FRAME
+(e.g., interpretation and application of content vs context modifications).
+
+While the FRAME served as a useful tool to outline the various components to adaptations that occurred during COVID-19, it might be more suitable in future studies to adapt some of the FRAME terminology a priori to fit that particular intervention or service. In the case of CMM, the five components
+(collect, assess, plan, implement, follow-up) presented in the Joint Commission of Pharmacy Practitioners (JCPP) Pharmacists' Patient Care Process,11
+ 
+for example, may be integrated into the FRAME to serve as more applicable categories to describe modifications that occurred.
+
+Even before the pandemic, community pharmacists were recognized as one of the most accessible health care providers with frequent opportunities to deliver patient care.15 With the emergence of COVID-19, some outpatient clinics had limited hours and/or access for patients, thus potentially increasing the patient need for CMM access during this time period. It is important to note that despite the disruptions that came with COVID-19 and the adaptations that needed to be made by community pharmacies, pharmacists were able to maintain fidelity to the CMM model during this time.
+
+## 5. Limitations 4
+
+There were a number of limitations to this study. This analysis was limited to field notes and survey responses; participating pharmacies may have experienced additional adaptations that were not captured in this report. In addition, the sample size was relatively small and may limit applicability of findings outside of the collaborative. Furthermore, a single representative from each organization completed the survey on behalf of the organization as a whole, and as such, there is a possibility that individual pharmacists within the organization may feel differently than the responses captured by the organizational lead.
+
+In addition, recall bias may have influenced participants' responses to the survey. This study was also limited to community pharmacies in Minnesota. Pharmacies outside of this geographic region may have had different public measures in place and variations in outbreak severity that may have impacted their adaptations differently.
+
+## 6. Conclusion
+
+This study is the first in pharmacy practice to systematically collect and report adaptations using the FRAME. Reactive adaptations due to COVID occurred during CMM implementation in community pharmacies. Despite these modifications, community pharmacies continued to provide access and care to patients when access to other health settings and services were limited.
+
+## 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.
+
+## Acknowledgements
+
+This work was funded by the National Association of Chain Drug Stores
+(NACDS) Foundation and the UNC Eshelman Institute for Innovation. We have no grant numbers to reference.
+
+## References
+
+1. Isetts BJ, Schondelmeyer SW, Artz MB, et al. Clinical and economic outcomes of medication therapy management services: the Minnesota experience. J Am Pharm Assoc 2008;48(2):203–211.
+
+2. Budlong H, Brummel A, Rhodes A, Nici H. Impact of comprehensive medication management on hospital readmission rates. Popul Health Manag 2018;21(5):395–400. https://
+doi.org/10.1089/pop.2017.0167.
+
+3. Ramalho de Oliveira D, Brummel AR, Miller DB. Medication therapy management: 10 years of experience in a large integrated health care system. J Manag Care Pharm 2010;16(3):185–195.
+
+4. Brummel AR, Soliman AM, Carlson AM, de Oliveira DR. Optimal diabetes care outcomes following face-to-face medication therapy management services. Popul Health Manag 2013;16(1):28–34. https://doi.org/10.1089/pop.2012.0023.
+
+5. Ferreri SP, Hughes TD, Snyder ME. Medication therapy management: current challenges.
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+Integr Pharm Res Pract 2020;9:71–81. https://doi.org/10.2147/IPRP.S179628.
+
+6. Stirman SW, Miller CJ, Toder K, Calloway A. Development of a framework and coding system for modifications and adaptations of evidence-based interventions. Implement Sci 2013;8:65. https://doi.org/10.1186/1748-5908-8-65.
+
+ 
+7. Wiltsey Stirman S, Baumann AA, Miller CJ. The FRAME: an expanded framework for reporting adaptations and modifications to evidence-based interventions. Implement Sci 2019;14(1):58. https://doi.org/10.1186/s13012-019-0898-y.
+
+8. Hohmeier KC, Wheeler JS, Turner K, et al. Targeting adaptability to improve Medication Therapy Management (MTM) implementation in community pharmacy. Implement Sci 2019;14(1):99. https://doi.org/10.1186/s13012-019-0946-7.
+
+9. Cadogan CA, Hughes CM. On the frontline against COVID-19: community pharmacists' contribution during a public health crisis. Res Social Adm Pharm 2021;17(1):2032–
+2035. https://doi.org/10.1016/j.sapharm.2020.03.015.
+
+10. Minnesota Department of Human Services. COVID-19. https://www.dhs.state.mn.us/
+main/idcplg?IdcService=GET_DYNAMIC_CONVERSION&RevisionSelectionMethod=
+LatestReleased&dDocName=DHS-320036 2021.(Accessed March 15, 2021).
+
+11. Joint Commission of Pharmacy Practitioners. Pharmacists' patient care process. http://
+www.pcpcc.org/sites/default/files/media/medmanagement.pdf 2014.
+
+12. Lin C-CC, Dievler A, Robbins C, Sripipatana A, Quinn M, Nair S. Telehealth in health centers: key adoption factors, barriers, and opportunities. Health Aff (Millwood) 2018;37
+(12):1967–1974. https://doi.org/10.1377/hlthaff.2018.05125.
+
+13. Scott Kruse C, Karem P, Shifflett K, Vegi L, Ravi K, Brooks M. Evaluating barriers to adopting telemedicine worldwide: a systematic review. J Telemed Telecare 2018;24 (1):4-12. https://doi.org/10.1177/1357633X16674087.
+
+14. Weinstein RS, Lopez AM, Joseph BA, et al. Telemedicine, telehealth, and mobile health applications that work: opportunities and barriers. Am J Med 2014;127(3):183–187.
+
+https://doi.org/10.1016/j.amjmed.2013.09.032.
+
+15. Berenbrok LA, Gabriel N, Coley KC, Hernandez I. Evaluation of frequency of encounters with primary care physicians vs visits to community pharmacies among medicare beneficiaries. JAMA Netw Open 2020;3(7):e209132. https://doi.org/10.1001/jama networkopen.2020.9132.

medical/md/PMC8572335.md

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+# Rapid Response To Mycophenolate Mofetil In Combination
+
+with romiplostim in a case of severe refractory immune thrombocytopenia post COVID-19 vaccination Snigdha Nutalapati | Garima Gupta | **Gerhard C. Hildebrandt**
+Division of Hematology and Oncology, University of Kentucky College of Medicine, Lexington, Kentucky, USA Correspondence Snigdha Nutalapati, Department of Hematology and Oncology, University of Kentucky, Markey Cancer Center CC402, 800 Rose Street, Lexington, KY 40536-0093, USA. Email: SNutalapati@uky.edu
+
+## Abstract
+
+Vaccine mediated immune mediated thrombocytopenia (ITP) is exceedingly rare. We present a case of a young female who developed severe refractory ITP with multiple bleeding sites post second dose of COVID-19 vaccination. She was treated with a combination of romiplostim and mycophenolate mofetil that resulted in rapid platelet count recovery.
+
+KEYWORDS
+immune thrombocytopenia, mycophenolate mofetil, romiplostim, SARS-CoV2 vaccination
+
+## 1 | **Introduction**
+
+Immune-mediated thrombocytopenia (ITP) is an autoimmune condition characterized by low platelet counts and wide range of clinical manifestations from asymptomatic presentation to major bleeding manifestations. Vaccinemediated secondary ITP is rare and is associated with influenza, measles mumps rubella (MMR), and hepatitis A
+and B vaccination. Most patients respond to steroids and intravenous immunoglobulin (IVIG).1 We hereby report a case of a 26-year-old woman with severe refractory ITP who presented with multiple active bleeding sites leading to a significant drop in hemoglobin postsecond dose of mRNA1273 SARS-CoV-2 vaccine. She was refractory to treatment with steroids and IVIG and was eventually treated with combination of romiplostim and mycophenolate mofetil (MMF) with which her counts improved dramatically.
+
+## 2 | **Case Discussion**
+
+A 25-year-old Caucasian woman with no significant past medical history except for well-controlled bronchial asthma presented to the hospital with two-day history of worsening petechiae that initially started on her face and lower extremities and eventually spread to her back, chest, abdomen, and upper extremities. She also complained of intermittent epistaxis.
+
+Vital signs on initial presentation including blood pressure, heart rate, and oxygen saturation were normal. Physical examination revealed extensive bruising over her back, chest, and extremities along with scattered petechiae all over the body. Laboratory findings on presentation included WBC of 11 × 109/L, hemoglobin (Hb) of 11 g/dl, and severe thrombocytopenia with a platelet count of 1
+× 109/L. Prothrombin time (PT/INR), activated partial thromboplastin time (aPTT), fibrinogen, lactate dehydrogenase (LDH), absolute reticulocyte count, and haptoglobin levels were within normal limits. Peripheral blood smear showed profound thrombocytopenia with no evidence of schistocytes. RT-PCR testing for SARS-CoV-2 infection was negative. Autoimmune and infectious workup including antinuclear antibody (ANA), coombs testing, HIV ELISA, and serology for hepatitis B and C were negative. Ultrasound of the abdomen did not show evidence of splenomegaly or liver parenchymal changes.
+
+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. © 2021 The Authors. *Clinical Case Reports* published by John Wiley & Sons Ltd.
+
+Her most recent laboratory testing including Hb and platelet count from three months ago during routine primary care visit, was within normal limits. She neither reported personal or family history of bleeding disorders nor had required blood transfusions prior to this presentation. 26 days prior to this presentation, she had received her second dose of mRNA-1273 SARS-CoV-2 vaccine.
+
+Given her clinical presentation, negative infectious and autoimmune workup, normal hemolysis and coagulation studies along with absence of other identifiable underlying etiology, she was diagnosed with ITP and was initiated on dexamethasone 40 mg daily for four days along with IVIG 1 g/kg for two days. She continued to have clinical worsening during the second and third day of hospitalization and developed multiple bleeding sites including gross hematuria, hematochezia, and subconjunctival hemorrhage. Her hemoglobin dropped from 11  g/dl on presentation to 6.4 g/dl. She was initiated on tranexamic acid for bleeding control and supportive care with platelet transfusions. Her platelet counts continued to remain low
+(5 × 109/L) despite receiving 4 days of dexamethasone, 2 days of IVIG, and platelet transfusion support. She continued to have intermittent epistaxis; however, hematuria and rectal bleeding started improving with tranexamic acid initiation.
+
+As there was no improvement in platelet counts with IVIG and dexamethasone, on day 7, she was initiated on MMF 1 g twice daily and romiplostim 1 mcg/kg weekly.
+
+On day 12, her platelet counts for the first time improved to 13 × 109/L. Hb remained stable around 11 g/dl. She was discharged after receiving second dose of romiplostim 3 mcg/kg on day 14. During her first outpatient visit on day 21, her platelet counts improved to 140 × 109/L. She went on to receive third dose of romiplostim at 3 mcg/kg. She had no evidence of active bleeding. During fourth week follow-up, platelet count recovered to 268 × 109/L and MMF was tapered down to 500 mg twice daily and romiplostim to 2  mcg/kg. By fifth-week, her platelet counts continued to improve to 324 × 109/L and MMF was tapered to 250 mg twice daily and romiplostim to 1 mcg/kg.
+
+She continued to have normal and stable platelet counts, and eventually, MMF and romiplostim were discontinued at her sixth-week follow-up visit. She continued follow-up in the hematology clinic for 3 monthsfrom discharge from the hospital after which she was advised to resume care with her primary care physician.
+
+## 3 | **Discussion**
+
+ITPis anautoimmune entity characterized by autoantibodies directed against platelet antigensleading to platelet destruction and thrombocytopenia. Clinical manifestations range from asymptomatic presentation, spontaneous petechiae and purpura, mucocutaneous hemorrhage to fatal complications such as gastrointestinal bleeding or intracranial hemorrhage, which usually occur when platelet count is below 10 × 109/L.2 Vaccination as an etiology for secondary ITP was previously reported with influenza, hepatitis A and B,
+measles, MMR, and diphtheria tetanus acellular pertussis (DTaP) vaccines. It is hypothesized that pathogenesis of ITP is mainly secondary to molecular mimicry, a phenomenon in which host antigens are recognized as antigens of immunization, provoking the development of autoantibodies. These antibody-coated platelets may undergo reticuloendothelial phagocytosis resulting in reduced platelet survival. It is also proposed that vaccine-associated autoimmunity can stem not only from antigen-mediated responses but also contributed by other constituents of vaccine such as adjuvants, preservatives, or diluents.1,3 Lee et al. described 20 patients who developed thrombocytopenia after receiving COVID vaccination with either mRNA-1273 or Pfizer-BioNTech BNT16B2b2 mRNA
+SARS-CoV-2 vaccine. Sixteen of these patients did not report history of thrombocytopenia prior to their vaccination.4 Onset of symptoms from the date of vaccination ranged from 1 to 23 days.4 In the other three individually published case reports, ITP developed 12 h to 5 days post Pfizer-BioNTech BNT16B2b2 mRNA SARS-CoV-2 vaccine in patients who previously had a normal platelet count.5–7 Similarly, our patient had normal platelet counts prior to this presentation. ITP developed on day 26 after the second vaccine was given, which differs from previously reported cases and, so far, is the most "delayed" onset reported in the literature. It is not clear how the immunologic mimicry happens after SARS-CoV2 vaccination and what the immunologic target is. Immune responses to vaccines are highly variable between different individuals and populations, and there may be other factors implicated such as the microbiome impact on vaccine immunity.8 Corticosteroids, IVIG with or without platelet transfusions, have been used successfully with responses noted within 4–6 days.5–7 In the study by Lee et al, outcomes were reported in 16 patients, of which 13 responded to the initial treatment with IVIG, steroids, or platelet transfusion. One patient received rituximab, romiplostim, vincristine along with steroids and IVIG. Another patient developed severe thrombocytopenia and intracranial hemorrhage despite receiving corticosteroids, IVIG, and Rituximab.4 In our case, thrombocytopenia was refractory to initial treatment with dexamethasone and IVIG. Due to her presentation with massive and multi-site bleeding, there was concern for imminent intracranial hemorrhage in this young and otherwise healthy female, and a rapid response to treatment of her refractory ITP seemed critical. We were hesitant to use commonly recommended options for refractory ITP, such as rituximab, single-agent thrombopoietin-stimulating agent (TSA), and spleen tyrosine kinase inhibitor fostamatinib due to acuity of this case.9 With rituximab, median time to response is 5.5 weeks,10 and TSAs and fostamatinib are approved for patients with chronic ITP who have failed conventional medical therapy or splenectomy.11–13 Although promising, there are limited data to support the use of single-agent TSAs in newly diagnosed ITP.14 In a pooled analysis of phase III clinical trials studying fostamatinib in patients with chronic ITP, only 18% of patients achieved a stable response.15 Splenectomy was prohibited due to bleeding risk and would increase lifelong susceptibility to encapsulated bacteria.9 Promising activity for MMF in the treatment of de novo and refractory ITP has emerged over the recent years, and after careful consideration and extensive discussion with the patient and her family, we decided to combine MMF
+with the TSA romiplostim. The combination of immunosuppressants with IVIG and TSA has been reported in a small series to be efficacious and safe.16,17 The response to this combination was rapid and vigorous.
+
+As SARS-CoV-2 pandemic persists, concerns for vaccine-related side effectsremain, both among caregivers and the general population. As of June 26, 2021, around 46.4% of the United States population has received full vaccination against SARS-CoV-2 and the risks of serious adverse events remain extremely low. Similar to primary ITP, vaccine-related ITP can have a broad spectrum of presentation ranging from mild bleeding and petechiae to fatal bleeding manifestations. When patients present with ITP, a thorough vaccination history is needed. ITP after SARS-CoV-2 vaccination is rare, and the potential association is often overlooked and missed. While neither of the clinical trials studying mRNA-1273 or Pfizer-BioNTech BNT16B2b2 mRNA SARS-CoV-2 vaccine had documented cases of thrombocytopenia,18 it is worthwhile to note that possibility of reported thrombocytopenia postvaccination could be merely a coincidence rather than a causative factor. Nevertheless, we are probably just now only at the beginning of our understanding of the plethora and complexity of side effects and immunological impact of the SARS-CoV-2-directed vaccination and the virus itself. Continued vigilance and reporting of the possible side effects of vaccinations are needed, and we believe this should not preclude individuals from getting vaccinated given rarity of these events and the proven effectiveness of vaccine in mitigating this horrendous pandemic.
+
+Further studies on the aggressive management with combination regimens including immunosuppressants and thrombopoietin agonists, especially among patients with refractory thrombocytopenia and bleeding complications, are warranted.
+
+## Conflict Of Interest
+
+The authors report no conflict of interest.
+
+## Author Contributions
+
+Snigdha Nutalapati contributed to the case management and manuscript writing. Garima Gupta contributed to the case management and manuscript writing. Gerhard C.
+
+Hildebrandt contributed to the case management, manuscript writing, and final approval.
+
+## Consent
+
+The authors confirm that the patient provided informed consent in accordance with the journal's patient consent policy.
+
+## Data Availability Statement
+
+Data sharing is not applicable as no new data were generated.
+
+## Orcid
+
+Snigdha Nutalapati https://orcid.
+
+org/0000-0002-4753-7177
+
+## References
+
+	 1.	 Perricone C, Ceccarelli F, Nesher G, et al. Immune thrombocytopenic purpura (ITP) associated with vaccinations: a review of reported cases. *Immunol Res*. 2014;60(2-3):226-235. https://doi.
+
+org/10.1007/s12026-014-8597-x
+	 2.	 Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. *Blood*. 2009;113(11):23862393. https://doi.org/10.1182/blood-2008-07-162503
+	 3.	 Rinaldi M, Perricone C, Ortega-Hernandez O-D, Perricone R,
+Shoenfeld Y. Immune thrombocytopaenic purpura: an autoimmune cross-link between infections and vaccines. *Lupus*.
+
+2014;23(6):554-567. https://doi.org/10.1177/0961203313499959
+	 4.	 Lee EJ, Cines DB, Gernsheimer T, et al. Thrombocytopenia following Pfizer and Moderna SARS-CoV-2 vaccination. Am J Hematol. 2021;96(5):534–537. https://doi.org/10.1002/ ajh.26132
+	 5.	 Fueyo-Rodriguez O, Valente-Acosta B, Jimenez-Soto R, et al.
+
+Secondary immune thrombocytopenia supposedly attributable to COVID-19 vaccination. *BMJ Case Rep*. 2021;14(5). https:// doi.org/10.1136/bcr-2021-242220
+	 6.	 Pasin F, Calabrese A, Pelagatti L. Immune thrombocytopenia following COVID-19 mRNA vaccine: casuality or causality? Intern Emerg Med. 2021. https://doi.org/10.1007/s11739-02102778-w
+	 7.	 Tarawneh O, Tarawneh H. Immune thrombocytopenia in a 22-year-old post Covid-19 vaccine. *Am J Hematol*. 2021;96(5):E1 33-E134. https://doi.org/10.1002/ajh.26106 4 of 4 | 
+	 8.	 de Jong SE, Olin A, Pulendran B. The impact of the microbiome on immunity to vaccination in humans. Cell Host Microbe. 2020;28(2):169-179. https://doi.org/10.1016/j.chom. 2020.06.014
+	 9.	 Cuker A, Prak ETL, Cines DB. Can immune thrombocytopenia be cured with medical therapy? *Semin Thromb Hemost*.
+
+2015;41(4):395-404.
+
+	10.	 Arnold DM, Dentali F, Crowther MA, et al. Systematic review:
+efficacy and safety of rituximab for adults with idiopathic thrombocytopenic purpura. *Ann Intern Med*. 2007;146(1):2533. https://doi.org/10.7326/0003-4819-146-1-200701020-00006 11.	 BusselJB, Arnold DM, Boxer MA, et al. Long-term fostamatinib treatment of adults with immune thrombocytopenia during the phase 3 clinical trial program. *Am J Hematol*. 2019;94(5):546553. https://doi.org/10.1002/ajh.25444 12.	 Bussel JB, Provan D, Shamsi T, et al. Effect of eltrombopag on platelet counts and bleeding during treatment of chronic idiopathic thrombocytopenic purpura: a randomised, doubleblind, placebo-controlled trial. *Lancet*. 2009;373(9664):641-648.
+
+https://doi.org/10.1016/S0140-6736(09)60402-5 13.	 Kuter DJ, Rummel M, Boccia R, et al. Romiplostim or standard of care in patients with immune thrombocytopenia. *N Engl J* 
+Med. 2010;363(20):1889-1899. https://doi.org/10.1056/NEJMo a1002625 14.	 González-López TJ, Fernández-Fuertes F, HernándezRivas JA, et al. Efficacy and safety of eltrombopag in persistent and newly diagnosed ITP in clinical practice. Int J 
+Hematol. 2017;106(4):508-516. https://doi.org/10.1007/s1218 5-017-2275-4 15.	 Connell NT, Berliner N. Fostamatinib for the treatment of chronic immune thrombocytopenia. *Blood*. 2019;133(19):20272030. https://doi.org/10.1182/blood-2018-11-852491 16.	 Gudbrandsdottir S, Leven E, Imahiyerobo A, Lee CS, Bussel J.
+
+Combination of thrombopoietin receptor agonists, immunosuppressants and intravenous immunoglobulin as treatment of severe refractory immune thrombocytopenia in adults and children. *Br J Haematol*. 2020;189(2):e37-e40. https://doi.org/
+10.1111/bjh.16426 17.	 Pell J, Greenwood R, Ingram J, et al. Trial protocol: a multicentre randomised trial of first-line treatment pathways for newly diagnosed immune thrombocytopenia: standard steroid treatment versus combined steroid and mycophenolate.
+
+The FLIGHT trial. *BMJ Open*. 2018;8(10):e024427. https://doi.
+
+org/10.1136/bmjopen-2018-024427 18.	 Walsh EE, Frenck RW, Falsey AR, et al. Safety and Immunogenicity of two RNA-based Covid-19 vaccine candidates. *N Engl J Med*. 2020;383(25):2439-2450. https://doi.org/
+10.1056/NEJMoa2027906 How to cite this article: Nutalapati S, Gupta G,
+Hildebrandt GC. Rapid response to mycophenolate mofetil in combination with romiplostim in a case of severe refractory immune thrombocytopenia post COVID-19 vaccination. *Clin Case Rep*.
+
+2021;9:e05035. https://doi.org/10.1002/ccr3.5035

medical/md/PMC8694087.md

@@ -0,0 +1,233 @@
+# Fibroblast To Myofibroblast Transition Is Enhanced By Increased Cell Density
+
+Mary T. Doolina, Ian M. Smitha, and Kimberly M. Strokaa,b,c,d,* aFischell Department of Bioengineering and bMaryland Biophysics Program, University of Maryland, College Park, College Park, MD, 20742; cCenter for Stem Cell Biology and Regenerative Medicine and dMarlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Baltimore, MD, 21201 ABSTRACT **Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a** 
+rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition 
+(FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. 
+
+We found that human lung fibroblasts (HLFs) express more α**-SMA and deposit more collagen** matrix, which are both characteristics of myofibroblasts, in response to TGF-β**1 when cells are** 
+seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β**1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This** 
+work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.
+
+Monitoring Editor Dennis Discher University of Pennsylvania Received: Aug 14, 2020 Revised: Oct 21, 2021 Accepted: Oct 25, 2021
+
+## Introduction
+
+Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). The excess ECM presents as scarring of the lung parenchyma and impairs gas exchange, making it difficult for patients to breathe (Martinez et al., 
+2017). There are no effective treatments for IPF, and the median survival time after diagnosis is ∼3 years, making IPF a critical disease to investigate and treat (Lan et al., 2015).
+
+Lung tissue explanted from patients diagnosed with IPF is stiffer than healthy lung tissue, due to the increased protein content of the ECM and altered collagen cross-linking (Burgstaller et al., 2017; Jones et al., 2018). Concomitant with increased ECM deposition This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E20-08-0536) on November 3, 2021. *Address correspondence to: Kimberly M. Stroka (kstroka@umd.edu). Abbreviations used: FMT, fibroblast-to-myofibroblast transition; HLF, human lung fibroblast; IPF, idiopathic pulmonary fibrosis.
+
+© 2021 Doolin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial-Share Alike 4.0 International Creative Commons License (https://creativecommons.org/licenses/by-nc-sa/4.0). "ASCB®," "The American Society for Cell Biology®," and "Molecular Biology of the Cell®" are registered trademarks of The American Society for Cell Biology.
+
+and cross-linking, there is increased confinement imposed on cells by the ECM. In concert with increased confinement imposed by the ECM, there is increased confinement imposed by increased cell density as fibroblastic foci develop. These fibroblastic foci within the lung are another characteristic feature of IPF and consist of activated fibroblasts and myofibroblasts (Burgstaller et al., 2017).
+
+Myofibroblasts are contractile matrix-depositing cells character-ized by α-smooth muscle actin (SMA)–positive stress fibers. Myofibroblasts are essential in the recovery of damaged tissues and critical in the inflammatory response, but cause pathology when they persist in tissue. Although the origin of myofibroblasts in IPF is con-tinually under investigation, there is evidence that resident fibro-blasts can differentiate into myofibroblasts, termed the fibroblastto-myofibroblast transition (FMT), when given certain physical and/ or chemical cues (Huang et al., 2012). For example, fibroblasts cultured on stiff substrates are more likely to differentiate into myofibroblasts than those on soft substrates, due to increased actomyosin contractility (Huang et al., 2012). Concomitantly, stiff matrices encourage stress fiber formation within cells, which compresses the nucleus (Swift et al., 2013). However, it is unknown to what degree, if any, direct nuclear compression in the absence of increased contractility alters FMT. We are able to induce nuclear deformation within low-contractile cells by confining cells within the micropillar 
+
+![1_image_0.png](1_image_0.png)
+
+assay (Doolin and Stroka, 2019). This micropillar array also allows us to investigate whether increased confinement, such as that imposed by the dense ECM within IPF, influences FMT.
+
+Confining forces can also come from neighboring cells within a tissue. In fibroblastic foci, fibroblasts and myofibroblasts are more densely packed than in healthy lung tissue. Cell density is known to affect the behavior of lung fibroblasts, such as how they con-tract a 3D collagen gel (Redden and Doolin, 2006). In this work, we investigated the role of mechanical and cell–cell imposed confine-ment in FMT. Our results enhance understanding of IPF progression at a mechanistic level and may potentially improve treatments for IPF.
+
+## Results And Discussion Tgf-Β1 Incubation Time Alters Α**-Sma Expression Within** Human Lung Fibroblasts
+
+TGF-β1 is a pro-inflammatory and pro-fibrotic cytokine that is upregulated in IPF (Fernandez and Eickelberg, 2012). TGF-β1 has been shown to induce FMT in vitro, as evidenced by increased αSMA expression within HLFs (Kaarteenaho-Wiik et al., 2009). Our first aim was to optimize the concentration and timing of TGF-β1 treatment to induce FMT by examining α-SMA expression via im-munofluorescent straining. We observed α-SMA within HLFs after a 48-h incubation with TGF-β1 at 2, 5, or 10 ng/ml, and to a much greater extent after 72 h (Figures 1, 3). However, there was a decrease in the percentage of cells expressing α-SMA when we seeded HLFs at ∼ 50% lower density (Figures 2, 3). At an even lower density, HLFs treated with 20 ng/ml TGF-β1 did not display the pronounced increase in α-SMA (Figure 4) that was observed in higherdensity HLFs. With the potential cytotoxic effects of the citric acid carrier in mind, we chose 10 ng/ml TGF-β1 as our standard treatment concentration for subsequent experiments.
+
+## Human Lung Fibroblast Seeding Density Alters Myofibroblastlike Expression Within Human Lung Fibroblasts
+
+We next investigated if HLF response to TGF-β1 remained densitydependent beyond 72 h. We seeded 500, 5000, or 50,000 cells/cm2
+(low, medium, and high density, respectively) and treated with 10 ng/ml TGF-β1 for 5 d. We then examined α-SMA expression via im-munofluorescent straining. We observed expression of α-SMA in HLFs seeded at medium or high density and treated with TGF-β1, but this effect was most prominent in HLFs seeded at high density (Figure 5A). Additionally, f-actin stress fibers were more prominent in medium and high-density groups. All groups treated with vehicle control and low-density cells treated with TGF-β1 were mostly devoid of α-SMA staining (Figure 5A). We confirmed these results via Western blot for the medium- and high-density groups, as the lowdensity group did not contain adequate protein for analysis (Figure 5, B and C). TGF-β1 induced much higher α-SMA protein expression in the high-density than in the medium-density group. However, the high-density group treated with vehicle control had higher levels of α-SMA than the medium-density vehicle control group by one order of magnitude. Consequently, TGF-β1 induced an ∼14fold increase in expression of α-SMA in the medium-density group and an ∼21-fold increase in the high-density group. The increased α-SMA expression was present in both the cytosol and cytoskeleton portions of the HLFs (unpublished data). We also quantified nucleus area (Figure 5D) as a function of seeding density and treatment for the image sets represented in Figure 5A; these results will be dis-cussed in more detail below. The observation here that HLFs seeded at high density expressed more α-SMA in response to TGF-β1 than those seeded at low or medium density is in contrast to other results using fibroblasts sourced from different tissues. For example, one group showed that α-SMA expression increased when corneal fibroblasts were plated 
+
+![2_image_0.png](2_image_0.png)
+
+at low density (500 cells/cm2) when compared with high density (50,000 cells/cm2), even in the absence of TGF-β1 (Masur et al., 
+1996). However, Masur et al. analyzed cells in each group once they reached confluence. Therefore, low-density cells were cultured for a longer time than high-density cells, on the order of days. This method for running the experiment could confound results, because substrate stiffness influences FMT, and traditional culture plastic is extremely stiff. Prolonged exposure to stiff plastic may induce FMT, and it has been shown that culture itself can induce FMT (Baranyi et al., 2019). Others have demonstrated that medium-density bronchial fibroblasts (5000 cells/cm2) undergo FMT more readily than high-density bronchial fibroblasts (50,000 cells/cm2; Michalik et al., 2011). However, this study investigated asthmatic bronchial fibroblasts, which is localized to a proximal site within the lung, while IPF affects distal alveoli. Again, Michalik et al. analyzed cells upon reaching confluence, with low-density cells cultured 8 d longer than high-density cells. Medium-density dermal fibroblasts undergo FMT 
+more readily than high-density cells, potentially involving the upregulation of OB-cadherin (Ehrlich et al., 2006).
+
+Despite being FBS-deprived, HLFs were still able to proliferate at a very low rate. HLFs seeded at low and medium density doubled 
+∼2–3 times in 6 d. HLFs seeded at high density doubled ∼1–2 times in 6 d. This decrease in proliferation in the high-density group may be a confounding factor. For example, others have shown that quiescent myofibroblasts have reduced α-SMA turnover compared with proliferating cells (Arora and McCulloch, 1999). We assessed mitotic fraction of cells (Supplemental Figure S1) and nuclear Hoechst staining intensity (Supplemental Figure S2) in an effort to provide support that cells at high density may be quiescent; how-ever, these data were inadequate to support this hypothesis.
+
+It has been proposed that a transition within fibroblasts from N-cadherin expression to OB-cadherin expression is a hallmark of FMT (Pittet et al., 2008). We seeded low-, medium-, and 
+
+![2_image_1.png](2_image_1.png)
+
+high-density HLFs and treated them with 10 ng/ml TGF-β1 for 5 d. We then performed a Western blot and noted that no group appeared to express N-cadherin (Figure 6A). Conversely, HLFs seeded at high density expressed more OB-cadherin than the medium-density group, and TGF-β1 treatment significantly increased OB-cadherin expression in the high-density group 
+(Figure 6, B and C). HLFs seeded at low density showed very low OB-cadherin expression and thus were excluded from Figure 6C.
+
+![3_image_0.png](3_image_0.png)
+
+There is a shift from N-cadherin to OB-cadherin expression in fibroblasts during wound healing (Hinz et al. , 2004). Previously, blocking N-cadherin in bronchial fibroblasts was shown to reduce FMT (Michalik et al. , 2011). FOXF1 was recently demonstrated to be a key protein in preventing the switch from N-cadherin to OB-cadherin expression in FMT (Black et al. , 2018). Adherens junctions have been implicated in transferring mechanical strain between myofibroblasts, thereby opening mechanosensitive ion channels, inducing a calcium ion influx, and subsequently inducing contraction in the neighbor cell (Follonier et al. , 2008). Our results suggest
+
+![3_image_1.png](3_image_1.png)
+
+![4_image_0.png](4_image_0.png)
+
+![4_image_1.png](4_image_1.png)
+
+that increased HLF density up-regulates OB-cadherin, perhaps making cells more responsive to TGF-β1 due to their increased capacity for force transmission.
+
+## A Combination Of High Seeding Density And Tgf-Β**1 Promotes Collagen Matrix** Deposition
+
+As mentioned above, it has been shown that myofibroblasts are matrix-depositing cells (Klingberg et al., 2013). To assess the effects of seeding density on collagen matrix deposition, we seeded low-, medium-, and high-density HLFs onto fibronectincoated glass slides and treated the cells with 10 ng/ml TGF-β1 for 5 d. Immunostaining of collagen type 1 motif alpha 1 (Col1A1) 
+in these samples revealed that there is a dramatic increase in COL1A1 fluorescence for HLFs seeded at high density and treated with TGF-β1 (Figure 7A). Glass slides coated with collagen and with fibronectin were used as positive and negative controls, respectively (Figure 7B). Quantification of the immunofluorescence images supported our qualitative observations (Figure 7C). These results suggest that the combination of high-density seeding and TGF-β1 treatment is necessary to promote COL1A1 matrix deposition.
+
+## Cell Confinement May Not Affect The
+
+fibroblast-to-myofibroblast transition Cell density may impact cell behavior through physical deformation of the cell nu-cleus. As mentioned above, we quantified cell nucleus projected (2D) area from images 
+(Figure 5A) of cells cultured at varying seed-ing densities. Cells (both control and TGF-β1 treated) seeded at medium and high density had significantly smaller nuclear ar-eas than cells seeded at low density (Figure 5D; statistics in Supplemental Table S1). This led to the investigation of whether nuclear compression may influence FMT. Fibroblasts cultured on stiff substrates are more likely to FIGURE 7: High-density HLFs deposit treated with TGF-β1 deposit more collagen matrix. (A) Representative immunostained images of COL1A1 (green) and the nucleus (blue). (B) Representative images of COL1A1 staining for collagen-coated and fibronectincoated slides. Scale bar on fibronectin-coated slide image represents 10 µm and applies to all images in panels A and B. (C) Fluorescence integrated density values from immunostaining images. Each dot in the dot plot represents the mean value of one sample and the center lines represent the median values. 
+
+*p<0.05, **p <0.01, ***p<0.001.
+
+![5_image_0.png](5_image_0.png)
+
+differentiate into myofibroblasts than those on soft substrates, due to increased actomyosin contractility and Yap/Taz signaling (Huang et al., 2012; Liu et al., 2015; Liang et al., 2017). Concomitantly, stiff matrices encourage stress fiber formation within cells, and these stress fibers compress the nucleus (Swift et al., 2013). However, it is unknown to what degree, if any, direct nuclear compression alters the FMT, in the absence of increased contractility.
+
+To decouple the possible effects of increased actomyosin contractility and increased nuclear deformation on the observed density-dependent FMT, we used the micropillar assay previously developed in our laboratory to confine cells (Doolin and Stroka, 2019). 
+
+We used PDMS micropillar arrays that are 5, 10, 20, or 50 µm apart from one another, as well as a 2D PDMS and 2D tissue culture polystyrene (TCPS) controls. This system allows nuclear deformation even in a reduced cell contractile state, that is, in the most confined micropillar array. TGF-β1 increased α-SMA expression at all levels of confinement by a small, though not statistically significant amount, with similar results on planar PDMS (Figure 8). Meanwhile, α-SMA expression was highest in HLFs treated with TGF-β1 on 2D TCPS. 
+
+When we visualized cells via immunofluorescent staining, we noted that HLFs treated with TGF-β1 began to grow over the micropillar tops in the 5-µm group (Figure 9A). Additionally, HLFs treated with TGF-β1 tended to have higher f-actin signal and form discrete clumps, while control HLFs were more evenly distributed (Figure 9, A and B). It has been suggested that the formation of small contractile units is the most effective way to induce a high net force on a matrix (Tomasek et al., 2002; Hinz et al., 2004). It should also be noted that the addition of TGF-β1 can cause human lung fibroblasts to form gaps in their monolayer, as seen in the PDMS condition of Figure 9, likely due to enhanced cell contractility (Liu et al., 2001; Epa et al., 2015). An inhibitor of myosin II activity could reduce this artifact, but this treatment would also prevent FMT, and thus future work could focus on approaches for investigating the effects of modulating local cell density (Southern et al., 2016; Sun et al., 2021). Furthermore, our analysis of number of nuclei per image (Figure 9C; 
+statistics in Supplemental Table S2) and mean nuclear area (Figure 9D; statistics in Supplemental Table S3) for cells in the mi-cropillar devices suggested that there were no systematic differences in these values between micropillar spacings, which may have created confounding effects between cell density and confinement, especially when considering the seeding densities of our low-, medium-, and high-density condi-tions were 10-fold different from each other.
+
+The above results indicate that there was no statistically significant effect of confinement on FMT within HLFs. Interestingly, our 2D PDMS control consistently had lower α-SMA expression than on 2D TCPS. We attribute this to several factors. PDMS is an innately hydrophobic material that we make hydrophilic via plasma treatment and then add a coating of collagen I. There is a possibility that collagen I attaches differently on plasma-treated PDMS vs TCPS. Additionally, there is the potential that PDMS may have adsorbed a portion of TGF-β1, a hy-drophobic molecule, making it inaccessible to the cells. We addressed this by changing media every 1–2 d, but the potential for PDMS acting as a TGF-β1 sink should be investigated further. Additionally, the PDMS used here is ∼ 1.75 MPa in stiffness, while TCPS 
+is ∼ 3 GPa (Johnston et al., 2014). While both materials are extremely stiff, the three orders of magnitude increase in stiffness of TCPS could have induced a greater incidence of FMT.
+
+Additionally, we note that the HLFs used in this study were iso-lated from a young, healthy female whose HLFs likely behave differently than those from an older or sick individual, because disease state has been shown to influence FMT. For example, bronchial fibroblasts derived from a patient with asthma underwent FMT with higher frequency than those from a non-asthmatic patient (Michalik et al., 2011). Hence, we acknowledge that the age and health of HLF donors should be considered in interpreting and comparing results from different research groups.
+
+IPF is a terminal disease with an average survival time of 3 years. 
+
+There are no effective treatments for IPF, which highlights the urgent need for improved understanding of disease progression to elucidate new drug targets. Here we investigated how confining forces from neighboring cells or from physical features influence the lung FMT. In summary, we found that HLFs express more α-SMA in re-sponse to TGF-β1 when seeded at high density than at medium or low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β1 when cells are densely packed. We speculate that this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modeling IPF in vitro and may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.
+
+## Materials And Methods
+
+Request a protocol through *Bio-protocol*.
+
+## Cell Culture
+
+HLFs were authenticated by and purchased from ATCC (Manassass, VA, USA) and cultured in fibroblast basal medium (ATCC) 
+
+![6_image_0.png](6_image_0.png)
+
+supplemented with 7.5 mM l-glutamine, 5 ng/ml FGF basic, 5 µg/ ml insulin, 1 µg/ml hydrocortisone, 50 µg/ml ascorbic acid, 2% fetal bovine serum (FBS, ATCC), and 1% penicillin–streptomycin 10,000 U/ml (Thermo Fisher Scientific, Waltham, MA, USA). HLFs were cultured and used until passage 5. Cells were washed with phosphatebuffered saline (PBS; VWR, Radnor, PA, USA), detached with trypsinEDTA for primary cells (ATCC), and resuspended in trypsin neutralizing solution (ATCC). All cells were cultured at 37°C and 50% 
+humidity under 5% CO2:95% air. Cells were checked for myco-plasma weekly by Hoechst staining and immunofluorescence microscopy.
+
+## Fibroblast-To-Myofibroblast Transition
+
+To induce the FMT, HLFs were seeded and allowed to attach overnight. The following day, cells were washed with PBS before FBSfree media supplemented with 10 ng/ml TGF-β1 (Peprotech, Rocky Hill, NJ, USA) or vehicle control (10 mM citric acid) were added to induce FMT. For density-dependent experiments, cells were seeded at 500, 5000, and 50,000 cells/cm2 for low, medium, and high densi-ties, respectively, and media were changed every 1–2 d.
+
+## Micropillar Array Fabrication
+
+Micropillar arrays were fabricated as previously described (Doolin and Stroka, 2019). Briefly, standard photolithographic techniques, as described previously, were used to create a silicon master of the micropillar array design, such that the pillars were 13–17 μm tall. All photolithography procedures were carried out in the University of Maryland Nanocenter FabLab. Polydimethylsiloxane (PDMS, Krayden, Denver, CO, USA) was mixed at a 10:1 base:curing agent ratio, poured over the silicon master, and baked to create a PDMS 
+master, which was then removed and silanized overnight under vac-uum using tridecafluoro-1,1,2,2,tetrahydrooctyl-1-trichlorosilane 
+(OTS, 97%; UCT, Bristol, PA, USA). Then PDMS was mixed at a 10:1 base:curing agent ratio, poured onto the PDMS master, baked at 80°C overnight, and removed, yielding the final PDMS micropillar arrays. The micropillar arrays were placed in a plasma cleaner (Harrick Plasma, Ithaca, NY, USA) and plasma-treated with air for 2.5 min in order to increase hydrophilicity. PDMS blocks were simultaneously coated in 8% Pluronic F127 (Sigma-Aldrich) solution for 1 h at room temperature, and then washed with DI water. Micropillar arrays were subsequently stamped with the Pluronic F127-coated PDMS blocks so that the pillar tops were rendered nonadhesive to cells and were placed in six-well plates. The micropillar-containing plates were UV sterilized for 10 min, and 20 μg/ml collagen I (SigmaAldrich) was added to all wells and incubated for at least 1 h at 37°C. 
+
+The collagen I solution was then removed, and devices were washed with PBS before cells were seeded at a density of 5 × 104 cells/well. 
+
+Cell media were changed every 1–2 d.
+
+## Immunofluorescence
+
+The following steps were carried out at room temperature, unless otherwise specified. Cells were fixed in 3.7% formaldehyde (Fisher Scientific) for 10 min, and then washed twice in PBS (VWR). Cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min, washed twice in PBS, and blocked for nonspecific binding in 2.5% goat serum (Abcam, Cambridge, MA, USA) for at least 1 h. 
+
+Mouse anti-α-smooth muscle actin antibody (Sigma-Aldrich, St. 
+
+Louis, MO, USA, \#A5228, 1:100) in 1% goat serum was added to cells and incubated at 4°C overnight. Cells were washed three times in PBS, blocked in 2.5% goat serum for at least 1 h, and then incubated with AlexaFluor 488 Phalloidin (Thermo Fisher Scientific, 1:500), Hoechst (Thermo Fisher Scientific, 1:2500), and AlexaFluor 568 goat anti-mouse (ThermoFisher Scientific \#A11004, 1:200) for 1 h. For collagen deposition experiments, mouse antiCollagen 1A1 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA, 3G3, 1:100) in 1% goat serum was added to cells and incubated at 4°C overnight. Cells were washed three times in PBS, 
+blocked in 2.5% goat serum for at least 1 h, and then incubated with Hoechst and AlexaFluor 568 for 1 h. Cells were washed three times in PBS and then imaged. Images were acquired on an Olympus IX83 microscope (Olympus, Center Valley, PA, USA) using a 10×, 20×, or 60× magnification objective. The settings for each fluorescent channel were maintained across all images acquired within a given experiment. Image intensity can be compared within figures (and between Figures 1 and 2) but should not be directly compared across other figures, since they are from different experiments.
+
+## Cell Lysis
+
+Cells were washed with cold PBS, placed on ice, and then incubated for 5 min in ice-cold RIPA lysis buffer (Thermo Fisher Scientific) sup-plemented with 1:100 protease inhibitor cocktail (Sigma-Aldrich) 
+and 1:500 10 mg/ml phenylmethylsulfonyl fluoride (PMSF) in ethanol. Cells were collected using a cell scraper, and samples were incubated on ice for 1 h, with vortexing every 15 min. Samples were centrifuged at 300 × g for 6 min, and the cell lysate supernatant was collected. A Pierce BCA assay (Thermo Fisher Scientific) was per-formed to determine total protein concentration of each cell lysate against bovine serum albumin (BSA) standard.
+
+## Western Blotting
+
+Cell lysates were diluted in RIPA so that each sample was at the same concentration and final volume. Lysates were mixed 1:1 with 2× Laemmli sample buffer with 1:20 β-mercaptoethanol (BioRad, Hercules, CA, USA) and then incubated for 10 min at 100°C. Samples were loaded into precast 10% polyacrylamide gels (BioRad) and subjected to SDS–PAGE at 120 V for 1 h. Protein was then transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad) at 100 V for 1 h. Membranes were blocked in Tris-buffered saline (TBS)-
+based blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature and then incubated in primary antibody overnight at 4°C. 
+
+Primary antibodies used include mouse anti-α-smooth muscle actin antibody (Sigma-Aldrich \#A5228, 1:1000), rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA, \#2118, 1:2000), rabbit anti-OB-cadherin (Cell Signaling Technology \#4442, 1:1000), rabbit anti-N-cadherin (Cell Signaling Technology \#4061, 1:1000), rabbit anti-vimentin (Cell Signaling Technology \#5741, 1:1000), and mouse anti-vimentin (Santa Cruz Biotechnology, Dallas, TX, USA, 1:2000). Membranes were then washed three times in TBS with 1:500 Tween 20 (TBST buffer, BioRad) at 4°C. Washed membranes were incubated in HRP linked secondary antibody for 1 h at room tempera-ture. Secondary antibodies used were anti-mouse IgG (Cell Signaling Technology, \#7076, 1:5000) and anti-rabbit IgG (Cell Signaling Technology, \#7074, 1:5000). Membranes were then washed three times in TBST buffer and once in TBS buffer. Clarity Western ECL substrate was mixed with 1:1 peroxide solution:luminol/enhancer (BioRad) and then added to the membrane and incubated for 5 min. Membranes were imaged using a FluorChem E gel imaging system. Imaging was performed in the BioWorkshop core facility in the Fischell Department of Bioengineering at the University of Maryland, College Park.
+
+## Data Analysis
+
+Images of Western blot membranes were analyzed in ImageJ. Band intensity was measured within the same area for each lane. The yaxis on the plots shows the ratio of the protein of interest to GAPDH. 
+
+A value of zero indicates that the protein is not expressed highly enough to be visualized via Western blot. Fluorescence images were quantified in ImageJ. Mean fluorescence intensity of each image was measured, and the TGF-β1 group was normalized to the vehicle control group. Fluorescence images were then adjusted in ImageJ for visualization. The brightness and contrast were adjusted in the same way for all images within a given fluorescent channel. For collagen staining, corrected fluorescence integrated density was determined by subtracting the background fluorescence from the total image fluorescence. Nuclear shape descriptions, including nucleus area, were determined using the analyze particles macro in ImageJ.
+
+## Statistical Analysis
+
+Data from at least three independent trials was pooled for statistical analysis in all experiments. A Kruskal–Wallis test with Dunn's multi-ple comparisons test was performed. A significance level of p = 0.05 was used.
+
+## Acknowledgments
+
+This work was supported by a Burroughs Wellcome Career Award at the Scientific Interface (to K.M.S.), a University of Maryland Research and Scholarship Award (to K.M.S.), a Clark doctoral fellowship (to I.M.S.), the Fischell Department of Bioengineering, and the University of Maryland. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award F31HL145991 (to M.T.D.). 
+
+The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We acknowledge the support of the Maryland NanoCenter and its FabLab for providing photolithography resources. We acknowledge the BioWorkshop core facility in the Fischell Department of Bioengineering at the University of Maryland for plate reader and gel imager resources.
+
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medical/md/PMC8844282.md

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+# Long-Term Effects Of Pm2.5 Components On Incident
+
+dementia in the northeastern United States Jing Li,1,* Yifan Wang,2 Kyle Steenland,2 Pengfei Liu,3 Aaron van Donkelaar,4 Randall V. Martin,4 Howard H. Chang,5 W. Michael Caudle,2 Joel Schwartz,1 Petros Koutrakis,1 and Liuhua Shi2,*
+*Correspondence: jingli@hsph.harvard.edu (J.L.); liuhua.shi@emory.edu (L.S.)
+Received: August 27, 2021; Accepted: January 13, 2022; Published Online: January 17, 2022; https://doi.org/10.1016/j.xinn.2022.100208 ª 2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+GRAPHICAL ABSTRACT
+
+![0_image_0.png](0_image_0.png) PUBLIC SUMMARY
+- A cohort study by resorting to the Medicare population and PM2.5 components data
+- Long-term exposure to specific PM2.5 components can accelerate dementia progression
+- Black carbon and sulfate showed the most stable associations
+
+![1_image_0.png](1_image_0.png)
+
+# Long-Term Effects Of Pm2.5 Components On Incident
+
+## Dementia In The Northeastern United States
+
+# Jing Li,1,* Yifan Wang,2 Kyle Steenland,2 Pengfei Liu,3 Aaron Van Donkelaar,4 Randall V. Martin,4 Howard H. Chang,5 W. Michael Caudle,2 Joel Schwartz,1
+
+## Petros Koutrakis,1 And Liuhua Shi2,*
+
+1Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA 2Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA 30322, USA 3School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA
+4Department of Energy, Environmental & Chemical Engineering, Washington University at St. Louis, MO 63130, USA
+5Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA 30322, USA
+*Correspondence: jingli@hsph.harvard.edu (J.L.); liuhua.shi@emory.edu (L.S.)
+Received: August 27, 2021; Accepted: January 13, 2022; Published Online: January 17, 2022; https://doi.org/10.1016/j.xinn.2022.100208 ª 2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+Citation: Li J., Wang Y., Steenland K., et al., (2022). Long-term effects of PM2.5 components on incident dementia in the northeastern United States. The Innovation 3(2), 100208.
+
+Growing evidence has linked long-term fine particulate matter (PM2.5) exposure to neurological disorders. Less is known about the individual effects of PM2.5 components. A population-based cohort study investigated the association between long-term (1-year average) exposure to PM2.5 components and dementia incidence among the elderly population (age, R65 years) in the United States. We used data from the Medicare Chronic Conditions Warehouse and a high-resolution PM2.5 components dataset of the northeastern United States (2000–2017). We identified dementia diagnoses from patients' hospital and medical insurance records and carried out Cox proportional hazards regression to investigate their association with PM2.5 components.
+
+Among 2 million participants, 15.1% developed dementia. From the single-pollutant models, hazard ratios per interquartile range increase were 1.10 (95% confidence interval [CI]: 1.09–1.11) for black carbon, 1.08 (95%
+CI: 1.07, 1.10) for inorganic nitrate, 1.03 (95% CI: 1.02, 1.04) for organic matter, 1.13 (95% CI: 1.11, 1.15) for sulfate, 1.07 (95% CI: 1.06, 1.07) for soil particles, and 1.04 (95% CI: 1.03, 1.05) for sea salt. Increase in exposure to black carbon and sulfate per interquartile range had the strongest associations with dementia incidence. Penalized spline models indicated that dementia incidence increased linearly with elevated black carbon concentrations, whereas the incidence of dementia was only elevated significantly following sulfate concentrations above 2 mg/m3. Our study suggests that long-term exposure to PM2.5 components is significantly associated with increased dementia incidence and that different components have different neurotoxicity.
+
+Reduction of PM2.5 emissions, especially for main sources of black carbon and sulfate, may reduce the burden of dementia in the aging United States population.
+
+## Introduction
+
+Fine particulate matter (PM2.5) is an important air pollutant worldwide and consists of a complex mixture of carbonaceous fractions, water-soluble ions, metals, crustal elements, and other constituents.1–3 Exposure to PM2.5 has been associated with adverse health effects, including cardiovascular disease, respiratory disease, lung cancer, and premature mortality.4–8 Several studies also suggest that long-term PM2.5 exposure is a risk factor for neurodegenerative diseases.9–11 Dementia is one of the most prevalent neurodegenerative diseases and a major public health concern. It affects more than 47 million people worldwide, resulting in substantial health and financial burdens.12 The prevalence of dementia has increased significantly in recent years.13 Therefore, identifying modifiable risk factors for dementia that can be addressed by interventions at the population level is a top research priority. Studies suggest that PM2.5 has the potential to induce dementia through biological mechanisms such as systemic inflammation, oxidative stress, and neuroinflammation.14–16 In addition, some evidence indicates that PM2.5 can exacerbate or accelerate existing diseases via these biological pathways.17 A growing body of epidemiological evidence suggests that particulate air pollution contributes to dementia, including several longitudinal studies conducted in the United States and around the world.11,18–28 The majority of these studies found positive associations between PM2.5 and dementia. A systematic review and meta-analysis also concluded that exposure to PM2.5 is associated with a 16% higher risk of dementia per 10 mg/m3 increase in PM2.5 concentration.29 Existing studies have often relied on inpatient hospitalization records as a measure of incidence. A recent study by Shi et al.,30 based on nationwide Medicare data in the United States including doctor visits, also provides evidence that long-term exposure to PM2.5 may increase the incidence of dementia.
+
+However, previous studies have almost exclusively focused on the effects of PM2.5 mass concentrations. As a complex mixture, the toxic effects of PM2.5 may be determined primarily by its chemical components. PM2.5 components, such as organic matter (OM), inorganic nitrate (NO3
+), inorganic sulfate
+(SO4 2), black carbon (BC), soil particles (SOILs), and sea salt (SS), emitted from specific sources, have different physicochemical and toxicological characteristics, resulting in various health effects.31–33 To date, the individual effects of PM2.5 components on dementia remain unclear. There is an urgent need to evaluate the effect of exposure to various PM2.5 components on dementia to determine which PM2.5 components are responsible for dementia, the possible biological mechanisms, and which emission sources are the most hazardous.32 To fill these knowledge gaps, we build on the study by Shi et al.30 by conducting a population-based cohort study of the Medicare dataset and a well-validated high-resolution (1 km 3 1 km) PM2.5 components dataset from 2000–2017.
+
+The latter includes data on OM, NO3
+, SO4 2, BC, SOILs, and SS in the northeastern United States, where better exposure estimates exist. First, we identified a diagnosis of all-cause dementia using all Medicare claims including doctor visits, with the requirement of a 5-year "clean" (i.e., dementia-free) period. PM2.5 component exposure was assigned to subjects based on resident ZIP code.
+
+The effects of long-term exposure to six major PM2.5 components (i.e., OM,
+NO3
+, SO4 2, BC, SOILs, and SS) on incident dementia were investigated using Cox proportional hazards models.
+
+## Results
+
+Study population characteristics and air pollution levels The dementia cohort with a "clean" period of 5 years had about 2 million subjects with a median follow-up of 7 years. Regarding the demographic data, 73.2%
+of the people entered the cohort between ages 65 and 74, 61.6% were female, 92% were white, and more than 90% were above the poverty level. Within the population cohort, 15.1% developed dementia. The descriptive information for the dementia cohort is listed in Table 1.
+
+Between 2000 and 2017, the northeastern United States had an average PM2.5 mass concentration of 8.8 mg/m3. The average concentrations of PM2.5 components were 0.7 mg/m3 (BC), 1.0 mg/m3 (NO3
+), 2.9 mg/m3
+(OM), 1.8 mg/m3 (SO4 2), 0.3 mg/m3 (SOILs), and 0.4 mg/m3 (SS). The interquartile ranges (IQRs) of BC, NO3
+, OM, SO4 2, SOILs, and SS were 0.3 mg/
+m3, 0.6 mg/m3, 1.0 mg/m3, 1.1 mg/m3, 0.2 mg/m3, and 0.2 mg/m3, respectively
+(Table S3). The Pearson correlations between each PM2.5 component and PM2.5 mass are listed in Table S4. The spatial distributions of each component at the ZIP code level over 2000–2017 are shown in Figure 1. The spatial distribution of PM2.5 mass is shown in Figure S1. The highest PM2.5 mass concentrations were distributed in southern areas of the northeast United States, including western and eastern Pennsylvania, New Jersey, and New York City. High BC concentrations were found in western Pennsylvania, New York City, and the border of eastern Pennsylvania and New Jersey, and the spatial distribution of OM was also similar. Compared with the other states, western Pennsylvania had higher average SO4 2 and SOIL levels. In contrast, high levels of NO3 were concentrated in eastern Pennsylvania.
+
+For SS, high concentrations occurred along the eastern coastline of Massachusetts, Rhode Island, Connecticut, and New York.
+
+ll   1
+
+| Report                                                             |                                                                            |            |
+|--------------------------------------------------------------------|----------------------------------------------------------------------------|------------|
+| www.the-innovation.org                                             | Table 1. Descriptive statistics for the dementia cohorts Variables Numbers | Percentage |
+| Number of events                                                   | 309,842                                                                    | 15.1       |
+| Total population                                                   | 2,051,550                                                                  | 100        |
+| Total person-years                                                 | 11,404,905                                                                 | 100        |
+| Median follow-up years                                             | 7                                                                          |            |
+| Age at entry (years) 65–74                                         | 1,481,574                                                                  | 73.2       |
+| 75–115                                                             | 569,976                                                                    | 27.8       |
+| Gender Male                                                        | 789,610                                                                    | 38.9       |
+| Female                                                             | 1,252,940                                                                  | 61.1       |
+| Race White                                                         | 1,888,347                                                                  | 92.0       |
+| Black                                                              | 88,327                                                                     | 4.3        |
+| Other                                                              | 74,876                                                                     | 3.6        |
+| Medicaid eligibility Eligible                                      | 136,283                                                                    | 6.6        |
+| Not eligible                                                       | 1,915,267                                                                  | 93.4       |
+| Air pollutants (mg/m3 ) a PM2.5 mass                               | 8.8 (2.4)                                                                  |            |
+| Black carbon                                                       | 0.7 (0.3)                                                                  |            |
+| Nitrate                                                            | 1.0 (0.6)                                                                  |            |
+| Organic matter                                                     | 2.9 (1.0)                                                                  |            |
+| Sulfate                                                            | 1.8 (1.1)                                                                  |            |
+| Soil particles                                                     | 0.3 (0.2)                                                                  |            |
+| Sea salt                                                           | 0.4 (0.2)                                                                  |            |
+| Area level variablesb Smoking rate, %                              | 47.6 (5.3)                                                                 |            |
+| Below poverty level, %                                             | 7.9 (5.7)                                                                  |            |
+| Not graduating from high school, %                                 | 20.2 (11.0)                                                                |            |
+| Population density, people/mile2                                   | 6,326.5 (17,086.9)                                                         |            |
+| Body mass index, kg/m2                                             | 27.2 (0.7)                                                                 |            |
+| Median household income, US $1,000                                 | 69.8 (27.7)                                                                |            |
+| Owner-occupied housing units, %                                    | 70.1 (18.0)                                                                |            |
+| Black population proportion, %                                     | 6.7 (12.5)                                                                 |            |
+| Number of hospitals                                                | 1.9 (4.3)                                                                  |            |
+| Number of medical doctors                                          | 261.6 (1,135.1)                                                            |            |
+| a Presented as mean concentration (IQR). b Presented as mean (SD). |                                                                            |            |
+
+## Main Analysis
+
+Health effect estimates. In the single-pollutant model, long-term exposure to PM2.5 mass and all PM2.5 major components had a statistically significant positive association with dementia (Table 2). A per-IQR increase in concentration was associated with an increase in dementia, with a hazard ratio (HR) of 1.10 (95%
+confidence interval [CI]: 1.09, 1.12) for PM2.5 mass, 1.10 (95% CI: 1.09, 1.11) for BC, 1.08 (95% CI: 1.07, 1.10) for NO3
+, 1.03 (95% CI: 1.02, 1.04) for OM, 1.13
+(95% CI: 1.11, 1.15) for SO4 2, 1.07 (95% CI: 1.06, 1.07) for SOILs, and 1.04
+(95% CI: 1.03, 1.05) for SS. In multi-pollutant models, the positive associations with long-term exposure to BC (HR = 1.05; 95% CI: 1.04, 1.07), NO3
+
+(HR = 1.02; 95% CI: 1.00, 1.03), SO4 2 (HR = 1.05, 95% CI: 1.03, 1.07), SOILs
+(HR = 1.03; 95% CI: 1.02, 1.04), and SS (HR = 1.04; 95% CI: 1.03, 1.05) remained, although they were notably reduced except for SO4 2. However, OM showed a negative association with dementia (HR = 0.98; 95% CI: 0.96, 0.99). Figure 2 shows the HRs for each subgroup in single-pollutant models. We found that males had higher HRs for BC, NO3
+, OM, SOILs, and SS, whereas females had higher HRs for SO4 2. Additionally, we observed higher HRs for BC, NO3
+,
+SO4 2, SOILs, and SS among those identified as Black than for those identified as white or other races. Regarding age, those younger than 75 had stronger associations between dementia and BC, OM, SOILs, and SS than those older than 75, whereas those older than 75 had stronger associations between dementia and NO3 and SO4 2.
+
+Concentration-Response (C-R) relationships. Next we evaluated the C-R relationships between each component of PM2.5 and dementia from singlepollutant models (Figure 3). A strong near-linear relationship was observed with BC and SOILs, with no sign of threshold. Near-linear relationships were observed with NO3 and OM at low concentrations, and then the C-R curves leveled off at 0.5 mg/m3 for NO3 and 3 mg/m3 for OM. The C-R curves showed null associations between dementia and SO4 2 at low concentrations; however, the relationship became significantly positive at high SO4 2 concentrations, with a steep rise at about 2 mg/m3. The pattern for SS was similar to SO4 2, with no significant relationship at low concentrations followed by a positive, linear relationship at high concentrations.
+
+Sensitivity analysis. The results of the single-pollutant models adjusting for PM2.5 mass are presented in Table S5. The patterns were similar with multipollutant models, with negative associations for OM and positive associations for the other components on dementia. The results of the non-mover cohort are very similar to the results of our main analysis (Table S6), suggesting little bias from residential mobility. The C-R curves from multi-pollutant models are shown in Figure S2. The C-R curves from multi-pollutant models for BC, NO3
+,
+SOILs, and SS are generally consistent with those from single-pollutant models. However, for OM, a null association was observed at high concentrations after adjusting for other PM2.5 components. We found that SO4 2 exposure did not increase the risk of dementia at levels below 2 mg/m3, followed by a monotonically increasing C-R relationship.
+
+## Discussion
+
+Using a large prospective cohort study from the northeastern United States, we found that long-term exposure to BC, NO3
+, SO4 2, SOILs, and SS elevated the risk of dementia among the elderly population (age, R65). OM has been shown to be associated with dementia only when the single-pollutant model was used, and a negative association was observed when PM2.5 mass or other major components of PM2.5 were adjusted. In single- and multi-pollutant models, per-IQR increases of BC and SO4 2 were associated with the highest dementia risk.
+
+BC primarily forms from incomplete combustion of fossil fuels, biofuels, and biomass.34 It is mainly associated with incomplete combustion processes, including traffic-related pollution and biomass burning.35 We found a positive association between BC and dementia. Despite limited studies investigating the associations between BC and neurodegenerative disorders, some researchers have observed associations between BC and quantifiable impairment of brain development in children and cognitive decline in the elderly.36,37 A possible explanation for the neurotoxicity of BC is that BC from traffic sources can be very small
+(50 nm) and may get into the bloodstream and reach the blood-brain barrier.38 Moreover, incomplete combustion can also co-emit polycyclic aromatic hydrocarbons (PAHs), which might be coated on BC and contribute to neurotoxicity.38 The OM in the atmosphere is a highly complex mixture of primary and secondary organic particles made mostly of organic carbon and trace amounts of hundreds of compounds such as PAH, alkanes, esters, and fatty acids.39 Combustion of biomass and fossil fuels is the major source of primary OM, but secondary production from oxidation of organic gases is also considered a major source of OM.40 In particular, secondary organic aerosols are generated by reactions of terpenes with nitrogen oxides. Numerous toxicological studies have implicated OM in cancer and cardiopulmonary illness; however, the effects of OM on the nervous system are largely unknown.41 There have been some 2   www.cell.com/the-innovation
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+studies that link exposure to PAHs to neurodevelopmental disorders in children as well as neurodegenerative disease-like symptoms in zebrafish.42,43 In our study, we observed an adverse effect of OM on dementia with the singlepollutant model, and a modest negative association was observed when PM2.5 mass or other components of PM2.5 were included in the model. According to the C-R relationships of the multi-pollutant model (Figure S2), for most of the OM distribution, the association was positive but became protective at high concentrations. The adjustments for other PM2.5 components somehow reduce the associations of OM and dementia, likely because of collinearity. Moreover, the effect sizes for all components in the multi-pollutant models decreased from their levels in the single-pollutant models. Similarly, Crouse et al.44 also observed a positive association between OM and mortality in the single-pollutant model, which reversed after adjusting for PM2.5 mass and other components.
+
+They also attributed it to collinearity among component concentrations. The effects of OM still need to be explored.
+
+NO3 is a secondary inorganic aerosol formed in the air; its major precursor gaseous species are ammonia and nitric acid.45 Agriculture is considered to be the major source of ammonia and nitric acid globally; however, in urban areas, traffic is considered to be the main source of nitrogen oxides.46 We found a significant positive association between NO3 and dementia. A linear C-R relationship was observed in low NO3 concentrations, and the relationships became stable at concentrations above 0.5 mg/m3. Although the adverse effects of NO3 on neurodegenerative disorders have been less explored, NO3 has been linked to oxidative stress, which is known to be a major pathological driver in dementia and neurodegenerative disease.47 In addition, there is some evidence that traffic-related air pollution increases the risk of neurodegenerative disorders.18,20,21 SO4 2 is also a secondary inorganic aerosol present in the atmosphere in the form of a mixture of ammonium sulfate, ammonium bisulfate, or sulfuric acid.
+
+Similar to our findings, van Wijngaarden et al.48 observed a positive association between SO4 2 and hospitalizations for neurodegenerative disorders with a relative risk of 1.09 (95% CI: 1.00–1.19) per IQR. In the C-R curves, we only observed a positive linear relationship between SO4 2 and dementia at high SO4 2 concentrations; a negative relationship was found at low SO4 2 concentrations when adjusting for other major components. This effect could be explained by sulfate precipitating the toxic compound deep in the lungs by creating an acidic environment in the microcirculation that promotes absorption of metal elements of particulate matter, but only after a certain threshold of lung accumulation occurs.49,50 SOILs are composed of oxides and carbonates of crustal elements such as Si, Al, Fe, Ca, and Ti. They are formed through mechanical processes such as eolian erosion, transportation, and industrial processes such as mining and cement production. We found a positive association between SOILs and dementia. It has been reported that accumulation of metals in the blood and bones can damage the nervous system.51 SS derived from sea spray is primarily composed of sodium chloride (NaCl) but also contains other chemical ions that are common in seawater. To date, no other studies have investigated the relationship between SS and neurodegenerative disorders. It has been reported that SS is associated with cardiovascular disease, such as hypertension.41 The effect of SS on dementia is worthy of attention based on our results.
+
+Our study has several strengths. To the best of our knowledge, this is the first population-based, large-scale cohort study characterizing associations between PM2.5 component exposure and incident dementia. PM2.5 dementia effects are currently mostly studied from the total mass perspective, and only a few studies have investigated specific PM2.5 components. Consequently, our study provides evidence for the association between PM2.5 components and dementia, which may have implications for PM2.5 component regulation. In addition, previous studies have almost exclusively focused on hospitalization data; in contrast, we used more comprehensive Medicare claims, including Medicare inpatient and outpatient claims, physician visits, skilled nursing facilities, and home health
+
+Table 2. Hazard Ratio of dementia associated with per-IQR increase in PM2.5 major
+
+components
+
+| components     | Hazard Ratio (95% CI)   |                       |
+|----------------|-------------------------|-----------------------|
+| Components     | Single-pollutant model  | Multi-pollutant model |
+| Black carbon   | 1.10 (1.09, 1.11)       | 1.05 (1.04, 1.07)     |
+| Nitrate        | 1.08 (1.07, 1.10)       | 1.02 (1.00, 1.03)     |
+| Organic matter | 1.03 (1.02, 1.04)       | 0.98 (0.96, 0.99)     |
+| Sulfate        | 1.13 (1.11, 1.15)       | 1.05 (1.03, 1.07)     |
+| Soil particles | 1.07 (1.06, 1.07)       | 1.03 (1.02, 1.04)     |
+| Sea salt       | 1.04 (1.03, 1.05)       | 1.04 (1.03, 1.05)     |
+| PM2.5 mass     | 1.10 (1.09,1.12)        | -                     |
+
+ll   3
+
+![4_image_0.png](4_image_0.png)
+
+care. Because many dementia cases might not result in hospitalization, our study can better capture disease incidence. The requirement of a 5-year "clean" period also enables us to obtain more representative results. Last, our Cox proportional hazards models employ a generalized estimating equation (GEE) to account for potential clustering within ZIP codes and are more robust when adjusting for numerous covariates at the neighborhood and individual level, including penalized splines.
+
+This study also has some limitations. First, for the exposure assessment, we were unable to obtain residential address information because of limitations of Medicare data. For each participant, we could only assign the annual average exposure data based on ZIP-code-level data. Therefore, exposure measurement error is likely. In addition, residential mobility could introduce some bias. Second, even with multiple covariates being controlled, we cannot fully rule out the possibility of residual confounding or unmeasured confounding bias, such as alcohol consumption, exercise frequency, and substance use, whichmay affect the development of dementia. Further studies taking into account the potential bias and unmeasured/residual confounding are encouraged. In addition, there were some covariates (e.g., education, income, and smoking) that were measured at the ZIP
+code or county level, which may not accurately reflect the differences between individuals in the cohort Third, multi-pollutant models and single-pollutant models with PM2.5mass adjustment have multicollinearity because of the high correlation among some PM2.5 components. To overcome the multicollinearity limitation, advanced computationally mixture analysis methods are urgently required.52 Fourth, the investigated six PM2.5 components contribute to more than half of the PM2.5 mass, whereas other trace components, such as metal elements, are not recorded because of the lack of exposure data for individual metal species.53 Therefore, the effect of other components on dementia cannot be estimated. To provide some inspiration for further research, we display the results for these other unmeasured components as a category called "other components (equal to total PM2.5 mass minus mass of six measured components)" in Table S7.
+
+## Conclusions
+
+Our study provides epidemiological evidence that long-term exposure to PM2.5 components could potentially accelerate dementia progression; the contributions of various PM2.5 chemical components to increased dementia incidence may vary. BC and SO4 2 showed the most stable associations with dementia risk, whereas OM, the largest component of PM2.5, has the lowest effects. Reducing PM2.5 pollution potentially has an important public health effect. We recommended that, besides PM2.5 mass, the effect of specific PM2.5 components on neurodegenerative disorders, especially for BC and SO4 2, also need to be considered for further analysis, leading to more targeted regulation of PM2.5 components in the near future.
+
+## Materials And Methods
+
+Study population This cohort study began on January 1, 2000 and ended on December 31, 2017. The Medicare denominator file and Medicare Chronic Conditions Warehouse (CCW) were used to construct this study population (age, R65), derived from the Centers for the Medicare and Medicaid Services (CMS). In the Medicare denominator file, Medicare beneficiaries are detailed by age, sex, race, date of death (if any), ZIP code of residence, and Medicaid eligibility status. Age, ZIP code of residence, and Medicaid eligibility were updated annually.
+
+The outcome variable in this study was time to first diagnosis of all-cause dementia. The CCW identifies pre-defined indicators of dementia through an algorithm (https://www2.
+
+ccwdata.org/web/guest/condition-categories) that leverages Medicare claims documents
+(such as hospital and physician files and health insurance files), including inpatient and outpatient claims, carrier file (primarily physician visits), skilled nursing facility, and home healthcare claims. The CCW provides the date of the first occurrence with a dementia diagnosis code across these Medicare claims (ICD codes are provided in Table S1). The abovementioned Medicare algorithm for defining dementia is primarily based on previous studies.54,55 These two Medicare databases enabled us to create a cohort of all Medicare fee-for-service (FFS) beneficiaries in nine northeastern states (Maine, New York, New Jersey, Vermont, Massachusetts, Rhode Island, Connecticut, New Hampshire, and Pennsylvania).
+
+To better ascertain disease incidence, our cohort required that (1) all subjects were always enrolled in the Medicare FFS program and in Medicare Part A (hospital insurance) and Part B
+(medical insurance) from 2000–2017 and (2) there was a "clean" period of 5 years after enrollment during which there was no dementia diagnosis logged. Other authors have also used a "clean" period while studying dementia or Alzheimer's disease.30,56 These requirements would increase the possibility that the individual did not have dementia prior 4   www.cell.com/the-innovation
+
+Figure 3. Concentration-Response curves from single-pollutant models for black carbon, nitrate, organic matter, sulfate, soil particles, and sea salt. The penalized spline regression models fit the concentrations of each PM2.5 component from the 0.1th to 99.9th percentiles.
+
+![5_image_0.png](5_image_0.png)
+
+to Medicare enrollment. Subjects were entered into the cohort on the first day of the year following the period of"clean" claims, and they were followed until the first diagnosis or death of an outcome of interest across all claims in Medicare or the end of follow up. This study is approved by Emory University's Institutional Review Board (\#STUDY00000316) and the CMS under the data use agreement (\#RSCH-2020-55733). The Medicare dataset was stored and analyzed in the Emory Rollins School secure cluster environment (HPC) with Health Insurance Portability and Accountability Act (HIPAA) compliance.
+
+## Exposure Assessment
+
+The annual average PM2.5 mass concentration and its six major components were estimated using well-validated PM2.5 composition prediction models for each year. The detailed methodology can be found elsewhere.2 Briefly, satellite-derived PM2.5 total mass concentrations were first produced by combining satellite retrievals of aerosol optical depth, chemical transport modeling (CTM), and ground-based observations made throughout 2000–2017.
+
+These PM2.5 values were then decomposed into individual chemical components according to CTM output and calibrated using ground-based observations from 2000–2017. Sources of compositional ground-based observations included the Clean Air Status and Trends Network, the National Core Network, the Chemical Speciation Network, and the Interagency Monitoring of Protected Visual Environments network. PM2.5 concentrations predicted by our model were available from the Washington University Atmospheric Compositional Analysis Group (https://sites.wustl.edu/acag/) with a series of available versions. Version V4.NA.03 total PM2.5 was used for total mass PM2.5 concentration. V4.NA.03 modified the V4.NA.02 geographically weighted regression described by van Donkelaar et al.2 to include new measurements of mass scattering efficiency and separate topographic and land-typebased predictor variables according to developments as part of the Mortality-Air Pollution Associations in Low-Exposure Environments (MAPLE) project. V4.NA.03 additionally utilized V4.GL.03 to provide its geophysical input.2,57 These modifications improved performance under low PM2.5 concentrations and enhanced consistency across the entire time series.
+
+The proportions of each component within total PM2.5 mass were then applied to V4.NA.03 total PM2.5 mass according to V4.NA.02, which demonstrated good spatial agreement with average cross-validated observations over the time series with an R2 value of 0.59 for BC, 0.86 for NO3
+, 0.57 for OM, 0.96 for SO4 2, 0.60 for SOILs, and 0.80 for SS;
+cross-validation was represented using a 10-fold holdback of 10% each during the inclusion of ground-based observations using geographically weighted regression.2 Using these data, we calculated the average PM2.5 mass and chemical components of each 1 km2 grid within each ZIP code in nine northeastern states and then averaged them annually between 2000 and 2017 as indicators of long-term exposure. Our exposure estimates were assigned depending on the ZIP code of the individual's residential address and the calendar year. We used annual average PM2.5 mass and components during the same year window because we had observed previously that PM2.5 in the current year could have higher estimate effects on dementia than alternative exposure windows, implying an acceleration of an existing dementia progression by PM2.5.
+
+30
+
+## Covariates
+
+Several individual-level and neighborhood-level covariates were collected to account for possible confounding. The individual-level covariates, including age at entry, race, sex, and Medicaid eligibility, were obtained from the Medicare denominator file. Neighborhood-level covariates included ZIP-code-level SES variables, county-level behavioral risk factors, and health care capacity variables. The ZIP-code-level variables (percentage of population above 65 years of age living below the poverty line, population density, Black population proportion, percentage of people older than 65 not graduated from high school, median household income, and percentage of owner-occupied housing units) were derived from the US Census and American Community Survey (ACS). County-level behavioral risk factors (smoking rate and body mass index) and health care capacity variables (number of hospitals and active medical doctors) were obtained from the Behavioral Risk Factor Surveillance System (BRFSS) and the Area Health Resources Files, respectively.
+
+ll   5 www.the-innovation.org Statistical analysis Single-pollutant and multi-pollutant (where we entered all six components) Cox proportional hazards models with GEE were used to quantify the association between annual mean exposure to PM2.5 components and incident dementia among the elderly. The models were adjusted for the abovementioned neighborhood-level status, behavioral risk factors, and health care capacity variables (Table S2). With GEE, all models were adjusted for residual autocorrelation within the ZIP code, allowing us to obtain statistically more robust confidence intervals for effect estimates.58 This resulted in our estimates being less likely to be affected by within-ZIP-code dependence of observations.
+
+The results from models were presented as HRs with 95% CIs per IQR increase in the annual average concentration of each PM2.5 component. To identify subpopulations that might be particularly susceptible, we performed subgroup analyses for the single-pollutant models based on race (white versus Blacks versus others), gender (men versus women),
+and age (>75 years versus %75 years). To account for possible nonlinearity between PM2.5 components and dementia, we fit penalized spline models59 by including a penalized thin spline term for each component in the computationally scalable stratified Cox-equivalent Poisson regressions.11 The spline term was included in the models one at a time; i.e., we fit single-pollutant models to assess the potential nonlinearity in the C-R relationships for each component. The models adjusted for the same covariates as our main model.11
+
+## Report Sensitivity Analysis
+
+To assess the robustness of our main results, we conducted several sensitivity analyses.
+
+First, we additionally adjusted for the total PM2.5 mass concentration in single-pollutant models. Second, we conducted a non-mover cohort analysis for subjects who did not move during the follow-up period to account for a potential bias related to change in residential address. Third, we fit multi-pollutant penalized spline models and investigated whether the C-R relationships were consistent.
+
+All computations for the analysis were conducted on the Rollins HPC Cluster at Emory University, with the statistical analysis done with R software, version 4.0.2. Statistical significance was determined by two-sided p < 0.05.
+
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+
+59. Meyer, M.C. (2012). Constrained penalized splines. Can. J. Stat. 40, 190–206.
+
+ACKNOWLEDGMENTS
+This study was supported by the National Institute on Aging (NIA/NIH R01 AG074357), the HERCULES Center (P30 ES019776) and the Goizueta Alzheimer's Disease Research Center
+(ADRC) of Emory University (P50 AG025688), the U.S. Environmental Protection Agency (RD835872), and NASA (80NSSC21K0508).
+
+## Author Contributions
+
+L.S. designed the research and directed its implementation. J.L. and L.S. analyzed data.
+
+J.L. and Y.W. made the figures and tables. L.S. prepared the health dataset. A.v.D. and R.V.M. prepared the exposure dataset. J.L., H.H.C., K.S., P.L., W.M.C., J.S., P.K., and L.S. interpreted the results. J.L. wrote the manuscript with input from all authors.
+
+DECLARATION OF INTERESTS
+The authors declare no competing interests.
+
+LEAD CONTACT WEBSITE
+https://sph.emory.edu/faculty/profile/index.php?FID=liuhua-shi-10928.
+
+SUPPLEMENTAL INFORMATION
+Supplemental information can be found online at https://doi.org/10.1016/j.xinn.2022.
+
+100208.

medical/md/PMC9033982.md

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+Cite this: RSC Adv., 2021, 11, 20836
+
+# Application Of Two Morphologies Of Mn2O3 For
+
+![0_Image_0.Png](0_Image_0.Png) Efficient Catalytic Ortho-Methylation Of 4Chlorophenol† Wenying Gui,A Chunlei Zhang,B Wanchun Zhu, A Li Zhang,A Xiaofei Liu,A Hongqiang Zhanga And Zhenlu Wang *A
+
+Vapor phase ortho-methylation of 4-chlorophenol with methanol was studied over Mn2O3 catalyst with two kinds of morphologies. Here, Mn2O3 was prepared by a precipitation and hydrothermal method, and showed the morphology of nanoparticles and nanowires, respectively. XRD characterization and BET
+results showed that, with the increase of calcination temperature, Mn2O3 had a higher crystallinity and a smaller specific surface area. N2 adsorption/desorption and TPD measurements indicated that Mn2O3 nanowires possessed larger external surface areas and more abundant acid and base sites.
+
+Simultaneously, in the fixed bed reactor, methanol was used as the methylation reagent for the orthomethylation reaction of 4-chlorophenol. XRD, XPS, TG-MS and other characterizations made it clear that methanol reduced 4-chlorophenol and its methide, which were the main side-reactions. And Mn3+ was reduced to Mn2+ under the reaction conditions. Changing the carrier gas N2 to a H2/Ar mixture further verified that the hydrogen generated by the decomposition of methanol was not the reason for dechlorination of 4-chlorophenol compounds. Here we summarized the progress of 4-chlorophenol methylation based on the methylation of phenol. Also, we proposed a mechanism of the 4-chlorophenol dechlorination effect which was similar to the Meerwein–Ponndorf–Verley-type (MPV) reaction. The crystal phase and carbon deposition were investigated in different reaction periods by XRD and TG-DTA.
+
+The reaction conditions for the two kinds of morphologies of the Mn2O3 catalyst such as calcination temperature, reaction temperature, phenol–methanol ratio and reaction space velocity were optimized.
+
+Received 8th February 2021 Accepted 28th May 2021 DOI: 10.1039/d1ra01062j rsc.li/rsc-advances
+
+## 1. Introduction
+
+4-Chlorophenol and 4-chlorophenol derivatives have many applications in the production and application of the chemical industry, especially the alkylation species of 4-chlorophenol.
+
+The derivatives of 4-chlorophenol are a kind of important organic chemical intermediate, which are widely used in pharmaceuticals, pesticides and dye intermediates, such as the brain stimulant chlorzoxazone,1 and the cure of coronary heart disease drug clobrate; pesticides fenamidone and fenpropion; dye intermediates 5-chloro-2-hydroxyaniline and 1,4-dihydroxyanthraquinone,2–4 etc. With the development of 4-chlorophenol into a variety of pharmaceutical and new dye intermediates in recent years, chemical industry and commercial demands for 4chlorophenol and its derivatives are increasing with passing year. Therefore, it is crucial to develop an appropriate catalyst with high efficiency of conversion, high selectivity, long operation lifetime and environmentally friendly, energy-saving reaction modes.
+
+The traditional way to synthesize alkylated 4-chlorophenol mainly depends on the chlorination of alkylated phenol. A large number of researchers have reported on the chlorination of phenol and its alkylated derivatives. For example, Gusevskaya's group used CuCl2 as the catalyst and chlorinating reagent (Cu2+
+is the catalyst and Cl are used as halogenating agents), and O2 is the oxidant. The result of chlorination and monohalogenation of aromatic phenols was satisfactory.5,6 Kee's group used SO2Cl2 as the chlorinating agent, and the ratio of pchlorine to o-chlorine in the chlorination products exceeded 50.7 Bovonsombat's used p-toluenesulfonic acid as the catalyst and N-chlorosuccinimide as the source of chlorine, which can react with para-chlorinated phenol analogs at room temperature.8 Sheldon's group applied montmorillonite clay and part of the cation-exchanged L-type molecular sieve as catalyst, SO2Cl2 as the chlorinating agent, and 2,2,4-trimethylpentane as the solvent. The conversion of phenol reached up to 96% at 25 C. The selectivity of 4-chlorophenol achieved 89%, and the ratio of para-chlorinated substitution to ortho-chlorinated substitution reached up to 8.0.2 Xiong et al. adopted microwave-assisted heating with CuCl2 as the catalyst and hydrochloric acid as the chlorine source to accomplish the complete chlorination of phenol within 60 min.9 Although the conversion rate and selectivity of the above methods are very considerable, it is inevitable to use strong acid and highly toxic SO2Cl2 as chlorination reagent. These problems will arise in the practical application of industrialization, whether in terms of the maintenance of industrial equipment or the safety of experimenters and environmental friendly. Accordingly, we converted train of thought and shied to the research about the methylation of 4chlorophenol. Moreover, the gas-phase methylation of phenol with methanol is one of the rst cases of a "green chemistry" applied in industry, in which an electrophilic substitution on an aromatic ring was carried out by applying an alcohol.
+
+As far as we know, the research on the ortho-methylation of 4chlorophenol has not been reported, but there is a lot of works on the ortho-methylation of phenol. Among them, metal oxide catalysts play a vital role, such as a-Fe2O3, 10 TiO2, 11 MgO,12 Mg–
+Al–hydrotalcite,13 Fe-doped ZrO2 composites,14 Si–Mn composite oxides,15 CoFe2O4 spinel,16 V2O5–ZrO2, 17 Mg/Me/O
+spinel (Me ¼ Al, Cr, Fe),18 CuxMn3xO4 spinel,19 Mg–Fe–O
+composite metal oxide.20 The reason why metal oxides show such high activity and selectivity for ortho-methylation of phenol was mainly based on the vertical adsorption of phenolate species on the surface of the catalyst and the formation of intermediate methyl species. Therefore, in chemical kinetics, methyl species are more likely to attack the ortho position of the phenol ring, forming ortho-substituted phenol (o-cresol) or di-ortho-substituted phenol (2,6-xylenol).
+
+Herein, two kinds of morphologies Mn2O3 catalysts were synthetized by two different approaches, and compared them through a series of characterization analysis, and also evaluated the activity of two kinds of Mn2O3 samples in ortho-methylation of 4-chlorophenol. The results of productivity were satised. At the same time, we also discussed the reaction mechanism of the ortho-methylation of 4-chlorophenol and the main side-reaction of this reaction: the dechlorination mechanism of 4-chlorophenol and methylated 4-chlorophenol, and provided a good reference and pavement to develop a series of the highperformance catalysts for ortho-methylation of 4-chlorophenol.
+
+## 2. Experimental 2.1 Chemicals And Materials
+
+Manganous acetate (Mn(Ac)2$4H2O), 25% ammonia solution
+(NH3$H2O), 4-chlorophenol and hexamethylenetetramine
+(C6H12N4) were purchased from Sinopharm Chemical Reagent Co., Ltd. Methanol, ethanol and acetone were purchased from Beijing Chemical Works. All chemicals and reagents were of analytical grade and were not puried before used. Water used in the experiments was ultrapure deionized water (resistance 18 MU cm). Commercial silicon carbide was washed by water and ethanol for 72 h, respectively. Aer desiccation, silicon carbide was calcined under 600 C for 6 h.
+
+## 2.2 Catalyst Preparation
+
+The nanoparticle morphology Mn2O3 catalyst (Mn2O3NP)
+prepared by the precipitation method was according to previously reported methodology with some minor improvements.15 In a typical procedure, 14.7 g Mn(Ac)2$4H2O was dissolved in 100 mL deionized water. Then the above solution was dropwise added into 200 mL 6.5 wt% NH3$H2O diluent at a rate of three drops per second with constant stirring. Aer the Mn(Ac)2 solution dripped out, aging of the mixture was carried out for 3 h at room temperature. Later, the above liquid was transferred to an evaporating dish, and heated in a water bath at 90 C for 12 h. Then it was dried in oven at 110 C for 15 h, and the dried precursor was calcined for 5 h with a 5 C min1 ramp rate. The available powders were ready for the o-methylation activity measurements aer being crushed and sieved (40–60 mesh).
+
+The synthesis of nanowire Mn2O3 catalyst (Mn2O3NW) is as follows. Briey, 2.45 g Mn(Ac)2$4H2O and 0.70 g C6H12N4 were dissolved in 40 mL deionized water. Finally, the mixture was transferred into a Teon-lined stainless steel autoclave and statically crystallized at 180 C for 15 h. The solid–liquid mixture was separated by vacuum ltration and washed with deionized water three times. The lter cake was dried at 80 C
+for 12 h, and the dried precursor was calcined for 6 h.
+
+## 2.3 Catalyst Characterizations
+
+The crystalline structure was characterized by X-ray diffraction analysis (XRD) with an Empyrean diffractometer from Rigaku equipped with a 9 kW rotating anode Cu source at 40 kV and 40 mA, from 10 to 80 with a scan rate of 10 min1. Nitrogen sorption experiment for the measurement of surface area and pore volume was carried out at 77 K on a Micromeritics ASAP
+2010N analyzer. The surface chemical composition analyzed by X-ray photoelectron spectrum (XPS) was conducted on a Thermo ESCA LAB 250 instrument equipped with Al Ka radiation (1486.6 eV). The 284.55 eV value of polluted carbon C
+1s was used as the nuclear effect of the internal standard calibration sample. Morphologies were tested on a eld-emission scanning electron microscope (SEM) instrument (HITACHI
+SU-8020). Simultaneous thermal analysis (STA) mass spectrometry (MS) analysis were performed on Germany NETZSCH
+STA499F3 QMS403D\Bruker V70 type simultaneous thermal analysis-mass spectrometry-infrared combined instrument at a heating rate of 10 C min1 from 40 C to 900 C in air.
+
+Temperature programmed desorption (TPD) and temperature programmed reduction (TPR) were recorded on a ChemBET Pulsar TPR/TPD instrument. The prole is obtained by deducting the blank signal of the sample. The acid and base sites were analyzed by using NH3 and CO2 as atmosphere respectively. Typically, 50 mg of catalysts were pretreated at 300 C in nitrogen ow for 30 min, and then cooled to 50 C
+under helium (He) gas. NH3 or CO2 adsorption was carried out at 50 C for 30 min under NH3 or CO2 gas. Aer samples were sweeped under He gas for 60 min, the temperature was heated to 600 C (10 C min1). The desorbed gas was determined by a GOW-MAC thermal conductivity detector (TCD). As for H2-
+TPR, 15 mg samples were treated in an nitrogen ow at 350 C
+
+![2_image_0.png](2_image_0.png)
+
+for 30 min. After the sample was cooled down to 50 °C, 5 vol%
+H 2 /Ar mixture was introduced to quartz tube for the catalysts reduction. The temperature was heated to 800 °C with a ramp rate of 10 °C min−¹.
+
+## 2.4 Catalytic Testing
+
+The continuous gas phase reaction of 4-chlorophenol with with temperature sensor at the top and bottom to read and control the reaction temperature. The reactor had a tube length of 400 mm and an inner diameter of 8 mm, which was vertically fixed on the heating bed. In a typical run, the lower part of the reaction tube was filled with rolled wire meshes and quartz wool. The mixture of 500 mg catalyst and silicon carbide were packed onto the quartz wool, and the upper portion of the reactor was filled with silicon carbide, which served as preheating devices. A mass flow controller was provided for continuous and constant flow of carrier gas (N 2 ) to influx the reactor. A HPLC pump was also equipped with reaction system for constant volume flow of reaction mixture. The effluents was collected by a glass bottle containing 1 mL of acetone and cooled with ice water bath. The exhaust was accessed to atmosphere for the pressure balance in the reactor. The reaction system is shown in the Fig. 1.
+
+The composition and content distribution of the products were analyzed by a gas chromatography (Shimadzu GC-14B)
+equipped with a flame ionization detector (FID) and a capillary column (PV-101; 60 m × 0.25 mm × 0.25 µ m). The product identification was analyzed by GC-MS (Shimadzu QP-2010 Plus). The major by products were phenol and o -cresol while others
+
+![2_image_1.png](2_image_1.png)
+
+such as 2,6-xylenol, 4-chloro-2,6-xylenol. 2,4-Xylenol and 2,4,6trimethylphenol were in trace amount.
+
+## 3. Results And Discussion
+
+![3_Image_0.Png](3_Image_0.Png) 3.1 Characterization Of The Catalyst
+
+Fig. 2A depicts the XRD patterns of Mn2O3NP at different calcination temperatures. It can be seen that when the calcination temperature was 400 C, the main diffraction peaks can be assigned as the phase of Mn3O4. The diffraction peaks with 2q values around 18.0, 28.9, 32.3, 36.1, 38.0, 44.4, 50.7, 58.5, 59.6, and 64.7 were attributed to the characteristic diffraction peaks of Mn3O4. Meanwhile, it can also be observed that the characteristic diffraction peaks of Mn2O3 appeared around 2q values of 32.9 and 55.1, indicating that a certain amount of Mn2O3 would be accompanied by calcination at this temperature. When the calcination temperature went up to 500 C, the diffraction peak of Mn3O4 disappeared and was replaced by Mn2O3, implied that the crystalline phase was transformed from Mn3O4 to Mn2O3. With the calcination temperature increased ulteriorly, the intensity of the diffraction peak gradually heightened. Meanwhile, the widths of the diffraction peaks became narrower, indicating that a clear increase in the crystallite size.15 Fig. 2C shows the XRD diffraction peaks of Mn2O3NW at different calcination temperatures. It can be seen that with the calcination temperature increased, the diffraction peak intensity of Mn2O3NW gradually increased, which was consistent with the tendency of Mn2O3NP. Under the calcination condition of 400 C, the XRD patterns of Mn2O3NW did not show the characteristic diffraction peak of Mn3O4, which proved that there was no Mn3O4 crystal phase in Mn2O3NW, there were all Mn2O3 phase.
+
+XRD characterization on the crystal phase composition of the two spent Mn2O3 catalysts was also performed. As shown in Fig. 2B and D, both Mn2O3NP and Mn2O3NW showed the same diffraction peak pattern aer an 8 h reaction. The 2q value at 15.1 was attributed to the characteristic diffraction peak of MnCl2, it was due to the dehalogenation effect of 4-chlorophenol. In addition, the 2q values of 34.9, 40.5 and 58.6 were the characteristic diffraction peaks of MnO, and the 2q values of 70.2 and 74.0 were the characteristic diffraction peaks of
+
+![4_image_0.png](4_image_0.png)
+
+Mn3O4. This was indicated that with the reaction proceeding, Mn3+ was reduced to Mn2+. One part of the reason was that during the methylation reaction, the H2 produced by the methanol decomposition reduced Mn3+ to Mn2+, which further formed a more stable M2+–M3+–O phase.21 Another part was that formaldehyde was an active specie formed by the dehydrogenation of methanol. During the formaldehyde formation process, part of the O2 from the surface of the catalyst was taken away.12 Signicantly, 3 h of reaction time, Mn2O3NP did not exhibit a MnCl2 diffraction peak but Mn2O3NW emerged.
+
+The main reason was that Mn2O3NW possessed more active sites in dehalogenation process. In addition, it can be conclude that the dehalogenation was a cumulative process on the Mn2O3 surface.
+
+The SEM images in Fig. 3 displayed the morphology of these fresh and spent samples. Fresh Mn2O3NP catalyst was sintered from nanoparticles, and its abundant porosities originated from the nanoparticles sintertion and stacking. Aer 3 h reaction, owing to coke deposition and the transformation of crystalline phase, it can be seen in Fig. 3C that the surface of catalyst became rough and the number of porosities decreased signicantly. With reaction to 8 h, in Fig. 3E, the porosity of the catalyst was further lost. The enlarged SEM image in Fig. 3E
+showed that there were some microcrystals neighboring the porosities. Integrating the results of XRD characterization, this may be the new generation of MnCl2. In Fig. 3B, Mn2O3NW
+exhibited a nanowire structure instead of nanorod, different synthesis methods made Mn2O3NW show different morphologies from Mn2O3NP. Mn2O3NW spent samples in 3 h and 8 h were displayed in Fig. 3D and F, respectively. The enlarged SEM
+image in Fig. 3D revealed that microcrystal had been formed on 3 h spent Mn2O3NW, that was different from Mn2O3NP but in agreement with the XRD results.
+
+In order to investigate the surface chemical composition and valence state of fresh and 8 h spent catalyst, XPS survey scan of fresh Mn2O3NP catalyst indicated the presence of Mn, O in Fig. 4A while aer the reaction mainly contained four elements:
+Mn, O, C and Cl (see Fig. 5A). The above results indicated that the physical and chemical state on the catalyst surface has changed signicantly aer the ortho-methylation reaction.
+
+Obviously the C (1s) peak appeared aer 8 h reaction, which can be attributed to carbon deposition. The emergence of Cl (2p)
+peak implied that the dehalogenation of 4-chlorophenol in pace with the reaction process. For fresh catalyst in Fig. 4B, the Mn 3s spectrogram contains two sets of multiplet splitting peak. The D-value of the binding energy (DE) was 5.15 eV, it was close to the DE ¼ 5.4 eV of Mn3+ reported in the literature,22 indicating
+
+![5_image_0.png](5_image_0.png)
+
+that the main valence of Mn in fresh Mn2O3NP catalyst was +3.
+
+As for the 8 h spent catalyst (Fig. 5B), the DE was 6.25 eV which closed to the reported 6.1 eV of Mn2+.
+
+22 It was proved that aer the ortho-methylation reaction of 4-chlorophenol, MnO and Mn3O4 were the major components of the catalyst. In addition, the Mn spectrogram embodies two peaks of Mn 2p3/2 and Mn 2p1/2 (Fig. 4C). Around Mn 2p doublet spectra of Mn 2p3/2 and Mn 2p1/2, the characteristic satellite peak of MnO appeared in the 8 h spent catalyst. It was indicated that the Mn in the Mn2O3NP catalyst was reduced from +3 valence to +2, which was consistent with the binding energy results of Mn 3s and the XRD
+patterns. Therefore, with the reaction proceeds, the Mn2O3 phase was gradually reduced to MnO and Mn3O4.
+
+The O 1s XPS spectra are presented in Fig. 4D and 5D. The XPS spectra show the broad doublet peaks in fresh and 8 h spent catalysts. For Fig. 4D, the rst peak at about 529.6–
+529.8 eV was attributed to oxygen ions (O2) in the crystal lattice oxygen23–25 while the second peak near 533 eV could be distinguished into three sets of peaks, which were referred to adsorbed oxygen species (531.4 eV),25 carbonate species and hydroxyl (532.4 eV), as well as adsorbed water (534.0 eV).26 The area integration of the above-mentioned tted peaks showed that in fresh catalyst, the lattice oxygen species (O2)
+accounted for 66.8% of the oxygen elements, and the adsorbed oxygen species (O/O2 2) accounted for 20.0%, the proportion of hydroxyl and carbonate species (OH/CO3 2) was 11.0%, and the ratio of adsorbed water was 2.2%. The lattice oxygen occupied a large number of all oxygen elements. It was obviously that the intensity of the O 1s high binding energy region was higher than the low part, which indicated that the lattice oxygen species on the catalyst surface gradually transformed into other forms of oxygen species with the reaction proceeding. Specically, the lattice oxygen decreased from 66.8% to 13.7%, the adsorbed oxygen species, hydroxyl and carbonate species increased to 52.5% and 31.2%, respectively. Almost, the content of adsorbed water was unchanged. This illustrated that the reactants could be destroy the lattice structure on the catalyst surface, causing the lattice oxygen change to other forms of oxygen species. It is worth noting that the increase in the content of carbonate could be due to the formation of carbon dioxide which produced by the methanol decomposed. Also, the hydroxyl may be formed by the dehalogenation process simultaneously. Similarly, Fig. 6 showed the binding energy of Cl 2p orbital electrons on the surface of the catalyst aer 8 h reaction.
+
+By tting the peaks of the Cl 2p XPS spectrum, in the low binding energy region, it can be obtained that the binding energy of the Cl 2p3/2 around 199.0 eV was the metal chloride, and the signal near 200.6 eV was metal chloride (Cl 2p1/2). The
+
+![6_image_0.png](6_image_0.png)
+
+spin orbit splitting energy of Cl 2p in metal chloride state was 1.6 eV, which was consistent with previous study. 27 In addition, it can be obtained by XPS peak differentiating analysis that the main form of Cl element on the surface of a 8 h spent catalyst was MnCl x , which accounted for 90.9% of all chlorine elements.
+
+It was illustrated that the source of Cl was from the reduction of 4-chlorophenol or its methylated derivatives (can be regarded as HCl), instead of 4-chlorophenol or its methides adsorbed on the surface of the catalyst. In the same way, Mn 2 O 3NW showed a similar distribution of chlorine elements (Fig. 6B).
+
+In Fig. S1 and S2, † Mn 2 O 3NW showed the same tendency as Mn 2 O 3NP in the XPS total spectrum and the 3s binding energy, 2p orbital binding energy and O 1s binding energy of Mn. The reason was completely consistent with the above explanation.
+
+Nitrogen adsorption/desorption measurements were performed to investigate the porosity of the obtained samples. As represented in Fig. 7A, all the isotherms were type IV as defined by IUPAC, characteristic of mesoporous structure. An obvious H3 hysteresis loop in the region of 0.8 < P/P o < 1.0 was observed.
+
+The adsorption/desorption isotherms show a sharp increase at P/P from 0.8 to 1.0 due to the capillary condensation of nitrogen within the mesoporous. 28 It can be seen that the amount of N 2 adsorbed gradually decreased with increasing the calcination temperature, which was due to the crystal particles sintering and growth accompanied by a significant decrease in surface area from 11.9 to 3.6 m 2 g − 1 (Table S1†). Assabumrungrat and Laosiripojana found that the specific surface area decreases with increasing calcination temperature. 29 The textural parameters are shown in Tables S1 and S2.† The low pore volume and pore diameter (4 V/S) of Mn 2 O 3 samples were due to the thermal sintering during the calcination procedure, which led to the blockage of pores. Notably, Mn 2 O 3NW samples possessed larger external surface areas (5.6–27.9 m² g– 1 ) and significantly increased pore volume (0.010–0.109 cm 3 g − 1 )
+compared with those of Mn 2 O 3NP samples. That was due to the hexamethylenetetramine and Mn 2+ build a sort of topological junction abundant supramolecular aggregates which proceed to 1-dimensional structural precursor. 30 -32 Thus, Mn 2 O 3 NW
+
+![6_image_1.png](6_image_1.png)
+
+possessed more channels and textural information than the
+
+![7_image_0.png](7_image_0.png)
+
+Mn2O3NP.
+
+It has been reported that both the acid and base sites are important for the ortho-methylation of phenol.14 Hence, the strength and qualitative analysis of the acid and base sites on the surface of the Mn2O3 catalysts were determined using stepwise TPD of NH3 and CO2. The desorption temperature revealed the acid and base strengths of the catalysts. The higher desorption temperature, the stronger acid or basic strength of the catalyst. The desorbed NH3 appearing at \#150 C should be corresponded to the hydrogen-bonded and physically adsorbed NH3.
+
+33 The ammonia molecules desorbed at higher temperature were attributed to acid-bonded NH3, and the acid sites could be dened as weak (\#150 C), medium (150–300 C), strong (300–
+450 C), and very strong ($450 C).34 From the NH3-TPD proles of the Mn2O3NP under different calcination temperatures in Fig. 8A, it can be seen that all the catalysts present a desorption peak at a range of 150–350 C. With the calcination temperature increased from 500 to 800 C, the TPD proles became inapparently. Thus, the amount of medium acid sites showed a decreasing trend. This explained that the particle size of the catalyst became larger and the specic surface area became smaller with the calcination temperature increased. This was consistent with the characterization results of XRD and N2 adsorption–desorption experiments. When baked at 400 C, the desorption peak of NH3 appeared in the temperature more than 500 C, indicating that there were very strong acid sites on the surface of the catalyst. This was due to the calcination of the catalyst at 400 C, the ambient temperature was not enough for the oxygen to completely oxidize the precursors to +3 valence, but a mixed valence state of +2 and +3. In principle, Mn2+ bound less O2 than Mn3+, and the Mn on the catalyst surface was in an oxygen-depleted state. Therefore, compared with Mn3+, Mn2+
+exposed more positions to bind with NH3 adsorption, and showed a very strong acid desorption peak on NH3-TPD. NH3-
+TPD curve of Mn2O3NW showed a completely different trend from that of Mn2O3NP. As shown in Fig. 8C, under the calcination conditions of 400–500 C, the number of acid sites of Mn2O3NW was numerous, and when calcination exceeded 600 C, the acid sites were signicantly reduced. Since Mn2O3NW
+catalyst formed a stable Mn2O3 phase at a calcination temperature of 400 C, the change in the number of acid sites can be attributed to the reduction of the catalyst surface area which caused by thermal sintering.
+
+From the CO2-TPD proles of the Mn2O3 with different calcination temperatures shown in Fig. 8B and D, it can be seen that all the catalysts present a desorption peak at a range of 100–
+300 C which corresponded to the weak base sites.35 From the desorption proles of CO2 (Fig. 8B), the amount of weak base sites decreased with the increase of calcination temperatures
+
+![8_image_0.png](8_image_0.png)
+
+(500–800 °C). When the calcination temperature increased from 400 to 500 °C, the number of weakly basic sites increased significantly, indicating that the basicity of the catalyst was increased. This was because the Mn 2 O 3NP calcined at 400 °C was a mixed phase of Mn 2 O 3 and Mn 3 O 4 . Since the average combined O 2 − of Mn 2 + in Mn 3 O 4 was less than the number of Mn 3+ , therefore, less O 2 - was exposed on the entire catalyst surface, so the alkalinity was less than that calcined at 500 °C.
+
+When calcined at a temperature above 500 °C, a stable Mn 2 O 3 crystal phase has been formed. With the temperature increased, the thermal sintering of the crystal particles intensified, the specific surface area of the catalyst decreased and the corresponding basic sites on the surface also decreased. The trends of the CO 2 -TPD profiles of Mn 2 O 3NW were similar to its NH 3 -
+TPD, which can also be attributed to the thermal sintering at high temperature. That was the reason why the number of basic
+
+![8_image_1.png](8_image_1.png)
+
+centers on the catalyst surface to decrease with the calcination temperature increased.
+
+The inuence of different morphologies was also reected on reducibility as depicted by Fig. 9A. The peak appearing at temperature below 550 C were assigned to hydrogen consumption by surcial oxygenic species that includes chemically adsorbed oxygen (OChem) at 372–439 C and surcial lattice oxygen (OLatt,S) at 454–507 C.36 Obviously, the reduction temperature of Mn2O3NP was lower than that Mn2O3NW, which indicated that the oxygen species on the surface of Mn2O3NP were more active. As shown in Fig. 9B, it is the H2-TPR spectra of the two catalysts aer 8 h reaction. Maybe the coke deposited on the surface of the catalysts, the OChem and OLatt,S were covered. Hence, the reduction temperatures for OChem and OLatt,S
+increased aer 8 h reaction. Also, there were two peaks of H2 consumption in the range (672–719 C) which was attributed to the reduction peak of the lattice oxygen in the bulk (OLatt,B).37 It is not difficult to nd that the reduction temperature of the OLatt,B of Mn2O3NW was lower than the Mn2O3NP. That indicates that the nanowire structure inhibited reactivity of oxygenic species on surface of Mn2O3, but the strong interaction between Mn3+ and OLatt,B accelerated migration of OLatt,B to the surface, which can promote the methanol catalytic oxidation to formaldehyde.
+
+In the methylation of 4-chlorophenol with methanol, with the reaction progressed, Mn3+ will be reduced to Mn2+ and the oxygen species will be consumed. In addition, the amount of coke deposited on the catalyst surface at different times would be signicantly different. In order to analyze the oxygen uptake behavior and coke deposition at different periods of the reaction, TG and DTA analysis were carried out in air. In Fig. 10A
+and C, it can be seen that the thermal stability of the two fresh Mn2O3 catalysts was very good. With the reaction progressed, it was found that the spent catalyst had two curves with weight decrease in the range of 50–200 C and 350–500 C. The former was attributed to the thermal weight loss of the catalyst adsorbing water, while the latter was due to the removing of the coke deposited on the catalyst surface. The longer the reaction carried out, the greater the thermal weight loss of the catalyst at 350–500 C, indicating that the amount of coke deposited increased with the progress of the reaction time. It can be seen that the spent catalyst has a weight increase at 200–350 C. It was reasonable that the weight increase caused by oxygen regeneration from air.38 Combined with the TG-MS chart of Mn2O3NP, it can be seen that in the main weight loss part of the catalyst (350–500 C), peaks of CO2 and HCl appeared (Fig. 11),
+which proved that CO2 and HCl were formed from the catalyst within this temperature range. There is no doubt that CO2 was generated by the complete combustion of coke deposited in an air atmosphere. Combined with the analysis of XPS and XRD, it can be concluded that the HCl was produced by MnCl2 in spent catalyst. When the temperature exceeded to 500 C, the catalyst showed a curve of weight increase. This was the fact that the oxygen in the air supplemented the anion vacancies aer the exit of the crystal lattice Cl under high temperature conditions, and was embedded in the crystal lattice of the catalyst in the
+
+![9_image_0.png](9_image_0.png)
+
+form of O2, which was also another type of oxygen weight increase.
+
+Fig. 10B and D displayed the DTA spectrum of the catalyst. It can be seen that the spent catalyst had an obvious exothermic peak in the temperature range of 300–400 C. This was because the catalyst emitted heat when the coke burned in an air atmosphere. The amount of coke deposited on the catalyst surface gradually increased with the progress of the reaction, and the intensity of the exothermic peak of the catalyst for 8 h should be stronger than the 3 h. However, the obtained thermal curves were the opposite conclusion. The reason may be that with the reaction progressed, the crystal lattice Cl on the catalyst surface gradually increased which made the crystal lattice structure more perfect, which was not conducive to the formation of coke deposited though the mass of coke increased. In other words, the increase in the crystal lattice Cl reduced the unsaturation sites exposed by the Mn element, that is, the acid sites decreased, so the formation of coke was hindered. In another aspect, the endothermic effect of decomposing MnCl2 was greater than the exothermic effect of coke combustion.
+
+## 3.2 Reaction Mechanism Of O-Methylation Of 4-Chlorophenol And The Dechlorination Mechanism Of Chlorophenol
+
+Regarding the research on the reaction mechanism of the orthomethylation of phenolic compounds catalyzed by metal oxides, Tanabe initiated pioneering work.39 By comparing the infrared spectra of phenol in acidic oxides and basic oxides, it was found that phenol dissociated and adsorbed on the surfaces of the two types of catalysts. The difference was that for acidic oxides, the aromatic ring of the phenate was adsorbed parallelly on the surface of the catalyst, so every position of the aromatic ring was easily attacked by the methylating agent. As for the basic oxides, due to the mutual repulsion between the large p-bonded electron cloud of the aromatic ring and the electron-rich Lewis base center (O2) on the surface of the catalyst, the aromatic ring was kept away from the surface of the catalyst and adsorbed vertically on the surface of the catalyst. Since the ortho position of the hydroxyl O was closer to the surface of the catalyst, it was more likely to be attacked by the methylating agent, thus ensuring extremely high ortho selectivity. Regarding the methyl active species in the methylation reaction of methanol and phenolic compounds, Mathew believed that the H+ generated by the dissociation and adsorption of phenol and protonated methanol, and then a molecule of H2O was le to form CH3 d+,
+which then attacked the aromatic ring.40 Cavani brought forward a completely different view, methanol was rst converted to formaldehyde on the surface of the catalyst, and formaldehyde as the actual methylated species attacked the ortho position of the phenolic hydroxyl group.12 Our work proved the correctness of Cavani's view through experiments and referred other similar works.
+
+The rst step was the dehydrogenation of methanol. The active site for methanol's dehydrogenation reaction was the terminal Mn]O site, which generated hydroxyl and methoxy species. The terminal oxygen was considered to be the most stable site for hydrogen adsorption41 (g ¼ gas phase, a ¼
+adsorbed species).
+
+$${\textbf{a}}^{\dagger}$$
+
+## Ch3Ohg + Ot / Ch3Oa + Oha
+
+Next, attacking of the bridging oxygen on the adsorbed methoxy species yielded a hydroxyl and adsorbed formaldehyde. The crack of the C–H bond of the methoxy group was deemed as the rate-determining step in formaldehyde formation, and required a higher temperature for activation than the rst process.
+
+$\text{p}_{\text{g}}+\text{O}$
+
+![10_image_1.png](10_image_1.png)
+
+## Ch3Oa + Ob / H2Cog + Oha
+
+Two hydroxyl groups recombination occurred to yield H2O
+from the surface of the catalyst.42 The remaining oxygen ion vacancies were lled by the migration of the bulk lattice oxygen inside the catalyst to ensure that the catalyst maintained regular activity and selectivity.
+
+The generated formaldehyde had good positive carbon electrophilic properties and the aromatic ring possessed the electron rich delocalized electron cloud.20 The carbon of formaldehyde attacked the 4-chlorophenate species, forming 5chloro-salicylic alcohol species with hydroxymethyl moieties. In detail, the positively charged carbon attacked the ortho position of the aromatic ring O to form a transition state intermediate species. Because of the aromatic ring was in an electrondecient state, the electron from the C–H covalent bond restores the aromatic ring original appearance. The generated H+ combined with the O of the aldehyde group to generate a hydroxymethyl moiety (Scheme 1).
+
+The resulting 5-chloro-salicylic alcohol generated 4-chloro-2cresol through a series of complex processes (Scheme 2).
+
+Signicantly, 5-chloro-salicylic alcohol can generate almost equimolar 5-chloro-salicylic aldehyde and 4-chloro-2-cresol through an intermolecular disproportionation reaction. A
+
+![10_image_0.png](10_image_0.png)
+
+slightly higher formation of 5-chloro-salicylic aldehyde can be attributed to the dehydrogenation of the alcohol.20 The same type of reaction has been widely reported, such as the intermolecular disproportionation of ethanol into ethane and acetaldehyde, catalyzed by reduced vanadium oxide.43 Also, benzyl alcohol disproportionated into toluene and benzoic aldehyde.44 Unlike the main side reactions of ortho-methylation of phenol with methanol (excessive methylation of phenol), the dechlorination of 4-chlorophenol compounds was the main side reaction. The dechlorination reaction of 4-chlorophenol compounds was most directly and easily associated with the hydrogen generated by the decomposition of methanol in high temperature. However, by changing the carrier gas N2 to H2/Ar mixture with 10% H2 content, we found that the selectivity of 4chlorophenol compounds has not changed, and even the selectivity has increased slightly (Table S11†). Therefore, it showed that the hydrogen generated by the decomposition of methanol was not the reason for dechlorination of 4-
+
+Scheme 3 A possible path way of 4-chlorophenol reduction.
+
+![11_image_1.png](11_image_1.png)
+
+chlorophenol compounds. We believed that methanol was used as a nucleophile and reducing reagent to reduce 4-chlorophenol compounds. This was due to a mechanism similar to Meerwein–Ponndorf–Verley-type (MPV) reaction.45 Next, it involved the substitution of chlorine from methanol hydrides (Scheme 3). Specically, methanol dissociated and adsorbed on the surface of Mn2O3, and the dissociated methoxy ions and H+
+were adsorbed on metal ions and oxygen ions, respectively.
+
+Chlorine in 4-chlorophenol compounds (take 4-chlorophenol as an example) was electron-rich, so it was easier to combine with oxygen-coordinated unsaturated Mn. At the same time, the negative hydrogen attacked the C–Cl bond, forming a thermodynamically stable six-membered cyclic transition state. Finally, negative hydrogen replaced the chlorine, and the adsorbed methoxy ions became formaldehyde and removed from the catalyst surface. Even though methanol was used as H transfer reagents, the MPV reaction usually occurred in the reduction of aromatic aldehydes and ketones. However, in the process of 4chlorophenol dechlorination, the manganese oxide catalyst introduced new chloride ions and hydrogen oxygen, this was consistent with the XPS characterization results of the catalyst aer the reaction (the increase in the content of hydroxide ions and carbonate ions and the chlorine element was a metal chloride), which further illustrated the rationality of the explanation of the dechlorination mechanism.
+
+## 3.3 Catalytic Activity Results
+
+In this reaction, the products were 4-chloro-2-cresol, phenol, 2cresol, 2,6-xylenol, 2,4-xylenol, 2,4,6-trimethylphenol, 4-chloro2,6-xylenol. Probably due to the catalyst and the reaction temperature range, this reaction did not generate 4-chloroanisole or anisole substances. In many previous work, phenol and methanol produced anisole instead of o-cresol under alkaline catalysts (such as MgO) and at low temperature.
+
+However, in this work, the formation of anisole or 4-chloroanisole was not detected.
+
+Table S3† showed the reaction activity and product selectivity of Mn2O3NP under different calcination temperatures. It can be seen that with the calcination temperature increased, the activity of the catalyst gradually decreased. The sintering effect was responsible for low catalytic activity. The sintering effect led to the decrease in the number of active centers on the surface of the catalyst, which was also proved by the NH3/CO2-TPD
+
+![11_image_0.png](11_image_0.png)
+
+characterization. With the increase of the calcination temperature, the number of medium acid and weak base centers decreased signicantly. The catalyst calcined at 400 C had more acid sites on the surface, so the dechlorination effect was more signicant. The selectivity of phenol and phenol methylate was higher than that of other calcination temperatures. In addition, the poly-methyl substituents of phenol (2,6-xylenol, 2,4-xylenol, 2,4,6-trimethylphenol) were also slightly higher than others, this conclusion was consistent with similar work.14 The insight was the stronger the acidity, the longer the average time the phenoxy anions were adsorbed at the acidic sites, then the easier it was for the resulting mono-substituted product to continue to react with the methyl species to generate di- or trisubstituted products.
+
+The result of Mn2O3NW was slightly different from Mn2O3NP
+(Table S4†). This was attribute to Mn2O3NW was the same Mn2O3 crystal phase under investigation. The difference was that the specic surface area (the number of medium acid and weak base centers), despite the specic surface area of Mn2O3NW was larger than that of Mn2O3NP, the two samples behaved quite similarly. In a previous paper, researchers had made the hypothesis that active sites in MgO were corner and steps at crystallite surface.12 Based on this, it might be expected that the high-surface-area Mn2O3NW possessed both a greater overall number and a greater density of defect sites, because of its wirelike structure. Nevertheless, this was in contrast with the experimental evidence. One possible explanation was that the particle efficiency in the high-surface-area Mn2O3NW was low, which was due to the internal diffusion limitations. This implied that only active sites located at the external surface of the catalyst particles contributed to reactivity. If this were the case, regardless of the difference in specic surface area, the reactivity of Mn2O3 would not differ too much.
+
+The reaction temperature played a crucial role in deciding the reactivity and the selectivity of products in either vapor phase or liquid phase reaction. In temperature studies, the reactions were carried out at temperature from 350 to 450 C. It was observed as given in Table S5 and S6,† the increased of temperature led to higher conversion of 4-chlorophenol. That was attribute to the process from methanol to formaldehyde was more likely to occur with the increase of reaction temperature. However, at 450 C, the conversion of 4-chlorophenol was slightly decreased (Table S5†). This was due to the coke generated during the reaction were deposited on the surface of the catalyst, which reduced the porosity of the catalyst.46 Simultaneously, the dechlorination effect of 4-chlorophenol also intensied with the increase of temperature. Maybe the increase of temperature enhanced the dissociation and adsorption of methanol on the catalyst surface, the hydride transfer reaction made 4-chlorophenol and 4-chlorophenol methyl compounds easier to dechlorine.
+
+The inuence of the molar ratio of chlorophenol to alcohol on this reaction is shown in Tables S7 and S8.† For the two morphologies catalysts, as the molar ratio increased from 1 : 1 to 1 : 20, the conversion of 4-chlorophenol generally increased, the selectivity of phenol poly-methylation species increased, and the dechlorination of chlorophenols has been inhibited.
+
+With the chlorophenol-to-alcohol molar ratio increased, the methanol densities increased, more formaldehyde intermediates produced, which increased the conversion of 4-chlorophenol. Meanwhile, the generated mono-substituted phenol or chlorophenol methyl compounds were more likely to react with formaldehyde to generate di- or multi-substituted methylated phenol or chlorophenol under an abundant formaldehyde condition. The dechlorination effect of chlorophenols was inferior, which was seems to contrary to the mechanism we proposed, because more methanol generated more hydrides to reduce the chlorophenols. Considering that the amount of methanol molecules was larger than the amount of dechlorination sites even in the ratio was 1 : 1, thus it was believed that increased methanol ratio could not be change dechlorination effect visibly. In other words, the dehydrogenation of methanol to formaldehyde and chlorophenol dechlorination was occurred on different sites, perhaps the number of the former is far greater than that of the latter.
+
+Tables S9 and S10† showed the reactivity and selectivity of the two Mn2O3 catalysts at different liquid hourly space velocity
+(LHSV). It can be observed that with the increase of volumetric space velocity, for Mn2O3NP catalyst, the conversion of 4-chlorophenol gradually decreased, the selectivity of phenol increased, the selectivity of 4-chloro-2-cresol slightly increased, as well the selectivity of o-cresol was basically unchanged, and the selectivity of poly-methyl substituted phenol decreased. The increase in LHSV indicated that the reactants owing through the catalyst per unit time increased. When it reached to an extreme level, the catalyst did not have the ability to deal with so many reactants, which caused the conversion dropped.
+
+Accordingly, the LHSV increased, especially too much methanol, which generated more coke depositions, and blocked the active sites on the catalyst surface, so the conversion decline. The selectivity of 4-chloro-2-cresol was increased due to more 4chlorophenol went through the surface of the catalyst per unit time. The chlorine removed by the dechlorination effect made the dechlorination site lled untimely. Therefore, when the sampling time was 3 h, the selectivity of 4-chloro-2-cresol would naturally increase. The variety in the selectivity of phenol and poly-methyl substituted phenol can be attributed to the increase in LHSV, the less contact time between phenol species and catalyst.46 Mn2O3NW showed a larger LHSV result than Mn2O3NP.
+
+That was because of the Mn2O3NW had a larger external surface area, which made it possible for more 4-chlorophenol dissociation adsorption.
+
+Without the presence of any catalyst, N2 and H2/Ar mixture were used to the carrier gas for this reaction. It was found that the conversion of 4-chlorophenol was very low, and the conversion in both cases were almost no difference (Table S11†). Even in the H2/Ar mixed gas atmosphere, phenol was formed, but the conversion was too low, indicating that there was no further conversion of 4-chlorophenol. That implied that the H2 produced by methanol decomposition did not the reason why chlorophenol dechlorination. Repeating the above experiment with the presence of catalyst, it was clear that the selectivity of 4-chloro-2-cresol remained largely the same, and the conversion of 4-chlorophenol was signicantly declined. This is because in the H2/Ar mixed gas condition, the lattice oxygen of the catalyst was easier to take away, and inhibited 4-chlorophenol dissociated and adsorbed on the surface of the catalyst.
+
+In addition, due to the removal of lattice oxygen, the cations on the catalyst surface were excessive and the acidity increased, and the adsorption effect of phenol oxygen anions on the catalyst surface became stronger, which aggravated the excessive methylation of phenol and 4-chlorophenol. Hence, the selectivity of phenol poly-methylation species was increased. Under the same conditions, when formalin solution was used as the methylating agent, the dechlorination of 4-chlorophenol was effectively inhibited, indicating that methanol was the cause of dechlorination of 4-chlorophenol and its analogues. In addition, thanks to formalin was directly used as the methylation reagent, the process of methanol to formaldehyde was skipped, so there were more methyl active species (formaldehyde) around the adsorbed phenol oxygen anions though traces of methanol. Therefore, the selectivity of phenol and o-cresol decreased, the poly-methyl substituted phenol increased.
+
+## 4. Conclusions
+
+Two kinds of morphologies Mn2O3 catalysts were prepared by precipitation and hydrothermal methods and calcined under different temperatures. Increasing calcination temperature increased the crystallinity of the catalysts. Mn2O3NP need higher calcination temperature to form a Mn2O3 phase than Mn2O3NW.
+
+As the reaction to, coke deposition on the catalyst and MnCl2 crystalline grain emerged, and the Mn3+ was reduce to Mn2+.
+
+The specic surface area of the Mn2O3NW catalyst was larger than that of the Mn2O3NP. And the specic surface area and pore volume of each catalyst decreased with the increase of the calcination temperature, which leaded to the decline of the number of acid–base centers on the catalyst surface. The catalytic activity and selectivity of the two catalysts had little difference, mainly because the methylation reaction of 4chlorophenol with methanol occurred on the outer surface of the catalyst. Temperature affected the process of methanol dehydrogenation to formaldehyde. High reaction temperature caused excessive coke deposition on the surface of the catalyst due to methanol decomposition, which affected the subsequent catalytic reaction. Increasing the ratio of alcohol to 4-chlorophenol produced more formaldehyde. Overabundance methanol could not promote the dechlorination of 4-chlorophenol and its methyl compounds, therefore, it did not cause excessive reduction of 4-chlorophenol, but enhanced the degree of polymethylation of 4-chlorophenol. The increase in the LHSV of the reactants reduced the conversion of 4-chlorophenol, and the selectivity of 4-chloro-2-cresol increased slightly. At the same time, the Mn2O3NW catalyst can maintain the reactivity at a higher LHSV. The conversion was corresponding to particle efficiency instead of simply specic surface area. The dechlorination effect of 4-chlorophenol and its analogues was due to the hydride produced by methanol on the surface of the catalyst substitute the chlorine on the phenol ring, rather than the hydrogen produced by methanol thermal decomposition.
+
+## Conflicts Of Interest
+
+The authors declare not competing nancial interest.
+
+## Acknowledgements
+
+This work was supported by Jilin Province Science and Technology Research Plan (Key Scientic Research Project) (No:
+20150204020GX).
+
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+78.

medical/md/PMC9680140.md

@@ -0,0 +1,342 @@
+# Let'S Talk About Antibiotics: A Randomised Trial Of Two Interventions To Reduce Antibiotic **Misuse**
+
+Kathy Goggin ,1,2,3 Emily A Hurley ,1,2 Brian R Lee,1,2 Andrea Bradley-Ewing,1,2 Carey Bickford,1 Kimberly Pina,1 Evelyn Donis de Miranda,1 David Yu,4 Kirsten Weltmer,2,5 Sebastian Linnemayr,6 Christopher C Butler ,7 Jason G Newland,8 Angela L Myers9 To cite: Goggin K, Hurley EA, 
+Lee BR, *et al*. Let's Talk About Antibiotics: a randomised trial of two interventions to reduce antibiotic misuse. *BMJ Open* 2022;12:e049258. doi:10.1136/ bmjopen-2021-049258
+► Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2021-049258). Received 19 January 2021 Accepted 08 November 2022 © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. For numbered affiliations see end of article. Correspondence to Dr Kathy Goggin; kgoggin@cmh.edu
+
+## Abstract
+
+Background Children with acute respiratory tract infections (ARTIs) receive ≈11.4 million unnecessary antibiotic prescriptions annually. A noted contributor is inadequate parent–clinician communication, however, efforts to reduce overprescribing have only indirectly targeted communication or been impractical.
+
+Objectives Compare two feasible (higher vs lower intensity) interventions for enhancing parent–clinician communication on the rate of inappropriate antibiotic prescribing. Design Multisite, parallel group, cluster randomised comparative effectiveness trial. Data collected between March 2017 and March 2019.
+
+Setting Academic and private practice outpatient clinics.
+
+Participants Clinicians (n=41, 85% of eligible approached) and 1599 parent–child dyads (ages 1–5 years with ARTI symptoms, 71% of eligible approached). Interventions All clinicians received 20 min ARTI diagnosis and treatment education. Higher intensity clinicians received an additional 50 min communication skills training. All parents viewed a 90 second antibiotic education video. Main outcome(s) and measure(s) Inappropriate antibiotic treatment was assessed via blinded medical record review by study clinicians and a priori defined as prescriptions for the wrong diagnosis or use of the wrong agent. Secondary outcomes were revisits, adverse drug reactions (both assessed 2weeks after the visit) and parent ratings of provider communication, shared decision-making and visit satisfaction (assessed at end of the visit on Likert-type scales). Results Most clinicians completed the study (n=38, 93%), were doctors (n=25, 66%), female (n=30, 78%) and averaged 8 years in practice. All parent–child dyad provided data for the main outcome (n=855 (54%) male, n=1043 (53%) <2 years). Inappropriate antibiotic prescribing was similar among patients who consulted with a higher intensity (54/696, 7.8%) versus a lower intensity (85/904, 9.4%) clinician. A generalised linear mixed effect regression model (adjusted for the twostage nested design, clinician type, clinic setting and clinician experience) revealed that the odds of receiving inappropriate antibiotic treatment did not significantly vary by group (AOR 0.99, 95%CI: 0.52 to 1.89, p=0.98). Secondary outcomes of revisits and adverse reactions did 
+
+## Strengths And Limitations Of This Study
+
+⇒ Large number of clinicians and parent–child dyads engaged.
+
+⇒ Feasible interventions modelled on prior successful interventions.
+
+⇒ Rigorous methods conducted in real world clinical settings.
+
+⇒ Lack of a control group or baseline antibiotic prescribing information.
+not vary between arms, and parent ratings of satisfaction with quality of parent–provider communication (5/5), shared decision making (9/10) and visit satisfaction (5/5) were similarly high in both arms. Conclusions and relevance Rate of inappropriate prescribing was low in both arms. Clinician education coupled with parent education may be sufficient to yield low inappropriate antibiotic prescribing rates. The absence of a significant difference between groups indicates that communication principles previously thought to drive inappropriate prescribing may need to be re-examined or may not have as much of an impact in practices where prescribing has improved in recent years.
+
+Trial registration number NCT03037112.
+
+In the USA, most antibiotic prescribing occurs in the outpatient setting1 where children with acute respiratory tract infections (ARTIs) receive 34million antibiotic prescriptions annually.2 Estimates indicate that at least 29% of these prescriptions are unnecessary.3 Antibiotic prescribing behaviour is a complex and multifaceted process, but the communication between parents or legal guardians (hereafter referred to as parents) and clinicians is central. Clinicians cite strong parent demand as a major cause of inappropriate prescribing.4–7 Clinicians often capitulate to this perceived pressure because they do not want parents/patients to leave 'emptyhanded',8–10 fear receiving poor encounter satisfaction scores from parents,11 and/or view explaining why antibiotics are not necessary as timeconsuming and unrewarding.8 9 Efforts to reduce inappropriate antibiotic prescribing in the paediatric setting have typically taken the form of educational interventions to increase antibiotic knowledge among clinicians and/or parents, electronic decision support systems and/or behavioural interventions informed by behavioural economics and psychological science.12–15 Many have been successful, with those that target parent–clinician communication and simultaneously intervene on parents and clinicians evidencing the strongest results.13 Of the communication interventions tested, only one has directly targeted clinicians' perceptions of parental expectations for antibiotics alongside antibiotic education and shared decision-making.16 This UK-based study provided intensive communication training for clinicians and a multipage patient–clinician interactive educational booklet to enhance shared decision making. Clinicians in the intervention arm demonstrated statistically and clinically significant reductions in antibiotic prescribing as compared with control clinicians. While impactful, the intervention was viewed impractical for most real-world settings.17 Effective interventions that are efficacious and feasible in routine outpatient paediatric care in the USA are lacking. Using the Francis *et al*16 intervention as our model, we sought to: (1) develop a version of this efficacious intervention that would enhance parent–clinician communication while being feasible in ambulatory paediatric care and (2) compare it to a feasible educational intervention on the rate of inappropriate antibiotic prescribing.
+
+## Methods Study Overview
+
+This was a multisite, parallel group, cluster randomised comparative effectiveness trial conducted in two paediatric outpatient clinics, with clinicians randomised (1:1) 
+to a higher intensity intervention (prescribing education and communication skills training) or a lower intensity intervention (prescribing education only). Parent–child dyads enrolled in the study were exposed to either intervention according to the clinician who conducting their clinic visit. We hypothesised that compared with parent– child dyads managed by clinicians randomised to the lower intensity intervention, parent–child dyads managed by clinicians randomised to the higher intensity intervention would evidence lower rates of inappropriate antibiotic prescriptions (see protocol paper for additional details).18
+
+## Patient And Public Involvement
+
+In the early planning stages for this study, we conducted focus groups and individual interviews with clinical, parent, payer and community stakeholders to assess the viability and inform the design of the study. We then recruited a parent research associate who became a core member of our research team, attended all study meetings and co-led our community advisory board (CAB). 
+
+Our CAB was composed of 15 parent, provider and community stakeholders and was diverse (ie, 3 males, 7 Latinx (3 exclusively Spanish speaking) and 3 African American members). CAB meetings occurred regularly throughout the study. All aspect of the study design, settings, participant burden, materials, procedures, interpretation of data and dissemination of study findings were informed by the CAB and community research associate. Study results were disseminated to all clinic providers. A parent summary of findings will be provided to study sites to share with parents after this paper is published.
+
+## Study Setting
+
+The study was conducted at an academic medical facility (CMH Primary Care Clinics) in Kansas City, Missouri, USA and both locations of a private practice (Heartland Primary Care) in Kansas City and Lenexa, Kansas, USA.
+
+## Participants
+
+Clinicians All clinicians at both clinics were screened for eligibility. Inclusion criteria were being a paediatrician (medical doctor (MD) or doctor of osteopathic medicine (DO)) or nurse practitioner (certified paediatric nurse practitioner (CPNP) or advanced practice registered nurse (APRN)) 
+and actively and independently conducting consultations with our target population. Eligible clinicians were recruited during study orientation sessions, where interested clinicians completed informed consent and were given a sealed envelope prepared by the study statistician that contained their group assignment. Clinicians were stratified by patient volume and clinic.
+
+## Parent–Child Dyads
+
+Parent–child dyads were recruited from March 2017 to March 2019. Study staff prescreened all potentially eligible parent–child dyads and provided a study flyer during check-in. Interested dyads were given information about the study and offered eligibility screening. If more than one caregiver was present, they were asked to designate one person who would independently complete the written informed consent and all assessments. Clinicians had no role in identifying potentially eligible dyads, screening, consenting or data collection. Clinic staff who scheduled patients' appointments were blinded to clinician randomisation. Dyads were eligible if the patient was between ages 1 and 5 years, had ARTI symptoms and his/her parent was fluent in English or Spanish.18 Children were not eligible if they had received an antibiotic in the last 30 days, had a concurrent probable bacterial non-respiratory infection, known immunocompromising conditions, complex chronic care condition,19 required hospitalisation during the visit or had previously participated in the study.
+
+| Table 1                                                                                                                                                                     | Diagnostic criteria for bacterial acute respiratory tract infections (ARTIs) and appropriate antibiotic selection                                                                                                                                                                                                                                           |                                                                                                                   |         |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------|---------|
+| Bacterial                                                                                                                                                                   | Primary                                                                                                                                                                                                                                                                                                                                                     | Secondary antibiotics for penicillin                                                                              |         |
+| ARTI                                                                                                                                                                        | Diagnostic criteria                                                                                                                                                                                                                                                                                                                                         | antibiotic                                                                                                        | allergy |
+| Acute Otitis                                                                                                                                                                | 1. Fever ≥38.3°C with either A or B:                                                                                                                                                                                                                                                                                                                        |                                                                                                                   |         |
+| Media                                                                                                                                                                       | A. Moderate to severe bulging of tympanic membrane on exam, or                                                                                                                                                                                                                                                                                              |                                                                                                                   |         |
+| (either criteria)                                                                                                                                                           | B. Mild bulging of TM and recent (<48hours) onset of ear pain                                                                                                                                                                                                                                                                                               |                                                                                                                   |         |
+| 2. New onset of otorrhea not due to acute otitis externa                                                                                                                    | Amoxicillin                                                                                                                                                                                                                                                                                                                                                 | Cefdinir, cefpodoxime, ceftriaxone,  cefuroxime, clindamycin                                                      |         |
+| Sinusitis (any of the  three criteria)                                                                                                                                      | 1. Daytime cough or nasal discharge for greater than 10 days 2. High fever (>39°C) with purulent nasal discharge or facial pain lasting  three consecutive days at the beginning of the illness 3. Worsening signs or symptoms characterised by the new onset of fever,  headache, or increase in nasal discharge following a typical viral URI Amoxicillin | Cefdinir, cefpodoxime, cefuroxime,  clindamycin                                                                   |         |
+| Community                                                                                                                                                                   | 1. Fever, tachypnoea and focal findings on pulmonary exam                                                                                                                                                                                                                                                                                                   |                                                                                                                   |         |
+| acquired                                                                                                                                                                    | 2. (A) Fever, (B) Tachypnoea, cough or retractions and (C) chest                                                                                                                                                                                                                                                                                            |                                                                                                                   |         |
+| Pneumonia                                                                                                                                                                   | radiograph consistent with a focal consolidation                                                                                                                                                                                                                                                                                                            |                                                                                                                   |         |
+| (either criteria)                                                                                                                                                           | Amoxicillin                                                                                                                                                                                                                                                                                                                                                 | Cefpodoxime, cefprozil, cefuroxime,  clindamycin                                                                  |         |
+| Streptococcal  pharyngitis (both criteria)                                                                                                                                  | Amoxicillin                                                                                                                                                                                                                                                                                                                                                 | Cephalexin (preferred unless previous  type I hypersensitivity reaction to  penicillin) clindamycin, azithromycin |         |
+| TM, tympanic membrane; URI, upper respiratory infection.1. Fever, pharyngitis and positive rapid streptococcal antigen test or  culture 2. Lack of viral signs and symptoms |                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                   |         |
+
+## Intervention Components And Description Of Arms Parent Video
+
+The 90s video used gain-framed messages20 21 to explain when antibiotics are and are not indicated while emphasising the risk of side effects and resistant organisms. 
+
+Research has shown that people react to the same trade-off in different ways depending on whether the possible outcomes are presented as losses or gains.20 We tailored all parent materials to highlight the gains of not using antibiotics (eg, staying safe from side effects) that might increase parents' comfort with not getting an antibiotic prescription for their child. The video also highlighted information that clinicians should provide (eg, estimate of illness duration, recommendations for system relief) during a visit. The video was successful in reducing parents' interest in obtaining an antibiotic for their child, especially among those with higher baseline interest.22 Parent educational trifold brochure The inside of the brochure provided 'gain-framed' information about when antibiotics are and are not necessary and the risks involved in taking antibiotics. The outside of the brochure included a place to write the child's first name and parent tips for communicating with clinicians. It also included check boxes for the clinician to indicate the diagnosis, if antibiotics were needed, recommended home care treatments and expected recovery time. Clinician general antibiotic education Using didactic and interactive learning strategies, study physicians (AM and JGN) provided a 20min, in-person general antibiotic education training on diagnosis and treatment of ARTI. The training covered pros and cons of antibiotics, impact of inappropriate use, Centers for Disease Control and Prevention antibiotic prescribing guidelines, common reasons for antibiotic misuse and viewing/discussing the 90s parent video. Refresher trainings for all study clinicians were provided twice during the study. Clinician communication skills training The in-person, 50min communication skills training provided by the study's behavioural psychologist (KG) used didactic and interactive learning strategies, including viewing/discussing motivational role model videos. The goal was to enhance clinicians' skills and confidence in the use of proven parent-centred communication strategies and the parent educational trifold brochure (see protocol paper for additional details).18 Clinicians were also trained to consider parents' ratings of their interest in obtaining antibiotics after viewed the video.
+
+## Lower Intensity Intervention
+
+This intervention was modelled on proven parent-focused and clinician-focused educational interventions.13 23 Clinicians completed the general antibiotic education described above.
+
+In exam rooms prior to the consultation, parents who saw a clinician trained in the Lower Intensity intervention completed the baseline survey, saw the video and the inside of the parent brochure, and rated their desire for antibiotics all via a tablet computer. They did not receive a hard copy of the study brochure and their interest in an antibiotic rating was not shared with their clinician.
+
+## Higher Intensity Intervention
+
+The higher intensity, Let's Talk About Antibiotics, intervention was informed by a series of evidence-based interventions conducted in the UK and Europe,16 24–29 Clinicians randomised to this arm completed the general antibiotic education and communication skills training described above. Before meeting with dyads, clinicians in this arm were provided with parents' ratings of their interest in obtaining antibiotics after watching the parent d
+
+![3_image_0.png](3_image_0.png)
+
+video via a sticky note on the exam room door. To assess fidelity to the communication skills and check for contamination between arms, a subsample of all visits (10%) were audio recorded and objectively coded by blinded raters using established methods. 38 In exam rooms prior to the consultation, parents who saw a clinician trained in the higher intensity intervention completed the baseline survey, saw the video and the inside of the parent brochure, rated their desire for antibiotics via a tablet computer and received a personalised (child's name written in) hard copy of the study brochure.
+
+Strategies to reduce the risk of contamination We employed several strategies to reduce the likelihood of contamination between study arms. Specifically, we (1)
+designed intervention components to not be easily transferred between clinicians (eg, the brochure was distributed by study staff to ensure that only parents who were consulted by clinicians in the Higher Intensity arm receive them), (2) ensured that all communication (written or in person) with clinicians in the lower intensity arm did not reveal any of the strategies from the higher intensity arm,
+(3) reviewed the importance of keeping intervention arms distinct during clinician training and asked higher intensity clinicians to pledge not to share any details or materials with their colleagues randomised to the lower intensity arm and (4) trained higher intensity arm clinicians in communication strategies for dealing with lower intensity arm colleagues who ask for more information.
+
+## Primary Outcome
+
+The primary outcome was rate of inappropriate antibiotic prescribing (ie, number of patients receiving an inappropriate prescription/number of patients in arm). Inappropriate prescribing was assessed by blinded study physicians
+(AM and JGN) who reviewed the medical record documentation for each patient. Prescriptions were considered inappropriate if they were, prescribed: (1) for a viral ARTI, (2) for a presumed bacterial ARTI that does not meet table 1 criteria, (3) broad-spectrum antibiotic for a bacterial ARTI in a child without a penicillin allergy or
+
+| Table 2                                                                                                                                  | Parent and patient demographics (N=1599) Higher  Lower  intensity intensity   |             |             |
+|------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------|-------------|-------------|
+| Gender of parent  Female                                                                                                                 | 584 (83.9%)                                                                   | 764 (84.6%) |             |
+| Ethnicity of parent  Hispanic                                                                                                            | 129 (18.5%)                                                                   | 171 (18.9%) |             |
+| Race of parent  Asian                                                                                                                    | 13 (1.9%)                                                                     | 10 (1.1%)   |             |
+|                                                                                                                                          | Black                                                                         | 90 (12.9%)  | 104 (11.5%) |
+|                                                                                                                                          | White                                                                         | 537 (77.2%) | 721 (79.8%) |
+|                                                                                                                                          | Multiracial                                                                   | 15 (2.2%)   | 24 (2.7%)   |
+|                                                                                                                                          | Other                                                                         | 10 (1.4%)   | 13 (1.4%)   |
+|                                                                                                                                          | Chose not to answer                                                           | 31 (4.5%)   | 31 (3.4%)   |
+| Preferred language  Spanish                                                                                                              | 41 (5.9%)                                                                     | 60 (6.6%)   |             |
+| Education  Less than high school                                                                                                         | 38 (5.5%)                                                                     | 49 (5.4%)   |             |
+|                                                                                                                                          | High school degree or GED                                                     | 151 (21.7%) | 183 (20.3%) |
+|                                                                                                                                          | Some college                                                                  | 228 (32.8%) | 335 (37.1%) |
+|                                                                                                                                          | Secondary degree                                                              | 196 (28.2%) | 240 (26.6%) |
+|                                                                                                                                          | Postsecondary degree                                                          | 79 (11.4%)  | 94 (10.4%)  |
+|                                                                                                                                          | Other/unknown                                                                 | 4 (0.6%)    | 2 (0.2%)    |
+| Patient age  1                                                                                                                           | 249 (35.8%)                                                                   | 307 (34.0%) |             |
+|                                                                                                                                          | 2                                                                             | 126 (18.1%) | 172 (19.0%) |
+|                                                                                                                                          | 3                                                                             | 104 (14.9%) | 145 (16.1%) |
+|                                                                                                                                          | 4                                                                             | 96 (13.8%)  | 136 (15.1%) |
+|                                                                                                                                          | 5                                                                             | 121 (17.4%) | 143 (15.8%) |
+| Patient sex  Female                                                                                                                      | 327 (47.1%)                                                                   | 414 (45.9%) |             |
+|                                                                                                                                          | Male                                                                          | 367 (52.9%) | 488 (54.1%) |
+| Patient diagnosis  Viral URI/pharyngitis/OME                                                                                             | 352 (50.6%)                                                                   | 440 (48.7%) |             |
+|                                                                                                                                          | Strep throat                                                                  | 20 (2.9%)   | 16 (1.8%)   |
+|                                                                                                                                          | AOM                                                                           | 126 (18.1%) | 162 (17.9%) |
+|                                                                                                                                          | Sinusitis                                                                     | 2 (0.3%)    | 4 (0.4%)    |
+|                                                                                                                                          | Pneumonia                                                                     | 6 (0.9%)    | 13 (1.4%)   |
+|                                                                                                                                          | Multiple or other diagnosis                                                   | 190 (27.3%) | 268 (29.7%) |
+| AOM, acute otitis media; GED, General Educational Development  Test; OME, otitis media with effusion; URI, upper respiratory  infection. |                                                                               |             |             |
+
+(4) non-recommended alternative antibiotic for a bacterial ARTI (see table 1) in a child with a penicillin allergy.
+
+To guard against the potential for clinicians to use diagnostic codes to support their antibiotic prescribing,7 study physicians reviewed detailed symptoms, physical examination findings and diagnostic tests to assess the appropriateness of the patient's diagnosis. Ten per cent of all chart reviews were verified by the other study physician blinded to the initial coding, inconsistencies were reconciled.
+
+## Secondary Outcomes
+
+Data on revisits and adverse drug reactions were collected via follow-up phone calls with parents 2weeks after the visit. Revisits were assessed by asking parents if they sought any additional treatment for their child for the same symptoms or complications from any treatment provided in the initial visit. Side effects and adverse drug reactions were assessed if antibiotics were prescribed.
+
+Shared decision-making was assessed immediately following the visit using an adapted version of the threeitem CollaboRATE questionnaire.32 Parents rated How much effort was made to: (1) help you understand your child's health issue?; (2) listen to the things that matter most to you about your child's health issues? and (3) include what matters most to you in choosing what to do next? on a 10-point response scale ranging from 'no effort was made' to 'every effort was made.' The scale's psychometric properties have been established.33 Quality of parent–clinician communication was assessed immediately following the visit with the question, 'How satisfied were you with the communication between you and your child's healthcare clinician?' Overall visit satisfaction was assessed with the question, 'Overall, how satisfied were you with the visit?' Both items were scored on a five-point scale ranging from 'very dissatisfied' to 'very satisfied'.
+
+## Analyses
+
+All analyses were conducted using an intention-to-treat strategy. We constructed a two-stage nested design, with parents nested within clinicians (level-1 units) and study site (level-2 units) generalised linear mixed-effect regression models (GLMM) to assess the impact of intervention type on our primary outcome of inappropriate antibiotic prescribing using Stata.34 Alternative covariance structures were investigated, but as hypothesised, the exchangeable structure was sufficient. We employed robust standard errors to help minimise misspecification and examine time as a potential random effect. 
+
+We examined the effects of the potential prespecified covariates on the outcomes with the goal of identifying the most parsimonious final models and we explored the heterogeneity of treatment effect. Variables considered included: clinic, clinician type, years of experience, patient age and sex, parent education level, race/
+ethnicity, preferred language, relationship to patient and insurance type. We created a binary indicator for each variable and included each as an interaction term in a separate regression model. We examined these interaction terms across intervention arms and explored within-arm differential trends in our primary and secondary outcomes over time.
+
+Sample size calculation and power Prior research examining our primary outcome showed 30% of the antibiotics prescribed in the outpatient ARTI visits were inappropriate.3 35 Prior intervention studies produced 20%–81% reductions in inappropriate prescribing.36 37 Based on the intraclass correlation coefficient (ICC) observed in the Meeker *et al* study,37 we assume an ICC of 0.04. With 40 clinicians, α of 0.05 and 80% power, we estimated that a sample size of 760 per arm would be needed to detect a 9% difference between arms. Allowing for an attrition rate of 5%, we aimed to recruit 1600 participants to ensure adequate power.
+
+## Results Clinicians
+
+All clinicians at both sites (N=51) were voluntarily screened for eligibility; five were ineligible, four failed to respond after multiple contacts and one declined to enrol in the study. All 41 clinicians enrolled (22 (54%) randomised to the higher intensity arm; 19 (46%) randomised to lower intensity arm) conducted clinic visits with enrolled participants. Three clinicians (all in the higher intensity arm) 
+left their practices during the study, leaving 38 clinicians who all completed the follow-up survey and interview. Most clinicians were female (n=30, 78%) and MD/DO physicians (n=25, 66%) with 8 median years in practice (IQR 4–14; range 1–40). Clinician demographics were similar across arms and between those who did and did not participate in the study.
+
+## Parent–Child Dyads
+
+Figure 1 illustrates the flow of parent–child dyads from prescreening through the 2-week follow-up visit. Table 2 displays demographics for the 1599 dyads included in the primary analysis. Demographic characteristics of parents and children were similar among those exposed to the higher or lower intensity intervention. Spanish-speaking parents and those who had more education were more likely to complete the 2-week assessment, no other differences were observed. Missing survey responses were rare overall (<1% for all variables) and did not exhibit any systematic patterns so we did not impute values.
+
+## Fidelity Assessment
+
+Analysis of 141 visit audio recordings confirmed a similar mean duration of visits (higher=11.7vs lower=10.2min) 
+and no evidence of contamination between arms. Compared with lower intensity clinicians, higher intensity clinicians consistently delivered more of the communication content they were taught in training and no higher intensity materials were used in sessions conducted by lower intensity clinicians.
+
+## Primary Outcome: Inappropriate Antibiotic Prescribing
+
+A total of 429 (26.8%) patients received an antibiotic prescription with 139 (32.4%) meeting criteria for being inappropriate (table 3). The most common reasons for an antibiotic to be considered inappropriate were being prescribed for a presumed bacterial ARTI that did not meet diagnostic criteria (n=109; 78.4%) and prescribing a broad-spectrum antibiotic for a child without a penicillin allergy (n=24; 17.3%). The rate of inappropriate prescriptions among all enrolled patients in each arm was similar among higher intensity (54 of 696; 7.8%) and lower intensity (85 of 903; 9.4%) clinicians. Inappropriate antibiotic prescribing also did not vary significantly based on study site or clinician type. In the unadjusted GLMM, 
+we found that the odds of receiving inappropriate antibiotic treatment for the higher intensity arm did not vary significantly when compared with the lower intensity arm (OR 1.09; 95% CI 0.56 to 2.10; ICC=0.11; table 4).
+
+## Heterogeneity Of Treatment Effect
+
+After adjusting for clinician type, clinic setting and clinician experience, there was still no significant intervention effect (Adjusted Odds Ratio (AOR) 0.99; 95%CI 0.52 to 1.89). However, the interaction of the treatment arm and clinician type was significant in the adjusted GLMM model (AOR 0.12; 95% CI 0.04 to 0.37; see online supplemental 
+
+| Table 3                                                                                                                               | χ2  comparison of overall and inappropriate antibiotic prescribing (N=1599) Any antibiotic prescribed Inappropriate antibiotic prescribed n Freq (%) P value n Freq (%) P value   |       |            |      |           |
+|---------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|------------|------|-----------|
+| Study arm                                                                                                                             | 0.291                                                                                                                                                                             | 0.244 |            |      |           |
+|                                                                                                                                       | Higher                                                                                                                                                                            | 696   | 196 (28.2) | 696  | 54 (7.8)  |
+|                                                                                                                                       | Lower                                                                                                                                                                             | 903   | 233 (25.8) | 903  | 85 (9.4)  |
+| Site                                                                                                                                  | 0.630                                                                                                                                                                             | 0.455 |            |      |           |
+|                                                                                                                                       | Private practice                                                                                                                                                                  | 1292  | 350 (27.1) | 1292 | 109 (8.4) |
+|                                                                                                                                       | Academic                                                                                                                                                                          | 307   | 79 (25.7)  | 307  | 30 (9.8)  |
+| Clinician type                                                                                                                        | 0.129                                                                                                                                                                             | 0.572 |            |      |           |
+|                                                                                                                                       | MD/DO                                                                                                                                                                             | 907   | 230 (25.4) | 907  | 82 (9.0)  |
+|                                                                                                                                       | CPNP/APRN                                                                                                                                                                         | 692   | 199 (28.8) | 692  | 57 (8.2)  |
+| APRN, advanced practice registered nurse; CPNP, certified paediatric nurse practitioner; MD/DO, medical doctor/doctor of osteopathic. |                                                                                                                                                                                   |       |            |      |           |
+
+| Table 4                                                                                                                                                                                                                                 | ORs of receiving inappropriate antibiotic (N=1599) Unadjusted   | Adjusted*   |       |       |         |      |      |      |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------|-------------|-------|-------|---------|------|------|------|
+| 95%CI                                                                                                                                                                                                                                   | 95%CI                                                           |             |       |       |         |      |      |      |
+| OR                                                                                                                                                                                                                                      | Lower                                                           | Upper       | Lower | Upper | P value |      |      |      |
+| P value                                                                                                                                                                                                                                 | OR                                                              |             |       |       |         |      |      |      |
+| Intervention arm  Higher intensity (vs lower)                                                                                                                                                                                           | 1.09                                                            | 0.56        | 2.10  | 0.81  | 0.99    | 0.52 | 1.89 | 0.98 |
+| Clinician type  CPNP/APRN (vs MD/DO)                                                                                                                                                                                                    | 1.24                                                            | 0.63        | 2.44  | 0.53  | 1.29    | 0.67 | 2.46 | 0.45 |
+| Practice type  Academic (vs private)                                                                                                                                                                                                    | 1.21                                                            | 0.63        | 2.30  | 0.56  | 1.13    | 0.60 | 2.16 | 0.70 |
+| Clinician years of experience  10+ years (vs <10years)                                                                                                                                                                                  | 1.40                                                            | 0.73        | 2.66  | 0.31  | 1.42    | 0.73 | 2.77 | 0.30 |
+| *Adjusting for intervention arm, clinician type, practice type and clinician years of experience. APRN, advanced practice registered nurse; CPNP, certified paediatric nurse practitioner; MD/OD, medical doctor/doctor of osteopathic. |                                                                 |             |       |       |         |      |      |      |
+
+table 1). Specifically, the MD/DO clinicians in the higher intensity intervention were less likely to prescribe an inappropriate antibiotic than MD/DO clinicians in the lower intensity intervention arm. The reverse was true for the CPNP/APRN clinicians. No interaction between intervention and gender or location was observed. No interaction between treatment and any of the parent–patient dyad variables (ie, patient age or sex, parent education level, race/ethnicity, preferred language, relationship to patient or insurance type) was observed.
+
+## Secondary Outcomes
+
+Revisits and adverse drug reactions: Approximately 12% of patients had a return visit within 2weeks of their index visit. The rate of revisits did not vary between patients seen by higher or lower intensity clinicians (12.2% vs 11.9%, p=0.879). Adverse reactions to the prescribed antibiotic were similar across arms (16.5% vs 12.8%, p=0.27). (Antibiotic prescription rates for different ARTI diagnoses are presented by arm in online supplemental table 2).
+
+Shared decision-making: Parents' CollaboRATE ratings were extremely high overall (likely evidencing a ceiling effect) and similar across higher and lower intensity arms (median 9.0 (IQR: 8.7–9.0) vs 9.0 (IQR: 8.3–9.0), p=0.85).
+
+Quality of parent–clinician communication and visit satisfaction: Parent rated satisfaction with their clinician's communication during the visit (median 5 (IQR: 5–5) vs 5 (IQR: 5–5), p=0.20) and their overall visit satisfaction (median 5 (IQR: 5–5) vs 5 (IQR: 5–5), p=0.38) were also very high overall and similar between arms.
+
+## Clinician Satisfaction And Feasibility
+
+Most clinicians (84%) reported being 'very satisfied' with the programme, thought it would be 'very' (71%) effective in reducing inappropriate prescribing and all would recommend it to other clinicians.
+
+## Parent Satisfaction
+
+The majority of parents who completed the 2-week follow-up survey (n=1337) reported being 'very' (92%) 
+satisfied with the programme and 93% reported that they would recommend it to others.
+
+## Discussion
+
+This randomised comparative effectiveness trial comparing two feasible interventions for enhancing parent–clinician communication found no evidence of a difference in inappropriate antibiotic prescriptions. Inappropriate antibiotic prescribing was lower than recently published estimates of inappropriate prescribing in the US Midwest (14.3%),38 which have been on the decline, especially among paediatricians.39 Nevertheless, it was still higher than findings from other successful intervention studies (rates from 1.5% to 3.9%).15 40 In the main outcome analysis, the odds of receiving an inappropriate antibiotic did not vary significantly between the higher and lower intensity arms, even after adjusting for clinician type, clinic setting and clinician experience.
+
+Secondary outcomes of revisits and adverse reactions did not vary between patients seen by higher or lower intensity clinicians. These findings indicate that there is no evidence that one of the interventions presented a greater risk to patients than the other. 
+
+Parent ratings of shared decision making, satisfaction with quality of parent–clinician communication and visit satisfaction were all very high and similar between arms. Ceiling effects on the measures were apparent and likely reduced our ability to observe any true differences between arms. Nevertheless, these findings indicate that both interventions were highly satisfactory to parents.
+
+In this study, higher intensity MD/DO clinicians were significantly less likely to prescribe inappropriate antibiotics than their MD/DO counterparts in the lower intensity arm. The reverse was true for the CPNP/APRN clinicians. The reasons for this difference among MD/DO clinicians are unclear and we likely have too few CPNP/APRN clinicians to draw any definitive conclusions about this subgroup, but there are data to support the notion that CPNP/APRN are simply more likely to adhere to guidelines41 so the educational training provided in both arms was likely sufficient to ensure similar low rates of inappropriate prescribing among CPNP/APRNs. Future studies should continue to explore difference in response to intervention between different types of clinicians.
+
+The lack of a statistically significant or clinically meaningful main effect may indicate that shared decision-making and the other communication factors targeted by the higher intensity intervention were not as strongly related to inappropriate prescribing as had been expected. This may indicate a cultural shift in parental expectations and/or clinician comfort in withholding unnecessary antibiotics, challenging the relevance of early literature to the social and communication dynamics at play today. It may be that the antibiotic education training for clinicians in both arms and study video that significantly reduced parents' desires for an antibiotic22 might have been enough to make a meaningful impact on prescribing. Other recent studies have found success focusing on clinician education about appropriate antibiotic prescribing and the effects of peer comparison.37 40 Clinician education interventions may be sufficient to yield long-term benefits, as parental expectations for antibiotics continue to decrease from an overall cultural shift or from exposure to a high-quality parent education video like the one used in this study.
+
+## Limitations
+
+The overall low rate of inappropriate antibiotic prescribing across interventions and sites is encouraging, but our design did not allow us to draw conclusions about the role of either intervention in these lower rates compared with usual care. Future studies should target settings with high rates of inappropriate prescribing. Higher intensity intervention clinicians may not have been given a sufficient 
+'dose' of the training. Lack of a control group or baseline antibiotic prescribing information limits our ability to understand how the rates of inappropriate prescribing we observed in the two intervention arms differs from usual practice at the study sites.
+
+## Conclusion
+
+Implementing evidence-based clinician and parent interventions to improve antibiotic prescribing can be acceptable to clinicians and parents and feasible in both academic and private paediatric outpatient settings. Clinician education coupled with high-quality parent education delivered via video may be sufficient to yield low inappropriate antibiotic prescribing rates.
+
+Author affiliations 1Health Services and Outcomes Research, Children's Mercy Hospitals and Clinics, Kansas City, Missouri, USA
+2School of Medicine, University of Missouri - Kansas City, Kansas City, MO, USA
+3School of Pharmacy, University of Missouri - Kansas City, Kansas City, MO, USA 4Sunflower Medical Group, Kansas City, Kansas, USA
+5General Academic Pediatrics, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA
+6RAND Corporation, Santa Monica, California, USA 7Nuffield Department of Primary Health Care Sciences, University of Oxford, Oxford, UK
+8Division of Infectious Diseases, Department of Pediatrics, Washington University in St. Louis, St. Louis, MO, USA
+9Pediatric Infectious Diseases, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA Twitter Christopher C Butler @ChrisColButler and Angela L Myers @DrMyersKC Acknowledgements Research reported in this publication was supported through a Patient-Centered Outcomes Research Institute (PCORI) Program Award (CDR1507-31759). All statements in this report, including its findings and conclusions, are solely those of the authors and do not necessarily represent the views of the Patient-Centered Outcomes Research Institute (PCORI), its Board of Governors or Methodology Committee. The authors wish to acknowledge the contributions to this study made by parent and child stakeholders, our Community Advisory Board members, clinical stakeholders at Children's Mercy Primary Care Clinics, Heartland Clinics, Alexander Mackenzie, Kirsten B. Delay, Sarah Schlachter, Areli Ramphal and Robert Finuf. Contributors KG conceptualised, designed and oversaw the study, designed and implemented the parent education and clinician communication training, designed and directed all study procedures and materials, directed the data analysis and interpretation of results, drafted the initial manuscript, and reviewed and revised the manuscript. EAH contributed to the design of the study, facilitated recruitment, intervention delivery and data collection, contributed to data analysis and interpretation of results, led qualitative data analysis and interpretation of results, drafted sections of the initial manuscript, and critically reviewed the manuscript for important intellectual content. BRL contributed to the design of the study, conducted the data analysis, contributed to the interpretation of results, drafted sections of the initial manuscript, and critically reviewed the manuscript for important intellectual content. AB-E and CB contributed to the design of all aspects of the study, co-led the engagement with the Community Advisory Board and critically reviewed the manuscript for important intellectual content. KP and EDdM contributed to the design study procedures, conducted recruitment and data collection, conducted the analysis and interpretation of qualitative results, drafted sections of the initial manuscript, and critically reviewed the manuscript for important intellectual content. DY, KW, SL and CCB contributed to the design of the study and critically reviewed the manuscript for important intellectual content. JGN and ALM designed the study, designed and implemented the ARTI diagnosis and treatment training, conducted the blind review of the EMR and critically reviewed the manuscript for important intellectual content. KG is responsible for the content as guarantor of the manuscript accepting full responsibility for the work and/or the conduct of the study. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.
+
+Funding Research reported in this publication was supported through a PatientCentered Outcomes Research Institute (PCORI) Program Award (CDR-1507-31759).
+
+Competing interests None declared. Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. Patient consent for publication Not applicable. Ethics approval This study involves human participants and was approved by Children's Mercy Hospital Pediatric Institutional Review Board (\#16060466). Participants gave informed consent to participate in the study before taking part.
+
+Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available on reasonable request. 
+
+Deidentified data limited to visit-based inappropriate prescribing rates, patient characteristics and dummy variables for clinic site will be shared. No protected health information will be shared. Data will be provided as a comma-separated values file with a data dictionary defining all variables included in the file and will be transferred via a secure file transfer protocol after establishing a data use agreement. No additional data or codes will be made available. The data will be made available after publication of the primary studies to researchers who provide a detailed methodologically sound proposal. Proposals should be submitted to KG (
+kgoggin@cmh.edu).
+
+Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iDs Kathy Goggin http://orcid.org/0000-0002-8679-9911 Emily A Hurley http://orcid.org/0000-0003-1806-075X Christopher C Butler http://orcid.org/0000-0002-0102-3453
+
+## References
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medical/md_images/PMC10367964/PMC10367964.md

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+# Rates Of Emotional Disturbance Among Children In Foster Care: Comparing Federal Child Welfare Data And Medicaid Records In Two States
+
+Child Maltreatment 2024, Vol. 29(1) 8–13
+© The Author(s) 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/10775595221118931 journals.sagepub.com/home/cmx Lindsey Palmer1,2, Sarah Font1,3, Toria Herd1, John Prindle2 and Emily Putnam-Hornstein2,4
+
+## Abstract
+
+The purpose of this study is to estimate the rate of emotional disturbance (ED) among children in foster care and assess the validity of the national foster care census data (AFCARS) measure of ED. This study used linked child protection and Medicaid records from 2014 and 2015, for the states of California and Wisconsin, as well as data from AFCARS, a federal population census of children in foster care which states are mandated to contribute to. ED is defined by AFCARS and includes an array of mental and behavioral health diagnoses. According to AFCARS, 13% of CA children in foster care and 15% of WI children in foster care had an ED, whereas Medicaid claims produce rates of 45% and 48%, respectively. Rates of ED among children in congregate care were underestimated by 43–46 percentage points, with substantial proportions having diagnoses of disruptive behavioral disorders. Despite the AFCARS ED measure being cited in congressional testimonies and its wide use in research, results from this study suggest that the AFCARS ED estimates are an unreliable metric for use in research, policy, or practice.
+
+## Keywords
+
+adoption and foster care reporting system, child welfare, foster care, congregate care, mental health Despite consensus that youth in foster care face elevated risks of mental illness compared with the general population
+(McMillen et al., 2005; Steele & Buchi, 2008), estimated rates vary considerably. Many researchers, as well as federal evaluation protocols (Administration for Children and Families, 2014), rely on the Adoption and Foster Care Reporting System (AFCARS), a federal population census of children in foster care to which states are mandated to contribute. AFCARS has a single indicator of "emotional disturbance" (ED), defined based on caseworker report of whether the child has one or more qualifying diagnoses, such as posttraumatic stress disorder or depression (AFCARS
+Foster Care Annual File Codebook, 2021). There have been, however, few attempts to verify the quality or reliability of the AFCARS ED measure, despite its continued relevance and use in research, evaluation, and policy (Finster &
+Norwalk, 2021; U.S. Children's Bureau, 2015).
+
+A recent example of the importance of validating estimated rates of ED comes from the Family First Prevention Services Act of 2018 (FFPSA) (Bipartisan Budget Act, 2018). Preceding the enactment of this law, Congressional hearings discussed the potential harms and overuse of congregate care. In the US, congregate care is typically used for adolescents and is supposed to be a placement option only where a family (kin or non-relative) foster home is unable to meet a child's needs. Citing a U.S. Children's Bureau analysis of AFCARS (U.S. Children's Bureau, 2015), it was asserted that only 21% of children living in congregate care –
+the vast majority of whom are teens— had an ED (No Place to Grow up: How to Safely Reduce Reliance on Foster Care Group Homes; Senate Hearing 114-273, 2015, p. 26). This estimate is 6 percentage points higher than the general population prevalence of depression, anxiety, or ADHD among children 6–11 years (15%) and equal to the general population prevalence for children ages 12–17 (21%)
+1Department of Human Development and Family Studies, Pennsylvania State University, University Park, PA, USA
+2Children's Data Network, University of Southern California, Los Angeles, CA, USA
+3Department of Sociology and Criminology, The Pennsylvania State University 4University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
+Corresponding Author: Lindsey Palmer, The Pennsylvania State University, University Park, PA
+16802, USA.
+
+Email: lnpalmer@psu.edu
+(Whitney & Peterson, 2019). The suggestion that rates of ED
+in the general population are no different than for youth in the most restrictive form of foster care should be viewed skeptically given abundant evidence linking exposure to child maltreatment - the primary antecedent to foster care entry—with mental health problems (Norman et al., 2012) as well as studies documenting that children in foster care have elevated rates of ED (McMillen et al., 2005; Steele & Buchi, 2008; Turney & Wildeman, 2016). Nevertheless, FFPSA, as enacted, includes provisions intended to drastically reduce use of congregate care by limiting federal funding for such placements. These changes have further incentivized states to transition a greater share of foster care youth into traditional foster or kinship placements. Although it is optimal to place children in the least restrictive setting in which their needs can be met, FFPSA came with little funding to improve recruitment, retention, or training of foster homes for youth with behavioral and emotional challenges. Efforts to scale down congregate care are motivated by several factors, including concerns that lack of a family environment is developmentally harmful to youth and the high cost of congregate care. Yet, the assertion that large proportions of children in congregate settings had no clinical justification for a restrictive placement motivated support for this legislation (Larson, 2018).
+
+An accurate understanding of children's mental health in foster care is crucial, both for the development of health policy and appropriate allocation of resources. Thus, this study sought to: (1) estimate rates of ED as defined by the AFCARS
+codebook, overall and by diagnosis group, from Medicaid claims records of children in care in Wisconsin and California;
+(2) compare the Medicaid claims-derived ED estimates to those reported in AFCARS; and (3) compare ED estimates for youth in congregate care versus family foster care settings.
+
+## Methods Data And Sample
+
+We draw primarily on foster care records from state child welfare information systems and Medicaid claims data in Wisconsin and California. Wisconsin's data were accessed via the Wisconsin Administrative Data Core (WADC) at the University of Wisconsin Madison and California's data were accessed from the Children's Data Network at the University of Southern California; both repositories hold data sharing agreements with the involved state entities. Data were probabilistically linked within state using available identifiers and then stripped of all direct identifiers prior to analysis. For comparison, we also draw upon the AFCARS child files, obtained through the National Data Archive on Child Abuse and Neglect (NDACAN), which are an annual national census of children in foster care.
+
+For the CA, WI, and AFCARS datasets, we selected the following analytic samples: children in foster care between January 1, 2014 through September 30, 2015, who entered care prior to their 18th birthday and spent at least 30 consecutive days in foster care. In AFCARS, we excluded children with missing or invalid information on birthdate or date of foster care entry.
+
+We then produced two datasets for each state: (1) linked foster care and Medicaid claims data (WI-Medicaid Linked and CAMedicaid Linked); and (2) records submitted to the federal AFCARS child files (WI-AFCARS and CA-AFCARS). A
+description of these datasets is shown in Supplementary Appendix A, along with the national AFCARS.
+
+## Measures
+
+AFCARS data files include an aggregate binary indicator of ED that is reported by the child's caseworker. A value of 1, according to the AFCARS codebook, should be assigned if the child has diagnoses that meet the definition of an ED,
+based on the Diagnostic and Statistical Manual of Mental Disorders IV (American Psychiatric Association, 2000). This list was mapped onto the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9)
+diagnostic codes that are used in medical claims. We create a narrow version, reflecting only the diagnoses stated explicitly in the AFCARS codebook, and an extended definition, which included all listed diagnoses in addition to conceptually relevant but not listed diagnoses (see Supplementary Appendix B). ED is equal to 1 if a child received any Medicaid-billed health care services billed under a qualifying diagnosis while in foster care and during the observation period. A qualifying claim could stem from inpatient or outpatient services provided by any health professional (e.g.,
+pediatrician, therapist, psychiatrist). Medicaid is a promising source of data on diagnoses given that it covers an estimated 99% of children in foster care (Libby et al., 2006; Medicaid and CHIP Payment and Access Commission, 2015).
+
+Using the Medicaid-linked data, we also categorized ED
+conditions into eight groups (World Health Organization, 2019): (1) Adjustment disorders, including acute reaction to stress and post-traumatic stress disorder; (2) Anxiety disorders; (3) Mood disorders; (4) Attachment disorders; (5) Autism; (6) Attention Deficit disorders; (7) Conduct disorders; and (8) other. The other category includes diagnoses that are too rare to generate estimates for individually and diagnoses that do not fall into any of the other groups. Appendix B lists the diagnoses by group and indicates inclusion in the narrow definition or only the definition only.
+
+Additionally, data from our linked state datasets are used to characterize the rates of any and specific types of EDqualifying diagnoses for congregate care placements and non-congregate care placements. Congregate care is defined as any group home or residential facility placement and family setting is defined as any kin or non-kin foster family home, pre-adoptive or guardian placement. As congregate care placements are rare for children under 9 years of age (<2% for children 0–8 years vs. 24% for children 9–17 years),
+
+![2_image_0.png](2_image_0.png)
+
+diagnostic breakdowns by placement type include only children aged 9–17 years. Placement type was indicated based on the child's last placement within the time frame.
+
+Descriptive data summaries are provided to illustrate and compare the percentage of children in foster care with an ED
+diagnosis overall and by state using both AFCARS and Medicaid derived estimates. Additional descriptive statistics are provided to outline ED estimates by age group (0–8 years; 9–17 years), type of diagnosis and placement setting.
+
+## Results
+
+Figure 1 displays the rates of ED identified in the AFCARS data files and those estimated from Medicaid-linked data. Rates of ED in CA and WI AFCARS are 13% and 15%, respectively. In both states, rates are very similar to that reported in AFCARS
+for the nation overall (15%), suggesting that neither state is an outlier with respect to AFCARS reporting. There is a 32percentage point gap for reported ED diagnoses between AFCARS and state data for CA (13% vs. 45%) and a 33-point difference between AFCARS and state data for WI (15% vs.
+
+48%). Even when using the conservative (narrow) ED definitions there remains a three-fold difference between AFCARS
+and state data estimates (CA 13% vs. 39%; WI 15% vs. 45%).
+
+In both states, majorities of children ages 9+ have an ED
+regardless of setting (Table 1). However, children in congregate care have significantly higher rates of ED compared to children in family settings. Again, rates of diagnoses for children in both family-like and congregate care settings are substantially lower in AFCARS than in Medicaid claims.
+
+Rates of disruptive disorder diagnoses are twice as high in WI and nearly three times higher in CA for children in congregate care compared to children placed in a family-like setting. In CA, one in four children in congregate care has an ADHD
+diagnosis, more than double the rate among children in a family-like setting, and in WI nearly half (45%) of children have an ADHD diagnosis. There is a 34-percentage point
+(CA) and 21 percentage point (WI) difference in proportion with mood disorder diagnoses between children in family-like versus congregate care settings. A minority of children eight and under had an ED, with Medicaid derived rates pointedly higher than those in AFCARS (Medicaid: CA 33%, WI 28%;
+AFCARS: CA 4%, WI 5%).
+
+## Discussion
+
+AFCARS data are used for federal performance monitoring of foster care systems under the Child and Family Service Reviews (Administration for Children and Families, 2014), in annual reports to Congress (U.S. Department of Health and Human Services, 2016), and to inform policy debates (No Place to Grow up: How to Safely Reduce Reliance on Foster Care Group Homes; Senate Hearing 114-273, 2015). States have long cited problems providing adequate care for children with mental and behavioral health concerns (Government Accountability Office, 2015), but their submitted AFCARS
+data imply that a small minority of children in their care have an ED, broadly defined to include most psychiatric diagnoses.
+
+This study leveraged linked foster care and Medicaid records in two states to ascertain the rates and types of psychiatric diagnoses among children in care and to evaluate whether AFCARS is a reliable source of information on ED prevalence. We find state estimates using linked Medicaid claims data - which largely align with the broader research (McMillen et al., 2005; Turney & Wildeman, 2016) - are three times higher than AFCARS estimates, signaling that AFCARS ED estimates are an unreliable metric for use in research, policy, or practice.
+
+| Table 1. Emotional Disturbance Estimates for Foster Care Population for 8 and Under and 9+ by State and Final Placement Setting. Children 0–8 years Children 9+ Children 9+ All settings Family settings Congregate care CA WI CA WI CA WI AFCARS data Caseworker recorded ED indicator 4% 5% 21% 25% 35% 35% Medicaid-linked data Constructed ED indicator - extended definition 33% 28% 56% 69% 78% 83% Diagnosis group Adjustment 19% 20% 34% 43% 39% 33% Anxiety 3% 3% 9% 14% 16% 19% Mood 2% 3% 23% 28% 57% 49% Attachment 1% 2% 1% 4% 2% 6% Autism 1% 2% 1% 3% 4% 7% ADD/ADHD 4% 9% 12% 31% 25% 45% Disruptive/conduct 5% 6% 12% 19% 36% 40% Other condition 10% 3% 6% 8% 22% 13% Note. Population of children 0–8 years in CA during study time frame was 53,034 and WI was 7,162. Population of children 9–17 years in foster family (kin or   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Note. Population of children 0–8 years in CA during study time frame was 53,034 and WI was 7,162. Population of children 9–17 years in foster family (kin or non-relative) settings in CA was 36,472 and WI was 4,365. Population of children 9–17 years in congregate care in CA was 6,003 and in WI was 1,555.
+
+Note. CA = California. WI = Wisconsin. ED = emotional disturbance. ADD/ADHD = Attention Deficit Disorder/Attention Deficit Hyperactivity Disorder.
+
+Low reliability of the AFCARS ED measure may have several causes. First, states have an array of medical information available to them for children in their care and need not rely on this specific data field for decision-making and evaluation.
+
+Thus, the accuracy of this specific field is inconsequential to agencies and may not be recorded at all or updated over time as children's mental health status changes or new information becomes available. Second, "emotionally disturbed" is a pejorative label that caseworkers may be reluctant to use, especially if a child's ED conditions are mild or adequately managed with current treatments. Although we found that most children in congregate care (78% in CA and 83% in WI) have one or more claims indicating a mental health diagnosis, it is challenging to ascertain whether their condition was severe enough to temporarily or permanently preclude placement in a familylike (less restrictive) environment. We note, however, that more than one-third of children in congregate care had a diagnosis of conduct, oppositional defiance, or impulse control disorder –
+conditions characterized by externalizing behaviors that are strongly predictive of placement disruption (Konijn et al.,
+2019). Thus, states may face serious challenges to moving children from congregate care to less-restrictive environments.
+
+Understating the prevalence of mental health needs in the congregate care population may also lead states to under-invest in the resources needed for children to safely and stably step down from or avoid congregate care.
+
+## Limitations
+
+Despite the high numbers of children overall, and especially older children, with diagnoses and claims for mental health services, this study may nevertheless undercount the incidence of ED. First, the probabilistic linking strategies used by both states may fail to link children where there were errors in their identifying information, such that they would be incorrectly identified as having no billed services during the time period. Second, some children may not be receiving mental health care despite a need (Shin, 2005) or may be receiving mental health care that is not billed through insurance (Department of Healthcare Services, 2020). Because both limitations mean that our Medicaid-based estimates are conservative (i.e., provide a lower-bound estimate of ED), we are potentially understating how unreliable the AFCARS
+estimates are. Additionally, this study is limited by an inability to characterize the onset, duration, or severity of ED
+and the extent to which a child's symptoms were effectively managed by the services they received. Future research may consider whether caseworkers' awareness of children's ED diagnoses affects the quality or intensity of services provided.
+
+## Implications
+
+The current study underscores shortcomings of data captured in state administrative child welfare systems and the significant inaccuracy of national statistics on mental health conditions among children in foster care. The implications of using inaccurate estimates to inform policy can be profound as exemplified by the congressional hearings on FFPSA and congregate care where inaccurate data contributed to the reduction in congregate care without investment in the recruitment, retention and training of foster families equipped to address the emotional and behavioral needs to youth stepping down from restrictive care. More broadly, however, children's mental and behavioral health both affects and is affected by many core foster care performance objectives and outcomes, including safety, stability, non-restrictive care, and permanency (Aarons et al., 2010; James et al., 2006; Rubin et al.,
+2007). Yet, there is currently no regular and systematic collection of this information. Prior studies have reported on rates of mental health conditions among children in foster care
+(McMillen et al., 2005; Steele & Buchi, 2008), but these data are typically confined to specific regions or points in time making generalizability and examinations of trends difficult.
+
+The lack of reliable information related to the mental health needs of children in foster care make it difficult to understand trends over time and accurately evaluate or compare system performance in providing quality care for children with ED,
+therefore, improving sound measurement of ED in administrative data is imperative. Given that all children in foster care are eligible for Medicaid, cross system information linking on a national level (AFCARS and Medicaid) could be utilized to improve measurement quality.
+
+## Declaration Of Conflicting Interests
+
+The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
+
+## Funding
+
+This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development grant R21HD091459 at the University of Wisconsin-Madison and P50HD096719 grant at the University of North Carolina Chapel Hill. Additional support was received in the form of research grant (R01HD095946), training grant
+(T32HD101390), and infrastructure grants (P50HD089922 and P2CHD041025) at Pennsylvania State University. Data infrastructure support for California records was provided by First 5 LA, the Conrad N. Hilton Foundation, and the Heising-Simons Foundation. The authors of this article are solely responsible for the content therein. The authors would like to thank the Wisconsin Department of Children and Families and Department of Health Services, as well as the California's Department of Social Services and Department of Healthcare Services for the use of data for this analysis, but these agencies do not certify the accuracy of the analyses presented. All conclusions drawn are those of the authors alone and do not necessarily reflect those of any agency partner or funding entity.
+
+## Orcid Ids
+
+Lindsey Palmer  https://orcid.org/0000-0001-5633-9976 Sarah Font  https://orcid.org/0000-0002-4022-5517 John Prindle  https://orcid.org/0000-0002-2641-5138
+
+## Supplemental Material
+
+Supplemental material for this article is available online.
+
+## References
+
+Aarons, G. A., James, S., Monn, A. R., Raghavan, R., Wells, R. S., &
+Leslie, L. K. (2010). Behavior problems and placement change in a national child welfare sample: A prospective study. Journal of the American Academy of Child & Adolescent Psychiatry, 49(1), 70–80. https://doi.org/10.1016/j.jaac.2009.09.005 Administration for Children, & Families (2014). Final notice of statewide data indicators and national standards for Child and Family Services Reviews. Children's Bureau.
+
+AFCARS Foster Care Annual File Codebook (2021). National data archive on child Abuse and Neglect. AFCARS Foster Care Annual File Codebook. https://www.ndacan.acf.hhs.gov/
+datasets/pdfs_user_guides/afcars-foster-care-file-codebook.
+
+pdf American Psychiatric Association (2000). Diagnostic and statistical manual of mental disorders: DSM-IV-TR. American Psychiatric Association.
+
+Bipartisan Budget Act, Pub. L. No. 115–123 (2018). https://www.
+
+congress.gov/bill/115th-congress/house-bill/253.
+
+Department of Healthcare Services (2020). Mental Health Services Act. Department of Healthcare Services. https://www.dhcs.ca.
+
+gov/services/mh/Pages/MH_Prop63.aspx Finster, H. P., & Norwalk, K. E. (2021). Characteristics, experiences, and mental health of children who re-enter foster care. Children and Youth Services Review, 129(1), Article 106165. https://doi. org/10.1016/j.childyouth.2021.106165 Government Accountability Office (2015). HHS could do more to support states' efforts to keep children in family-based care. (GAO-16-85). https://www.gao.gov/assets/gao-16-85.pdf James, S., Leslie, L. K., Hurlburt, M. S., Slymen, D. J., Landsverk, J.,
+Davis, I., Mathiesen, S. G., & Zhang, J. (2006). Children in outof-home care: Entry into intensive or restrictive mental health and residential care placements. Journal of Emotional and Behavioral Disorders, 14(4), 196–208. https://doi.org/10.1177/
+10634266060140040301 Konijn, C., Admiraal, S., Baart, J., van Rooij, F., Stams, G.-J.,
+Colonnesi, C., Lindauer, R., & Assink, M. (2019). Foster care placement instability: A meta-analytic review. Children and Youth Services Review, 96(1), 483–499. https://doi.org/10.1016/
+j.childyouth.2018.12.002 Larson, J. B. (2018). Text - H.R.1892 - 115th congress (2017-2018.
+
+Bipartisan Budget Act of 2018 (2017/2018) [Legislation] https://www.congress.gov/bill/115th-congress/house-bill/1892/
+text Libby, A. M., Kelleher, K. J., Leslie, L. K., O'connell, J., Wood, P. A.,
+Rolls, J. A., & Landsverk, J. (2006). Child welfare systems policies and practices affecting medicaid health insurance for children. Journal of Social Service Research, 33(2), 39–49.
+
+https://doi.org/10.1300/J079v33n02_04 McMillen, J. C., Zima, B. T., Scott, L. D., Auslander, W., Munson, M., Ollie, M., & Spitznagel, E. (2005). Prevalence of psychiatric disorders among older youths in the foster care system. Journal of the American Academy of Child & Adolescent Psychiatry, 44(1), 88–95. https://doi.org/10.1097/01.chi.0000145806. 24274.d2 Medicaid and CHIP Payment and Access Commission (2015). Intersection of Medicaid and child welfare (p. 33). Medicaid and CHIP Payment and Access Commission.
+
+No place to grow up: How to safely reduce reliance on foster care group homes; Senate Hearing, 114-273, 114th Congress, 1(2015).
+
+Norman, R. E., Byambaa, M., De, R., Butchart, A., Scott, J., & Vos, T. (2012). The long-term health consequences of child physical abuse, emotional abuse, and neglect: A systematic review and meta-analysis. PLoS Medicine, 9(11), Article e1001349. https://
+doi.org/10.1371/journal.pmed.1001349 Rubin, D. M., O'Reilly, A. L., Luan, X., & Localio, A. R. (2007). The impact of placement stability on behavioral well-being for children in foster care. Pediatrics, 119(2), 336–344. https://doi. org/10.1542/peds.2006-1995 Shin, S. H. (2005). Need for and actual use of mental health service by adolescents in the child welfare system. Children and Youth Services Review, 27(10), 1071–1083. https://doi.org/10.1016/j.
+
+childyouth.2004.12.027 Steele, J. S., & Buchi, K. F. (2008). Medical and mental health of children entering the Utah foster care system. Pediatrics, 122(3), e703–e709. https://doi.org/10.1542/peds.2008-0360 Turney, K., & Wildeman, C. (2016). Mental and physical health of children in foster care. Pediatrics, 138(5), Article e20161118. https://doi.org/10.1542/peds.2016-1118 U.S. Children's Bureau (2015). A national look at the use of congregate care in child welfare. https://www.acf.hhs.gov/cb/
+resource/congregate-care-brief U.S. Department of Health and Human Services (2016). Child welfare outcomes 2016: Report to congress (p. 95). Retrieved from: https://www.acf.hhs.gov/sites/default/files/cb/cwo2016.
+
+pdf\#page=57 Whitney, D. G., & Peterson, M. D. (2019). US national and statelevel prevalence of mental health disorders and disparities of mental health care use in children. JAMA Pediatrics, 173(4), 389–391. https://doi.org/10.1001/jamapediatrics.
+
+2018.5399 World Health Organization (2019). International classification of Diseases, Ninth Revision. CDC. https://www.cdc.gov/nchs/icd/ icd9.htm

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medical/md_images/PMC10407207/PMC10407207.md

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+Contents lists available at ScienceDirect 
+
+![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_4.png](0_image_4.png)
+
+![0_image_5.png](0_image_5.png)
+
+![0_image_6.png](0_image_6.png)
+
+Evaluating the cost-effectiveness of low-level HBV DNA screening 
+
+![0_image_2.png](0_image_2.png)
+
+in occult hepatitis B infection donors: A study from Shandong Blood Center, China Jianfeng Chen *****, Zili Ma, Dandan Wu, Qi Zuo, Fengtian Wang ****, Chen Xiao ***, Fuqiang Chen **, Peng Li * 
+Blood Center of Shandong Province, No 22 Shanshi East Road, Lixia District, Jinan, Shandong, China 
+
+| ARTICLE INFO  Keywords:  Nucleic acid technology  Occult HBV infection  Transfusion transmitted HBV  Hepatitis B virus   |
+|--------------------------------------------------------------------------------------------------------------------------|
+
+| ABSTRACT   |
+|------------|
+
+Objective: This study aimed to assess the efficacy of individual-donation nucleic acid testing (IDNAT) in detecting occult hepatitis B virus infection (OBI) among *anti*-HBc positive blood donors, compared to minipool nucleic acid testing (MP-NAT). 
+
+Methods: The present study analyzed data from the Shandong Blood Center in China during the period from January 2018 to June 2022, where HBsAg-negative blood donors were screened using the 6-sample minipool nucleic acid testing (6-sample MP NAT) method. NAT-positive samples underwent subsequent *anti*-HBc and *anti*-HBs testing. Approximately 5000 samples that passed the nucleic acid mixing test were randomly selected for *anti*-HBc testing, and over 100 anti-HBc positive samples underwent individual donor nucleic acid testing (ID-NAT). Any HBV 
+DNA positive samples detected by ID-NAT were subsequently confirmed using alternative nucleic acid testing methods. Results: Among 220,445 HBsAg-negative blood donors, the positivity rate of HBV DNA detection using the 6-sample minipool nucleic acid testing (MP NAT) method was found to be 0.031% (69/ 220,445). Of the 67 HBV DNA positive samples, 55 (82.09%) and 25 (37.31%) were found to be positive for *anti*-HBc and *anti*-HBs, respectively, using the supplementary chemiluminescent microparticle immunoassay (CMIA). Among the 4797 HBsAg-negative/MP NAT-negative samples, 909 (18.95%) tested positive for *anti*-HBc. Further NAT testing was performed on 164 arbitrarily selected *anti*-HBc-positive/MP HBV DNA-negative samples, revealing a HBV DNA positivity rate of 1.22% (2/164). 
+
+Conclusion: Using individual donation nucleic acid testing can significantly increase the detection rate of occult hepatitis B virus infection in *anti*-HBc-positive blood donors, resulting in a detection rate of 0.22% (1.22 × 0.1895). This rate is 8.10 times higher than the detection rate achieved by mixed testing methods (0.031%) [calculated as (0.22 + 0.031)/0.031]. Therefore, it is recommended to perform single HBV DNA testing on *anti*-HBc-positive blood donors, discard plasma 
+* Corresponding author. 
+
+** Corresponding author. *** Corresponding author. 
+
+**** Corresponding author. 
+
+***** Corresponding author. 
+
+E-mail addresses: guoguo8818@163.com (J. Chen), wangft82@163.com (F. Wang), 448782241@qq.com (C. Xiao), cfqcfqcfq@163.com 
+(F. Chen), lipengmm123@163.com (P. Li). 
+
+https://doi.org/10.1016/j.heliyon.2023.e18609 Received 10 March 2023; Received in revised form 14 July 2023; Accepted 24 July 2023 Available online 25 July 2023 2405-8440/© 2023 The Authors. 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/).
+
+## 1. **Introduction** 2. **Materials And Methods** 2.1. Blood Samples 2.2. Elisa Screening 2.3. Nucleic Acid Amplification Testing (Nat) Assays
+
+To minimize the residual risk of transfusion-transmitted infections, several blood centers in China initiated a pilot program of nucleic acid testing (NAT) in early 2010. This screening method is more sensitive than the conventional methods for detecting hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus 1/2 (HIV 1/2). By 2015, China had achieved full coverage of NAT screening for all unpaid blood donors. To control costs, a common practice in China is to perform NAT screening by pooling 6 or 8 blood samples together. Liu et al calculated the total number of NAT screenings performed in China using NAT reagents from 2010 to 2015, which utilized eight different reagents [1]. Among the top four reagents utilized, the Procleix Ultrio Assay had a utilization rate of 40.19% for single-sample testing, while Roche's 6-pooling had a utilization rate of 33.17% (Cobas Taqscreen MPX at 21.74%, Cobas Taqscreen MPX Test, Version 2.0 at 11.43%), Haoyuan's 8-pooling had a utilization rate of 12.21%, and Kehua's 8-pooling had a utilization rate of 12.06%. The second, third, and fourth reagents were all based on pooling methods, and their combined utilization rate exceeded 57%. Although the latter three NAT systems can also be used for single-sample testing, the cost is 6–8 times higher than that of the pooling method. The sensitivity of pooling analysis is much lower than that of single-sample testing, for example, Roche's NAT system has a limit of detection of 2.3 IU/mL for single-sample testing, which would translate to approximately 14 IU/mL for 6-pooling testing. Currently, the blood supply and demand in China are tight, with a high prevalence of previous HBV infection, making it difficult to eliminate *anti*-HBc positive blood. In addition, NAT pooling screening cannot detect occult HBV infection (OBI) 
+with low viral load (2.3–14 IU/mL), which poses a risk of infection to susceptible individuals. We evaluated the proportion of these low viral load HBV infections in eligible blood donors and the screening cost, and discussed the optimization of screening strategies by adding *anti*-HBc testing. It is worth noting that this study is part of a larger body of research on HBV DNA testing of blood donors in China. Numerous similar studies have been conducted and published previously. However, this study focuses specifically on the cost of testing a sample with low levels of HBV DNA, an area that has been rarely reported on in previous studies. 
+
+From January 1st, 2020 to June 30th, 2022, a total of 220,445 blood donor samples were collected from the Shandong Blood Center and tested using the Roche NAT system. Prior to sample collection, all donors provided informed consent for blood donation and underwent a health history questionnaire, physical examination, ALT rapid test, ABO blood group test, HBsAg rapid test nonresponsiveness test, and hemoglobin test to ensure their eligibility. Blood samples were stored in a refrigerator at a temperature of 2~8 ◦C after collection. Nucleic acid samples were centrifuged within 4 h, while ELISA and blood group samples were centrifuged within 12 h. The ALT rate method, blood group forward and reverse typing, ELISA, and NAT tests were completed in the laboratory within 72 h. 
+
+The blood samples were assayed for HBsAg, *anti*-TP, *anti*-HCV, and HIV/*anti*-HIV through the use of ELISA reagents obtained from two distinct manufacturers. Any samples that were negative with either ELISA reagent or positive with one reagent underwent nucleic acid testing (NAT). 
+
+In the initial detection mode of Roche NAT, 167 μL of plasma were extracted from 6 tubes using the STAR sampling instrument and placed into an S-tube, resulting in a total volume of 1.002 mL as a reaction pool. The plasma was then extracted and amplified using the Roche Cobas s 201 system (Cobas TaqScreen MPX test, version 2.0, USA). If the mixed sample tube showed a positive result, 1 mL of plasma was taken from each corresponding tube and tested individually, with the result of the single sample test serving as the final result of routine testing. Roche analyzed the WHO viral (NIBSC code 97/746) standard in single-test mode with a detection volume of 1.0 mL, and the results showed a detection sensitivity of 2.3 IU/mL (average 95% LOD). In addition, 1 IU/mL is equivalent to 5.6 copies/mL. In Roche nucleic acid testing, the critical criterion for determining a negative result is a cycle threshold (CT) value exceeding 60, indicating the absence of amplification within the specified number of cycles. To ensure quality control, we incorporated three weak positive control sera in each testing batch, including HBV DNA, HCV RNA, and HIV RNA control materials. The concentration of the HBV DNA control material was maintained at 50 IU/mL. Over the course of 19 testing batches conducted in 2023, the average CT value was determined to be 33.5 (Due to a six-fold dilution of the 50 IU/ML control material, the actual concentration is 8.33 IU/ML). Notably, for the negative control, the CT value surpassed 60, signifying the absence of amplification. 
+
+## 2.4. Anti-Hbc Screening
+
+Some samples with qualified test results were randomly selected for *anti*-HBc ELISA (Wantai Diagnostics, Beijing, China) screening test,all *anti*-HBc positive samples were retested for *anti*-HBc by another domestic EIA kit (InTec PRODUCTS, INC),only samples reactive to both tests were considered as *anti*-HBc positive samples. 
+
+## 2.5. Nat Single-Sample Testing
+
+Random selection of *anti*-HBc positive samples for Roche NAT single-sample testing. 
+
+## 2.6. Nat Test Result Review
+
+Roche single-sample test positive samples were further reviewed in both single-sample and mixed-sample modes using the Huayimei detection system (1.2 mL, LOD: 4.2 IU/mL for HBV DNA, 95% confidence interval: 2.0–2.8 IU/mL). A positive result in either Huayimei test pattern was considered positive, a negative result was considered indeterminate, and an indeterminate result was considered negative, and such samples were not included in this study. 
+
+## 2.7. Nat Quantitative Test
+
+NAT positive samples were sent to Jinan KingMed Laboratory Center and subjected to quantitative detection of HBV DNA using the cobas 4800 system (Roche PCR-fluorescence method). 
+
+## 2.8. Cmia Testing
+
+We selected samples with HBV DNA mixed-sample mode positive and single-sample mode positive reactions, and performed HBsAg neutralization testing, HBsAg, *anti*-HBs, and *anti*-HBc CMIA testing using the Abbott Architect i2000 chemiluminescence detection system. Additionally, we repeated testing on some of the samples using HBsAg, *anti*-HBs, and *anti*-HBc CMIA reagents produced by Wantai (Beijing, China). 
+
+## 2.9. Statistical Analysis
+
+Statistical analyses were performed using SPSS 22.0 software. Differences between groups or years were assessed using the chisquare test, and statistical significance was defined as p < 0.05. 
+
+## 3. **Results** 3.1. Total Number Of Roche Hbv Nat Blood Donation Tests
+
+Between January 2018 and June 2022, a total of 220,445 samples from seronegative donors at the Shandong Blood Center were screened using Roche NAT. Over these five years, the positive detection rate of Roche HBV DNA has increased annually, from 0.023% 
+in 2018 to 0.062% in 2022, with an average annual detection rate of 0.031%. No significant differences were observed between groups 
+(χ2 = 6.784, p > 0.05) (Table 1). 
+
+## 3.2. Anti-Hbc Screening
+
+4797 samples with qualifying test results were randomly selected and subjected to additional testing with the *anti*-HBc ELISA test, which revealed a positive rate of 18.95% (Table 2). 
+
+| Roche HBV DNA test results(n = 220,445).  Number of test samples Number of positive pools of 6   | Number of HBV DNA-positive samples upon retesting individuals   | HBV DNA positive rate   |    |       |
+|----------------------------------------------------------------------------------------------------|-----------------------------------------------------------------|-------------------------|----|-------|
+| 2018                                                                                               | 56,016                                                          | 19                      | 13 | 0.023 |
+| 2019                                                                                               | 69,663                                                          | 39                      | 17 | 0.024 |
+| 2020                                                                                               | 53,245                                                          | 29                      | 19 | 0.036 |
+| 2021                                                                                               | 35,039                                                          | 33                      | 16 | 0.046 |
+| 2022                                                                                               | 6482                                                            | 7                       | 4  | 0.062 |
+| total                                                                                              | 220,445                                                         | 127                     | 69 | 0.031 |
+
+| HBV DNA test results of Roche (n = 4797).   | Number of test samples   | Number of anti-HBc positive cases   | The positive rate of anti-HBc   |
+|---------------------------------------------|--------------------------|-------------------------------------|---------------------------------|
+| College student                             | 1045                     | 47                                  | 4.50                            |
+| General population (non-college students)   | 3752                     | 862                                 | 22.97                           |
+| total                                       | 4797                     | 909                                 | 18.95                           |
+
+3.3. *NAT single-sample result* A random selection of 164 *anti*-HBc positive samples were tested using the Roche NAT single-test system, resulting in the detection of 3 samples positive for HBV DNA (Table 3). 
+
+## 3.4. Nat Recheck Testing
+
+Three Roche single-test reactive samples were retested using both the Huayimei system's 8-mixed tests and single-sample tests. One sample was positive in both single-sample mode and mixed-sample mode tests, another sample was positive only in single-sample mode test, and the third sample was non-reactive in both single and mixed tests and was deemed an indeterminate result, and therefore excluded from statistical analysis (Table 4). However, we still discussed the case of the third sample during our analysis. By conducting NAT single-sample testing on 164 HBV core-positive blood donors, two HBV DNA-positive donors were identified, resulting in a detection rate of 1.22% for HBV DNA low-level positive blood donors. 
+
+## 3.5. Hbv Dna Quantitative Testing
+
+The two Roche single-test reactive samples were sent to Jinan KingMed Medical Testing Center for analysis, and the results indicated that no HBV DNA was detected (Table 4). 
+
+## 3.6. Serological Characteristics Of Seronegative And Hbv Dna-Positive Blood Samples
+
+Out of the 164 samples that tested positive for *anti*-HBc, additional testing was conducted using ELISA and Abbott CMIA to assess anti-HBs levels. Results indicated that both *anti*-HBs and *anti*-HBc were present in 75.61% of the samples, while the rate of samples that tested positive for only *anti*-HBc was 24.39%. 
+
+## 3.7. Cmia Testing For Hbv Dna-Positive Blood Donors Of Mix Mode
+
+During the period of 2018–2022, 67 samples were identified as HBV DNA positive but ELISA negative. These samples underwent hepatitis B five-item testing at the National Clinical Laboratory (NCCL) using Abbott CMIA, and 25 of them underwent confirmatory testing using Wantai CMIA, with both yielding consistent results. To better understand the serological pattern of these samples, we evaluated the results and presented them in Table 5. The results showed that in the 67 HBV DNA positive samples, both HBsAg and HBsAg neutralization tests were negative in CMIA testing. However, 55 samples (82.09%) were positive for *anti*-HBc (OBI), while 12 samples (17.91%) were negative for *anti*-HBc (window period infection, WP). Among the 67 samples, 17 (25.37%) carried both *anti*HBc and *anti*-HBs, 38 (56.72%) only had *anti*-HBc, 8 (11.94%) only had *anti*-HBs, and 4 were completely negative. Table 6 shows the anti-HBs levels of the 67 HBV DNA positive samples. The results showed that 3 samples (4.48%) had *anti*-HBs levels greater than 200 IU/L, 7 samples (10.45%) had *anti*-HBs levels greater than 100 IU/L, and 19 samples (28.36%) had *anti*-HBs levels greater than 14 IU/ 
+L. 
+
+## 3.8. Cost Calculation
+
+The average cost to detect one HBV DNA positive sample using the NAT mixed test mode is 212,777 RMB. 
+
+The average cost of nucleic acid reagents for the detection of one HBV DNA positive sample in this study was 32,767 RMB (the number after the decimal point is ignored), plus the cost of *anti*-HBc ELISA reagents, totaling 35,017 RMB. 
+
+Cost of *anti*-HBc ELISA reagent: 75 RMB/box * 60 boxes = 4500 RMB. 
+
+Cost of detecting 1 HBV DNA positive sample (decimal numbers ignored): (4500 + 65,534)/2 = 35,017 (RMB). 
+
+Note: This article only calculates the basic costs of reagents and does not include additional costs such as positive and negative 
+
+| Single nucleic acid test results of 164 anti-HBc positive samples.  Number of anti-HBc positive samples Number of HBV DNA positive samples   | Detection rate of HBV DNA positive samples(%)   |    |      |
+|----------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------|----|------|
+| anti-HBs negative                                                                                                                            | 41                                                | 1  | 1.64 |
+| anti-HBs positive                                                                                                                            | 123                                               | 2  | 1.94 |
+| 4                                                                                                                                            |                                                   |    |      |
+
+| Table 4  Supplementary test results of positive HBV DNA single sample(n = 3).  Roche ID-NAT(CT  HYM ID-NAT(CT  Sample  Roche 6-  HYM 8-   | antiHBc          |         |          |          |      |                                            |                              |
+|-----------------------------------------------------------------------------------------------------------------------------------------------|----------|---------|----------|----------|------|--------------------------------------------|------------------------------|
+| number                                                                                                                                        | MP       | value) | MP       | value)  | S/CO | HBV DNA  Quantitative detection(IU/  ml) |                              |
+| 202202241                                                                                                                                     | negative | 33.9    | negative | 37.51    | 0.01 | 0.15                                       | -                            |
+| 202202242                                                                                                                                     | negative | 37.3    | 38.36    | 36.63    | 0.01 | 28.57                                      | -                            |
+| 202203051                                                                                                                                     | negative | 37.4    | negative | negative | 0.01 | 28.57                                      | Not submitted for inspection |
+| Note: "–" indicates that no HBV DNA is detected.                                                                                              | antiHBs  S/CO          |         |          |          |      |                                            |                              |
+
+| Serological characteristics of the HBV DNA positive and seronegative blood donations (n = 67).  HBsAg anti-HBs anti-HBc   | quantity   | Percent (%)   |    |       |
+|---------------------------------------------------------------------------------------------------------------------------|------------|---------------|----|-------|
+| -                                                                                                                         | -          | -             | 4  | 5.97  |
+| -                                                                                                                         | +          | -             | 8  | 11.94 |
+| -                                                                                                                         | +          | +             | 17 | 25.37 |
+| -                                                                                                                         | -          | +             | 38 | 56.72 |
+
+Table 6 
+
+Anti-HBs and *anti*-HBc characteristics of HBV DNA positive blood donors (n = 67). 
+
+anti-HBs(IU/L) Number of *anti*-HBs(%) Number of *anti*-HBc positives(%) Number of *anti*-HBc negative samples(%) 
+
+200–500 3(4.48) 1 2 
+
+100–200 4(5.97) 2 2 
+
+20–100 12(17.91) 8 4 10–20 6(8.96) 6 0 
+
+<10 42(62.69) 38 4 
+
+total 67 (100) 55 12 
+
+Note: *Anti*-HBs concentration ≥10 mIU/mL is considered positive. 
+
+quality control, in-house quality control, labor costs, utilities, and equipment depreciation. 
+
+Cost comparison: Approximately 1 in 5 samples will test positive for *anti*-HBc. With a 20% positivity rate for *anti*-HBc, the cost of a single test using a mixed test approach is twice that of a pure mixed test [(4 + 6)/5 = 2](Table 7). 
+
+## 4. **Discussion**
+
+HBV and HCV infections are major public health concerns in China, with China having the highest number of HBV and HCV infections in the world. In 2018, it was estimated that there were approximately 80 million cases of chronic HBV infection in China [2]. 
+
+The economic burden of these infections is also significant, with the average annual cost of hospitalization for each HBV-infected patient estimated to be 4454.0 USD in direct costs, 924.3 USD in indirect costs, and 6611.10 USD in intangible costs [3]. 
+
+In 2010, the Shandong Blood Center participated in a pilot project organized by the National Ministry of Health to introduce nucleic acid testing (NAT) as a screening modality for enzyme immunoassay negative samples. By 2015, the national blood collection and supply system had fully implemented NAT screening technology, enabling testing of all blood donors to mitigate the risk of transmitting HBV, HCV, and HIV through blood donations. 
+
+According to a study conducted by Chao Liu et al. from 2010 to 2015, a total of 20,084,187 seronegative blood donors underwent NAT screening in China. Among these, the number of HBV DNA-positive samples detected in seronegative blood donations was 1/ 1,482, resulting in a detection rate of 0.0675% (6.75 per 10,000) [1]. Our investigation unveiled that in Jinan, Shandong Province, there was a detection of 1 HBV DNA-positive blood donor for every 3195 (6-MP), equating to a detection rate of 0.031% (310 per 
+
+Table 7 
+
+NAT detection cost. 
+
+Nucleic acid detection model 
+
+unit price 
+
+(RMB) 
+
+Test semples 
+
+total 
+
+(RMB) 
+
+Number of HBV DNA positive cases 
+
+1 case of HBV DNA positive cost was detected 
+
+(RMB) 
+
+6-MP 66.6 220,445 14,681,637 69 212,777 Single sample detection 399.6 164 65,534.4 2 32,767.2 
+
+Note: Roche charges based on the number of individuals for whom test reports are issued, at a rate of RMB 66.6 per sample. Roche's nucleic acid 
+
+reagent can simultaneously detect HBV DNA, HIV RNA, and HCV RNA using a 3-in-1 specification, with a conventional detection mode of mixing 6 samples in one reaction pool for testing. In this study, the single detection reagent was also 3-in-1, therefore the cost was calculated as 66.6*7 RMB per 
+
+unit. 
+
+million). This rate was slightly lower than the detection rate of 0.037% (103/280,818) from 2015 to 2017 [4], less than half of the national average, and higher than the rate of 0.025% in India (20/79,938) [5]. Comparing these findings to other regions, the detection rate of HBV DNA-positive blood donors from 2008 to 2010 was 20.9 per million in Japan [6], while in Europe, the detection rate was generally lower. For instance, the detection rates in Germany [7], France [8], and the Netherlands [9] were 0.64 per million, 0.88 per million, and 0.9 per million, respectively. The aforementioned findings further support the positive correlation between the positive detection rate of HBV DNA testing and the prevalence of *anti*-HBc in the population. For example, in our study, 18.95% of HBsAg-negative blood donors from Shandong, China were found to be positive for *anti*-HBc, while only 0.71% of blood donors from the Netherlands were identified as positive for *anti*-HBc during initial screening [9]. In Germany, there were 0.55 cases of *anti*-HBc positivity per one million blood donations [7]. 
+
+Anti-HBc can be detected during asymptomatic infection and after recovery from HBV infection [10]. In contrast, *anti*-HBs may not always be produced as a result of HBV infection. Therefore, *anti*-HBc is the most critical serum marker for OBI, and screening for anti-HBc can exclude most OBI that cannot be detected by NAT [6,11–13]. Liu et al.'s research indicates that 77.36% of blood donors who tested negative for HBsAg but positive for HBV DNA were positive for *anti*-HBc [1]. This suggests that screening for *anti*-HBc can effectively prevent the clinical use of up to 77.36% of HBV DNA-positive blood products. In this study, the proportion of OBI among HBV DNA positive blood donors in Jinan, Shandong Province was 82.09% (55/67), which is higher than the national average and very close to the 83.57% (229/274) reported by the Blood Center of Zhejiang Province [14]. 
+
+In accordance with the Blood Donation Law of the People's Republic of China, screening for *anti*-HBc is not a mandatory test for blood donors. This is mainly because China is an endemic country for HBV infection. Based on the results of a national epidemiological survey on viral hepatitis from 1992 to 1995, the prevalence of HBV infection in the Chinese population was 57.6%, with a HBsAg positive rate of 9.75% and an *anti*-HBc positive rate of 49.8%. Universal hepatitis B vaccination for newborns was implemented in China in 1992, resulting in a rapid decrease in the prevalence of hepatitis B. By 2006, the prevalence of HBV infection in the adult population had decreased to between 42.40% and 80.77%, with a HBsAg positive rate of 7.18%, representing a decrease of 26.36% 
+[15]. Ling Ouyang et al.'s survey on HBV infection in blood donors showed that the prevalence of *anti*-HBc positivity was 63.4% among blood donors in Shenzhen between 1998 and 2000 [16]. In 2013, the prevalence rates of HBsAg and *anti*-HBc among children aged 1–14 years in Guangdong Province were 1.16% and 2.35%, respectively [17]. The fourth national hepatitis B serological survey in 2014 showed that the HBsAg positivity rate among children aged 1–4 years was 0.3%, a decrease of over 60% compared to 2006 [18]. 
+
+In the present study, it was observed that the HBV core antibody positivity rate among non-student blood donors was 22.97% during the period from 2021 to 2022, which is 5.1 times greater than that observed among university students (4.50%). This reflects the benefits of universal hepatitis B vaccination in China since 1992. However, the overall positivity rate of core antibodies among the entire blood donor population was still 18.95%. Discarding blood that tests positive for HBV core antibody could exacerbate the existing shortage of blood products in Shandong Province. 
+
+To prevent transmission of OBI through blood transfusion, developed countries like those in Europe and the United States routinely screen blood donors for *anti*-HBc and discard blood from donors who test positive for *anti*-HBc. According to a WHO report, countries around the world employ three main strategies for screening blood donors for HBV: (a) 56% (98/176) of countries employ HBsAg testing; (b) 17.6% (31/176) of countries employ both HBsAg and *anti*-HBc testing (with 5 countries, including selective *anti*-HBc testing); (c) Another 8 countries implemented a 3-testing strategy that included selective *anti*-HBc testing [19]. In Sao ˜ Paulo, Brazil, 0.6% (6/976) of *anti*-HBc positive blood donors who were negative for HBsAg and 6 MP-NAT were positive for single-sample HBV DNA 
+testing [20]. This rate is half as low as that found in Shandong Province, China, where 1.2% (2/164) of donors were positive for HBV DNA testing. The NAT single screening mode detects HBV DNA-positive samples in serologically negative cases with a detection rate of 0.22%, calculated as the product of the *anti*-HBV core antibody positive rate of 18.95% and the HBV DNA-positive rate of 1.22%. 
+
+However, in mixed samples, the detection rate is markedly lower at 0.031%, suggesting that 85.90%【(0.22–0.031)/0.22】 of samples with a low viral load of occult HBV infection (OBI) were not detected in mixed tests. These findings were similar to those of a study by the American Red Cross. In that study, nucleic acid testing was performed on 142 OBI-positive blood donors, and 121 (85%) samples were negative for HBV DNA by multiplex nucleic acid testing using single-well, but positive by single-sample testing [21]. The Japanese Red Cross (JRC) analyzed blood from 4742 donors with low *anti*-HBc and *anti*-HBs antibody titers using individual-donation nucleic acid testing (ID-NAT), which increased the OBI detection rate from 3.9 to 15.2 per million, and confirmed the OBI transmission rate from 0.67 to 1.49 per million [6]. A study by Spreafico M et al. in Italy performed supplemental ID-NAT on previously retained blood from HBV DNA positive donors screened for 6-MP, and the results showed that 6-MP HBV DNA screening did not detect 14/28 (50%) viremic donations that were released for transfusion. HBV marker testing of the recipients of these blood products identified 2 cases of transfusion-transmitted HBV infection, recorded as donor-recipient genetic identity [22]. A study by Daniel Candotti et al. also demonstrated that HBV viral load as low as 2.3 IU/mL or even lower in serum could still cause 29% (9/31) of transfusion infections, including 7 infected individuals transfused with plasma and 2 infected individuals transfused with red blood cells containing 20 mL plasma [23]. In addition to false negatives for HBV MP-NAT, HIV and HCV also have infectious donations in which MP-NAT is not detected but ID-NAT is reactive [24]. The cost of one sample in Roche single-test mode is about 399.6 RMB. In 2021, Shandong Blood Center tested about 140,000 samples (100,000 whole blood samples and 40,000 apheresis platelet samples). 
+
+With a grant of RMB 3 million per year from the financial department, if individual-donation supplemental ID-NAT was performed for all samples using Roche reagents, it would cost an additional RMB 52.94 million. However, Shandong Blood Center, which is a public charity relying solely on financial contributions, cannot afford the additional cost. 
+
+In this study, we first screened for *anti*-HBc and subsequently performed NAT single testing on *anti*-HBc positive samples. If we calculate the total cost of performing single testing on 20% of the samples (this study used two ELISA kits for detecting HBc antibodies, with a dual-kit positivity rate of 18.95%, but approximately 1% of single-kit positive samples were not included in the statistical 6 analysis, hence we used a positivity rate of 20% for the calculation) and mixed testing on 80% of the samples, the expected cost will double. (The cost of the *Anti*-HBc ELISA kit is very low, less than 0.1 yuan per person, and is not included in the total cost.) Based on the collection of 100,000 samples per year by the Shandong Blood Center, routine screening could detect only 31 HBV DNA positive samples (Table 1). However, theoretically, using the *anti*-HBc positive and NAT single testing mode, an additional 244 HBV DNA 
+positive OBI samples could be detected (calculated as 100000*0.20*0.0122), resulting in a detection rate of 7.87 times higher than routine screening. Although this method incurs twice the cost, it is relatively cost-effective. Furthermore, according to the detailed data from this study, the prevalence of occult hepatitis B infection (OBI) among blood donors should be approximately 8.9 times higher than the detection rate of current screening methods. This was determined by the calculated detection rate of a single screening mode, which was found to be 8.9 times greater than that of mixed screening (calculated using formula (0.22 + 0.031)/0.031). It should be noted that the prevalence of occult hepatitis B infection (OBI) may vary depending on the specific population and screening methods used. For instance, in this study, confirmation was not performed on a sample that tested positive by Roche single assay but negative by other testing methods. Moreover, most of these low-level samples were below 10 IU/mL, which could potentially be missed even by the most sensitive single-sample NAT (Roche LOD 1.6 IU/mL). Additionally, the small sample size used in our single testing approach may have contributed to significant statistical errors. Therefore, further research is warranted to confirm these findings. 
+
+In situations where blood collection and supply institutions face financial constraints and cannot perform both *anti*-HBc screening and NAT single tests simultaneously, screening for *anti*-HBc alone can be sufficient. *Anti*-HBc negative blood can be transfused to recipients who are negative for *anti*-HBs and have low levels of *anti*-HBs antibodies. Furthermore, *anti*-HBc positive samples can be screened with supplemental *anti*-HBs testing, and blood products with high levels of *anti*-HBs can be allocated to clinical recipients. It is advisable to avoid giving blood that is only *anti*-HBc positive and/or has low levels of *anti*-HBs antibodies to recipients with weaker immune systems against HBV. For instance, Allain JP et al. demonstrated that in recipients who were unvaccinated and received *anti*HBs-negative occult bloodborne infection (OBI) blood products, the prevalence of *anti*-HBc increased to 63.8% (28/44) in a background of 15% *anti*-HBc-positive recipients. In contrast, *anti*-HBc positivity was observed in only 15.4% (4/26) of *anti*-HBs-positive blood product recipients. The presence of *anti*-HBs (titer: 20–160 IU/L) in donors reduced the risk of HBV transmission via transfusion by approximately five-fold [25]. In Japan, blood centers discard blood products with low *anti*-HBc and *anti*-HBs titers, which represent 1.3% of total donations. Conversely, *anti*-HBc positive blood containing *anti*-HBs levels of 200 IU/L and above is considered to pose a low risk of transmitting HBV [6,26]. However, some countries like Germany and Austria consider blood with *anti*-HBs levels greater than 100 IU/L to pose a low risk of transmitting HBV [27]. Employing the criteria utilized by countries like Germany and Austria to ascertain low-risk blood, only 10% (Table 6,7/67) of occult blood-borne infections (OBI) cases in our study exhibited *anti*-HBs levels exceeding 100 IU/L, signifying their classification as low-risk samples. These findings imply that the presence of detectable *anti*-HBs in blood may not guarantee complete safety. Additionally, 17.91% (Table 6, 12/67) of HBV DNA positive patients were found to be in the window period, with 80% of those infected during this period being *anti*-HBc-negative individuals. Therefore, a single test for screening the low-load window period may not be cost-effective in *anti*-HBc-negative populations. A study by Ramachandran et al. in the USA also demonstrated that using the more sensitive Ultrio Plus assay (3.4 IU/mL) instead of Procleix Ultrio (10.4 IU/mL) for screening HBV DNA did not significantly reduce residual blood infectivity. However, individual nucleic acid detection is required for detecting occult bloodborne infection (OBI) in 92% (537/583) of OBI blood donors [28]. The choice of nucleic acid single or mixed detection has a significant impact on the detection rate of OBI, but has little impact on the detection of the window period. Unfortunately, due to limited funding, we did not examine the detection rate of the window period in *anti*-HBc-negative samples with HBV 
+DNA levels ranging from 2.3 to 14 IU/mL in this study. 
+
+Currently, there is limited available data on the prevalence of HBV infections and corresponding statistics regarding blood donors in China with an HBV DNA load ranging from 2.3 to 14 IU/mL.Our study suggests that a conservative estimate of 509 blood donors with low-load HBV DNA levels (220,445*0.1895*0.0122) may have gone undetected over the past five years. It is important to note that this figure does not account for individuals with inconclusive test results or those in the window period of initial HBV infection. This may be due to inadequate awareness of the infection risks associated with low-load HBV DNA, as well as insufficient follow-up of blood recipients who received such blood units. However, transfusion-related infections caused by occult HBV infection (OBI) with very low viral loads are associated with the amount of transfused blood and blood components, and can be prevented by *anti*-HBs. 
+
+Therefore, a rational transfusion strategy can be developed by conducting *anti*-HBc and *anti*-HBs tests in combination with NAT single tests for blood donors, as well as *anti*-HBc and *anti*-HBs tests for blood recipients, in order to minimize the risk of HBV transfusional transmission. 
+
+## Author Contribution Statement
+
+Jianfeng Chen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. 
+
+Zili Ma: Performed the experiments; Wrote the paper. 
+
+Dandan Wu, Qi Zuo: Performed the experiments. 
+
+Fengtian Wang, Chen Xiao, Fuqiang Chen, Peng Li: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data. 
+
+## Data Availability Statement
+
+Data included in article/supp. material/referenced in article. 
+
+## Funding Statement
+
+This research was generously supported by the Shandong Province Humanities and Social Science Project (2021-ZXJK-24), as well as the Shandong Province Projects of Medical and Health Technology Development Program (2019WS537, 2019WS538, 2019WS541, 2019WS542, 202011000934). 
+
+## Declaration Of Competing Interest
+
+I, Chen Jianfeng, hereby declare the following potential conflicts of interest associated with the submitted manuscript titled 
+"Evaluating the Cost-effectiveness of Low-level HBV DNA Screening in Occult Hepatitis B Infection Donors: A Study from Shandong Blood Center, China" to the journal Heliyon: 
+1. The research conducted for this study does not involve any conflicts of interest that could bias or distort the research findings. 
+
+This study received funding from the Shandong Health Science and Technology Development Plan and the Research Fund of Blood Screening Technology from the Chinese Medical Equipment Association Transfusion Medical Equipment Technology Professional Committee. The researchers involved in this study have no financial interests related to the research. 
+
+2. The data and analysis methods used in this study are accurate and reliable, and the conclusions are objective and unbiased. There was no manipulation of the research results during the study process. 
+
+3. All authors of this study have reviewed and approved the final version of the manuscript. The corresponding author, Chen Jianfeng, on behalf of all authors, has authorized the submission of the manuscript to Heliyon journal for review. 
+
+4. This manuscript has not been previously published in any other journal, nor is it under consideration for publication elsewhere. 
+
+The content of this manuscript is original and does not involve any plagiarism or duplication of other research findings. 
+
+5. If the manuscript is accepted for publication in Heliyon journal, the copyright will be granted exclusively to the publisher for global publication. The authors agree that the abstract of the paper may be included in other journals and indexed in databases. 
+
+## Acknowledgements
+
+We express our heartfelt gratitude to Dr. Wang Lunan, the Director and Researcher at the Blood Safety and Immunohematology Laboratory, National Center for Clinical Laboratories, for her invaluable aid and guidance. We also extend our sincere appreciation to Dr. Chang Le for her generous provision of technical assistance. Furthermore, we would like to express our sincere thanks to Dr. Huang Liqin of the Shenzhen Blood Center Laboratory for his invaluable aid in facilitating our access to research reagents and providing technical consultation. Finally, we extend our profound appreciation to the esteemed companies Abbott and Roche for their unwavering support in providing scientific reagents and technical assistance. We are grateful for their assistance and collaboration. 
+
+## Appendix A. **Supplementary Data**
+
+Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e18609. 
+
+## References
+
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